Tachykinin receptors in the guinea-pig isolated bronchi.

PubMed

Maggi, C A; Patacchini, R; Quartara, L; Rovero, P; Santicioli, P

1991-05-17

The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery.

Activation of neurokinin NK(2) receptors by tachykinin peptides causes contraction of uterus in pregnant women near term.

PubMed

Patak, E N; Ziccone, S; Story, M E; Fleming, A J; Lilley, A; Pennefather, J N

2000-06-01

The aim of this study was firstly to elucidate whether the mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)-regulated contractility of myometrium obtained from near-term pregnant women, and secondly to investigate the receptor subtype(s) responsible. In the presence of peptidase inhibitors, i.e. thiorphan (3 micromol/l; endopeptidase 24.11 inhibitor), captopril (10 micromol/l; angiotensin converting enzyme inhibitor) and bestatin (10 micromol/l; aminopeptidase inhibitor); all three mammalian tachykinins elicited concentration-related contractions of isolated myometrial preparations. The rank order of agonist potency of the mammalian tachykinins in the presence of the peptidase inhibitors was NKA > SP = NKB, indicating that the contractile effects were mediated by activation of an NK(2) receptor. The NK(2) receptor-selective agonist, [Lys(5), MeLeu(9), Nle(10)]NKA(4-10), produced concentration-related contractile responses, while the respective NK(1) and NK(3) receptor-selective agonists, [Sar(9), Met(O(2))(11)]SP and [N-MePhe(7)]NKB, had no effect either in the absence or presence of the peptidase inhibitors. The NK(2) receptor-selective antagonist, SR48968, produced concentration-related rightward shift in the log concentration curve to [Lys(5), MeLeu(9), Nle(10)]NKA(4-10). This study shows that tachykinins elicit contractile effects on human myometrium obtained from pregnant women near term, and that these effects are mediated by an NK(2) receptor. An excitatory effect of the tachykinins on these preparations could indicate a physiological role for these peptides in enhancing contractility of the uterus in women at term.

NK2 tachykinin receptors and contraction of circular muscle of the human colon: characterization of the NK2 receptor subtype.

PubMed

Giuliani, S; Barbanti, G; Turini, D; Quartara, L; Rovero, P; Giachetti, A; Maggi, C A

1991-10-22

The contractile effect of substance P, neurokinin A, receptor selective agonists for tachykinin receptors and NK2 tachykinin receptor antagonists was investigated in mucosa-free circular strips of the human isolated colon. Neurokinin A and substance P produced concentration-dependent contractions which approached 80-90% of the maximal response to carbachol. Neurokinin A was about 370 times more potent than substance P. The action of neurokinin A and substance P was not modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The NK2 receptor selective agonist, [beta-Ala8]neurokinin A-(4-10) closely mimicked the response to neurokinin A while NK1 and NK3 receptor selective agonists were active only at microM concentrations. The pseudopeptide, MDL 28,564, which is one of the most selective NK2 ligands available, behaved as a full agonist. Responses to [beta-Ala8]neurokinin A were antagonized by NK2 receptor selective antagonists, with the rank order of potency MEN 10,376 greater than L 659,877 much greater than R 396. These data indicate that NK2 tachykinin receptors play a dominant role in determining the contraction of the circular muscle of the human colon to peptides of this family. The NK2 receptor subtype responsible for this effect belongs to the same subtype (NK2A) previously identified in the rabbit pulmonary artery and guinea-pig bronchi.

Parainfluenza virus type 3 induced alterations in tachykinin NK1 receptors, substance P levels and respiratory functions in guinea pig airways.

PubMed

Kudlacz, E M; Shatzer, S A; Farrell, A M; Baugh, L E

1994-08-03

We have investigated the effects of parainfluenza virus type 3 (PI-3) on sensory neuropeptide levels, tachykinin receptors and their functions in guinea pig airways during the course of respiratory viral infection. PI-3 infected guinea pigs were hyperresponsive to methacholine and substance P aerosols as determined by earlier onset of dyspnea in these animals as compared with control on post-inoculation day (PID) 7 but not 19. In addition, plasma protein extravasation produced in response to the tachykinin was increased in infected airways during the first week post inoculation. Infected guinea pig trachea did not respond any differently to methacholine when smooth muscle contraction and [3H]inositol phosphate accumulation were measured although the magnitude of substance P effects using in vitro tests was significantly greater than control on post-inoculation day 7 but not 19. Trachea from PI-3 infected animals were characterized by reductions in substance P-like immunoreactivity, tachykinin NK1 receptor number and agonist affinity during the first post-inoculation week. Substance P levels or tachykinin NK1 receptor numbers or affinity were not altered in trachea of guinea pigs 4 days after treatment with lipopolysaccharide. These data suggest substance P release occurs during critical periods of respiratory viral infection which are temporally correlated with airway hyperresponsiveness. Despite apparent down-regulation of tachykinin NK1 receptors, substance P-mediated functions remained enhanced suggesting some alterations in post-receptor mechanisms.

Tachykinin receptors in the guinea-pig renal pelvis: activation by exogenous and endogenous tachykinins.

PubMed Central

Maggi, C. A.; Patacchini, R.; Eglezos, A.; Quartara, L.; Giuliani, S.; Giachetti, A.

1992-01-01

1. The contractile response to substance P, neurokinin A, selective agonists for the NK1, NK2 and NK3 tachykinin receptors and the activity of receptor-selective antagonists has been investigated in circular muscle strips of the guinea-pig isolated renal pelvis in the presence of indomethacin (3 microM). 2. Neurokinin A was the most potent agonist tested, being about 32 times more potent than substance P. The action of both substance P and neurokinin A was enhanced by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The selective NK2 receptor agonist [beta Ala8] neurokinin A (4-10), was slightly less potent and effective than neurokinin A itself. The selective NK1 receptor agonist [Sar9] substance P sulphone was effective at low (nM) concentrations but its maximal effect did not exceed 30% of maximal response to substance P or neurokinin A. The NK3-selective agonist [MePhe7] neurokinin B was effective only at high (microM) concentrations. 3. The pseudopeptide derivative of neurokinin A(4-10), MDL 28,564, displayed a clear-cut agonist character, although it was less potent than neurokinin A. 4. The responses to roughly equieffective (25-35% of maximal response) concentrations of [beta Ala8] neurokinin A (4-10), MDL 28,564 and [MePhe7] neurokinin B were antagonized to a similar extent by MEN 10,376 (3 microM), a selective NK2 tachykinin receptor antagonist, while the response to [Sar9] substance P sulphone was unchanged. 5. The response to [Sar9] substance P sulphone was inhibited by the NK1 receptor-selective antagonist, GR 82,334 (3 microM) while the response to [beta Ala8] neurokinin A (4-10) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1384907

Tachykinins mediate contraction of the human lower esophageal sphincter in vitro via activation of NK2 receptors.

PubMed

Huber, O; Bertrand, C; Bunnett, N W; Pellegrini, C A; Nadel, J A; Nakazato, P; Debas, H T; Geppetti, P

1993-08-03

The contractile response to natural tachykinins and selective peptide agonists for tachykinin receptors was studied in strips of circular smooth muscle of human lower esophageal sphincter in vitro. The effects of phosphoramidon, which inhibits neutral endopeptidase (EC.3.4.24.11) and of the non-peptide compounds, SR 48968 and CP-96,345, which selectively block NK1 and NK2 receptors, respectively, were also investigated. Substance P, neurokinin A and neurokinin B produced a concentration-dependent contractile response. The rank order of potency was neurokinin A > neurokinin B > substance P. Phosphoramidon (1 microM) potentiated the response to substance P without changing the order of potency of natural tachykinins. The NK2-selective agonist, ([ beta Ala8]neurokinin A-(4-10)), produced a concentration-dependent contraction. The NK1 ([Sar9,Met(O2)11]substance P, 1 microM) and NK3 ([MePhe7]neurokinin B, 1 microM) selective agonists, however, did not exert any contractile effect. The selective NK2 antagonist, SR 48968, potently inhibited in a concentration-dependent (10 nM-1 microM) manner the response to neurokinin A, without affecting the response to carbachol. The selective NK1 antagonist, CP-96,345 (1 microM), did not affect the response to neurokinin A. These results indicate that tachykinins contract the circular muscle of human lower esophageal sphincter, and that this effect is mediated by NK2 receptor stimulation. Moreover, a phosphoramidon-sensitive mechanism plays a role in the regulation of the response to substance P.

Facilitatory effects of selective agonists for tachykinin receptors on cholinergic neurotransmission: evidence for species differences.

PubMed Central

Belvisi, M. G.; Patacchini, R.; Barnes, P. J.; Maggi, C. A.

1994-01-01

1. Exogenous tachykinins modulate cholinergic neurotransmission in rabbit and guinea-pig airways. We have investigated the effect of selective tachykinin receptor agonists and antagonists on cholinergic neurotransmission evoked by electrical field stimulation (EFS) of bronchial rings in rabbit, guinea-pig and human airways in vitro to assess which type of tachykinin receptor is mediating this facilitatory effect. 2. Bronchial rings were set up for isometric tension recording. Contractile responses to EFS (60 V, 0.4 ms, 2 Hz for 10 s every min) and exogenous acetylcholine (ACh) were obtained and the effects of selective tachykinin agonists and antagonists were investigated. 3. In rabbit bronchi the endogenous tachykinins, substance P (SP) and neurokinin A (NKA) (10 nM) potentiated cholinergic responses to EFS (by 287.6 +/- 121%, P < 0.01 and 181.4 +/- 56.5%, P < 0.001 respectively). 4. The NK1 receptor selective agonist, [Sar9]SP sulphone (10 nM) evoked a maximal facilitatory action on cholinergic responses of 334.9 +/- 63% (P < 0.01) (pD2 = 8.5 +/- 0.06) an effect which was blocked by the selective NK1-receptor antagonist, CP 96,345 (100 nM) (P < 0.05) but not by the NK2 receptor antagonist, MEN 10,376 (100 nM). The NK2 receptor selective agonist, [beta Ala8]NKA(4-10) (10 nM), produced a maximum enhancement of 278 +/- 83.5% (P < 0.01) (pD2 = 8.7 +/- 0.1) an effect which was blocked by MEN 10,376 (100 nM) (P < 0.05) and not by CP 96,345. [MePhe7]NKB, an NK3 receptor selective agonist was without effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7516799

Effects of tachykinins on uterine smooth muscle.

PubMed

Patak, E N; Pennefather, J N; Story, M E

2000-11-01

1. Sensory nerves supplying the mammalian uterus have been shown to contain substance P (SP) and neurokinin (NK)A. This review presents some of the advances that have led to a greater understanding of the effects of tachykinins on uterine smooth muscle. 2. The cell-surface peptidase neprilysin (EC.3 24.11, endopeptidase 24.11, enkephalinase, CALLA, CD10) has been shown to play a major role in regulating the actions of tachykinins on both rat and human myometrium. Because this peptidase is known to be regulated by steroids and pregnancy, its effects may be of physiological relevance. 3. Tachykinins produce contractions of isolated myometrial preparations from non-pregnant rats and mice. The NK2 receptor mediates these effects in rat uterus, while the NK1 receptor may mediate these effects in the mouse uterus. 4. The effects of tachykinins have been examined on myometrial preparations obtained at Caesarean section from near-term pregnant women. In the presence of the peptidase inhibitors (thiorphan, captopril and bestatin), the mammalian tachykinins SP, NKA and NKB produced concentration-dependent uterine contractions. 5. The order of agonist potency NKA > SP = NKB suggested that NK2 receptors mediate uterine contractions in the human. This was confirmed using the stable analogues [Sar9,Met(O2)11]SP, [Lys5MeLeu9Nle10]NKA(4-10) and [N-MePhe7]NKB, which are NK1, NK2 and NK3 receptor selective, respectively. Only [Lys5MeLeu9Nle10]NKA(4-10) produced concentration-related contractions of human uterine smooth muscle. 6. The experimental findings described in the present review, taken together with results published previously in the literature, indicate that tachykinin peptides may play a physiological or pathophysiological role in regulating uterine smooth muscle activity. However, more extensive research will be required to confirm such a role for these peptides.

Autocrine regulation of human sperm motility by tachykinins

PubMed Central

2010-01-01

Background We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. Methods Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). Results The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). Conclusion These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins. PMID:20796280

Autocrine regulation of human sperm motility by tachykinins.

PubMed

Pinto, Francisco M; Ravina, Cristina G; Subiran, Nerea; Cejudo-Román, Antonio; Fernández-Sánchez, Manuel; Irazusta, Jon; Garrido, Nicolas; Candenas, Luz

2010-08-26

We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

Tachykinin receptors in the guinea-pig renal pelvis: activation by exogenous and endogenous tachykinins.

PubMed

Maggi, C A; Patacchini, R; Eglezos, A; Quartara, L; Giuliani, S; Giachetti, A

1992-09-01

1. The contractile response to substance P, neurokinin A, selective agonists for the NK1, NK2 and NK3 tachykinin receptors and the activity of receptor-selective antagonists has been investigated in circular muscle strips of the guinea-pig isolated renal pelvis in the presence of indomethacin (3 microM). 2. Neurokinin A was the most potent agonist tested, being about 32 times more potent than substance P. The action of both substance P and neurokinin A was enhanced by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The selective NK2 receptor agonist [beta Ala8] neurokinin A (4-10), was slightly less potent and effective than neurokinin A itself. The selective NK1 receptor agonist [Sar9] substance P sulphone was effective at low (nM) concentrations but its maximal effect did not exceed 30% of maximal response to substance P or neurokinin A. The NK3-selective agonist [MePhe7] neurokinin B was effective only at high (microM) concentrations. 3. The pseudopeptide derivative of neurokinin A(4-10), MDL 28,564, displayed a clear-cut agonist character, although it was less potent than neurokinin A. 4. The responses to roughly equieffective (25-35% of maximal response) concentrations of [beta Ala8] neurokinin A (4-10), MDL 28,564 and [MePhe7] neurokinin B were antagonized to a similar extent by MEN 10,376 (3 microM), a selective NK2 tachykinin receptor antagonist, while the response to [Sar9] substance P sulphone was unchanged. 5. The response to [Sar9] substance P sulphone was inhibited by the NK1 receptor-selective antagonist, GR 82,334 (3 microM) while the response to [beta Ala8] neurokinin A (4-10) was unchanged. 6. The selective NK2 receptor antagonists MEN 10,376, L 659,877 and R 396 antagonized competitively the response to [PAla8] neurokinin A (4-10) with the following rank order of potency (pA2 values in parentheses): MEN 10,376 (7.41)>L 659,877 (7.15)>R 396 (6.43). MEN 10,376 and L 659,877 also competitively antagonized the response to neurokinin A, although with lower potency as compared to the selective NK2 receptor agonist.7. MEN 10,376, L 659,877 and R 396 reduced in a concentration-dependent manner the contractile response produced by electrical field stimulation (1 Hz, 100 V, 0.25 ms pulse width, trains of 10 s). The rank order of potency of NK2 receptor antagonists in blocking the response to electrical stimulation (MEN 10,376> L 659,877> R 396) closely mimicked their potency in antagonizing exogenous tachykinins.8. The inhibitory effect of MEN 10,376 toward responses produced by electrical field stimulation was significantly reduced when tested in the presence of peptidase inhibitors, which increased significantly the response to nerve stimulation.9. GR 82,334 (3 pM) did not significantly affect the response to nerve stimulation in untreated preparations and slightly reduced it in the presence of peptidase inhibitors.10. We conclude that both NK, and NK2 receptors mediate the contractile effect of tachykinins in the circular muscle of the guinea-pig renal pelvis and that the response ascribable to NK2 receptor stimulation is larger than that ascribed to NK, receptor stimulation. The NK2 receptor in the guinea-pig renal pelvis belongs to the same subtype previously identified in the rabbit pulmonary artery. NK2 receptors play a dominant role in the physiological response determined by the release of endogenous tachykinins and a contribution of NKI receptors becomes evident after inhibition of peptide degradation.

Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

PubMed

Hastrup, H; Schwartz, T W

1996-12-16

The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

Tachykinin-induced contraction of the guinea-pig isolated oesophageal mucosa is mediated by NK2 receptors

PubMed Central

Kerr, Karen P; Thai, Binh; Coupar, Ian M

2000-01-01

The tachykinin receptor present in the guinea-pig oesophageal mucosa that mediates contractile responses of the muscularis mucosae has been characterized, using functional in vitro experiments. The NK1 receptor-selective agonist, [Sar9(O2)Met11]SP and the NK3 receptor-selective agonists, [MePhe7]-NKB and senktide, produced no response at submicromolar concentrations. The NK2 receptor-selective agonists, [Nle10]-NKA(4–10), and GR 64,349 produced concentration-dependent contractile effects with pD2 values of 8.20±0.16 and 8.30±0.15, respectively. The concentration-response curve to the non-selective agonist, NKA (pD2=8.13±0.04) was shifted significantly rightwards only by the NK2 receptor-selective antagonist, GR 159,897 and was unaffected by the NK1 receptor-selective antagonist, SR 140,333 and the NK3 receptor-selective antagonist, SB 222,200. The NK2 receptor-selective antagonist, GR 159,897, exhibited an apparent competitive antagonism against the NK2 receptor-selective agonist, GR 64,349 (apparent pKB value=9.29±0.16) and against the non-selective agonist, NKA (apparent pKB value=8.71±0.19). The NK2 receptor-selective antagonist, SR 48,968 exhibited a non-competitive antagonism against the NK2 receptor-selective agonist, [Nle10]-NKA(4–10). The pKB value was 10.84±0.19. It is concluded that the guinea-pig isolated oesophageal mucosa is a useful preparation for studying the effects of NK2 receptor-selective agonists and antagonists as the contractile responses to various tachykinins are mediated solely by NK2 receptors. PMID:11090121

Tachykinin receptors and noncholinergic bronchoconstriction in the guinea-pig isolated bronchi.

PubMed

Maggi, C A; Patacchini, R; Rovero, P; Santicioli, P

1991-08-01

The aim of the study was to assess which type(s) of tachykinin receptor mediate the noncholinergic bronchoconstriction produced by activation (electrical field stimulation) of capsaicin-sensitive primary afferents in epithellum-denuded guinea-pig isolated bronchi. Experiments with natural and synthetic tachykinin agonists indicated the presence of both NK-1 and NK-2 receptors at this level. Experiments with the putative NK-1 (L668, 169) or NK-2 (MEN 10,207, MEN 10,376, L659,877, and R396) selective antagonists against NK-1 and NK-2 selective agonists further supported this conclusion. All the tachykinin antagonists tested reduced the noncholinergic bronchoconstriction to field stimulation with the order of potency MEN 10,207 = MEN 10,376 greater than L659,877 greater than L668,169 congruent to R396. In the presence of peptidase inhibitors, the activity of MEN 10,376 toward the noncholinergic bronchoconstriction was slightly reduced, whereas that of L668,169 was increased. These findings demonstrate that both NK-1 and NK-2 receptors mediate the noncholinergic constriction produced by endogenous tachykinins in guinea-pig bronchi and that the relative contribution of NK-2 receptors is greater than that of NK-1. These findings implicate a major role for neurokinin A rather than for substance P as an endogenous bronchoconstrictor in the guinea-pig isolated bronchi. In the presence of peptidase inhibitors, the relative contribution of NK-1 receptors is increased.

Facilitation and inhibition by capsaicin of cholinergic neurotransmission in the guinea-pig small intestine.

PubMed

Geber, Christian; Mang, Christian F; Kilbinger, Heinz

2006-01-01

The effects of capsaicin on [3H]acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [3H]choline. Capsaicin concentration-dependently increased both basal [3H]acetylcholine release (pEC50 7.0) and muscle tone (pEC50 6.1). The facilitatory effects of capsaicin were antagonized by 1 microM capsazepine (pK (B) 7.0 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 microM). This suggests that stimulation by capsaicin of TRPV1 receptors on primary afferent fibres causes a release of tachykinins which, in turn, mediate via NK1 and NK3 receptors an increase in acetylcholine release. The capsaicin-induced acetylcholine release was significantly enhanced by the NO synthase inhibitor L-NG-nitroarginine (100 microM). This indicates that tachykinins released from sensory neurons also stimulate nitrergic neurons and thus lead, via NO release, to inhibition of acetylcholine release. Capsaicin concentration-dependently reduced the electrically-evoked [3H]acetylcholine release (pEC50 6.4) and twitch contractions (pEC50 5.9). The inhibitory effects were not affected by either capsazepine, NK1 and NK3 receptor antagonists, the cannabinoid CB1 antagonist SR141716A or by L-NG-nitroarginine. Desensitization of TRPV1 receptors by a short exposure to 3 microM capsaicin abolished the facilitatory responses to a subsequent administration, but did not modify the inhibitory effects. In summary, capsaicin has a dual effect on cholinergic neurotransmission. The facilitatory effect is indirect and involves tachykinin release and excitation of NK1 and NK3 receptors on cholinergic neurons. The inhibition of acetylcholine release may be due to a decrease of Ca2+ influx into cholinergic neurons.

NK-2 is the predominant tachykinin receptor subtype in the swine ureter.

PubMed

Jerde, T J; Saban, R; Bjorling, D E; Nakada, S Y

1999-02-01

To determine which of the known tachykinin receptor subtypes is predominant in the swine ureter. Ureters from adult pigs were harvested, cut into longitudinal strips and placed in 10 mL tissue baths containing Krebs buffer, under 4 g of initial tension. The magnitude and frequency of contractions were recorded. Tissues were incubated with 1 micromol/L solutions of peptidase inhibitors (phosphoramidon and captopril) for 1 h to inhibit degradation of peptides and treated with either CP 96,345 (NK-1 receptor antagonist), SR 48,968 (NK-2 receptor antagonist) or saline (control). Concentration-response curves to the tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) were determined. Ureteric segments showed a concentration-dependent response to all tachykinins; NKA stimulated increased contractions at a lower concentration than either SP or NKB (P<0.05). This was reflected by the difference in the effective concentration required to obtain half the maximal response (EC50 ) for each of the peptides. The mean (sd) EC50 values were (micromol/L): NKA, 0.2 (0.02); SP, 3.5 (0.7); and NKB, 4.5 (1.7). In addition, the selective NK-2 antagonist (SR 48,968) significantly reduced contractile responses to all peptides, as indicated by a 10-fold rightward shift of the concentration-response curves (P<0. 05), whereas the NK-1 antagonist (CP 96,345) had no significant effect. These results indicate that NK-2 is the predominant tachykinin receptor subtype responsible for contraction of ureteric smooth muscle. The use of mediators which act on NK-2 receptors may have clinical applications for the treatment of ureteric disease.

Tachykinin receptors in the circular muscle of the guinea-pig ileum.

PubMed

Maggi, C A; Patacchini, R; Giachetti, A; Meli, A

1990-12-01

1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family. The response to activation of NK3-receptors is totally neurogenic and partially mediated by endogenous acetylcholine, the response to activation of NK1-receptors is partly neurogenic and largely myogenic and the response to activation of NK2-receptors is totally myogenic.

Tachykinin receptors in rabbit airways--characterization by functional, autoradiographic and binding studies.

PubMed Central

Black, J. L.; Diment, L. M.; Alouan, L. A.; Johnson, P. R.; Armour, C. L.; Badgery-Parker, T.; Burcher, E.

1992-01-01

1. In many species, both NK1 and NK2 tachykinin receptors appear to be important in mediating the contraction of airway smooth muscle. We have examined the distribution and characterization of receptors for tachykinins in rabbit airways using functional length tension studies, autoradiography and radioligand binding studies. 2. Contractile responses to tachykinins were elicited in four different areas of the respiratory tree--trachea, and three progressively more distal areas of the right bronchus. The NK2 receptor-preferring agonists, neurokinin A (NKA), neuropeptide gamma (NP gamma) and the NK2-selective [Lys5 MeLeu9, Nle10]-NKA(4-10) [NKA (4-10) analogue] produced similar contraction in all four areas. Substance P (SP) and the NK1-selective [Sar9,Met(O2)11]-SP (Sar-SP) exhibited a marked location-dependence in the magnitude of contraction, producing minimal contraction in the trachea and more proximal bronchi with contractions becoming progressively larger in the more distal airways. Senktide (which is selective for the NK3 receptor) produced negligible contraction in all areas. 3. The NK2-selective antagonist, MDL29,913, was a weak antagonist of NKA and NKA(4-10) analogue. At a concentration of 2 microM, it produced a small but significant shift in the response curve to NKA and a greater shift (8 fold) in the curve to NKA(4-10) analogue, but it had no effect on responses to Sar-SP. The non peptide NK1 receptor antagonist, CP-96,345, was also unexpectedly weak in this preparation. The pD2 value for Sar-SP was decreased 27 fold by CP-96,345 at a concentration of 1 microM, without alteration in the maximum response.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 PMID:1384914

Tachykinin receptors in the circular muscle of the guinea-pig ileum.

PubMed Central

Maggi, C. A.; Patacchini, R.; Giachetti, A.; Meli, A.

1990-01-01

1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1707710

Mechanisms underlying the inhibitory effects of tachykinin receptor antagonists on eosinophil recruitment in an allergic pleurisy model in mice

PubMed Central

Alessandri, Ana Letícia; Pinho, Vanessa; Souza, Danielle G; Castro, Maria Salete de A; Klein, André; Teixeira, Mauro M

2003-01-01

The activation of tachykinin NK receptors by neuropeptides may induce the recruitment of eosinophils in vivo. The aim of the present study was to investigate the effects and underlying mechanism(s) of the action of tachykinin receptor antagonists on eosinophil recruitment in a model of allergic pleurisy in mice. Pretreatment of immunized mice with capsaicin partially prevented the recruitment of eosinophils after antigen challenge, suggesting the potential contribution of sensory nerves for the recruitment of eosinophils Local (10–50 nmol per pleural cavity) or systemic (100–300 nmol per animal) pretreatment with the tachykinin NK1 receptor antagonist SR140333 prevented the recruitment of eosinophils induced by antigen challenge of immunized mice. Neither tachykinin NK2 nor NK3 receptor antagonists suppressed eosinophil recruitment. Pretreatment with SR140333 failed to prevent the antigen-induced increase of interleukin-5 concentrations in the pleural cavity. Similarly, SR140333 failed to affect the bone marrow eosinophilia observed at 48 h after antigen challenge of immunized mice. SR140333 induced a significant increase in the concentrations of antigen-induced eotaxin at 6 h after challenge. Antigen challenge of immunized mice induced a significant increase of Leucotriene B4 (LTB4) concentrations at 6 h after challenge. Pretreatment with SR140333 prevented the antigen-induced increase of LTB4 concentrations. Our data suggest an important role for NK1 receptor activation with consequent LTB4 release and eosinophil recruitment in a model of allergic pleurisy in the mouse. Tachykinins appear to be released mainly from peripheral endings of capsaicin-sensitive sensory neurons and may act on mast cells to facilitate antigen-driven release of LTB4. PMID:14585802

Effects of two novel tachykinin antagonists, FK224 and FK888, on neurogenic airway plasma exudation, bronchoconstriction and systemic hypotension in guinea-pigs in vivo.

PubMed Central

Hirayama, Y.; Lei, Y. H.; Barnes, P. J.; Rogers, D. F.

1993-01-01

1. We compared the effects of two novel tachykinin receptor antagonists, FK888 (selective at the tachykinin NK1 receptor) and FK224 (dual antagonist at NK1 and NK2 tachykinin receptors) on stimulus-evoked airway plasma exudation, bronchoconstriction and systemic hypotension in guinea-pigs in vivo. Plasma exudation was induced by substance P (SP), synthetic tachykinin receptor agonists, platelet activating factor (PAF), electrical stimulation of the cervical vagus nerves or by inhalation of cigarette smoke. Changes in airway tone and in carotid artery blood pressure (BP) were induced by synthetic tachykinin agonists, PAF and vagal stimulation. 2. Both FK224 and FK888 dose-dependently inhibited SP-induced plasma exudation in the lower trachea and main bronchi (ID50 values respectively of 1.1 and 0.1 mumol kg-1 in lower trachea, and of 0.5 and 0.1 mumol kg-1 in main bronchi) with complete inhibition at both airway levels at 10 mumol kg-1 for FK224 and at 2 mumol kg-1 for FK888. 3. The NK1-selective tachykinin receptor agonist, [Sar9,Met(O2)11]substance P ([Sar]SP), induced plasma exudation, a response which was blocked by both FK888 and FK224. The NK2-selective agonist, [beta-Ala8]neurokinin A-(4-10) ([beta-Ala]NKA), did not induce plasma exudation: neither FK888 nor FK224 affected this lack of response to [beta-Ala]NKA. 4. [beta-Ala]NKA induced bronchoconstriction, a response which was blocked by FK224 but which was completely unaffected by FK888. [Sar]SP induced a small but significant bronchoconstriction which was completely inhibited by both tachykinin antagonists. 5. In animals pretreated with capsaicin to deplete sensory neuropeptides, PAF induced both plasma exudation and bronchoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7682142

Tachykinin-mediated respiratory effects in conscious guinea pigs: modulation by NK1 and NK2 receptor antagonists.

PubMed

Kudlacz, E M; Logan, D E; Shatzer, S A; Farrell, A M; Baugh, L E

1993-09-07

Tachykinins, in particular neurokinin A and substance P, produce a number of airway effects which may contribute to respiratory diseases such as asthma. We examined the ability of aerosolized substance P, neurokinin A or capsaicin to produce respiratory alterations in conscious guinea pigs using modified whole body plethysmography. Substance P-mediated dyspnea and significant respiratory events were inhibited by the NK1 receptor antagonist, CP-96,345. Neurokinin A-mediated respiratory effects were ablated by the NK2 receptor antagonists: MEN 10207, MDL 29,913 and SR 48,968, the latter being the most potent. The peptide-based antagonist, MEN 10207, produced respiratory effects itself suggesting partial agonist activity. The cyclic hexapeptide, MDL 29,913, relaxed airway smooth muscle via mechanisms other than tachykinin antagonism. NK2 but not NK1 receptor antagonists were able to delay the onset of capsaicin-induced dyspnea, although alone they did not usually (in approximately 10% of the animals) eliminate the response. However, when NK2 receptor antagonists were combined with CP-96,345, the incidence of dyspnea induced by capsaicin decreased significantly (40%) suggesting that both tachykinins contribute to dyspnea in this system.

Substance P relaxes rat bronchial smooth muscle via epithelial prostanoid synthesis.

PubMed

Bodelsson, M; Blomquist, S; Caverius, K; Törnebrandt, K

1999-01-01

Substance P is present in bronchial nerve fibres. The physiological actions of substance P are mediated via tachykinin NK(1) receptors. Immunochemical studies have demonstrated tachykinin NK(1) receptors in the rat airway epithelium. To elucidate how epithelial tachykinin NK(1) receptors affect smooth muscle response to substance P. Contractile response of isolated rat bronchial trunk with or without epithelium was recorded. In intact segments precontracted by 5-hydroxytryptamine, relaxation was induced by substance P and the nitric oxide donor, sodium nitroprusside. Removal of the epithelium abolished relaxation induced by substance P but did not affect relaxation induced by sodium nitroprusside. The cyclo-oxygenase inhibitor, indomethacin, but not the nitric oxide synthase inhibitor, L-N(G)-monomethylarginine, reduced the relaxation in response to substance P. Epithelial tachykinin NK(1) receptors mediate substance-P-induced relaxation of rat bronchial smooth muscle via release of prostanoids but not nitric oxide.

Pharmacological analysis of [3H]-senktide binding to NK3 tachykinin receptors in guinea-pig ileum longitudinal muscle-myenteric plexus and cerebral cortex membranes.

PubMed Central

Guard, S.; Watson, S. P.; Maggio, J. E.; Too, H. P.; Watling, K. J.

1990-01-01

1. The binding properties and pharmacological specificity of the selective NK3 tachykinin receptor agonist [3H))-senktide [( 3H]-succinyl[Asp6,MePhe8] substance P (6-11] have been examined in homogenates of guinea-pig ileum longitudinal muscle-myenteric plexus (LM/MP) and cerebral cortex. 2. Scatchard analysis of saturation binding studies in guinea-pig ileum LM/MP and cerebral cortex membranes indicated that [3H]-senktide bound to a single site with apparent high affinity, KD = 2.21 +/- 0.65 nM; Bmax = 13.49 +/- 0.04 fmol mg-1 protein in ileum and KD = 8.52 +/- 0.45 nM; Bmax = 76.3 +/- 1.6 fmol mg-1 protein in cortex (values are means +/- ranges; n = 2). 3. The pharmacological profile for tachykinins and analogues in displacing [3H]-senktide from ileum membranes was: [MePhe7] neurokinin B greater than neurokinin B (NKB) congruent to senktide greater than eledoisin greater than substance P (SP) greater than neurokinin A(NKA) greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than [Nle10]NKA(4-10) = [Glp6,L-Pro9]-SP(6-11) greater than substance P methyl ester, consistent with [3H]-senktide binding to an NK3 subtype of tachykinin receptor. A similar rank order of affinity was obtained for these peptides in displacing [3H]-senktide from cortex membranes. 4. Several tachykinin receptor agonists were tested for their ability to displace [3H]-senktide from ileal and cortical NK3 binding sites and were found to be either weak displacers (pIC50 less than 5.00) or inactive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1694464

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

PubMed

Maggi, C A; Patacchini, R; Giuliani, S; Rovero, P; Dion, S; Regoli, D; Giachetti, A; Meli, A

1990-07-01

1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA. Similar pA2 values were obtained after 15 or 90min of incubation with the antagonists. Peptides I, II and III had no inhibitory effect on contractions produced by noradrenaline in the RPA or by carbachol in the HT. 5. Peptides I, II and III showed weak or no antagonistic activity toward the vasodilatator effect of substance P in the dog carotid artery (NK, receptor-mediated) or toward the contractile effect of neurokinin B in the rat portal vein (NK3 receptor-mediated). 6. These results provide pharmacological evidence for heterogeneity of NK2 receptors in the RPA and HT. The NK2 receptors present in these tissues are not discriminated by natural tachykinins or selective agonists, but are recognized with very different affinity by NK2 receptor antagonists.

A pharmacological study of NK1 and NK2 tachykinin receptor characteristics in the rat isolated urinary bladder.

PubMed Central

Hall, J. M.; Flowers, J. M.; Morton, I. K.

1992-01-01

1. We have estimated potencies of tachykinin receptor agonist and antagonist analogues in order to determine the recognition characteristics of tachykinin receptors mediating phasic contractile responses of the rat isolated urinary bladder in vitro. 2. The NK1-selective synthetic agonists, substance P methyl ester and GR73632, the synthetic NK2-selective agonists [beta-Ala8]-NKA(4-10) and GR64349, and the mammalian tachykinins, neurokinin A and neurokinin B, were assayed relative to substance P and were found to be approximately equipotent. The NK3-selective agonist, senktide, was inactive (10 microM). 3. Potencies of all these agonists were not significantly different (P > 0.05) when experiments were carried out in the presence of the neutral endopeptidase inhibitor, phosphoramidon, and the kininase II inhibitor, enalaprilat (both 1 microM). 4. The NK1-selective antagonist, GR82334, inhibited responses to substance P methyl ester in a competitive manner in the rat urinary bladder and the rat ileum, and also in the guinea-pig ileum. Markedly different pKB estimates were obtained in the rat bladder (6.38) and rat ileum (6.56) compared to the guinea-pig ileum (7.42). GR82334 (3 microM) was inactive against responses of the rat bladder to [beta-Ala8]-NKA(4-10). 5. The NK1-selective antagonist (+/-)-CP-96,345 also inhibited responses of the rat bladder and guinea-pig ileum to substance P methyl ester; however, in the rat bladder at 1 microM, this antagonist reversibly inhibited responses both to the NK2-selective agonist [beta-Ala8]-NKA(4-10) and to the muscarinic agonist carbachol (P < or = 0.01), thus showing evidence of some non-selective depressant actions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1282072

Tachykinin receptors in the small intestine of the cane toad (Bufo marinus): a radioligand binding and functional study.

PubMed

Burcher, E; Warner, F J

1998-06-01

In this study, we have used radioligand binding and functional techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [125I]Bolton-Hunter substance P ([125I]BHSP) was saturable, of high affinity (Kd 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin approximately neurokinin A (NKA) > or = SP(5-11) > or = neuropeptide gamma > or = scyliorhinin II > scyliorhinin I > or = [Sar9]-SP > or = neurokinin B approximately physalaemin approximately carassin > SP(7-11) approximately eledoisin > or = SP(4-11) approximately SP(6-11). Binding was also inhibited by Gpp[NH]p > or = GTPgammaS > App[NH]p, indicating a G-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC50 1.4 nM) > eledoisin approximately SP > or = physalaemin > or = ranakinin > SP(6-11) > scyliorhinin II > or = neuropeptide gamma > neurokinin B approximately NKA approximately scyliorhinin I > or = SP(4-11) > or = SP(5-11) > [Sar9]SP > SP(7-11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar9,Met(O2)11]SP, [Lys5,Me-Leu9,Nle10]NKA(4-10) and senktide were weak or ineffective. There was a strong positive correlation between the pD2 and pIC50 values for mammalian tachykinins and analogues (r = 0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar9,Met(O2)11]-SP(pD2 5.7) was approximately 25-fold less potent as an agonist than [Sar9]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP (spantide; 1 microM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 microM) were ineffective in both functional and binding studies. Tetrodotoxin (1 microM) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [125I]BHSP prefers SP and ranakinin. This toad "NK-1-like receptor" differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.

Tachykinins Stimulate a Subset of Mouse Taste Cells

PubMed Central

Grant, Jeff

2012-01-01

The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca2+-imaging on isolated taste cells, it was observed that SP induces Ca2+ -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca2+-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca2+-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca2+-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca2+ responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods. PMID:22363709

Cyclooxygenase-dependent alterations in substance P-mediated contractility and tachykinin NK1 receptor expression in the colonic circular muscle of patients with slow transit constipation.

PubMed

Liu, Lu; Shang, Fei; Morgan, Matthew J; King, Denis W; Lubowski, David Z; Burcher, Elizabeth

2009-04-01

Tachykinins are important neurotransmitters regulating intestinal motility. Slow transit constipation (STC) represents an extreme colonic dysmotility with unknown etiology that predominantly affects women. We examined whether the tachykinin system is involved in the pathogenesis of STC. Isolated sigmoid colon circular muscle from female STC and control patients was studied using functional and quantitative reverse transcriptase-polymerase chain reaction methods. A possible alteration of neurotransmission was investigated by electrical field stimulation (EFS) and ganglionic stimulation by dimethylphenylpiperazinium (DMPP). Substance P (SP)-mediated contractions in circular muscle strips were significantly diminished in STC compared with age-matched control (P < 0.001). In contrast, contractile responses to neurokinin A, the selective tachykinin NK(2) receptor agonist, [Lys(5),MeLeu(9),Nle(10)]NKA(4-10), and acetylcholine were unaltered in STC. The reduced responses to SP in STC were fully restored by indomethacin, partially reversed by tetrodotoxin (TTX), but unaffected by atropine or hexamethonium. The restoration by indomethacin was blocked by the NK(1) receptor antagonist CP99994 [(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] and TTX. In STC colonic muscle, there was a significant increase of NK(1) receptor mRNA expression, but no difference in NK(2) mRNA level. DMPP generated biphasic responses, relaxation at lower and contraction at higher concentrations. Although the responses to DMPP were similar in STC and control, an altered contractile pattern in response to EFS was observed in STC circular muscle. In conclusion, we postulate that the diminished contractile response to SP in STC is due to an increased release of inhibitory prostaglandins through activation of up-regulated NK(1) receptors. Our results also indicate some malfunction of the enteric nervous system in STC.

Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis.

PubMed

Jenkinson, K M; Southwell, B R; Furness, J B

1999-01-01

1. In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r. 2. We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum. 3. SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis. 4. The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leu,[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-betaAla) were both more potent in inhibiting endocytosis (50 x and 8 x greater respectively) against septide than against SP. 5. The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor.

Electrophysiological effects of tachykinins and capsaicin on guinea-pig bronchial parasympathetic ganglion neurones.

PubMed Central

Myers, A C; Undem, B J

1993-01-01

1. We evaluated the effects of neurokinins, tachykinin analogues, or capsaicin on passive membrane properties of guinea-pig bronchial parasympathetic neurones using intracellular recording techniques. 2. Substance P (SP) and the tachykinin analogue, acetyl-[Arg6,Sar9,Met(O2)11]-SP(6-11) (ASMSP), at concentrations selective for the neurokinin (NK)-1 receptor subtype, depolarized the resting potential (3 and 5 mV, respectively) with no change in input resistance. Neurokinin A and beta Ala8NKA(4-10), at concentrations selective for the NK-2 receptor subtype (0.1 microM), were without effect. 3. Neurokinin B (NKB) and [Asp5,6,methyl-Phe8]SP(5-11) (senktide analogue), at concentrations selective for NK-3 receptor subtype, elicited maximum depolarizations of 16 +/- 2 mV for both agonists. The peak of the depolarization was associated with an decrease in membrane resistance (35 +/- 4 and 50 +/- 7%, respectively). 4. Capsaicin (1 microM) elicited a 3-24 mV depolarization of the resting potential of thirteen of eighteen bronchial ganglion neurones and decreased the input resistance of seven of thirteen of these neurones. The effects of capsaicin were reduced by desensitization with senktide analogue at a concentration selective for the NK-3 receptor subtype, whereas a non-peptide NK-1 receptor antagonist had no effect. 5. Using voltage clamp analysis, capsaicin and senktide analogue evoked an inward current and an increase in membrane conductance at the resting membrane potential. The reversal potential for senktide analogue was estimated to be + 4 mV. 6. These data support the hypothesis that neurokinin-containing nerve terminals are localized within guinea-pig bronchial parasympathetic ganglia and, when released, the predominant effect of the neurokinins is by activation of NK-3 receptors. PMID:7508508

The third intracellular loop and carboxyl tail of neurokinin 1 and 3 receptors determine interactions with beta-arrestins.

PubMed

Schmidlin, Fabien; Roosterman, Dirk; Bunnett, Nigel W

2003-10-01

Tachykinins interact with three neurokinin receptors (NKRs) that are often coexpressed by the same cell. Cellular responses to tachykinins depend on the NKR subtype that is activated. We compared the colocalization of NK1R and NK3R with beta-arrestins 1 and 2, which play major roles in receptor desensitization, endocytosis, and signaling. In cells expressing NK1R, the selective agonist Sar-Met-substance P induced rapid translocation of beta-arrestins 1 and 2 from the cytosol to the plasma membrane and then endosomes, indicative of interaction with both isoforms. In contrast, the NK3R interacted transiently with only beta-arrestin 2 at the plasma membrane. Despite these differences, both NK1R and NK3R similarly desensitized, internalized, and activated MAP kinases. Because interactions with beta-arrestins can explain differences in the rate of receptor resensitization, we compared resensitization of agonist-induced Ca2+ mobilization. The NK1R resensitized greater than twofold more slowly than the NK3R. Replacement of intracellular loop 3 and the COOH tail of the NK1R with comparable domains of the NK3R diminished colocalization of the NK1R with beta-arrestin 1 and accelerated resensitization to that of the NK3R. Thus loop 3 and the COOH tail specify colocalization of the NK1R with beta-arrestin 1 and determine the rate of resensitization.

Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis

PubMed Central

Jenkinson, Karl M; Southwell, Bridget R; Furness, John B

1999-01-01

In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r.We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum.SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis.The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leuψ[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-βAla) were both more potent in inhibiting endocytosis (50× and 8× greater respectively) against septide than against SP.The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor. PMID:10051129

Defensive and pathological functions of the gastrointestinal NK3 receptor.

PubMed

Sanger, Gareth J; Tuladhar, Bishwa R; Bueno, Lionel; Furness, John B

2006-10-01

In general, normal gut functions are unaffected by selective NK(3) receptor antagonists such as talnetant (SB-223412), osanetant (SR 142901) or SB-235375. However, NK(3) receptors may mediate certain defensive or pathological intestinal processes. The precise mechanisms, by which this role is achieved, are not fully understood. In summary, intense stimulation of the intrinsic primary afferent neurones (IPANs) of the enteric nervous system is thought to release tachykinins from these neurones, to induce slow excitation (slow EPSPs) of connecting IPANs. This is hypothesised to cause hypersensitivity and disrupt intestinal motility, at least partly explaining why NK(3) receptor antagonism can reduce the level of disruption caused by supramaximal distension pressures in vitro. Tachykinin release from IPANs may also increase C-fibre sensitivity, directly or indirectly. Thus, NK(3) receptor antagonists can inhibit nociception associated with intestinal distension, in normal animals or after pre-sensitisation by restraint stress. Importantly, such inhibition has been found with SB-235375, a peripherally restricted antagonist. SB-235375 can also reduce a visceromotor response to brief colorectal distension without affecting similar responses to skin pinch, providing additional evidence for intestinal-specific activity. NK(3) receptor biology is, therefore, revealing a novel pathway by which disruptions in intestinal motility and nociception can be induced.

Tachykinin receptors in the rat isolated uterus.

PubMed

Fisher, L; Pennefather, J N; Hall, S

1993-07-02

Tachykinin receptors mediating uterotonic effects were examined in preparations from oestrogen-primed rats. In the absence of peptidase inhibitors [Lys5-MeLeu9-Nle10] NKA (4-10) was 14-fold more potent than neurokinin A (NKA), but the two peptides were equipotent in the presence of phosphoramidon alone and in combination with amastatin. The NK-2 receptor antagonist SR 48968 antagonised responses to the tachykinins. These findings indicate that an NK-2 receptor is present in the oestrogen-primed rat uterus and that endopeptidase 24.11 plays a major role to inactivate NKA in this tissue.

Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung.

PubMed Central

Walsh, D A; Salmon, M; Featherstone, R; Wharton, J; Church, M K; Polak, J M

1994-01-01

1. The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]-Bolton Hunter-labelled substance P ([125I]-BH-SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung. 2. [125I]-BH-SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung. 3. Specific binding of [125I]-BH-SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150-800 microns and was less dense in trachea than in main bronchi. 4. [125I]-BH-SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125I]-BH-SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors. 5. In both human and guinea pig lung, [125I]-BH-SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]-BH-SP binding sites in both guinea pig arteries and bronchi.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:7534186

Distribution pattern of tachykinin NK2 receptors in human colon: involvement in the regulation of intestinal motility.

PubMed

Jaafari, Nadia; Khomitch-Baud, Alexandra; Christen, Marie-Odile; Julé, Yvon

2007-07-20

Although a number of pharmacological studies have shown the involvement of tachykinin type 2 receptors (NK2r) in the regulation of human colonic motility, few data are available so far on their pattern of expression. In this study this pattern was investigated in the myenteric plexuses, the longitudinal and circular muscle layers (external muscular layers), and the interstitial cells of Cajal (ICCs) using confocal microscopy immunofluorescence methods. NK2r immunoreactivity (NK2r-IR) was detected in the soma of myenteric neurons and in nerve varicosities located in myenteric plexuses as well as in external muscular layers. Colocalization analysis of NK2r-IR and synaptophysin-IR, showed significant regional differences in the distribution of NK2r-expressing nerve varicosities, the rate of occurrence was found to be 56.08% +/- 3% (mean +/- SE) in the external muscular layers and 30.22% +/- 1% (mean +/- SE) in the myenteric plexuses. NK2r-IR was found in membranes of most muscle cells previously incubated with a selective NK2r agonist, [beta-Ala(8)] neurokinin A fragment 4-10, at 4 degrees C, and then mainly relocated in the cytoplasm when heated to 37 degrees C. A number of NK2r-IR nerve varicosities were close to NK2r-expressing neurons and muscle cells. Some of NK2r-expressing neurons and nerves were tachykinin-IR. No NK2r-IR was detected in ICCs. The present data indicate that presynaptic and postsynaptic neuroneuronal and neuromuscular regulatory processes mediated by tachykinins via NK2r may occur for modulating human colonic motility.

Further investigation into the mechanism of tachykinin NK(2) receptor-triggered serotonin release from guinea-pig proximal colon.

PubMed

Kojima, Shu-Ichi; Ikeda, Masashi; Kamikawa, Yuichiro

2009-05-01

The effects of the monoamine oxidase A (MAO-A) inhibitor clorgyline, the L-type calcium-channel blocker nicardipine, the syntaxin inhibitor botulinum toxin type C, and the potent thiol-oxidant phenylarsine oxide (PAO) on the selective tachykinin NK(2)-receptor agonist [beta-Ala(8)]-neurokinin A(4-10) [betaAla-NKA-(4-10)]-evoked 5-hydroxytryptamine (5-HT) outflow from colonic enterochromaffin (EC) cells was investigated in vitro using isolated guinea-pig proximal colon. The betaAla-NKA-(4-10)-evoked outflow of 5-HT from clorgyline-treated colonic strips was markedly higher than that from clorgyline-untreated colonic strips. The betaAla-NKA-(4-10)-evoked 5-HT outflow from the clorgyline-treated colonic strips was sensitive to nicardipine or botulinum toxin type C. Moreover, PAO concentration-dependently suppressed the betaAla-NKA-(4-10)-evoked 5-HT outflow from the clorgyline-treated colonic strips. The suppressant action of PAO was reversed by the reducing agent dithiothrietol, but was not blocked by the protein tyrosine kinase inhibitor genistein. These results suggest that the tachykinin NK(2) receptor-triggered 5-HT release from guinea-pig colonic EC cells is mediated by syntaxin-related exocytosis mechanisms and that colonic mucosa MAO-A activity has the important function of modulating the tachykinin NK(2) receptor-triggered 5-HT release. It also appears that PAO-mediated sulfhydryl oxidation plays a role in modulating the tachykinin NK(2) receptor-triggered 5-HT release through a mechanism independent of inhibition of protein tyrosine phosphatase activity.

Neurokinin receptors in the rabbit iris sphincter characterised by novel agonist ligands.

PubMed

Hall, J M; Mitchell, D; Morton, I K

1991-06-18

We have used novel selective agonist ligands to examine neurokinin receptors mediating the contractile response to tachykinins in the rabbit iris sphincter preparation in vitro. The selective NK-1 receptor agonist delta-amino valeryl-[L-Pro9,N-Me Leu10]SP-(7-11) (GR73632) and the NK-3 receptor-selective agonist succ-[Asp6,N-Me-Phe8] SP-(6-11) (senktide) were both very active (concentration range 0.032 pM-10 nM and 0.1 pM-32 nM respectively), and were 933 and 16.6 times more potent than substance P, respectively, in contracting the iris. In contrast, the NK-2 selective agonist [Lys3,Gly8-R-gamma-lactam,Leu9]NKA-(3-10) (GR64349) was active only at the highest concentrations tested (3.2 nM-32 microM), and had 0.054 the activity of substance P. The presence of several peptidase inhibitors was without effect on the concentration-response relationship to substance P, GR73632, GR64349 or senktide. Tachykinins differed in their offset kinetics. Responses to GR73632, GR64349 and senktide were rapid in offset (times to reach half maximal responses were 1.5, 1.1 and 5.1 min, respectively), whereas responses to substance P were very much more prolonged in duration (time to reach half maximal response was 35.3 min). These results suggest the presence of both NK-1 and NK-3 receptors mediating contraction of the rabbit iris sphincter preparation. In addition, differences in response offset kinetics seem not to be due to differences in peptide metabolism, and suggest a property of substance P not shared by the other tachykinins used in this study.

In vivo evidence for the involvement of tachykinin NK3 receptors in the hexamethonium-resistant inhibitory transmission in the rat colon.

PubMed

Lecci, A; Giuliani, S; Tramontana, M; Meini, S; De Giorgio, R; Maggi, C A

1996-05-01

In urethane-anaesthetized rats, moderate colonic distention (0.5 ml) induced reflex rhythmic contractions (5 mm Hg amplitude and 1.1 cycles/min frequency). Senktide (1-10 nmol/kg, i.v.), a tachykinin NK3 receptor selective agonist, transiently suppressed distension-induced contractions. SR 142,801 (1-10 mumol/kg i.v.), a non-peptide tachykinin NK3 receptor antagonist, had no effect on distension-induced contractions but prevented the inhibitory effect of senktide. Infusion of N-omega-nitro-1-arginine methyl esther hydrochloride (L-NAME, 20 mumol/ml/h, i.v) increased the amplitude of colonic contractions and decreased the inhibitory effect of senktide. Hexamethonium (15 mumol/ml/h, i.v.) or atropine (1 mumol/ml/h, i.v.) inhibited the distension-induced contractions. In hexamethonium- or atropine-treated rats, senktide (10 nmol/kg) transiently and selectively enhanced the amplitude of contractions. Also SR 142,801 (10 mumol/kg), but not its inactive enantiomer SR 142,806, increased both amplitude and frequency of contractions. During continuous infusion of L-NAME and hexamethonium or atropine both frequency and amplitude of distension-induced colonic contractions were higher than when in hexamethonium or atropine only. Senktide (10 nmol/kg) had no effect and SR 142,801 (10 mumol/kg) produced a slight enhancement of colonic contractions. Infusion of sodium nitroprusside (3 mumol/ml/h, i.v.) decreased amplitude and frequency of distension-induced contractions. SR 142,801 had no effect in the presence of the nitric oxide (NO) donor. We conclude that tachykinins acting through NK3 receptors exert at least four different actions on colonic motility activated by distension: 1) a hexamethonium-resistant, NO-dependent, suppressant effect on contractions; 2) a hexamethonium-sensitive, NO-independent inhibitory effect on the amplitude of contractions; 3) a hexamethonium-resistant, NO-independent inhibitory effect on the amplitude of contractions and 4) a hexamethonium resistant and L-NAME-sensitive excitatory effect on amplitude of contractions. The prevalent inhibitory effect evoked in normal conditions along with the excitatory activity induced by SR 142,801 on hexamethonium-resistant colonic motility indicates that tachykinins, acting through neuronal NK3 receptors, activate NO-dependent and NO-independent inhibitory neurotransmission in the rat colon.

Identification of both NK1 and NK2 receptors in guinea-pig airways.

PubMed Central

McKee, K. T.; Millar, L.; Rodger, I. W.; Metters, K. M.

1993-01-01

1. NK1 and NK2 receptors have been characterized in guinea-pig lung membrane preparations by use of [125I-Tyr8]-substance P and [125I]-neurokinin A binding assays in conjunction with tachykinin-receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [beta Ala8]neurokinin A (4-10) for NK2) and antagonists (CP-99,994 for NK1 and SR48968 for NK2). 2. The presence of high affinity, G-protein-coupled NK1 receptors in guinea-pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance P > substance P-methyl ester = physalaemin > neurokinin A = neurokinin B >> [beta Ala8]neurokinin A (4-10). The novel NK1 antagonist CP-99,994 has a Ki of 0.4 nM at this NK1 site. 3. In order to characterize [125I]-neurokinin A binding to guinea-pig lung, the number of [125I]-neurokinin A specific binding sites was increased 3-4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1- and NK2-receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]-neurokinin A specific binding was also to the NK1 receptor. 4. Under conditions where the guinea-pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 microM) or CP-99,994 (2.7 microM), residual [125I]-neurokinin A specific binding was inhibited in a concentration-dependent manner by both [beta Ala8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7694756

Characterization of species-related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3.

PubMed

Leffler, Agnes; Ahlstedt, Ingela; Engberg, Susanna; Svensson, Arne; Billger, Martin; Oberg, Lisa; Bjursell, Magnus K; Lindström, Erik; von Mentzer, Bengt

2009-05-01

Tachykinin NK receptors (NKRs) differ to a large degree among species with respect to their affinities for small molecule antagonists. The aims of the present study were to clone NKRs from gerbil (NK2R and NK3R) and dog (NK1R, NK2R and NK3R) in which the sequence was previously unknown and to investigate the potency of several NKR antagonists at all known human, dog, gerbil and rat NKRs. The NKR protein coding sequences were cloned and expressed in CHO cells. The inhibitory concentrations of selective and non-selective NKR antagonists were determined by inhibition of agonist-induced mobilization of intracellular Ca2+. Receptor homology models were constructed based on the rhodopsin crystal structure to investigate and identify the antagonist binding sites and interaction points in the transmembrane (TM) regions of the NKRs. Data collected using the cloned dog NK1R confirmed that the dog NK1R displays similar pharmacology as the human and the gerbil NK1R, but differs greatly from the mouse and the rat NK1R. Despite species-related amino acid (AA) differences located close to the antagonist binding pocket of the NK2R, they did not affect the potency of the antagonists ZD6021 and saredutant. Two AA differences located close to the antagonist binding site of NK3R likely influence the NK3R antagonist potency, explaining the 3-10-fold decrease in potency observed for the rat NK3R. For the first time, detailed pharmacological experiments in vitro with cloned NKRs demonstrate that not only human, but also dog and gerbil NKR displays similar antagonist pharmacology while rat diverges significantly with respect to NK1R and NK3R.

Characterization of tachykinin receptors mediating bronchomotor and vasodepressor responses to neuropeptide gamma and substance P in the anaesthetized rabbit.

PubMed

Yuan, L; Burcher, E; Nail, B S

1998-02-01

The effects of i.v. injections of two endogenous tachykinins, substance P (SP) and neuropeptide gamma and the highly selective tachykinin agonists [Sar9,Met(O2)11]-SP, [Lys5,MeLeu9, Nle10]-NKA(4-10) and senktide, on total lung resistance (RL), dynamic lung compliance (Cdyn) and systemic blood pressure, were compared in the anaesthetized rabbit. Senktide, the NK-3 receptor selective agonist, had no effect on RL, Cdyn or blood pressure. The other four agonists caused dose-dependent increases in RL and Cdyn, with [Sar9,Met(O2)11]-SP being the most potent agonist in producing changes in the absence of phosphoramidon. This suggested that NK-1 receptors play an important role in these responses. [Sar9, Met(O2)11]-SP, SP and neuropeptide gamma also decreased blood pressure. Phosphoramidon (1 mg/kg) potentiated the changes in RL and Cdyn evoked by [Sar9,Met(O2)11]-SP and SP, with very marked enhancement of responses to neuropeptide gamma. Responses to [Lys5, MeLeu9,Nle10]-NKA(4-10) were unaffected, suggesting that this NK-2 selective agonist may not be catabolized by neutral endopeptidase (NEP). In the presence of phosphoramidon, the non-peptide tachykinin NK-1 receptor selective antagonist CP 96345 (80 nmol/kg) reduced all responses to [Sar9,Met(O2)11]-SP and SP, whereas the NK-2 selective antagonist SR 48968 (40 nmol/kg) inhibited the bronchomotor but not the vasodepressor responses to neuropeptide gamma and [Lys5,MeLeu9, Nle10]-NKA(4-10). The fall in blood pressure induced by neuropeptide gamma was diminished by CP 96345, whereas bronchoconstriction was unaffected, indicating possible differences in NK-1 receptors in the vasculature and airways. Electrical stimulation of the distal ends of vagus nerves caused increases in RL which were abolished by atropine (1 mg/kg). Copyright 1998 Academic Press Limited

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

PubMed Central

Maggi, C. A.; Patacchini, R.; Giuliani, S.; Rovero, P.; Dion, S.; Regoli, D.; Giachetti, A.; Meli, A.

1990-01-01

1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2167737

Regulation of the stimulant actions of neurokinin a and human hemokinin-1 on the human uterus: a comparison with histamine.

PubMed

Pennefather, Jocelyn N; Patak, Eva; Ziccone, Sebastian; Lilley, Alison; Pinto, Francisco M; Page, Nigel M; Story, Margot E; Grover, Sonia; Candenas, M Luz

2006-09-01

Regulation of the contractile effects of tachykinins and histamine on the human uterus was investigated with biopsy sections of the outer myometrial layer. The effects of neurokinin A (NKA) and human hemokinin-1 (hHK-1) in tissues from pregnant but not from nonpregnant women were enhanced by the inhibition of neprilysin. The effects of NKA and eledoisin were blocked by the NK2 receptor antagonist SR 48968 but not by the NK1 receptor antagonist SR 140333 in tissues from both groups of women. Human HK-1 acted as a partial agonist blocked by SR 48968 and, to a lesser extent, by SR 140333; endokinin D was inactive. In tissues from pregnant women, responses to high potassium-containing Krebs solution were 2-3-fold higher than those from nonpregnant women. Mepyramine-sensitive maximal responses to histamine were similarly enhanced. The absolute maximum responses to NKA and its stable NK2 receptor-selective analogue, [Lys5MeLeu9Nle10]NKA(4-10), were increased in pregnancy, but their efficacies relative to potassium responses were decreased. Tachykinin potencies were lower in tissues from pregnant women than in those from nonpregnant women. These data 1) show for the first time that hHK-1 is a uterine stimulant in the human, 2) confirm that the NK2 receptor is predominant in mediating tachykinin actions on the human myometrium, and 3) indicate that mammalian tachykinin effects are tightly regulated during pregnancy in a manner that would negate an inappropriate uterotonic effect. The potencies of these peptides in tissues from nonpregnant women undergoing hysterectomy are consistent with their possible role in menstrual and menopausal disorders.

Tachykinin NK2 receptor and functional mechanisms in human colon: changes with indomethacin and in diverticular disease and ulcerative colitis.

PubMed

Burcher, Elizabeth; Shang, Fei; Warner, Fiona J; Du, Qin; Lubowski, David Z; King, Denis W; Liu, Lu

2008-01-01

Neurokinin A (NKA) is an important spasmogen in human colon. We examined inflammatory disease-related changes in the tachykinin NK(2) receptor system in human sigmoid colon circular muscle, using functional, radioligand binding, and quantitative reverse transcription-polymerase chain reaction methods. In circular muscle strips, indomethacin enhanced contractile responses to NKA (p < 0.01) and to the NK(2) receptor-selective agonist [Lys(5),MeLeu(9),Nle(10)]-NKA(4-10) (p < 0.05) in both normal and acute diverticular disease (DD) specimens, indicating NK(2) receptor-mediated release of relaxant prostanoids. Contractile responses to both tachykinins were reduced in strips from DD (p < 0.001) and ulcerative colitis (UC) (p < 0.05) specimens. Responses to acetylcholine were no different in other strips from the same disease patients, demonstrating that the change in responsiveness to tachykinins in disease is specifically mediated by the NK(2) receptor. In membranes from UC specimens, receptor affinity for (125)I-NKA (median K(D) 0.91 nM, n = 16) was lower (p < 0.01) than that in age-matched control specimens (K(D) 0.55 nM, n = 40), whereas K(D) (0.65 nM, n = 28) in DD was no different from control. No disease-related changes in receptor number (B(max)) were found (mean, 2.0-2.5 fmol/mg of wet weight tissue), suggesting that the reduced contractile responses in disease are not due to a loss of receptor number. Different mechanisms may account for the reduced contractility in DD compared with UC. A gender-related difference in receptor density was seen in controls, with B(max) lower in females (1.77 fmol/mg, n = 15) than in males (2.60 fmol/mg, n = 25, p = 0.01). In contrast, no gender-related differences were seen in NK(2) receptor mRNA in control colonic muscle, indicating that the gender difference is a post-translational event.

Involvement of tachykinin receptors in Clostridium perfringens beta-toxin-induced plasma extravasation

PubMed Central

Nagahama, Masahiro; Morimitsu, Shinsuke; Kihara, Atsushi; Akita, Masahiko; Setsu, Koujun; Sakurai, Jun

2003-01-01

Clostridium perfringens beta-toxin causes dermonecrosis and oedema in the dorsal skin of animals. In the present study, we investigated the mechanisms of oedema induced by the toxin. The toxin induced plasma extravasation in the dorsal skin of Balb/c mice. The extravasation was significantly inhibited by diphenhydramine, a histamine 1 receptor antagonist. However, the toxin did not cause the release of histamine from mouse mastocytoma cells. Tachykinin NK1 receptor antagonists, [D-Pro2, D-Trp7,9]-SP, [D-Pro4, D-Trp7,9]-SP and spantide, inhibited the toxin-induced leakage in a dose-dependent manner. Furthermore, the non-peptide tachykinin NK1 receptor antagonist, SR140333, markedly inhibited the toxin-induced leakage. The leakage induced by the toxin was markedly reduced in capsaicin-pretreated mouse skin but the leakage was not affected by systemic pretreatment with a calcitonin gene-related peptide receptor antagonist (CGRP8-37). The toxin-induced leakage was significantly inhibited by the N-type Ca2+ channel blocker, ω-conotoxin MVIIA, and the bradykinin B2 receptor antagonist, HOE140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin), but was not affected by the selective L-type Ca2+ channel blocker, verapamil, the P-type Ca2+ channel blocker, ω-agatoxin IVA, tetrodotoxin (TTX), the TTX-resistant Na+ channel blocker, carbamazepine, or the sensory nerve conduction blocker, lignocaine. These results suggest that plasma extravasation induced by beta-toxin in mouse skin is mediated via a mechanism involving tachykinin NK1 receptors. PMID:12522069

In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist.

PubMed

Emonds-Alt, X; Doutremepuich, J D; Heaulme, M; Neliat, G; Santucci, V; Steinberg, R; Vilain, P; Bichon, D; Ducoux, J P; Proietto, V

1993-12-21

(S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pip eridin-3- yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR140333) is a new non-peptide antagonist of tachykinin NK1 receptors. SR140333 potently, selectively and competitively inhibited substance P binding to NK1 receptors from various animal species, including humans. In vitro, it was a potent antagonist in functional assays for NK1 receptors such as [Sar9,Met(O2)11]substance P-induced endothelium-dependent relaxation of rabbit pulmonary artery and contraction of guinea-pig ileum. Up to 1 microM, it had no effect in bioassays for NK2 ([beta Ala8]neurokinin A-induced contraction of endothelium-deprived rabbit pulmonary artery) and NK3 ([MePhe7]neurokinin B-induced contraction of rat portal vein) receptors. The antagonism exerted by SR140333 toward NK1 receptors was apparently non-competitive, with pD2' values (antagonism potency evaluated by the negative logarithm of the molar concentration of antagonist that produces a 50% reduction of the maximal response to the agonist) between 9.65 and 10.16 in the different assays. SR140333 also blocked in vitro [Sar9,Met(O2)11]substance P-induced release of acetylcholine from rat striatum. In vivo, SR140333 exerted highly potent antagonism toward [Sar9,Met(O2)11]substance P-induced hypotension in dogs (ED50 = 3 micrograms/kg i.v.), bronchoconstriction in guinea-pig (ED50 = 42 micrograms/kg i.v.) and plasma extravasation in rats (ED50 = 7 micrograms/kg i.v.). Finally, it also blocked the activation of rat thalamic neurons after nociceptive stimulation (ED50 = 0.2 micrograms/kg i.v.).

Do Substance P and Neurokinin A Play Important Roles in the Control of LH Secretion in Ewes?

PubMed Central

Fergani, Chrysanthi; Mazzella, Leanne; Coolen, Lique M.; McCosh, Richard B.; Hardy, Steven L.; Newcomb, Nora; Grachev, Pasha; Lehman, Michael N.

2016-01-01

There is now general agreement that neurokinin B (NKB) acts via neurokinin-3-receptor (NK3R) to stimulate secretion of GnRH and LH in several species, including rats, mice, sheep, and humans. However, the roles of two other tachykinins, substance P (SP) and neurokinin A, which act primarily via NK1R and NK2R, respectively, are less clear. In rodents, these signaling pathways can stimulate LH release and substitute for NKB signaling; in humans, SP is colocalized with kisspeptin and NKB in the mediobasal hypothalamus. In this study, we examined the possible role of these tachykinins in control of the reproductive axis in sheep. Immunohistochemistry was used to describe the expression of SP and NK1R in the ovine diencephalon and determine whether these proteins are colocalized in kisspeptin or GnRH neurons. SP-containing cell bodies were largely confined to the arcuate nucleus, but NK1R-immunoreactivity was more widespread. However, there was very low coexpression of SP or NK1R in kisspeptin cells and none in GnRH neurons. We next determined the minimal effective dose of these three tachykinins that would stimulate LH secretion when administered into the third ventricle of ovary-intact anestrous sheep. A much lower dose of NKB (0.2 nmol) than of neurokinin A (2 nmol) or SP (10 nmol) consistently stimulated LH secretion. Moreover, the relative potency of these three neuropeptides parallels the relative selectivity of NK3R. Based on these anatomical and pharmacological data, we conclude that NKB-NK3R signaling is the primary pathway for the control of GnRH secretion by tachykinins in ewes. PMID:27704950

Do Substance P and Neurokinin A Play Important Roles in the Control of LH Secretion in Ewes?

PubMed

Fergani, Chrysanthi; Mazzella, Leanne; Coolen, Lique M; McCosh, Richard B; Hardy, Steven L; Newcomb, Nora; Grachev, Pasha; Lehman, Michael N; Goodman, Robert L

2016-12-01

There is now general agreement that neurokinin B (NKB) acts via neurokinin-3-receptor (NK3R) to stimulate secretion of GnRH and LH in several species, including rats, mice, sheep, and humans. However, the roles of two other tachykinins, substance P (SP) and neurokinin A, which act primarily via NK1R and NK2R, respectively, are less clear. In rodents, these signaling pathways can stimulate LH release and substitute for NKB signaling; in humans, SP is colocalized with kisspeptin and NKB in the mediobasal hypothalamus. In this study, we examined the possible role of these tachykinins in control of the reproductive axis in sheep. Immunohistochemistry was used to describe the expression of SP and NK1R in the ovine diencephalon and determine whether these proteins are colocalized in kisspeptin or GnRH neurons. SP-containing cell bodies were largely confined to the arcuate nucleus, but NK1R-immunoreactivity was more widespread. However, there was very low coexpression of SP or NK1R in kisspeptin cells and none in GnRH neurons. We next determined the minimal effective dose of these three tachykinins that would stimulate LH secretion when administered into the third ventricle of ovary-intact anestrous sheep. A much lower dose of NKB (0.2 nmol) than of neurokinin A (2 nmol) or SP (10 nmol) consistently stimulated LH secretion. Moreover, the relative potency of these three neuropeptides parallels the relative selectivity of NK3R. Based on these anatomical and pharmacological data, we conclude that NKB-NK3R signaling is the primary pathway for the control of GnRH secretion by tachykinins in ewes.

Osanetant Sanofi-Synthélabo.

PubMed

Kamali, F

2001-07-01

Osanetant is a neurokinin (NK3) receptor antagonist under development by Sanofi-Synthélabo (formerly Sanofi) as a potential treatment for schizophrenia [328910]. Sanofi was originally investigating its potential use as a treatment for psychosis and anxiety [169511]. Following phase IIa clinical trials [307656], [328910], [359231], osanetant entered phase IIb development in February 2001 [409432]. Osanetant was the first potent and selective non-peptide antagonist described for the NK3 tachykinin receptor [176305]. It has a higher affinity for human and guinea pig NK3 receptors than for rat NK3 receptors [176305]. In October 1999, Lehman Brothers predicted that the probability of the product reaching the market was 10%, with a possible launch in 2003 and potential peak sales of US $200 million in 2011 [346267].

Evidence that tachykinins relax the guinea-pig trachea via nitric oxide release and by stimulation of a septide-insensitive NK1 receptor.

PubMed Central

Figini, M.; Emanueli, C.; Bertrand, C.; Javdan, P.; Geppetti, P.

1996-01-01

1. This study investigated the possibility that tachykinins relax the guinea-pig isolated trachea by releasing nitric oxide (NO) from the epithelium. The types of tachykinin receptor mediating both relaxation and contraction of the trachea were also studied. Isometric tension was recorded in isolated tracheal tube preparations precontracted with acetylcholine (10 microM) in which compounds were administered intraluminally in the presence of phosphoramidon and indomethacin (both 1 microM) and the tachykinin NK2 receptor antagonist, SR 48,968 ((S)-N-methyl-N[4-(4-acetyl amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide), 0.1 microM). 2. In the presence of the inactive enantiomer of an NO-synthase inhibitor, NG-monomethyl-D-arginine (D-NMMA, 100 microM), substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and the selective NK1 receptor agonist, [Sar9, Met(O2)11]-SP, (0.1-10 nM) relaxed tracheal tube preparations. This relaxation was changed into a contraction by pretreatment with the NO-synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA, 100 microM). The effect of L-NMMA on SP- and [Sar9, Met(O2)11]-SP-induced responses was reversed by L-arginine (L-Arg, 1 mM), but not by D-Arg (1 mM). After removal of the epithelium SP, NKA and NKB and [Sar9, Met(O2)11]-SP (0.1-10 nM) evoked contractile responses in the presence of either L-NMMA (100 microM) or D-NMMA (100 microM). The effects of SP and [Sar9, Met(O2)11]-SP obtained in the presence of another NO-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) or its inactive enantiomer, NG-nitro-D-arginine methyl ester (D-NAME, 100 microM) were similar to those observed with L-NMMA or D-NMMA, respectively. 3. The selective NK1 receptor agonist, [pGlu6, Pro9]-SP(6-11) (septide, 0.1-10 nM) evoked contractile responses of tracheal tube preparations in the presence of either D-NMMA (100 microM) or L-NMMA (100 microM). The log concentration-response curve to septide obtained in the presence of L-NMMA was similar to that obtained in the presence of D-NMMA. [Sar9, Met(O2)11]-SP (0.1-10 nM) relaxed tracheal tube preparations precontracted with septide (1 microM), whereas septide (0.1 nM-1 microM) further contracted tracheal tube preparations precontracted with [Sar9, Met(O2)11]-SP (1 microM). 4. Relaxant and contractile responses evoked by SP, NKA, NKB and by [Sar9, Met(O2)11]-SP (0.1-10 nM) were not affected by a combination of the histamine H1 (pyrilamine, 1 microM) and H2 (cimetidine, 1 microM) receptor antagonists, but were abolished by the tachykinin NK1 receptor antagonist, CP-99,994 ((2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine, 1 microM), though not by its inactive enantiomer CP-100,263 (1 microM). Contractile responses evoked by septide (10 nM and 1 microM) were also abolished by CP-99,994 (1 microM) but not by CP-100,263 (1 microM). 5. These results demonstrate that tachykinins relax guinea-pig tracheal tube preparations by releasing NO via the stimulation of epithelial NK1 receptors by a mechanism independent of histamine release. The NK1 receptor type involved is sensitive to SP, NKA, NKB and [Sar9, Met(O2)11]-SP but not to septide, and is pharmacologically distinct from the NK1 receptor that mediates contraction, which is stimulated by all the agonists, including septide. PMID:8882625

Tachykinin control of ferret airways: mucus secretion, bronchoconstriction and receptor mapping.

PubMed

Meini, S; Mak, J C; Rohde, J A; Rogers, D F

1993-02-01

The effects of synthetic tachykinin receptor agonists on mucus secretion by ferret trachea was determined in vitro in Ussing chambers using 35SO4 as a mucus marker and the synthetic peptides [Sar9,Met(O2)11]substance P (SarSP), [beta Ala8]neurokinin A-(4-10) and [MePhe7] neurokinin B which are selective for NK1, NK2 and NK3 tachykinin-receptors respectively. The bronchomotor effects of the same agonists were also studied in vitro and tachykinin receptors were localized by autoradiographic mapping. SarSP was the only synthetic agonist able to elicit a concentration-dependent increase in mucus secretion and was much more potent than SP. The EC50 for SarSP was 1.7 x 10(-6) M. Moreover, the maximal increase in release of 35SO4 produced by SarSP 10(-5) M was 95% of the increase produced by methacholine 10(-4) M indicating that this concentration of SarSP induced a near maximal secretory response. There was no significant difference in the secretory action of SP administered from the luminal or the submucosal side of the tissue. Only the NK2 agonist was able to produce a concentration-dependent contractility of bronchial ring preparations and its effect was relatively weak (EC50 6.4 x 10(-6) M). Capsaicin (10(-5) M) produced only a slight increase in tracheal mucus secretion (28 +/- 5%; n = 6) and was completely ineffective in inducing bronchoconstriction. Binding sites for [125I]-Bolton Hunter SP were more evident than sites for [125I]-NKA on submucosal glands and epithelium. In contrast, only binding sites to NKA could be observed over the smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of NK1 and NK2 tachykinin receptors in guinea-pig and rat bronchopulmonary and vascular systems.

PubMed Central

Floch, A.; Fardin, V.; Cavero, I.

1994-01-01

1. NK1 and NK2 tachykinin receptors were characterized in guinea-pig and rat bronchopulmonary systems and in the vasculature of the rat by use of radioligand binding and/or functional studies. 2. The radioligands for NK1 and NK2 receptors ([3H]-SP and [3H]-pNKA, respectively) did not label tachykinin receptors in homogenates of rat lungs or bronchi. In contrast, in the guinea-pig, [3H]-SP bound with high affinity to these tissues (KD = 0.23 +/- 0.08 nM and 0.34 +/- 0.05 nM, for lungs and bronchi, respectively). The total number of binding sites was 4.6 fold greater in bronchus (Bmax = 135 +/- 27 fmol mg-1 protein) than in lung homogenates (Bmax = 29.3 +/- 0.1 fmol mg-1 protein). Furthermore, this binding was markedly displaced by CP-96,345 (pKi = 9.5 +/- 0.1) and RP 67580 (pKi = 7.6 +/- 0.1), antagonists of NK1 receptors, slightly displaced by SR 48968 (pKi = 6.6 +/- 0.1), but not affected by actinomycin D or L-659,877, antagonists of NK2 receptors. Specific binding of [3H]-pNKA, detected in guinea-pig bronchi (KD = 5.2 +/- 0.1 nM, and Bmax = 203 +/- 19 fmol mg-1 protein) but not in lungs, was similarly (40 to 53%) displaced by RP 67580 (1 microM), CP-96,345 (10 and 100 nM) or SR 48968 (10 and 100 nM). The displacement approximately doubled (87 to 91%) when SR 48968 (10 nM) was combined with either RP 67580 (1 microM) or CP-96,345 (10 nM), but not when RP 67580 was combined with CP-96,345. 3. In urethane-anaesthetized guinea-pigs, i.v. injections of the NK1 receptor agonists SP, [Pro9]-SP, [Sar9,Met(O2)11]-SP and septide, as well as the NK2 receptor agonists NKA and [Lys5,MeLeu9,NLeu10]-NKA(4-10) (0.1-10 micrograms kg-1, i.v.), dose-dependently increased lung inflation pressure. The most potent of these peptides were septide and [Lys5, MeLeu9,NLeu10]-NKA(4-10) (EC50 = 0.38 +/- 0.07 and 0.07 +/- 0.02 microgram kg-1, respectively). Interestingly, septide was 130 fold less potent than SP in displacing [3H]-SP from its binding sites in the guinea-pig lung, whereas it was 14 fold more potent than SP as a bronchoconstrictor. RP 67580 (0.3-5 mg kg-1, i.v.) and CP-96,345 (0.01-3 mg kg-1, i.v.) dose-dependently reduced the bronchoconstriction produced by the NK1 receptor agonists.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7517328

Effect of thiorphan on response of the guinea-pig gallbladder to tachykinins.

PubMed

Maggi, C A; Patacchini, R; Renzi, D; Santicioli, P; Regoli, D; Rovero, P; Drapeau, G; Surrenti, C; Meli, A

1989-06-08

Tachykinins produced a concentration-related contraction of the isolated guinea-pig gallbladder, with a rank order of potency neurokinin A (NKA) greater than Arg-neurokinin B = neurokinin B (NKB) greater than substance P (SP). Only the effect of SP was potentiated by thiorphan (0.1-10 microM). A significant enhancement of the response to SP was also produced by captopril (1 microM). [Nle10]NKA-(4-10) and [beta-Ala8]NKA-(4-10), selective NK-2 receptor agonists, were active, whereas [Pro9]SP sulfone (selective NK-1 agonist) was almost ineffective. [MePhe7]NKB (selective NK-3 agonist) had some activity but only at high concentrations. Septide was almost ineffective and DiMeC7 had an action comparable to that of [MePhe7]NKB. None of the effects induced by these synthetic tachykinin analogs were significantly potentiated by thiorphan. Capsaicin (10 microM) produced a contraction which was unaffected by thiorphan. Both capsaicin and NKA-induced contractions were antagonized by Spantide at concentrations (5-10 microM) which had no effect against the atropine-sensitive contractions produced by electrical field stimulation. Capsaicin (1 microM) produced a consistent release of SP-like immunoreactivity (SP-LI) and a second application of the drug had no further effect, indicating complete desensitization. SP-LI release by capsaicin was almost doubled in the presence of thiorphan. These findings indicate that NK-2 and possibly some NK-3 receptors mediate the contractile response of the guinea-pig gallbladder to tachykinins. Both exogenous and endogenous (released by capsaicin) SP were degraded to a significant extent in this organ via a thiorphan-sensitive mechanism, the identity of which remains to be established.

Expression and function of human hemokinin-1 in human and guinea pig airways.

PubMed

Grassin-Delyle, Stanislas; Naline, Emmanuel; Buenestado, Amparo; Risse, Paul-André; Sage, Edouard; Advenier, Charles; Devillier, Philippe

2010-10-07

Human hemokinin-1 (hHK-1) and endokinins are peptides of the tachykinin family encoded by the TAC4 gene. TAC4 and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study. RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages. In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK1-and NK2-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK2-receptors, which blockade unmasked a NK1-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK1-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages. We demonstrate endogenous expression of TAC4 in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.

Effect of single point mutations of the human tachykinin NK1 receptor on antagonist affinity.

PubMed

Lundstrom, K; Hawcock, A B; Vargas, A; Ward, P; Thomas, P; Naylor, A

1997-10-15

Molecular modelling and site-directed mutagenesis were used to identify eleven amino acid residues which may be involved in antagonist binding of the human tachykinin NK1 receptor. Recombinant receptors were expressed in mammalian cells using the Semliki Forest virus system. Wild type and mutant receptors showed similar expression levels in BHK and CHO cells, verified by metabolic labelling. Binding affinities were determined for a variety of tachykinin NK1 receptor antagonists in SFV-infected CHO cells. The binding affinity for GR203040, CP 99,994 and CP 96,345 was significantly reduced by mutant Q165A. The mutant F268A significantly reduced the affinity for GR203040 and CP 99,994 and the mutant H197A had reduced affinity for CP 96,345. All antagonists seemed to bind in a similar region of the receptor, but do not all rely on the same binding site interactions. Functional coupling to G-proteins was assayed by intracellular Ca2+ release in SFV-infected CHO cells. The wild type receptor and all mutants except A162L and F268A responded to substance P stimulation.

The rabbit iris sphincter contains NK1 and NK3 but not NK2 receptors: a study with selective agonists and antagonists.

PubMed

Wang, Z Y; Håkanson, R

1993-04-08

Tachykinin analogues, claimed to be selective NK1, NK2 and NK3 receptor agonists, contracted the isolated rabbit iris sphincter muscle in a concentration-dependent manner. The contractions were not modified by the enkephalinase inhibitor thiorphan and the angiotensin-converting enzyme inhibitor captopril (10(-5) M of each). The pD2 values for (Sar9,Met(O2)11)SP (NK1 receptor agonist), (Nle10)NKA(4-10) (NK2 receptor agonist) and (MePhe7)NKB (NK3 receptor agonist) were 8.3, 6.1 and 8.2, respectively. (Sar9,Met(O2)11)SP was the most efficacious of the three agonists. The results are compatible with the presence of NK1 and NK3 receptors. The low pD2 value for the NK2 agonist may reflect a lack of NK2 receptors and interaction of the NK2 agonist with NK1 receptors. The contraction caused by the NK1 receptor agonist was inhibited competitively by the highly selective NK1 receptor antagonist (+/-) CP-96,345; the pA2 value was 5.5. Also the contraction caused by the NK2 receptor agonist was inhibited competitively by (+/-) CP-96,345 with a pA2 value of 5.7, supporting the view that the two agonists (Sar9,Met(O2)11)SP and (Nle10)NKA(4-10) interact with the same receptor. The selective NK2 receptor antagonist actinomycin D did not affect the contraction caused by the NK2 receptor agonist. We conclude that the rabbit iris sphincter muscle contains NK1 and probably NK3 receptors. We obtained no evidence for the presence of NK2 receptors.

Augmentation of neurally evoked cholinergic bronchoconstrictor responses by prejunctional NK2 receptors in the guinea-pig.

PubMed

Hey, J A; Danko, G; del Prado, M; Chapman, R W

1996-02-01

1. We examined the effect of exogenously administered tachykinins, neurokinin A (NKA), substance P (SP) and neurokinin B (NKB) on neurally mediated cholinergic bronchoconstrictor responses in guinea-pigs. 2. Electrical stimulation of regions in the dorsal medulla oblongata produced a cholinergic bronchospasm that was not affected by depletion of endogenous tachykinins with capsaicin pretreatment (50 mg kg-1, s.c., 1 week earlier) or by pretreatment with the neutral endopeptidase inhibitor, phosphoramidon (3 mg kg-1, i.v.). 3. Infusion of NKA (0.03-0.1 microgram kg-1 min-1), SP (1 microgram kg-1 min-1) or NKB (1 microgram kg-1 min-1) potentiated the bronchoconstrictor response to electrical stimulation of the dorsal medulla. The doses of tachykinins tested were subthreshold for direct activation of airway smooth muscle, because they were devoid of direct bronchoconstrictor effects. The relative rank order potency for augmentation of centrally induced bronchospasm was NKA > NKB approximately SP, suggesting activation of the NK2 receptor subtype. 4. Infusion of NKA, SP and NKB had no effect on bronchoconstrictor responses to i.v. methacholine (1 microgram kg-1) indicating that a prejunctional neural mechanism of action was responsible for the effects on CNS stimulation-induced bronchospasm. 5. Potentiation of the bronchoconstrictor response to dorsal medullary stimulation produced by infusion of NKA was blocked by pretreatment with the NK2 antagonist SR 48968 (1 mg kg-1, i.v.) but not by the NK1 antagoinst CP 96,345 (1 mg kg-1, i.v.). 6. The potentiation of CNS-induced bronchospasm produced by infusion of SP was partially inhibited by CP 96,345 (1 mg kg-1, i.v.) but not by SR 48968 (1 mg kg-1, i.v.). Treatment with combined SR 48968 (1 mg kg-1, i.v.) and CP 96,345 (1 mg kg-1, i.v.) completely blocked the SP-induced potentiation of CNS-stimulated bronchospasm. 7. These results identify an important modulatory role for NK2 receptors, located at prejunctional sites on parasympathetic nerves, on cholinergic bronchoconstrictor responses in guinea-pigs. 8. It is proposed that substances that release tachykinins from airway sensory nerves, e.g. inflammatory mediators or irritants, may induce hyperresponsiveness of cholinergic bronchomotor responses by activation of NK2-receptors on parasympathetic airway nerves. Furthermore, these studies indicate that endogenous tachykinins are not involved in the maintenance of basal cholinergic bronchomotor tone in the intact guinea-pig.

UTILIZATION OF THE LEAST SHREW AS A RAPID AND SELECTIVE SCREENING MODEL FOR THE ANTIEMETIC POTENTIAL AND BRAIN PENETRATION OF SUBSTANCE P AND NK1 RECEPTOR ANTAGONISTS

PubMed Central

Darmani, Nissar A.; Wang, Yaozhi; Abad, Joseph; Ray, Andrew P.; Thrush, Gerald R.; Ramirez, Juan

2008-01-01

Substance P (SP) is thought to play a cardinal role in emesis via the activation of central tachykinin NK1 receptors during the delayed phase of vomiting produced by chemotherapeutics. Although the existing supportive evidence is significant, due to lack of an appropriate animal model, the evidence is indirect. As yet, no study has confirmed that emesis produced by SP or a selective NK1 receptor agonist is sensitive to brain penetrating antagonists of either NK1, NK2, or NK3 receptors. The goals of this investigation were to demonstrate: 1) whether intraperitoneal (i.p.) administration of either SP, a brain penetrating (GR73632) or non-penetrating (e.g. SarMet – SP) NK1 receptor agonist, an NK2 receptor agonist (GR64349), or an NK3 receptor agonist (Pro7-NKB), would induce vomiting and/or scratching in the least shrew (Cryptotis parva) in a dose-dependent manner; and whether these effects are sensitive to the above selective receptor antagonists; 2) whether an exogenous emetic dose of SP (50 mg/kg, i.p.) can penetrate into the shrew brain stem and frontal cortex; 3) whether GR73632 (2.5 mg/kg, i.p.)-induced activation of NK1 receptors increases Fos-measured neuronal activity in the neurons of both brain stem emetic nuclei and the enteric nervous system of the gut; and 4) whether selective ablation of peripheral NK1 receptors can affect emesis produced by GR73632. The results clearly demonstrated that while SP produced vomiting only, GR73632 caused both emesis and scratching behavior dose-dependently in shrews, and these effects were sensitive to NK1-, but not NK2- or NK3-receptor antagonists. Neither the selective, non-penetrating NK1 receptor agonists, nor the selective NK2- or NK3-receptor agonists, caused a significant dose-dependent behavioral effect. An emetic dose of SP selectively and rapidly penetrated the brain stem but not the frontal cortex. Systemic GR73632 increased Fos expression in the enteric nerve plexi, the medial subnucleus of nucleus tractus solitarius, and the dorsal motor nucleus of the vagus, but not the area postrema. Ablation of peripheral NK1 receptors attenuated the ability of GR73632 to induce a maximal frequency of emesis and shifted its percent animals vomiting dose-response curve to the right. The NK1-ablated shrews exhibited scratching behavior after systemic GR73632-injection. These results, for the first time, affirm a cardinal role for central NK1 receptors in SP-induced vomiting, and a facilitatory role for gastrointestinal NK1 receptors. In addition, these data support the validation of the least shrew as a specific and rapid behavioral animal model to screen concomitantly both the CNS penetration and the antiemetic potential of tachykinin NK1 receptor antagonists. PMID:18471804

Tachykinin NK2 receptors predominantly mediate tachykinin-induced contractions in ovine trachea.

PubMed

Reynolds, A M; Reynolds, P; Holmes, M; Scicchitano, R

1998-01-12

In vitro studies were conducted to characterize the contractile effects of tachykinins in normal ovine trachea with a view in the future to compare tachykinin contractile responses in allergic tissue. Tracheal smooth muscle strips were prepared for in vitro studies of isometric contraction in response to cumulative addition of carbachol, acetylcholine, histamine, neuropeptide gamma, substance P, neurokinin A, neurokinin B, [Sar9, Met(O2)11]substance P, [Nle10]neurokinin A-(4-10), and [Succinyl-Asp6, Me-Phe8]substance P-(6-11) (senktide). The rank order of potency was neuropeptide gamma > carbachol > neurokinin A > or = [Nle10]neurokinin A-(4-10) > acetylcholine > or = histamine. Phosphoramidon enhanced the contractile response to neurokinin A and substance P, but not to neuropeptide gamma, [Sar9, Met(O2)11]substance P or senktide. Repeated cumulative concentration responses for acetylcholine, substance P, neurokinin A, [Sar9, Met(O2)11]substance P and histamine were also conducted to test for tachyphylaxis. No tachyphylaxis to acetylcholine, substance P, or neurokinin A was observed, however, [Sar9, Met(O2)11]substance P and histamine did exhibit tachyphylaxis. Atropine had no effect on tracheal contractions to neurokinin A and substance P, while [Sar9, Met(O2)11]substance P contractions were atropine sensitive. Pyrilamine did not affect substance P-induced tracheal smooth muscle contractions, indicating that the response to substance P was not mediated by histamine release. These results show that, in vitro, natural tachykinins induce tracheal smooth muscle contraction predominantly by a direct effect mediated by tachykinin NK2 receptors, and a small tachykinin NK1 receptor mediated cholinergic mechanism.

Evidence that antidromically stimulated vagal afferents activate inhibitory neurones innervating guinea-pig trachealis.

PubMed Central

Canning, B J; Undem, B J

1994-01-01

1. We recently described a capsaicin-sensitive vagal pathway mediating non-adrenergic, non-cholinergic (NANC) relaxations of an isolated, innervated rostral guinea-pig tracheal preparation. These afferent fibres are carried by the superior laryngeal nerves and relaxations elicited by their activation are insensitive to autonomic ganglion blockers such as hexamethonium. In the present study this vagal relaxant pathway was further characterized. 2. Relaxations of the trachealis elicited by electrical stimulation of capsaicin-sensitive vagal afferents were mimicked by bath application of capsaicin. Relaxations elicited by both methods were abolished when the tissue between the trachea and the adjacent oesophagus was disrupted. Indeed, separating the trachea from the oesophagus uncovered a contractile effect of capsaicin administration on the trachealis. 3. Capsaicin-induced, oesophagus-dependent relaxations of the trachealis were blocked by pretreatment with the fast sodium channel blocker tetrodotoxin (TTX). By contrast, capsaicin-induced contractions of the trachealis (obtained in the absence of the oesophagus) were unaffected by tetrodotoxin. 4. Substance P, neurokinin A (NKA) and neurokinin B (NKB) also elicited NANC relaxations of precontracted trachealis that were abolished by separating the trachea from the oesophagus or by TTX pretreatment. Like capsaicin, the tachykinins elicited only contractions of the trachealis following TTX pretreatment or separation of the trachea from the adjacent oesophagus. 5. Relaxations elicited by stimulation of the capsaicin-sensitive nerves were unaffected by a concentration of the tachykinin NK2 receptor-selective antagonist, SR 48968, that is selective for NK2 receptor blockade and were not mimicked by the NK2 receptor-selective agonist [beta-Ala8]-NKA(4-10). This suggests that NK2 receptors are not responsible for these relaxations. By contrast, the NK3 receptor-selective agonist, senktide analogue, and the NK1 receptor-selective agonist, acetyl-[Arg6, Sar9, Met (O2)11]-SP(6-11), elicited oesophagus-dependent relaxations of the trachealis that were abolished by oesophagus removal. Furthermore, pretreatment with the NK1-selective antagonists, CP 96345 and CP 99994, or pretreatment with a concentration of SR 48968 that also blocks NK3 receptors, markedly attenuated relaxations elicited by stimulation of the capsaicin-sensitive vagal pathways. 6. The data are consistent with the hypothesis that relaxations elicited by stimulation of capsaicin-sensitive vagal afferents involve tachykinin-mediated activation of peripheral NANC inhibitory neurones that are in some way associated with the oesophagus. The data also indicate that airway smooth muscle tone might be regulated by peripheral reflexes initiated by activation of capsaicin-sensitive afferent fibres. PMID:7869272

The Integrated Hypothalamic Tachykinin-Kisspeptin System as a Central Coordinator for Reproduction

PubMed Central

Bosch, Martha A.; León, Silvia; Simavli, Serap; True, Cadence; Pinilla, Leonor; Carroll, Rona S.; Seminara, Stephanie B.; Tena-Sempere, Manuel; Rønnekleiv, Oline K.; Kaiser, Ursula B.

2015-01-01

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r−/− mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreased LH release in ovariectomized-sham replaced females, as documented for NK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons and GnRH neurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons. PMID:25422875

The integrated hypothalamic tachykinin-kisspeptin system as a central coordinator for reproduction.

PubMed

Navarro, Víctor M; Bosch, Martha A; León, Silvia; Simavli, Serap; True, Cadence; Pinilla, Leonor; Carroll, Rona S; Seminara, Stephanie B; Tena-Sempere, Manuel; Rønnekleiv, Oline K; Kaiser, Ursula B

2015-02-01

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r(-/-) mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreased LH release in ovariectomized-sham replaced females, as documented for NK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons and GnRH neurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons.

Neurokinin NK1 and NK3 receptors as targets for drugs to treat gastrointestinal motility disorders and pain.

PubMed

Sanger, Gareth J

2004-04-01

NK1 and NK3 receptors do not appear to play significant roles in normal GI functions, but both may be involved in defensive or pathological processes. NK1 receptor antagonists are antiemetic, operating via vagal sensory and motor systems, so there is a need to study their effects on other gastro-vagal functions thought to play roles in functional bowel disorders. Interactions between NK1 receptors and enteric nonadrenergic, noncholinergic motorneurones suggest a need to explore the role of this receptor in disrupted colonic motility. NK1 receptor antagonism does not exert consistent analgesic activity in humans, but similar studies have not been carried out against pain of GI origin, where NK1 receptors may have additional influences on mucosal inflammatory or "irritant" processes. NK3 receptors mediate certain disruptions of intestinal motility. The activity may be driven by tachykinins released from intrinsic primary afferent neurones (IPANs), which induce slow EPSP activity in connecting IPANs and hence, a degree of hypersensitivity within the enteric nervous system. The same process is also proposed to increase C-fibre sensitivity, either indirectly or directly. Thus, NK3 receptor antagonists inhibit intestinal nociception via a "peripheral" mechanism that may be intestine-specific. Studies with talnetant and other selective NK3 receptor antagonists are, therefore, revealing an exciting and novel pathway by which pathological changes in intestinal motility and nociception can be induced, suggesting a role for NK3 receptor antagonism in irritable bowel syndrome.

Qualitative and quantitative analysis of tachykinin NK2 receptors in chemically defined human colonic neuronal pathways.

PubMed

Jaafari, Nadia; Khomitch-Baud, Alexandra; Gilhodes, Jean-Claude; Hua, Guoqiang; Julé, Yvon

2008-04-01

The involvement of NK2 receptors (NK2r) in the neuroregulation of human colonic motility has been mainly assessed by using pharmacological approaches. The aim of this study was to characterize the intramural neurons and nerve varicosities expressing NK2r in human colonic neuronal pathways. Neuronal coding in the myenteric plexus and external muscle layers was identified on the basis of the patterns of colocalization of tachykinins (TK), vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), glutamate decarboxylase (GAD), and vasoactive intestinal peptide (VIP) with NK2r immunoreactivity. The proportions of chemically defined synaptophysin-immunoreactive nerve varicosities were accurately determined by using specific software. NK2r immunoreactivity was detected in the soma of many myenteric neurons (71.8%). A large proportion of these neurons was immunoreactive to VAChT (39.3%), TK (30%), and GAD (23.5%) and, to a lesser extent, to NOS and VIP. The proportions of nerve varicosities expressing NK2r showed significant regional differences: the highest proportion (59.8%) was located in the myenteric plexus. High proportions of the myenteric nerve varicosities expressing NK2r were immunoreactive to VIP (80.9%) and NOS (77.9%), and lower proportions were recorded with VAChT, TK, and GAD. In the circular and longitudinal muscle layers, the proportions of nerve varicosities expressing NK2r were 49.6% and 45.3%, respectively. The chemically defined intramuscular varicosities were closely apposed to smooth muscle cells expressing NK2r. In conclusion, the data obtained in this study, in which the expression of NK2r was mapped in the human colonic neuronal pathways, confirm that these receptors are involved in the neuroneuronal and neuromuscular processes regulating human colonic motility. Copyright 2008 Wiley-Liss, Inc.

Effects and interactions of tachykinins and dynorphin on FSH and LH secretion in developing and adult rats.

PubMed

Ruiz-Pino, F; Garcia-Galiano, D; Manfredi-Lozano, M; Leon, S; Sánchez-Garrido, M A; Roa, J; Pinilla, L; Navarro, V M; Tena-Sempere, M

2015-02-01

Kisspeptin/neurokinin B/dynorphin (KNDy) neurons, which coexpress kisspeptins (Kps), neurokinin B (NKB), and dynorphin (Dyn), regulate gonadotropin secretion. The KNDy model proposes that NKB (a stimulator, through NK3R) and Dyn (an inhibitor, through κ-opioid receptor) shape Kp secretion onto GnRH neurons. However, some aspects of this paradigm remain ill defined. Here we aimed to characterize the following: 1) the effects of NKB signaling on FSH secretion and 2) the role of Dyn in gonadotropin secretion after NK3R activation; 3) additionally, we explored the roles of other tachykinin receptors, NK1R and NK2R, on gonadotropin release. Thus, the effects of the NK3R agonist, senktide, on FSH release were explored across postnatal development in male and female rats; gonadotropin responses to agonists of NK1R substance P and NK2R [neurokinin A (NKA)] were also monitored. Moreover, the effects of senktide on gonadotropin secretion were assessed after antagonizing Dyn actions by nor-binaltorphimine didydrochloride. Before puberty, rats of both sexes showed increased FSH secretion to senktide (and Kp-10). Conversely, adult female rats were irresponsive to senktide in terms of FSH, despite proven LH responses, whereas the adult males did not display FSH or LH responses to senktide, even at high doses. In turn, substance P and NKA stimulated gonadotropin secretion in prepubertal rats, whereas in adults modest gonadotropin responses to NKA were detected. By pretreatment with a Dyn antagonist, adult males became responsive to senktide in terms of LH secretion and displayed elevated basal LH and FSH levels; nor-binaltorphimine didydrochloride treatment uncovered FSH responses to senktide in adult females. Furthermore, the expression of Pdyn and Opkr1 (encoding Dyn and κ-opioid receptor, respectively) in the mediobasal hypothalamus was greater in males than in females at prepubertal ages. Overall, our data contribute to refining our understanding on how the elements of the KNDy node and related factors (ie, other tachykinins) differentially participate in the control of gonadotropins at different stages of rat postnatal maturation.

Genetic control of indirect airway responsiveness in the rat.

PubMed

Pauwels, R A; Germonpré, P R; Kips, J C; Joos, G F

1995-11-01

Many of the airway responses to endogenous and exogenous stimuli are caused by indirect mechanisms such as the activation of neurons and/or inflammatory cells. In the present study we compare the bronchoconstrictor and the plasma protein extravasation response to adenosine and tachykinins in two highly inbred rat strains, F344 and BDE. BDE-rats have a bronchoconstrictor response to adenosine at lower doses. Challenge with the A3-adenosine receptor agonist APNEA demonstrates that the difference in airway responsiveness to adenosine between BDE- and F344-rats is probably related to a higher number of A3-receptors on the airway mast cells of BDE-rats. In contrast, F344-rats have a higher airway responsiveness to tachykinins than BDE-rats. Tachykinins cause bronchoconstriction in F344-rats mainly by an indirect mechanism, involving stimulation of NK1-receptors and mast cell activation. In BDE-rats they cause bronchoconstriction by a direct effect on airway smooth muscle via activation of NK2-receptors. Finally we also observed a difference between F344- and BDE-rats with regard to the mechanisms involved in the plasma protein extravasation in the airways caused by substance P or capsaicin. In F344-rats but not in BDE-rats mast cell activation and the release of 5-hydroxytryptamine is partly responsible for this plasma protein extravasation.

TAC1 Gene Products Regulate Pituitary Hormone Secretion and Gene Expression in Prepubertal Grass Carp Pituitary Cells.

PubMed

Hu, Guangfu; He, Mulan; Ko, Wendy K W; Wong, Anderson O L

2017-06-01

Tachykinin-1 (TAC1) is known to have diverse functions in mammals, but similar information is scarce in fish species. Using grass carp as a model, the pituitary actions, receptor specificity and postreceptor signaling of TAC1 gene products, namely substance P (SP) and neurokinin A (NKA), were examined. TAC1 encoding SP and NKA as well as tachykinin receptors NK1R and NK2R were cloned in the carp pituitary. The newly cloned receptors were shown to be functional with properties similar to mammalian counterparts. In carp pituitary cells, SP and NKA could trigger luteinizing hormone (LH), prolactin (PRL), and somatolactin α (SLα) secretion, with parallel rises in PRL and SLα transcripts. Short-term SP treatment (3 hours) induced LH release, whereas prolonged induction (24 hours) could attenuate LHβ messenger RNA (mRNA) expression. At pituitary cell level, LH, PRL, and SLα regulation by TAC1 gene products were mediated by NK1R, NK2R, and NK3R, respectively. Apparently, SP- and NKA-induced LH and SLα secretion and transcript expression were mediated by adenylyl cyclase/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholiphase C (PLC)/inositol 1,4,5-triphosphate/protein kinase C (PKC), and Ca2+/calmodulin (CaM)/CaM-dependent protein kinase-II pathways. The signal transduction for PRL responses was similar, except for the absence of a PKC component. Regarding SP inhibition of LHβ mRNA expression, the cAMP/PKA- and PLC/PKC-dependent (but not Ca2+/CaM-dependent) cascades were involved. These results, as a whole, suggest that TAC1 gene products play a role in LH, PRL, and SLα regulation via overlapping postreceptor signaling coupled to different subtypes of tachykinin receptor expressed in the carp pituitary. Copyright © 2017 Endocrine Society.

Proinflammatory tachykinins that signal through the neurokinin 1 receptor promote survival of dendritic cells and potent cellular immunity.

PubMed

Janelsins, Brian M; Mathers, Alicia R; Tkacheva, Olga A; Erdos, Geza; Shufesky, William J; Morelli, Adrian E; Larregina, Adriana T

2009-03-26

Dendritic cells (DCs) are the preferred targets for immunotherapy protocols focused on stimulation of cellular immune responses. However, regardless of initial promising results, ex vivo generated DCs do not always promote immune-stimulatory responses. The outcome of DC-dependent immunity is regulated by proinflammatory cytokines and neuropeptides. Proinflammatory neuropeptides of the tachykinin family, including substance P (SP) and hemokinin-1 (HK-1), bind the neurokinin 1 receptor (NK1R) and promote stimulatory immune responses. Nevertheless, the ability of pro-inflammatory tachykinins to affect the immune functions of DCs remains elusive. In the present work, we demonstrate that mouse bone marrow-derived DCs (BMDCs) generated in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), express functional NK1R. Signaling via NK1R with SP, HK-1, or the synthetic agonist [Sar(9)Met(O(2))(11)]-SP rescues DCs from apoptosis induced by deprivation of GM-CSF and IL-4. Mechanistic analysis demonstrates that NK1R agonistic binding promotes DC survival via PI3K-Akt signaling cascade. In adoptive transfer experiments, NK1R-signaled BMDCs loaded with Ag exhibit increased longevity in draining lymph nodes, resulting in enhanced and prolonged effector cellular immunity. Our results contribute to the understanding of the interactions between the immune and nervous systems that control DC function and present a novel approach for ex vivo-generation of potent immune-stimulatory DCs.

Tachykinin NK2 receptors and enhancement of cholinergic transmission in the inflamed rat colon: an in vivo motility study

PubMed Central

Carini, F; Lecci, A; Tramontana, M; Giuliani, S; Maggi, C A

2001-01-01

In the gastrointestinal tract, tachykinin NK2 receptors are localized both on smooth muscle and nerve fibres. NK2 receptor antagonists reduce exaggerated intestinal motility in various diarrhoea models but the site of action contributing to this effect is unknown. In this study we investigated the effects of atropine (1.4 μmol kg−1, i.v.), hexamethonium (13.5 μmol kg−1, i.v.), and nepadutant (0.1 μmol kg−1, i.v.), a selective tachykinin NK2 receptor antagonist, on distension (0.5 and 1 ml)-, or irritation (acetic acid, 0.5 ml of 7.5% v v−1)-induced motility in the rat distal colon in vivo. The effects of atropine, hexamethonium or Nω-nitro-L-argininemethylester (L-NAME, 1.85 μmol kg−1, i.v.) on [βAla8]NKA(4-10) (10 nmol kg−1, i.v.)-induced colonic contractions were also investigated.When the colonic balloon was filled with a subthreshold volume (0.5 ml), the intraluminal instillation of acetic acid triggered a high-amplitude phasic colonic motility which was partially reduced by nepadutant and suppressed by either hexamethonium or atropine. Filling of the balloon with 1 ml evoked reflex (hexamethonium-sensitive), atropine-sensitive phasic colonic motility: nepadutant had no significant effect on the distension-evoked motility.Neither hexamethonium nor atropine significantly reduced [βAla8]NKA(4-10)-induced colonic contractions, whereas nepadutant suppressed them. Following L-NAME pretreatment, [βAla8]NKA(4-10)-induced colonic contractions were inhibited by both atropine and hexamethonium. In hexamethonium-pretreated animals, an atropine-sensitive component of [βAla8]NKA(4-10)-induced colonic contractions was also evident.These results indicate that the application of irritants onto the colonic mucosa induces the release of endogenous tachykinins which enhance excitatory cholinergic mechanisms through the stimulation of NK2 receptors. PMID:11487522

Role of nitric oxide and septide-insensitive NK1 receptors in bronchoconstriction induced by aerosolised neurokinin A in guinea-pigs

PubMed Central

Ricciardolo, Fabio L M; Trevisani, Marcello; Geppetti, Pierangelo; Nadel, Jay A; Amadesi, Silvia; Bertrand, Claude

2000-01-01

The tachykinin, neurokinin A (NKA), contracts guinea-pig airways both in vitro and in vivo, preferentially activating smooth muscle NK2 receptors, although smooth muscle NK1 receptors may also contribute. In vitro evidence suggests that NKA activates epithelial NK1 receptors, inducing the release of nitric oxide (NO) and subsequent smooth muscle relaxation. A number of selective NK1 receptor agonists have been reported to activate both smooth muscle and epithelial NK1 receptors, however septide appears only to activate smooth muscle NK1 receptors. The aim of the present study was to investigate whether NKA-induced bronchoconstriction in guinea-pigs in vivo may be limited by NO release via NK1 receptor activation, and whether selective NK1 receptor agonists may activate this mechanism differently. Aerosolized NKA caused an increase in total pulmonary resistance (RL) that was markedly reduced by the NK2 receptor antagonist, SR 48968, and abolished by the combination of SR 48968 and the NK1 receptor antagonist, CP-99,994. The increase in RL evoked by NKA was potentiated by pretreatment with the NO synthase (NOs) inhibitor, L-NAME, but not by the inactive enantiomer D-NAME. Potentiation by L-NAME of NKA-induced increase in RL was reversed by L-Arginine, but not by D-Arginine. Pretreatment with L-NAME did not affect the increase in RL induced by the selective NK2 receptor agonist, [β-Ala8]NKA(4-10), and by the selective NK1 receptor agonist, septide, whereas it markedly potentiated the increase in RL caused by a different NK1 selective agonist, [Sar9,Met(O2)11]SP. Dose-response curves showed that septide was a more potent bronchoconstrictor than [Sar9,Met(O2)11]SP to cause bronchoconstriction. Pretreatment with the NK1 receptor antagonist, CP-96,994, abolished the ability of L-NAME to increase bronchoconstriction to aerosolized NKA. Bronchoconstriction to aerosolized NKA was increased by L-NAME, after pretreatment with the NK3 receptor antagonist, SR 142801. The present study shows that in vivo bronchoconstriction in response to the aerosolized naturally occurring tachykinin, NKA, is limited by its own ability to release relaxant NO via NK1 receptor activation. This receptor is apparently insensitive to septide, thus justifying, at least in part, the high potency of septide to cause bronchoconstriction in guinea-pigs. PMID:10696090

Role of nitric oxide and septide-insensitive NK(1) receptors in bronchoconstriction induced by aerosolised neurokinin A in guinea-pigs.

PubMed

Ricciardolo, F L; Trevisani, M; Geppetti, P; Nadel, J A; Amadesi, S; Bertrand, C

2000-03-01

The tachykinin, neurokinin A (NKA), contracts guinea-pig airways both in vitro and in vivo, preferentially activating smooth muscle NK(2) receptors, although smooth muscle NK(1) receptors may also contribute. In vitro evidence suggests that NKA activates epithelial NK(1) receptors, inducing the release of nitric oxide (NO) and subsequent smooth muscle relaxation. A number of selective NK(1) receptor agonists have been reported to activate both smooth muscle and epithelial NK(1) receptors, however septide appears only to activate smooth muscle NK(1) receptors. The aim of the present study was to investigate whether NKA-induced bronchoconstriction in guinea-pigs in vivo may be limited by NO release via NK(1) receptor activation, and whether selective NK(1) receptor agonists may activate this mechanism differently. Aerosolized NKA caused an increase in total pulmonary resistance (RL) that was markedly reduced by the NK(2) receptor antagonist, SR 48968, and abolished by the combination of SR 48968 and the NK(1) receptor antagonist, CP-99, 994. The increase in RL evoked by NKA was potentiated by pretreatment with the NO synthase (NOs) inhibitor, L-NAME, but not by the inactive enantiomer D-NAME. Potentiation by L-NAME of NKA-induced increase in RL was reversed by L-Arginine, but not by D-Arginine. Pretreatment with L-NAME did not affect the increase in RL induced by the selective NK(2) receptor agonist, [beta-Ala(8)]NKA(4-10), and by the selective NK(1) receptor agonist, septide, whereas it markedly potentiated the increase in RL caused by a different NK(1) selective agonist, [Sar(9),Met(O(2))(11)]SP. Dose-response curves showed that septide was a more potent bronchoconstrictor than [Sar(9),Met(O(2))(11)]SP to cause bronchoconstriction. Pretreatment with the NK(1) receptor antagonist, CP-96,994, abolished the ability of L-NAME to increase bronchoconstriction to aerosolized NKA. Bronchoconstriction to aerosolized NKA was increased by L-NAME, after pretreatment with the NK(3) receptor antagonist, SR 142801. The present study shows that in vivo bronchoconstriction in response to the aerosolized naturally occurring tachykinin, NKA, is limited by its own ability to release relaxant NO via NK(1) receptor activation. This receptor is apparently insensitive to septide, thus justifying, at least in part, the high potency of septide to cause bronchoconstriction in guinea-pigs.

Radioligand binding, autoradiographic and functional studies demonstrate tachykinin NK-2 receptors in dog urinary bladder.

PubMed

Mussap, C J; Stamatakos, C; Burcher, E

1996-10-01

Tachykinin receptors in the dog bladder were characterized using radioligand binding, functional and autoradiographic techniques. In detrusor muscle homogenates, specific binding of [125l]iodohistidyl neurokinin A (INKA) and [125l]Bolton Hunter eledoisin was reversible, saturable and, to a single class of sites of Kd, 3,6 and 27 nM, respectively. No specific binding of [125l]Bolton Hunter[Sar9, Met (O2)11] substance P occurred. INKA binding was reduced by the peptidase inhibitor bacitracin. The rank potency order of agonists competing for binding of both radioligands indicated interaction at NK-2 sites. NK-2-selective antagonists also competed for INKA binding, with SR 48968, GR 94800, MDL 29913 and the selective agonist [Lys5, MeLeu9, Nle10]-NKA(4-10) showing biphasic binding profiles. Autoradiographic studies revealed specific binding of INKA and [125l]Bolton Hunter eledoisin over detrusor muscle and small arteries. [125l]Bolton Hunter [Sar9, Met (O2)11] SP labeled the intima of arteries and arterioles, but not the detrusor muscle. Tachykinins contracted detrusor muscle strips, with potency order at the carbachol EC15 NKA = kassinin > [Lys5, MeLeu9, Nle10]-NKA(4-10) = neuropeptide gamma = neuropeptide K = NKB > > MDL 28564, with [Sar9, Met(O2)11]-SP ineffective. Shallow concentration-response curves, variable efficacies and inhibition by atropine and mepyramine suggest that other mechanisms may influence contractile responses. Responses to [Lys5, MeLeu9, Nle10]-NKA(4-10) were inhibited competitively by MDL 29913 and MEN 10207 (pA2 values: 6.4 and 5.3, respectively). Antagonism by SR 48968 and GR 94800 was noncompetitive (both pK8 values 8.9). In summary, NK-2-preferring ligands showed superior potency as both binding competitors and contractile agonists, demonstrating that NK-2 receptors mediate detrusor muscle contraction, similar to the human detrusor. Tachykinins may play important roles in the micturition reflex and in regulating detrusor muscle blood flow in the dog.

Bronchodilatation by tachykinins and capsaicin in the mouse main bronchus.

PubMed

Manzini, S

1992-04-01

1. The effect of sensory neuropeptides and capsaicin on basal and stimulated tone of mouse bronchial smooth muscle has been evaluated. 2. In basal conditions neither sensory neuropeptides (substance P, neurokinin A or calcitonin gene-related peptide (CGRP) nor capsaicin exerted any contractile effects. However, when a tonic contraction was induced with carbachol (1 microM) a prompt relaxation was induced by substance P (1- 100 nM) and by neurokinin A (1- 100 nM), with substance P being more potent. A second application of substance P was without effect. CGRP (10 nM) produced only a very small and erratic relaxation. Relaxation was also induced by capsaicin (1 microM), and this response could be evoked only once in each preparation. In 4 out of 6 preparations a cross-desensitization between substance P and capsaicin was observed. 3. The selective NK1 tachykinin agonist, [Pro9]-SP sulphone (1 microM), exerted potent bronchodilator actions on carbachol-contracted mouse bronchial preparations. In contrast, neither [beta Ala8]-NKA (4-10) nor [MePhe7]-NKB (both at a concentration of 1 microM), selective synthetic agonists for NK2 and NK3 receptors, exerted significant relaxant effects. Furthermore, the selective NK1 tachykinin antagonist, (+/-)-CP 96,345 (1 microM), abolished substance P (1 nM)- but not isoprenaline (0.1 microM)-induced relaxations. 4. Application of electrical field stimulation (EFS) (20 Hz, supramaximal voltage, 0.5 ms for 10 s) to carbachol-contracted preparations evoked a transient contraction followed by a relaxation. The tetrodotoxin-sensitive slow component of this relaxation was reduced following capsaicin desensitization. 5. In the presence of indomethacin (5 microM) the relaxation induced by substance P, capsaicin or EFS was suppressed.6. In conclusion, the mouse main bronchus appears to be a monoreceptorial tissue containing only NK, receptors which subserve bronchodilator functions. Such receptors could be activated by exogenous or endogenously (capsaicin or EFS) released tachykinins and the consequent relaxation is probably mediated by the generation of prostanoids.

Sensory neuropeptides and the human lower airways: present state and future directions.

PubMed

Joos, G F; Germonpre, P R; Kips, J C; Peleman, R A; Pauwels, R A

1994-06-01

The sensory neuropeptides, substance P and neurokinin A, are present in human airway nerves, beneath and within the epithelium, around blood vessels and submucosal glands, and within the bronchial smooth muscle layer. Studies on autopsy tissue, bronchoalveolar lavage and sputum suggest that in asthma the substance P content of the airways may be increased. Neurokinin A is a more potent bronchoconstrictor than substance P. Asthmatics are hyperresponsive to neurokinin A and substance P. The neuropeptide degrading enzyme, neutral endopeptidase is present in the airways and is involved in the degradation of endogenously released and exogenously administered substance P and neurokinin A, both in normal and asthmatic subjects. As for other indirect bronchoconstrictor stimuli, the effect of neurokinin A on airway calibre in asthmatics can be inhibited by pretreatment with nedocromil sodium. Evidence is accumulating, not only from studies in animals but also from experiments on human airways, that tachykinins may also cause mucus secretion and plasma extravasation. They also have important proinflammatory effects, such as the chemoattraction of eosinophils and neutrophils, the adhesion of neutrophils, and the stimulation of lymphocytes, macrophages and mast cells. The tachykinins interact with the targets on the airways by specific tachykinin receptors. The NK1 and the NK2 receptor have been characterized in human airways, both pharmacologically and by cloning. The NK2 receptor is responsible for the in vitro contraction of normal airways, whilst the NK1 receptor is responsible for most of the other airway effects. Because of their presence in the airways and because of their ability to mimic the various pathophysiological features of asthma, substance P and neurokinin A are presently considered as possible mediators of asthma. The present development of potent and selective tachykinin antagonists will allow us to further define the role of tachykinins in the pathogenesis of asthma.

Three days after a single exposure to ozone, the mechanism of airway hyperreactivity is dependent on substance P and nerve growth factor.

PubMed

Verhein, Kirsten C; Hazari, Mehdi S; Moulton, Bart C; Jacoby, Isabella W; Jacoby, David B; Fryer, Allison D

2011-02-01

Ozone causes persistent airway hyperreactivity in humans and animals. One day after ozone exposure, airway hyperreactivity is mediated by release of eosinophil major basic protein that inhibits neuronal M(2) muscarinic receptors, resulting in increased acetylcholine release and increased smooth muscle contraction in guinea pigs. Three days after ozone, IL-1β, not eosinophils, mediates ozone-induced airway hyperreactivity, but the mechanism at this time point is largely unknown. IL-1β increases NGF and the tachykinin substance P, both of which are involved in neural plasticity. These experiments were designed to test whether there is a role for NGF and tachykinins in sustained airway hyperreactivity following a single ozone exposure. Guinea pigs were exposed to filtered air or ozone (2 parts per million, 4 h). In anesthetized and vagotomized animals, ozone potentiated vagally mediated airway hyperreactivity 24 h later, an effect that was sustained over 3 days. Pretreatment with antibody to NGF completely prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone and significantly reduced the number of substance P-positive airway nerve bundles. Three days after ozone, NK(1) and NK(2) receptor antagonists also blocked this sustained hyperreactivity. Although the effect of inhibiting NK(2) receptors was independent of ozone, the NK(1) receptor antagonist selectively blocked vagal hyperreactivity 3 days after ozone. These data confirm mechanisms of ozone-induced airway hyperreactivity change over time and demonstrate 3 days after ozone that there is an NGF-mediated role for substance P, or another NK(1) receptor agonist, that enhances acetylcholine release and was not present 1 day after ozone.

Three days after a single exposure to ozone, the mechanism of airway hyperreactivity is dependent on substance P and nerve growth factor

PubMed Central

Verhein, Kirsten C.; Hazari, Mehdi S.; Moulton, Bart C.; Jacoby, Isabella W.; Jacoby, David B.

2011-01-01

Ozone causes persistent airway hyperreactivity in humans and animals. One day after ozone exposure, airway hyperreactivity is mediated by release of eosinophil major basic protein that inhibits neuronal M2 muscarinic receptors, resulting in increased acetylcholine release and increased smooth muscle contraction in guinea pigs. Three days after ozone, IL-1β, not eosinophils, mediates ozone-induced airway hyperreactivity, but the mechanism at this time point is largely unknown. IL-1β increases NGF and the tachykinin substance P, both of which are involved in neural plasticity. These experiments were designed to test whether there is a role for NGF and tachykinins in sustained airway hyperreactivity following a single ozone exposure. Guinea pigs were exposed to filtered air or ozone (2 parts per million, 4 h). In anesthetized and vagotomized animals, ozone potentiated vagally mediated airway hyperreactivity 24 h later, an effect that was sustained over 3 days. Pretreatment with antibody to NGF completely prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone and significantly reduced the number of substance P-positive airway nerve bundles. Three days after ozone, NK1 and NK2 receptor antagonists also blocked this sustained hyperreactivity. Although the effect of inhibiting NK2 receptors was independent of ozone, the NK1 receptor antagonist selectively blocked vagal hyperreactivity 3 days after ozone. These data confirm mechanisms of ozone-induced airway hyperreactivity change over time and demonstrate 3 days after ozone that there is an NGF-mediated role for substance P, or another NK1 receptor agonist, that enhances acetylcholine release and was not present 1 day after ozone. PMID:21056958

In vitro characterization of the effects of rat/mouse hemokinin-1 on mouse colonic contractile activity: a comparison with substance P.

PubMed

Kong, Zi-Qing; Han, Min; Yang, Wen-Le; Zhao, You-Li; Fu, Cai-Yun; Tao, Yan; Chen, Qiang; Wang, Rui

2009-06-01

Rat/mouse hemokinin-1 (r/m HK-1) has been identified as a member of the tachykinin family and its effect in colonic contractile activity remains unknown. We investigated the effects and mechanisms of actions of r/m HK-1 on the mouse colonic contractile activity in vitro by comparing it with that of substance P (SP). R/m HK-1 induced substantial contractions on the circular muscle of mouse colon. The maximal contractile responses to r/m HK-1 varied significantly among proximal-, mid- and distal-colon, suggesting that the action of r/m HK-1 was region-specific in mouse colon. The contractile response induced by r/m HK-1 is primarily via activation of tachykinin NK(1) receptors leading to activation of cholinergic excitatory pathways and with a minor contribution of NK(2) receptors, which may be on the smooth muscle itself. A direct action on colonic smooth muscles may be also involved. In contrast, SP induced biphasic colonic responses (contractile and relaxant responses) on the circular muscle, in which the contractile action of SP was equieffective with r/m HK-1. SP exerted its contractile effect predominantly through neural and muscular tachykinin NK(1) receptors, but unlike r/m HK-1 did not appear to act via NK(2) receptors. The relaxation induced by SP was largely due to release of nitric oxide (NO) produced via an action on neural NK(1) receptors. These results indicate that the receptors and the activation properties involved in r/m HK-1-induced mouse colonic contractile activity are different from those of SP.

Characterization of the [125I]-neurokinin A binding site in the circular muscle of human colon

PubMed Central

Warner, Fiona J; Comis, Alfio; Miller, Robert C; Burcher, Elizabeth

1999-01-01

Neurokinin A (NKA) is a potent contractile agonist of human colon circular muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [125I]-NKA has been used to characterize binding sites in this tissue, using tachykinin agonists and antagonists. 125INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [125I]-NKA was saturable (KD 0.47±0.05 nM), of high capacity (Bmax 2.1±0.1 fmol mg−1 wet weight tissue) and reversible (kinetically derived KD 0.36±0.07 nM). The rank order of agonists competing for the [125I]-NKA binding site was neuropeptide γ (NPγ)≥NKA≥[Lys5,MeLeu9,Nle10]NKA (4–10) (NK2 agonist)>>substance P (SP)>neurokinin B (NKB)≥[Pro9]SP (NK1 agonist)>>senktide (NK3 agonist), indicating binding to an NK2 site. The nonpeptide selective NK2 antagonist SR48968 showed higher affinity for the [125I]-NKA site than selective peptide NK2 antagonists. The rank order of potency for NK2 antagonists was SR48968≥MEN11420>GR94800≥MEN10627>MEN10376≥R396. The NK1 antagonist SR140333 was a weak competitor. The competition curve for SP could be resolved into two sites. When experiments were repeated in the presence of SR140333 (0.1 μM), the curve for SP became monophasic and showed a significant shift to the right, whereas curves to NKA and NKB were unaffected. In conclusion, binding of the radioligand [125I]-NKA to membranes from circular muscle is predominantly to the NK2 receptor. There may be a small component of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscle is unclear. PMID:10455255

The role of tachykinin NK1 and NK2 receptors in atropine-resistant colonic propulsion in anaesthetized guinea-pigs.

PubMed

Lecci, A; Giuliani, S; Tramontana, M; Giorgio, R D; Maggi, C A

1998-05-01

1. The role of endogenous tachykinins on guinea-pig colonic propulsion was investigated by using potent and selective tachykinin NK1 and NK2 receptor antagonists. Colonic propulsion and contractions were determined by means of a balloon-catheter device, inserted into the rectum of guanethidine (68 micromol kg(-1), s.c., 18 and 2 h before)-pretreated, urethane-anaesthetized guinea-pigs. Propulsion of the device (dynamic model) was determined by measuring the length of the catheter expelled during 60 min filling of the balloon (flow rate 5 microl min(-1)). 2. In control conditions the tachykinin NK1 receptor antagonist SR 140333 (1 micromol kg(-1), i.v.) did not affect either colonic propulsion or the amplitude of contractions. The tachykinin NK2 receptor antagonists MEN 10627 and MEN 11420 (1 micromol kg(-1), i.v.) increased colonic propulsion at 10 min (+120% and 150%, respectively) but at 60 min the effect was significant only for MEN 10627 (+84%). SR 48968 (1 micromol kg(-1), i.v.) did not significantly enhance the colonic propulsion. None of these tachykinin NK2 receptor antagonists modified the amplitude of colonic contractions. In contrast, both atropine (6 micromol kg(-1), i.v., plus infusion of 1.8 micromol h(-1)) and hexamethonium (55 micromol kg(-1), i.v., plus infusion of 17 micromol h(-1)) abolished propulsion (81% and 87% inhibition, respectively) and decreased the amplitude of contractions (68% inhibition for either treatment). 3. In atropine-treated animals (6 micromol kg(-1), i.v., plus infusion of 1.8 micromol h(-1)), apamin (30 nmol kg(-1), i.v.) restored colonic propulsion (+416%) and increased the amplitude of contractions (+367% as compared to atropine alone). Hexamethonium (55 micromol kg(-1), i.v., plus infusion of 17 micromol h(-1)) abolished the apamin-induced, atropine-resistant colonic propulsion (97% inhibition) and reduced the amplitude of the atropine-resistant contractions (52% inhibition). 4. The apamin-induced, atropine-resistant colonic propulsion was inhibited by SR 140333 (-69% at 1 micromol kg(-1)), SR 48968 (-78% at 1 micromol kg(-1)), MEN 11420 (-59% at 1 micromol kg(-1)) and MEN 10627 (-50% at 1 micromol kg(-1)), although the latter effect was not statistically significant. The combined administration of SR 140,333 and MEN 10,627 (1 micromol kg(-1) for each antagonist) almost completely abolished colonic propulsion (90% inhibition). The amplitude of colonic contractions was also reduced by SR 140333 (-42%), SR 48968 (-29%), MEN 11420 (-45%) but not by MEN 10627 (-16%). The combined administration of SR 140333 and MEN 10,627 reduced the amplitude of contractions by 47%. SR 140603 (1 micromol kg(-1), i.v.), the less potent enantiomer of SR 140333, was inactive. 5. In control animals, apamin (30 nmol kg(-1), i.v.) enhanced colonic propulsion (+84%) and increased the amplitude of contractions (+68%), as compared to the vehicle. Hexamethonium (55 micromol kg(-1), i.v. plus infusion of 17 micromol h(-1)) inhibited propulsion (86% inhibition) and decreased the amplitude of contractions (49% inhibition). SR 140333, SR 48968, MEN 11420, MEN 10627, or the coadministration of SR 140333 and MEN 10627 had no effect. 6. In a separate series of experiments, the mean amplitude of colonic contractions was also recorded under isovolumetric conditions through the balloon-catheter device kept in place at 75 mm from the anal sphincter (static model). In control conditions, neither SR 140333 nor MEN 11420 modified the amplitude of contractions. In atropine-pretreated guinea-pigs, SR 140333 and MEN 11420 (0.1-1 micromol kg(-1)) dose-dependently decreased the amplitude of contractions. In apamin- and atropine-pretreated animals, only the highest (1 micromol kg(-1)) dose of SR 140333 or MEN 11420 significantly decreased the amplitude of contractions. The inhibitory potency of atropine (0.3-1 micromol kg(-1)) was similar in apamin-pretreated animals and in controls. 7. It was concluded that, in anaesthetized guinea-pigs, endogenous tachykinins, acting through both NK(1) and NK(2) receptors, act as non-cholinergic excitatory neurotransmitters in promoting an apamin-evoked reflex propulsive activity of the distal colon.

Use of NK1 receptor antagonists in the exploration of physiological functions of substance P and neurokinin A.

PubMed

Otsuka, M; Yoshioka, K; Yanagisawa, M; Suzuki, H; Zhao, F Y; Guo, J Z; Hosoki, R; Kurihara, T

1995-07-01

Tachykinin NK1 receptor antagonists were used to explore the physiological functions of substance P (SP) and neurokinin A (NKA). Pharmacological profiles of three NK1 receptor antagonists, GR71251, GR82334, and RP 67580, were examined in the isolated spinal cord preparation of the neonatal rat. These tachykinin receptor antagonists exhibited considerable specificities and antagonized the actions of both SP and NKA to induce the depolarization of ventral roots. Electrical stimulation of the saphenous nerve with C-fiber strength evoked a depolarization lasting about 30 s of the ipsilateral L3 ventral root. This response, which is referred to as saphenous-nerve-evoked slow ventral root potential (VRP), was depressed by these NK1 receptor antagonists. In contrast, the saphenous-nerve-evoked slow VRP was potentiated by application of a mixture of peptidase inhibitors, including thiorphan, actinonin, and captopril in the presence of naloxone, but not after further addition of GR71251. Likewise, in the isolated coeliac ganglion of the guinea pig, electrical stimulation of the mesenteric nerves evoked in some ganglionic cells slow excitatory postsynaptic potentials (EPSPs), which were depressed by GR71251 and potentiated by peptidase inhibitors. These results further support the notion that SP and NKA serve as neurotransmitters producing slow EPSPs in the neonatal rat spinal cord and guinea pig prevertebral ganglia.

Heterogeneity of NK-2 tachykinin receptors in hamster and rabbit smooth muscles.

PubMed

Maggi, C A; Eglezos, A; Quartara, L; Patacchini, R; Giachetti, A

1992-01-23

The possible existence of NK-2 receptor subtypes in peripheral smooth muscle preparations from rabbit and hamster was investigated by studying the effect of neurokinin A, the selective NK-2 receptor agonist [beta Ala8] neurokinin A (4-10), the selective NK-2 tachykinin receptor antagonists, MEN 10,376, L 659,877 and R 396, and the pseudopeptide derivative of neurokinin A (4-10), MDL 28,564. All experiments were performed in the presence of peptidase inhibitors (captopril, bestatin and thiorphan, 1 microM each). Both neurokinin A and [beta Ala8] neurokinin A (4-10) produced concentration-dependent contractions of the rabbit isolated bronchus and hamster isolated stomach and colon, as well as enhancement of the nerve-mediated twitches of rabbit isolated vas deferens (pars prostatica). MEN 10,376, L 659,877 and R 396 antagonized the effect of the NK-2 receptor selective agonist in all four tissues under study, although marked differences in antagonist potency were evident for the three antagonists. Thus MEN 10,376 was distinctly more potent (about 100 times) in rabbit than in hamster preparations while L 659,877 and R 396 were more potent in hamster than rabbit preparations. MDL 28,564 showed a distinct agonist character in rabbit preparations while it was virtually inactive in hamster preparations, where it antagonized the effect of the NK-2 receptor selective agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that tachykinin NK2 receptors modulate resting tone in the rat isolated small intestine.

PubMed Central

Maggi, C. A.; Giuliani, S.

1996-01-01

1. In the progress of experiments aimed at evaluating the role of tachykinins as enteric nonadrenergic noncholinergic (NANC) transmitters, we noted that certain tachykinin receptor antagonists produce a relaxation of circular muscle strips in the rat small intestine. This study aimed to assess the nature of this response and to determine the receptor type involved. The majority of the experiments were performed in capsaicin- (10 microM for 15 min) pretreated mucosa-free circular muscle strips from the rat small intestine, in the presence of atropine (1 microM), guanethidine (3 microM) and indomethacin (10 microM). 2. Under isometric recording of mechanical activity, the tachykinin NK1 receptor antagonist SR 140,333 (0.1 microM) had no effect on resting tone or spontaneous activity in duodenal or ileal circular muscle strips. The NK2 receptor antagonists, MEN 10,627 (0.1 microM) and GR 94,800 (0.1 microM) produced, after a delay of 10-15 min, a relaxation which averaged 61 +/- 3 and 57 +/- 6% (n = 6 and 4, respectively) of the maximal response (Emax) to isoprenaline (1 microM). The effect of maximal concentrations of MEN 10,627 and GR 94,800 when applied together was non-additive. The relaxant effect of MEN 10,627 (0.1 microM) was similar in the absence and presence of apamin (0.3 microM) and L-nitroarginine (100 microM). 3. Under isotonic recording of mechanical activity, MEN 10,627 (10 nM-1 microM) produced a concentration- and time-related relaxation of duodenal strips. The maximal relaxation averaged 72 +/- 4 and 69 +/- 4% (n = 5 each) of Emax to isoprenaline (1 microM) and was achieved 15-20 or 20-30 min after application of 1.0 or 0.1 microM MEN 10,627, respectively. 4. Duodenal strips were relaxed by other NK2 receptor selective antagonists (values in parentheses are % of Emax to isoprenaline at the given concentration of antagonist) GR 94,800 (69 +/- 3% at 1 microM, n = 4), SR 48,968 (60 +/- 3% at 1 microM, n = 4) and MDL 29,913 (66 +/- 4% at 1 microM, n = 4). SR 48,965 (1 microM), the inactive enantiomer of SR 48,968, was without effect. The NK1 receptor selective antagonists, SR 140,333 (0.1 microM), FK 888 (10 microM) RP 67,580 (1 microM) and GR 82,334 (10 microM) were also without effect (n = 4-5). 5. A cocktail of peptidase inhibitors, thiorphan, bestatin and captopril (1 microM each) had no significant effect on tone or spontaneous activity of duodenal strips. In the presence of peptidase inhibitors, MEN 10,627 (1 microM) produced a relaxation of duodenal strips (72 +/- 6% of Emax to isoprenaline, n = 5), whilst GR 82,334 (10 microM, n = 6) had no significant effect. 6. The relaxant response to MEN 10,627 was preserved in mucosa-free strips not pre-exposed to capsaicin. Tetrodotoxin (1 microM), saxitoxin (1 microM), hexamethonium (100 microM) and omega-conotoxin (0.1 microM) had no significant effect on the resting tone of duodenal strips nor did they affect the relaxation to MEN 10,627. L-Nitroarginine (100 microM) increased the tone of the strips but did not affect the response to MEN 10,627. Nifedipine (1 microM) relaxed the strips by 62 +/- 4% (n = 4), but in its presence a small relaxant effect to MEN 10,627 (26 +/- 5%, n = 4) was still evident. 7. Under isotonic recording of mechanical activity along the longitudinal axis, MEN 10,627 (1 microM) produced a slowly developing relaxation (39 +/- 3% of Emax to isoprenaline; n = 6) of whole segments of rat duodenum. When similar experiments were performed on whole segments of rat proximal colon MEN 10,627 had no effect. 8. The present findings document the observation that tachykinin NK2 receptors contribute to the maintenance of resting tone of the rat isolated small intestine. We found no evidence to suggest that this effect follows the blockade of the contractile effect of spontaneously released endogenous tachykinins. The present findings raise the possibility that constitutively active NK2 receptors account for the relaxant effect produced by NK2 receptor ant PMID:8818352

Evidence that tachykinin NK2 receptors modulate resting tone in the rat isolated small intestine.

PubMed

Maggi, C A; Giuliani, S

1996-07-01

1. In the progress of experiments aimed at evaluating the role of tachykinins as enteric nonadrenergic noncholinergic (NANC) transmitters, we noted that certain tachykinin receptor antagonists produce a relaxation of circular muscle strips in the rat small intestine. This study aimed to assess the nature of this response and to determine the receptor type involved. The majority of the experiments were performed in capsaicin- (10 microM for 15 min) pretreated mucosa-free circular muscle strips from the rat small intestine, in the presence of atropine (1 microM), guanethidine (3 microM) and indomethacin (10 microM). 2. Under isometric recording of mechanical activity, the tachykinin NK1 receptor antagonist SR 140,333 (0.1 microM) had no effect on resting tone or spontaneous activity in duodenal or ileal circular muscle strips. The NK2 receptor antagonists, MEN 10,627 (0.1 microM) and GR 94,800 (0.1 microM) produced, after a delay of 10-15 min, a relaxation which averaged 61 +/- 3 and 57 +/- 6% (n = 6 and 4, respectively) of the maximal response (Emax) to isoprenaline (1 microM). The effect of maximal concentrations of MEN 10,627 and GR 94,800 when applied together was non-additive. The relaxant effect of MEN 10,627 (0.1 microM) was similar in the absence and presence of apamin (0.3 microM) and L-nitroarginine (100 microM). 3. Under isotonic recording of mechanical activity, MEN 10,627 (10 nM-1 microM) produced a concentration- and time-related relaxation of duodenal strips. The maximal relaxation averaged 72 +/- 4 and 69 +/- 4% (n = 5 each) of Emax to isoprenaline (1 microM) and was achieved 15-20 or 20-30 min after application of 1.0 or 0.1 microM MEN 10,627, respectively. 4. Duodenal strips were relaxed by other NK2 receptor selective antagonists (values in parentheses are % of Emax to isoprenaline at the given concentration of antagonist) GR 94,800 (69 +/- 3% at 1 microM, n = 4), SR 48,968 (60 +/- 3% at 1 microM, n = 4) and MDL 29,913 (66 +/- 4% at 1 microM, n = 4). SR 48,965 (1 microM), the inactive enantiomer of SR 48,968, was without effect. The NK1 receptor selective antagonists, SR 140,333 (0.1 microM), FK 888 (10 microM) RP 67,580 (1 microM) and GR 82,334 (10 microM) were also without effect (n = 4-5). 5. A cocktail of peptidase inhibitors, thiorphan, bestatin and captopril (1 microM each) had no significant effect on tone or spontaneous activity of duodenal strips. In the presence of peptidase inhibitors, MEN 10,627 (1 microM) produced a relaxation of duodenal strips (72 +/- 6% of Emax to isoprenaline, n = 5), whilst GR 82,334 (10 microM, n = 6) had no significant effect. 6. The relaxant response to MEN 10,627 was preserved in mucosa-free strips not pre-exposed to capsaicin. Tetrodotoxin (1 microM), saxitoxin (1 microM), hexamethonium (100 microM) and omega-conotoxin (0.1 microM) had no significant effect on the resting tone of duodenal strips nor did they affect the relaxation to MEN 10,627. L-Nitroarginine (100 microM) increased the tone of the strips but did not affect the response to MEN 10,627. Nifedipine (1 microM) relaxed the strips by 62 +/- 4% (n = 4), but in its presence a small relaxant effect to MEN 10,627 (26 +/- 5%, n = 4) was still evident. 7. Under isotonic recording of mechanical activity along the longitudinal axis, MEN 10,627 (1 microM) produced a slowly developing relaxation (39 +/- 3% of Emax to isoprenaline; n = 6) of whole segments of rat duodenum. When similar experiments were performed on whole segments of rat proximal colon MEN 10,627 had no effect. 8. The present findings document the observation that tachykinin NK2 receptors contribute to the maintenance of resting tone of the rat isolated small intestine. We found no evidence to suggest that this effect follows the blockade of the contractile effect of spontaneously released endogenous tachykinins. The present findings raise the possibility that constitutively active NK2 receptors account for the relaxant effect produced by NK2 receptor ant

Role of capsaicin-sensitive nerves and tachykinins in mast cell tryptase-induced inflammation of murine knees.

PubMed

Borbély, Éva; Sándor, Katalin; Markovics, Adrienn; Kemény, Ágnes; Pintér, Erika; Szolcsányi, János; Quinn, John P; McDougall, Jason J; Helyes, Zsuzsanna

2016-09-01

Mast cell tryptase (MCT) is elevated in arthritic joints, but its direct effects are not known. Here, we investigated MCT-evoked acute inflammatory and nociceptive mechanisms with behavioural, in vivo imaging and immunological techniques. Neurogenic inflammation involving capsaicin-sensitive afferents, transient receptor potential vanilloid 1 receptor (TRPV1), substance P (SP), neurokinin A (NKA) and their NK1 tachykinin receptor were studied using gene-deleted mice compared to C57Bl/6 wildtypes (n = 5-8/group). MCT was administered intraarticularly or topically (20 μl, 12 μg/ml). Capsaicin-sensitive afferents were defunctionalized with the TRPV1 agonist resiniferatoxin (RTX; 30-70-100 μg/kg s.c. pretreatment). Knee diameter was measured with a caliper, synovial perfusion with laser Doppler imaging, mechanonociception with aesthesiometry and weight distribution with incapacitance tester over 6 h. Cytokines and neuropeptides were determined with immunoassays. MCT induced synovial vasodilatation, oedema, impaired weight distribution and mechanical hyperalgesia, but cytokine or neuropeptide levels were not altered at the 6-h timepoint. Hyperaemia was reduced in RTX-treated and TRPV1-deleted animals, and oedema was absent in NK1-deficient mice. Hyperalgesia was decreased in SP/NKA- and NK1-deficient mice, weight bearing impairment in RTX-pretreated, TRPV1- and NK1-deficient animals. MCT evokes synovial hyperaemia, oedema, hyperalgesia and spontaneous pain. Capsaicin-sensitive afferents and TRPV1 receptors are essential for vasodilatation, while tachykinins mediate oedema and pain.

Eledoisin and Kassinin, but not Enterokassinin, stimulate ion transport in frog skin.

PubMed

Lippe, Claudio; Bellantuono, Vito; Ardizzone, Concetta; Cassano, Giuseppe

2004-11-01

In frog skin, tachykinins stimulate the ion transport, estimated by measuring the short-circuit current (SCC) value, by interacting with NK1-like receptors. In this paper we show that Kassinin (NK2 preferring in mammals) increases the SCC, while Enterokassinin has no effect. Therefore, either 2 Pro residues or 1 Pro and 1 basic amino acid must be present in the part exceeding the C-terminal pentapeptide. Eledoisin (NK3 preferring in mammals) stimulation of SCC is reduced by CP99994 and SR48968 (NK1 and NK2 antagonists) and not affected by SB222200 (NK3 antagonist). None of the three antagonists affects Kassinin stimulation of SCC.

Bidirectional regulation of human colonic smooth muscle contractility by tachykinin NK(2) receptors.

PubMed

Nakamura, Akihiro; Tanaka, Takahiro; Imanishi, Akio; Kawamoto, Makiko; Toyoda, Masao; Mizojiri, Gaku; Tsukimi, Yasuhiro

2011-01-01

In this study, we attempted to clarify the mechanism of tachykinin-induced motor response in isolated smooth muscle preparations of the human colon. Fresh specimens of normal colon were obtained from patients suffering from colonic cancer. Using mucosa-free smooth muscle strips, smooth muscle tension with circular direction was monitored isometrically. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) produced marked contraction. All of these contractions were inhibited by saredutant, a selective NK(2)-R antagonist, but not by CP122721, a selective NK(1)-R antagonist or talnetant, a selective NK(3)-R antagonist. βAla(8)-NKA(4-10) induced concentration-dependent contraction similar to NKA, but Sar(9)-Met(11)-SP and Met-Phe(7)-NKB did not cause marked contraction. Colonic contraction induced by βAla(8)-NKA(4-10) was completely blocked by saredutant, but not by atropine. Tetrodotoxin or N(G)-nitro-L-arginine methyl ester pretreatment significantly enhanced βAla(8)-NKA(4-10)-induced contraction. Immunohistochemical analysis showed that the NK(2)-R was expressed on the smooth muscle layers and myenteric plexus where it was also co-expressed with neuronal nitric oxide synthase in the myenteric plexus. These results suggest that the NK(2)-R is a major contributor to tachykinin-induced smooth muscle contraction in human colon and that the NK(2)-R-mediated response consists of an excitatory component via direct action on the smooth muscle and an inhibitory component possibly via nitric oxide neurons.

The common C-terminal sequences of substance P and neurokinin A contact the same region of the NK-1 receptor.

PubMed

Bremer, A A; Leeman, S E; Boyd, N D

2000-12-01

Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK-1 receptor (NK-1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high-affinity interaction with this receptor. To document the site of interaction between NKA and the NK-1R, we have used a photoreactive analogue of NKA containing p-benzoyl-L-phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by (125)I-iodohistidyl(1)-Bpa(7)NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEWPEHPNR). We have previously shown that (125)I-BH-Bpa(8)SP, a photoreactive analogue of SP, covalently attaches to M(181) within this same receptor sequence. Importantly, both of these peptides ((125)I-iodohistidyl(1)-Bpa(7)NKA and (125)I-BH-Bpa(8)SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carboxyl-terminal tail. In this report, we also show that site-directed mutagenesis of M(181) to A(181) in the NK-1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK-1R, contact the same residue.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

PubMed

Wu, Zining; Graybill, Todd L; Zeng, Xin; Platchek, Michael; Zhang, Jean; Bodmer, Vera Q; Wisnoski, David D; Deng, Jianghe; Coppo, Frank T; Yao, Gang; Tamburino, Alex; Scavello, Genaro; Franklin, G Joseph; Mataruse, Sibongile; Bedard, Katie L; Ding, Yun; Chai, Jing; Summerfield, Jennifer; Centrella, Paolo A; Messer, Jeffrey A; Pope, Andrew J; Israel, David I

2015-12-14

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

Characterization of the tachykinin NK2 receptor in the human bronchus: influence of amastatin-sensitive metabolic pathways.

PubMed Central

Astolfi, M.; Treggiari, S.; Giachetti, A.; Meini, S.; Maggi, C. A.; Manzini, S.

1994-01-01

1. The aim of this study was to characterize the tachykinin NK2 receptor subtype mediating the spasmogenic response in the human isolated bronchus. The motor response to neurokinin A (NKA) and the selective NK2 agonist [beta Ala8]NKA(4-10), as well as the antagonistic effects of cyclic (L659,877) and linear (MEN 10376) peptide NK2 antagonists were assessed in the presence or absence of amastatin (an inhibitor of aminopeptidases A and M). 2. NKA was more potent than [beta Ala8]NKA(4-10) in eliciting bronchoconstriction (pD2 being 7,43 and 6,87 respectively). In the presence of amastatin (1 microM), the estimated affinity of [beta Ala8]NKA(4-10), but not that of NKA, was significantly increased to yield a pD2 of 7,44. 3. L659,877 and MEN 10376 inhibited [beta Ala8]NKA(4-10)-induced contraction with similar affinities; pA2 values were 5.7 +/- 0.22 and 6.3 +/- 0.32, respectively. Amastatin (1 microM) increased the potency of MEN 10376 to 7.28 +/- 0.46, whereas that of L659,877 was unaffected. 4. In the presence of amastatin the pseudopeptide MDL 28,564 behaved as a partial agonist. 5. We conclude that the NK2 receptor subtype present in the human bronchus has properties similar to those described for the circular muscle of the human colon and thus may be classified as a 'NK2A' subtype. We show that the apparent potency of peptides, bearing N-terminal acidic residues, is influenced by an amastatin-sensitive peptidase, possibly aminopeptidase A. PMID:8004400

Characterization of the tachykinin NK2 receptor in the human bronchus: influence of amastatin-sensitive metabolic pathways.

PubMed

Astolfi, M; Treggiari, S; Giachetti, A; Meini, S; Maggi, C A; Manzini, S

1994-02-01

1. The aim of this study was to characterize the tachykinin NK2 receptor subtype mediating the spasmogenic response in the human isolated bronchus. The motor response to neurokinin A (NKA) and the selective NK2 agonist [beta Ala8]NKA(4-10), as well as the antagonistic effects of cyclic (L659,877) and linear (MEN 10376) peptide NK2 antagonists were assessed in the presence or absence of amastatin (an inhibitor of aminopeptidases A and M). 2. NKA was more potent than [beta Ala8]NKA(4-10) in eliciting bronchoconstriction (pD2 being 7,43 and 6,87 respectively). In the presence of amastatin (1 microM), the estimated affinity of [beta Ala8]NKA(4-10), but not that of NKA, was significantly increased to yield a pD2 of 7,44. 3. L659,877 and MEN 10376 inhibited [beta Ala8]NKA(4-10)-induced contraction with similar affinities; pA2 values were 5.7 +/- 0.22 and 6.3 +/- 0.32, respectively. Amastatin (1 microM) increased the potency of MEN 10376 to 7.28 +/- 0.46, whereas that of L659,877 was unaffected. 4. In the presence of amastatin the pseudopeptide MDL 28,564 behaved as a partial agonist. 5. We conclude that the NK2 receptor subtype present in the human bronchus has properties similar to those described for the circular muscle of the human colon and thus may be classified as a 'NK2A' subtype. We show that the apparent potency of peptides, bearing N-terminal acidic residues, is influenced by an amastatin-sensitive peptidase, possibly aminopeptidase A.

[Increased expressions of substance P and neurokinin/tachykinin receptor 1 in eosinophils of patients with psoriasis].

PubMed

Zuo, Zhe; Wang, Junling; Zhang, Huiyun; Zheng, Wenjiao; Zhang, Zenan; He, Shaoheng

2017-07-01

Objective To investigate the expressions of substance P (SP) and its receptor neurokinin/tachykinin receptor 1 (NK1R) in peripheral blood eosinophils of patients with psoriasis. Methods The levels of SP and NK1R in the peripheral blood of both patients with psoriasis and healthy people were detected by flow cytometry. This method was again used to detect the levels of SP and NK1R in the peripheral blood eosinophils of patients with psoriasis after stimulated with the crude extracts of Artemisia pollen, dust mite and Platanus pollen (all at concentrations of 0.1 and 1.0 μg/mL). Results Compared with the healthy controls, the percentages of SP + and NK1R + eosinophils in psoriasis patients increased up to 2.7 and 0.5 folds, respectively. Moreover, the mean fluorescence intensity (MFI) of SP + and NK1R + eosinophils of psoriasis patients were elevated by 1.5 and 0.2 folds, respectively. The percentage of SP + eosinophils in psoriasis were down-regulated by 60% after the stimulation with Platanus pollen extract (1 μg/mL), while 0.1 μg/mL Platanus pollen extract induced a 0.6-fold increase in the percentage of NK1R + eosinophis. Conclusion The expressions of SP and NK1R are up-regulated in peripheral blood eosinophils of patients with psoriasis.

Characterization of the tachykinin neurokinin-2 receptor in the human urinary bladder by means of selective receptor antagonists and peptidase inhibitors.

PubMed

Giuliani, S; Patacchini, R; Barbanti, G; Turini, D; Rovero, P; Quartara, L; Giachetti, A; Maggi, C A

1993-11-01

The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

Contribution of NK(2) tachykinin receptors to propulsion in the rabbit distal colon.

PubMed

Onori, L; Aggio, A; Taddei, G; Tonini, M

2000-01-01

The role of the tachykinin neurokinin (NK)(2) receptors on rabbit distal colon propulsion was investigated by using two selective NK(2)-receptor antagonists, MEN-10627 and SR-48968. Experiments on colonic circular muscle strips showed that contractile responses to [beta-Ala(8)]NKA-(4-10) (1 nM-1 microM), a selective NK(2)-receptor agonist, were competitively antagonized by MEN-10627 (1-100 nM), whereas SR-48968 (0.1-10 nM) caused an insurmountable antagonism, thus confirming the difference in the mode of action of the two compounds. Colonic propulsion was elicited by distending a mobile rubber balloon with 0.3 ml (submaximal stimulus) or 1.0 ml (maximal stimulus) of water. The velocity of anal displacement of the balloon (mm/s) was considered the main propulsion parameter. At low concentrations (1.0-100 nM and 0.1-10 nM, respectively), MEN-10627 and SR-48968 facilitated the velocity of propulsion, whereas at high concentrations (100 nM and 1 microM, respectively) they decelerated propulsion. The excitatory and inhibitory effects of both antagonists were observed only with submaximal stimulus. We focused on the hypothesis that the facilitatory effect on propulsion may result from blockade of neuronal NK(2) receptors and the inhibitory effect from suppression of the excitatory transmission mediated by NK(2) receptors on smooth muscle cells. In the presence of N(G)-nitro-L-arginine (300 microM), a nitric oxide synthase inhibitor, MEN-10627, at a concentration (10 nM) that was found to accelerate propulsion in control experiments inhibited the velocity of propulsion. In the presence of threshold (1-10 nM) or full (1 microM) concentration of atropine, which inhibited to a great extent the velocity of propulsion, the inhibitory effect of MEN-10627 (1 microM) was markedly increased. In conclusion, in the rabbit distal colon NK(2) receptors may decelerate propulsion by activating a nitric oxide-dependent neuronal mechanism and may accelerate it by a postjunctional synergistic interaction with cholinergic muscarinic receptors.

Bladder inflammatory transcriptome in response to tachykinins: Neurokinin 1 receptor-dependent genes and transcription regulatory elements

PubMed Central

Saban, Ricardo; Simpson, Cindy; Vadigepalli, Rajanikanth; Memet, Sylvie; Dozmorov, Igor; Saban, Marcia R

2007-01-01

Background Tachykinins (TK), such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could block the tachykinin system, we set forth to determine the regulatory network downstream of NK1 receptor activation. First, NK1R-dependent transcripts were determined and used to query known databases for their respective transcription regulatory elements (TREs). Methods An expression analysis was performed using urinary bladders isolated from sensitized wild type (WT) and NK1R-/- mice that were stimulated with saline, LPS, or antigen to provoke inflammation. Based on cDNA array results, NK1R-dependent genes were selected. PAINT software was used to query TRANSFAC database and to retrieve upstream TREs that were confirmed by electrophoretic mobility shift assays. Results The regulatory network of TREs driving NK1R-dependent genes presented cRel in a central position driving 22% of all genes, followed by AP-1, NF-kappaB, v-Myb, CRE-BP1/c-Jun, USF, Pax-6, Efr-1, Egr-3, and AREB6. A comparison between NK1R-dependent and NK1R-independent genes revealed Nkx-2.5 as a unique discriminator. In the presence of NK1R, Nkx2-5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF) and inflammation (PAR-3, IL-1R, IL-6, α-NGF, TSP2). In the absence of NK1R, the matrix Nkx2-5_02 had a predominant participation driving 8 transcripts, which includes those involved in cancer (EYA1, Trail, HSF1, and ELK-1), smooth-to-skeletal muscle trans-differentiation, and Z01, a tight-junction protein, expression. Electrophoretic mobility shift assays confirmed that, in the mouse urinary bladder, activation of NK1R by substance P (SP) induces both NKx-2.5 and NF-kappaB translocations. Conclusion This is the first report describing a role for Nkx2.5 in the urinary tract. As Nkx2.5 is the unique discriminator of NK1R-modulated inflammation, it can be imagined that in the near future, new based therapies selective for controlling Nkx2.5 activity in the urinary tract may be used in the treatment in a number of bladder disorders. PMID:17519035

CGRP potentiates excitatory transmission to the circular muscle of guinea-pig colon.

PubMed

Maggi, C A; Giuliani, S; Santicioli, P

1997-04-30

We aimed to assess whether calcitonin gene-related peptide (CGRP) can modulate the release of tachykinins which are the main nonadrenergic noncholinergic (NANC) excitatory transmitters to the circular muscle of the guinea-pig proximal colon. In organ bath experiments, electrical field stimulation (EFS) in the presence of atropine (1 microM) and guanethidine (3 microM) evoked twitch phasic NANC contractions which were abolished by the combined administration of tachykinin NK1 and NK2 receptor antagonists. Human alphaCGRP (CGRP, 1-100 nM) produced a concentration-dependent potentiation of the amplitude of the NANC contractions induced by EFS while salmon calcitonin (up to 1 microM) had no effect. The potentiating effect of CGRP was unaffected by in vitro capsaicin pretreatment (10 microM for 15 min), peptidase inhibitors (captopril, bestatin and thiorphan, 1 microM each), apamin (0.3 microM) plus L-nitroarginine (L-NOARG, 100 microM) and by the CGRP1 receptor antagonist, the C-terminal fragment CGRP(8-37) (1 microM). The NK2 receptor antagonist MEN 10627 which, when administered alone, had only a partial inhibitory effect on the amplitude of NANC twitches, concentration-dependently (10 nM-1 microM) inhibited the potentiating effect of CGRP. CGRP (1-100 nM) produced a concentration-dependent potentiation of the atropine-sensitive cholinergic contractions evoked by EFS in the presence of guanethidine and of tachykinin NK1 and NK2 receptor antagonists. Similar to the effect of CGRP, application of capsaicin (0.1-1 microM) potentiated the amplitude of the NANC contraction to EFS, an effect undergoing complete desensitization upon a second application of the drug. CGRP (0.1 microM) did not affect the contractile action of a submaximally effective concentration of neurokinin A (2 nM) while it inhibited that induced by substance P (2 nM). In sucrose gap, single pulse EFS in the presence of atropine (1 microM) and guanethidine (3 microM) induced an inhibitory junction potential (i.j.p.) and a small excitatory junction potential (e.j.p.). CGRP (0.1 microM) produced membrane hyperpolarization and relaxation without affecting i.j.p. amplitude but concomitantly increased the e.j.p. amplitude to induce a contraction in correspondence to each electrical pulse. In the presence of the NK1 receptor antagonist, GR 82334 (3 microM), the membrane hyperpolarization and relaxation produced by CGRP and the EFS-evoked i.j.p. were unaffected, while the potentiating effect of CGRP on the EFS-evoked NANC e.j.p. and the corresponding contraction were abolished. We conclude that, in addition to the previously characterized direct smooth muscle relaxant action via CGRP1 receptors (Maggi et al. Regulatory Peptides 61, 27-36, 1996), CGRP also induces a remarkable potentiation of excitatory neurotransmission to the circular muscle of the guinea-pig colon via CGRP2 receptors. The latter effect, documented in this study, is evidenced on both the atropine-sensitive and the atropine-resistant (tachykinin-mediated) components of excitatory transmission: this effect does not involve mediator(s) release from capsaicin-sensitive primary afferent nerves, nor inhibition of peptide degradation or modulation of NANC inhibitory transmission.

Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission.

PubMed

Maggi, C A; Patacchini, R; Santicioli, P; Giuliani, S

1991-06-01

1. The possible involvement of tachykinins (TKs) in the contraction produced by capsaicin in the rat isolated urinary bladder was addressed on the hypothesis that co-release of substance P (SP) and neurokinin A (NKA) occurs from sensory nerve terminals. 2. A low concentration of SP (30 nM) produced a rapid contraction which faded to baseline within 10 min. A low concentration of NKA (10 nM) produced a slowly developing contraction which was still evident at 10 min. Capsaicin (1 microM) produced a rapid phasic response and a tonic response (late response to capsaicin). Co-administration of SP and NKA mimicked the response to capsaicin more than each TK alone. 3. Fading of the response to SP was not caused by receptor desensitization and was partially prevented by peptidase inhibitors. 4. Spantide (3 microM) selectively antagonized the SP-induced contraction while L-659,877 (3-10 microM) or MEN 10,376 (10-30 microM) which are NK2 receptor selective antagonists selectively blocked the response to NKA. Co-administration of spantide and L-659,877 inhibited the response to both SP and NKA by an amount not greater than that produced by each antagonist alone. 5. Spantide selectively reduced the peak response to capsaicin, while leaving the late response unaffected. L-659,877 (3 microM) and MEN 10,376 (10 microM) selectively inhibited the late response to capsaicin while, at higher concentrations, also reduced the peak response to capsaicin. Co-administration of spantide and L-659,877 reduced the peak response to capsaicin more than that produced by each antagonist alone. 6. Bombesin (10 nM) produced a tonic contraction similar to that induced by NKA. The response to bombesin was not affected by spantide, L-659,877 or MEN 10,376. 7 P2. purinoceptor desensitization by repeated administration of alpha,betal-methylene ATP depressed the twitch response to electrical stimulation of postganglionic nerves but did not affect the peak or the late response to capsaicin. 8. We conclude that multiple TKs are coreleased by capsaicin in the rat bladder and mediate the capsaicin-induced contraction by activating both NKI and NK2 receptors. Endogenous TK with preferential affinity for the NK, receptor (putatively SP) are selectively involved in the peak response to capsaicin while endogenous TK with preferential affinity for the NK2 receptor (putatively NKA) are selectively involved in the late response to capsaicin and partly contribute to the peak response. These findings provide pharmacological evidence for tachykinin-mediated cotransmission in the rat urinary bladder. ATP is unlikely to be involved in the efferent function of capsaicin-sensitive sensory nerves in the rat bladder.

Diminished pheromone-induced sexual behavior in neurokinin-1 receptor deficient (TACR1(-/-)) mice.

PubMed

Berger, A; Tran, A H; Dida, J; Minkin, S; Gerard, N P; Yeomans, J; Paige, C J

2012-07-01

Studies in mice with targeted deletions of tachykinin genes suggest that tachykinins and their receptors influence emotional behaviors such as aggression, depression and anxiety. Here, we investigated whether TAC1- and TAC4-encoded peptides (substance P and hemokinin-1, respectively) and the neurokinin-1 receptor (NK-1R) are involved in the modulation of sexual behaviors. Male mice deficient for the NK-1R (TACR1 (-/-)) exhibited decreased exploration of female urine in contrast to C57BL/6 control mice and mice deficient for NK-1R ligands such as TAC1 (-/-), TAC4 (-/-) and the newly generated TAC1 (-/-) /TAC4 (-/-) mice. In comparison to C57BL/6 mice, mounting frequency and duration were decreased in male TACR1 (-/-) mice, while mounting latency was increased. Decreased preference for sexual pheromones was also seen in female TACR1 (-/-) mice. Furthermore, administration of the NK-1R-antagonist L-703,606 decreased investigation of female urine by male C57BL/6 mice, suggesting an involvement of NK-1R in urine sniffing behavior. Our results provide evidence for the NK-1R in facilitating sexual approach behavior, as male TACR1 (-/-) mice exhibited blunted approach behavior toward females following the initial interaction compared with C57BL/6 mice. NK-1R signaling may therefore play an important role in pheromone-induced sexual behavior. © 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Evidence of NK1 and NK2 Tachykinin Receptors and their Involvement in Histamine Release in a Murine Mast Cell Line

DTIC Science & Technology

1992-01-01

either human p ~ulmo(nary,. Delectaible in the absence of estrmcclular CaCI’. i’Potent 4.23ug/105 cells, or rat peritoneal mast cells. bousbesin...ABSTRACT (Maximum 200 words) Abstract-Binding of )kH substance P (SP) and histamine release were examined using a cloned mouse mast cell line SP binding...the cells with the NK2 antagonist peptide A reduced NKA-induced histamine release ID.Arg’,D.Phe’,D-Trp 0 3 .Leu t )nsu b s tance P , a putative SP

Trypsin induces biphasic muscle contraction and relaxation via transient receptor potential vanilloid 1 and neurokinin receptors 1/2 in porcine esophageal body.

PubMed

Xiaopeng, Bai; Tanaka, Yoshimasa; Ihara, Eikichi; Hirano, Katsuya; Nakano, Kayoko; Hirano, Mayumi; Oda, Yoshinao; Nakamura, Kazuhiko

2017-02-15

Duodenal reflux of fluids containing trypsin relates to refractory gastroesophageal reflux disease (GERD). Esophageal peristalsis and clearance are important factors in GERD pathogenesis. However, the function of trypsin in esophageal body contractility is not fully understood. In this study, effects of trypsin on circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of the porcine esophageal body were examined. Trypsin elicited a concentration dependent biphasic response, a major contraction and a subsequent relaxation only in CSM. In CSM, contraction occurred at trypsin concentrations of 100nM and relaxation at 1μM. A proteinase-activated receptor (PAR)2 activating peptide, SLIGKV-NH 2 (1mM), induced a monophasic contraction. Those responses were unaffected by tetrodotoxin though abolished by the gap junction uncouplers carbenoxolone and octanol. They were also partially inhibited by a transient receptor potential vanilloid type 1 (TRPV1) antagonist and abolished by combination of neurokinin receptor 1 (NK 1 ) and NK 2 antagonists, but not by an NK 3 antagonist, suggesting a PAR2-TRPV1-substance P pathway in sensory neurons. Substance P (100nM), an agonist for various NK receptors (NK 1 , NK 2 and NK 3 ) with differing affinities, induced significant contraction in CSM, but not in LSM. The contraction was also blocked by the combination of NK 1 and NK 2 antagonists, but not by the NK 3 antagonist. Moreover, substance P-induced contractions were unaffected by the TRPV1 antagonist, but inhibited by a gap junction uncoupler. In conclusion, trypsin induced a biphasic response only in CSM and this was mediated by PAR2, TRPV1 and NK 1/2 . Gap junctions were indispensable in this tachykinin-induced response. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea-pig ileum and proximal colon.

PubMed

Maggi, C A; Patacchini, R; Meini, S; Quartara, L; Sisto, A; Potier, E; Giuliani, S; Giachetti, A

1994-05-01

1. The aim of this study was the pharmacological characterization of tachykinin NK1 and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [Sar9]SP sulphone, [Glp6,Pro9]SP(6-11) (septide) and [beta Ala8]NKA(4-10) was investigated. The affinities of various peptide and nonpeptide antagonists for the NK1 and NK2 receptor was estimated by use of receptor selective agonists. 2. The natural agonists, SP and NKA, produced concentration-dependent contraction in both preparations. EC50 values were 100 pM and 5 nM for SP, 1.2 nM and 19 nM for NKA in the ileum and colon, respectively. The action of SP and NKA was not significantly modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). 3. Synthetic NK1 and NK2 receptor agonists produced concentration-dependent contraction of the circular muscle of the ileum and proximal colon. EC50 values were 83 pM, 36 pM and 10 nM in the ileum, 8 nM, 0.7 nM and 12 nM in the colon for [Sar9]SP sulphone, septide and [beta Ala8]NKA-(4-10), respectively. The pseudopeptide derivative of NKA(4-10), MDL 28,564 behaved as a full or near-to-full agonist in both preparations, its EC50s being 474 nM and 55 nM in the ileum and colon, respectively. 4. Nifedipine (1 microM) abolished the response to septide and [Sar9]SP sulphone in the ileum and produced a rightward shift and large depression of the response in the colon. The response to [beta Ala8]NKA(4-10) was abolished in the ileum and largely unaffected in the colon. 5. The NK1 receptor antagonists, (+/-)-CP 96,34, FK 888 and GR 82,334 competitively antagonized the response to septide and [Sar9]SP sulphone in both preparations without affecting that to [beta Ala8]NKA(4-10). In general, the NK1 receptor antagonists were significantly more potent toward septide than [Sar9]SP sulphone in both preparations. 6. The NK2 receptor antagonists, GR 94,800 and SR 48,968 selectively antagonized the response to [beta Ala8]NKA(4-10) without affecting that to [Sar9]SP sulphone or septide in the ileum and colon. SR 48,968 produced noncompetitive antagonism of the response to the NK2 receptor agonist in the ileum and competitive antagonism in the colon. 7. MEN 10,376 and the cyclic pseudopeptide MEN 10,573 antagonized in a competitive manner the response to [beta Ala8]NKA(4-10) in the ileum and colon. While MEN 10,573 was equipotent in both preparations, MEN 10,376 was significantly more potent in the colon than in the ileum. MEN 10,376was also effective against septide in both preparations, without affecting the response to [Sar9] SP sulphone. MEN 10,573 antagonized the response to [Sar9]SP sulphone and septide in both preparations,pKB values against septide being intermediate, and significantly different from, those measured against[Beta Ala 8]NKA(4-10) and [Sa9]lSP sulphone.8. These findings show that tachykinin NK1 and NK2 receptors mediate contraction of the circular muscle of the guinea-pig ileum and colon. In both preparations NK1 receptor antagonists display higher apparent affinity when tested against septide than [Sar9]SP sulphone. These findings are compatible with the proposed existence of NK1 receptor subtypes in guinea-pig, although alternative explanations (e.g.agonist binding to different epitopes of the same receptor protein) cannot be excluded at present.Furthermore, an intraspecies heterogeneity of the NK2 receptor in the circular muscle of the guinea-pig ileum and colon is suggested.

Inducible nitric oxide synthase evoked nitric oxide counteracts capsaicin-induced airway smooth muscle contraction, but exacerbates plasma extravasation.

PubMed

Li, Ping-Chia; Shaw, Chen-Fu; Kuo, Tin-Fan; Chien, Chiang-Ting

2005-04-18

The contribution of nitric oxide (NO) to capsaicin-evoked airway responses was investigated in rats. The measurement of plasma NO level, airway dynamics, airway smooth muscle electromyogram, and plasma extravasation by India ink and Evans blue leakage technique was adapted. Capsaicin-evoked hypotension, bronchoconstriction, trachea plasma extravasation as well as increases in plasma NO level in a dose-dependent manner. L-732138 (NK1 receptor antagonist) or SR-48968 (NK2 receptor antagonist) pretreatment reduced capsaicin-enhanced hypotension, bronchoconstriction, plasma extravasation, and plasma NO level. N(G)-nitro-L-Arginine methyl ester (L-NAME, 10 mg/kg, i.v.), a non-selective NO synthase (NOS) inhibitor, or aminoguanidine (10 mg/kg, i.v.), a selective inducible NOS (iNOS) inhibitor, reduced capsaicin-induced increases in plasma NO level and protected against capsaicin-induced plasma extravasation, whereas L-arginine (150 mg/kg, i.v.), a NO precursor, enhanced capsaicin-evoked plasma NO level and plasma extravasation. L-Arginine pretreatment ameliorated capsaicin-induced bronchoconstriction, whereas L-NAME and aminoguanidine exaggerated capsaicin-induced bronchoconstriction. In summary, NK1 and NK2 receptors and iNOS play a role in NO formation and on capsaicin-induced bronchoconstriction and plasma extravasation. NO generated by iNOS counteracts tachykinin-mediated bronchoconstriction, but exacerbates tachykinin-mediated plasma extravasation.

Receptor binding sites for substance P in surgical specimens obtained from patients with ulcerative colitis and Crohn disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mantyh, C.R.; Gates, T.S.; Zimmerman, R.P.

1988-05-01

Several lines of evidence indicate that tachykinin neuropeptides (substance P (SP), substance K (SK), and neuromedin K (NK)) play a role in regulating the inflammatory and immune responses. To test this hypothesis in a human inflammatory disease, quantitative receptor autoradiography was used to examine possible abnormalities in tachykinin binding sites in surgical specimens from patients with inflammatory bowel disease. In all cases, specimens were processed for quantitative receptor autoradiography by using /sup 125/I-labeled Bolton-Hunter conjugates of NK, SK, and SP. In colon tissue obtained from ulcerative colitis and Crohn disease patients, very high concentrations of SP receptor binding sites aremore » expressed by arterioles and venules located in the submucosa, muscalairs mucosa, external circular muscle, external longitudinal muscle, and serosa, in contrast to control patients. These results demonstrate that receptor binding sites for SP, but not SK or NK, are ectopically expressed in high concentrations by cells involved in mediating inflammatory and immune responses. These data suggest that SP may be involved in the pathophysiology of inflammatory bowel disease and might provide some insight into the interaction between the nervous system and the regulation of inflammation and the immune response in human inflammatory disease.« less

Tachykinin NK-1 and NK-3 selective agonists induce analgesia in the formalin test for tonic pain following intra-VTA or intra-accumbens microinfusions.

PubMed

Altier, N; Stewart, J

1997-12-01

Experiments were designed to examine the analgesic effects induced by selective tachykinin receptor agonists microinfused into either the ventral tegmental area (VTA) or nucleus accumbens septi (NAS). Rats were tested in the formalin test for tonic pain following an injection of 0.05 ml of 2.5% formalin into one hind paw immediately after bilateral intra-VTA infusions of either the NK-1 agonist, GR-73632 (0.005, 0.05 or 0.5 nmol/side), the NK-3 agonist, senktide (0.005, 0.5 or 1.5 nmol/side), or saline. Two weeks later, the saline-treated rats were assessed in the tail-flick test for phasic pain after infusions of the tachykinin agonists. Tail-flick latencies were recorded following immersion of the tail in 55 degrees C hot water at 10 min intervals for 1 h immediately after intra-VTA infusions of either GR-73632 (0.5 nmol/side), senktide (1.5 nmol/side) or saline. In a second group of rats, the same effects were studied after infusions into the nucleus accumbens (NAS) of GR-73632 (0.005, 0.5 or 1.5 nmol/side), senktide (0.005, 0.5 or 1.5 nmol/side), or saline. In both the VTA and NAS, the NK-1 and the NK-3 agonists caused significant analgesia in the formalin test, although the NK-1 agonist appeared to be more effective. Naltrexone (2.0 mg/kg) pretreatment failed to reverse the analgesic effects in the formalin test induced by intra-VTA infusions of the substance P (SP) analog, DiMe-C7 (3.0 microg/side), GR-73632 (0.5 nmol/side), or senktide (1.5 nmol/side). Neither compound given at either site was effective in the tail-flick test. These findings suggest that SP-dopamine (DA) interactions within the mesolimbic DA system play an important role in the inhibition of tonic pain. Furthermore, they support our earlier ideas that activation of midbrain DA systems by SP might play a role in stress- and/or pain-induced analgesia.

The pharmacology of GR203040, a novel, potent and selective non-peptide tachykinin NK1 receptor antagonist.

PubMed Central

Beattie, D. T.; Beresford, I. J.; Connor, H. E.; Marshall, F. H.; Hawcock, A. B.; Hagan, R. M.; Bowers, J.; Birch, P. J.; Ward, P.

1995-01-01

1. The in vitro and in vivo pharmacology of GR203040 ((2S, 3S)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), a novel, highly potent and selective non-peptide tachykinin NK1 receptor antagonist, was investigated in the present study. 2. GR203040 potently inhibited [3H]-substance P binding to human NK1 receptors expressed in Chinese hamster ovary (CHO) and U373 MG astrocytoma cells, and NK1 receptors in ferret and gerbil cortex (pKi values of 10.3, 10.5, 10.1 and 10.1 respectively). GR203040 had lower affinity at rat NK1 receptors (pKi = 8.6) and little affinity for human NK2 receptors (pKi < 5.0) in CHO cells and NK3 receptors in guinea-pig cortex (pKi < 6.0). With the exception of the histamine H1 receptor (pIC50 = 7.5). GR203040 had little affinity (pIC50 < 6.0) at all non-NK1 receptors and ion channels examined. Furthermore, GR203040 produced only weak inhibition of Na+ currents in SH-SY5Y neuroblastoma and superior cervical ganglion cells (pIC50 values < 4.0). GR203040 produced only weak antagonism of Ca(2+)-evoked contractions of rat isolated portal vein (pKn = 4.1). The enantiomer of GR203040, GR205608 (2R, 3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), had 10,000 fold lower affinity at the human NK1 receptor expressed in CHO cells (pKi = 6.3). 3. In gerbil ex vivo binding experiments, GR203040 produced a dose-dependent inhibition of the binding of [3H]-substance P to cerebral cortical membranes (ED50 = 15 micrograms kg-1 s.c. and 0.42 mg kg-1 p.o.). At 10 micrograms kg-1 s.c., the inhibition of [3H]-substance P binding was maintained for > 6 h. In the rat, GR203040 was less potent (ED50 = 15.4 mg kg-1 s.c.) probably reflecting, at least in part, its lower affinity at the rat NK1 receptor. 4. In guinea-pig isolated ileum and dog isolated middle cerebral and basilar arteries, GR203040 produced a rightward displacement of the concentration-effect curves to substance P methyl ester (SPOMe) with suppression of the maximum agonist response (apparent pKB values of 11.9, 11.2 and 11.1 respectively). 5. In anaesthetized rabbits, GR203040 antagonized reductions in carotid arterial vascular resistance evoked by SPOMe, injected via the lingual artery (DR10 (i.e. the dose producing a dose-ratio of 10) = 1.1 micrograms kg-1, i.v.). At a dose 20 fold greater than its DR10 value (i.e. 22 micrograms kg-1, i.v.), significant antagonism was evident more than 2 h after GR203040 administration. 6. In anaesthetized rats, GR203040 (3 and 10 mg kg-1, i.v.) produced a dose-dependent inhibition of plasma protein extravasation in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. 7. It is concluded that GR203040 is one of the most potent and selective NK1 receptor antagonists yet described, and as such, has considerable potential as a pharmacological tool to characterize the physiological and pathological roles of substance P and NK1 receptors. GR203040 may also have potential as a novel therapeutic agent for the treatment of conditions such as migraine, emesis and pain. PMID:8719789

Antagonist profile of ibodutant at the tachykinin NK2 receptor in guinea pig isolated bronchi.

PubMed

Santicioli, Paolo; Meini, Stefania; Giuliani, Sandro; Lecci, Alessandro; Maggi, Carlo Alberto

2013-10-24

In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK 2 receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK 2 receptor agonist [βAla 8 ]NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300nM) induced a concentration-dependent rightward shift of the [βAla 8 ]NKA(4-10) concentration-response curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65-1.25), thus indicating a surmountable behaviour. The calculated apparent antagonist potency as pK B value was 8.31±0.05. Ibodutant (0.3-100nM), produced a concentration-dependent inhibition of the nonadrenergic-noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100nM) ibodutant almost abolished the EFS-induced bronchoconstriction (95±4% inhibition), the calculated IC 50 value was 2.98nM (95% c.l. 1.73-5.16nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrate that ibodutant is a potent NK 2 receptor antagonist in guinea pig airways. © 2013 Published by Elsevier B.V.

A tachykinin NK1 receptor antagonist, CP-122,721-1, attenuates kainic acid-induced seizure activity.

PubMed

Zachrisson, O; Lindefors, N; Brené, S

1998-10-01

Substance P (SP) can play an important role in neuronal survival. To analyze the role of SP in excitotoxicity, kainic acid (KA) was administered to rats and in situ hybridization was used to analyze the levels of the SP encoding preprotachykinin-A (PPT-A) mRNA in striatal and hippocampal subregions 1, 4, and 24 h and 7 days after KA. In striatum and piriform cortex, PPT-A mRNA peaked 4 h after KA while in hippocampus, levels peaked after 24 h. KA caused seizures and neuronal toxicity as indicated by a reduction of the number of neurons in the hippocampal CA1 subregion after 7 days. KA was later administered alone or following pretreatment with the tachykinin NK1 receptor antagonist CP-122,721-1 (0.3 mg/kg). The pretreatment decreased seizure activity and a negative correlation was found between seizure activity and survival of CA1 neurons. Conclusively, treatment with CP-122,721-1 has a seizure inhibiting property and may possibly counteract KA-induced nerve cell death in CA1. Copyright 1998 Elsevier Science B.V.

Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea-pig ileum and proximal colon.

PubMed Central

Maggi, C. A.; Patacchini, R.; Meini, S.; Quartara, L.; Sisto, A.; Potier, E.; Giuliani, S.; Giachetti, A.

1994-01-01

1. The aim of this study was the pharmacological characterization of tachykinin NK1 and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [Sar9]SP sulphone, [Glp6,Pro9]SP(6-11) (septide) and [beta Ala8]NKA(4-10) was investigated. The affinities of various peptide and nonpeptide antagonists for the NK1 and NK2 receptor was estimated by use of receptor selective agonists. 2. The natural agonists, SP and NKA, produced concentration-dependent contraction in both preparations. EC50 values were 100 pM and 5 nM for SP, 1.2 nM and 19 nM for NKA in the ileum and colon, respectively. The action of SP and NKA was not significantly modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). 3. Synthetic NK1 and NK2 receptor agonists produced concentration-dependent contraction of the circular muscle of the ileum and proximal colon. EC50 values were 83 pM, 36 pM and 10 nM in the ileum, 8 nM, 0.7 nM and 12 nM in the colon for [Sar9]SP sulphone, septide and [beta Ala8]NKA-(4-10), respectively. The pseudopeptide derivative of NKA(4-10), MDL 28,564 behaved as a full or near-to-full agonist in both preparations, its EC50s being 474 nM and 55 nM in the ileum and colon, respectively. 4. Nifedipine (1 microM) abolished the response to septide and [Sar9]SP sulphone in the ileum and produced a rightward shift and large depression of the response in the colon. The response to [beta Ala8]NKA(4-10) was abolished in the ileum and largely unaffected in the colon. 5. The NK1 receptor antagonists, (+/-)-CP 96,34, FK 888 and GR 82,334 competitively antagonized the response to septide and [Sar9]SP sulphone in both preparations without affecting that to [beta Ala8]NKA(4-10). In general, the NK1 receptor antagonists were significantly more potent toward septide than [Sar9]SP sulphone in both preparations. 6. The NK2 receptor antagonists, GR 94,800 and SR 48,968 selectively antagonized the response to [beta Ala8]NKA(4-10) without affecting that to [Sar9]SP sulphone or septide in the ileum and colon. SR 48,968 produced noncompetitive antagonism of the response to the NK2 receptor agonist in the ileum and competitive antagonism in the colon. 7. MEN 10,376 and the cyclic pseudopeptide MEN 10,573 antagonized in a competitive manner the response to [beta Ala8]NKA(4-10) in the ileum and colon.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7518302

Bronchodilatation by tachykinins and capsaicin in the mouse main bronchus.

PubMed Central

Manzini, S.

1992-01-01

1. The effect of sensory neuropeptides and capsaicin on basal and stimulated tone of mouse bronchial smooth muscle has been evaluated. 2. In basal conditions neither sensory neuropeptides (substance P, neurokinin A or calcitonin gene-related peptide (CGRP) nor capsaicin exerted any contractile effects. However, when a tonic contraction was induced with carbachol (1 microM) a prompt relaxation was induced by substance P (1- 100 nM) and by neurokinin A (1- 100 nM), with substance P being more potent. A second application of substance P was without effect. CGRP (10 nM) produced only a very small and erratic relaxation. Relaxation was also induced by capsaicin (1 microM), and this response could be evoked only once in each preparation. In 4 out of 6 preparations a cross-desensitization between substance P and capsaicin was observed. 3. The selective NK1 tachykinin agonist, [Pro9]-SP sulphone (1 microM), exerted potent bronchodilator actions on carbachol-contracted mouse bronchial preparations. In contrast, neither [beta Ala8]-NKA (4-10) nor [MePhe7]-NKB (both at a concentration of 1 microM), selective synthetic agonists for NK2 and NK3 receptors, exerted significant relaxant effects. Furthermore, the selective NK1 tachykinin antagonist, (+/-)-CP 96,345 (1 microM), abolished substance P (1 nM)- but not isoprenaline (0.1 microM)-induced relaxations. 4. Application of electrical field stimulation (EFS) (20 Hz, supramaximal voltage, 0.5 ms for 10 s) to carbachol-contracted preparations evoked a transient contraction followed by a relaxation. The tetrodotoxin-sensitive slow component of this relaxation was reduced following capsaicin desensitization. 5. In the presence of indomethacin (5 microM) the relaxation induced by substance P, capsaicin or EFS was suppressed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1380376

MicroRNAs May Mediate the Down-Regulation of Neurokinin-1 Receptor in Chronic Bladder Pain Syndrome

PubMed Central

Sanchez Freire, Veronica; Burkhard, Fiona C.; Kessler, Thomas M.; Kuhn, Annette; Draeger, Annette; Monastyrskaya, Katia

2010-01-01

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B1 receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor. PMID:20008142

Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells

PubMed Central

Mizuta, Kentaro; Gallos, George; Zhu, Defen; Mizuta, Fumiko; Goubaeva, Farida; Xu, Dingbang; Panettieri, Reynold A.; Yang, Jay; Emala, Charles W.

2013-01-01

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [β-Ala8]-neurokinin A(4–10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase. PMID:18203813

Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells.

PubMed

Mizuta, Kentaro; Gallos, George; Zhu, Defen; Mizuta, Fumiko; Goubaeva, Farida; Xu, Dingbang; Panettieri, Reynold A; Yang, Jay; Emala, Charles W

2008-03-01

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [beta-Ala8]-neurokinin A(4-10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase.

Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats

PubMed Central

YAMAMURA, Takashi; WAKABAYASHI, Yoshihiro; OHKURA, Satoshi; NAVARRO, Victor M.; OKAMURA, Hiroaki

2014-01-01

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats. PMID:25345909

Role for NK(1) and NK(2) receptors in the motor activity in mouse colon.

PubMed

Mulè, Flavia; Amato, Antonella; Serio, Rosa

2007-09-10

The present study examined the effects induced by endogenous and exogenous activation of NK(1) and NK(2) receptors on the mechanical activity of mouse proximal colon. Experiments were performed in vitro recording the changes in intraluminal pressure from isolated colonic segments. Electrical field stimulation in the presence of atropine and guanethidine produced a small relaxation, followed by nonadrenergic noncholinergic (NANC) contraction. SR140333, NK(1) receptor antagonist, or SR48968, NK(2) receptor antagonist, significantly reduced the contraction, although SR48968 appeared more efficacious. The co-administration of SR140333 and SR48968 virtually abolished the NANC contraction. [Sar(9), Met(O(2))(11)]-substance P, selective NK(1) receptor agonist, induced a concentration-dependent biphasic effect, contraction followed by reduction of the mechanical spontaneous activity. Both effects were antagonized by SR140333, but not by SR48968. [beta-Ala(8)]-neurokinin A (4-10), selective NK(2) receptor agonist, evoked concentration-dependent contraction, which was antagonized by SR48968, but not by SR140333. The contraction induced by [Sar(9), Met(O(2))(11)]-substance P, but not by [beta-Ala(8)]-neurokinin A (4-10), was reduced by tetrodotoxin or atropine, and increased by N(omega)-nitro-L-arginine methyl ester (L-NAME), inhibitor of nitric oxide synthase. The inhibitory effects induced by [Sar(9), Met(O(2))(11)]-substance P were abolished by tetrodotoxin or L-NAME. The results of the present study suggest that in mouse colon both NK(1) and NK(2) receptors are junctionally activated by endogenous tachykinins to cause an additive response. NK(1) receptors appear to be located on cholinergic and on nitrergic neurons as well as on smooth muscle cells, whereas NK(2) receptors seem to be present exclusively on smooth muscle cells.

Effects of tachykinins and capsaicin on the mechanical and electrical activity of the guinea-pig isolated trachea

PubMed Central

Girard, Valerie; Félétou, Michel; Advenier, Charles; Canet, Emmanuel

1997-01-01

The effects of tachykinins and capsaicin were studied by means of intracellular membrane potential and isometric tension recordings in the isolated trachea of the guinea-pig. The basal membrane potential averaged −51 mV, and most preparations demonstrated spontaneous slow waves. Tetraethylammonium (TEA), a potassium channel blocker (8×10−3 M), depolarized the membrane potential to −44 mV and induced a rhythmic activity. In control solution, substance P (10−8–10−6 M), [Nle10]-neurokinin A(4–10) (10−8–10−6 M) and capsaicin (10−7–10−6 M) induced concentration-dependent depolarizations which were statistically significant at the highest concentration tested (depolarization by 10−6 M: 8, 11 and 16 mV for the NK1 agonist, the NK2 agonist and capsaicin, respectively). In the presence of TEA (8×10−3 M), the three substances induced depolarizations which were statistically significant at the highest concentration tested for substance P (10−6 M) and at 10−7 and 10−6 M for both [Nle10]-neurokinin A(4–10) and capsaicin (depolarization by 10−6 M: 11, 17 and 10 mV for substance P, [Nle10]neurokinin A(4–10) and capsaicin, respectively). In the presence or absence of tetraethylammonium, [MePhe7]-neurokinin B (10−8–10−6 M) did not induce any significant changes in membrane potential. The depolarizing effects of substance P (10−6 M) and [Nle10]-neurokinin A(4–10) (10−6 M) were blocked only by the specific antagonists for NK1 and NK2 receptors, SR 140333 (10−7 M) and SR 48968 (10−7 M), respectively. The effects of capsaicin (10−6 M) were partially inhibited by each antagonist and fully blocked by their combination. Substance P (10−9 to 10−4 M), [Nle10]-neurokinin A(4–10) (10−10 to 10−5 M), [MePhe7]-neurokinin B and capsaicin (10−7 to 10−5 M) evoked concentration-dependent contractions. The contractions to substance P were significantly inhibited by SR 140333 (10−8 to 10−6 M) but unaffected by SR 48968 (10−8 to 10−6 M). Furthermore, the response to [Nle10]-neurokinin A(4–10) was significantly inhibited by SR 48968 and unaffected by SR 140333 at the same concentrations. Although SR 48968 (10−7 M) alone did not influence the effects of substance P, it potentiated the inhibitory effect of SR 140333 (10−7 M). A similar synergetic effect of these two compounds was observed in the inhibition of the contractile response to [Nle10]-neurokinin A(4–10). Neither SR 140333 (10−7 M) nor SR 48968 (10−7 M) alone influenced the contractions to [MePhe7]-neurokinin B and capsaicin. However, the combination of the two antagonists abolished the contractions to either peptide. These results demonstrate that the stimulation of both NK1 and NK2 tachykinin-receptors induced contraction and depolarization of the guinea-pig tracheal smooth muscle and that both receptors were stimulated during the endogenous release of tachykinins by capsaicin. There was no evidence for a major role of NK3 receptors in the contractile and electrical activity of the guinea-pig isolated trachea. PMID:9384499

Renal effects of intrathecally injected tachykinins in the conscious saline-loaded rat: receptor and mechanism of action

PubMed Central

Ding Yuan, Yi; Couture, Réjean

1997-01-01

The effects of intrathecally (i.t.) injected substance P (SP), neurokinin A (NKA), [β-Ala8]NKA (4–10) and [MePhe7]neurokinin B (NKB) at T13 thoracic spinal cord level were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. Antagonists selective for NK1 (RP 67580), NK2 (SR 48968) and NK3 (R 820; 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) receptors were used to characterize the spinal effect of SP on renal function. Saline gavage (4.5% of the body weight) enhanced renal excretion of water, sodium and potassium over the subsequent hour of measurement. Whereas these renal responses were not affected by 0.65 nmol SP, the dose of 6.5 nmol SP blocked the natriuretic response (aCSF value 3.9±0.8; SP value 0.7±0.3 μmol min−1, P<0.01) as well as the renal excretion of water (aCSF value 48.9±5.8; SP value 14.5±4.0 μl min−1, P<0.01) and potassium (aCSF value 4.8±0.6; SP value 1.5±0.6 μmol min−1, P<0.01) at 30 min post-injection. SP had no significant effect on urinary osmolality. The SP-induced renal inhibitory effects during the first 30 min were abolished in rats subjected to bilateral renal denervation 1 week earlier or in rats injected i.t. 5 min earlier with 6.5 nmol RP 67580. In contrast, the co-injection of SR 48968 and R 820 (6.5 nmol each) did not affect the inhibitory responses to SP. On their own, these antagonists had no direct effect on renal excretion function. Since SP induced only transient changes in mean arterial blood pressure (−18.8±3.8 mmHg at 1 min and +6.3±2.4 mmHg at 5 min post-injection), it is unlikely that the renal effects of SP are due to systemic haemodynamic changes. NKA (6.5 nmol but not 0.65 nmol) produced a transient drop in renal excretion of water (aCSF value 31.2±5.1; NKA value 11.3±4.2 μl min−1, P<0.05), sodium (aCSF value 1.7±0.8; NKA value 0.4±0.2 μmol min−1, P<0.05) and potassium (aCSF value 4.1±0.7; NKA value 1.5±0.4 μmol min−1, P<0.05) at 15 min post-injection. However, the same doses (6.5 nmol) of selective agonists for tachykinin NK2 ([β-Ala8]NKA(4-10)) and NK3 ([MePhe7]NKB) receptors were devoid of renal effects. This study provided functional evidence that tachykinins may be involved in the renal control of water and electrolyte excretion at the level of the rat spinal cord through the activation of NK1 receptors and the sympathetic renal nerve. PMID:9249250

Inhibition of emesis by tachykinin NK1 receptor antagonists in Suncus murinus (house musk shrew).

PubMed

Rudd, J A; Ngan, M P; Wai, M K

1999-02-05

The anti-emetic potential of CP-122,721 ((+)-2S,3S)-3-(2-methoxy-5-trifluoromethoxybenzyl)amino-2-phenylpi peridine), CP-99,994 ((+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine), CP-100,263 ((-)-(2R,3R)-3-(2-methoxybenzylamino)-2-phenylpiperidine), RP 67580 ((3R, 7aR)-7,7-diphenyl-2-[1-imino-2-(2-methoxyphenyl)ethyl] po-hydroisoindol-4-one), FK 888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-in-dole-3-yl)carbonyl-L-propyl] -N-methyl-N-phenylmethyl-1-3-(2-naphthyl)-alaninamide) and GR 82334 ([D-Pro9[spiro-g-lactam]Leu10]-physalaemin-(1-11)) was investigated to inhibit nicotine (5 mg/kg, s.c.)-, copper sulphate pentahydrate (120 mg/kg, intragastric)- and motion (4 cm horizontal displacement at 1 Hz for 5 min)-induced emesis in Suncus murinus. A 30 min intraperitoneal pre-treatment with CP-122,721, CP-99,994, RP 67580 and FK 888 significantly (P < 0.05) antagonized nicotine-induced emesis with ID50 values of 2.1, 2.3, 13.5 and 19.2 mg/kg, respectively CP-100,263, the less active enantiomer of CP-99,994, was inactive at doses up to 10 mg/kg. Infusion of GR 82334, CP-122,721, CP-99,994 and FK 888 into the dorsal vagal complex of the hindbrain also antagonized nicotine-induced emesis yielding ID50 values of 1.1, 3.0, 3.3 and 58.0 microg/dorsal vagal complex, respectively RP 67580 and CP-100,263 were inactive. RP 67580 and FK 888 failed to antagonize copper sulphate-induced emesis but CP-122,721 and CP-99,994 were active yielding ID50 values of 2.2 and 3.0 mg/kg, i.p., respectively. CP-99,994 also completely prevented motion-induced emesis at 10 mg/kg, i.p. (P < 0.05) and RP 67580 produced a significant reduction of motion-induced emesis at 10 mg/kg, i.p. (P < 0.05). These studies provide evidence of a central site of action of tachykinin NK1 receptor antagonists to inhibit nicotine-induced emesis in S. murinus and confirm the broad profile of inhibitory action. The rank order of potency of the antagonists following the intra-dorsal vagal complex administration suggests that the S. murinus tachykinin NK1 receptor has a unique pharmacological profile.

Endothelin-converting enzyme 1 degrades neuropeptides in endosomes to control receptor recycling.

PubMed

Roosterman, Dirk; Cottrell, Graeme S; Padilla, Benjamin E; Muller, Laurent; Eckman, Christopher B; Bunnett, Nigel W; Steinhoff, Martin

2007-07-10

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with beta-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide-receptor-arrestin complex is sequestered into endosomes. Although dissociation of beta-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide-receptor-arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of beta-arrestins followed by trafficking of the SP-NK1R-beta-arrestin complex to early endosomes containing ECE-1a-d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; beta-arrestins returned to the cytosol, and the NK1R, freed from beta-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide-receptor-arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control beta-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

The N-terminal domain of substance P is required for complete homologous desensitization but not phosphorylation of the rat neurokinin-1 receptor.

PubMed

Vigna, S R

2001-02-01

The agonist activity of substance P (SP) is a function of the C-terminal domain of the peptide. A C-terminal SP fragment (SP(6-11)) and analog (septide) and neurokinin A (NKA; a related tachykinin with a divergent N-terminal amino acid sequence) were found to be full neurokinin-1 receptor (NK-1R) agonists, but were not able to desensitize the receptor maximally as much as SP. Substance P caused 95.6 +/- 0.9% maximal desensitization of the NK-1R whereas SP(6-11), septide, and NKA(only)caused 74 +/- 3.5, 50.6 +/- 8, and 71.5 +/- 4.4% maximal desensitization, respectively (mean +/- SEM; P < 0.001 vs SP). When a series of SP C-terminal fragment peptides were tested for their NK-1R desensitizing activity, it was found that SP(5-11)and SP(6-11)caused significantly less maximal NK-1R desensitization than SP. SP N-terminal fragment peptides had no effect on the ability of SP(6-11)to compete with(3)H-SP binding, generate an IP(3)response, or cause NK-1R desensitization when tested with or without SP(6-11). SP, SP(6-11), septide, and NKA all maximally stimulated 8-9-fold increases in NK-1R phosphorylation. When attached to the C-terminal domain of SP responsible for NK-1R binding and agonism, the N-terminus of SP is responsible for 25-50% of homologous desensitization and this may occur via a mechanism other than NK-1R phosphorylation. Copyright 2001 Harcourt Publishers Ltd.

The plasma protein extravasation induced by adenosine and its analogues in the rat dorsal skin: evidence for the involvement of capsaicin sensitive primary afferent neurones and mast cells.

PubMed

Esquisatto, L C; Costa, S K; Camargo, E A; Ribela, M T; Brain, S D; de Nucci, G; Antunes, E

2001-09-01

1. The contribution of sensory neurons and mast cells to the oedema evoked by adenosine A1 (N(6)-cyclopentyladenosine, CPA, 3 - 30 nmol site(-1)), A2 (5'N-ethylcarboxamidoadenosine, NECA, 1 - 10 nmol site(-1)) and A3 receptor agonists (N6-[3-iodobenzyl]-N-methyl-5'-carboxiamidoadenosine, IB-MECA, 0.01 - 3 nmol site(-1)) was investigated in the rat skin microvasculature, by the extravascular accumulation of intravenously-injected (i.v.) 125I-albumin. 2. Intradermal (i.d.) injection of adenosine and analogues induced increased microvascular permeability in a dose-dependent manner (IB-MECA > NECA > CPA > adenosine). The non-selective adenosine receptor antagonist theophylline (5 - 50 nmol site(-1)) markedly inhibited adenosine, CPA or NECA but not IB-MECA-induced plasma extravasation. The A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.3 - 3 micromol kg(-1), i.v.) significantly reduced CPA-induced plasma extravasation whereas responses to adenosine, NECA or IB-MECA were unchanged. The A2 receptor antagonist 3,7-dymethyl-1-proprargylxanthine (DMPX, 0.5 - 50 nmol site(-1)) significantly reduced NECA-induced plasma extravasation without affecting responses to adenosine, CPA and IB-MECA. 3. The tachykinin NK1 receptor antagonist (S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl) piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2]octane chloride (SR140333), but not the NK2 receptor antagonist (S)-N-methyl-N[4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl]-benzamide (SR48968), significantly inhibited the plasma extravasation evoked by higher doses of adenosine (100 nmol site(-1)), CPA (100 nmol site(-1)), NECA (1 nmol site(-1)) and IB-MECA (0.1 - 1 nmol site(-1)). In rats treated with capsaicin to destroy sensory neurons, the response to higher doses of adenosine, CPA and NECA, but not IB-MECA, was significantly inhibited. 4. The effects of adenosine and analogues were largely inhibited by histamine and 5-hydroxytryptamine (5-HT) antagonists and by compound 48/80 pretreatment. 5. In conclusion, our results provide evidence that adenosine A1 and A2, but not A3, receptor agonists may function as cutaneous neurogenic pro-inflammatory mediators; acting via microvascular permeability-increasing mechanisms that can, depending on dose of agonist and purine receptor under study, involve the tachykinin NK1 receptor and mast cell amines.

Substance P mediates inflammatory oedema in acute pancreatitis via activation of the neurokinin-1 receptor in rats and mice

PubMed Central

Grady, Eileen F; Yoshimi, Shandra K; Maa, John; Valeroso, Dahlia; Vartanian, Robert K; Rahim, Shamila; Kim, Edward H; Gerard, Craig; Gerard, Norma; Bunnett, Nigel W; Kirkwood, Kimberly S

2000-01-01

Pancreatic oedema occurs early in the development of acute pancreatitis, and the overall extent of fluid loss correlates with disease severity. The tachykinin substance P (SP) is released from sensory nerves, binds to the neurokinin-1 receptor (NK1-R) on endothelial cells and induces plasma extravasation, oedema, and neutrophil infiltration, a process termed neurogenic inflammation. We sought to determine the importance of neurogenic mechanisms in acute pancreatitis.Pancreatic plasma extravasation was measured using the intravascular tracers Evans blue and Monastral blue after administration of specific NK1-R agonists/antagonists in rats and NK1-R(+/+)/(−/−) mice. The effects of NK1-R genetic deletion/antagonism on pancreatic plasma extravasation, amylase, myeloperoxidase (MPO), and histology in cerulein-induced pancreatitis were characterized.In rats, both SP and the NK1-R selective agonist [Sar9 Met(O2)11]SP stimulated pancreatic plasma extravasation, and this response was blocked by the NK1-R antagonist CP 96,345. Selective agonists of the NK-2 or NK-3 receptors had no effect.In rats, cerulein stimulated pancreatic plasma extravasation and serum amylase. These responses were blocked by the NK1-R antagonist CP 96,345.In wildtype mice, SP induced plasma extravasation while SP had no effect in NK1-R knockout mice.In NK1-R knockout mice, the effects of cerulein on pancreatic plasma extravasation and hyperamylasemia were reduced by 60%, and pancreatic MPO by 75%, as compared to wildtype animals.Neurogenic mechanisms of inflammation are important in the development of inflammatory oedema in acute interstitial pancreatitis. PMID:10821777

Tachykinin actions on deep dorsal horn neurons in vitro: an electrophysiological and morphological study in the immature rat.

PubMed

King, A E; Slack, J R; Lopez-Garcia, J A; Ackley, M A

1997-05-01

To assess whether functional neurokinin receptors exist in the deep dorsal horn of the rat, the actions of the selective neurokinin-1 receptor (NK1R) agonist [Sar9,Met(O2)11]substance P ([Sar9,Met(O2)11]SP), the neurokinin-2 receptor (NK2R) agonists [beta-Ala8]NKA(4-10) and GR64349 and the neurokinin-3 receptor (NK3R) agonist senktide were examined intracellularly in vitro. [Sar9,Met(O2)11]SP (1-4 microM) and senktide (1-2 microM) elicited slow depolarizations (<10 mV) associated with increased synaptic activity and cell firing. [beta-Ala8]NKA(4-10) (10-20 microM) and GR64349 (0.25-10 microM) caused small depolarizations (<2.0 mV) and no firing. Neurons were categorized as either 'tonic' or 'phasic' depending on their firing response to direct current step depolarizations. Tonic neurons, which, unlike phasic neurons, display no spike firing accommodation, generated a significantly larger depolarization to the NK1R and NK3R agonists. The putative contribution of these receptors to primary afferent-mediated synaptic transmission was assessed by testing the NK1R antagonist GR82334 (1 microM), the NK2R antagonist MEN10,376 (1 microM) and the NK3R antagonist [Trp7,beta-Ala8]NKA(4-10) (1 microM) against the dorsal root-evoked excitatory postsynaptic potential (DR-EPSP). GR82334 and [Trp7,beta-Ala8]NKA(4-10) significantly reduced (P < or = 0.05) the duration but not the amplitude of the DR-EPSP. MEN10,376 (1 microM) had no effect on DR-EPSP amplitude or duration. Morphological detail was obtained for seven biocytin-filled deep dorsal horn neurons tested with [Sar9,Met(O2)11]SP. Five neurons responded to the NK1R agonist, and two of these had dorsally directed dendrites into the substantia gelatinosa. The other three [Sar9,Met(O2)11]SP-sensitive neurons had dendrites within deeper laminae. These data support the existence of functional NK1Rs and NK3Rs in the deep dorsal horn which may be involved in mediating sensory afferent inputs from nociceptors.

The Neuropeptide Tac2 Controls a Distributed Brain State Induced by Chronic Social Isolation Stress.

PubMed

Zelikowsky, Moriel; Hui, May; Karigo, Tomomi; Choe, Andrea; Yang, Bin; Blanco, Mario R; Beadle, Keith; Gradinaru, Viviana; Deverman, Benjamin E; Anderson, David J

2018-05-17

Chronic social isolation causes severe psychological effects in humans, but their neural bases remain poorly understood. 2 weeks (but not 24 hr) of social isolation stress (SIS) caused multiple behavioral changes in mice and induced brain-wide upregulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function and suggest potential new therapeutic applications for Nk3R antagonists. Copyright © 2018 Elsevier Inc. All rights reserved.

Contractile properties of the pig bladder mucosa in response to neurokinin A: a role for myofibroblasts?

PubMed Central

Sadananda, P; Chess-Williams, R; Burcher, E

2008-01-01

Background and purpose: The bladder urothelium is now known to have active properties. Our aim was to investigate the contractile properties of the urinary mucosa in response to the tachykinin neurokinin A (NKA) and carbachol. Experimental approach: Discrete concentration–response curves for carbachol and NKA were obtained in matched strips of porcine detrusor, mucosa and intact bladder, suspended in organ baths. The effects of inhibitors and tachykinin receptor antagonists were studied on NKA-mediated contractions in mucosal strips. Intact sections of bladder and experimental strips were processed for histology and immunohistochemistry. Key results: All types of strips contracted to both carbachol and NKA. Mucosal responses to NKA (pD2 7.2) were higher than those in intact strips and were inhibited by the NK2 receptor antagonist SR48968 (pKB 9.85) but not the NK1 receptor antagonist SR140333, tetrodotoxin or indomethacin. Immunostaining for smooth muscle actin and vimentin occurred under the urothelium and on blood vessels. Desmin immunostaining and histological studies showed only sparse smooth muscle to be present in the mucosal strips. Removal of smooth muscle remnants from mucosal strips did not alter the responses to NKA. Conclusions and implications: This study has shown both functional and histological evidence for contractile properties of the mucosa, distinct from the detrusor. Mucosal contractions to NKA appear to be directly mediated via NK2 receptors. The main cell type mediating mucosal contractions is suggested to be suburothelial myofibroblasts. Mucosal contractions may be important in vivo for matching the luminal surface area to bladder volume. PMID:18264120

Contribution of NK3 tachykinin receptors to propulsion in the rabbit isolated distal colon.

PubMed

Onori, L; Aggio, A; Taddei, G; Ciccocioppo, R; Severi, C; Carnicelli, V; Tonini, M

2001-06-01

The role of NK3 receptors in rabbit colonic propulsion has been investigated in vitro with the selective agonist, senktide, and two selective antagonists, SR142801 and SB222200. Peristalsis was elicited by distending a rubber balloon with 0.3 and 1.0 mL of water leading to a velocity of 2.2 and 2.8 mm s-1, respectively. At concentrations of 1 nM, senktide inhibited propulsion evoked by both distensions (range 25-40%), whereas at 6 and 60 nmol L-1 facilitated 'submaximal' propulsion by 30%. In the presence of Nomega-nitro-L-arginine (L-NNA, 200 micromol L-1), which per se caused a slight prokinetic effect, 1 nmol L-1 senktide markedly accelerated propulsion (range 35-50%). Hexamethonium (200 micromol L-1) had minor effects on propulsion. In its presence, 60 nmol L-1 senktide significantly inhibited propulsion induced by both stimuli (range 20-50%). SR142801 (0.3, 3 nmol L-1) and SB222200 (30, 300 nmol L-1) facilitated 'submaximal' propulsion (range 20-40%). Conversely, higher antagonist concentrations (SR142801: 30, 300 nM; SB222200: 1, 10 micromol L-1) inhibited propulsion to both distensions by 20%. A combination of SR142801 (300 nmol L-1) plus hexamethonium (200 micromol L-1) induced an approximately four-fold greater inhibition of propulsion than that induced by SR142801 alone. In conclusion, in the rabbit-isolated distal colon, a subset of NK3 receptors located on descending pathways mediates an inhibitory effect on propulsion by activating a NO-dependent mechanism. Another subset of NK3 receptors, located on ascending pathways mediates a facilitative effect involving a synergistic interaction with cholinergic nicotinic receptors.

Effect of [D-Phe6] bombesin (6-13) methylester, a bombesin receptor antagonist, towards bombesin-induced contractions in the guinea-pig and rat isolated urinary bladder.

PubMed

Maggi, C A; Coy, D H; Giuliani, S

1992-08-01

1. The effect of [D-Phe6] bombesin (6-13) methylester (OMe), a newly developed potent antagonist of bombesin receptors, has been investigated against bombesin-induced contractions of the guinea-pig and rat isolated urinary bladder. 2. Bombesin (0.1 nM-10 microM) produced a concentration-dependent contraction of the guinea-pig isolated bladder which approached the same maximum response as KCl (80 mM). The response to bombesin was antagonized in a competitive manner (rightward shift of the concentration-response curve without depression of the maximal response) by [D-Phe6] bombesin (6-13) OMe (0.3-10 microM). Degree of antagonism was concentration-dependent between 0.3 and 3 microM (dose ratios = 2.4, 9 and 39 in the presence of 0.3, 1, 3 microM of the antagonist). However, a larger concentration (10 microM) of the antagonist was not more effective (dose ratio = 36) than 3 microM. 3. Neither the action of bombesin nor the activity of the antagonist was influenced by peptidase inhibitors (bestatin, captopril and thiorphan 3 microM each) or by atropine, indomethacin, chlorpheniramine and desensitization of P2x purinoceptors by alpha, beta methylene ATP. 4. The bombesin antagonist was ineffective against contraction of the guinea-pig urinary bladder produced by the NK-1 tachykinin receptor-selective agonist, [Sar9] substance P sulphone. The action of the NK-1 receptor agonist was antagonized by L 668, 169 (3 microM), a cyclic peptide tachykinin antagonist. L 668, 169 had no effect toward bombesin-induced contraction. 5. The bombesin antagonist (1-10 microM) had no effect against the non-adrenergic non-cholinergic response of the guinea-pig isolated urinary bladder to electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitation and contraction in the circular muscle of guinea-pig duodenum.

PubMed Central

Zagorodnyuk, V.; Santicioli, P.; Maggi, C. A.; Giachetti, A.

1995-01-01

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), apamin (0.1 microM) and L-nitroarginine (L-NOARG, 30 microM), electrical field simulation (EFS) produced a nonadrenergic, noncholinergic (NANC) excitatory junctional potential (e.j.p.), action potentials and contraction of the circular muscle of the guinea-pig proximal duodenum, recorded by the single sucrose gap technique. 2. The selective tachykinin (TK) NK1 receptor antagonist, GR 82,334 (30 nM-3 microM) produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. Similarly, the selective NK2 receptor antagonists, MEN 10,627 (30 nM-3 microM) and GR 94,800 (100 nM-10 microM), both produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. GR 82,334 inhibited the electrical and mechanical NANC responses to EFS in an almost parallel manner, while MEN 10,627 and GR 94,800 were more effective in inhibiting the mechanical than the electrical response to EFS. 3. Activation of the NK1 or NK2 receptor by the selective agonists, [Sar9]substance P (SP) sulphone and [beta Ala8]neurokinin A (NKA) (4-10), respectively (0.3 microM each), produced depolarization, action potentials and contractions. GR 82,334 selectively inhibited the responses to [Sar9]SP sulphone, without affecting the responses to [beta Ala8]NKA (4-10). MEN 10,627 and GR 94,800 inhibited or abolished the responses to [beta Ala8]NKA (4-10), without affecting the responses to [Sar9]SP sulphone. 4. Nifedipine (1 microM) abolished the action potentials and contraction produced either by EFS or by the TK receptor agonists [Sar9]SP sulphone or [beta Ala8]NKA (4-10). 5. In the presence of nifedipine, the NANC e.j.p. produced by EFS was biphasic: in the majority of strips tested (21 out of 29) an early fast phase of depolarization was followed by a second slow component. The combined administration of GR 82,334 and GR 94,800 (3 microM each) reduced both components, the slow phase being inhibited to a greater extent than the fast phase. 6. The P2 purinoreceptor antagonist, suramin (100 microM) reduced the fast phase of the e.j.p. produced by EFS in the presence of nifedipine, without affecting the slow phase. The combined administration of suramin, GR 82,334 and GR 94,800 produced a nearly complete blockade of the e.j.p. produced by EFS in the presence of nifedipine. 7. When tested in the absence of apamin and L-NOARG, EFS induced a NANC inhibitory junction potential (i.j.p.) followed by an e.j.p., and the selective P2Y receptor agonist, adenosine-5'-O-(2-thiodiphosphate) (ADP beta S, 10 microM), produced membrane hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7545517

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

Comparison of the phenotype of NK1R-/- mice with pharmacological blockade of the substance P (NK1 ) receptor in assays for antidepressant and anxiolytic drugs.

PubMed

Rupniak, N M; Carlson, E J; Webb, J K; Harrison, T; Porsolt, R D; Roux, S; de Felipe, C; Hunt, S P; Oates, B; Wheeldon, A

2001-11-01

The phenotype of NK1R-/- mice was compared with that of acute pharmacological blockade of the tachykinin NK1 receptor on sensorimotor function and in assays relevant to depressive illness and anxiety. The dose range for L-760735 and GR205171 that was associated with functional blockade of central NK1 receptors in the target species was established by antagonism of the behavioural effects of intracerebroventricular NK1 agonist challenge in gerbils, mice and rats. The caudal grooming and scratching response to GR73632 was absent in NK1R-/- mice, confirming that the receptor had been genetically ablated. There was no evidence of sedation or motor impairment in NK1R-/- mice or following administration of L-760735 to gerbils, even at doses in excess of those required for central NK1 receptor occupancy. In the resident-intruder and forced swim test, the behaviour of NK1R-/- mice, or animals treated acutely with L-760735 or GR205171, resembled that seen with the clinically used antidepressant drug fluoxetine. However, the effects of GR205171 were not clearly enantioselective in mice. In contrast, although NK1R-/- mice also exhibited an increase in the duration of struggle behaviour in the tail suspension test, this was not observed following pharmacological blockade with L-760735 in gerbils or GR205171 in mice, suggesting that this may reflect a developmental alteration in the knockout mouse. There was no effect of NK1 receptor blockade with L-760735 in guinea-pigs or GR205171 in rats, or deletion of the NK1 receptor in mice, on behaviour in the elevated plus-maze test for anxiolytic activity. These findings extend previous observations on the phenotype of the NK1R-/- mouse and establish a broadly similar profile following acute pharmacological blockade of the receptor. These studies also serve to underscore the limitations of currently available antagonists that are suitable for use in rat and mouse behavioural assays.

Role of Substance P in the Regulation of Glucose Metabolism via Insulin Signaling-Associated Pathways

PubMed Central

Bakirtzi, Kyriaki; Kokkotou, Efi; Stavrakis, Dimitris; Margolis, Kara Gross; Thomou, Thomas; Giorgadze, Nino; Kirkland, James L.

2011-01-01

Substance P (SP), encoded by the tachykinin 1 (Tac1) gene, is the most potent tachykinin ligand for the high-affinity neurokinin-1 receptor (NK-1R). We previously reported that NK-1R-deficient mice show less weight gain and reduced circulating levels of leptin and insulin in response to a high-fat diet (HFD) and demonstrated the presence of functional NK-1R in isolated human preadipocytes. Here we assessed the effects of SP on weight gain in response to HFD and determined glucose metabolism in Tac1-deficient (Tac1−/−) mice. The effect of SP on the expression of molecules that may predispose to reduced glucose uptake was also determined in isolated human mesenteric, omental, and sc preadipocytes. We show that although weight accumulation in response to HFD was similar between Tac1−/− mice and wild-type littermates, Tac1−/− mice demonstrated lower glucose and leptin and increased adiponectin blood levels and showed improved responses to insulin challenge after HFD. SP stimulated phosphorylation of c-Jun N-terminal kinase, protein kinase Cθ, mammalian target of rapamycin, and inhibitory serine insulin receptor substrate-1 phosphorylation in human preadipocytes in vitro. Preincubation of human mesenteric preadipocytes with the protein kinase Cθ pseudosubstrate inhibitor reduced insulin receptor substrate 1 phosphorylation in response to SP. Lastly, SP also induced insulin receptor substrate-1 phosphorylation in mature human sc adipocytes. Our results demonstrate an important role for SP in adipose tissue responses and obesity-associated pathologies. These novel SP effects on molecules that enhance insulin resistance at the adipocyte level may reflect an important role for this peptide in the pathophysiology of type 2 diabetes. PMID:22009727

Role of substance P in the regulation of glucose metabolism via insulin signaling-associated pathways.

PubMed

Karagiannides, Iordanes; Bakirtzi, Kyriaki; Kokkotou, Efi; Stavrakis, Dimitris; Margolis, Kara Gross; Thomou, Thomas; Giorgadze, Nino; Kirkland, James L; Pothoulakis, Charalabos

2011-12-01

Substance P (SP), encoded by the tachykinin 1 (Tac1) gene, is the most potent tachykinin ligand for the high-affinity neurokinin-1 receptor (NK-1R). We previously reported that NK-1R-deficient mice show less weight gain and reduced circulating levels of leptin and insulin in response to a high-fat diet (HFD) and demonstrated the presence of functional NK-1R in isolated human preadipocytes. Here we assessed the effects of SP on weight gain in response to HFD and determined glucose metabolism in Tac1-deficient (Tac1(-/-)) mice. The effect of SP on the expression of molecules that may predispose to reduced glucose uptake was also determined in isolated human mesenteric, omental, and sc preadipocytes. We show that although weight accumulation in response to HFD was similar between Tac1(-/-) mice and wild-type littermates, Tac1(-/-) mice demonstrated lower glucose and leptin and increased adiponectin blood levels and showed improved responses to insulin challenge after HFD. SP stimulated phosphorylation of c-Jun N-terminal kinase, protein kinase C, mammalian target of rapamycin, and inhibitory serine insulin receptor substrate-1 phosphorylation in human preadipocytes in vitro. Preincubation of human mesenteric preadipocytes with the protein kinase C pseudosubstrate inhibitor reduced insulin receptor substrate 1 phosphorylation in response to SP. Lastly, SP also induced insulin receptor substrate-1 phosphorylation in mature human sc adipocytes. Our results demonstrate an important role for SP in adipose tissue responses and obesity-associated pathologies. These novel SP effects on molecules that enhance insulin resistance at the adipocyte level may reflect an important role for this peptide in the pathophysiology of type 2 diabetes.

Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes.

PubMed

Im, Hee-Jeong; Li, Xin; Muddasani, Prasuna; Kim, Gun-Hee; Davis, Francesca; Rangan, Jayanthi; Forsyth, Christopher B; Ellman, Michael; Thonar, Eugene J M A

2008-05-01

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK(1)-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1beta accelerate matrix degradation via a neural pathway upregulation of substance P and NK(1)-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK(1)-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK(1)-R is, in part, through an IL-1beta-dependent pathway. (c) 2007 Wiley-Liss, Inc.

Basic Fibroblast Growth Factor Accelerates Matrix Degradation Via a Neuro-Endocrine Pathway in Human Adult Articular Chondrocytes

PubMed Central

IM, HEE-JEONG; LI, XIN; MUDDASANI, PRASUNA; KIM, GUN-HEE; DAVIS, FRANCESCA; RANGAN, JAYANTHI; FORSYTH, CHRISTOPHER B.; ELLMAN, MICHAEL; THONAR, EUGENE JMA

2010-01-01

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK1-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1β accelerate matrix degradation via a neural pathway upregulation of substance P and NK1-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK1-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK1-R is, in part, through an IL-1β-dependent pathway. PMID:17960584

Tachykininergic slow depolarization of motoneurones evoked by descending fibres in the neonatal rat spinal cord.

PubMed

Kurihara, T; Yoshioka, K; Otsuka, M

1995-06-15

1. In the isolated spinal cord of the neonatal rat, repetitive electrical stimulation of the upper cervical region elicited a prolonged depolarization of lumbar motoneurones (L3-5) lasting 1-2 min, which was recorded extracellularly from ventral roots, or intracellularly. 2. This depolarizing response was markedly depressed by the excitatory amino acid receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (D-APV, 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). The remaining response was further depressed by a 5-hydroxytryptamine (5-HT) receptor antagonist, ketanserin (3 microM). 3. In the presence of these antagonists, a small part of the depolarizing response of slow time course remained, and this response was partially blocked by the tachykinin NK1 receptor antagonists GR71251 (0.3-5 microM) and RP67580 (0.3-1 microM). In contrast, RP68651 (0.3-1 microM), the inactive enantiomer of RP67580, had no effect on the depolarizing response. 4. The slow depolarizing response in the presence of D-APV, CNQX and ketanserin was markedly potentiated by a peptidase inhibitor, thiorphan (1 microM). 5. This descending fibre-evoked slow depolarization became smaller after prolonged treatment (5-7 h) with 5,7-dihydroxytryptamine (10 microM), a neurotoxin for 5-HT neurones. Under such conditions, the effects of thiorphan and GR71251 on the slow depolarization were virtually absent. 6. Under the action of D-APV, CNQX and ketanserin, applications of tachykinins, substance P and neurokinin A produced depolarizing responses of lumbar motoneurones, and the responses were depressed by GR71251 and potentiated by thiorphan. 7. These results suggest that tachykinins contained in serotonergic fibres serve as neurotransmitters mediating the descending fibre-evoked slow excitatory postsynaptic potentials in motoneurones.

Immunohistochemical localization of cardio-active neuropeptides in the heart of a living fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata).

PubMed

Springer, J; Ruth, P; Beuerlein, K; Westermann, B; Schipp, R

2004-01-01

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neurosecretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).

Novel selective agonists and antagonists confirm neurokinin NK1 receptors in guinea-pig vas deferens.

PubMed Central

Hall, J. M.; Morton, I. K.

1991-01-01

1. This study investigated the recognition characteristics of neurokinin receptors mediating potentiation of the contractile response to field stimulation in the guinea-pig vas deferens. 2. A predominant NK1 receptor population is strongly suggested by the relative activities of the common naturally-occurring tachykinin agonists, which fall within less than one order of magnitude. This conclusion is supported by the relative activities of the synthetic NK1 selective agonists substance P methyl ester, [Glp6,L-Pro9]-SP(6-11) and delta-aminovaleryl-[L-Pro9,N-MeLeu10]- SP(7-11) (GR73632) which were 0.78, 9.3 and 120 as active as substance P, respectively. Furthermore, the NK2 selective agonist [Lys3, Gly8,-R-gamma-lactam-Leu9]-NKA(3-10) (GR64349) was active only at the highest concentrations tested (greater than 10 microM), and the NK3 selective agonist, succ-[Asp6,N-MePhe8]-SP(6-11) (senktide) was essentially inactive (10 nM-32 microM). 3. [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP(1-11) antagonized responses to neurokinin A, neurokinin B, physalaemin, eledoisin, [Glp6,D-Pro9]-SP(6-11), GR73632 and GR64349 (apparent pKB s 5.6-6.2), but was less potent in antagonizing responses to substance P, substance P methyl ester and [Glp6,L-Pro9]-SP(6-11) (apparent pKB s less than or equal to 5.0-5.0). 4. In contrast, the recently developed NK1-selective receptor antagonist [D-Pro9[Spiro-gamma-lactam]Leu10,Trp11]-SP(1-11) (GR71251) did not produce agonist-dependent pKB estimates. Schild plot analysis indicated a competitive interaction with a single receptor population where the antagonist had an estimated overall pKB of 7.58 +/- 0.13 for the four agonists of differing subtype selectivity tested (GR73632, GR64349, substance P methyl ester and neurokinin B).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1707714

Effect of tachykinins on the need-free sodium intake of female rats: a continuous intracerebroventricular infusion study.

PubMed

Polidori, C; Ciccocioppo, R; Epstein, A N; de Caro, G; Massi, M

1994-11-01

The present study investigated the effect of 24-h continuous ICV infusion of four different tachykinins on the enhanced need-free sodium intake induced by previous repeated sodium depletions in female rats. Female rats were employed because, in response to sodium depletions, they develop a higher need-free sodium intake than male rats. The following tachykinins were used: eledoisin, substance P (SP), [Sar9,Met(O2)11]SP and [Asp5,6,MePhe8]SP(5-11), also referred to as NH2-senktide, all at the same doses of 300 or 600 ng/h x 24 h. Food pellets, water, and 3% NaCl sodium solution were freely available. Eledoisin and NH2-senktide were more potent than SP in reducing the need-free sodium intake. On the other hand, [Sar9,Met(O2)11]SP had no effect. None of the tachykinins employed completely blocked the intake. Water intake was reduced, but this reduction was apparently a consequence of reduced intake of hypertonic sodium chloride solution, because at the same doses TKs did not inhibit water intake in a single-bottle test. Food intake remained unchanged at either dose used. These findings confirm previous studies in which pulse injection of the same drugs potently inhibited sodium intake. They also demonstrate that tachykinins endowed with high affinity for the NK3 receptor are the most potent in inhibiting sodium intake. Furthermore, these findings indicate that the tachykinins reduce the need-free sodium intake only during the infusion period, indicating that in these conditions they do not evoke either aversion for salt, or toxic consequences in the follow-up period.

Receptor Binding Sites for Substance P, but not Substance K or Neuromedin K, are Expressed in High Concentrations by Arterioles, Venules, and Lymph Nodules in Surgical Specimens Obtained from Patients with Ulcerative Colitis and Crohn Disease

NASA Astrophysics Data System (ADS)

Mantyh, Christopher R.; Gates, Troy S.; Zimmerman, Robert P.; Welton, Mark L.; Passaro, Edward P.; Vigna, Steven R.; Maggio, John E.; Kruger, Lawrence; Mantyh, Patrick W.

1988-05-01

Several lines of evidence indicate that tachykinin neuropeptides [substance P (SP), substance K (SK), and neuromedin K (NK)] play a role in regulating the inflammatory and immune responses. To test this hypothesis in a human inflammatory disease, quantitative receptor autoradiography was used to examine possible abnormalities in tachykinin binding sites in surgical specimens from patients with inflammatory bowel disease. Surgical specimens of colon were obtained from patients with ulcerative colitis (n = 4) and Crohn disease (n = 4). Normal tissue was obtained from uninvolved areas of extensive resections for carcinoma (n = 6). In all cases, specimens were obtained <5 min after removal to minimize influences associated with degradation artifacts and were processed for quantitative receptor autoradiography by using 125I-labeled Bolton--Hunter conjugates of NK, SK, and SP. In the normal colon a low concentration of SP receptor binding sites is expressed by submucosal arterioles and venules and a moderate concentration is expressed by the external circular muscle, whereas SK receptor binding sites are expressed in low concentrations by the external circular and longitudinal muscle. In contrast, specific NK binding sites were not observed in any area of the human colon. In colon tissue obtained from ulcerative colitis and Crohn disease patients, however, very high concentrations of SP receptor binding sites are expressed by arterioles and venules located in the submucosa, muscularis mucosa, external circular muscle, external longitudinal muscle, and serosa. In addition, very high concentrations of SP receptor binding sites are expressed within the germinal center of lymph nodules, whereas the concentrations of SP and SK binding sites expressed by the external muscle layers are not altered significantly. These results demonstrate that receptor binding sites for SP, but not SK or NK, are ectopically expressed in high concentrations (1000-2000 times normal) by cells involved in mediating inflammatory and immune responses. These data suggest that SP may be involved in the pathophysiology of inflammatory bowel disease and might provide some insight into the interaction between the nervous system and the regulation of inflammation and the immune response in human inflammatory disease.

Cardiovascular and behavioural effects of centrally administered neuropeptide K in the rat: receptor characterization.

PubMed Central

Prat, A.; Picard, P.; Couture, R.

1994-01-01

1. The cardiovascular and behavioural responses to intracerebroventricularly (i.c.v.) administered neuropeptide K (NPK) were studied in conscious rats. The central effects of NPK were characterized by pretreatment (i.c.v.) with selective antagonists for the NK1 ((+/-)-CP 96345 and RP 67580), NK2 (SR 48968) and NK3 (R 487) receptors. 2. NPK (10-65 pmol) induced tachycardia and dose-dependent increases of mean arterial blood pressure. The cardiovascular responses reached a maximum within 3 min post-injection and lasted for more than 1 h. Concurrently, NPK produced dose-dependent increases of face washing, head scratching, grooming, walking and wet dog shakes. 3. A desensitization of most of the behavioural responses (except head scratching) but not of the cardiovascular response was shown when two consecutive injections of 25 pmol NPK were given 24 h apart. 4. Both the cardiovascular and behavioural responses (except the head scratching) to 25 pmol NPK were blocked by pre-administration (i.c.v.) of 6.5 nmol (+/-)-CP 96345 or RP 67580 given 5 min earlier. No inhibition of NPK responses was observed when 6.5 nmol SR 48968 or R 487 were used in a similar study. Additionally, NPK effects were significantly reduced 24 h after the prior injection of (+/-)-CP 96345 but not of RP 67580. 5. These results support the involvement of NK1 receptors in the cardiovascular and behavioural effects of i.c.v. NPK. Thus, this peptide may play a putative role in central cardiovascular regulation as it is the most potent endogenous tachykinin described centrally, to date. PMID:7518305

Airway extravasation induced by increasing airway temperature in ovalbumin-sensitized rats

PubMed Central

Hsu, Chun-Chun; Tapia, Reyno J.; Lee, Lu-Yuan

2015-01-01

This study was carried out to determine whether hyperventilation of humidified warm air (HWA) induced airway extravasation in ovalbumin (Ova)-sensitized rats. Our results showed: 1) After isocapnic hyperventilation with HWA for 2 min, tracheal temperature (Ttr) was increased to 40.3°C, and the Evans blue contents in major airways and lung tissue were elevated to 651% and 707%, respectively, of that after hyperventilation with humidified room air in Ova-sensitized rats; this striking effect of HWA was absent in control rats. 2) The HWA-induced increase in Evans blue content in sensitized rats was completely prevented by a pretreatment with either L-732138, a selective antagonist of neurokinin type 1 (NK-1) receptor, or formoterol, a selective agonist of β2 adrenoceptor. This study demonstrated that an increase in airway temperature induced protein extravasation in the major airways and lung tissue of sensitized rats, and an activation of the NK-1 receptor by tachykinins released from bronchopulmonary C-fiber nerve endings was primarily responsible. PMID:25864799

Expression of neurokinin B/NK3 receptor and kisspeptin/KISS1 receptor in human granulosa cells.

PubMed

García-Ortega, J; Pinto, F M; Fernández-Sánchez, M; Prados, N; Cejudo-Román, A; Almeida, T A; Hernández, M; Romero, M; Tena-Sempere, M; Candenas, L

2014-12-01

Are neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells? The NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells. The NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their well-recognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown. This study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. The samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus-oocyte complexes were collected after transvaginal ultrasound-guided oocyte retrieval. RT-PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca(2+) levels, [Ca(2+)]i, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured. NKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca(2+)]i in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca(2+)]i. However, the [Ca(2+)]i response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB. The granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells. Our data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells. This work was supported by grants from Ministerio de Economía y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andalucía (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis.

PubMed

Holst, B; Hastrup, H; Raffetseder, U; Martini, L; Schwartz, T W

2001-06-08

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.

Comparative, general pharmacology of SDZ NKT 343, a novel, selective NK1 receptor antagonist

PubMed Central

Walpole, C S J; Brown, M C S; James, I F; Campbell, E A; McIntyre, P; Docherty, R; Ko, S; Hedley, L; Ewan, S; Buchheit, K-H; Urban, L A

1998-01-01

The in vitro and in vivo pharmacology of SDZ NKT 343 (2-nitrophenyl-carbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benzyl-N-methylamide), a novel tachykinin NK1 receptor antagonist was investigated.SDZ NKT 343 inhibited [3H]-substance P binding to the human NK1 receptor in transfected Cos-7 cell membranes (IC50=0.62±0.11 nM). In comparison, in the same assay Ki values for FK888, CP 99,994, SR 140,333 and RPR 100,893 were 2.13±0.04 nM, 0.96±0.20 nM, 0.15±0.06 nM and 1.77±0.41 nM, respectively. SDZ NKT 343 showed a markedly lower affinity at rat NK1 receptors in whole forebrain membranes (IC50=451±139 nM).SDZ NKT 343 caused an increase in EC50 as well as reduction in the number of binding sites (Bmax) determined for [3H]-substance P, suggesting a non-competitive interaction at the human NK1 receptor. SDZ NKT 343 also caused a reduction in the maximum elevation of [Ca2+]i evoked by substance P (SP) in human U373MG cells and depressed the maximum [Sar9]SP sulphone-induced contraction of the guinea-pig isolated ileum. The antagonism of SP effects on U373MG cells by SDZ NKT 343 was reversible.SDZ NKT 343 showed weak affinity to human NK2 and NK3 receptors in transfected Cos-7 cells (Ki of 0.52±0.04 μM and 3.4±1.2 μM, respectively). SDZ NKT 343 was inactive in a broad array of binding assays including the bradykinin B2 receptor the histamine H1 receptor, opiate receptors and adrenoceptors. SDZ NKT 343 only weakly inhibited the voltage-activated Ca2+ and Na+currents in guinea-pig dorsal root ganglion neurones. The enantiomer of SDZ NKT 343, (R,R)-SDZ NKT 343 was about 1000 times less active at human NK1 receptors expressed in Cos-7 cell membranes.Contractions of the guinea-pig ileum by [Sar9]SP sulphone were inhibited by SDZ NKT 343 in a concentration-dependent manner, with an IC50=1.60±0.94 nM, while the enantiomer (R,R)-SDZ NKT 343 was 100 times less active (IC50=162±26 nM). In comparison, in the same assay IC50 values for other NK1 receptor antagonists CP 99,994, SR 140,333, RPR 100,893 and FK 888 were 2.90±07 nM, 0.14±0.02 nM, 11.4±2.9 nM and 2.4±0.83 nM, respectively.In anaesthetized guinea-pigs i.v. administered SDZ NKT 343 antagonized [Sar9]SP sulphone-evoked bronchoconstriction (70% reduction at 0.4 mg kg−1, i.v.). Basal airway resistance, mean arterial blood pressure and heart rate were not affected.In conclusion, SDZ NKT 343 is a highly selective NK1 receptor antagonist with high potency at the human and guinea-pig receptors. SDZ NKT 343 may be used as a potential novel therapeutic agent in human diseases where NK1 receptor hyperfunction is involved. PMID:9630347

Roles of neuronal NK1 and NK3 receptors in synaptic transmission during motility reflexes in the guinea-pig ileum

PubMed Central

Johnson, P J; Bornstein, J C; Burcher, E

1998-01-01

The role of NK1 and NK3 receptors in synaptic transmission between myenteric neurons during motility reflexes in the guinea-pig ileum was investigated by recording intracellularly the reflex responses of the circular muscle to distension or compression of the mucosal villi. Experiments were performed in a three-chambered organ bath that enabled drugs to be selectively applied to different sites along the reflex pathways.When applied in the recording chamber, an NK1 receptor antagonist, SR140333 (100 nM), reduced by 40–50% the amplitudes of inhibitory junction potentials (i.j.ps) evoked in the circular muscle by activation of descending reflex pathways. This effect was abolished when synaptic transmission in the stimulus region was blocked with physiological saline containing 0.1 mM Ca2+ plus 10 mM Mg2+, leaving only the component of the descending reflex pathway conducted via long anally directed collaterals of intrinsic sensory neurons.SR140333 (100 nM) had no effect on descending reflex i.j.ps when applied to the stimulus region. Ascending reflexes were also unaffected by SR140333 in the stimulus region or between the stimulus and recording sites.Septide (10 nM), an NK1 receptor agonist, enhanced descending reflexes by 30–60% when in the recording chamber. [Sar9,Met(O2)11]substance P had no effect at 10 nM, but potentiated distension-evoked reflexes at 100 nM.A selective NK3 receptor antagonist, SR142801 (100 nM), when applied to the stimulus region, reduced the amplitude of descending reflex responses to compression by 40%, but had no effect on responses to distension. SR142801 (100 nM) had no effect when applied to other regions of the descending reflex pathways.SR142801 (100 nM) only inhibited ascending reflexes when applied at the recording site. However, after nicotinic transmission in the stimulus region was blocked, SR142801 (100 nM) at this site reduced responses to compression.Contractions of the circular muscle of isolated rings of ileum evoked by low concentrations of septide, but not [Sar9,Met(O2)11]substance P, were potentiated by tetrodotoxin (300 nM).Contractile responses evoked by an NK3 receptor agonist, senktide, were non-competitively inhibited by SR142801. After excitatory neuromuscular transmission was blocked, senktide produced inhibitory responses that were also antagonised by SR142801, but to a lesser extent and in an apparently competitive manner.These results indicate that tachykinins acting via NK1 receptors partly mediate transmission to inhibitory motor neurons. NK3 receptors play a role in transmission from intrinsic sensory neurons and from ascending interneurons to excitatory motor neurons during motility reflexes. PMID:9723948

Non-specific actions of the non-peptide tachykinin receptor antagonists, CP-96,345, RP 67580 and SR 48968, on neurotransmission.

PubMed Central

Wang, Z. Y.; Tung, S. R.; Strichartz, G. R.; Håkanson, R.

1994-01-01

1. Three non-peptide tachykinin receptor antagonists, CP-96,345, RP 67580 and SR 48968, were found to inhibit the electrically-evoked, tachykinin-mediated contractile responses of the rabbit iris sphincter in a concentration-dependent fashion; the pIC50 values were 5.6 +/- 0.01, 5.4 +/- 0.07 and 4.8 +/- 0.03, respectively. 2. These antagonists also inhibited the electrically-evoked, parasympathetic response of the rabbit iris sphincter and the sympathetic response of the guinea-pig vas deferens in a concentration-dependent manner; the pIC50 values were 0.3-1.2 log units lower than those recorded for the tachykinin-mediated responses. 3. Two local anaesthetics, bupivacaine and oxybuprocaine, were also found to inhibit the tachykinin-mediated, cholinergic and sympathetic contractile responses in these tissues in a concentration-dependent manner; the concentration ranges for producing the inhibition were similar to those of the non-peptide tachykinin receptor antagonists. 4. On the sciatic nerves of frogs, the tachykinin receptor antagonists inhibited action potentials in a concentration-dependent manner; the potency of the three drugs was similar to that of bupivacaine. 5. Our results suggest that, in addition to blocking tachykinin receptors, the non-peptide tachykinin receptor antagonists, CP-96,345, RP 67580 and SR 48968, may exert non-specific inhibitory effects on neurotransmission. PMID:8012694

Substance P and the Neurokinin-1 Receptor: The New CRF.

PubMed

Schank, Jesse R; Heilig, Markus

2017-01-01

Substance P (SP) is an 11-amino acid neuropeptide of the tachykinin family that preferentially activates the neurokinin-1 receptor (NK1R). First isolated 85 years ago and sequenced 40 years later, SP has been extensively studied. Early studies identified a role for SP and the NK1R in contraction of intestinal smooth muscle, central pain processing, and neurogenic inflammation. An FDA-approved NK1R antagonist, aprepitant, is used clinically for the treatment of chemotherapy-induced nausea, as the NK1R influences the activity of the brain stem emesis centers. More recently, SP and the NK1R have gained attention for their role in complex psychiatric processes including stress, anxiety, and depression. However, clinical development of NK1R antagonists for these indications has so far been unsuccessful. Several preclinical studies have also demonstrated a role of the NK1R in drug taking and drug seeking, especially as it relates to escalated consumption and stress-elicited seeking. This line of research developed in parallel with findings supporting a role of corticotropin-releasing factor (CRF) in stress-induced drug seeking. Over this time, CRF arguably gained more attention as a target for development of addiction pharmacotherapies. However, this effort has not resulted in a viable drug for use in human populations. Given promising clinical findings for the efficacy of NK1R antagonists on craving in alcoholics, along with recent data suggesting that a number of negative results from NK1R trials were likely due to insufficient receptor occupancy, the NK1R merits being revisited as a target for the development of novel pharmacotherapeutics for addiction. © 2017 Elsevier Inc. All rights reserved.

Structure-activity studies of bufokinin, substance P and their C-terminal fragments at bufokinin receptors in the small intestine of the cane toad, Bufo marinus.

PubMed

Liu, Lu; Murray, Michael; Burcher, Elizabeth

2002-01-15

Bufokinin is a substance P-related tachykinin peptide with potent spasmogenic actions, isolated from the intestine of the cane toad, Bufo marinus. Bufokinin acts via a tachykinin receptor with similarities to the mammalian NK(1) receptor. In this structure-activity study of bufokinin, substance P (SP) and their C-terminal fragments, we have used isolated segments and homogenates of toad small intestine to compare the contractile potencies and abilities to compete for the binding of [125I]-Bolton-Hunter bufokinin. In general, potency was very similar in both studies (r=0.956) and was primarily related to peptide length, with the natural undecapeptide tachykinins bufokinin - ranakinin>SP- cod SP -trout SP being most potent. The weakest peptides were [Pro(9)]SP, BUF(7-11) and SP(7-11). Bufokinin fragments (BUF) were approximately equipotent to the corresponding SP fragments, with only BUF(5-11) showing unexpectedly low binding affinity. Data obtained with SP, bufokinin and fragments were subjected to quantitative structure--activity (QSAR) analysis which demonstrated that molecular connectivity and shape descriptors yielded significant regression equations (r approximately 0.90). The predictive capacity of the equations was confirmed using ranakinin, trout SP and cod SP, but not using the synthetic analogs [Pro(9)]SP and [Sar(9)]SP. The study suggests that the full undecapeptide sequence of bufokinin is required for optimal activity, with high potency conferred by Lys(1), Pro(2), Gly(9) and probably Tyr(8). The finding that receptor-ligand interactions were correlated with the shape descriptor 2kappa(alpha) and favored by basic and rigid residues at position 1-3 is consistent with an important role of conformation at the N-terminus of bufokinin.

PepPat, a pattern-based oligopeptide homology search method and the identification of a novel tachykinin-like peptide.

PubMed

Jiang, Ying; Gao, Ge; Fang, Gang; Gustafson, Eric L; Laverty, Maureen; Yin, Yanbin; Zhang, Yong; Luo, Jingchu; Greene, Jonathan R; Bayne, Marvin L; Hedrick, Joseph A; Murgolo, Nicholas J

2003-05-01

PepPat, a hybrid method that combines pattern matching with similarity scoring, is described. We also report PepPat's application in the identification of a novel tachykinin-like peptide. PepPat takes as input a query peptide and a user-specified regular expression pattern within the peptide. It first performs a database pattern match and then ranks candidates on the basis of their similarity to the query peptide. PepPat calculates similarity over the pattern spanning region, enhancing PepPat's sensitivity for short query peptides. PepPat can also search for a user-specified number of occurrences of a repeated pattern within the target sequence. We illustrate PepPat's application in short peptide ligand mining. As a validation example, we report the identification of a novel tachykinin-like peptide, C14TKL-1, and show it is an NK1 (neuokinin receptor 1) agonist whose message is widely expressed in human periphery. PepPat is offered online at: http://peppat.cbi.pku.edu.cn.

Tachykininergic slow depolarization of motoneurones evoked by descending fibres in the neonatal rat spinal cord.

PubMed Central

Kurihara, T; Yoshioka, K; Otsuka, M

1995-01-01

1. In the isolated spinal cord of the neonatal rat, repetitive electrical stimulation of the upper cervical region elicited a prolonged depolarization of lumbar motoneurones (L3-5) lasting 1-2 min, which was recorded extracellularly from ventral roots, or intracellularly. 2. This depolarizing response was markedly depressed by the excitatory amino acid receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (D-APV, 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). The remaining response was further depressed by a 5-hydroxytryptamine (5-HT) receptor antagonist, ketanserin (3 microM). 3. In the presence of these antagonists, a small part of the depolarizing response of slow time course remained, and this response was partially blocked by the tachykinin NK1 receptor antagonists GR71251 (0.3-5 microM) and RP67580 (0.3-1 microM). In contrast, RP68651 (0.3-1 microM), the inactive enantiomer of RP67580, had no effect on the depolarizing response. 4. The slow depolarizing response in the presence of D-APV, CNQX and ketanserin was markedly potentiated by a peptidase inhibitor, thiorphan (1 microM). 5. This descending fibre-evoked slow depolarization became smaller after prolonged treatment (5-7 h) with 5,7-dihydroxytryptamine (10 microM), a neurotoxin for 5-HT neurones. Under such conditions, the effects of thiorphan and GR71251 on the slow depolarization were virtually absent. 6. Under the action of D-APV, CNQX and ketanserin, applications of tachykinins, substance P and neurokinin A produced depolarizing responses of lumbar motoneurones, and the responses were depressed by GR71251 and potentiated by thiorphan. 7. These results suggest that tachykinins contained in serotonergic fibres serve as neurotransmitters mediating the descending fibre-evoked slow excitatory postsynaptic potentials in motoneurones. PMID:7562617

Phosphoramidon potentiates mammalian tachykinin-induced biting, licking and scratching behaviour in mice.

PubMed

Sakurada, T; Tan-No, K; Yamada, T; Sakurada, S; Kisara, K

1990-12-01

The effects of peptidase inhibitors were examined upon behavioural responses including scratch, bite and lick produced by intrathecal (IT) injection of substance P (SP) and neurokinin A (NK A) in mice. Phosphoramidon (0.002-2.0 nmol), an endopeptidase-24.11 inhibitor, simultaneously injected with SP or NK A, remarkably enhanced and prolonged SP- or NK A-induced behavioural response in a dose-dependent manner. The behavioural response to SP was significantly increased by 2.0 nmol of bestatin, an aminopeptidase inhibitor, but not by 1.0 nmol. Captopril, an angiotensin-converting enzyme inhibitor, was without effect on both tachykinin-induced responses. When phosphoramidon was injected together with bestatin and captopril which have no significant effect alone, SP- or NK A-induced behavioral response was significantly increased. These data suggest that endopeptidase-24.11 may be an important enzyme responsible for terminating of SP- or NK A-induced behavioral response at the spinal cord level.

Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission.

PubMed Central

Maggi, C. A.; Patacchini, R.; Santicioli, P.; Giuliani, S.

1991-01-01

1. The possible involvement of tachykinins (TKs) in the contraction produced by capsaicin in the rat isolated urinary bladder was addressed on the hypothesis that co-release of substance P (SP) and neurokinin A (NKA) occurs from sensory nerve terminals. 2. A low concentration of SP (30 nM) produced a rapid contraction which faded to baseline within 10 min. A low concentration of NKA (10 nM) produced a slowly developing contraction which was still evident at 10 min. Capsaicin (1 microM) produced a rapid phasic response and a tonic response (late response to capsaicin). Co-administration of SP and NKA mimicked the response to capsaicin more than each TK alone. 3. Fading of the response to SP was not caused by receptor desensitization and was partially prevented by peptidase inhibitors. 4. Spantide (3 microM) selectively antagonized the SP-induced contraction while L-659,877 (3-10 microM) or MEN 10,376 (10-30 microM) which are NK2 receptor selective antagonists selectively blocked the response to NKA. Co-administration of spantide and L-659,877 inhibited the response to both SP and NKA by an amount not greater than that produced by each antagonist alone. 5. Spantide selectively reduced the peak response to capsaicin, while leaving the late response unaffected. L-659,877 (3 microM) and MEN 10,376 (10 microM) selectively inhibited the late response to capsaicin while, at higher concentrations, also reduced the peak response to capsaicin. Co-administration of spantide and L-659,877 reduced the peak response to capsaicin more than that produced by each antagonist alone. 6. Bombesin (10 nM) produced a tonic contraction similar to that induced by NKA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1715797

Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells.

PubMed

Wan, Ying; Meng, Fanyin; Wu, Nan; Zhou, Tianhao; Venter, Julie; Francis, Heather; Kennedy, Lindsey; Glaser, Trenton; Bernuzzi, Francesca; Invernizzi, Pietro; Glaser, Shannon; Huang, Qiaobing; Alpini, Gianfranco

2017-08-01

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct-ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R -/- ) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2 -/- ) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2 -/- mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2 -/- mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R -/- mice with BDL surgery or Mdr2 -/- mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528-541). © 2017 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Involvement of tumour necrosis factor-α in Clostridium perfringens β-toxin-induced plasma extravasation in mice

PubMed Central

Nagahama, M; Kihara, A; Kintoh, H; Oda, M; Sakurai, J

2008-01-01

Background and purpose: Clostridium perfringens beta-toxin, an important agent of necrotic enteritis, causes plasma extravasation due to the release of a tachykinin NK1 receptor agonist in mouse skin. In this study, we investigated the role of cytokines in beta-toxin-induced plasma extravasation. Experimental approach: Male Balb/c, C3H/HeN and C3H/HeJ mice were anaesthetized with pentobarbitone and beta-toxin was injected i.d. into shaved dorsal skin. SR140333, capsaicin, chlorpromazine and pentoxifylline were given as pretreatment when required before the injection of the toxin. Cytokines in the dorsal skin were measured by ELISA. Key results: Injection (i.d.) of beta-toxin induced a dose-dependent increase in dermal TNF-α and interleukin (IL)-1β levels with a concomitant increase in plasma extravasation, but not the release of IL-6. SR140333 and capsaicin significantly inhibited the toxin-induced release of TNF-α and IL-1β. The plasma extravasation and the release of TNF-α induced by beta-toxin were significantly inhibited by chlorpromazine and pentoxifylline which inhibit the release of TNF-α. The toxin-induced plasma extravasation in mouse skin was attenuated by pretreatment with a monoclonal antibody against TNF-α, but not anti-IL-1β. Furthermore, the toxin caused an increase in plasma extravasation in both C3H/HeN (TLR4-intact) and C3H/HeJ (TLR4-deficient) mice. In C3H/HeN mice, the toxin-induced leakage was not inhibited by pretreatment with anti-TLR4/MD-2 antibody. Conclusions and implications: These observations show that beta-toxin-induced plasma extravasation in mouse skin is related to the release of TNF-α via the mechanism involving tachykinin NK1 receptors, but not via TLR4. PMID:18264118

Expression of the neurokinin type 1 receptor in the human colon.

PubMed

Boutaghou-Cherid, Hikma; Porcher, Christophe; Liberge, Martine; Jule, Yvon; Bunnett, Nigel W; Christen, Marie-Odile

2006-01-30

The distribution of the neurokinin type 1 receptor (NK1r) in human intestine, mapped in a few immunohistochemical investigations in the antrum and the duodenum, is comparable to that widely studied in rodents. Importantly, despite pharmacological evidence of their presence in mammalian intestinal muscle, their immunohistochemical visualization in smooth muscle cells remains to be determined in human digestive tract. In the present work, we studied the distribution of NK1r in the human colon, with a particular view to visualize their expression in muscle cells. With this aim, part of colonic segments were incubated with nicardipine and TTX in order to induce accumulation of the NK1r on cell membrane. NK1r were visualized by using immunohistochemistry combined with fluorescence and confocal microscopy. Without incubation, NK1r-IR was clearly observed on the membrane and the cytoplasm of myenteric and submucous neurons and interstitial cells of Cajal, but could not be clearly determined in the longitudinal and circular muscle. NK1r-IR-expressing neurons and interstitial cells were closely surrounded by substance P (SP) immunoreactive nerves. Incubation of colonic segments with nicardipine and TTX at 4 degrees C for 1 h with SP allowed to reveal a strong NK1r-IR at the surface of muscle cells. Incubation with SP (10(-6) M) at 37 degrees C for 1 min induced a relocation of NK1r-IR into the cytoplasm of muscle. This is interpreted as an internalization of NK1r induced by the binding of SP on muscular NK1r. The present data contribute to emphasize the role of NK1r in tachykinin-mediated neuronal processes regulating intestinal motility.

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways*

PubMed Central

Lecat, Sandra; Matthes, Hans W.D.; Pepperkok, Rainer; Simpson, Jeremy C.; Galzi, Jean-Luc

2015-01-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

PubMed

Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

2015-05-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A novel activity for substance P: stimulation of peroxisome proliferator-activated receptor-γ protein expression in human monocytes and macrophages

PubMed Central

Amoruso, A; Bardelli, C; Gunella, G; Ribichini, F; Brunelleschi, S

2008-01-01

Background and purpose: Substance P (SP) and peroxisome proliferator-activated receptor-γ (PPAR-γ) play important roles in different inflammatory conditions and are both expressed in human monocytes and macrophages. However, it is not known whether or not they interact. This study was undertaken to evaluate the effects of SP on PPAR-γ protein expression in monocytes and macrophages (MDMs: monocyte-derived macrophages) from healthy smokers and non-smokers. Experimental approach: PPAR-γ protein was detected by western blot and quantified by calculating the ratio between PPAR-γ and β-actin protein expression. Constitutive tachykinin NK1 receptor expression in monocytes and MDMs from healthy smokers and non-smokers was evaluated by western blot. Cytokine release was evaluated by ELISA. Key results: In the concentration range 10−10–10−6 M, SP stimulated PPAR-γ protein expression in monocytes and MDMs, being more effective in cells from healthy smokers. Moreover, in these cells there was a constitutively increased expression of NK1 receptors. SP-induced expression of the PPAR-γ protein was receptor-mediated, as it was reproduced by the NK1 selective agonist [Sar9Met(O2)11]SP and reversed by the competitive NK1 antagonist GR71251. SP-induced maximal effects were similar to those evoked by 15-deoxy-Δ12,14-prostaglandin J2; an endogenous PPAR-γ agonist, and were significantly reduced by a PPAR-γ antagonist. NK1 and PPAR-γ agonists exerted opposite effects on TNF-α release from monocytes and MDMs. Conclusions and implications: Enhancement of PPAR-γ protein expression represents a novel activity for SP, which could contribute to a range of chronic inflammatory disorders. PMID:18278062

The relationship between the agonist-induced activation and desensitization of the human tachykinin NK2 receptor expressed in Xenopus oocytes

PubMed Central

Maudsley, S; Gent, J P; Findlay, J B C; Donnelly, D

1998-01-01

Repeated applications of neurokinin A (NKA) to oocytes injected with 25 ng wild-type hNK2 receptor cRNA caused complete attenuation of second and subsequent NKA-induced responses while analogous experiments using repeated applications of GR64349 and [Nle10]NKA(4–10) resulted in no such desensitization. This behaviour has been previously attributed to the ability of the different ligands to stabilize different active conformations of the receptor that have differing susceptibilities to receptor kinases (Nemeth & Chollet, 1995).However, for Xenopus oocytes injected (into the nucleus) with 10 ng wild-type hNK2 receptor cDNA, a single 100 nM concentration of any of the three ligands resulted in complete desensitization to further concentrations.On the other hand, none of the ligands caused any desensitization in oocytes injected with 0.25 ng wild-type hNK2 receptor cRNA, even at concentrations up to 10 μM.The two N-terminally truncated analogues of neurokinin A have a lower efficacy than NKA and it is likely that it is this property which causes the observed differences in desensitization, rather than the formation of alternative active states of the receptor.The peak calcium-dependent chloride current is not a reliable measure of maximal receptor stimulation and efficacy is better measured in this system by studying agonist-induced desensitization.The specific adenylyl cyclase inhibitor SQ22536 can enhance NKA and GR64349-mediated desensitization which suggests that agonist-induced desensitization involves the inhibition of adenylyl cyclase and the subsequent down-regulation of the cyclic AMP-dependent protein kinase, possibly by cross-talk to a second signalling pathway. PMID:9690859

Role of tachykinins in bronchial hyper-responsiveness.

PubMed

Reynolds, P N; Holmes, M D; Scicchitano, R

1997-01-01

1. Sensory afferent fibres mediate important protective reflexes in the lung. Small, unmyelinated C-fibre nerves have both sensory afferent and effector functions. C-fibres contain a number of neuropeptides, including the tachykinins, which have pro-inflammatory effects in the airways. Following stimulation with capsaicin and other stimuli, neuropeptides are released from the nerve endings, either directly or by axonal reflexes. 2. Important tachykinin effects include smooth muscle contraction, vasodilatation and oedema, mucus secretion and inflammatory cell activation. There are also trophic effects, including proliferation of fibroblasts, smooth muscle and epithelial cells. 3. Tachykinins mediate their effects by binding to G-proteinlinked receptors. Receptor-specific agonists and antagonists are available, which have helped clarify the effects of tachykinins. These agents may have therapeutic potential. 4. Tachykinins are degraded by the enzyme neutral endo-peptidase. 5. Studies in humans in vivo show an increase in airways resistance following challenge with tachykinins. There is some evidence for an increase in tachykinins and their receptors in airway inflammation, but this has not been found in all studies. A reduction in neutral endopeptidase has been seen in some animal models of airway inflammation, but this has not been shown in human disease. 6. Trials of tachykinin receptor antagonists in human asthma have begun, but it is too early to say what their therapeutic impact will be.

Pharmacology of Bradykinin-Evoked Coughing in Guinea Pigs

PubMed Central

Hewitt, Matthew M.; Adams, Gregory; Mazzone, Stuart B.; Mori, Nanako; Yu, Li

2016-01-01

Bradykinin has been implicated as a mediator of the acute pathophysiological and inflammatory consequences of respiratory tract infections and in exacerbations of chronic diseases such as asthma. Bradykinin may also be a trigger for the coughing associated with these and other conditions. We have thus set out to evaluate the pharmacology of bradykinin-evoked coughing in guinea pigs. When inhaled, bradykinin induced paroxysmal coughing that was abolished by the bradykinin B2 receptor antagonist HOE 140. These cough responses rapidly desensitized, consistent with reports of B2 receptor desensitization. Bradykinin-evoked cough was potentiated by inhibition of both neutral endopeptidase and angiotensin-converting enzyme (with thiorphan and captopril, respectively), but was largely unaffected by muscarinic or thromboxane receptor blockade (atropine and ICI 192605), cyclooxygenase, or nitric oxide synthase inhibition (meclofenamic acid and NG-nitro-L-arginine). Calcium influx studies in bronchopulmonary vagal afferent neurons dissociated from vagal sensory ganglia indicated that the tachykinin-containing C-fibers arising from the jugular ganglia mediate bradykinin-evoked coughing. Also implicating the jugular C-fibers was the observation that simultaneous blockade of neurokinin2 (NK2; SR48968) and NK3 (SR142801 or SB223412) receptors nearly abolished the bradykinin-evoked cough responses. The data suggest that bradykinin induces coughing in guinea pigs by activating B2 receptors on bronchopulmonary C-fibers. We speculate that therapeutics targeting the actions of bradykinin may prove useful in the treatment of cough. PMID:27000801

Inhibitors of endopeptidase and angiotensin-converting enzyme lead to an amplification of the morphological changes and an upregulation of the substance P system in a muscle overuse model.

PubMed

Song, Yafeng; Stål, Per S; Yu, Ji-Guo; Lorentzon, Ronny; Backman, Clas; Forsgren, Sture

2014-04-11

We have previously observed, in studies on an experimental overuse model, that the tachykinin system may be involved in the processes of muscle inflammation (myositis) and other muscle tissue alterations. To further evaluate the significance of tachykinins in these processes, we have used inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), substances which are known to terminate the activity of various endogenously produced substances, including tachykinins. Injections of inhibitors of NEP and ACE, as well as the tachykinin substance P (SP), were given locally outside the tendon of the triceps surae muscle of rabbits subjected to marked overuse of this muscle. A control group was given NaCl injections. Evaluations were made at 1 week, a timepoint of overuse when only mild inflammation and limited changes in the muscle structure are noted in animals not treated with inhibitors. Both the soleus and gastrocnemius muscles were examined morphologically and with immunohistochemistry and enzyme immunoassay (EIA). A pronounced inflammation (myositis) and changes in the muscle fiber morphology, including muscle fiber necrosis, occurred in the overused muscles of animals given NEP and ACE inhibitors. The morphological changes were clearly more prominent than for animals subjected to overuse and NaCl injections (NaCl group). A marked SP-like expression, as well as a marked expression of the neurokinin-1 receptor (NK-1R) was found in the affected muscle tissue in response to injections of NEP and ACE inhibitors. The concentration of SP in the muscles was also higher than that for the NaCl group. The observations show that the local injections of NEP and ACE inhibitors led to marked SP-like and NK-1R immunoreactions, increased SP concentrations, and an amplification of the morphological changes in the tissue. The injections of the inhibitors thus led to a more marked myositis process and an upregulation of the SP system. Endogenously produced substances, out of which the tachykinins conform to one substance family, may play a role in mediating effects in the tissue in a muscle that is subjected to pronounced overuse.

Amygdala-Dependent Molecular Mechanisms of the Tac2 Pathway in Fear Learning

PubMed Central

Andero, Raül; Daniel, Sarah; Guo, Ji-Dong; Bruner, Robert C; Seth, Shivani; Marvar, Paul J; Rainnie, Donald; Ressler, Kerry J

2016-01-01

Recently we determined that activation of the tachykinin 2 (Tac2) pathway in the central amygdala (CeA) is necessary and sufficient for the modulation of fear memories. The Tac2 pathway includes the Tac2 gene, which encodes the neuropeptide neurokinin B and its corresponding receptor neurokinin 3 receptor (NK3R). In this study, using Tac2–cre and Tac2–GFP mice, we applied a combination of in vivo (optogenetics) and multiple in vitro techniques to further explore the mechanisms of action within the Tac2 pathway. In transgenic mice that express ChR2 solely in Tac2 neurons, in vivo optogenetic stimulation of CeA Tac2-expressing neurons during fear acquisition enhanced fear memory consolidation and drove action potential firing in vitro. In addition, Tac2–CeA neurons were shown to co-express striatal-enriched protein tyrosine phosphatase, which may have an important role in regulating Nk3R signaling during fear conditioning. These data extend our current understanding for the underlying mechanism(s) for the role of the Tac2 pathway in the regulation of fear memory, which may serve as a new therapeutic target in the treatment of fear-related disorders. PMID:27238620

C-terminal substance P fragments elicit histamine release from a murine mast cell line.

PubMed

Krumins, S A; Broomfield, C A

1993-01-01

Incubation of mouse mast cells with C-terminal substance P fragments in the micromolar range caused a release of histamine. Maximum release was observed with the tetrapeptide SP(8-11), followed by the tripeptide SP(9-11). SP(6-11) and SP(5-11) were nearly equipotent, while SP(4-11) caused only a slight histamine release. The substance P parent molecule and the N-terminal substance P fragments SP(1-4), SP(1-6) and SP(1-7) evoked no release of histamine. In confirmation of our previous findings, incubation with neurokinin A caused a release comparable to that of SP(8-11). Whereas neurokinin A-induced release was partially preventable by pretreating the cells with the NK2 receptor-selective antagonist cyclo(Gln-Trp-Phe-(R)Gly[ANC-2]Leu-Met), SP(8-11)-induced release was completely abolished by such treatment. The results provide the first evidence for the involvement of NK2 tachykinin receptors in the release of histamine by C-terminal substance P fragments.

Pharmacology of Bradykinin-Evoked Coughing in Guinea Pigs.

PubMed

Hewitt, Matthew M; Adams, Gregory; Mazzone, Stuart B; Mori, Nanako; Yu, Li; Canning, Brendan J

2016-06-01

Bradykinin has been implicated as a mediator of the acute pathophysiological and inflammatory consequences of respiratory tract infections and in exacerbations of chronic diseases such as asthma. Bradykinin may also be a trigger for the coughing associated with these and other conditions. We have thus set out to evaluate the pharmacology of bradykinin-evoked coughing in guinea pigs. When inhaled, bradykinin induced paroxysmal coughing that was abolished by the bradykinin B2 receptor antagonist HOE 140. These cough responses rapidly desensitized, consistent with reports of B2 receptor desensitization. Bradykinin-evoked cough was potentiated by inhibition of both neutral endopeptidase and angiotensin-converting enzyme (with thiorphan and captopril, respectively), but was largely unaffected by muscarinic or thromboxane receptor blockade (atropine and ICI 192605), cyclooxygenase, or nitric oxide synthase inhibition (meclofenamic acid and N(G)-nitro-L-arginine). Calcium influx studies in bronchopulmonary vagal afferent neurons dissociated from vagal sensory ganglia indicated that the tachykinin-containing C-fibers arising from the jugular ganglia mediate bradykinin-evoked coughing. Also implicating the jugular C-fibers was the observation that simultaneous blockade of neurokinin2 (NK2; SR48968) and NK3 (SR142801 or SB223412) receptors nearly abolished the bradykinin-evoked cough responses. The data suggest that bradykinin induces coughing in guinea pigs by activating B2 receptors on bronchopulmonary C-fibers. We speculate that therapeutics targeting the actions of bradykinin may prove useful in the treatment of cough. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

The Therapeutic Potential of Targeting Substance P/NK-1R Interactions in Inflammatory CNS Disorders

PubMed Central

Johnson, M. Brittany; Young, Ada D.; Marriott, Ian

2017-01-01

The inflammatory responses of resident central nervous system (CNS) cells are now known to play a critical role in the initiation and progression of an array of infectious and sterile neuroinflammatory disorders such as meningitis, encephalitis, Parkinson’s disease, Alzheimer’s disease and multiple sclerosis (MS). Regulating glial inflammatory responses in a timely manner is therefore critical in preserving normal CNS functions. The neuropeptide substance P is produced at high levels within the CNS and its selective receptor, the neurokinin 1 receptor (NK-1R), is abundantly expressed by neurons and is present on glial cell types including microglia and astrocytes. In addition to its functions as a neurotransmitter in the perception of pain and its essential role in gut motility, this tachykinin is widely recognized to exacerbate inflammation at peripheral sites including the skin, gastrointestinal tract and the lungs. Recently, a number of studies have identified a role for substance P and NK-1R interactions in neuroinflammation and described the ability of this neuropeptide to alter the immune functions of activated microglia and astrocytes. In this review article, we describe the expression of substance P and its receptor by resident CNS cells, and we discuss the ability of this neuropeptide to exacerbate the inflammatory responses of glia and immune cells that are recruited to the brain during neurodegenerative diseases. In addition, we discuss the available data indicating that the NK-1R-mediated augmentation of such responses appears to be detrimental during microbial infection and some sterile neurodegenerative disorders, and propose the repurposed use of NK-1R antagonists, of a type that are currently approved as anti-emetic and anti-anxiolytic agents, as an adjunct therapy to ameliorate the inflammatory CNS damage in these conditions. PMID:28101005

Chronic treatment with otilonium bromide induces changes in L-type Ca²⁺ channel, tachykinins, and nitric oxide synthase expression in rat colon muscle coat.

PubMed

Traini, C; Cipriani, G; Evangelista, S; Santicioli, P; Faussone-Pellegrini, M-S; Vannucchi, M-G

2013-11-01

Otilonium bromide (OB) is a quaternary ammonium derivative used for the treatment of intestinal hypermotility and is endowed with neurokinin2 receptor (NK2r) antagonist and Ca²⁺ channel blocker properties. Therefore, the possibility that OB might play a role in the neurokinin receptor/Substance-P/nitric oxide (NKr/SP/NO) circuit was investigated after chronic exposition to the drug. Rats were treated with OB 2-20 mg kg⁻¹ for 10 and 30 days. In the proximal colon, the expression and distribution of muscle NOsynthase 1 (NOS1), NK1r, NK2r, SP and Cav 1.2 subunit (for L-type Ca²⁺ channel) and the spontaneous activity and stimulated responses to NK1r and NK2r agonists were investigated. Immunohistochemistry showed a redistribution of NK1r and L-type Ca²⁺ channel in muscle cells with no change of NK2r at 30 days, a significant increase in muscle NOS1 expression at 10 days and a significant decrease in the SP content early in the ganglia and later in the intramuscular nerve fibers. Functional studies showed no change in spontaneous activity but a significant increase in maximal contraction induced by NK1r agonist. Chronic exposition to OB significantly affects the NKr/SP/NO circuit. The progressive decrease in SP-expression might be the consequence of the persistent presence of OB, the increase of NOS1 expression in muscle cells at 10 days in an attempt to guarantee an adequate NO production, and, at 30 days, the redistribution of the L-type Ca²⁺ channel and NK1r as a sign to compensate the drug channel block by re-cycling both of them. The physiological data suggest NK1r hypersensitivity. © 2013 John Wiley & Sons Ltd.

Substance P and the neurokinin-1 receptor expression in dog ileum with and without inflammation.

PubMed

Polidoro, Giulia; Giancola, Fiorella; Fracassi, Federico; Pietra, Marco; Bettini, Giuliano; Asti, Martina; Chiocchetti, Roberto

2017-10-01

In the gastrointestinal tract, the tachykinin Substance P (SP) is involved in motility, fluid and electrolyte secretion, and blood flow and regulation of immunoinflammatory response. SP exerts its biological activity on target cells by interacting mainly with the neurokinin-1 receptor (NK 1 R). The present study aims to quantify the percentage of SP-immunoreactive (SP-IR) enteric neurons and the density of SP-IR nerve fibers in the ileum of control dogs (CTRL-dogs; n=7) vs dogs with spontaneous ileal inflammation (INF-dogs; n=8). In addition, the percentage of enteric neurons bearing NK 1 R, and nitrergic neurons (nNOS-IR) expressing NK 1 R immunoreactivity were evaluated in both groups. The percentages of SP-IR neurons were similar in CTRL- and INF-dogs, in either the myenteric (MP) (15±8% vs. 16±7%, respectively) and submucosal plexus (SMP) (26±7% vs. 24±14%, respectively). In INF-dogs, the density of SP-IR mucosal nerve fibers showed a trend to decrease (P=0.07). Myenteric neurons of CTRL- and INF-dogs expressed similar percentages of NK 1 R-immunoreactivity (39±5% vs. 38±20%, respectively). Submucosal NK 1 R-IR neurons were occasionally observed in a CTRL-dog. MP nitrergic neurons bearing NK 1 R showed a trend to decrease in INF-dogs vs. CTRL- dogs (41±22% vs. 65±10%, respectively; P=0.11). In INF-dogs, muscle cells and immune cells overexpressed NK 1 R immunoreactivity. These findings should be taken as a warning for possible intestinal motility disorders, which might occur during administration of NK 1 R-antagonist drugs. Conversely, the strong expression of NK 1 R immunoreactivity observed in muscle and mucosal immune cells of inflamed tissues may provide a rationale for the use of NK 1 R antagonist drugs in the treatment of intestinal inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tachykinin substance P depletion by capsaicin exacerbates inflammatory response to sidestream cigarette smoke in rats.

PubMed

Sun, Nina N; Wong, Simon S; Keith, Ingegerd; Witten, Mark L

2004-09-01

To evaluate the role of substance P (SP)-containing C-fiber nerves in the development of the inflammatory responses to sidestream cigarette smoke (SSCS), female Fischer 344 rats were randomly assigned into vehicle and capsaicin groups, respectively. Then, half the number in each group (N = 24) was nose-only exposed to air or 0.4 mg/m3 total particulate matter of SSCS for 4 h/day for 7 days. Exposure of the vehicle rats to SSCS induced obvious pulmonary neurogenic inflammation as indicated by elevations in plasma extravasation and proinflammatory cytokine secretions [interieukin (IL)-1beta and IL-12]. In addition, except for SP release, SSCS exposure significantly induced the tachykininergic toxicities at the gene level: upregulation of beta-preprotachykinin-I (beta-PPT-I) mRNA. However, neither SSCS exposure nor capsaicin pretreatment affects the immunolabeling density of neurokinin-1 receptor (NK-1R) in airway epithelium. SSCS also significantly inactivated pulmonary neutral endopeptidase (NEP) in lung tissue. Moreover, pretreatment with capsaicin significantly exacerbated the SSCS-induced inflammatory responses mentioned above as well as the release of plasma protein. Considering that capsaicin did not affect the normal control baselines of these parameters except for a decrease in NK-1R mRNA, we conclude that the degree of SSCS-induced inflammatory response was exacerbated because of the depletion of stored SP and/or inactivation of capsaicin-sensitive C-fiber nerves. Our data suggest the loss of afferent tachykinin SP signaling may lead to dysfunction of the sensory C-fiber nerve reflexes during exposure to SSCS, suggesting that SP serves a protective role.

Inhibitors of endopeptidase and angiotensin-converting enzyme lead to an amplification of the morphological changes and an upregulation of the substance P system in a muscle overuse model

PubMed Central

2014-01-01

Background We have previously observed, in studies on an experimental overuse model, that the tachykinin system may be involved in the processes of muscle inflammation (myositis) and other muscle tissue alterations. To further evaluate the significance of tachykinins in these processes, we have used inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), substances which are known to terminate the activity of various endogenously produced substances, including tachykinins. Methods Injections of inhibitors of NEP and ACE, as well as the tachykinin substance P (SP), were given locally outside the tendon of the triceps surae muscle of rabbits subjected to marked overuse of this muscle. A control group was given NaCl injections. Evaluations were made at 1 week, a timepoint of overuse when only mild inflammation and limited changes in the muscle structure are noted in animals not treated with inhibitors. Both the soleus and gastrocnemius muscles were examined morphologically and with immunohistochemistry and enzyme immunoassay (EIA). Results A pronounced inflammation (myositis) and changes in the muscle fiber morphology, including muscle fiber necrosis, occurred in the overused muscles of animals given NEP and ACE inhibitors. The morphological changes were clearly more prominent than for animals subjected to overuse and NaCl injections (NaCl group). A marked SP-like expression, as well as a marked expression of the neurokinin-1 receptor (NK-1R) was found in the affected muscle tissue in response to injections of NEP and ACE inhibitors. The concentration of SP in the muscles was also higher than that for the NaCl group. Conclusions The observations show that the local injections of NEP and ACE inhibitors led to marked SP-like and NK-1R immunoreactions, increased SP concentrations, and an amplification of the morphological changes in the tissue. The injections of the inhibitors thus led to a more marked myositis process and an upregulation of the SP system. Endogenously produced substances, out of which the tachykinins conform to one substance family, may play a role in mediating effects in the tissue in a muscle that is subjected to pronounced overuse. PMID:24725470

Mechanisms mediating substance P-induced contraction in the rat iris in vitro.

PubMed

Grumann-Júnior, A; Dias, M A; Alves, R V; Boteon, J E; Calixto, J B

2000-06-01

To determine some of the mechanisms by which substance P (SP) induces contraction in the isolated rat iris. Rings of rat iris were mounted in a 5-ml organ chamber containing Krebs solution at 37 degrees C under basal tension of 75 mg, and isometric tension was recorded. Substance P produced graded contraction in the rat iris, being approximately 40-fold more potent than carbachol. Peptidase inhibitors (captopril, phosphoramidon, thiorphan) did not affect the SP response. The SP contraction was dependent on external Ca2+ by a mechanism resistant to both nifedipine and omega-conotoxin GVIA. Atropine and tetrodotoxin significantly shifted the SP response to the right (three- and fivefold, respectively). Neither phorbol nor genistein altered the SP-induced contraction, whereas staurosporine caused a weak inhibition. Indomethacin, pyrilamine, guanethidine, 8-37 calcitonin gene-related peptide (CGRP) fragment, and NG-nitro-L-arginine methyl ester had no effect on SP response. All the natural tachykinin agonists caused concentration-dependent contraction in rat iris with similar maximal responses. The NK3 selective agonist senktide caused graded contraction, being approximately 150-fold more active than the NK2 selective agonist [beta-ala] NKA. The NK1 selective agonist SP methyl ester induced a small contraction. The NK3 and NK2 antagonists SR 142801 and SR 48968 shifted the SP response to the right. Schilds plots gave pA2 (negative logarithm of the molar concentration of antagonist causing a twofold rightward displacement of the concentration response curves) values of 9.37 and 7.97 and slopes of 0.70 and 1.02, respectively. Substance P produces a potent contraction in the isolated rat iris that seems to depend on the neural release of acetylcholine by tetrodotoxin-sensitive mechanisms. Its response relies largely on external Ca2+, through mechanisms independent of activation of L- or N-type Ca2+ channels, and is probably mediated via activation of NK3 and NK2 receptors.

KW-4679-induced inhibition of tachykininergic contraction in the guinea-pig bronchi by prejunctional inhibition of peripheral sensory nerves.

PubMed

Ikemura, T; Okarmura, K; Sasaki, Y; Ishi, H; Ohmori, K

1996-03-01

1. Sensory mechanisms play an important role in the vagal regulation of tracheobronchial smooth muscle tone. We examined the effect of KW-4679, an anti-allergic drug, on guinea-pig tachykinin-mediated contractile responses induced by electrical field stimulation (EFS) in guinea-pig bronchial muscles. 2. EFS (8 Hz, 0.5 ms, 15 V, for 15 s) evoked biphasic contractile responses in the guinea-pig isolated main bronchus in the presence of 5 microM indomethacin. The contractions consisted of a fast phase of an atropine-sensitive transient contraction and a slow phase of a sustained contraction which was inhibited by a combination of the tachykinin NK1 receptor antagonist, (+/-)-CP-96,345 (1 microM) and the NK2 receptor antagonist, SR 48969 (0.1 microM). 3. KW-4679 preferentially inhibited the slow phase in a concentration-dependent manner by 43.2 +/- 7.7% at 10 microM, whereas the drug had no effect on the fast phase at concentrations up to 10 microM. KW-4679, at a concentration of 100 microM, inhibited not only the slow phase by 49.2 +/- 11.4%, but also the fast phase by 36.8 +/- 9.3% [corrected]. 4. KW-4679 (10 microM and 100 microM) did not affect the substance P-induced or neurokinin A-induced contraction. Against the acetylcholine-induced contractile responses, 100 microM KW-4679 had a marked effect producing a 10.2 fold shift to the right in the curve. 5. The inhibitory effect of KW-4679 (10 microM) on the slow phase contraction was not influenced by treatment with naloxone (100 nM), propranolol (1 microM), thioperamide (1 microM), saclofen (50 microM), yohimbine (1 microM), methiothepin (1 microM) or methysergide (1 microM). 6. The inhibitory effect of KW-4679 (10 microM) on the slow phase contraction was not influenced by treatment with intermediate or large conductance Ca(2+)-activated K+ channel blockers (charybdotoxin (10 nM) or iberiotoxin (10 nM)), but suppressed by treatment with small conductance Ca(2+)-activated K+ channel blockers, apamin (500 nM) or scyllatoxin (300 nM). Apamin or scyllatoxin per se did not influence the slow phase contractions. 7. The results suggest that KW-4679 preferentially inhibits the release of tachykinins from the bronchial sensory nerves through activation of small conductance Ca(2+)-activated K+ channels.

Reviewing current and emerging antiemetics for chemotherapy-induced nausea and vomiting prophylaxis.

PubMed

Natale, James J

2015-01-01

This review provides background information on chemotherapy-induced nausea and vomiting (CINV) classification and pathophysiology and reviews various antiemetic agents for CINV prophylaxis, including corticosteroids, serotonin receptor antagonists (5-HT3 RAs), tachykinin NK1 receptor antagonists (NK1 RAs), and olanzapine. Other less commonly used agents are briefly discussed. Practical considerations are reviewed as well, including emetogenicity of chemotherapeutic regimens, patient-specific risk factors for CINV, principles of CINV management, health economics outcome research, and quality of life. Available data on the newly FDA-approved antiemetic combination netupitant/palonosetron (NEPA) is also reviewed. Prevention of CINV is an important goal in managing patients with cancer and is especially difficult with respect to nausea and delayed CINV. Corticosteroids are a mainstay of CINV prophylaxis and are usually given in combination with other therapies. The 5-HT3 RA palonosetron has shown increased efficacy over other agents in the same class for prevention of delayed emesis with moderately emetogenic chemotherapy and NK1 RAs improve emesis prevention in combination with 5-HT3 RAs and dexamethasone. Olanzapine has shown efficacy for CINV prophylaxis and the treatment of breakthrough CINV. The new combination therapy, NEPA, has been shown to be efficacious for the prevention of acute, delayed, and overall CINV. Risk factors that have been identified for CINV include gender, age, and alcohol intake. It is important to assess the emetogenicity of chemotherapy regimens as well as the potential impact of patient risk factors in order to provide adequate prophylaxis. Acute and delayed CINV are severe, burdensome side effects of chemotherapy; however, new data on prevention and the discovery of new agents can further improve CINV control.

The role of substance P in inflammatory disease.

PubMed

O'Connor, Terence M; O'Connell, Joseph; O'Brien, Darren I; Goode, Triona; Bredin, Charles P; Shanahan, Fergus

2004-11-01

The diffuse neuroendocrine system consists of specialised endocrine cells and peptidergic nerves and is present in all organs of the body. Substance P (SP) is secreted by nerves and inflammatory cells such as macrophages, eosinophils, lymphocytes, and dendritic cells and acts by binding to the neurokinin-1 receptor (NK-1R). SP has proinflammatory effects in immune and epithelial cells and participates in inflammatory diseases of the respiratory, gastrointestinal, and musculoskeletal systems. Many substances induce neuropeptide release from sensory nerves in the lung, including allergen, histamine, prostaglandins, and leukotrienes. Patients with asthma are hyperresponsive to SP and NK-1R expression is increased in their bronchi. Neurogenic inflammation also participates in virus-associated respiratory infection, non-productive cough, allergic rhinitis, and sarcoidosis. SP regulates smooth muscle contractility, epithelial ion transport, vascular permeability, and immune function in the gastrointestinal tract. Elevated levels of SP and upregulated NK-1R expression have been reported in the rectum and colon of patients with inflammatory bowel disease (IBD), and correlate with disease activity. Increased levels of SP are found in the synovial fluid and serum of patients with rheumatoid arthritis (RA) and NK-1R mRNA is upregulated in RA synoviocytes. Glucocorticoids may attenuate neurogenic inflammation by decreasing NK-1R expression in epithelial and inflammatory cells and increasing production of neutral endopeptidase (NEP), an enzyme that degrades SP. Preventing the proinflammatory effects of SP using tachykinin receptor antagonists may have therapeutic potential in inflammatory diseases such as asthma, sarcoidosis, chronic bronchitis, IBD, and RA. In this paper, we review the role that SP plays in inflammatory disease. Copyright 2004 Wiley-Liss, Inc.

Central respiratory effects of substance P in neonatal mice: an in vitro study.

PubMed

Ptak, K; Hilaire, G

1999-05-14

Experiments were performed on neonatal mice to know whether substance P (SP) modified the rhythm and the amplitude of the phrenic bursts generated in vitro in brainstem-cervical cord preparations. In OF1 and C3H neonatal preparations, SP or the tachykinin NK1 receptor agonist [Sar9,Met(O2)11] substance P both increased significantly phrenic burst amplitude (10(-7) M) but had no significant effect on respiratory rhythm unless used at concentrations 10 times larger. In neonates from the monoamine oxidase-A deficient transgenic Tg8 line, SP increased phrenic burst amplitude but had no effect on the respiratory rhythm at the tested concentrations. The role of SP in regulating neonatal respiratory activity is discussed on the basis of rat and mouse results.

Cardiorespiratory action of opioid/tachykinin agonist peptide hybrid in anaesthetized rats: Transduction pathways.

PubMed

Wojciechowski, Piotr; Szereda-Przestaszewska, Małgorzata; Lipkowski, Andrzej Wojciech

2017-09-05

AWL3106 composed of opioid (dermorphin) and tachykinin (substance P 7-11 ) pharmacophores is a new compound with high analgesic potency and markedly reduced ability to induce tolerance and dependence. The present study aimed to determine the respiratory and cardiovascular responses evoked by this peptide in urethane-chloralose anaesthetized, spontaneously breathing rats in the presence or absence of vagal connection. Intravenous injection of AWL3106 at a dose of 0.3μmol/kg in intact rats resulted in apnoea lasting 5.1 ± 0.7s. Breathing that followed was of diminished frequency (F) and augmented tidal volume (V T ) with no significant impact on minute ventilation. AWL3106-challenge induced biphasic fall in arterial blood pressure with no effect on heart rate. Midcervical and supranodosal sectioning the vagal nerves prevented the occurrence of the apnoea and abrogated the post-AWL3106 reduction in F but failed to eliminate the increase in V T . Hypotensive response appeared to be less profound following supranodose vagotomy. NaloxoneHCl abolished solely the occurrence of apnoea. However additional blockade of tachykinin NK 1 receptors with SR140333 was required to abolish V T increase, deceleration of breathing and to markedly suppress AWL3106-induced hypotension. The present study shows that extravagally controlled stimulation of V T maintains fairly regular ventilation by levelling the bradypnoeic effects. Although the peptide showed no cardiac effects, hypotension occurring beyond the vagal loop may limit future therapeutic benefits of this chimeric compound. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of excitatory and inhibitory neurotransmission on motor patterns of human sigmoid colon in vitro

PubMed Central

Aulí, M; Martínez, E; Gallego, D; Opazo, A; Espín, F; Martí-Gallostra, M; Jiménez, M; Clavé, P

2008-01-01

Background and purpose: To characterize the in vitro motor patterns and the neurotransmitters released by enteric motor neurons (EMNs) in the human sigmoid colon. Experimental approach: Sigmoid circular strips were studied in organ baths. EMNs were stimulated by electrical field stimulation (EFS) and through nicotinic ACh receptors. Key results: Strips developed weak spontaneous rhythmic contractions (3.67±0.49 g, 2.54±0.15 min) unaffected by the neurotoxin tetrodotoxin (TTX; 1 μM). EFS induced strong contractions during (on, 56%) or after electrical stimulus (off, 44%), both abolished by TTX. Nicotine (1–100 μM) inhibited spontaneous contractions. Latency of off-contractions and nicotine responses were reduced by NG-nitro-L-arginine (1 mM) and blocked after further addition of apamin (1 μM) or the P2Y1 receptor antagonist MRS 2179 (10 μM) and were unaffected by the P2X antagonist NF279 (10 μM) or α-chymotrypsin (10 U mL−1). Amplitude of on- and off-contractions was reduced by atropine (1 μM) and the selective NK2 receptor antagonist Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (1 μM). MRS 2179 reduced the amplitude of EFS on- and off-contractions without altering direct muscular contractions induced by ACh (1 nM–1 mM) or substance P (1 nM–10 μM). Conclusions and implications: Latency of EFS-induced off-contractions and inhibition of spontaneous motility by nicotine are caused by stimulation of inhibitory EMNs coreleasing NO and a purine acting at muscular P2Y1 receptors through apamin-sensitive K+ channels. EFS-induced on- and off-contractions are caused by stimulation of excitatory EMNs coreleasing ACh and tachykinins acting on muscular muscarinic and NK2 receptors. Prejunctional P2Y1 receptors might modulate the activity of excitatory EMNs. P2Y1 and NK2 receptors might be therapeutic targets for colonic motor disorders. PMID:18846038

Effect of experimental diabetes on cholinergic, purinergic and peptidergic motor responses of the isolated rat bladder to electrical field stimulation or capsaicin.

PubMed

Benkó, Rita; Lázár, Zsófia; Pórszász, Róbert; Somogyi, George T; Barthó, Loránd

2003-09-30

An attempt has been made to pharmacologically isolate cholinergic, P(2) purinoceptor-mediated and peptidergic (capsaicin-sensitive, tachykinin-mediated) contraction of the guanethidine-treated rat bladder detrusor preparation, in vitro. The effect of experimental diabetes was assessed on these types of contraction. Responses were evoked by electrical field stimulation (single shocks or 1 Hz for 30 s or 10 Hz for 40 s). Single shocks and 1-Hz stimulation were applied in the presence of (a). atropine (1 microM) or (b). P(2) purinoceptor antagonists (50 microM pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) [PPADS] plus 100 microM suramin. Long-term electrical field stimulation (10 Hz for 40 s) (c). was applied with both atropine and the P(2) purinoceptor antagonists present in the organ bath. The effects of capsaicin (d). and ATP (e). were also studied. Three groups of experimental animals were used: streptozotocin-treated (50 mg.kg(-1) i.p., 8 weeks before the experiment), parallel solvent-treated and untreated rats. (a). Responses to electrical field stimulation in the presence of atropine were reduced by half by PPADS plus suramin, but were resistant to capsaicin tachyphylaxis. They were enhanced in preparations taken from diabetic rats. (b). Contractions to electrical field stimulation in the presence of PPADS plus suramin were reduced by 2/3 by atropine, but were left unchanged by capsaicin or diabetes. (c). Contractions to long-term stimulation had a quick and a sustained phase. Especially the latter was inhibited by capsaicin tachypyhlaxis; it was also strongly reduced in preparations taken from diabetic rats. (d). Contractions to capsaicin (30 nM and 1 microM) were resistant to tetrodotoxin, strongly reduced by a combination of tachykinin NK(1) and NK(2) receptor antagonists, and slightly reduced in preparations from diabetic animals. Capsaicin (1 microM) had no acute inhibitory action on cholinergic or purinergic responses, nor did it cause relaxation in precontracted preparations treated with tachykinin receptor antagonists. (e) ATP-induced contractions were strongly reduced by PPADS plus suramin (50 plus 100 microM) and to a similar degree by 100 plus 200 microM, respectively. It is concluded that experimental diabetes selectively impairs peptidergic, capsaicin-sensitive responses (especially those that involve impulse conduction) in the rat detrusor preparation. The contractile response to electrical field stimulation that remains after atropine plus the P(2) purinoceptor antagonists has a yet unknown transmitter background.

Neurogenic mediators contribute to local edema induced by Micrurus lemniscatus venom

PubMed Central

2017-01-01

Background/Aims Micrurus is one of the four snake genera of medical importance in Brazil. Coral snakes have a broad geographic distribution from the southern United States to Argentina. Micrurine envenomation is characterized by neurotoxic symptoms leading to dyspnea and death. Moreover, various local manifestations, including edema formation, have been described in patients bitten by different species of Micrurus. Thus, we investigated the ability of Micrurus lemniscatus venom (MLV) to induce local edema. We also explored mechanisms underlying this effect, focusing on participation of neuropeptides and mast cells. Methodology/Principal findings Intraplantar injection of MLV (1–10 μg/paw) in rats caused dose- and time-dependent edema with a peak between 15 min and 1 h after injection. MLV also induced degranulation of peritoneal mast cells (MCs). MC depletion by compound 48/80 markedly reduced MLV-induced edema. Pre-treatment (30 min) of rats with either promethazine a histamine H1 receptor antagonist or methysergide, a nonselective 5-HT receptor antagonist, reduced MLV-induced edema. However, neither thioperamide, a histamine H3/H4 receptor antagonist, nor co-injection of MLV with HOE-140, a BK2 receptor antagonist, altered the response. Depletion of neuropeptides by capsaicin or treatment of animals with NK1- and NK2-receptor antagonists (SR 140333 and SR 48968, respectively) markedly reduced MLV-induced edema. Conclusions/Significance In conclusion, MLV induces paw edema in rats by mechanisms involving activation of mast cells and substance P-releasing sensory C-fibers. Tachykinins NKA and NKB, histamine, and serotonin are major mediators of the MLV-induced edematogenic response. Targeting mast cell- and sensory C-fiber-derived mediators should be considered as potential therapeutic approaches to interrupt development of local edema induced by Micrurus venoms. PMID:29161255

Interaction of tachykinins with their receptors studied with cyclic analogues of substance P and neurokinin B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ploux, O.; Lavielle, S.; Chassaing, G.

1987-11-01

The activities of two groups of cyclic agonists of substance P (SP) have been studied. The disulfide bridge constraints have been designed on the basis of conformational studies on SP and physalaemin indicating an ..cap alpha..-helical structure for the core of these two tachykinins (group I) and a folding of the C-terminal carboxamide towards the side chains of the glutamines 5 and 6 (group II). Only peptides simulating the ..cap alpha..-helix present substantial potencies. (Cys/sup 3,6/)SP is as active as SP in inhibiting /sup 125/I-labeled Bolton and Hunter SP-specific binding on rat brain synaptosomes and on dog carotid bioassay, twomore » assays specific for the neurokinin 1 receptor. Moreover, (Cys/sup 3,6/)SP is a potent as neurokinin B in inhibiting /sup 125/I-labeled Bolton and Hunter eledoisin-specific binding on rat cortical synaptosomes as well as in stimulating rat portal vein, two tests specific for the neurokinin 3 receptor. Interestingly, in contrast to neurokinin B, (Cys/sup 3,6/)SP is a weak agonist of the neurokinin 2 receptor subtype, as evidenced by its binding potency in inhibiting /sup 3/H-labeled neurokinin A-specific binding on rat duodenum and in inducing the contractions of the rabbit pulmonary artery, a neurokinin 2-type bioassay. To increase the specificity of the cyclic analogue (Cys/sup 3,6/)SP positions 8 and 9 were modified. Collectively, these results suggest that the neurokinin 1 and neurokinin 3 tachykinin receptors may recognize a similar three-dimensional structure of the core of the tachykinins. Different orientations of the common C-terminal tripeptide may be related to the selectivity for the different receptor subtypes.« less

TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction.

PubMed

Topaloglu, A Kemal; Reimann, Frank; Guclu, Metin; Yalin, Ayse Serap; Kotan, L Damla; Porter, Keith M; Serin, Ayse; Mungan, Neslihan O; Cook, Joshua R; Imamoglu, Sazi; Akalin, N Sema; Yuksel, Bilgin; O'Rahilly, Stephen; Semple, Robert K

2009-03-01

The timely secretion of gonadal sex steroids is essential for the initiation of puberty, the postpubertal maintenance of secondary sexual characteristics and the normal perinatal development of male external genitalia. Normal gonadal steroid production requires the actions of the pituitary-derived gonadotropins, luteinizing hormone and follicle-stimulating hormone. We report four human pedigrees with severe congenital gonadotropin deficiency and pubertal failure in which all affected individuals are homozygous for loss-of-function mutations in TAC3 (encoding Neurokinin B) or its receptor TACR3 (encoding NK3R). Neurokinin B, a member of the substance P-related tachykinin family, is known to be highly expressed in hypothalamic neurons that also express kisspeptin, a recently identified regulator of gonadotropin-releasing hormone secretion. These findings implicate Neurokinin B as a critical central regulator of human gonadal function and suggest new approaches to the pharmacological control of human reproduction and sex hormone-related diseases.

Interaction of tachykinins with phospholipid membranes: A neutron diffraction study

NASA Astrophysics Data System (ADS)

Darkes, Malcolm J. M.; Davies, Sarah M. A.; Bradshaw, Jeremy P.

Tachykinins are a group of peptides which bind to G-protein-coupled receptors. Receptor affinity appears to depend on different secondary structures of tachykinin which share the same hydrophobic carboxy-terminal sequence, FXGLM. Receptor activation is thought to be due to the carboxy-terminal submerging into the bilayer and the amino-terminal binding on the surface. Binding of tachykinins to phospholipid bilayers may take place both on the aqueous membrane surface and in the hydrophobic region. The two-state equilibrium appears to depend on the surface charge of the membrane. Deuterating substance P and neurokinin A at their carboxy-terminals, our results show two populations of label for each peptide. One is very close to the water-hydrocarbon interface, the other some 13 Å deeper. We report that the bilayer location of the two tachykinins is remarkably similar, thereby inferring that receptor specifity must be controlled by finer levels of structure.

Neurogenic airway microvascular leakage induced by toluene inhalation in rats.

PubMed

Sakamoto, Tatsuo; Kamijima, Michihiro; Miyake, Mio

2012-06-15

Toluene is a representative airborne occupational and domestic pollutant that causes eye and respiratory tract irritation. We investigated whether a single inhalation of toluene elicits microvascular leakage in the rat airway. We also evaluated the effects of CP-99,994, a tachykinin NK(1) receptor antagonist, and ketotifen, a histamine H1 receptor antagonist with mast cell-stabilizing properties, on the airway response. The content of Evans blue dye that extravasated into the tissues was measured as an index of plasma leakage. Toluene (18-450 ppm, 10 min) concentration-dependently induced dye leakage into the trachea and main bronchi of anesthetized and mechanically ventilated rats. Toluene at concentrations of ≥ 50 and ≥ 30 ppm caused significant responses in the trachea and main bronchi, respectively, which both peaked after exposure to 135 ppm toluene for 10 min. This response was abolished by CP-99,994 (5 mg/kg i.v.), but not by ketotifen (1mg/kg i.v.). Nebulized phosphoramidon (1 mM, 1 min), a neutral endopeptidase 24.11 inhibitor, significantly enhanced the response induced by toluene (135 ppm, 10 min) compared with nebulized 0.9% saline (1 min). These results show that toluene can rapidly increase airway plasma leakage that is predominantly mediated by tachykinins endogenously released from airway sensory nerves. However, mast cell activation might not be important in this airway response. Copyright © 2012 Elsevier B.V. All rights reserved.

Tachykinins and Their Receptors: Contributions to Physiological Control and the Mechanisms of Disease

PubMed Central

Steinhoff, Martin S.; von Mentzer, Bengt; Geppetti, Pierangelo; Pothoulakis, Charalabos; Bunnett, Nigel W.

2014-01-01

The tachykinins, exemplified by substance P, are one of the most intensively studied neuropeptide families. They comprise a series of structurally related peptides that derive from alternate processing of three Tac genes and are expressed throughout the nervous and immune systems. Tachykinins interact with three neurokinin G protein-coupled receptors. The signaling, trafficking, and regulation of neurokinin receptors have also been topics of intense study. Tachykinins participate in important physiological processes in the nervous, immune, gastrointestinal, respiratory, urogenital, and dermal systems, including inflammation, nociception, smooth muscle contractility, epithelial secretion, and proliferation. They contribute to multiple diseases processes, including acute and chronic inflammation and pain, fibrosis, affective and addictive disorders, functional disorders of the intestine and urinary bladder, infection, and cancer. Neurokinin receptor antagonists are selective, potent, and show efficacy in models of disease. In clinical trials there is a singular success: neurokinin 1 receptor antagonists to treat nausea and vomiting. New information about the involvement of tachykinins in infection, fibrosis, and pruritus justifies further trials. A deeper understanding of disease mechanisms is required for the development of more predictive experimental models, and for the design and interpretation of clinical trials. Knowledge of neurokinin receptor structure, and the development of targeting strategies to disrupt disease-relevant subcellular signaling of neurokinin receptors, may refine the next generation of neurokinin receptor antagonists. PMID:24382888

Tac1 Signaling Is Required for Sexual Maturation and Responsiveness of GnRH Neurons to Kisspeptin in the Male Mouse.

PubMed

Maguire, Caroline A; Song, Yong Bhum; Wu, Min; León, Silvia; Carroll, Rona S; Alreja, Meenakshi; Kaiser, Ursula B; Navarro, Víctor M

2017-07-01

The tachykinins substance P (SP) and neurokinin A (Tac1) have emerged as novel regulators of kisspeptin/GnRH release. Recently, we documented that SP modulates reproductive function in the female mouse. Here, we extended this characterization to the male mouse. Tac1-/- male mice showed delayed puberty onset. They also presented significantly decreased expression levels of Pdyn (dynorphin) and Nos1 (nitric oxide synthase) in the mediobasal hypothalamus and elevated Gnrh1 levels. Unexpectedly, the response of Tac1-/- mice to central kisspeptin or senktide (neurokinin B receptor-agonist) administration was significantly decreased compared with controls, despite the preserved ability of GnRH neurons to stimulate luteinizing hormone release as demonstrated by central N-methyl-D-aspartate receptor administration, suggesting a deficit at the GnRH neuron level. Importantly, we demonstrated that kisspeptin receptor and SP receptor (NK1R) heterodimerize, indicating that changes in the SP tone could alter the responsiveness of GnRH neurons to kisspeptin. Finally, electrophysiological recordings from arcuate Kiss1 neurons showed that, although virtually all Kiss1 neurons responded to NKB and senktide, only half responded to an NK1R agonist and none to the neurokinin A receptor agonist at a 1-μM dose. In summary, we provide compelling evidence for a role of Tac1 in the control of reproductive function in the male mouse, suggesting a predominant central action that may involve a change in the balance of neural factors that control GnRH expression. Copyright © 2017 Endocrine Society.

Tachykinin activation of human alveolar macrophages in tobacco smoke and sarcoidosis: a phenotypical and functional study.

PubMed

Brunelleschi, S; Guidotto, S; Viano, I; Fantozzi, R; Pozzi, E; Ghio, P; Albera, C

1996-10-01

Substance P (SP) and neurokinin A (NKA), which exert bronchoconstrictor effects on human airways, are known to interact with inflammatory and immune cells, including monocyte macrophages. We have evaluated the effects of SP, NKA and the NK2 selective agonist [beta-Ala8]-NKA(4-10) on alveolar macrophages (AM) isolated from 4 healthy smokers and 4 non-smoker active pulmonary sarcoid patients. An accumulation of activated mononuclear phagocytes, as well as elevated angiotensin-converting enzyme (ACE) activity, has been evidenced in both clinical conditions. The phenotype of AMs in the studied subjects was characterized by an elevated expression of CD68+, HLA-DR+ and CD14+, CD14+ being significantly less in sarcoidosis as compared to smokers. SP, NKA and the NK2 selective agonist evoked superoxide anion (O2-) production in AMs obtained from sarcoid patients or healthy smokers. While SP acted in a non-dose-dependent manner in both conditions, NKA and [beta-Ala8]-NKA(4-10) evoked a dose-dependent respiratory burst (ED50 = 0.25 and 0.26 nM, respectively) in smokers, but not in sarcoidosis. The more marked phenotypical expression correlated well with the ability of NK2 receptors to activate AMs in smoker subjects.

Overexpression of membrane metalloendopeptidase inhibits substance P stimulation of cholangiocarcinoma growth.

PubMed

Meng, Fanyin; DeMorrow, Sharon; Venter, Julie; Frampton, Gabriel; Han, Yuyan; Francis, Heather; Standeford, Holly; Avila, Shanika; McDaniel, Kelly; McMillin, Matthew; Afroze, Syeda; Guerrier, Micheleine; Quezada, Morgan; Ray, Debolina; Kennedy, Lindsey; Hargrove, Laura; Glaser, Shannon; Alpini, Gianfranco

2014-05-01

Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). This study aimed to evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. NK1R, Tac1, and MME expression and SP secretion were assessed in human CCA cells and nonmalignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and of L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated by using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared with nonmalignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19, and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared with controls. The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.

Abnormalities in substance P neurokinin-1 receptor binding in key brainstem nuclei in sudden infant death syndrome related to prematurity and sex.

PubMed

Bright, Fiona M; Vink, Robert; Byard, Roger W; Duncan, Jhodie R; Krous, Henry F; Paterson, David S

2017-01-01

Sudden infant death syndrome (SIDS) involves failure of arousal to potentially life threatening events, including hypoxia, during sleep. While neuronal dysfunction and abnormalities in neurotransmitter systems within the medulla oblongata have been implicated, the specific pathways associated with autonomic and cardiorespiratory failure are unknown. The neuropeptide substance P (SP) and its tachykinin neurokinin-1 receptor (NK1R) have been shown to play an integral role in the modulation of homeostatic function in the medulla, including regulation of respiratory rhythm generation, integration of cardiovascular control, and modulation of the baroreceptor reflex and mediation of the chemoreceptor reflex in response to hypoxia. Abnormalities in SP neurotransmission may therefore result in autonomic dysfunction during sleep and contribute to SIDS deaths. [125I] Bolton Hunter SP autoradiography was used to map the distribution and density of the SP, NK1R to 13 specific nuclei intimately related to cardiorespiratory function and autonomic control in the human infant medulla of 55 SIDS and 21 control (non-SIDS) infants. Compared to controls, SIDS cases exhibited a differential, abnormal developmental profile of the SP/NK1R system in the medulla. Furthermore the study revealed significantly decreased NK1R binding within key medullary nuclei in SIDS cases, principally in the nucleus tractus solitarii (NTS) and all three subdivisions of the inferior portion of the olivo-cerebellar complex; the principal inferior olivary complex (PIO), medial accessory olive (MAO) and dorsal accessory olive (DAO). Altered NK1R binding was significantly influenced by prematurity and male sex, which may explain the increased risk of SIDS in premature and male infants. Abnormal NK1R binding in these medullary nuclei may contribute to the defective interaction of critical medullary mechanisms with cerebellar sites, resulting in an inability of a SIDS infant to illicit appropriate respiratory and motor responses to life threatening challenges during sleep. These observations support the concept that abnormalities in a multi-neurotransmitter network within key nuclei of the medullary homeostatic system may underlie the pathogenesis of a subset of SIDS cases.

Abnormalities in substance P neurokinin-1 receptor binding in key brainstem nuclei in sudden infant death syndrome related to prematurity and sex

PubMed Central

Vink, Robert; Byard, Roger W.; Duncan, Jhodie R.; Krous, Henry F.; Paterson, David S.

2017-01-01

Sudden infant death syndrome (SIDS) involves failure of arousal to potentially life threatening events, including hypoxia, during sleep. While neuronal dysfunction and abnormalities in neurotransmitter systems within the medulla oblongata have been implicated, the specific pathways associated with autonomic and cardiorespiratory failure are unknown. The neuropeptide substance P (SP) and its tachykinin neurokinin-1 receptor (NK1R) have been shown to play an integral role in the modulation of homeostatic function in the medulla, including regulation of respiratory rhythm generation, integration of cardiovascular control, and modulation of the baroreceptor reflex and mediation of the chemoreceptor reflex in response to hypoxia. Abnormalities in SP neurotransmission may therefore result in autonomic dysfunction during sleep and contribute to SIDS deaths. [125I] Bolton Hunter SP autoradiography was used to map the distribution and density of the SP, NK1R to 13 specific nuclei intimately related to cardiorespiratory function and autonomic control in the human infant medulla of 55 SIDS and 21 control (non-SIDS) infants. Compared to controls, SIDS cases exhibited a differential, abnormal developmental profile of the SP/NK1R system in the medulla. Furthermore the study revealed significantly decreased NK1R binding within key medullary nuclei in SIDS cases, principally in the nucleus tractus solitarii (NTS) and all three subdivisions of the inferior portion of the olivo-cerebellar complex; the principal inferior olivary complex (PIO), medial accessory olive (MAO) and dorsal accessory olive (DAO). Altered NK1R binding was significantly influenced by prematurity and male sex, which may explain the increased risk of SIDS in premature and male infants. Abnormal NK1R binding in these medullary nuclei may contribute to the defective interaction of critical medullary mechanisms with cerebellar sites, resulting in an inability of a SIDS infant to illicit appropriate respiratory and motor responses to life threatening challenges during sleep. These observations support the concept that abnormalities in a multi-neurotransmitter network within key nuclei of the medullary homeostatic system may underlie the pathogenesis of a subset of SIDS cases. PMID:28931039

Neutral endopeptidase inhibitor potentiates the tachykinin-induced increase in ciliary beat frequency in rabbit trachea.

PubMed

Kondo, M; Tamaoki, J; Takizawa, T

1990-08-01

We used cultured rabbit tracheal epithelium to determine the effect of mammalian-derived tachykinin on airway ciliary activity and its modulation by neutral endopeptidase EC 3.4.24.11 (NEP). Neurokinin A (NKA) caused dose-dependent increases in ciliary beat frequency (CBF), as measured by a photoelectric method, with the maximal increase from the baseline 15.7 +/- 1.7% (mean +/- SEM, p less than 0.01), whereas substance P (SP) had no effect. The NKA-induced increase in CBF was not inhibited by phentolamine, propranolol, or atropine, but it was abolished by the tachykinin antagonist [D-Pro2, D-Trp7,9]SP. Pretreatment of tissue with thiorphan (10(-5) M), a NEP inhibitor, had little effect on CBF responses to NKA; however, it significantly potentiated the responses to SP (14.9 +/- 3.0%, p less than 0.01). Other peptidase inhibitors, including captopril, bestatin, and leupeptin, did not alter the tachykinin-induced CBF response, suggesting that angiotensin converting enzyme, aminopeptidases, and serine proteinases do not modulate ciliary activity in response to tachykinins. These results suggest that NKA increases CBF by acting directly on tachykinin receptors and that NEP may play a role in modulating the tachykinin-induced stimulatory effects on CBF.

Effect of substance P and other tachykinins on arterial pressure in guinea-pigs.

PubMed

Hancock, J C; Hoover, D B

1985-03-01

The blood pressure and respiratory effects of i.v. administration of the tachykinins substance P (SP), physalaemin (P), eledoisin (E) and kassinin (K) and purported antagonists of SP were compared in guinea-pigs anaesthetized with sodium pentobarbital. Tachykinins caused a dose-dependent decrease in diastolic and systolic pressure with systolic pressure decreased more than diastolic. Heart rate was not affected. Duration of response was directly related to dosage. These data are in agreement with observations that tachykinins decrease peripheral vascular resistance in other species. Tachyphylaxis did not develop to the vascular actions of tachykinins. Comparison of ED50's demonstrated a rank order of potency of SP greater than P congruent to E greater than K suggesting that the vascular receptor for SP is of the SP-P type. Analysis of the regression lines for log dose of tachykinin vs. percent decrease in diastolic blood pressure revealed similar slopes for SP and E and for P and K. The maximal response caused by P was greater than that caused by SP, E or K. These observations are not consistent with postulated classifications of tachykinins or tachykinin receptors suggesting that undefined tissue factors may have affected the relative in vivo potencies of these peptides. Apnoea occurred with K and E throughout the effective dosage range. SP caused apnoea only in doses in excess of those causing maximal vasodilation. P did not cause apnoea. These observations suggest that the SP-receptor mediating respiratory depression is of the SP-E type.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeting tachykinin receptors in neuroblastoma.

PubMed

Henssen, Anton G; Odersky, Andrea; Szymansky, Annabell; Seiler, Marleen; Althoff, Kristina; Beckers, Anneleen; Speleman, Frank; Schäfers, Simon; De Preter, Katleen; Astrahanseff, Kathy; Struck, Joachim; Schramm, Alexander; Eggert, Angelika; Bergmann, Andreas; Schulte, Johannes H

2017-01-03

Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma.

Neurokinin receptor modulation of respiratory activity in the rabbit.

PubMed

Bongianni, Fulvia; Mutolo, Donatella; Cinelli, Elenia; Pantaleo, Tito

2008-06-01

The respiratory role of neurokinin (NK) receptors was investigated in alpha-chloralose-urethane-anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30-50 nL) of NK receptor agonists and antagonists. Microinjections were performed in a region located just caudal to the rostral expiratory neurons. This region displayed features similar to those of the pre-Bötzinger complex (pre-BötC) of adult cats and rats, and proved to produce excitatory respiratory effects in response to microinjections of D,L-homocysteic acid. We used as agonists (0.1, 0.5 and 5 mM) substance P (SP), the NK1 receptor agonists [Sar(9), Met(O2)(11)]-SP and GR 73632, the NK2 receptor agonist NKA, the NK3 receptor agonist senktide, and as antagonists (5 mM) the NK1 receptor antagonist CP-99,994 and the NK2 receptor antagonist MEN 10376. SP always increased respiratory frequency, but NK1 receptor agonists did not change respiratory variables. NKA and senktide at 5 mm increased respiratory frequency. CP-99,994 caused increases in respiratory frequency and did not antagonize the effects of SP. MEN 10376 prevented the respiratory responses induced by NKA and reduced those provoked by SP. SP or the NK1 receptor agonists (5 mM) injected (1 microL) into the IV ventricle caused marked excitatory effects on respiration. The results suggest that NK2 and NK3, but not NK1, receptors are involved in the excitatory modulation of inspiratory activity within the investigated region and are consistent with the notion that the pre-BötC neurons are important components of the inspiratory rhythm-generating mechanisms.

Development of novel neurokinin 3 receptor (NK3R) selective agonists with resistance to proteolytic degradation.

PubMed

Misu, Ryosuke; Oishi, Shinya; Yamada, Ai; Yamamura, Takashi; Matsuda, Fuko; Yamamoto, Koki; Noguchi, Taro; Ohno, Hiroaki; Okamura, Hiroaki; Ohkura, Satoshi; Fujii, Nobutaka

2014-10-23

Neurokinin B (NKB) regulates the release of gonadotropin-releasing hormone (GnRH) via activation of the neurokinin-3 receptor (NK3R). We evaluated the biological stability of NK3R selective agonists to develop novel NK3R agonists to regulate reproductive functions. On the basis of degradation profiles, several peptidomimetic derivatives were designed. The modification of senktide with (E)-alkene dipeptide isostere generated a novel potent NK3R agonist with high stability and prolonged bioactivity.

The contractile effect of anandamide in the guinea-pig small intestine is mediated by prostanoids but not TRPV1 receptors or capsaicin-sensitive nerves.

PubMed

Dékány, András; Benko, Rita; Szombati, Veronika; Bartho, Lorand

2013-05-01

Although exogenous and endogenous cannabinoid receptor agonists have well-documented inhibitory effects on gastrointestinal motility, a TRPV1 receptor-mediated excitatory action of anandamide (arachidonoyl ethanolamide, AEA) in the guinea-pig ileum strip has also been described. We used in vitro capsaicin desensitization for assessing the possible participation of sensory neurons in the contractile effect of anandamide on the guinea-pig whole ileum, as well as autonomic drugs and a cyclooxygenase inhibitor for characterizing this response. Isolated organ experiments were used with isotonic recording. Contractions induced by anandamide (1 or 10 μM) were strongly inhibited by tetrodotoxin, indomethacin or atropine plus a tachykinin NK(1) receptor antagonist, but weakly to moderately reduced by atropine alone and partly diminished by the fatty acid amide hydrolase inhibitor URB 597. Neither capsaicin pre-treatment nor the TRPV1 receptor antagonist BCTC, the ganglionic blocking drug hexamethonium or cannabinoid (CB1 or CB2 ) receptor antagonists, influenced the effect of anandamide. It is concluded that the capsaicin-insensitive, neuronal excitatory effect of anandamide in the intestine is most probably mediated by cyclooxygenase products. Such a mechanism may also play a role at other sites in the mammalian body. © 2012 Nordic Pharmacological Society. Published by Blackwell Publishing Ltd.

Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction in ferret trachea.

PubMed

Sekizawa, K; Tamaoki, J; Graf, P D; Basbaum, C B; Borson, D B; Nadel, J A

1987-12-01

To determine the roles of endogenous enkephalinase (EC.3.4.24.11) in regulating tachykinin-induced contraction of airway smooth muscle, the authors studied the effects of the enkephalinase inhibitor leucine-thiorphan on the contractile responses to substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) in isolated ferret tracheal smooth muscle segments. Leucine-thiorphan shifted, in concentration-dependent fashions, the dose-response curves to all tachykinins to lower concentrations. Leucine-thiorphan changed the rank order of tachykinin potency from NKA greater than SP greater than NKB to NKA = NKB greater than SP. Removal of the epithelium slightly enhanced the contractile responses to SP and NKA but not to NKB. Atropine shifted the dose-response curves of all tachykinins to higher concentrations. Each tachykinin increased the contractile response to electrical field stimulation (5 Hz, 20 sec of duration, 20 V) in a dose-dependent fashion. This effect was not altered by hexamethonium, indomethacin, BW755C or naloxone but was potentiated by leucine-thiorphan and inhibited by the tachykinin receptor antagonist (D-Pro2, D-Trp7,9)-SP and by atropine. Because tachykinins did not affect contractile responses to acetylcholine significantly, their effects were probably on presynaptic postganglionic nerves. Captopril, bestatin and leupeptin did not alter contractile responses, suggesting that angiotensin converting enzyme, aminopeptidases and serine proteinases did not modulate tachykinin-induced effects. Enkephalinase immunofluorescence was found in the smooth muscle and epithelium and confirmed the authors' finding of enkephalinase-like activity in the muscle. The results suggest that tracheal enkephalinase is an important modulator of tachykinin-induced effects.

Bilateral increase in expression and concentration of tachykinin in a unilateral rabbit muscle overuse model that leads to myositis

PubMed Central

2013-01-01

Background Tachykinins can have pro-inflammatory as well as healing effects during tissue reorganization and inflammation. Recent studies report an up-regulation in the expression of the substance P (SP)-preferred receptor, the neurokinin-1 receptor, in marked muscle inflammation (myositis). There is, however, only very little information on the expression patterns and levels of tachykinins in this situation. Methods The tachykinin system was analyzed using a rabbit experimental model of muscle overuse, whereby unilateral muscle exercise in combination with electrical stimulation led to muscle derangement and myositis in the triceps surae muscle (experimental length 1–6 weeks). Evaluations were made for both parts of the muscle (soleus and gastrocnemius muscles) in experimental and non-experimental (contralateral) sides. Morphologic evaluation, immunohistochemistry, in situ hybridization and enzyme immunoassay (EIA) analyses were applied. Results Myositis and muscle derangement occurred focally not only in the experimental side but also in the non-experimental side. In the inflammatory areas (focal myositis areas), there were frequent nerve fibers showing tachykinin-like immunoreactivity and which were parts of nerve fascicles and which were freely dispersed in the tissue. Cells in the inflammatory infiltrates showed tachykinin-like immunoreactivity and tachykinin mRNA expression. Specific immunoreactivity and mRNA expression were noted in blood vessel walls of both sides, especially in focally affected areas. With increasing experimental length, we observed an increase in the degree of immunoreactivity in the vessel walls. The EIA analyses showed that the concentration of tachykinin in the tissue on both sides increased in a time-dependent manner. There was a statistical correlation in the concentration of tachykinin and the level of tachykinin immunoreactivity in the blood vessel walls between experimental and non-experimental sides. Conclusions The observations show an up-regulation of the tachykinin system bilaterally during muscle derangement/myositis in response to pronounced unilateral muscle overuse. This up-regulation occurred in inflammatory areas and was related not only to increased tachykinin innervation but also to tachykinin expression in blood vessel walls and inflammatory cells. Importantly, the tachykinin system appears to be an important factor not only ipsilaterally but also contralaterally in these processes. PMID:23587295

Bilateral increase in expression and concentration of tachykinin in a unilateral rabbit muscle overuse model that leads to myositis.

PubMed

Song, Yafeng; Stål, Per S; Yu, Ji-Guo; Forsgren, Sture

2013-04-12

Tachykinins can have pro-inflammatory as well as healing effects during tissue reorganization and inflammation. Recent studies report an up-regulation in the expression of the substance P (SP)-preferred receptor, the neurokinin-1 receptor, in marked muscle inflammation (myositis). There is, however, only very little information on the expression patterns and levels of tachykinins in this situation. The tachykinin system was analyzed using a rabbit experimental model of muscle overuse, whereby unilateral muscle exercise in combination with electrical stimulation led to muscle derangement and myositis in the triceps surae muscle (experimental length 1-6 weeks). Evaluations were made for both parts of the muscle (soleus and gastrocnemius muscles) in experimental and non-experimental (contralateral) sides. Morphologic evaluation, immunohistochemistry, in situ hybridization and enzyme immunoassay (EIA) analyses were applied. Myositis and muscle derangement occurred focally not only in the experimental side but also in the non-experimental side. In the inflammatory areas (focal myositis areas), there were frequent nerve fibers showing tachykinin-like immunoreactivity and which were parts of nerve fascicles and which were freely dispersed in the tissue. Cells in the inflammatory infiltrates showed tachykinin-like immunoreactivity and tachykinin mRNA expression. Specific immunoreactivity and mRNA expression were noted in blood vessel walls of both sides, especially in focally affected areas. With increasing experimental length, we observed an increase in the degree of immunoreactivity in the vessel walls. The EIA analyses showed that the concentration of tachykinin in the tissue on both sides increased in a time-dependent manner. There was a statistical correlation in the concentration of tachykinin and the level of tachykinin immunoreactivity in the blood vessel walls between experimental and non-experimental sides. The observations show an up-regulation of the tachykinin system bilaterally during muscle derangement/myositis in response to pronounced unilateral muscle overuse. This up-regulation occurred in inflammatory areas and was related not only to increased tachykinin innervation but also to tachykinin expression in blood vessel walls and inflammatory cells. Importantly, the tachykinin system appears to be an important factor not only ipsilaterally but also contralaterally in these processes.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

PubMed

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W; Vanden Broeck, Jozef; Tourwé, Dirk

2011-04-14

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

Photoaffinity labeling the substance P receptor using a derivative of substance P containing para-benzoylphenylalanine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyd, N.D.; White, C.F.; Leeman, S.E.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine (L-Phe(pBz)) in place of the Phe{sup 8} residue of SP. (Phe{sup 8}(OpBz))SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the {sup 125}I-labeled Bolton-Hunter conjugate of (Phe{sup 8}(pBz))SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites with an affinity K{sub D} = 0.4 nM. The binding of {sup 125}I-(Phe{sup 8}(pBz))SP was inhibited competitivelymore » by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70{percent} of the specifically bound {sup 125}I-(Phe{sup 8}(pBz))SP underwent covalent linkage to two polypeptides of M{sub r} = 53 000 and 46 000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on {sup 125}I-(Phe{sup 8}(pBz))SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies. The highly specific and remarkably efficient photolabeling achieved with {sup 125}I-(Phe{sup 8}(pBz))SP suggests that this photoaffinity probe will be of considerable value for the characterization of the molecular structure of the SP receptor.« less

Behavioural and biochemical responses following activation of midbrain dopamine pathways by receptor selective neurokinin agonists.

PubMed

Elliott, P J; Mason, G S; Stephens-Smith, M; Hagan, R M

1991-06-01

Preferential activation of mesolimbic and nigro-striatal dopamine (DA) pathways by receptor-selective and peptidase-resistant neurokinin (NK) agonists is reported. The DA cell body region of the mesolimbic pathway appears to be activated by NK agonists selective for NK-1 and NK-3 receptors whereas the DA cell bodies in the substantia nigra are under an excitatory NK-2 receptor-mediated influence. Stimulation of the mesolimbic DA pathway by NK-1 (Ava[L-Pro9,N-Me-Leu10]SP (7-11) [GR73632]) or NK-3 (Senktide) agonists increase locomotor activity. Additional studies showed that this elevated motor response observed after intra-VTA infusion of GR73632 was accompanied by a corresponding increase in DA turnover in the terminal fields of this pathway. Similarly, unilateral activation of the nigro-striatal DA pathway by NK-2 selective agonists (Ava (D-Pro9) SP (7-11) [GR51667] or [Lys3,Gly8,R-Lac-Leu9]NKA (3-10) [GR64349]) elicit contralateral rotational activity and an increase in DA turnover in the ipsilateral striatum. The rotational response was attenuated by prior administration of an NK-2 antagonist (cyclo (Gln, Trp, Phe, Gly, Leu, Met)] L-659877]) into the nigra. Peripheral injection of haloperidol, a DA antagonist, also blocked the NK-2 agonist induced rotations.

Pharmacological blockade or genetic deletion of substance P (NK(1)) receptors attenuates neonatal vocalisation in guinea-pigs and mice.

PubMed

Rupniak, N M; Carlson, E C; Harrison, T; Oates, B; Seward, E; Owen, S; de Felipe, C; Hunt, S; Wheeldon, A

2000-06-08

The regulation of stress-induced vocalisations by central NK(1) receptors was investigated using pharmacological antagonists in guinea-pigs, a species with human-like NK(1) receptors, and transgenic NK1R-/- mice. In guinea-pigs, i.c.v. infusion of the selective substance P agonist GR73632 (0.1 nmol) elicited a pronounced vocalisation response that was blocked enantioselectively by the NK(1) receptor antagonists CP-99,994 and L-733,060 (0.1-10 mg/kg). GR73632-induced vocalisations were also markedly attenuated by the antidepressant drugs imipramine and fluoxetine (30 mg/kg), but not by the benzodiazepine anxiolytic diazepam (3 mg/kg) or the 5-HT(1A) agonist buspirone (10 mg/kg). Similarly, vocalisations in guinea-pig pups separated from their mothers were blocked enantioselectively by the highly brain-penetrant NK(1) receptor antagonists L-733,060 and GR205171 (ID(50) 3 mg/kg), but not by the poorly brain-penetrant compounds LY303870 and CGP49823 (30 mg/kg). Separation-induced vocalisations were also blocked by the anxiolytic drugs diazepam, chlordiazepoxide and buspirone (ID(50) 0.5-1 mg/kg), and by the antidepressant drugs phenelzine, imipramine, fluoxetine and venlafaxine (ID(50) 3-8 mg/kg). In normal mouse pups, GR205171 attenuated neonatal vocalisations when administered at a high dose (30 mg/kg) only, consistent with its lower affinity for the rat than the guinea-pig NK(1) receptor. Ultrasound calls in NK1R-/- mouse pups were markedly reduced compared with those in WT pups, confirming the specific involvement of NK(1) receptors in the regulation of vocalisation. These observations suggest that centrally-acting NK(1) receptor antagonists may have clinical utility in the treatment of a range of anxiety and mood disorders.

Colonic smooth muscle responses in patients with diverticular disease of the colon: effect of the NK2 receptor antagonist SR48968.

PubMed

Maselli, M A; Piepoli, A L; Guerra, V; Caruso, M L; Pezzolla, F; Lorusso, D; Demma, I; De Ponti, F

2004-05-01

Little is known about the pathophysiology of diverticular disease. To compare passive and active stress and the response to carbachol of colonic smooth muscle specimens from patients with diverticular disease and patients with colon cancer. The effect of the NK2 receptor antagonist, SR48968, on electrically evoked contractions of circular muscle was also investigated. Sigmoid colon segments were obtained from 16 patients (51-83 years) undergoing elective sigmoid resection for diverticular disease and 39 patients (50-88 years) undergoing left hemicolectomy for non-obstructive sigmoid colon cancer. Isometric tension was measured on circular or longitudinal taenial muscle. Strips were stretched gradually to Lo (length allowing the development of optimal active tension with carbachol) and were also exposed to increasing carbachol concentrations. The effects of atropine, tetrodotoxin and SR48968 on electrically evoked (supramaximal strength, 0.3 ms, 0.1-10 Hz) contractions of circular strips from 8 patients with diverticular disease and 19 patients with colon cancer were also studied. Both passive and active stress in circular muscle strips obtained from patients with diverticular disease was higher than in patients with colon cancer (P < 0.05). Electrically evoked contractions were significantly reduced by atropine in all preparations and were virtually suppressed by combined SR48968 and atropine. Tetrodotoxin suppressed electrically evoked contractions only in patients with colon cancer, whereas a tetrodotoxin-resistant component was identified in patients with diverticular disease. The changes in both passive and active stress in specimens from patients with diverticular disease may reflect circular smooth muscle dysfunction. Acetylcholine and tachykinins are the main excitatory neurotransmitters mediating electrically evoked contractions in human sigmoid colon circular muscle.

Both exogenous 5-HT and endogenous 5-HT, released by fluoxetine, enhance distension evoked propulsion in guinea-pig ileum in vitro.

PubMed

Gwynne, Rachel M; Clarke, Amanda J; Furness, John B; Bornstein, Joel C

2014-01-01

The roles of 5-HT3 and 5-HT4 receptors in the modulation of intestinal propulsion by luminal application of 5-HT and augmentation of endogenous 5-HT effects were studied in segments of guinea-pig ileum in vitro. Persistent propulsive contractions evoked by saline distension were examined using a modified Trendelenburg method. When 5-HT (30 nM), fluoxetine (selective serotonin reuptake inhibitor; 1 nM), 2-methyl-5-HT (5-HT3 receptor agonist; 1 mM), or RS 67506 (5-HT4 receptor agonist, 1 μM) was infused into the lumen, the pressure needed to initiate persistent propulsive activity fell significantly. A specific 5-HT4 receptor antagonist, SB 207266 (10 nM in lumen), abolished the effects of 5-HT, fluoxetine, and RS 67506, but not those of 2-methyl-5-HT. Granisetron (5-HT3 receptor antagonist; 1 μM in lumen) abolished the effect of 5-HT, fluoxetine, RS 67506, and 2-methyl-5-HT. The NK3 receptor antagonist SR 142801 (100 nM in lumen) blocked the effects of 5-HT, fluoxetine, and 2-methyl-5-HT. SB 207266, granisetron, and SR 142801 had no effect by themselves. Higher concentrations of fluoxetine (100 and 300 nM) and RS 67506 (3 and 10 μM) had no effect on the distension threshold for propulsive contractions. These results indicate that luminal application of exogenous 5-HT, or increased release of endogenous mucosal 5-HT above basal levels, acts to lower the threshold for propulsive contractions in the guinea-pig ileum via activation of 5-HT3 and 5-HT4 receptors and the release of tachykinins. The results further indicate that basal release of 5-HT is insufficient to alter the threshold for propulsive motor activity.

Evidence for the presence of NK1 and NK3 receptors on cholinergic neurones in the guinea-pig ileum.

PubMed

Legat, F J; Althuber, P; Maier, R; Griesbacher, T; Lembeck, F

1996-03-29

In a guinea-pig ileum longitudinal muscle preparation, substance P (SP) (> or = 6 nM) caused an initial contraction followed by a sustained plateau contraction of about 20-50% of the initial response. This plateau contraction is caused by the SP-induced activation of cholinergic motoneurones which contract the smooth muscles by the released acetylcholine (ACh). We investigated the contribution of neurokinin NK1 and NK3 receptors during this 'plateau phase' of contraction. The plateau contraction induced by SP (60 nM) was significantly reduced by the NK1 receptor antagonist CP-96,345 (200 nM) added 5 min after SP, but was not affected by its inactive enantiomer CP-96,344 (200 nM). The NK1 receptor antagonist CP-99,994 (100 nM) significantly reduced the plateau contraction induced by SP (60 nM and 600 nM) and that induced by the NK1 receptor agonist substance P-O-methylester (SPOMe; 100 nM). CP-99,994 (100 nM), however did not affect the plateau contraction induced by the NK3 receptor agonist [Asp5,6, MePhe8]-SP(5-11) (100 nM). The plateau contraction induced by SP (600 nM) was not affected by the NK3 receptor antagonist SR-142,801 (100 nM), added 5 min after SP. Pre-incubation of the ileum with SR-142,801 (100 nM) 30 min prior to the addition of SP (600 nM) also had no significant effect on the plateau contraction. However, it significantly reduced the ileal contraction in the first minutes after the initial spasmogenic contraction. We suggest that SP induces the plateau contraction of the guinea-pig ileum longitudinal muscle mainly by the activation of NK1 receptors on cholinergic neurones.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist

PubMed Central

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N.; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W.; Broeck, Jozef Vanden; Tourwé, Dirk

2011-01-01

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3′,5′-(CF3)2-Bn], 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn] and 23 [Ac-Tic-NMe-3′,5′-(CF3)2-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], which combines the N-terminus of the established Dmt1-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH2) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, i.e. Dmt-D-Arg-Aba-Gly-NH2 36, also proved to be an extremely potent and balanced μ- and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity. PMID:21413804

Characterization of Substance P processing in mouse spinal cord S9 fractions using high-resolution Quadrupole-Orbitrap mass spectrometry.

PubMed

Saidi, Mouna; Kamali, Soufiane; Beaudry, Francis

2016-10-01

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins and Substance P (SP) levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinins from neurons. Proteolysis is suspected to regulate extracellular SP concentrations but few studies were conducted on the metabolism of proneuropeptides and neuropeptides. Here, we provide evidence that proteolysis controls SP levels in the spinal cord leading to the formation of active C-terminal fragments. Using high-resolution mass spectrometry, specific tachykinins fragments were characterized and quantified. The metabolic stability of β-Tachykinin 58-71 and SP were very short resulting in half-life of 5.7 and 3.5min respectively. Several C-terminal fragments were identified, including SP 3-11 , SP 5-11 and SP 8-11 , which conserve affinity for the Neurokinin 1 receptor. Interestingly, the metabolic stability of C-terminal fragments was significantly superior. Two specific Prolyl endopeptidase inhibitors were used and showed a significant reduction in the rate of formation of SP 3-11 and SP 5-11 providing strong evidence that Prolyl endopeptidase is involved into N-terminal processing of SP in the spinal cord. Copyright © 2016 Elsevier Ltd. All rights reserved.

Role of tachykinins in airway responses to ozone in rats.

PubMed

Takebayashi, T; Abraham, J; Murthy, G G; Lilly, C; Rodger, I; Shore, S A

1998-08-01

Previous studies that used neonatal capsaicin (Cap) treatment to ablate C fibers indicate that C fibers act to inhibit lung damage and airway hyperresponsiveness after ozone (O3) exposure in rats. The purpose of this study was to determine 1) the role of tachykinins in these protective effects and 2) whether differences in minute ventilation (VE) during O3 exposure might account for the effect of Cap. In the first study, male Sprague-Dawley rats were exposed to 1 part/million O3 or air for 3 h. Four hours later, a bronchoalveolar lavage (BAL) was performed or airway responsiveness was measured. Rats were treated with CP-99994 and SR-48968, selective neurokinin-1- and -2-receptor antagonists, respectively, or with vehicle (Veh). O3 caused an increase in the number of neutrophils recovered from BAL fluid in both the Veh-treated and tachykinin-receptor antagonist (TKRA)-treated rats, but the number of neutrophils was approximately twofold greater in the TKRA-treated rats. In contrast, TKRA treatment had no effect on baseline pulmonary mechanics or airway responsiveness. After O3 exposure, the number of neutrophils in BAL fluid was also greater in Cap- than in Veh-treated rats. O3 reduced VE in both Veh- and Cap-treated rats, but the response was greater (reduction of 44.7 +/- 3.7 vs. 27.8 +/- 6.8%) and occurred earlier (10 vs. 70 min) in Cap- than in Veh-treated rats (P < 0.02). These results suggest that tachykinins mediate protective effects of C fibers against O3-induced lung inflammation. The results also indicate that the more pronounced effect of O3 on BAL neutrophils in Cap-treated rats is not the result of a greater inhaled dose of O3 resulting from greater VE.

The effect of compound 48/80 on contractions induced by toluene diisocyanate in isolated guinea-pig bronchus.

PubMed

Mapp, C E; Boniotti, A; Papi, A; Chitano, P; Coser, E; Di Stefano, A; Saetta, M; Ciaccia, A; Fabbri, L M

1993-06-01

We have investigated the ability of compound 48/80 and of histamine H1 and H2 receptor antagonists to inhibit toluene diisocyanate-induced contractions in isolated guinea-pig bronchi. Compound 48/80 (100 micrograms/ml) significantly inhibited toluene diisocyanate-induced contractions. By contrast, the two histamine H1 and H2 receptor antagonists, chlorpheniramine (10 microM) and cimetidine, (10 microM) did not affect toluene diisocyanate-induced contractions, but significantly inhibited contractions induced by exogenously applied histamine (100 microM) and by 48/80. We investigated which mechanisms 48/80 used to inhibit toluene diisocyanate-induced contractions, paying particular attention to the possible involvement of capsaicin-sensitive primary afferents. In vitro capsaicin desensitization (10 microM for 30 min followed by washing) significantly reduced compound 48/80-induced contractions. A capsaicin-resistant component of contraction was also evident. Ruthenium red (3 microM), an inorganic dye which acts as a selective functional antagonist of capsaicin, did not affect 48/80-induced contraction. MEN 10,207 (Tyr5,D-Trp6,8,9,Arg10)-neurokinin A (4-10) (3 microM) a selective antagonist of NK2-tachykinin receptors significantly reduced 48/80-induced contractions. These results show that compound 48/80 inhibits toluene diisocyanate-induced contractions in isolated guinea-pig bronchi. It is likely that two mechanisms are involved in the inhibition: (1) the release of mediators other than histamine by mast cells, (2) an effect of 48/80 on sensory nerves.

Specific neurokinin receptors mediate plasma extravasation in the rat knee joint.

PubMed Central

Lam, F. Y.; Ferrell, W. R.

1991-01-01

1 Plasma extravasation in the rat knee joint was induced by intra-articular injection of neurokinins and specific neurokinin receptor agonists. 2 Pronounced plasma extravasation was produced by substance P (SP, 4-185 microM) and to a lesser extent by neurokinin-B (NKB, 83-413 microM), whereas neurokinin-A (NKA, 88-440 microM) and calcitonin gene-related peptide (CGRP, 26-130 microM) had no significant effect. 3 The specific neurokinin1 receptor agonist [Sar9, Met(O2)11]-substance P (NK1 agonist) in doses of 0.4-70 microM appeared to be more potent than SP in eliciting plasma extravasation. The neurokinin2 receptor agonist [Nle10]-neurokinin A4-10 (NK2 agonist) was not effective at 70 microM but produced a small and significant effect at 330 microM, whereas the neurokinin3 receptor agonist [MePhe7]-neurokinin B (NK3 agonist) was without effect at 40 microM or 400 microM. 4 Injections of SP or NKA into the synovial cavity of the rat knee were equally effective in producing marked plasma extravasation in remote sites such as the forelimb and hindlimb paws. 5 Co-administration experiments showed that the effects of SP were synergistic with NKA or the NK1 receptor agonist, but not with CGRP or the NK2 receptor agonist. 6 The rank order of potency was NK1 agonist greater than or equal to SP greater than NKB greater than NK2 agonist suggesting that NK1 receptors mediate plasma extravasation in the rat knee joint. PMID:1715229

Receptor-selective, peptidase-resistant agonists at neurokinin NK-1 and NK-2 receptors: new tools for investigating neurokinin function.

PubMed

Hagan, R M; Ireland, S J; Jordan, C C; Beresford, I J; Deal, M J; Ward, P

1991-06-01

The pharmacological profiles of two novel neurokinin agonists have been investigated. delta Ava[L-Pro9,N-MeLeu10]SP(7-11) (GR73632) and [Lys3,Gly8-R-gamma-lactam-Leu9] NKA(3-10) (GR64349) are potent and selective agonists at NK-1 and NK-2 receptors respectively. In the guinea-pig isolated trachea preparation, contractions induced by these agonists were largely unaffected by inclusion of peptidase inhibitors in the bathing medium, indicating that these agonists are resistant to metabolism by peptidases. In the anaesthetised guinea-pig, both agonists were more potent bronchoconstrictor agents than either NKA or the SP analogue, SP methylester. In the anaesthetised rat, the NK-1 agonist, GR73632 was more potent than SP, NKA or NKB at causing the histamine-independent extravasation of plasma proteins into the skin after intradermal administration. The NK-2 agonist, GR64349 and the NK-3 agonist, senktide were without significant effect in this model. These agonists are useful tools for characterizing neurokinin receptor-mediated actions both in vitro and in vivo.

Ava[L-Pro9,N-MeLeu10] substance P(7-11) (GR 73632) and Sar9, Met(O2)11 increase distention-induced peristalsis through activation of neurokinin-1 receptors on smooth muscle and interstitial cells of cajal.

PubMed

Nieuwmeyer, Florentine; Ye, Jing; Huizinga, Jan D

2006-04-01

Substance P is generally considered an excitatory neurotransmitter related to gut motor activity, although an inhibitory influence of neurokinin-1 (NK1) receptor activation on peristalsis has also been reported. With an optimized in vitro method to assess distention-induced peristalsis, our aim was to clarify the effect of NK1 receptor activation on peristaltic activity and to reveal the mechanisms by which NK1 activation alters peristalsis. Distention of the small intestine of the mouse and guinea pig induced periodic occurrence of rhythmic waves of propagating rings of circular muscle contraction, associated with slow waves and superimposed action potentials, that propelled intestinal contents aborally. Activation of NK1 receptors by Ava[l-Pro(9),N-MeLeu10] substance P(7-11) (GR 73632) and Sar(9), Met(O(2))(11) on smooth muscle cells resulted in prolongation of the activity periods and increased action potential generation occurring superimposed on the intestinal slow wave activity. Activation of NK1 receptors on interstitial cells of Cajal resulted in an increase in slow wave frequency. Slow wave amplitude increased, likely by increased cell-to-cell coupling. The NK1 antagonist (S)-1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR 140333) induced a decrease in the slow wave frequency and duration of the activity periods evoked by distention, which makes it likely that NK1 receptor activation plays a role in the normal physiological distention-induced generation of peristaltic motor patterns. In summary, NK1 receptors play a role in normal development of peristalsis and NK1 receptor activation markedly increases propulsive peristaltic contractile activity.

NK cell activation: distinct stimulatory pathways counterbalancing inhibitory signals.

PubMed

Bakker, A B; Wu, J; Phillips, J H; Lanier, L L

2000-01-01

A delicate balance between positive and negative signals regulates NK cell effector function. Activation of NK cells may be initiated by the triggering of multiple adhesion or costimulatory molecules, and can be counterbalanced by inhibitory signals induced by receptors for MHC class I. A common pathway of inhibitory signaling is provided by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domains of these receptors which mediate the recruitment of SH2 domain-bearing tyrosine phosphate-1 (SHP-1). In contrast to the extensive progress that has been made regarding the negative regulation of NK cell function, our knowledge of the signals that activate NK cells is still poor. Recent studies of the activating receptor complexes have shed new light on the induction of NK cell effector function. Several NK receptors using novel adaptors with immunoreceptor tyrosine-based activation motifs (ITAMs) and with PI 3-kinase recruiting motifs have been implicated in NK cell stimulation.

Role of Neurokinin 3 Receptor Signaling in Oral Squamous Cell Carcinoma.

PubMed

Obata, Kyoichi; Shimo, Tsuyoshi; Okui, Tatsuo; Matsumoto, Kenichi; Takada, Hiroyuki; Takabatake, Kiyofumi; Kunisada, Yuki; Ibaragi, Soichiro; Yoshioka, Norie; Kishimoto, Koji; Nagatsuka, Hitoshi; Sasaki, Akira

2017-11-01

The neurokinin 3 receptor (NK-3R) is differentially expressed in the central nervous system including cases of human oral squamous cell carcinoma. However, the role of NK-3R signaling in oral squamous cell carcinoma is not well known. NK-3R expression in surgically resected oral squamous cell carcinoma was examined immunohistochemically and the strength of the expression was quantified. We evaluated the function of NK-3R signaling using NK-3R antagonist in human oral squamous cell carcinoma bone invasion mouse model. NK-3R was significantly expressed in tumor cells that had invaded the bone matrix compared to the oral side tumor cells. SB222200, a selective antagonist of NK-3R, significantly suppressed the radiographic osteolytic lesion and tumorigenesis. NK-3R signaling is a potential target for the treatment of oral squamous cell carcinoma in cases of bone destruction. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Neurokinin-induced changes in pial artery diameter in the anaesthetized guinea-pig.

PubMed Central

Beattie, D. T.; Stubbs, C. M.; Connor, H. E.; Feniuk, W.

1993-01-01

1. The effects of selective neurokinin agents on pial artery diameter, measured with an on-line image analyser, have been studied in anaesthetized guinea-pigs in order to characterize the neurokinin receptors present on pial arteries. 2. Perivascular injection of either substance P (0.01-1 microM) or the selective NK1 receptor agonists, substance P methyl ester (SPOMe, 0.01-1 microM) and GR73632 (0.1 microM), increased pial artery diameter. 3. In contrast, the selective NK2 receptor agonist, GR64349 (1 microM), produced a small vasoconstriction while the NK3 receptor-selective agonist, senktide (1 microM) was inactive. 4. Co-administration of GR82334 (1 microM), a selective NK1 receptor antagonist, inhibited the vasodilatation produced by SPOMe (0.1 microM) but not that caused by calcitonin gene-related peptide (CGRP, 0.01 microM). 5. The results are consistent with an involvement of NK1 receptors in the neurokinin-induced increase in guinea-pig pial artery diameter. PMID:7679026

Association between Kinin B1 Receptor Expression and Leukocyte Trafficking across Mouse Mesenteric Postcapillary Venules

PubMed Central

McLean, Peter G.; Ahluwalia, Amrita; Perretti, Mauro

2000-01-01

Using intravital microscopy, we examined the role played by B1 receptors in leukocyte trafficking across mouse mesenteric postcapillary venules in vivo. B1 receptor blockade attenuated interleukin (IL)-1β–induced (5 ng intraperitoneally, 2 h) leukocyte–endothelial cell interactions and leukocyte emigration (∼50% reduction). The B1 receptor agonist des-Arg9bradykinin (DABK), although inactive in saline- or IL-8–treated mice, caused marked neutrophil rolling, adhesion, and emigration 24 h after challenge with IL-1β (when the cellular response to IL-1β had subsided). Reverse transcriptase polymerase chain reaction and Western blot revealed a temporal association between the DABK-induced response and upregulation of mesenteric B1 receptor mRNA and de novo protein expression after IL-1β treatment. DABK-induced leukocyte trafficking was antagonized by the B1 receptor antagonist des-arg10HOE 140 but not by the B2 receptor antagonist HOE 140. Similarly, DABK effects were maintained in B2 receptor knockout mice. The DABK-induced responses involved the release of neuropeptides from C fibers, as capsaicin treatment inhibited the responses. Treatment with the neurokinin (NK)1 and NK3 receptor antagonists attenuated the responses, whereas NK2, calcitonin gene-related peptide, or platelet-activating factor receptor antagonists had no effect. Substance P caused leukocyte recruitment that, similar to DABK, was inhibited by NK1 and NK3 receptor blockade. Mast cell depletion using compound 48/80 reduced DABK-induced leukocyte trafficking, and DABK treatment was shown histologically to induce mast cell degranulation. DABK-induced trafficking was inhibited by histamine H1 receptor blockade. Our findings provide clear evidence that B1 receptors play an important role in the mediation of leukocyte–endothelial cell interactions in postcapillary venules, leading to leukocyte recruitment during an inflammatory response. This involves activation of C fibers and mast cells, release of substance P and histamine, and stimulation of NK1, NK3, and H1 receptors. PMID:10934225

A CONSTITUTIVELY ACTIVE FORM OF NEUROKININ 1 RECEPTOR AND NEUROKININ 1 RECEPTOR-MEDIATED APOPTOSIS IN GLIOBLASTOMAS

PubMed Central

Akazawa, Toshimasa; Kwatra, Shawn G.; Goldsmith, Laura E.; Richardson, Mark D.; Cox, Elizabeth A.; Sampson, John H.; Kwatra, Madan M.

2009-01-01

Previous studies have shown that neurokinin 1 receptor (NK1R) occurs naturally in human glioblastomas and its stimulation causes cell proliferation. In the present study we show that stimulation of NK1R in human U373 glioblastoma cells by substance P (SP) increases Akt phosphorylation by 2.5-fold, with an EC50 of 57 nM. Blockade of NK1R lowers basal phosphorylation of Akt, indicating the presence of a constitutively active form of NK1R; similar results are seen in U251 MG and DBTRG-05 glioblastoma cells. Linkage of NK1R to Akt implicates NK1R in apoptosis of glioblastoma cells. Indeed, treatment of serum-starved U373 cells with SP reduces apoptosis by 53 ± 1% (P < 0.05), and treatment with NK1R antagonist L-733,060 increases apoptosis by 64 ± 16 % (P < 0.01). Further, the blockade of NK1R in human glioblastoma cells with L-733,060 causes cleavage of Caspase-3 and proteolysis of poly (ADP-ribose) polymerase (PARP). Experiments designed to elucidate the mechanism of NK1R-mediated Akt phosphorylation revealed total involvement of non-receptor tyrosine kinase Src and PI-3-kinase, a partial involvement of epidermal growth factor receptor (EGFR), and no involvement of MEK. Taken together, the results of the present study indicate a key role for NK1R in glioblastoma apoptosis. PMID:19519779

N- and C-terminal substance P fragments: differential effects on striatal [3H]substance P binding and NK1 receptor internalization.

PubMed

Michael-Titus, A T; Blackburn, D; Connolly, Y; Priestley, J V; Whelpton, R

1999-07-13

N- and C-terminal substance P (SP) fragments increase striatal dopamine outflow at nanomolar concentrations. This contrasts with their low affinity for NK1 receptors. To explore this discrepancy, we investigated the interaction of SP and SP fragments with NK1 sites in fresh striatal slices, the same model used in the functional studies on dopamine outflow. [3H]SP bound specifically to one site (Kd = 6.6 +/- 0.9 nM; Bmax = 12.6 +/- 0.7 fmol/mg protein). [3H]SP binding was displaced by SP (IC50 = 11.8 nM), but not by SP(1-7) or SP(5-11), up to 10 microM. In contrast, 10 nM SP(1-7) or SP(5-11) induced significant internalization of the NK1 receptor, similar to that induced by SP. We suggest that SP fragments have high affinity for an NK1 receptor conformer which is different from that labelled by [3H]SP.

Soluble HLA-G dampens CD94/NKG2A expression and function and differentially modulates chemotaxis and cytokine and chemokine secretion in CD56bright and CD56dim NK cells.

PubMed

Morandi, Fabio; Ferretti, Elisa; Castriconi, Roberta; Dondero, Alessandra; Petretto, Andrea; Bottino, Cristina; Pistoia, Vito

2011-11-24

Soluble HLA-G (sHLA-G) inhibits natural killer (NK) cell functions. Here, we investigated sHLA-G-mediated modulation of (1) chemokine receptor and NK receptor expression and function and (2) cytokine and chemokine secretion in CD56bright and CD56dim NK cells. sHLA-G-treated or untreated peripheral blood (PB) and tonsil NK cells were analyzed for chemokine receptor and NK receptor expression by flow cytometry. sHLA-G down-modulated (1) CXCR3 on PB and tonsil CD56bright and CD56dim, (2) CCR2 on PB and tonsil CD56bright, (3) CX3CR1 on PB CD56dim, (4) CXCR5 on tonsil CD56dim, and (5) CD94/NKG2A on PB and tonsil CD56brigh) and CD56dim NK cells. Such sHLA-G-mediated down-modulations were reverted by adding anti-HLA-G or anti-ILT2 mAbs. sHLA-G inhibited chemotaxis of (1) PB NK cells toward CXCL10, CXCL11, and CX3CL1 and (2) PB CD56bright NK cells toward CCL2 and CXCL10. IFN-γ secretion induced by NKp46 engagement was inhibited by NKG2A engagement in untreated but not in sHLA-G-treated NK cells. sHLA-G up-regulated secretion of (1) CCL22 in CD56bright and CD56dim and (2) CCL2, CCL8, and CXCL2-CXCL3 in CD56dim PB NK cells. Signal transduction experiments showed sHLA-G-mediated down-modulation of Stat5 phosphorylation in PB NK cells. In conclusion, our data delineated novel mechanisms of sHLA-G-mediated inhibition of NK-cell functions.

Neurokinin subtype receptors mediating substance P contraction in immature rabbit airways.

PubMed

Kazem, E; John, C; Tanaka, D T

1996-01-01

Two-week-old rabbit tracheal smooth muscle (TSM) and bronchial smooth muscle (BSM) segments were placed in organ baths, and isometric contractions to substance P (SP) were obtained. In the presence of phosphoramidon (PHOS), a neutral endopeptidase inhibitor, BSM segments were significantly more reactive and sensitive to SP than TSM segments. Neither neostigmine (NEO) nor atropine (ATR) eliminated these regional differences. Airway contractile responses to: 1) Senktide (NK-3 agonist); 2) neurokinin A (NKA, a NK-2 agonist); and 3) Septide (a highly selective NK-1 agonist) were separately obtained. In the presence of PHOS and NEO, Senktide was virtually inactive in both BSM and TSM. In the presence of PHOS, NEO, and ATR, NKA was equipotent in all airway segments; in contrast, the Septide response was significantly more reactive in BSM than in TSM segments. After inhibition of NK-1 activity with GR 82334, a competitive NK-1 receptor antagonist, the regional differences in SP reactivity were greatly diminished. This latter indication of a NK-1 contribution was confirmed using Septide-mediated inactivation of NK-1 receptors whereby the regional differences in airway sensitivity to SP were eliminated. These findings indicate that both endogenous neutral endopeptidase activity as well as NK-1 and NK-2 receptor influences may modulate the contractile responses to SP in immature rabbit airways.

Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand.

PubMed

Laufer, R; Gilon, C; Chorev, M; Selinger, Z

1986-08-05

We have recently characterized a tachykinin receptor subtype (SP-N) whose preferred ligand is the mammalian neuropeptide, neurokinin B (Laufer, R., Wormser, U., Friedman, Z. Y., Gilon, C., Chorev, M., and Selinger, Z. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7444-7448). To investigate this novel tachykinin receptor, we have now prepared a radiolabeled peptide, N alpha-[( 125I]desamino-3-iodotyrosyl)-[Asp5,6, N-methyl-Phe8]substance P (5-11) heptapeptide (125I-BH-NH-Senktide), which selectively interacts with the SP-N receptor subtype. The binding of 125I-BH-NH-Senktide to rat cerebral cortex membranes was studied under conditions that minimized nonspecific binding. Unlike other tachykinin receptor probes, this radioligand is not degraded during the binding experiment. Binding of 125I-BH-NH-Senktide is reversible, saturable, and of high affinity (KD = 0.9 nM). The radioligand labels a single class of binding site (122 fmol binding sites/mg of protein), as indicated by a linear Scatchard plot and a Hill coefficient close to unity (nH = 1.05). The pharmacological specificity of this binding site corresponds to that of the neuronal SP-N receptor in guinea pig ileum myenteric plexus, which was determined by a functional bioassay. Among various rat brain regions, the highest binding was observed in the cerebral cortex, olfactory bulb, hypothalamus, and hippocampus. These results suggest the existence and specific distribution of a neurokinin B receptor site of the SP-N type in rat brain. 125I-BH-NH-Senktide is the first selective and potent probe for this receptor and is thus an important tool for further studies of its distribution, regulation, and functional role.

Lidocaine Stimulates the Function of Natural Killer Cells in Different Experimental Settings.

PubMed

Cata, Juan P; Ramirez, Maria F; Velasquez, Jose F; Di, A I; Popat, Keyuri U; Gottumukkala, Vijaya; Black, Dahlia M; Lewis, Valerae O; Vauthey, Jean N

2017-09-01

One of the functions of natural killer (NK) cells is to eliminate cancer cells. The cytolytic activity of NK cells is tightly regulated by inhibitory and activation receptors located in the surface membrane. Lidocaine stimulates the function of NK cells at clinically relevant concentrations. It remains unknown whether this effect of lidocaine has an impact on the expression of surface receptors of NK cells, can uniformly stimulate across different cancer cell lines, and enhances the function of cells obtained during oncological surgery. NK cells from healthy donors and 43 patients who had undergone surgery for cancer were isolated. The function of NK cells was measured by lactate dehydrogenase release assay. NK cells were incubated with clinically relevant concentrations of lidocaine. By flow cytometry, we determined the impact of lidocaine on the expression of galactosylgalactosylxylosylprotein3-beta-glucuronosytranferase 1, marker of cell maturation (CD57), killer cell lectin like receptor A, inhibitory (NKG2A) receptors and killer cell lectin like receptor D, activation (NKG2D) receptors of NK cells. Differences in expression at p<0.05 were considered statistically significant. Lidocaine increased the expression of NKG2D receptors and stimulated the function of NK cells against ovarian, pancreatic and ovarian cancer cell lines. Lidocaine also increased the cytolytic activity of NK cells from patients who underwent oncological surgery, except for those who had orthopedic procedures. Lidocaine showed an important stimulatory activity on NK cells. Our findings suggest that lidocaine might be used perioperatively to minimize the impact of surgery on NK cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.

PubMed

Garland, A M; Grady, E F; Payan, D G; Vigna, S R; Bunnett, N W

1994-10-01

Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.

Mouse natural killer cell development and maturation are differentially regulated by SHIP-1.

PubMed

Banh, Cindy; Miah, S M Shahjahan; Kerr, William G; Brossay, Laurent

2012-11-29

The SH2-containing inositol phosphatase-1 (SHIP-1) is a 5' inositol phosphatase known to negatively regulate the product of phosphoinositide-3 kinase (PI3K), phosphatidylinositol-3.4,5-trisphosphate. SHIP-1 can be recruited to a large number of inhibitory receptors expressed on natural killer (NK) cells. However, its role in NK cell development, maturation, and functions is not well defined. In this study, we found that the absence of SHIP-1 results in a loss of peripheral NK cells. However, using chimeric mice we demonstrated that SHIP-1 expression is not required intrinsically for NK cell lineage development. In contrast, SHIP-1 is required cell autonomously for NK cell terminal differentiation. These findings reveal both a direct and indirect role for SHIP-1 at different NK cell development checkpoints. Notably, SHIP-1-deficient NK cells display an impaired ability to secrete IFN-γ during cytokine receptor-mediated responses, whereas immunoreceptor tyrosine-based activation motif containing receptor-mediated responses is not affected. Taken together, our results provide novel insights on how SHIP-1 participates in the development, maturation, and effector functions of NK cells.

Role of substance P and neurokinin A in toluene diisocyanate-induced increased airway responsiveness in rabbits.

PubMed

Marek, W; Potthast, J J; Marcynski, B; Baur, X

1996-01-01

The aim of the present study was to examine the role of neuropeptides, especially substance P (SP) and neurokinin A (NKA), in toluene diisocyanate (TDI)-induced airway hyperresponsiveness (AHR) to acetylcholine aerosols. Thirty parts per billion of TDI in air administered over 4 hours caused a significant increase in the airway constrictive response to acetylcholine (ACH) aerosols in rabbits (DeltaRI: 245 +/- 30%, p < 0.005) without altering basic values of respiratory, cardiovascular or blood gas parameters. Inhalation of the aerosolized neuropeptides SP and NKA resulted in a similar increase in airway responsiveness (AR) to ACH as exposure to 30 ppb TDI. To determine whether neuropeptides contribute to TDI-induced AHR, we studied their effects after systemic treatment with capsaicin as well as after infusion of specific synthetic antagonists for SP and NK2 (NKA) receptors. CAPS treatment performed on 4 consecutive days as well as antagonists' infusion only moderately (p > 0.05) decreased airway responses to ACH. CAPS application prevented the TDI-induced increase in AR to ACH in all rabbits. The increase in airway resistance to ACH did not significantly change after TDI exposure (98 +/- 22% of the control response before TDI, p > 0.05). Simultaneous infusion of specific synthetic SP and NK2 receptor antagonists also abolished the TDI-induced increase in airway responses to ACH in all animals investigated (p > 0.05). The results of this study demonstrate that neuropeptides, especially the tachykinins SP and NKA, are important mediators in TDI-induced AHR in rabbits.

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy

PubMed Central

Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

2005-01-01

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with ≤ 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3− CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0·001) but increased to a normal level during the 24 months’ follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0·001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0·05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells. PMID:16045743

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy.

PubMed

Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

2005-09-01

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with < or = 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3- CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0.001) but increased to a normal level during the 24 months' follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0.001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0.05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells.

Tissue-Specific Education of Decidual NK Cells

PubMed Central

Xiong, Shiqiu; Kennedy, Philippa R.; Gardner, Lucy; Farrell, Lydia E.; Chazara, Olympe; Ivarsson, Martin A.; Hiby, Susan E.; Colucci, Francesco; Moffett, Ashley

2015-01-01

During human pregnancy, fetal trophoblast cells invade the decidua and remodel maternal spiral arteries to establish adequate nutrition during gestation. Tissue NK cells in the decidua (dNK) express inhibitory NK receptors (iNKR) that recognize allogeneic HLA-C molecules on trophoblast. Where this results in excessive dNK inhibition, the risk of pre-eclampsia or growth restriction is increased. However, the role of maternal, self–HLA-C in regulating dNK responsiveness is unknown. We investigated how the expression and function of five iNKR in dNK is influenced by maternal HLA-C. In dNK isolated from women who have HLA-C alleles that carry a C2 epitope, there is decreased expression frequency of the cognate receptor, KIR2DL1. In contrast, women with HLA-C alleles bearing a C1 epitope have increased frequency of the corresponding receptor, KIR2DL3. Maternal HLA-C had no significant effect on KIR2DL1 or KIR2DL3 in peripheral blood NK cells (pbNK). This resulted in a very different KIR repertoire for dNK capable of binding C1 or C2 epitopes compared with pbNK. We also show that, although maternal KIR2DL1 binding to C2 epitope educates dNK cells to acquire functional competence, the effects of other iNKR on dNK responsiveness are quite different from those in pbNK. This provides a basis for understanding how dNK responses to allogeneic trophoblast affect the outcome of pregnancy. Our findings suggest that the mechanisms that determine the repertoire of iNKR and the effect of self-MHC on NK education may differ in tissue NK cells compared with pbNK. PMID:26320253

Behavioural effects of tachykinins and related peptides.

PubMed

Elliott, P J; Iversen, S D

1986-08-27

Substance P (SP) and related tachykinins administered either intracerebroventricularly or directly into the ventral tegmental area of the mesencephalon of rat brain caused increased locomotor activity, grooming behaviour and wet dog shakes. Kassinin, eledoisin, neurokinin A and DiMe-C7, agonists with some selectivity for the SP-E-receptor elicited the greatest locomotor activity and wet dog shake responses, whereas SP and physalaemin which are more selective for the SP-P-receptor were most effective in eliciting the grooming response.

Neuropeptide K potently stimulates salivary gland secretion and potentiates substance P-induced salivation.

PubMed Central

Takeda, Y; Krause, J E

1989-01-01

Neuropeptide K (NPK) is an N-terminally extended derivative of neurokinin A (NKA) that can be a final product in the posttranslational processing of beta-preprotachykinin. A rat salivation bioassay was used to demonstrate potent effects of NPK at low doses, while effects due to NKA were much weaker at higher doses. The rank order of potency of beta-preprotachykinin-derived peptides on salivation responses was NPK greater than substance P greater than NKA much greater than beta-preprotachykinin-(72-96)-peptide. The time course of the NPK response was longer than that observed with substance P. The responses elicited by NPK were blocked by the tachykinin antagonist [D-Pro2,D-Trp7,9]substance P but not by atropine. In peptide coinfusion studies, NPK strikingly potentiated the salivation responses elicited by substance P. NPK in vitro displayed a 100 times lower potency than substance P in displacing 3H-labeled substance P binding in submandibular gland membranes, a tissue rich in SP-P type (NK-1) receptors. The possible cellular mechanisms by which NPK stimulates salivary gland secretion are discussed. We conclude that NPK and substance P may be cotransmitters derived by posttranslational processing of beta-preprotachykinin. Images PMID:2463627

The role of Gαs in activation of NK92-MI cells by neuropeptide substance P.

PubMed

Diandong, Hou; Kefeng, Sun; Weixin, Fu; Moran, Wang; Jiahui, Wang; Zaifu, Liang

2014-02-01

Substance P (SP) is well known for its immunoregulatory influence on NK cells. The biological actions of SP are mediated primarily through the high-affinity neurokinin-1 receptor (NK-1R), a G protein-coupled receptor (GPCR). Receptor binding triggers a cAMP signaling pathway and intracellular levels of cAMP are regulated via Gαs and Gαi. In this study NF449, a Gαs-selective G protein antagonist, was used to study the role of Gαs in the activation of NK92-MI cells by SP. Results show that 10(-12)M SP enhances the expression of Gαs and Gαi3 in NK92-MI cells promoting a cytotoxic phenotype characterized by expression of perforin and granzyme B. Development of a cytotoxic phenotype in NK92-MI cells stimulated with SP is blunted by inhibition of Gαs by NF449. In summary, SP signaling through NK-1R promotes a cytotoxic phenotype in NK92-MI cells characterized by upregulation of both Gαs and Gαi3. NF449 inhibits Gαs, blunts SP-induced expression of perforin and granzyme B, and represents a potential therapeutic avenue for reducing NK-cell mediated cytotoxicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Modification of Expanded NK Cells with Chimeric Antigen Receptor mRNA for Adoptive Cellular Therapy.

PubMed

Chu, Yaya; Flower, Allyson; Cairo, Mitchell S

2016-01-01

NK cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors and cells infected by viruses. The regulation of NK activation vs inhibition is regulated by the expression of a variety of NK receptors (NKRs) and specific NKRs' ligands expressed on their targets. However, factors limiting NK therapy include small numbers of active NK cells in unexpanded peripheral blood and lack of specific tumor targeting. Chimeric antigen receptors (CAR) usually include a single-chain Fv variable fragment from a monoclonal antibody, a transmembrane hinge region, and a signaling domain such as CD28, CD3-zeta, 4-1BB (CD137), or 2B4 (CD244) endodimers. Redirecting NK cells with a CAR will circumvent the limitations of the lack of NK targeting specificity. This chapter focuses on the methods to expand human NK cells from peripheral blood by co-culturing with feeder cells and to modify the expanded NK cells efficiently with the in vitro transcribed CAR mRNA by electroporation and to test the functionality of the CAR-modified expanded NK cells for use in adoptive cellular immunotherapy.

Zika Virus Escapes NK Cell Detection by Upregulating Major Histocompatibility Complex Class I Molecules.

PubMed

Glasner, Ariella; Oiknine-Djian, Esther; Weisblum, Yiska; Diab, Mohammad; Panet, Amos; Wolf, Dana G; Mandelboim, Ofer

2017-11-15

NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus infection is largely unknown. Here we demonstrate that Zika virus infection is almost undetected by NK cells, as evidenced by the fact that the expression of activating ligands for NK cells is not induced following Zika infection. We identified a mechanism whereby Zika virus sensing via the RIGI-IRF3 pathway resulted in IFN-β-mediated upregulation of MHC-I molecules and inhibition of NK cell activity. Countering MHC class I upregulation and boosting NK cell activity may be employed as prophylactic measures to combat Zika virus infection. Copyright © 2017 American Society for Microbiology.

Zika Virus Escapes NK Cell Detection by Upregulating Major Histocompatibility Complex Class I Molecules

PubMed Central

Glasner, Ariella; Oiknine-Djian, Esther; Weisblum, Yiska; Diab, Mohammad; Panet, Amos; Wolf, Dana G.

2017-01-01

ABSTRACT NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus infection is largely unknown. Here we demonstrate that Zika virus infection is almost undetected by NK cells, as evidenced by the fact that the expression of activating ligands for NK cells is not induced following Zika infection. We identified a mechanism whereby Zika virus sensing via the RIGI-IRF3 pathway resulted in IFN-β-mediated upregulation of MHC-I molecules and inhibition of NK cell activity. Countering MHC class I upregulation and boosting NK cell activity may be employed as prophylactic measures to combat Zika virus infection. PMID:28878071

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors

PubMed Central

Anton, Olga M.; Vielkind, Susina; Peterson, Mary E.; Tagaya, Yutaka; Long, Eric O.

2015-01-01

IL-15 bound to the IL-15 receptor α chain (IL-15Rα) is presented in trans to cells bearing the IL-2 receptor β and γc chains. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor–ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR–HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to down-regulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A+ cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Co-engagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis. PMID:26453750

Lack of efficacy of L-759274, a novel neurokinin 1 (substance P) receptor antagonist, for the treatment of generalized anxiety disorder.

PubMed

Michelson, David; Hargreaves, Richard; Alexander, Robert; Ceesay, Paulette; Hietala, Jarmo; Lines, Christopher; Reines, Scott

2013-02-01

Preclinical studies suggest that substance P acting at neurokinin 1 (NK1) receptors may be involved in stress responses and NK1 receptor antagonists show activity in tests of anxiety. These data raise the possibility that NK1 receptor antagonists could be potential anxiolytic treatments in humans. We evaluated this hypothesis clinically using the NK1 antagonist L-759274. This is a randomized, double-blind, placebo- and active-controlled, multicentre, proof-of-concept trial. Patients with generalized anxiety disorder were randomized 1:1:1 to 6 wk of treatment with 40 mg L-759274 (n = 73), 1-6 mg lorazepam (n = 69) or placebo (n = 71). Efficacy was assessed using the Hamilton Anxiety Scale (HAMA). A positron emission tomography (PET) study was also performed in 16 healthy subjects to determine the relationship between NK1 receptor occupancy and plasma levels of L-759274 to verify adequate target engagement by the doses tested during the clinical trial. No statistically significant difference in mean change from baseline HAMA score at 6 wk was seen for L-759274 vs. placebo [difference = 1.0 (95% confidence intervals (CI) -1.2 to 3.2), p = 0.359] whereas the lorazepam group did show a significant improvement vs. placebo (difference = -2.7, 95% CI -5.0 to -0.4, p = 0.020) and L-759274 (difference = 3.7, 95% CI 1.5-6.0, p = 0.001]. Results from the PET study indicated that the L-759274 dosing regimen used in the clinical trial likely provided high levels of NK1 receptor occupancy (>90%), supporting the view that it was an adequate proof-of-concept trial. The NK1 receptor antagonist L-759274 does not appear to be efficacious for the treatment of generalized anxiety disorder.

Effects of receptor-selective neurokinin agonists and a neurokinin antagonist on the electrical activity of spinal cord neurones in culture.

PubMed

Wienrich, M; Reuss, K; Harting, J

1989-11-01

1. Rat spinal cord neurones grown in tissue culture were used to examine the electrophysiological effects of the neurokin in (NK)-selective agonists (pGlu6, Pro9) substance P(6-11) (septide; NK1, 10(-6)M) and (pGlu5, MePhe8, MeGly9)SP(1-7) (DiMe-C7; NK3, 10(-6)M). In addition, the effect of the neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP (10(-5)M) on the neurokinin-evoked responses was investigated. 2. Neurokinin-evoked responses consisted of an increase in neuronal activity with or without long-lasting (mean: 50s) depolarizations of the membrane potential of up to 25mV. The latter also occurred in the presence of tetrodotoxin (10(-7)M) (direct response). 3. In a number of spinal cord neurones (n = 17) only septide induced a membrane depolarization while DiMe-C7 elicited no response. On the other hand, in 2 neurones a response was exclusively evoked by DiMe-C7. 4. The neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP had no effect of its own but blocked the septide- and DiMe-C7-induced depolarizations. It had no effect on the glutamate (10(-5)M)-evoked depolarization. 5. It is concluded that by the use of neurokinin receptor-selective agonists, subpopulations of spinal cord neurones in primary dissociated cell culture can be differentiated which express the NK1 or the NK3 receptor. Cells expressing only the NK1 receptor outnumber those expressing only the NK3 receptor subtype. Both receptors can be blocked by the neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP.

Evidence for involvement of NK₃ receptors in the anxiogenic-like effect of SP6-11(C-terminal), a metabolite of substance P, in rats evaluated in the elevated plus-maze.

PubMed

Duarte, Filipe Silveira; Duzzioni, Marcelo; Leme, Leandro Rinaldi; Smith, Saulo de Paiva; De Lima, Thereza C M

2016-04-15

Substance P (SP) is a neuropeptide widely expressed throughout the fear-processing pathways of the brain. SP is cleaved by several proteolytic enzymes in amino (N-) and carboxy (C-) terminal sequences, which can have biological activities per se. We have previously shown that the anxiogenic-like effects elicited by SP6-11(C-terminal), a specific metabolite of SP, are mediated via NK1 and NK2 receptors. Nevertheless, there are evidences that C-terminal fragments may have a greater affinity for NK3 receptors. The aim of the present study was to further investigate the possible involvement of NK3 receptors in the anxiogenic-like effects induced by SP6-11(C-terminal). Adult male Wistar rats were intracerebroventricularly (i.c.v.) treated with SR142801 (NK3 receptors antagonist) or vehicle one minute to prior SP6-11(C-terminal) or vehicle. Other experimental groups received SP6-11(C-terminal) or vehicle i.c.v. one minute prior to senktide (NK3 receptors agonist) or vehicle. After five minutes, the animals were behaviorally evaluated in the elevated plus-maze test (EPM). SR142801 (100 pmol) or SP6-11(C-terminal) (10 pmol) reduced all the parameters of open-arms exploration and increased the number of protected stretch-attend postures in the EPM, indicating an anxiogenic-like effect. Senktide (10 pmol) promoted an opposite effect on these behavioral parameters, characterizing an anxiolytic-like profile. Pretreatment with SR142801, in an ineffective dose, potentiated the SP6-11-induced anxiety, especially in the unprotected head-dipping and protected stretch-attend postures behaviors. Moreover, the anxiolytic-like effect induced by senktide (1 pmol) was prevented by SP6-11. Our results give support to the involvement of NK3 receptors in the anxiogenic-like actions of SP6-11(C-terminal), where this metabolite seems to behave as an antagonist, in a way similar to SR142801. Copyright © 2016 Elsevier B.V. All rights reserved.

Bronchoconstriction induced by increasing airway temperature in ovalbumin-sensitized rats: role of tachykinins.

PubMed

Hsu, Chun-Chun; Lin, Ruei-Lung; Lin, You Shuei; Lee, Lu-Yuan

2013-09-01

This study was carried out to determine the effect of allergic inflammation on the airway response to increasing airway temperature. Our results showed the following: 1) In Brown-Norway rats actively sensitized by ovalbumin (Ova), isocapnic hyperventilation with humidified warm air (HWA) for 2 min raised tracheal temperature (Ttr) from 33.4 ± 0.6°C to 40.6 ± 0.1°C, which induced an immediate and sustained (>10 min) increase in total pulmonary resistance (Rl) from 0.128 ± 0.004 to 0.212 ± 0.013 cmH2O·ml(-1)·s (n = 6, P < 0.01). In sharp contrast, the HWA challenge caused the same increase in Ttr but did not generate any increase in Rl in control rats. 2) The increase in Rl in sensitized rats was reproducible when the same HWA challenge was repeated 60-90 min later. 3) This bronchoconstrictive effect was temperature dependent: a slightly smaller increase in peak Ttr (39.6 ± 0.2°C) generated a significant but smaller increase in Rl in sensitized rats. 4) The HWA-induced bronchoconstriction was not generated by the humidity delivered by the HWA challenge alone, because the same water content delivered by saline aerosol at room temperature had no effect. 5) The HWA-evoked increase in Rl in sensitized rats was not blocked by atropine but was completely prevented by pretreatment either with a combination of neurokinin (NK)-1 and NK-2 antagonists or with formoterol, a β2 agonist, before the HWA challenge. This study showed that increasing airway temperature evoked a pronounced and reversible increase in airway resistance in sensitized rats and that tachykinins released from the vagal bronchopulmonary C-fiber endings were primarily responsible.

Oxygen Modulates Human Decidual Natural Killer Cell Surface Receptor Expression and Interactions with Trophoblasts1

PubMed Central

Wallace, Alison E.; Goulwara, Sonu S.; Whitley, Guy S.; Cartwright, Judith E.

2014-01-01

Decidual natural killer (dNK) cells have been shown to both promote and inhibit trophoblast behavior important for decidual remodeling in pregnancy and have a distinct phenotype compared to peripheral blood NK cells. We investigated whether different levels of oxygen tension, mimicking the physiological conditions of the decidua in early pregnancy, altered cell surface receptor expression and activity of dNK cells and their interactions with trophoblast. dNK cells were isolated from terminated first-trimester pregnancies and cultured in oxygen tensions of 3%, 10%, and 21% for 24 h. Cell surface receptor expression was examined by flow cytometry, and the effects of secreted factors in conditioned medium (CM) on the trophoblast cell line SGHPL-4 were assessed in vitro. SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 10% were significantly more invasive (P < 0.05) and formed endothelial-like networks to a greater extent (P < 0.05) than SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 3% or 21%. After 24 h, a lower percentage of dNK cells expressed CD56 at 21% oxygen (P < 0.05), and an increased percentage of dNK cells expressed NKG2D at 10% oxygen (P < 0.05) compared to other oxygen tensions, with large patient variation. This study demonstrates dNK cell phenotype and secreted factors are modulated by oxygen tension, which induces changes in trophoblast invasion and endovascular-like differentiation. Alterations in dNK cell surface receptor expression and secreted factors at different oxygen tensions may represent regulation of function within the decidua during the first trimester of pregnancy. PMID:25232021

Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

Mechanism of the contractile effects of galantide and Galanin(1-14) [alpha-aminobutyric acid]scyliorhinin-I in rat isolated fundus strips.

PubMed

Korolkiewicz, Roman P; Konstanski, Zdziaław; Rekowski, Piotr; Szyk, Agnieszka; Ruczyński, Jaroslaw; Dabrowski, Jaroslaw; Ujda, Marek; Korolkiewicz, Konstanty Zbigniew; Petrusewicz, Jacek

2002-01-01

The study was undertaken to establish the pharmacological basis of the stimulatory activity of galantide (M15) and galanin(1-14)-(a-aminobutyric acid8-[Abu8])scyliorhinin-I [Scy-I] in gastric smooth muscle. Isotonic contractions of the isolated, longitudinal rat gastric fundus strips were recorded. Galanin, galanin(1-15)-NH2, M15 and galanin(1-14)-[Abu8]Scy-I elicited concentration-dependent contractions. Their EC50s equaled 12.95, 174, 70.06 and 187 nM respectively. Hill's coefficients for galanin and galanin(1-15)-NH2 were not different from unity, indicating an interaction of one peptide molecule with one receptor according to the principles of classical receptor theory. Hill's coefficients were 0.73 and 1.56 for M15 and galanin (1-14)-[Abu8]Scy-I, respectively, a value significantly different from unity. Cold-storage denervation, tetrodotoxin-TTX (1 mM), spantide (100 mM) and NK1-3 receptor antagonists SR140333, 48968, 142801 (up to 10 mM) notably diminished M15, galanin(1-14)-[Abu8]Scy-I, SP(5-11) and [Abu8]-Scy-I evoked contractions without affecting activities of galanin and galanin(1-15)-NH2. Additionally, cross-desensitization experiments attenuated activity of M15 and galanin(1-14)-[Abu8]Scy-I without any noticeable action on galanin or galanin(1-15)-NH2. The action of chimeric peptides on the smooth muscle of the rat gastrointestinal tract depended not only on the myogenic interaction of those peptides with galanin binding sites, but also on the activation of tachykinin receptors or the release of endogenous mediators from the presynaptic terminals.

Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells

PubMed Central

Akazawa, Takashi; Ebihara, Takashi; Okuno, Manabu; Okuda, Yu; Shingai, Masashi; Tsujimura, Kunio; Takahashi, Toshitada; Ikawa, Masahito; Okabe, Masaru; Inoue, Norimitsu; Okamoto-Tanaka, Miki; Ishizaki, Hiroyoshi; Miyoshi, Jun; Matsumoto, Misako; Seya, Tsukasa

2007-01-01

Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1−/− or IFNAR−/− mice, whereas a normal NK antitumor response was induced in PKR−/−, MyD88−/−, IFN-β−/−, and wild-type mice. IFNAR was a prerequisite for the induction of IFN-α/β and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR−/− mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1−/− mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors. PMID:17190817

Education of human natural killer cells by activating killer cell immunoglobulin-like receptors.

PubMed

Fauriat, Cyril; Ivarsson, Martin A; Ljunggren, Hans-Gustaf; Malmberg, Karl-Johan; Michaëlsson, Jakob

2010-02-11

Expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self-major histocompatibility complex (MHC) class I molecules provides an educational signal that generates functional natural killer (NK) cells. However, the effects of activating KIRs specific for self-MHC class I on NK-cell education remain elusive. Here, we provide evidence that the activating receptor KIR2DS1 tunes down the responsiveness of freshly isolated human NK cells to target cell stimulation in donors homozygous for human leukocyte antigen (HLA)-C2, the ligand of KIR2DS1. The tuning was apparent in KIR2DS1(+) NK cells lacking expression of inhibitory KIRs and CD94/NKG2A, as well as in KIR2DS1(+) NK cells coexpressing the inhibitory MHC class I-specific receptors CD94/NKG2A and KIR2DL3, but not KIR2DL1. However, the tuning of responsiveness was restricted to target cell recognition because KIR2DS1(+) NK cells responded well to stimulation with exogenous cytokines. Our results provide the first example of human NK-cell education by an activating KIR and suggest that the education of NK cells via activating KIRs is a mechanism to secure tolerance that complements education via inhibitory KIRs.

Binding characteristics of [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P, a new selective radioligand for the NK1 receptor.

PubMed

Lew, R; Geraghty, D P; Drapeau, G; Regoli, D; Burcher, E

1990-08-02

The selective tachykinin agonist [Sar9,Met(O2)11]substance P (Sar-SP) was radioiodinated with [125I]Bolton-Hunter reagent and the product [125I]Bolton-Hunter-[Sar9,Met(O)2)11]SP (BHSar-SP) purified using reverse phase HPLC. Autoradiographic studies showed dense specific binding of BHSar-SP over the rat submandibular gland and over several regions in rat brain, with very low nonspecific binding, identical with the pattern of binding sites seen in a parallel study with [125I]Bolton-Hunter SP (BHSP). In homogenate binding experiments, BHSar-SP bound with high affinity to a single site in membranes from rat brain (KD 261 pM) and rat submandibular gland (KD 105 pM). Comparative values for BHSP were 495 and 456 pM, i.e. of two and four fold lower affinity than BHSar-SP. Association of BHSar-SP to membranes from brain (k+1 3.7 x 10(9) M-1 min-1) was faster than to membranes from salivary gland (k+1 5.6 x 10(8) M-1 min-1). In competition studies, BHSar-SP was displaced from salivary gland membranes by substance P (SP) approximately physalaemin greater than or equal to Sar-SP approximately SP-(3-11) greater than SP-(5-11) much greater than neurokinin A (NKA) approximately eledoisin = kassinin = SP-methyl ester greater than or equal to neurokinin B (NKB) much greater than [Nle10]NKA-(4-10) greater than [MePhe7]NKB-(4-10). In brain membranes, the rank potency order was SP greater than Sar-SP greater than or equal to physalaemin greater than SP-(3-11) greater than SP-(5-11) greater than NKA greater than or equal to eledoisin much greater than NKB greater than kassinin greater than SP-methyl ester: however [MePhe7]NKB-(4-10) and [Nle10]NKA-(4-10) were ineffective competitors at concentrations up to 1 microM. Both binding patterns are consistent with BHSar-SP binding to an NK1 site. With the exception of SP, Sar-SP, SP-(3-11) and physalaemin, all competitors were 5 to 54 times less potent at BHSar-SP binding sites in brain than in salivary gland. These data reveal some differences in characteristics of NK1 binding sites in brain and submandibular gland. Although of higher affinity, BHSar-SP does not appear greatly more selective than BHSP in its ability to define NK1 binding sites.

A decrease of regulatory T cells and altered expression of NK receptors are observed in subacute sclerosing panencephalitis.

PubMed

Yentur, Sibel P; Gurses, Candan; Demirbilek, Veysi; Adin-Cinar, Suzan; Kuru, Umit; Uysal, Serap; Yapici, Zuhal; Yilmaz, Gülden; Cokar, Ozlem; Onal, Emel; Gökyigit, Aysen; Saruhan-Direskeneli, Güher

2014-12-01

Subacute sclerosing panencephalitis (SSPE) is caused by a persistent measles virus infection. Regulatory mechanisms can be responsible for a failure of immunosurveillance in children with SSPE. In this study, peripheral blood cells of 71 patients with SSPE and 57 children with other diseases were compared phenotypically. The proportions of CD4(+), CD8(+) T, and NK cells were homogenous, whereas total CD3(+) T and Treg (CD4(+)CD25(+)CD152(+)) cells were decreased in patients with SSPE. The proportion of CD8(+) T cells expressing the inhibitory NKG2A(+) receptor was also decreased (1.7% ± 1.7% vs. 2.6% ± 1.9%, p = 0.007) in patients with SSPE, whereas the proportion of NK cells expressing activating NKG2C was increased compared with the control group (30.0% ± 17.3% vs. 22.2% ± 17.0%, p = 0.039). The decrease in the number of cells with regulatory phenotype, the lower presence of the inhibitory NK receptors on CD8(+) cells, and higher activating NK receptors on NK cells in SSPE indicate an upregulation of these cell types that favors their response. This state of active immune response may be caused by chronic stimulation of viral antigens leading to altered regulatory pathways.

Neurogenic plasma leakage in mouse airways

PubMed Central

Baluk, Peter; Thurston, Gavin; Murphy, Thomas J; Bunnett, Nigel W; McDonald, Donald M

1999-01-01

This study sought to determine whether neurogenic inflammation occurs in the airways by examining the effects of capsaicin or substance P on microvascular plasma leakage in the trachea and lungs of male pathogen-free C57BL/6 mice. Single bolus intravenous injections of capsaicin (0.5 and 1 μmol kg−1, i.v.) or substance P (1, 10 and 37 nmol kg−1, i.v.) failed to induce significant leakage in the trachea, assessed as extravasation of Evans blue dye, but did induce leakage in the urinary bladder and skin. Pretreatment with captopril (2.5 mg kg−1, i.v.), a selective inhibitor of angiotensin converting enzyme (ACE), either alone or in combination with phosphoramidon (2.5 mg kg−1, i.v.), a selective inhibitor of neutral endopeptidase (NEP), increased baseline leakage of Evans blue in the absence of any exogenous inflammatory mediator. The increase was reversed by the bradykinin B2 receptor antagonist Hoe 140 (0.1 mg kg−1, i.v.). After pretreatment with phosphoramidon and captopril, capsaicin increased the Evans blue leakage above the baseline in the trachea, but not in the lung. This increase was reversed by the tachykinin (NK1) receptor antagonist SR 140333 (0.7 mg kg−1, i.v.), but not by the NK2 receptor antagonist SR 48968 (1 mg kg−1, i.v.). Experiments using Monastral blue pigment as a tracer localized the leakage to postcapillary venules in the trachea and intrapulmonary bronchi, although the labelled vessels were less numerous in mice than in comparably treated rats. Blood vessels of the pulmonary circulation were not labelled. We conclude that neurogenic inflammation can occur in airways of pathogen-free mice, but only after the inhibition of enzymes that normally degrade inflammatory peptides. Neurogenic inflammation does not involve the pulmonary microvasculature. PMID:10077247

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells.

PubMed

Müller, Tina; Uherek, Christoph; Maki, Guitta; Chow, Kai Uwe; Schimpf, Annemarie; Klingemann, Hans-Georg; Tonn, Torsten; Wels, Winfried S

2008-03-01

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

Confinement of activating receptors at the plasma membrane controls natural killer cell tolerance.

PubMed

Guia, Sophie; Jaeger, Baptiste N; Piatek, Stefan; Mailfert, Sébastien; Trombik, Tomasz; Fenis, Aurore; Chevrier, Nicolas; Walzer, Thierry; Kerdiles, Yann M; Marguet, Didier; Vivier, Eric; Ugolini, Sophie

2011-04-05

Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.

Low gene expression levels of activating receptors of natural killer cells (NKG2E and CD94) in patients with fulminant type 1 diabetes.

PubMed

Nakata, Shinsuke; Imagawa, Akihisa; Miyata, Yugo; Yoshikawa, Atsushi; Kozawa, Junji; Okita, Kohei; Funahashi, Tohru; Nakamura, Seiji; Matsubara, Kenichi; Iwahashi, Hiromi; Shimomura, Iichiro

2013-01-01

Fulminant type 1 diabetes is an independent subtype of type 1 diabetes characterized by extremely rapid onset and absence of islet-related autoantibodies. However, detailed pathophysiology of this subtype is poorly understood. In this study, a comprehensive approach was applied to understand the pathogenesis of fulminant type 1 diabetes. We determined the genes that were differentially expressed in fulminant type 1 diabetes compared with type 1A diabetes and healthy control, using gene expression microarray in peripheral blood cells. Using volcano plot analysis, we found reduced expression of killer cell lectin-like receptor subfamily C, member 3 (KLRC3) which encodes NKG2E, a natural killer (NK) cell activating receptor, in fulminant type 1 diabetes, compared with healthy controls. This difference was confirmed by real-time RT-PCR among NK-enriched cells. The expression of KLRD1 (CD94), which forms heterodimer with NKG2E (KLRC3), was also reduced in NK-enriched cells in fulminant type 1 diabetes. Furthermore, flow cytometry showed significantly lower proportion of NK cells among peripheral blood mononuclear cells (PBMCs) in fulminant type 1 diabetes than in healthy controls. In patients with fulminant type 1 diabetes, the relative proportion of NK cells correlated significantly with the time period between onset of fever to the appearance of hyperglycemic-related symptoms. We conclude the presence of reduced NK activating receptor gene expression and low proportion of NK cells in fulminant type 1 diabetes. Copyright © 2013 Elsevier B.V. All rights reserved.

In vitro contractile effects of neurokinin receptor blockade in the human ureter.

PubMed

Nakada, S Y; Jerde, T J; Bjorling, D E; Saban, R

2001-10-01

We identified the predominance of neurokinin-2 receptors and evaluated the inhibition of spontaneous contraction via the blockade of neurokinin-2 receptors in human ureteral segments. Excess ureteral segments from human subjects undergoing donor nephrectomy or reconstructive procedures were suspended in tissue baths containing Krebs buffer. After spontaneous contractions were recorded, tissues were incubated with 1 microM. solutions of phosphoramidon and captopril (to inhibit peptide degradation) and either the neurokinin-1 receptor antagonist CP 99,994, the neurokinin-2 receptor antagonist SR 48,968, the neurokinin-3 receptor antagonist SR 142,801 or dimethyl sulfoxide (control) for 1 hour. Contraction magnitude and frequency were again recorded and compared with spontaneous levels. Concentration-response curves to the tachykinins substance P, and neurokinins A and B were determined in the presence and absence of antagonists. Neurokinin A increased contractility at lower concentrations than substance P or neurokinin B (p <0.013). Neurokinin-2 receptor blockade produced a 100-fold rightward shift of the concentration-response curves (p <0.013), while neurokinins 1 and 3 receptor blockade had no effect. SR 48,968 significantly reduced contractility during the 1-hour incubation period, causing a 97% reduction in spontaneous rates compared with a 29% reduction in control tissues. CP 99,994 and SR 142,801 had no significant effect. Neurokinin-2 is the predominant receptor subtype responsible for tachykinin induced contraction of human ureteral smooth muscle. In vitro treatment with the neurokinin-2 antagonist SR 48,968 reduces the spontaneous contraction rate by 97% in vitro. Neurokinin-2 receptor antagonists may have clinical applications for ureteral disease.

Endometrial natural killer (NK) cells reveal a tissue-specific receptor repertoire.

PubMed

Feyaerts, D; Kuret, T; van Cranenbroek, B; van der Zeeuw-Hingrez, S; van der Heijden, O W H; van der Meer, A; Joosten, I; van der Molen, R G

2018-02-13

Is the natural killer (NK) cell receptor repertoire of endometrial NK (eNK) cells tissue-specific? The NK cell receptor (NKR) expression profile in pre-pregnancy endometrium appears to have a unique tissue-specific phenotype, different from that found in NK cells in peripheral blood, suggesting that these cells are finely tuned towards the reception of an allogeneic fetus. NK cells are important for successful pregnancy. After implantation, NK cells encounter extravillous trophoblast cells and regulate trophoblast invasion. NK cell activity is amongst others regulated by C-type lectin heterodimer (CD94/NKG2) and killer cell immunoglobulin-like (KIR) receptors. KIR expression on decidual NK cells is affected by the presence of maternal HLA-C and biased towards KIR2D expression. However, little is known about NKR expression on eNK cells prior to pregnancy. In this study, matched peripheral and menstrual blood (a source of endometrial cells) was obtained from 25 healthy females with regular menstrual cycles. Menstrual blood was collected during the first 36 h of menstruation using a menstrual cup, a non-invasive technique to obtain endometrial cells. KIR and NKG2 receptor expression on eNK cells was characterized by 10-color flow cytometry, and compared to matched pbNK cells of the same female. KIR and HLA-C genotypes were determined by PCR-SSOP techniques. Anti-CMV IgG antibodies in plasma were measured by chemiluminescence immunoassay. KIR expression patterns of eNK cells collected from the same female do not differ over consecutive menstrual cycles. The percentage of NK cells expressing KIR2DL2/L3/S2, KIR2DL3, KIR2DL1, LILRB1 and/or NKG2A was significantly higher in eNK cells compared to pbNK cells, while no significant difference was observed for NKG2C, KIR2DL1/S1, and KIR3DL1. The NKR repertoire of eNK cells was clearly different from pbNK cells, with eNK cells co-expressing more than three NKR simultaneously. In addition, outlier analysis revealed 8 and 15 NKR subpopulation expansions in eNK and pbNK cells, respectively. In contrast to the pbNK cell population, the expansions present in the eNK cell population were independent of CMV status and HLA-C genotype. Moreover, the typical NKG2C imprint induced by CMV infection on pbNK cells was not observed on eNK cells from the same female, suggesting a rapid local turnover of eNK cells and/or a distinct licensing process. Based on our previous work and the parameters studied here, menstrual blood-derived eNK cells closely resemble biopsy-derived eNK cells. However, sampling is not done at the exact same time during the menstrual cycle, and therefore we cannot exclude some, as yet undetected, differences. Our data reveals that NK cells in the pre-implantation endometrium appear to have a dedicated tissue-specific phenotype, different from NK cells in peripheral blood. This may indicate that eNK cells are finely tuned to receive an allogeneic fetus. Studying the endometrial NKR repertoire of women with pregnancy related problems could provide clues to understand the pathogenesis of pregnancy complications. No external funding was obtained for the present study. None of the authors has any conflict of interest to declare. NA. © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.

PubMed

Anton, Olga M; Vielkind, Susina; Peterson, Mary E; Tagaya, Yutaka; Long, Eric O

2015-11-15

IL-15 bound to the IL-15Rα-chain (IL-15Rα) is presented in trans to cells bearing the IL-2Rβ-chain and common γ-chain. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor-ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR-HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to downregulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A(+) cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Coengagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across, inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15-dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis. Copyright © 2015 by The American Association of Immunologists, Inc.

Regulation of NK92-MI cell cytotoxicity by substance P.

PubMed

Fu, W X; Qin, B; Zhou, A P; Yu, Q Y; Huang, Q J; Liang, Z F

2011-08-01

The neuropeptide substance P (SP) can regulate a number of immunological functions in vitro and in vivo and may regulate natural killer (NK) cell activity. Here, we investigated whether SP has a role in regulating NK92-MI cell function in vitro, and how it influences NK cell activity. We found that SP dose dependently increased the cytotoxicity of NK92-MI cells and had a maximal effect at a concentration of 10(-12) and 10(-10) m. Furthermore, the expression of cytotoxic-associated molecules (perforin, granzyme) and activating receptor NKp46 [a member of natural cytotoxicity receptors (NCRs)] was observed to be upregulated by SP at optimal concentration, at which SP enhanced the cytotoxicity of NK92-MI cells. Neurokinin-1 receptor (NK-1R), a functional receptor of SP, was found on NK92-MI cells, and the observed effects of SP on NK92-MI cells could be more partially blocked by an NK-1R antagonist. Our data suggest that SP induces NK92-MI cell cytotoxicity by directly increasing the expression of cytotoxic granules and upregulates NK92-MI cell receptor-mediated functions indirectly. Thus, SP may regulate NK cell function mainly through NK-1R. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

Substance P (SP) enhances CCL5-induced chemotaxis and intracellular signaling in human monocytes, which express the truncated neurokinin-1 receptor (NK1R)

PubMed Central

Chernova, Irene; Lai, Jian-Ping; Li, Haiying; Schwartz, Lynnae; Tuluc, Florin; Korchak, Helen M.; Douglas, Steven D.; Kilpatrick, Laurie E.

2009-01-01

Substance P (SP) is a potent modulator of monocyte/macrophage function. The SP-preferring receptor neurokinin-1 receptor (NK1R) has two forms: a full-length NK1R (NK1R-F) isoform and a truncated NK1R (NK1R-T) isoform, which lacks the terminal cytoplasmic 96-aa residues. The distribution of these receptor isoforms in human monocytes is not known. We previously identified an interaction among SP, NK1R, and HIV viral strains that use the chemokine receptor CCR5 as a coreceptor, suggesting crosstalk between NK1R and CCR5. The purpose of this study was to determine which form(s) of NK1R are expressed in human peripheral blood monocytes and to determine whether SP affects proinflammatory cellular responses mediated through the CCR5 receptor. Human peripheral blood monocytes were found to express NK1R-T but not NK1R-F. SP interactions with NK1R-T did not mobilize calcium (Ca2+), but SP mobilized Ca2+ when the NK1R-F was transfected into monocytes. However, the NK1R-T was functional in monocytes, as SP enhanced the CCR5 ligand CCL5-elicited Ca2+ mobilization, a response inhibited by the NK1R antagonist aprepitant. SP interactions with the NK1R-T also enhanced CCL5-mediated chemotaxis, which was ERK1/2-dependent. NK1R-T selectively activated ERK2 but increased ERK1 and ERK2 activation by CCL5. Activation of NK1R-T elicited serine phosphorylation of CCR5, indicating that crosstalk between CCL5 and SP may occur at the level of the receptor. Thus, NK1R-T is functional in human monocytes and activates select signaling pathways, and the NK1R-T-mediated enhancement of CCL5 responses does not require the NK1R terminal cytoplasmic domain. PMID:18835883

Insights into seven and single transmembrane-spanning domain receptors and their signaling pathways in human natural killer cells.

PubMed

Maghazachi, Azzam A

2005-09-01

Human natural killer (NK) cells are important cells of the innate immune system. These cells perform two prominent functions: the first is recognizing and destroying virally infected cells and transformed cells; the second is secreting various cytokines that shape up the innate and adaptive immune re-sponses. For these cells to perform these activities, they express different sets of receptors. The receptors used by NK cells to extravasate into sites of injury belong to the seven transmembrane (7TM) family of receptors, which characteristically bind heterotrimeric G proteins. These receptors allow NK cells to sense the chemotactic gradients and activate second messengers, which aid NK cells in polarizing and migrating toward the sites of injured tissues. In addition, these receptors determine how and why human resting NK cells are mainly found in the bloodstream, whereas activated NK cells extravasate into inflammatory sites. Receptors for chemokines and lysophospholipids belong to the 7TM family. On the other hand, NK cells recognize invading or transformed cells through another set of receptors that belong to the single transmembrane-spanning domain family. These receptors are either inhibitory or activating. Inhibitory receptors contain the immune receptor tyrosine-based inhibitory motif, and activating receptors belong to either those that associate with adaptor molecules containing the immune receptor tyrosine-based activating motif (ITAM) or those that associate with adaptor molecules containing motifs other than ITAM. This article will describe the nature of these receptors and examine the intracellular signaling pathways induced in NK cells after ligating both types of receptors. These pathways are crucial for NK cell biology, development, and functions.

Substance P activates both contractile and inflammatory pathways in lymphatics through the neurokinin receptors NK1R and NK3R.

PubMed

Chakraborty, Sanjukta; Nepiyushchikh, Zhanna; Davis, Michael J; Zawieja, David C; Muthuchamy, Mariappan

2011-01-01

The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics. A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP-mediated signaling pathways in lymphatics. We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs. SP stimulation increased phosphorylation of myosin light chain 20 (MLC₂₀) as well as p38 mitogen associated protein kinase (p38-MAPK) and extracellular signal regulated kinase (ERK1/2) indicating activation of both a contractile and a pro-inflammatory MAPK pathway. Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Inhibition of ERK1/2 decreased levels of p-MLC₂₀ after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways. These data provide the first evidence that SP-mediated crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation. © 2010 John Wiley & Sons Ltd.

Substance P activates both contractile and inflammatory pathways in lymphatics through the neurokinin receptors NK1R and NK3R

PubMed Central

Chakraborty, Sanjukta; Nepiyushchikh, Zhanna; Davis, Michael J.; Zawieja, David C.; Muthuchamy, Mariappan

2010-01-01

Objective The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics. Methods A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP mediated signaling pathways in lymphatics. Results We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs. SP stimulation increased phosphorylation of myosin light chain 20 (MLC20) as well as p38 mitogen associated protein kinase (p38-MAPK) and extracellular signal regulated kinase (ERK1/2) indicating activation of both a contractile and a pro-inflammatory MAPK pathway. Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Inhibition of ERK1/2 decreased levels of p-MLC20 after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways. Conclusions These data provide the first evidence that SP mediated crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation. PMID:21166923

Chemokine Receptor Expression on Normal Blood CD56+ NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population

PubMed Central

Queirós, Maria Luís; Gonçalves, Marta; Fonseca, Sónia; Moura, João

2015-01-01

Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56+low CD16+ and CD56+high  CD16−/+low NK-cells. Conventional CD56+low and CD56+high NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56+low NK-cells are mainly CXCR1/CXCR2+ and CXCR3/CCR5−/+, whereas mostly CD56+high NK-cells are CXCR1/CXCR2− and CXCR3/CCR5+. Both NK-cell subsets have variable CXCR4 expression and are CCR4− and CCR6−. The CKR repertoire of the CD56+low NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56+high NK-cells mimics that of Th1+ T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56+int NK-cells. These NK-cells are CXCR3/CCR5+, they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57− and CD158a−. In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56+high and CD56+low NK-cells populations. PMID:26543875

Direct Actions of Kisspeptins on GnRH Neurons Permit Attainment of Fertility but are Insufficient to Fully Preserve Gonadotropic Axis Activity

PubMed Central

León, Silvia; Barroso, Alexia; Vázquez, María J.; García-Galiano, David; Manfredi-Lozano, María; Ruiz-Pino, Francisco; Heras, Violeta; Romero-Ruiz, Antonio; Roa, Juan; Schutz, Günther; Kirilov, Milen; Gaytan, Francisco; Pinilla, Leonor; Tena-Sempere, Manuel

2016-01-01

Kisspeptins, ligands of the receptor, Gpr54, are potent stimulators of puberty and fertility. Yet, whether direct kisspeptin actions on GnRH neurons are sufficient for the whole repertoire of their reproductive effects remains debatable. To dissect out direct vs. indirect effects of kisspeptins on GnRH neurons in vivo, we report herein the detailed reproductive/gonadotropic characterization of a Gpr54 null mouse line with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54−/−Tg; rescued). Despite preserved fertility, adult rescued mice displayed abnormalities in gonadal microstructure, with signs of precocious ageing in females and elevated LH levels with normal-to-low testosterone secretion in males. Gpr54−/−Tg rescued mice showed also altered gonadotropin responses to negative feedback withdrawal, while luteinizing hormone responses to various gonadotropic regulators were variably affected, with partially blunted relative (but not absolute) responses to kisspeptin-10, NMDA and the agonist of tachykinin receptors, NK2R. Our data confirm that direct effects of kisspeptins on GnRH cells are sufficient to attain fertility. Yet, such direct actions appear to be insufficient to completely preserve proper functionality of gonadotropic axis, suggesting a role of kisspeptin signaling outside GnRH cells. PMID:26755241

Suppression of natural killer cell cytotoxicity in postpartum women: time course and potential mechanisms.

PubMed

Groer, Maureen W; El-Badri, Nagwa; Djeu, Julie; Williams, S Nicole; Kane, Bradley; Szekeres, Karoly

2014-07-01

Little is known about the recovery of the immune system from normal pregnancy and whether the postpartum period is a uniquely adapted immune state. This report extends previous observations from our group of decreased natural killer (NK) cell cytotoxicity in the postpartum period. NK cytotoxicity was measured from 1 week through 9 months postpartum. In addition, NK cytotoxicity was assayed in the presence or absence of pooled plasmas collected from either postpartum or nonpostpartum women. Samples of cells were stained for inhibitory receptors and analyzed by flow cytometry. NK cytotoxicity remained decreased in postpartum women compared to controls through the first 6 postpartum months, returned to normal levels by 9 months, and remained normal at 12 months. NK cytotoxicity during the first 6 months was further inhibited by the addition of pooled plasma to NK cultures from postpartum women, but the addition of pooled plasma from the control group did not affect that group's NK cultures. There were differences in inhibitory receptor staining between the two groups, with decreased CD158a and CD158b and increased NKG2A expression on postpartum NK cells during the first 3 postpartum months. These data suggest that NK cytotoxicity postpartum inhibition lasts 6 months and is influenced by unidentified postpartum plasma components. The effect may also involve receptors on NK cells. © The Author(s) 2013.

Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes

PubMed Central

Norman, Paul J.; Abi-Rached, Laurent; Gendzekhadze, Ketevan; Hammond, John A.; Moesta, Achim K.; Sharma, Deepti; Graef, Thorsten; McQueen, Karina L.; Guethlein, Lisbeth A.; Carrington, Christine V.F.; Chandanayingyong, Dasdayanee; Chang, Yih-Hsin; Crespí, Catalina; Saruhan-Direskeneli, Güher; Hameed, Kamran; Kamkamidze, Giorgi; Koram, Kwadwo A.; Layrisse, Zulay; Matamoros, Nuria; Milà, Joan; Park, Myoung Hee; Pitchappan, Ramasamy M.; Ramdath, D. Dan; Shiau, Ming-Yuh; Stephens, Henry A.F.; Struik, Siske; Tyan, Dolly; Verity, David H.; Vaughan, Robert W.; Davis, Ronald W.; Fraser, Patricia A.; Riley, Eleanor M.; Ronaghi, Mostafa; Parham, Peter

2009-01-01

Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric “half” was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family. PMID:19411600

AHR prevents human IL-1R1hi ILC3 differentiation to natural killer cells

PubMed Central

Hughes, Tiffany; Briercheck, Edward L.; Freud, Aharon G.; Trotta, Rossana; McClory, Susan; Scoville, Steven D.; Keller, Karen; Deng, Youcai; Cole, Jordan; Harrison, Nicholas; Mao, Charlene; Zhang, Jianying; Benson, Don M.; Yu, Jianhua; Caligiuri, Michael A.

2014-01-01

SUMMARY Accumulating evidence indicates that human natural killer (NK) cells develop in secondary lymphoid tissue (SLT) through a so-called “stage 3” developmental intermediate minimally characterized by a CD34-CD117+CD94- immunophenotype that lacks mature NK cell function. This stage 3 population is heterogeneous, potentially composed of functionally distinct innate lymphoid cell (ILC) types that includes interleukin-1 receptor (IL-1R1) positive, IL-22-producing ILC3s. Whether human ILC3s are developmentally related to NK cells is a subject of ongoing investigation. Here we show that antagonism of the aryl hydrocarbon receptor (AHR) or silencing of AHR gene expression promotes differentiation of tonsillar IL-22-producing IL-1R1hi human ILC3s to CD56brightCD94+ IFN-gamma-producing cytolytic mature NK cells expressing eomesodermin (EOMES) and T-Box Protein 21 (TBX21 or TBET). Hence, AHR is a transcription factor that prevents human IL-1R1hi ILC3s from differentiating into NK cells. PMID:24953655

Neurokinin B receptor antagonism decreases luteinising hormone pulse frequency and amplitude and delays puberty onset in the female rat.

PubMed

Li, S Y; Li, X F; Hu, M H; Shao, B; Poston, L; Lightman, S L; O'Byrne, K T

2014-08-01

The neural mechanisms controlling puberty onset remain enigmatic. Humans with loss of function mutations in TAC3 or TACR3, the genes encoding neurokinin B (NKB) or its receptor, neurokinin-3 receptor (NK3R), respectively, present with severe congenital gonadotrophin deficiency and pubertal failure. Animal studies have shown ambiguous actions of NKB-NK3R signalling with respect to controlling puberty onset. The present study aimed to determine the role of endogenous NKB-NK3R signalling in the control of pulsatile luteinising hormone (LH) secretion and the timing of puberty onset, and also whether precocious pubertal onset as a result of an obesogenic diet is similarly regulated by this neuropeptide system. Prepubertal female rats, chronically implanted with i.c.v. cannulae, were administered SB222200, a NK3R antagonist, or artificial cerebrospinal fluid via an osmotic mini-pump for 14 days. SB222200 significantly delayed the onset of vaginal opening and first oestrus (as markers of puberty) compared to controls in both normal and high-fat diet fed animals. Additionally, serial blood sampling, via chronic indwelling cardiac catheters, revealed that the increase in LH pulse frequency was delayed and that the LH pulse amplitude was reduced in response to NK3R antagonism, regardless of dietary status. These data suggest that endogenous NKB-NK3R signalling plays a role in controlling the timing of puberty and the associated acceleration of gonadotrophin-releasing hormone pulse generator frequency in the female rat. © 2014 British Society for Neuroendocrinology.

Expression patterns of lectin-like natural killer receptors, inhibitory CD94/NKG2A, and activating CD94/NKG2C on decidual CD56bright natural killer cells differ from those on peripheral CD56dim natural killer cells.

PubMed

Kusumi, Maki; Yamashita, Takahiro; Fujii, Tomoyuki; Nagamatsu, Takeshi; Kozuma, Shiro; Taketani, Yuji

2006-06-01

The balance of inhibitory and activating natural killer (NK) receptors on maternal decidual NK cells, most of which are CD56bright, is thought to be crucial for the proper growth of trophoblasts in placenta. A lectin-like NK receptor, CD94/NKG2, is the receptor for human leukocyte antigen (HLA)-E, which is expressed on trophoblasts. To clarify the mechanism regulating the activity of decidual NK cells during pregnancy, we investigated the expression patterns of inhibitory NK receptor, CD94/NKG2A, and activating receptor, CD94/NKG2C, on decidual NK cells in an early stage of normal pregnancy and compared them with those on peripheral NK cells, most of which are CD56dim. The rate of NKG2A-positive cells was significantly higher for decidual CD56bright NK cells than for peripheral CD56dim NK cells, but the rates of NKG2C-positive cells were comparable between the two cell types. Interestingly, peripheral CD56dim NK cells reciprocally expressed inhibitory NKG2A and activating NKG2C, but decidual CD56bright NK cells that expressed activating NKG2C simultaneously expressed inhibitory NKG2A. The co-expression of inhibitory and activating NKG2 receptors may fine-tune the immunoregulatory functions of the decidual NK cells to control the trophoblast invasion in constructing placenta.

Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor

PubMed Central

Lai, Jian-Ping; Lai, Saien; Tuluc, Florin; Tansky, Morris F.; Kilpatrick, Laurie E.; Leeman, Susan E.; Douglas, Steven D.

2008-01-01

The neurokinin-1 receptor (NK1R) has two naturally occurring forms that differ in the length of the carboxyl terminus: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. We examined whether there are differential signaling properties attributable to the carboxyl terminus of this receptor by using stably transfected human embryonic kidney (HEK293) cell lines that express either full-length or truncated NK1R. Substance P (SP) specifically triggered intracellular calcium increase in HEK293 cells expressing full-length NK1R but had no effect in the cells expressing the truncated NK1R. In addition, in cells expressing full-length NK1R, SP activated NF-κB and IL-8 mRNA expression, but in cells expressing the truncated NK1R, SP did not activate NF-κB, and it decreased IL-8 mRNA expression. In cells expressing full-length NK1R, SP stimulated phosphorylation of PKCδ but inhibited phosphorylation of PKCδ in cells expressing truncated NK1R. There are also differences in the timing of SP-induced ERK activation in cells expressing the two different forms of the receptor. Full-length NK1R activation of ERK was rapid (peak within 1–2 min), whereas truncated NK1R-mediated activation was slower (peak at 20–30 min). Thus, the carboxyl terminus of NK1R is the structural basis for differences in the functional properties of the full-length and truncated NK1R. These differences may provide important information toward the design of new NK1R receptor antagonists. PMID:18713853

Tachykinin antagonist FK224 inhibits neurokinin A-, substance P- and capsaicin-induced human bronchial contraction.

PubMed

Honda, I; Kohrogi, H; Yamaguchi, T; Hamamoto, J; Hirata, N; Iwagoe, H; Fujii, K; Goto, E; Ando, M

1997-01-01

To determine the roles of endogenously released tachykinins (substance P [SP] and neurokinin A [NKA]) in the human bronchial tissues, we studied the effects of tachykinin antagonist FK224 on bronchial smooth muscle contraction induced by SP, NKA and capsaicin in an organ bath. FK224 (10(-6) M and 10(-5) M, respectively) significantly inhibited NKA-induced contraction and 10(-5) M FK224 shifted the dose-response curve to more than one log unit higher concentration. Because SP- and capsaicin-induced contractions were small, we pretreated the tissues with the neutral endopeptidase inhibitor phosphoramidon (10(-5) M), which inhibits degradation of exogenous tachykinins in order to potentiate the contractions. FK224 (10(-5) M) significantly inhibited SP-induced contraction and it shifted the dose-response curves to about one log unit higher concentration. FK224 (10(-5) M) also significantly inhibited capsaicin-induced contraction and it shifted the dose-response curves to more than one log unit higher concentration. In contrast, FK224 (10(-5) M) did not affect on acetylcholine-, histamine-, and leukotriene D4-induced contraction. These results suggest that FK224 is a tachykinin receptor antagonist in the human bronchial smooth muscle, and that capsaicin-induced contraction is due to endogenously released tachykinin-like substances in the human bronchus.

CD3+/CD19+-depleted grafts in HLA-matched allogeneic peripheral blood stem cell transplantation lead to early NK cell cytolytic responses and reduced inhibitory activity of NKG2A.

PubMed

Eissens, D N; Schaap, N P M; Preijers, F W M B; Dolstra, H; van Cranenbroek, B; Schattenberg, A V M; Joosten, I; van der Meer, A

2010-03-01

Natural killer (NK) cells have an important function in the anti-tumor response early after stem cell transplantation (SCT). As part of a prospective randomized phase III study, directly comparing the use of CD3(+)/CD19(+)-depleted peripheral blood stem cell (PBSC) harvests with CD34(+)-selected PBSC harvests in allogeneic human leukocyte antigen-matched SCT, we here show that the use of CD3(+)/CD19(+)-depleted PBSC grafts leads to early NK cell repopulation and reconstitution of the CD56(dim) and CD56(bright) NK cell subsets, with concomitant high cytolytic capacity. In the CD34 group, this process took significantly longer. Moreover, in the CD3/19 group after reconstitution, a higher percentage of killer immunoglobulin-like receptor-positive NK cells was found. Although similar percentages of CD94-positive NK cells were found in both groups, in the CD34 group, almost all expressed the inhibitory CD94:NKG2A complex, whereas in the CD3/19 group, the inhibitory CD94:NKG2A and the activating CD94:NKG2C complex were equally distributed. This preferential development of NKG2C-expressing NK cells in the CD3/19 group was paralleled by a loss of NKG2A-mediated inhibition of NK cell degranulation. These results show that the use of CD3(+)/CD19(+)-depleted grafts facilitates strong NK cell cytolytic responses directly after SCT, and the rapid emergence of an NK cell receptor phenotype that is more prone to activation.

A role for the substance P/NK-1 receptor complex in cell proliferation and apoptosis in oral lichen planus.

PubMed

González Moles, M A; Esteban, F; Ruiz-Avila, I; Gil Montoya, J A; Brener, S; Bascones-Martínez, A; Muñoz, M

2009-03-01

To determine whether substance P (SP) and NK-1 receptor (NK-1R) are expressed in oral lichen planus (OLP) and are related to cell proliferation and apoptosis in this disease. Tissue samples from 50 OLP patients and 26 healthy controls were studied. Immunohistochemistry was performed with anti-SP, anti-NK-1R, anti-ki-67 and anti-caspase-3 monoclonal antibodies and the clinical and pathological data of the OLP patients were evaluated. With the exception of NK-1R expression in epithelial cell membrane and cytoplasm, all markers were more frequently present in OLP patients than in controls (P < 0.05). Higher cytoplasmatic expression of NK-1R was associated with higher epithelial expression of caspase-3 (P < 0.05). Higher epithelial expression of NK-1R and SP was associated with higher suprabasal and basal epithelial expression of ki-67 (P < 0.05 and P < 0.005, respectively). Actions of the SP/NK-1R complex may contribute to the immune disorder underlying OLP and trigger stimuli to induce cell proliferation. These results indicate that this complex might play a role in the malignant transformation of OLP.

Identification of natural killer cell receptor clusters in the platypus genome reveals an expansion of C-type lectin genes.

PubMed

Wong, Emily S W; Sanderson, Claire E; Deakin, Janine E; Whittington, Camilla M; Papenfuss, Anthony T; Belov, Katherine

2009-08-01

Natural killer (NK) cell receptors belong to two unrelated, but functionally analogous gene families: the immunoglobulin superfamily, situated in the leukocyte receptor complex (LRC) and the C-type lectin superfamily, located in the natural killer complex (NKC). Here, we describe the largest NK receptor gene expansion seen to date. We identified 213 putative C-type lectin NK receptor homologs in the genome of the platypus. Many have arisen as the result of a lineage-specific expansion. Orthologs of OLR1, CD69, KLRE, CLEC12B, and CLEC16p genes were also identified. The NKC is split into at least two regions of the genome: 34 genes map to chromosome 7, two map to a small autosome, and the remainder are unanchored in the current genome assembly. No NK receptor genes from the LRC were identified. The massive C-type lectin expansion and lack of Ig-domain-containing NK receptors represents the most extreme polarization of NK receptors found to date. We have used this new data from platypus to trace the possible evolutionary history of the NK receptor clusters.

Nervous control of photophores in luminescent fishes.

PubMed

Zaccone, Giacomo; Abelli, Luigi; Salpietro, Lorenza; Zaccone, Daniele; Macrì, Battesimo; Marino, Fabio

2011-07-01

Functional studies of the autonomic innervation in the photophores of luminescent fishes are scarce. The majority of studies have involved either the stimulation of isolated photophores or the modulatory effects of adrenaline-induced light emission. The fish skin is a highly complex organ that performs a wide variety of physiological processes and receives extensive nervous innervations. The latter includes autonomic nerve fibers of spinal sympathetic origin having a secretomotor function. More recent evidence indicates that neuropeptide-containing nerve fibers, such as those that express tachykinin and its NK1 receptor, neuropeptide Y, or nitric oxide, may also play an important role in the nervous control of photophores. There is no anatomical evidence that shows that nNOS positive (nitrergic) neurons form a population distinct from the secretomotor neurons with perikarya in the sympathetic ganglia. The distribution and function of the nitrergic nerves in the luminous cells, however, is less clear. It is likely that the chemical properties of the sympathetic postganglionic neurons in the ganglia of luminescent fishes are target-specific, such as observed in mammals. Copyright © 2010 Elsevier GmbH. All rights reserved.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival.

PubMed

Genßler, Sabrina; Burger, Michael C; Zhang, Congcong; Oelsner, Sarah; Mildenberger, Iris; Wagner, Marlies; Steinbach, Joachim P; Wels, Winfried S

2016-04-01

Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

Disordered expression of inhibitory receptors on the NK1-type natural killer (NK) leukaemic cells from patients with hypersensitivity to mosquito bites

PubMed Central

Seo, N; Tokura, Y; Ishihara, S; Takeoka, Y; Tagawa, S; Takigawa, M

2000-01-01

Recent studies have revealed the existence of a distinct type of NK cell leukaemia of the juvenile type, which presents with hypersensitivity to mosquito bites (HMB) as an essential clinical manifestation and is infected with clonal Epstein–Barr virus (EBV). This disorder is thus called HMB-EBV-NK disease and has been reported in Orientals, mostly from Japan. We investigated the profile of cytokine production and the expression of both types of NK inhibitory receptors, i.e. CD94 lectin-like dimers and killer-cell immunoglobulin-like receptors, in NK leukaemic cells from three patients with HMB-EBV-NK disease. It was found that freshly isolated NK leukaemic cells expressed mRNA for interferon-gamma (IFN-γ) and additionally produced IL-10 upon stimulation with IL-2, indicating that the NK cells were of NK1 type. More than 98% of NK cells from the patients bore CD94 at a higher level than did normal NK cells, whereas p70 or NKAT2, belonging to immunoglobulin-like receptor, was not expressed in those NK cells. Freshly isolated leukaemic NK cells transcribed mRNA for CD94-associated molecule NKG2C at an abnormally high level, and upon stimulation with IL-2 and/or IL-12 they expressed NKG2A as well. The disordered expression of these inhibitory receptors not only provides some insights into the pathogenesis of HMB-EBV-NK disease but also can be used as phenotypic markers for the diagnosis of this type of NK cell leukaemia. PMID:10844517

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells.

PubMed

Paolino, Magdalena; Choidas, Axel; Wallner, Stephanie; Pranjic, Blanka; Uribesalgo, Iris; Loeser, Stefanie; Jamieson, Amanda M; Langdon, Wallace Y; Ikeda, Fumiyo; Fededa, Juan Pablo; Cronin, Shane J; Nitsch, Roberto; Schultz-Fademrecht, Carsten; Eickhoff, Jan; Menninger, Sascha; Unger, Anke; Torka, Robert; Gruber, Thomas; Hinterleitner, Reinhard; Baier, Gottfried; Wolf, Dominik; Ullrich, Axel; Klebl, Bert M; Penninger, Josef M

2014-03-27

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

Cattle NK Cell Heterogeneity and the Influence of MHC Class I

PubMed Central

Allan, Alasdair J.; Sanderson, Nicholas D.; Gubbins, Simon; Ellis, Shirley A.

2015-01-01

Primate and rodent NK cells form highly heterogeneous lymphocyte populations owing to the differential expression of germline-encoded receptors. Many of these receptors are polymorphic and recognize equally polymorphic determinants of MHC class I. This diversity can lead to individuals carrying NK cells with different specificities. Cattle have an unusually diverse repertoire of NK cell receptor genes predicted to encode receptors that recognize MHC class I. To begin to examine whether this genetic diversity leads to a diverse NK cell population, we isolated peripheral NK cells from cattle with different MHC homozygous genotypes. Cytokine stimulation differentially influenced the transcription of five receptors at the cell population level. Using dilution cultures, we found that a further seven receptors were differentially transcribed, including five predicted to recognize MHC class I. Moreover, there was a statistically significant reduction in killer cell lectin-like receptor mRNA expression between cultures with different CD2 phenotypes and from animals with different MHC class I haplotypes. This finding confirms that cattle NK cells are a heterogeneous population and reveals that the receptors creating this diversity are influenced by the MHC. The importance of this heterogeneity will become clear as we learn more about the role of NK cells in cattle disease resistance and vaccination. PMID:26216890

Development and Function of CD94-Deficient Natural Killer Cells

PubMed Central

Orr, Mark T.; Wu, Jun; Fang, Min; Sigal, Luis J.; Spee, Pieter; Egebjerg, Thomas; Dissen, Erik; Fossum, Sigbjørn; Phillips, Joseph H.; Lanier, Lewis L.

2010-01-01

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions. PMID:21151939

Development and function of CD94-deficient natural killer cells.

PubMed

Orr, Mark T; Wu, Jun; Fang, Min; Sigal, Luis J; Spee, Pieter; Egebjerg, Thomas; Dissen, Erik; Fossum, Sigbjørn; Phillips, Joseph H; Lanier, Lewis L

2010-12-03

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.

Human liver-resident CD56(bright)/CD16(neg) NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways.

PubMed

Hudspeth, Kelly; Donadon, Matteo; Cimino, Matteo; Pontarini, Elena; Tentorio, Paolo; Preti, Max; Hong, Michelle; Bertoletti, Antonio; Bicciato, Silvio; Invernizzi, Pietro; Lugli, Enrico; Torzilli, Guido; Gershwin, M Eric; Mavilio, Domenico

2016-01-01

The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56(bright) lr-NK cells that localize within hepatic sinusoids. CD56(bright) lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids express CCL3(pos) Kupffer cells, CXCL16(pos) endothelial cells and CCL5(pos) T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56(bright) NK cells that constitutively express CCR5 and CXCR6. CD56(bright) lr-NK cells co-exist with CD56(dim) conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56(dim) c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56(bright) lr-NK cells will require modification of hepatic CCR5 and CXCR6. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human liver-resident CD56bright/CD16neg NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways

PubMed Central

Hudspeth, Kelly; Donadon, Matteo; Cimino, Matteo; Pontarini, Elena; Tentorio, Paolo; Preti, Max; Hong, Michelle; Bertoletti, Antonio; Bicciato, Silvio; Invernizzi, Pietro; Lugli, Enrico; Torzilli, Guido; Gershwin, M. Eric; Mavilio, Domenico

2015-01-01

Rationale The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. Objectives We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. Findings Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56bright lr-NK cells that localize within hepatic sinusoids. Further, CD56bright lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. In addition, hepatic sinusoids express CCL3pos Kupffer cells, CXCL16pos endothelial cells and CCL5pos T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a tissue niche for lr-CD56bright NK cells that constitutively express CCR5 and CXCR6. CD56bright lr-NK cells co-exist with CD56dim conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56dim c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. Conclusion Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56bright lr-NK cells will require modification of hepatic CCR5 and CXCR6. PMID:26330348

Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.

PubMed

Shah, Nina; Martin-Antonio, Beatriz; Yang, Hong; Ku, Stephanie; Lee, Dean A; Cooper, Laurence J N; Decker, William K; Li, Sufang; Robinson, Simon N; Sekine, Takuya; Parmar, Simrit; Gribben, John; Wang, Michael; Rezvani, Katy; Yvon, Eric; Najjar, Amer; Burks, Jared; Kaur, Indreshpal; Champlin, Richard E; Bollard, Catherine M; Shpall, Elizabeth J

2013-01-01

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56(+)/CD3(-)) and less than 1% CD3(+) cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Bridging innate NK cell functions with adaptive immunity.

PubMed

Marcenaro, Emanuela; Carlomagno, Simona; Pesce, Silvia; Moretta, Alessandro; Sivori, Simona

2011-01-01

Killer Ig-like receptors (KIRs) are major human NK receptors displaying either inhibitory or activating functions which recognize allotypic determinants of HLA-class I molecules. Surprisingly, NK cell treatment with CpG-ODN (TLR9 ligands) results in selective down-modulation of KIR3DL2, its co-internalization with CpG-ODN and its translocation to TLR9-rich early endosomes. This novel KIR-associated function may offer clues to better understand the possible role of certain KIRs and also emphasizes the involvement of NK cells in the course of microbial infections. NK cells are involved not only in innate immune responses against viruses and tumors but also participate in the complex network of cell-to cell interaction that leads to the development of adaptive immune responses. In this context the interaction of NK cells with DC appears to play a crucial role in the acquisition of CCR7, a chemokine receptor that enables NK cells to migrate towards lymph nodes in response to CCL19 and/or CCL21. Analysis of NK cell clones revealed that KIR-mismatched but not KIR-matched NK cells acquire CCR7. These data have important implications in haploidentical haematopoietic stem cell transplantation (HSCT), in which KIR-mismatched NK cells may acquire the ability to migrate to secondary lymphoid compartments (SLCs), where they can kill recipient's antigen presenting cells (APCs) and T cells thus preventing graft versus host (and host vs. graft) reactions.

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

PubMed Central

Schafer, Jamie L.; Ries, Moritz; Guha, Natasha; Connole, Michelle; Colantonio, Arnaud D.; Wiertz, Emmanuel J.; Wilson, Nancy A.; Kaur, Amitinder; Evans, David T.

2015-01-01

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses. PMID:26333068

Expression of activating natural killer-cell receptors is a hallmark of the innate-like T-cell neoplasm in peripheral T-cell lymphomas.

PubMed

Uemura, Yu; Isobe, Yasushi; Uchida, Akiko; Asano, Junko; Nishio, Yuji; Sakai, Hirotaka; Hoshikawa, Masahiro; Takagi, Masayuki; Nakamura, Naoya; Miura, Ikuo

2018-04-01

Peripheral T- or natural killer (NK)-cell lymphomas are rare and difficult-to-recognize diseases. It remains arduous to distinguish between NK cell- and cytotoxic T-lymphocyte-derived lymphomas through routine histological evaluation. To clarify the cells of origin, we focused on NK-cell receptors and examined the expression using immunohistochemistry in 22 cases with T- and NK-cell neoplasms comprising angioimmunoblastic T-cell lymphoma, anaplastic lymphoma kinase (ALK)-positive and -negative anaplastic large-cell lymphomas, extranodal NK/T-cell lymphoma, nasal type, monomorphic epitheliotropic intestinal T-cell lymphoma, aggressive NK-cell leukemia, and other peripheral T-cell lymphomas. Inhibitory receptor leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) was detected in 14 (64%) cases, whereas activating receptors DNAM1, NKp46, and NKG2D were expressed in 7 (32%), 9 (41%), and 5 (23%) cases, respectively. Although LILRB1 was detected regardless of the disease entity, the activating NK-cell receptors were expressed predominantly in TIA-1-positive neoplasms (DNAM1, 49%; NKp46, 69%; and NKG2D, 38%). In addition, NKp46 and NKG2D were detected only in NK-cell neoplasms and cytotoxic T-lymphocyte-derived lymphomas including monomorphic epitheliotropic intestinal T-cell lymphoma. One Epstein-Barr virus-harboring cytotoxic T-lymphocyte-derived lymphoma mimicking extranodal NK/T-cell lymphoma, nasal type lacked these NK-cell receptors, indicating different cell origin from NK and innate-like T cells. Furthermore, NKG2D expression showed a negative impact on survival among the 22 examined cases, which mainly received the standard chemotherapy regimen (log-rank test, P = .024). We propose that the presence of activating NK-cell receptors may provide new insights into understanding peripheral T-cell lymphomas and characterizing them as innate-like T-cell neoplasm. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Restoring Natural Killer Cell Immunity against Multiple Myeloma in the Era of New Drugs

PubMed Central

Pittari, Gianfranco; Vago, Luca; Festuccia, Moreno; Bonini, Chiara; Mudawi, Deena; Giaccone, Luisa; Bruno, Benedetto

2017-01-01

Transformed plasma cells in multiple myeloma (MM) are susceptible to natural killer (NK) cell-mediated killing via engagement of tumor ligands for NK activating receptors or “missing-self” recognition. Similar to other cancers, MM targets may elude NK cell immunosurveillance by reprogramming tumor microenvironment and editing cell surface antigen repertoire. Along disease continuum, these effects collectively result in a progressive decline of NK cell immunity, a phenomenon increasingly recognized as a critical determinant of MM progression. In recent years, unprecedented efforts in drug development and experimental research have brought about emergence of novel therapeutic interventions with the potential to override MM-induced NK cell immunosuppression. These NK-cell enhancing treatment strategies may be identified in two major groups: (1) immunomodulatory biologics and small molecules, namely, immune checkpoint inhibitors, therapeutic antibodies, lenalidomide, and indoleamine 2,3-dioxygenase inhibitors and (2) NK cell therapy, namely, adoptive transfer of unmanipulated and chimeric antigen receptor-engineered NK cells. Here, we summarize the mechanisms responsible for NK cell functional suppression in the context of cancer and, specifically, myeloma. Subsequently, contemporary strategies potentially able to reverse NK dysfunction in MM are discussed. PMID:29163516

Spinal action of neurokinins producing cardiovascular responses in the conscious freely moving rat: evidence for a NK-1 receptor mechanism.

PubMed

Hasséssian, H; Drapeau, G; Couture, R

1988-12-01

This study was initiated to characterize the receptors which mediate the cardiovascular responses elicited by the intrathecal (i.th.) administration of neurokinins (NK) in the conscious freely moving rat. The dose response profile for substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) was determined over 0.065-65 nmol doses of the peptides. After i.th. administration at the T8-T10 thoracic level, only SP elicited a dose dependent pressor response. However, all NK elicited a dose dependent increase in heart rate (HR), and the following rank order of potency was observed: SP greater than NKA greater than NKB. SP (6.5 nmol) produced cardiovascular responses markedly greater than an equimolar dose of any of the seven SP fragments which were studied. The C-terminal sequences SP (4-11), [pGlu5]SP (5-11), [pGlu6]SP (6-11), and SP (7-11), as a group were slightly more potent than the N-terminal fragments, SP (1-4), SP (1-7) and SP (1-9) which were almost inactive. The NK-1 receptor selective agonists [Pro9, Met(O2)11]SP and [beta-Ala4, Sar9, Met(O2)11]SP (4-11), produced pressor and positive chronotropic responses equal to or greater in intensity than SP. With up to 6.5 nmol of the NK-2 receptor selective agonist [Nle10]NKA (4-10), no dose dependent cardiovascular response was produced and the NK-3 receptor selective agonist senktide (succinyl-[Asp6, MePhe8]SP (6-11], produced neither a cardiac nor pressor response when 6.5 nmol was administered. These results are consistent with the hypothesis that, receptors of the NK-1 subtype mediate the cardiovascular responses evoked by the spinal action of NK.

NK1R/5-HT1AR interaction is related to the regulation of melanogenesis.

PubMed

Wu, Huali; Zhao, Yucheng; Huang, Qiaoling; Cai, Minxuan; Pan, Qi; Fu, Mengsi; An, Xiaohong; Xia, Zhenjiang; Liu, Meng; Jin, Yu; He, Ling; Shang, Jing

2018-06-01

Substance P (SP) is a candidate mediator along the brain-skin axis and can mimic the effects of stress to regulate melanogenesis. Previously, we and others have found that the regulation of SP for pigmentary function was mediated by neurokinin 1 receptor (NK1R). Emerging evidence has accumulated that psychologic stress can induce dysfunction in the cutaneous serotonin 5-hydroxytryptamine (5-HT)-5-HT1A/1B receptor system, thereby resulting in skin hypopigmentation. Moreover, NK1R and 5-HTR (except 5-HT3) belong to GPCR. The present study aimed at assessing the possible existence of NK1R-5-HTR interactions and related melanogenic functions. Western blot and PCR detection revealed that SP reduced expression of 5-HT1A receptor via the NK1 receptor. Biochemical analyses showed that NK1R and 5-HT1AR could colocalize and interact in a cell and in the skin. When the N terminus of the NK1R protein was removed NK1R surface targeting was prevented, the interaction between NK1R-5-HT1AR decreased, and the depigmentation caused by SP and WAY100635 could be rescued. Importantly, pharmaceutical coadministration of NK1R agonist (SP) and 5-HT1A antagonist (WAY100635) enhanced the NK1-5-HT1A receptor coimmunoprecipitation along with the depigmentary response. SP and WAY100635 cooperation elicited activation of a signaling cascade (the extracellular, regulated protein kinase p-JNK signaling pathway) and inhibition of p70S6K1 phosphorylation and greatly reduced melanin production in vitro and in vivo in mice and zebrafish. Moreover, the SP-induced depigmentation response did not be occur in 5-htr1aa +/- zebrafish embryos. Taken together, the results of our systemic study increases our knowledge of the roles of NK1R and 5-HT1AR in melanogenesis and provides possible, novel therapeutic strategies for treatment of skin hypo/hyperpigmentation.-Wu, H., Zhao, Y., Huang, Q., Cai, M., Pan, Q., Fu, M., An, X., Xia, Z., Liu, M., Jin, Y., He, L., Shang, J. NK1R/5-HT1AR interaction is related to the regulation of melanogenesis.

THE AP-2 CLATHRIN ADAPTOR MEDIATES ENDOCYTOSIS OF AN INHIBITORY KILLER CELL Ig-LIKE RECEPTOR (KIR) IN HUMAN NK CELLS1

PubMed Central

Purdy, Amanda K.; Alvarez-Arias, Diana A.; Oshinsky, Jennifer; James, Ashley M.; Serebriiskii, Ilya; Campbell, Kerry S.

2014-01-01

Stable surface expression of human inhibitory killer cell immunoglobulin-like receptors (KIR) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC-I+ cells. Our recent experiments, however, have found that antibody-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the μ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/μ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of μ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon antibody engagement with the receptor, as compared to untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIR though their ITIM domains. Based upon our results, we propose a model in which non-engaged KIR are internalized by this mechanism, whereas engagement with MHC-I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors. PMID:25238755

Metabolic stability of some tachykinin analogues to cell-surface peptidases: roles for endopeptidase-24.11 and aminopeptidase N.

PubMed

Medeiros, M D; Turner, A J

1995-01-01

The metabolism of several tachykinin antagonists by membrane peptidases has been examined. [beta Ala8]NKA(4-10) was not stabilized against degradation by endopeptidase-24.11 and this was the major activity in renal brush border membranes hydrolyzing this peptide. The antagonist MEN 10263 was much more resistant to hydrolysis by endopeptidase-24.11, although hydrolysis of the C-terminal Leu-Phe bond was detectable. Three other tachykinin receptor antagonists (MEN 10208, MEN 10207, and MEN 10376), by virtue of D-Trp substitutions, were rendered resistant to endopeptidase-24.11 but were still susceptible to aminopeptidase action. These studies provide further insight into design features necessary to produce metabolically stable peptide analogues.

A novel system of polymorphic and diverse NK cell receptors in primates.

PubMed

Averdam, Anne; Petersen, Beatrix; Rosner, Cornelia; Neff, Jennifer; Roos, Christian; Eberle, Manfred; Aujard, Fabienne; Münch, Claudia; Schempp, Werner; Carrington, Mary; Shiina, Takashi; Inoko, Hidetoshi; Knaust, Florian; Coggill, Penny; Sehra, Harminder; Beck, Stephan; Abi-Rached, Laurent; Reinhardt, Richard; Walter, Lutz

2009-10-01

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.

A Novel System of Polymorphic and Diverse NK Cell Receptors in Primates

PubMed Central

Rosner, Cornelia; Neff, Jennifer; Roos, Christian; Eberle, Manfred; Aujard, Fabienne; Münch, Claudia; Schempp, Werner; Carrington, Mary; Shiina, Takashi; Inoko, Hidetoshi; Knaust, Florian; Coggill, Penny; Sehra, Harminder; Beck, Stephan; Abi-Rached, Laurent; Reinhardt, Richard; Walter, Lutz

2009-01-01

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in “higher” primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire. PMID:19834558

Substance P inhibits natural killer cell cytotoxicity through the neurokinin-1 receptor.

PubMed

Monaco-Shawver, Linda; Schwartz, Lynnae; Tuluc, Florin; Guo, Chang-Jiang; Lai, Jian Ping; Gunnam, Satya M; Kilpatrick, Laurie E; Banerjee, Pinaki P; Douglas, Steven D; Orange, Jordan S

2011-01-01

SP is a potent neuroimmunomodulator that functions through ligating members of the neurokinin receptor family, one of which, NK1R, is widely expressed in immune cells. As in humans, circulating SP levels are increased in pathologic states associated with impairment of NK cell functions, such as depression and HIV infection, we hypothesized that SP has a direct, inhibitory effect upon NK cells. We have studied a clonal human NK cell line (YTS) as well as ex vivo human NK cells and have determined that truncated and full-length NK1R isoforms are expressed in and SP bound by ex vivo NK cells and the YTS NK cell line. Incubation of YTS cells with 10⁻⁶ M SP and ex vivo NK cells with 10⁻⁵ M SP inhibited cytotoxic ability by ∼20% and reduced degranulation. This inhibitory effect upon cytotoxicity was partially prevented by the NK1R antagonist CP96,345. The treatment of YTS or ex vivo NK cells with SP neither down-modulated NCR expression nor affected triggering receptor-induced NF-κB activation. Preincubation of YTS cells with SP, however, did abbreviate the typically prolonged intracellular calcium increase induced by target cell engagement and reduced triggering receptor-induced pERK. Thus, SP has the potential to regulate NK cell functions and acts downstream from neurokinin receptors to modulate NK cell activation signaling. This mechanism may contribute to impairment of NK cell function in certain disease states associated with increased circulating SP. Antagonism of this system may present an opportunity to augment NK cell function therapeutically in selected human diseases.

Neurokinin-2 receptor levels correlate with intensity, frequency, and duration of pain in chronic pancreatitis.

PubMed

Michalski, Christoph W; Shi, Xin; Reiser, Carolin; Fachinger, Patrick; Zimmermann, Arthur; Büchler, Markus W; Di Sebastiano, Pierluigi; Friess, Helmut

2007-11-01

Generation and maintenance of pain in chronic pancreatitis (CP) have been shown to be partially attributable to neuroimmune interactions, which involve neuropeptides such as substance P (SP). So far, expression of SP receptors NK-2R, NK-3R, the SP-encoding gene preprotachykinin A (PPT-A), and the SP degradation enzyme neutral endopeptidase (NEP) and their relation to pain in CP have not been determined. Tissue samples from patients with CP (n = 25) and from healthy donors (n = 20) were analyzed for PPT-A, NK-2R, NK-3R, and NEP expression using quantitative RT-PCR. NEP protein levels were examined by immunoblot analysis and its localization was determined using immunohistochemistry. A scoring system was used to grade the extent of fibrosis on hematoxylin and eosin- and Masson-Trichrome-stained sections. Messenger RNA levels and the extent of pain were analyzed for correlations. In CP tissues, NK-2R and PPT-A expression was increased, whereas NK-3R and NEP mRNA levels were comparable with normal pancreas. Overexpression of NK-2R was related to the intensity, frequency, and duration of pain in CP patients. NK-1R and NEP expression was significantly related to the extent of fibrosis. Expression of NK-2R and PPT-A is increased in CP and is associated with pain. Failure to up-regulate NEP may contribute to the disruption of the neuropeptides loop balance in CP and thus may exacerbate the severe pain syndrome.

NK cells and their receptors in naive and rituximab-treated patients with anti-MAG polyneuropathy.

PubMed

Benedetti, Luana; Facco, Monica; Franciotta, Diego; Dalla Torre, Chiara; Campagnolo, Marta; Lucchetta, Marta; Boscaro, Elisa; Ermani, Mario; Del Sette, Massimo; Berno, Tamara; Candiotto, Laura; Zambello, Renato; Briani, Chiara

2013-08-15

Natural killer (NK) cells can bridge innate and acquired immunity, and play a role in autoimmunity. A few studies evaluated the distribution of NK cells and the expression of their receptors in chronic immune-mediated demyelinating polyneuropathies. We investigated NK cell distribution and NK cell receptor expression in 20 naïve patients with anti-MAG polyneuropathy (MAG-PN). Using flow cytometry, we analysed NK cells and a series of NK cell receptors in the peripheral blood of patients with MAG-PN, and, as controls, in patients with chronic inflammatory demyelinating peripheral polyradiculoneuropathy (CIDP) and in healthy subjects. Six MAG-PN patients were also tested after rituximab treatment. At baseline the percentage of NK cells did not differ among the groups. KIR2DL2 receptor expression in MAG-PN patients was higher, andCD94/NKG2A receptor expression in both MAG-PN and CIDP patients was lower than in healthy controls. These abnormalities did not correlate with any clinical or demographic variable. No modification was found after rituximab therapy. The data suggest that MAG-PN shows abnormalities in NK cell receptors that characterise other autoimmune diseases, and cannot help in differential diagnosis with CIDP. The impairment of the relevant CD94/NKG2A inhibitory pathway, which might play a central role in the development and perpetuation of MAG-PN, warrants further functional investigations. Copyright © 2013 Elsevier B.V. All rights reserved.

Electrophysiological effects of tachykinin analogues on ganglion cell activity in cyprinid fish retina.

PubMed

Downing, J E; Djamgoź, M B

1993-02-01

Electrical spike activity of ganglion cells has been recorded extracellularly in the teleost (roach) retina, and effects of a variety of tachykinins studied at a working concentration of 1 microM. Application of substance P mostly caused a slow and prolonged increase in background activity. In contrast, the response to carbachol was very brisk and short-lasting. Substance P and physalaemin predominantly induced an enhancement of 'On' and 'Off' components of light-evoked responses, whilst eledoisin and neurokinin A were mostly inhibitory. All effects were independent of chromatic and spatial aspects of the responses. Interestingly, in the presence of a tachykinin antagonist, 'Spantide' [D-Arg1,D-Pro2, D-Trp7.9, Leu11]SP, the profile of the effect of substance P reversed, inhibitory actions becoming much more common. Taken together, the results suggest that a tachykinin system utilising two subtypes of the receptor may be active in the roach retina and these may be involved in differential control of visual sensitivity.

The effect of mifepristone on the peripheral blood natural killer cell's cytotoxicity and expression of CD94/NKG2A and NKG2D during the implantation phase.

PubMed

Chen, Xiu-Ying; Zhuang, Ya-Ling; Li, Li; Zhang, Wu-Wen; Huang, Li-Li

2010-05-15

To investigate the effect of mifepristone on peripheral blood natural killer cell's (pbNK) cytotoxicity and the expression of the inhibitory receptor CD94/NKG2A and the activated receptor NKG2D on pbNK cells. In vitro study. University hospital and research laboratory. Twenty healthy nonpregnant women. Detected the cytolytic activity of pbNK to K562 target cells; measured the expression of CD94/NKG2A and NKG2D on pbNK. Cytotoxicity of pbNK was detected by Methyl thiazolyl tetrazolium. The expression of CD94/NKG2A and NKG2D receptor on pbNK cells were detected by flow cytometry. The NK cell cytotoxicity and the expression of inhibitory receptor CD94/NKG2A during the proliferative phase (81.71 +/- 11.5, 86.6 +/- 9.0) was significantly higher than the secretory phase (60.16 +/- 19.2, 60.15 +/- 31.0). The NK cells cytotoxicity, after being treated with mifepristone and the expression of inhibitory receptor CD94/NKG2A on pbNK cells treated with 200 nmol/L mifepristone, were significantly increased. Mifepristone had no effect on the expression of activating receptor NKG2D. These data suggest that Mifepristone maybe exert its anti-implantation function by increasing NK cytotoxicity. The increasing NK cytotoxicity of mifepristone is not related to CD94/NKG2A and NKG2D. In the secretory phase down-regulated CD94/NKG2A, NKG2D, and NK cytotoxicity may benefit with embryo implantation. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

Trigeminal Medullary Dorsal Horn Neurons Activated by Nasal Stimulation Coexpress AMPA, NMDA, and NK1 Receptors

PubMed Central

McCulloch, P. F.; DiNovo, K. M.; Westerhaus, D. J.; Vizinas, T. A.; Peevey, J. F.; Lach, M. A.; Czarnocki, P.

2013-01-01

Afferent information initiating the cardiorespiratory responses during nasal stimulation projects from the nasal passages to neurons within the trigeminal medullary dorsal horn (MDH) via the anterior ethmoidal nerve (AEN). Central AEN terminals are thought to release glutamate to activate the MDH neurons. This study was designed to determine which neurotransmitter receptors (AMPA, kainate, or NMDA glutamate receptor subtypes or the Substance P receptor NK1) are expressed by these activated MDH neurons. Fos was used as a neuronal marker of activated neurons, and immunohistochemistry combined with epifluorescent microscopy was used to determine which neurotransmitter receptor subunits were coexpressed by activated MDH neurons. Results indicate that, during nasal stimulation with ammonia vapors in urethane-anesthetized Sprague-Dawley rats, activated neurons within the superficial MDH coexpress the AMPA glutamate receptor subunits GluA1 (95.8%) and GluA2/3 (88.2%), the NMDA glutamate receptor subunits GluN1 (89.1%) and GluN2A (41.4%), and NK1 receptors (64.0%). It is therefore likely that during nasal stimulation the central terminals of the AEN release glutamate and substance P that then produces activation of these MDH neurons. The involvement of AMPA and NMDA receptors may mediate fast and slow neurotransmission, respectively, while NK1 receptor involvement may indicate activation of a nociceptive pathway. PMID:24967301

Dok1 and Dok2 proteins regulate natural killer cell development and function

PubMed Central

Celis-Gutierrez, Javier; Boyron, Marilyn; Walzer, Thierry; Pandolfi, Pier Paolo; Jonjić, Stipan; Olive, Daniel; Dalod, Marc; Vivier, Eric; Nunès, Jacques A

2014-01-01

Natural killer (NK) cells are involved in immune responses against tumors and microbes. NK-cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Here, we show that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK-cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK-cell-activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK-cell maturation defect and leads to increased IFN-γ production induced by activating receptors. Taken together, these results reveal that Dok1 and Dok2 proteins are involved in an intrinsic negative feedback loop downstream of NK-cell-activating receptors in mouse and human. PMID:24963146

Cannabinoid CB1 receptor facilitation of substance P release in the rat spinal cord, measured as neurokinin 1 receptor internalization

PubMed Central

Zhang, Guohua; Chen, Wenling; Lao, Lijun; Marvizón, Juan Carlos G.

2010-01-01

The contribution of CB1 receptors in the spinal cord to cannabinoid analgesia is still unclear. The objective of this study was to investigate the effect of CB1 receptors on substance P release from primary afferent terminals in the spinal cord. Substance P release was measured as NK1 receptor internalization in lamina I neurons. It was induced in spinal cord slices by dorsal root stimulation and in live rats by a noxious stimulus. In spinal cord slices, the CB1 receptor antagonists AM251, AM281 and rimonabant partially but potently inhibited NK1 receptor internalization induced by electrical stimulation of the dorsal root. This was due to an inhibition of substance P release and not of NK1 receptor internalization itself, because AM251 and AM281 did not inhibit NK1 receptor internalization induced by exogenous substance P. The CB1 receptor agonist ACEA increased NK1 receptor internalization evoked by dorsal root stimulation. The effects of AM251 and ACEA cancelled each other. In vivo, AM251 injected intrathecally decreased NK1 receptor internalization in spinal segments L5 and L6 induced by noxious hind paw clamp. Intrathecal AM251 also produced analgesia to radiant heat stimulation of the paw. The inhibition by AM251 of NK1 receptor internalization was reversed by antagonists of μ-opioid and GABAB receptors. This indicates that CB1 receptors facilitate substance P release by inhibiting the release of GABA and opioids next to primary afferent terminals, producing disinhibition. This results in a pronociceptive effect of CB1 receptors in the spinal cord. PMID:20074214

Two classes of binding sites for [3H]substance P in rat cerebral cortex.

PubMed

Geraghty, D P; Burcher, E

1993-01-22

The binding characteristics of [3H]substance P ([3H]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [3H]SP reached equilibrium after 50 min at 25 degrees C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, Kd, 0.22 nM) and low affinity sites (Kd, 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [3H]SP binding. The rank order of potency for inhibition of [3H]SP (2 nM) binding was SP > or = [Sar9, Met(O2)11]-SP > or = physalaemin > SP(3-11) > NP gamma = [Ala3]-SP > or = SP(4-11) > or = NPK > or = SP(5-11) > or = NKB approximately NKA > SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [3H]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9, Met(O2)11]-SP could be resolved into two components.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of NK cells in HIV-1 protection: autologous, allogeneic or both?

PubMed

Hens, Jef; Jennes, Wim; Kestens, Luc

2016-01-01

Natural killer (NK) cells specialize in killing virally infected- or tumor cells and are part of the innate immune system. The activational state of NK cells is determined by the balance of incoming activating and inhibitory signals mediated by receptor-ligand binding with the target cell. These receptor-ligand bonds mainly consist of the killer immunoglobulin-like receptors (KIR), which are expressed at the cell surface of NK cells, and their ligands: the highly variable human leukocyte antigen -class I molecules (HLA). Absence of an inhibitory receptor-ligand bond lowers the NK cell activation threshold, whereas an activating receptor-ligand bond stimulates the cell, potentially overcoming this threshold and triggering NK cell activation. NK cells influence the course of infection as well as the acquisition of HIV-1. Several lines of evidence relate the activating NK cell receptor KIR3DS1, in the presence or absence of its putative ligand HLA-Bw4, with slower disease progression as well as resistance to HIV-1 infection. Overall, resistance to HIV-1 infection predominantly correlates with activating KIR/HLA profiles, consisting of e.g. activating KIRs, group B haplotypes, or inhibitory KIRs in absence of their ligands. Such a conclusion is less evident for studies of HIV-1 disease progression, with studies reporting beneficial as well as detrimental effects of activating KIR/HLA genotypes. It is likely that KIR/HLA association studies are complicated by the complexity of the KIR and HLA loci and their mutual interactions, as well as by additional factors like route of HIV exposure, immune activation, presence of co-infections, and the effect of anti-HIV-1 antibodies. One newly discovered NK cell activation pathway associated with resistance to HIV-1 infection involves the presence of an iKIR/HLA mismatch between partners. The absence of such an iKIR/HLA bond renders donor-derived allogeneic HIV-1 infected cells vulnerable to NK cell responses during HIV-1 transmission. Therefore, theoretically, HIV-1 would be eliminated before it has the chance to infect the autologous cells in the recipient. While this "alloreactive" NK cell mechanism is especially relevant to HIV transmission in monogamous couples, it would be interesting to investigate how it could influence resistance to HIV in other settings. The objective of this review is to summarize the knowledge about these autologous and alloreactive NK cell responses with regard to HIV-1 outcome.

Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair.

PubMed

Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

2008-09-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

Ly49 Receptors: Innate and Adaptive Immune Paradigms

PubMed Central

Rahim, Mir Munir A.; Tu, Megan M.; Mahmoud, Ahmad Bakur; Wight, Andrew; Abou-Samra, Elias; Lima, Patricia D. A.; Makrigiannis, Andrew P.

2014-01-01

The Ly49 receptors are type II C-type lectin-like membrane glycoproteins encoded by a family of highly polymorphic and polygenic genes within the mouse natural killer (NK) gene complex. This gene family is designated Klra, and includes genes that encode both inhibitory and activating Ly49 receptors in mice. Ly49 receptors recognize class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as altered cells. Their functional homologs in humans are the killer cell immunoglobulin-like receptors, which recognize HLA class I molecules as ligands. Classically, Ly49 receptors are described as being expressed on both the developing and mature NK cells. The inhibitory Ly49 receptors are involved in NK cell education, a process in which NK cells acquire function and tolerance toward cells that express “self-MHC-I.” On the other hand, the activating Ly49 receptors recognize altered cells expressing activating ligands. New evidence shows a broader Ly49 expression pattern on both innate and adaptive immune cells. Ly49 receptors have been described on multiple NK cell subsets, such as uterine NK and memory NK cells, as well as NKT cells, dendritic cells, plasmacytoid dendritic cells, macrophages, neutrophils, and cells of the adaptive immune system, such as activated T cells and regulatory CD8+ T cells. In this review, we discuss the expression pattern and proposed functions of Ly49 receptors on various immune cells and their contribution to immunity. PMID:24765094

Effects of age and clustered hypoxia on [(125)I] substance P binding to neurotachykinin-1 receptors in brainstem of developing swine.

PubMed

Rodier, M E; Laferrière, A; Moss, I R

2001-03-29

This work focused on the postnatal development of substance P-bound neurotachykinin-1 (NK-1) receptors in the porcine brainstem using 2-3-, 6-11-, 16-18-, and 21-28-day-old piglets versus adult, and on alterations in these receptors after single and six-daily repeated clustered hypoxia using 6-11- and 21-28-day-old piglets. NK-1 receptor localization and densities were determined by quantitative autoradiography using mono-iodinated Bolton-Hunter substance P ([(125)I]BHSP). Slide-mounted brainstem sections, incubated in [(125)I]BHSP and then exposed to film, have shown [(125)I]BHSP binding throughout many brainstem nuclei and tracts, including the ambigual/periambigual (nAmb), dorsal motor vagal (dmnv), gigantocellular (nGC), hypoglossal (nHyp), medial parabrachial (nPBM), lateral reticular (nRL), raphe magnus (nRMg), raphe obscurus (nROb) and solitary tract (nTS) nuclei. NK-1 receptor densities decreased with age. As compared to normoxia, NK-1 receptor densities increased significantly after the six-daily hypoxia protocol in nAmb, dmnv, nHyp, nRL, nRMg, nROb, and nTS of both the young and older age groups. This increase may represent receptor upregulation as an adaptation to repeated hypoxia.

Synergistic inhibition of natural killer cells by the nonsignaling molecule CD94.

PubMed

Cheent, Kuldeep S; Jamil, Khaleel M; Cassidy, Sorcha; Liu, Mengya; Mbiribindi, Berenice; Mulder, Arend; Claas, Frans H J; Purbhoo, Marco A; Khakoo, Salim I

2013-10-15

Peptide selectivity is a feature of inhibitory receptors for MHC class I expressed by natural killer (NK) cells. CD94-NKG2A operates in tandem with the polymorphic killer cell Ig-like receptors (KIR) and Ly49 systems to inhibit NK cells. However, the benefits of having two distinct inhibitory receptor-ligand systems are not clear. We show that noninhibitory peptides presented by HLA-E can augment the inhibition of NKG2A(+) NK cells mediated by MHC class I signal peptides through the engagement of CD94 without a signaling partner. Thus, CD94 is a peptide-selective NK cell receptor, and NK cells can be regulated by nonsignaling interactions. We also show that KIR(+) and NKG2A(+) NK cells respond with differing stoichiometries to MHC class I down-regulation. MHC-I-bound peptide functions as a molecular rheostat controlling NK cell function. Selected peptides which in isolation do not inhibit NK cells can have different effects on KIR and NKG2A receptors. Thus, these two inhibitory systems may complement each other by having distinct responses to bound peptide and surface levels of MHC class I.

Coengagement of CD16 and CD94 receptors mediates secretion of chemokines and induces apoptotic death of naive natural killer cells.

PubMed

Jewett, Anahid; Cacalano, Nicholas A; Head, Christian; Teruel, Antonia

2006-04-01

Down-modulation of CD16 (FcgammaRIII) receptors and loss of natural killer (NK) cell function have been observed in oral cancer patients. However, neither the mechanisms nor the significance of the decrease in CD16 receptors have been fully understood. The cytotoxic activity and survival of NK cells are negatively regulated by antibodies directed against CD16 surface receptor. The addition of anti-CD94 antibody in combination with either F(ab')(2) fragment or intact anti-CD16 antibody to NK cells resulted in significant inhibition of NK cell cytotoxic function and induction of apoptosis in resting human peripheral blood NK cells. Addition of interleukin-2 to anti-CD16 and/or anti-CD94 antibody-treated NK cells significantly inhibited apoptosis and increased the function of NK cells. There was a significant increase in tumor necrosis factor-alpha (TNF-alpha) but not IFN-gamma secretion in NK cells treated either with anti-CD16 antibody alone or in combination with anti-CD94 antibodies. Consequently, the addition of anti-TNF-alpha antibody partially inhibited apoptosis of NK cells mediated by the combination of anti-CD94 and anti-CD16 antibodies. Increase in apoptotic death of NK cells also correlated with an increase in type 2 inflammatory cytokines and in the induction of chemokines. Thus, we conclude that binding of antibodies to CD16 and CD94 NK cell receptors induces death of the NK cells and signals for the release of chemokines.

Chronic intermittent hypoxia reduces neurokinin-1 (NK(1)) receptor density in small dendrites of non-catecholaminergic neurons in mouse nucleus tractus solitarius.

PubMed

Lessard, Andrée; Coleman, Christal G; Pickel, Virginia M

2010-06-01

Chronic intermittent hypoxia (CIH) is a frequent concomitant of sleep apnea, which can increase sympathetic nerve activity through mechanisms involving chemoreceptor inputs to the commissural nucleus of the solitary tract (cNTS). These chemosensory inputs co-store glutamate and substance P (SP), an endogenous ligand for neurokinin-1 (NK(1)) receptors. Acute hypoxia results in internalization of NK(1) receptors, suggesting that CIH also may affect the subcellular distribution of NK(1) receptors in subpopulations of cNTS neurons, some of which may express tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis (TH). To test this hypothesis, we examined dual immunolabeling for the NK(1) receptor and TH in the cNTS of male mice subjected to 10days or 35days of CIH or intermittent air. Electron microscopy revealed that NK(1) receptors and TH were almost exclusively localized within separate somatodendritic profiles in cNTS of control mice. In dendrites, immunogold particles identifying NK(1) receptors were prevalent in the cytoplasm and on the plasmalemmal surface. Compared with controls, CIH produced a significant region-specific decrease in the cytoplasmic (10 and 35days, P<0.05, unpaired Student t-test) and extrasynaptic plasmalemmal (35days, P<0.01, unpaired Student t-test) density of NK(1) immunogold particles exclusively in small (<0.1microm) dendrites without TH immunoreactivity. These results suggest that CIH produces a duration-dependent reduction in the availability of NK(1) receptors preferentially in small dendrites of non-catecholaminergic neurons in the cNTS. The implications of our findings are discussed with respect to their potential involvement in the slowly developing hypertension seen in sleep apnea patients. Copyright (c) 2009 Elsevier Inc. All rights reserved.

Chronic intermittent hypoxia reduces neurokinin-1 (NK1) receptor density in small dendrites of non-catecholaminergic neurons in mouse nucleus tractus solitarius

PubMed Central

Lessard, Andrée; Coleman, Christal G.; Pickel, Virginia M.

2010-01-01

Chronic intermittent hypoxia (CIH) is a frequent concomitant of sleep apnea, which can increase sympathetic nerve activity through mechanisms involving chemoreceptor inputs to the commissural nucleus of the solitary tract (cNTS). These chemosensory inputs co-store glutamate and substance P (SP), an endogenous ligand for neurokinin-1 (NK1) receptors. Acute hypoxia results in internalization of NK1 receptors, suggesting that CIH also may affect the subcellular distribution of NK1 receptors in subpopulations of cNTS neurons, some of which may express tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis (TH). To test this hypothesis, we examined dual immunolabeling for the NK1 receptor and TH in the cNTS of male mice subjected to 10 days or 35 days of CIH or intermittent air. Electron microscopy revealed that NK1 receptors and TH were almost exclusively localized within separate somatodendritic profiles in cNTS of control mice. In dendrites, immunogold particles identifying NK1 receptors were prevalent in the cytoplasm and on the plasmalemmal surface. Compared with controls, CIH produced a significant region-specific decrease in the cytoplasmic (10 and 35 days, P< 0.05, unpaired Student t-test) and extrasynaptic plasmalemmal (35 days, P< 0.01, unpaired Student t-test) density of NK1 immunogold particles exclusively in small (<0.1 µm) dendrites without TH immunoreactivity. These results suggest that CIH produces a duration-dependent reduction in the availability of NK1 receptors preferentially in small dendrites of non-catecholaminergic neurons in the cNTS. The implications of our findings are discussed with respect to their potential involvement in the slowly developing hypertension seen in sleep apnea patients. PMID:20206166

Engineering NK Cells Modified With an EGFRvIII-specific Chimeric Antigen Receptor to Overexpress CXCR4 Improves Immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma.

PubMed

Müller, Nadja; Michen, Susanne; Tietze, Stefanie; Töpfer, Katrin; Schulte, Alexander; Lamszus, Katrin; Schmitz, Marc; Schackert, Gabriele; Pastan, Ira; Temme, Achim

2015-06-01

Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell-resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII glioblastoma cells in vitro and to established subcutaneous U87-MG tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor-engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.

Markers of nonselective and specific NK cell activation.

PubMed

Fogel, Leslie A; Sun, Michel M; Geurs, Theresa L; Carayannopoulos, Leonidas N; French, Anthony R

2013-06-15

NK cell activation is controlled by the integration of signals from cytokine receptors and germline-encoded activation and inhibitory receptors. NK cells undergo two distinct phases of activation during murine CMV (MCMV) infection: a nonselective phase mediated by proinflammatory cytokines and a specific phase driven by signaling through Ly49H, an NK cell activation receptor that recognizes infected cells. We sought to delineate cell surface markers that could distinguish NK cells that had been activated nonselectively from those that had been specifically activated through NK cell receptors. We demonstrated that stem cell Ag 1 (Sca-1) is highly upregulated during viral infections (to an even greater extent than CD69) and serves as a novel marker of early, nonselective NK cell activation. Indeed, a greater proportion of Sca-1(+) NK cells produced IFN-γ compared with Sca-1(-) NK cells during MCMV infection. In contrast to the universal upregulation of Sca-1 (as well as KLRG1) on NK cells early during MCMV infection, differential expression of Sca-1, as well as CD27 and KLRG1, was observed on Ly49H(+) and Ly49H(-) NK cells late during MCMV infection. Persistently elevated levels of KLRG1 in the context of downregulation of Sca-1 and CD27 were observed on NK cells that expressed Ly49H. Furthermore, the differential expression patterns of these cell surface markers were dependent on Ly49H recognition of its ligand and did not occur solely as a result of cellular proliferation. These findings demonstrate that a combination of Sca-1, CD27, and KLRG1 can distinguish NK cells nonselectively activated by cytokines from those specifically stimulated through activation receptors.

Targeting NK-1 Receptors to Prevent and Treat Pancreatic Cancer: A New Therapeutic Approach

PubMed Central

Muñoz, Miguel; Coveñas, Rafael

2015-01-01

Pancreatic cancer (PC) is the fourth leading cause of cancer related-deaths in both men and women, and the 1- and 5-year relative survival rates are 25% and 6%, respectively. It is known that smoking, alcoholism and psychological stress are risk factors that can promote PC and increase PC progression. To date, the prevention of PC is crucial because there is no curative treatment. After binding to the neurokinin-1 (NK-1) receptor (a receptor coupled to the stimulatory G-protein Gαs that activates adenylate cyclase), the peptide substance P (SP)—at high concentrations—is involved in many pathophysiological functions, such as depression, smoking, alcoholism, chronic inflammation and cancer. It is known that PC cells and samples express NK-1 receptors; that the NK-1 receptor is overexpressed in PC cells in comparison with non-tumor cells, and that nanomolar concentrations of SP induce PC cell proliferation. By contrast, NK-1 receptor antagonists exert antidepressive, anxiolytic and anti-inflammatory effects and anti-alcohol addiction. These antagonists also exert an antitumor action since in vitro they inhibit PC cell proliferation (PC cells death by apoptosis), and in a xenograft PC mouse model they exert both antitumor and anti-angiogenic actions. NK-1 receptor antagonists could be used for the treatment of PC and hence the NK-1 receptor could be a new promising therapeutic target in PC. PMID:26154566

Peristalsis and fecal pellet propulsion do not require nicotinic, purinergic, 5-HT3, or NK3 receptors in isolated guinea pig distal colon.

PubMed

Nicholas, Sarah; Spencer, Nick J

2010-06-01

The neuronal mechanism by which distension of the colon triggers peristalsis and the propulsion of colonic contents is incompletely understood. In this study, we used video imaging and spatiotemporal mapping techniques to investigate the neuroneuronal mechanisms underlying peristalsis in isolated guinea pig distal colon. In direct contrast to previous studies, we found that hexamethonium (100 muM-1 mM) or mecamylamine (20 muM) never abolished peristalsis or fecal pellet propulsion, although a temporary blockade of peristalsis was common, giving the impression perhaps that peristalsis was blocked permanently. During the initiation of peristalsis, the intraluminal propulsive force applied to an inserted fecal pellet was significantly reduced by hexamethonium 100 muM, even though, once initiated, the propagation velocity of fecal pellets was never reduced by nicotinic antagonists. In the presence of hexamethonium or mecamylamine, further addition of PPADS (10 muM), ondansetron (1 muM), and SR 142801 (300 nM) had no inhibitory effect on the propagation velocity of fecal pellets. In these preparations, antagonists for nicotinic, purinergic (P2), serotonergic (5-HT3), or tachykinergic (NK3) receptors always abolished responses to the agonists for these receptors, confirming that when peristalsis occurred, nicotinic, P2, 5-HT3, and NK3 receptors were blocked. Tetrodotoxin abolished nonnicotinic peristalsis. In summary, nicotinic transmission contributes to excitatory neuroneuronal transmission underlying peristalsis and fecal pellet propulsion but is not required for peristalsis, nor fecal pellet propulsion, as once thought. These observations could be explained by an excitatory nonnicotinic neuroneuronal pathway that can generate peristalsis and induce normal fecal pellet propagation velocities but does not require nicotinic, P2, 5-HT3, or NK3 receptors.

Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ-opioid receptors and GABAB receptors, but not α2 adrenergic receptors

PubMed Central

Zhang, Guohua; Chen, Wenling; Marvizón, Juan Carlos G.

2010-01-01

GABAB, μ-opioid, and adrenergic α2 receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ-opioid and GABAB receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage-gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high frequency dorsal root stimulation was inhibited by the μ-opioid agonist DAMGO and the GABAB agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α2 adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ-opioid and GABAB receptors, but not by α2 receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels. PMID:20726886

Cellular and synaptic effect of substance P on neonatal phrenic motoneurons.

PubMed

Ptak, K; Konrad, M; Di Pasquale, E; Tell, F; Hilaire, G; Monteau, R

2000-01-01

Experiments were carried out on the in vitro brainstem-spinal cord preparation of the newborn rat to analyse the effects of substance P (SP) on phrenic motoneuron (PMN) activity. In current-clamp mode, SP significantly depolarized PMNs, increased their input resistance, decreased the rheobase current and shifted the firing frequency-intensity relationships leftwards, but did not affect spike frequency adaptation or single spike configuration. The neurokinin receptor agonist NK1 had SP-mimetic effects, whereas the NK3 and NK2 receptor agonists were less effective and ineffective, respectively. In a tetrodotoxin-containing aCSF, only SP or the NK1 receptor agonist were still active. No depolarization was observed when the NK1 receptor agonist was applied in the presence of muscarine. In voltage-clamp mode, SP or the NK1 receptor agonist produced an inward current (ISP) which was not significantly reduced by extracellular application of tetraethylammonium, Co2+, 4-aminopyridine or Cs+. In aCSF containing tetrodotoxin, Co2+ and Cs+, ISP was blocked by muscarine. No PMN displayed any M-type potassium current but only a current showing no voltage sensitivity over the range -100 to 0 mV, reversing near the expected EK +, hence consistent with a leak current. SP application to the spinal cord only (using a partitioned chamber) significantly increased the phrenic activity. Pretreatment with the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) decreased the C4 discharge duration and blocked the effect of SP, thus exhibiting an NMDA potentiation by SP. In conclusion, SP modulates postsynaptically the response of phrenic motoneurons to the inspiratory drive through the reduction of a leak conductance and the potentiation of the NMDA component of the synaptic input.

A tachykinin-like neuroendocrine signalling axis couples central serotonin action and nutrient sensing with peripheral lipid metabolism

PubMed Central

Palamiuc, Lavinia; Noble, Tallie; Witham, Emily; Ratanpal, Harkaranveer; Vaughan, Megan; Srinivasan, Supriya

2017-01-01

Serotonin, a central neuromodulator with ancient ties to feeding and metabolism, is a major driver of body fat loss. However, mechanisms by which central serotonin action leads to fat loss remain unknown. Here, we report that the FLP-7 neuropeptide and its cognate receptor, NPR-22, function as the ligand-receptor pair that defines the neuroendocrine axis of serotonergic body fat loss in Caenorhabditis elegans. FLP-7 is secreted as a neuroendocrine peptide in proportion to fluctuations in neural serotonin circuit functions, and its release is regulated from secretory neurons via the nutrient sensor AMPK. FLP-7 acts via the NPR-22/Tachykinin2 receptor in the intestine and drives fat loss via the adipocyte triglyceride lipase ATGL-1. Importantly, this ligand-receptor pair does not alter other serotonin-dependent behaviours including food intake. For global modulators such as serotonin, the use of distinct neuroendocrine peptides for each output may be one means to achieve phenotypic selectivity. PMID:28128367

Atomoxetine reduces hyperactive/impulsive behaviours in neurokinin-1 receptor 'knockout' mice.

PubMed

Pillidge, Katharine; Porter, Ashley J; Vasili, Temis; Heal, David J; Stanford, S Clare

2014-12-01

Mice with functional ablation of the neurokinin-1 receptor gene (NK1R(-/-)) display behavioural abnormalities which resemble the hyperactivity, inattention and impulsivity seen in Attention Deficit Hyperactivity Disorder (ADHD). Here, we investigated whether the established ADHD treatment, atomoxetine, alleviates these abnormalities when tested in the light/dark exploration box (LDEB) and 5-Choice Serial Reaction-Time Task (5-CSRTT). Separate cohorts of mice were tested in the 5-CSRTT and LDEB after treatment with no injection, vehicle or atomoxetine (5-CSRTT: 0.3, 3 or 10mg/kg; LDEB: 1, 3 or 10mg/kg). Atomoxetine reduced the hyperactivity displayed by NK1R(-/-) mice in the LDEB at a dose (3mg/kg) which did not affect the locomotor activity of wildtypes. Atomoxetine (10mg/kg) also reduced impulsivity in NK1R(-/-) mice, but not wildtypes, in the 5-CSRTT. No dose of drug affected attention in either genotype. This evidence that atomoxetine reduces hyperactive/impulsive behaviours in NK1R(-/-) mice consolidates the validity of using NK1R(-/-) mice in research of the aetiology and treatment of ADHD. Copyright © 2014. Published by Elsevier Inc.

Atomoxetine reduces hyperactive/impulsive behaviours in neurokinin-1 receptor ‘knockout’ mice

PubMed Central

Pillidge, Katharine; Porter, Ashley J.; Vasili, Temis; Heal, David J.; Stanford, S. Clare

2014-01-01

Background Mice with functional ablation of the neurokinin-1 receptor gene (NK1R−/−) display behavioural abnormalities which resemble the hyperactivity, inattention and impulsivity seen in Attention Deficit Hyperactivity Disorder (ADHD). Here, we investigated whether the established ADHD treatment, atomoxetine, alleviates these abnormalities when tested in the light/dark exploration box (LDEB) and 5-Choice Serial Reaction-Time Task (5-CSRTT). Methods Separate cohorts of mice were tested in the 5-CSRTT and LDEB after treatment with no injection, vehicle or atomoxetine (5-CSRTT: 0.3, 3 or 10 mg/kg; LDEB: 1, 3 or 10 mg/kg). Results Atomoxetine reduced the hyperactivity displayed by NK1R−/− mice in the LDEB at a dose (3 mg/kg) which did not affect the locomotor activity of wildtypes. Atomoxetine (10 mg/kg) also reduced impulsivity in NK1R−/− mice, but not wildtypes, in the 5-CSRTT. No dose of drug affected attention in either genotype. Conclusions This evidence that atomoxetine reduces hyperactive/impulsive behaviours in NK1R−/− mice consolidates the validity of using NK1R−/− mice in research of the aetiology and treatment of ADHD. PMID:25450119

Identification, characterisation and expression analysis of natural killer receptor genes in Chlamydia pecorum infected koalas (Phascolarctos cinereus).

PubMed

Morris, Katrina M; Mathew, Marina; Waugh, Courtney; Ujvari, Beata; Timms, Peter; Polkinghorne, Adam; Belov, Katherine

2015-10-15

Koalas (Phascolarctos cinereus), an iconic Australian marsupial, are being heavily impacted by the spread of Chlamydia pecorum, an obligate intracellular bacterial pathogen. Koalas vary in their response to this pathogen, with some showing no symptoms, while others suffer severe symptoms leading to infertility, blindness or death. Little is known about the pathology of this disease and the immune response against it in this host. Studies have demonstrated that natural killer (NK) cells, key components of the innate immune system, are involved in the immune response to chlamydial infections in humans. These cells can directly lyse cells infected by intracellular pathogens and their ability to recognise these infected cells is mediated through NK receptors on their surface. These are encoded in two regions of the genome, the leukocyte receptor complex (LRC) and the natural killer complex (NKC). These two families evolve rapidly and different repertoires of genes, which have evolved by gene duplication, are seen in different species. In this study we aimed to characterise genes belonging to the NK receptor clusters in the koala by searching available koala transcriptomes using a combination of search methods. We developed a qPCR assay to quantify relative expression of four genes, two encoded within the NK receptor cluster (CLEC1B, CLEC4E) and two known to play a role in NK response to Chalmydia in humans (NCR3, PRF1). We found that the NK receptor repertoire of the koala closely resembles that of the Tasmanian devil, with minimal genes in the NKC, but with lineage specific expansions in the LRC. Additional genes important for NK cell activity, NCR3 and PRF1, were also identified and characterised. In a preliminary study to investigate whether these genes are involved in the koala immune response to infection by its chlamydial pathogen, C. pecorum, we investigated the expression of four genes in koalas with active chlamydia infection, those with past infection and those without infection using qPCR. This analysis revealed that one of these four, CLEC4E, may be upregulated in response to chlamydia infection. We have characterised genes of the NKC and LRC in koalas and have discovered evidence that one of these genes may be upregulated in koalas with chlamydia, suggesting that these receptors may play a role in the immune response of koalas to chlamydia infection.

NK cell-based immunotherapy for malignant diseases

PubMed Central

Cheng, Min; Chen, Yongyan; Xiao, Weihua; Sun, Rui; Tian, Zhigang

2013-01-01

Natural killer (NK) cells play critical roles in host immunity against cancer. In response, cancers develop mechanisms to escape NK cell attack or induce defective NK cells. Current NK cell-based cancer immunotherapy aims to overcome NK cell paralysis using several approaches. One approach uses expanded allogeneic NK cells, which are not inhibited by self histocompatibility antigens like autologous NK cells, for adoptive cellular immunotherapy. Another adoptive transfer approach uses stable allogeneic NK cell lines, which is more practical for quality control and large-scale production. A third approach is genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc receptors and/or chimeric tumor-antigen receptors. Therapeutic NK cells can be derived from various sources, including peripheral or cord blood cells, stem cells or even induced pluripotent stem cells (iPSCs), and a variety of stimulators can be used for large-scale production in laboratories or good manufacturing practice (GMP) facilities, including soluble growth factors, immobilized molecules or antibodies, and other cellular activators. A list of NK cell therapies to treat several types of cancer in clinical trials is reviewed here. Several different approaches to NK-based immunotherapy, such as tissue-specific NK cells, killer receptor-oriented NK cells and chemically treated NK cells, are discussed. A few new techniques or strategies to monitor NK cell therapy by non-invasive imaging, predetermine the efficiency of NK cell therapy by in vivo experiments and evaluate NK cell therapy approaches in clinical trials are also introduced. PMID:23604045

Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction.

PubMed

Mathew, Stephen O; Chaudhary, Pankaj; Powers, Sheila B; Vishwanatha, Jamboor K; Mathew, Porunelloor A

2016-10-18

Prostate cancer is the most common type of cancer diagnosed and the second leading cause of cancer-related death in American men. Natural Killer (NK) cells are the first line of defense against cancer and infections. NK cell function is regulated by a delicate balance between signals received through activating and inhibitory receptors. Previously, we identified Lectin-like transcript-1 (LLT1/OCIL/CLEC2D) as a counter-receptor for the NK cell inhibitory receptor NKRP1A (CD161). Interaction of LLT1 expressed on target cells with NKRP1A inhibits NK cell activation. In this study, we have found that LLT1 was overexpressed on prostate cancer cell lines (DU145, LNCaP, 22Rv1 and PC3) and in primary prostate cancer tissues both at the mRNA and protein level. We further showed that LLT1 is retained intracellularly in normal prostate cells with minimal cell surface expression. Blocking LLT1 interaction with NKRP1A by anti-LLT1 mAb on prostate cancer cells increased the NK-mediated cytotoxicity of prostate cancer cells. The results indicate that prostate cancer cells may evade immune attack by NK cells by expressing LLT1 to inhibit NK cell-mediated cytolytic activity through LLT1-NKRP1A interaction. Blocking LLT1-NKRP1A interaction will make prostate cancer cells susceptible to killing by NK cells and therefore may be a new therapeutic option for treatment of prostate cancer.

Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

PubMed

Kepler, Christopher K; Markova, Dessislava Z; Koerner, John D; Mendelis, Joseph; Chen, Chiu-Ming; Vaccaro, Alexander R; Risbud, Makarand V; Albert, Todd J; Anderson, D Greg

2015-08-15

Laboratory study. To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1β, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1β, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. 4.

Substance P and central respiratory activity: a comparative in vitro study in NK1 receptor knockout and wild-type mice.

PubMed

Ptak, K; Hunt, S P; Monteau, R

2000-07-01

Neurokinin-1 receptors (NK1) are present within the respiratory medullary network and in the phrenic nucleus, which controls the diaphragm. We compared the efficacy of substance P (SP) at inducing changes in respiratory frequency or the amplitude of the respiratory motor output between NK1 knockout (NK1-/-) and wild-type mice, using the in vitro brainstem-spinal cord preparation. The in vitro respiratory frequency, as well as the variability of the rhythm and the amplitude of the motor output were similar in both lines. In wild-type mice, application of exogenous SP induced either an increase in respiratory frequency (superfusion of the medulla) or an increase of the inspiratory motor output, as defined by the integral of C4 cervical ventral root activity (superfusion of the spinal cord). These two effects were not apparent in NK1-/- mice. In conclusion, NK1 receptors mediate the respiratory responses to SP but the lack of NK1 receptors in newborn NK1-/- mice does not change the respiratory activity.

The impact of chronic nandrolone decanoate administration on the NK1 receptor density in rat brain as determined by autoradiography.

PubMed

Hallberg, Mathias; Kindlundh, Anna M S; Nyberg, Fred

2005-07-01

Adult male Sprague-Dawley rats were treated with the anabolic androgenic steroid nandrolone decanoate (15 mg/kg day) or oil vehicle (sterile arachidis oleum) during 14 days. The effect on the densities of the neurokinin NK1 receptor in brain was examined with autoradiography. An overall tendency of attenuation of NK1 receptor density was observed after completed treatment with nandrolone decanoate. The density of the NK1 receptor was found to be significantly lower compared to control animals in the nucleus accumbens core (37% density reduction), in dentate gyrus (26%), in basolateral amygdaloid nucleus (23%), in ventromedial hypothalamic nucleus (36%), in dorsomedial hypothalamic nucleus (43%) and finally in the periaqueductal gray (PAG) (24%). In the cortex region, no structures exhibited any significant reduction of NK1 receptor density. This result provides additional support to the hypothesis that substance P and the NK1 receptor may be involved as important components that participate in mediating physiological responses including the adverse behaviors often associated with chronically administrated anabolic androgenic steroids in human.

Differences in neurokinin receptor pharmacology between rat and guinea-pig superior cervical ganglia.

PubMed

Seabrook, G R; Main, M; Bowery, B; Wood, N; Hill, R G

1992-04-01

1. The depolarizations elicited by seven neurokinin receptor agonists were examined in both rat and guinea-pig superior cervical ganglia by use of grease-gap methodology in the presence of tetrodotoxin (0.1 microM). Responses were normalised with respect to 1 microM eledoisin. 2. The rank order of agonist potency in the rat ganglia was senktide greater than substance P greater than substance P methyl ester = eleidosin = Sar-Met-substance P greater than neurokinin B greater than neurokinin A, whereas in guinea-pig superior cervical ganglion (SCG) the rank order was senktide greater than Sar-Met-substance P greater than neurokinin B = eledoisin = substance P methyl ester. The concentration-effect curves for substance P and neurokinin A in guinea-pig ganglia were biphasic which precluded the determination of meaningful potency values. 3. The maximal depolarization achieved by subtype selective ligands was different between these two species. On rat and guinea-pig SCG, the NK3-selective ligand, senktide, produced a maximal depolarization of 27% and 274% respectively, whereas the NK1-selective ligand, substance P methyl ester, produced depolarizations of 77% and 64% respectively. 4. The depolarizations induced by substance P methyl ester and senktide in either species were unaffected by atropine (1 microM), suggesting a lack of involvement of presynaptic neurokinin receptors in the generation of the response. 5. The potency of substance P methyl ester, senktide, and neurokinin A were unaffected by pretreating ganglia with the peptidase inhibitors bacitracin (40 micrograms ml-1), leupeptin (4 micrograms ml-1), and chymostatin (2 micrograms ml-1). Similarly, these peptidase inhibitors had no effect on the maximal depolarizations achieved by any of these agonists.6. It is evident that rat and guinea-pig superior cervical ganglia possess both NK, and NK3 receptors, but that their net contribution to depolarizations are different between the two species. The depolarizations in guinea-pig SCG are mediated predominantly by an NK3 subtype and in rat SCG by an NK, receptor subtype.

Genome-wide siRNA screen reveals a new cellular partner of NK cell receptor KIR2DL4: heparan sulfate directly modulates KIR2DL4-mediated responses

PubMed Central

Brusilovsky, Michael; Cordoba, Moti; Rosental, Benyamin; Hershkovitz, Oren; Andrake, Mark D.; Pecherskaya, Anna; Einarson, Margret B.; Zhou, Yan; Braiman, Alex

2013-01-01

KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56high subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cells surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0-domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4) and induces differential localization of KIR2DL4 to rab5+ and rab7+ endosomes, thus leading to down-regulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS Proteo-Glycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking. PMID:24127555

Role of Neurokinin B in the Control of Female Puberty and Its Modulation by Metabolic Status

PubMed Central

Navarro, Víctor M.; Ruiz-Pino, Francisco; Sánchez-Garrido, Miguel A.; García-Galiano, David; Hobbs, Samuel J.; Manfredi-Lozano, María; León, Silvia; Sangiao-Alvarellos, Susana; Castellano, Juan M.; Clifton, Donald K.; Pinilla, Leonor; Steiner, Robert A.; Tena-Sempere, Manuel

2012-01-01

Human genetic studies have revealed that neurokinin B (NKB) and its receptor, NK3R, are essential elements for normal reproduction; however, the precise role of NKB-NK3R signaling in the initiation of puberty remains un known. We investigated here the regulation of Tac2 and Tacr3 mRNAs (encoding NKB and NK3R, respectively) in female rats and demonstrated that their hypothalamic expression is increased along postnatal maturation. At puberty, both genes were widely expressed throughout the brain, including the lateral hypothalamic area (LHA) and the arcuate nucleus (ARC)/medial basal hypothalamus, where the expression of Tacr3 increased across pubertal transition. We showed that central administration of senktide (NK3R agonist), induced LH secretion in pre- and peri-pubertal females. Conversely, chronic infusion of an NK3R antagonist during puberty moderately delayed the timing of vaginal opening (VO) and tended to decrease LH levels. The expression of NKB and its receptor was sensitive to changes in metabolic status during puberty, as reflected by a reduction in Tacr3 (and to a lesser extent, Tac2) expression in the ARC after a 48-h fast. Yet, acute LH responses to senktide in pubertal females were preserved, if not augmented under fasting conditions, suggesting sensitization of the NKB-NK3R-GnRH signaling pathway under metabolic distress. Moreover, repeated administration of senktide to female rats with pubertal arrest due to chronic undernutrition rescued VO (in ~50% of animals) and potently elicited LH release. Altogether, our observations suggest that NKB-NK3R signaling plays a role in pubertal maturation and that its alterations may contribute to pubertal disorders linked to metabolic stress and negative energy balance. PMID:22396413

The activating receptor NKG2D of natural killer cells promotes resistance against enterovirus-mediated inflammatory cardiomyopathy.

PubMed

Klingel, Karin; Fabritius, Cornelia; Sauter, Martina; Göldner, Katrin; Stauch, Diana; Kandolf, Reinhard; Ettischer, Nicole; Gahlen, Sabine; Schönberger, Tanja; Ebner, Susanne; Makrigiannis, Andrew P; Bélanger, Simon; Diefenbach, Andreas; Polić, Bojan; Pratschke, Johann; Kotsch, Katja

2014-10-01

In enterovirus-induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b(high) NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D-dependent cytotoxicity compared to CVB3-susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D-deficient mice but was shown to be independent of CD8(+) T-cell-dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre- (day -1) as well as post-infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D-activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus-induced myocarditis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Human NK cells: From surface receptors to clinical applications.

PubMed

Moretta, Lorenzo; Pietra, Gabriella; Vacca, Paola; Pende, Daniela; Moretta, Francesca; Bertaina, Alice; Mingari, Maria Cristina; Locatelli, Franco; Moretta, Alessandro

2016-10-01

Natural killer (NK) cells play a major role in innate defenses against pathogens, primarily viruses, and are also thought to be part of the immunosurveillance against tumors. They express an array of surface receptors that mediate NK cell function. The human leukocytes antigen (HLA) class I-specific inhibitory receptors allow NK cells to detect and kill cells that have lost or under-express HLA class I antigens, a typical feature of tumor or virally infected cells. However, NK cell activation and induction of cytolytic activity and cytokine production depends on another important checkpoint, namely the expression on target cells of ligands recognized by activating NK receptors. Despite their potent cytolytic activity, NK cells frequently fail to eliminate tumors. This is due to mechanisms of tumor escape, determined by the tumor cells themselves or by tumor-associated cells (i.e. the tumor microenvironment) via the release of soluble suppressive factors or the induction of inhibitory loops involving induction of regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells. The most important clinical application involving NK cells is the cure of high-risk leukemias in the haplo-identical hematopoietic stem cell transplant (HSCT) setting. NK cells originated from hematopoietic stem cells (HSC) of HLA-haploidentical donors may express Killer Immunoglobulin-like receptors (KIRs) that are mismatched with the HLA class I alleles of the recipient. This allows NK cells to kill leukemia blasts residual after the conditioning regimen, while sparing normal cells (that do not express ligands for activating NK receptors). More recent approaches based on the specific removal of TCR α/β(+) T cells and of CD19(+) B cells, allow the infusion, together with CD34(+) HSC, of mature KIR(+) NK cells and of TCR γ/δ(+) T cells, both characterized by a potent anti-leukemia activity. This greatly reduces the time interval necessary to obtain alloreactive, KIR(+) NK cells derived from donor HSC. Another promising approach is based on the use of anti-KIR blocking monoclonal antibodies (mAbs), rendering alloreactive any KIR(+) NK cells. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma.

PubMed

Kremer, Veronika; Ligtenberg, Maarten A; Zendehdel, Rosa; Seitz, Christina; Duivenvoorden, Annet; Wennerberg, Erik; Colón, Eugenia; Scherman-Plogell, Ann-Helén; Lundqvist, Andreas

2017-09-19

Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer. Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays. We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells. To increase the success of NK cell-based therapies of solid tumors, it is of great importance to promote their homing to the tumor site. In this study, we show that stable engineering of human primary NK cells to express a chemokine receptor thereby enhancing their migration is a promising strategy to improve anti-tumor responses following adoptive transfer of NK cells.

Mechanical Conflict System: A Novel Operant Method for the Assessment of Nociceptive Behavior

PubMed Central

Harte, Steven E.; Meyers, Jessica B.; Donahue, Renee R.; Taylor, Bradley K.; Morrow, Thomas J.

2016-01-01

A new operant test for preclinical pain research, termed the Mechanical Conflict System (MCS), is presented. Rats were given a choice either to remain in a brightly lit compartment or to escape to a dark compartment by crossing an array of height-adjustable nociceptive probes. Latency to escape the light compartment was evaluated with varying probe heights (0, .5, 1, 2, 3, and 4 mm above compartment floor) in rats with neuropathic pain induced by constriction nerve injury (CCI) and in naive control rats. Escape responses in CCI rats were assessed following intraperitoneal administration of pregabalin (10 and 30 mg/kg), morphine (2.5 and 5 mg/kg), and the tachykinin NK1 receptor antagonist, RP 67580 (1 and 10 mg/kg). Results indicate that escape latency increased as a function of probe height in both naive and CCI rats. Pregabalin (10 and 30 mg/kg) and morphine (5 mg/kg), but not RP 67580, decreased latency to escape in CCI rats suggesting an antinociceptive effect. In contrast, morphine (10 mg/kg) but not pregabalin (30 mg/kg) increased escape latency in naive rats suggesting a possible anxiolytic action of morphine in response to light-induced fear. No order effects following multiple test sessions were observed. We conclude that the MCS is a valid method to assess behavioral signs of affective pain in rodents. PMID:26915030

Viral Evasion of Natural Killer Cell Activation

PubMed Central

Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

2016-01-01

Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections. PMID:27077876

Viral Evasion of Natural Killer Cell Activation.

PubMed

Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

2016-04-12

Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections.

Activation of Neurokinin-1 Receptors during Ozone Inhalation Contributes to Epithelial Injury and Repair

PubMed Central

Oslund, Karen L.; Hyde, Dallas M.; Putney, Leialoha F.; Alfaro, Mario F.; Walby, William F.; Tyler, Nancy K.; Schelegle, Edward S.

2008-01-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2′-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2′-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress–positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways. PMID:18390473

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

PubMed Central

Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

2011-01-01

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization*

PubMed Central

Arapulisamy, Obulakshmi; Mannangatti, Padmanabhan; Jayanthi, Lankupalle D.

2013-01-01

Neurokinin-1 receptor (NK1R) mediates down-regulation of human norepinephrine (NE) transporter (hNET) via protein kinase C (PKC). However, native NET regulation by NK1R and the mechanism by which NK1R targets NET among other potential effectors are unknown. Effect of NK1R activation on native NET regulation and NET/NK1R interaction were studied using rat brain synaptosomes expressing native NET and NK1R as well as human placental trophoblast (HTR) cells coexpressing WT-hNET or NK1R/PKC-resistant hNET-T258A,S259A double mutant (NET-DM) and hNK1R. The selective NK1R agonist, GR73632, and Substance-P (SP) inhibited NE transport and reduced plasma membrane expression of NET and NK1R. Pretreatment with the NK1R antagonist, EMEND (aprepitant) prevented these NK1R-mediated effects. Immunoprecipitation experiments showed that NET forms stable complexes with NK1R. In HTR cells, combined biotinylation and immunoprecipitation studies revealed plasma membrane localization of NET·NK1R complexes. Receptor activation resulted in the internalization of NET·NK1R complexes. Lipid raft and immunoprecipitation analyses revealed the presence of NET·NK1R complexes exclusively in non-raft membrane fractions under basal/unstimulated conditions. However, NK1R activation led to translocation of NET·NK1R complexes to raft-rich membrane fractions. Importantly, PKCα was found in association with raft-localized NET following SP treatment. Similar to WT-NET, PKC-resistant NET-DM was found in association with NK1R exclusively in non-raft fractions. However, SP treatment failed to translocate NET-DM·NK1R complexes from non-raft fractions to raft fractions. Collectively, these results suggest that NK1R forms physical complexes with NET and that the receptor-mediated Thr258 + Ser259 motif-dependent translocation of NET·NK1R complexes into raft-rich microdomains facilitates NET/NK1R interaction with PKCα to coordinate spatially restricted NET regulation. PMID:23979140

Regulated norepinephrine transporter interaction with the neurokinin-1 receptor establishes transporter subcellular localization.

PubMed

Arapulisamy, Obulakshmi; Mannangatti, Padmanabhan; Jayanthi, Lankupalle D

2013-10-04

Neurokinin-1 receptor (NK1R) mediates down-regulation of human norepinephrine (NE) transporter (hNET) via protein kinase C (PKC). However, native NET regulation by NK1R and the mechanism by which NK1R targets NET among other potential effectors are unknown. Effect of NK1R activation on native NET regulation and NET/NK1R interaction were studied using rat brain synaptosomes expressing native NET and NK1R as well as human placental trophoblast (HTR) cells coexpressing WT-hNET or NK1R/PKC-resistant hNET-T258A,S259A double mutant (NET-DM) and hNK1R. The selective NK1R agonist, GR73632, and Substance-P (SP) inhibited NE transport and reduced plasma membrane expression of NET and NK1R. Pretreatment with the NK1R antagonist, EMEND (aprepitant) prevented these NK1R-mediated effects. Immunoprecipitation experiments showed that NET forms stable complexes with NK1R. In HTR cells, combined biotinylation and immunoprecipitation studies revealed plasma membrane localization of NET·NK1R complexes. Receptor activation resulted in the internalization of NET·NK1R complexes. Lipid raft and immunoprecipitation analyses revealed the presence of NET·NK1R complexes exclusively in non-raft membrane fractions under basal/unstimulated conditions. However, NK1R activation led to translocation of NET·NK1R complexes to raft-rich membrane fractions. Importantly, PKCα was found in association with raft-localized NET following SP treatment. Similar to WT-NET, PKC-resistant NET-DM was found in association with NK1R exclusively in non-raft fractions. However, SP treatment failed to translocate NET-DM·NK1R complexes from non-raft fractions to raft fractions. Collectively, these results suggest that NK1R forms physical complexes with NET and that the receptor-mediated Thr(258) + Ser(259) motif-dependent translocation of NET·NK1R complexes into raft-rich microdomains facilitates NET/NK1R interaction with PKCα to coordinate spatially restricted NET regulation.

NK1 receptor activation in rat rostral ventrolateral medulla selectively attenuates somato-sympathetic reflex while antagonism attenuates sympathetic chemoreflex.

PubMed

Makeham, John M; Goodchild, Ann K; Pilowsky, Paul M

2005-06-01

The effects of activation and blockade of the neurokinin 1 (NK1) receptor in the rostral ventrolateral medulla (RVLM) on arterial blood pressure (ABP), splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, the somato-sympathetic reflex, baroreflex, and chemoreflex were studied in urethane-anesthetized and artificially ventilated Sprague-Dawley rats. Bilateral microinjection of either the stable substance P analog (pGlu5, MePhe8, Sar9)SP(5-11) (DiMe-SP) or the highly selective NK1 agonist [Sar9, Met (O(2))11]SP into the RVLM resulted in an increase in ABP, sSNA, and heart rate and an abolition of phrenic nerve activity. The effects of [Sar9, Met (O(2))11]SP were blocked by the selective nonpeptide NK1 receptor antagonist WIN 51708. NK1 receptor activation also dramatically attenuated the somato-sympathetic reflex elicited by tibial nerve stimulation, while leaving the baroreflex and chemoreflex unaffected. This effect was again blocked by WIN 51708. NK1 receptor antagonism in the RVLM, with WIN 51708 significantly attenuated the sympathoexcitatory response to hypoxia but had no effect on baseline respiratory function. Our findings suggest that substance P and the NK1 receptor play a significant role in the cardiorespiratory reflexes integrated within the RVLM.

Revving up Natural Killer Cells and Cytokine-Induced Killer Cells Against Hematological Malignancies.

PubMed

Pittari, Gianfranco; Filippini, Perla; Gentilcore, Giusy; Grivel, Jean-Charles; Rutella, Sergio

2015-01-01

Natural killer (NK) cells belong to innate immunity and exhibit cytolytic activity against infectious pathogens and tumor cells. NK-cell function is finely tuned by receptors that transduce inhibitory or activating signals, such as killer immunoglobulin-like receptors, NK Group 2 member D (NKG2D), NKG2A/CD94, NKp46, and others, and recognize both foreign and self-antigens expressed by NK-susceptible targets. Recent insights into NK-cell developmental intermediates have translated into a more accurate definition of culture conditions for the in vitro generation and propagation of human NK cells. In this respect, interleukin (IL)-15 and IL-21 are instrumental in driving NK-cell differentiation and maturation, and hold great promise for the design of optimal NK-cell culture protocols. Cytokine-induced killer (CIK) cells possess phenotypic and functional hallmarks of both T cells and NK cells. Similar to T cells, they express CD3 and are expandable in culture, while not requiring functional priming for in vivo activity, like NK cells. CIK cells may offer some advantages over other cell therapy products, including ease of in vitro propagation and no need for exogenous administration of IL-2 for in vivo priming. NK cells and CIK cells can be expanded using a variety of clinical-grade approaches, before their infusion into patients with cancer. Herein, we discuss GMP-compliant strategies to isolate and expand human NK and CIK cells for immunotherapy purposes, focusing on clinical trials of adoptive transfer to patients with hematological malignancies.

PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors.

PubMed

Zhao, Aiping; Shea-Donohue, Terez

2003-10-01

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 microM), but maximal responses were similar (12.3 +/- 1.6 vs. 13.7 +/- 1.3 N/cm2). Trypsin-evoked contraction (1 microM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 microM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.

Modulation of basal and stress-induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L-822429.

PubMed

Singewald, Nicolas; Chicchi, Gary G; Thurner, Clemens C; Tsao, Kwei-Lan; Spetea, Mariana; Schmidhammer, Helmut; Sreepathi, Hari Kishore; Ferraguti, Francesco; Singewald, Georg M; Ebner, Karl

2008-09-01

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self-regulating role of SP, we used a selective neurokinin-1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress-evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L-822429 represents a potent and selective rat NK1 receptor antagonist. Intra-amygdaloid administration of L-822429 via inverse microdialysis enhanced basal, but attenuated swim stress-induced SP release, while the low-affinity enantiomer of L-822429 had no effect. Using light and electron microscopy, synaptic contacts between SP-containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self-regulatory capacity of SP-mediated neurotransmission that differs in the effect on basal and stress-induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor-mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.

Viral antigen mediated NKp46 activation of NK cells results in tumor rejection via NK-DC crosstalk

PubMed Central

Chinnery, Fay; King, Catherine A.; Elliott, Tim; Bateman, Andrew R.; James, Edward

2012-01-01

Natural killer (NK) cells play a critical role in antitumor immunity, their activation being regulated through NK cell receptors. Although the endogenous ligands for these receptors are largely unknown, viral ligands have been identified. We investigated the ability of an activating NK receptor ligand derived from the mumps virus, haemagglutinin-neuraminidase (HN) to enhance NK activation against tumor cells. HN-expressing B16.OVA tumor cells induced stronger activation of NK cells compared with B16.OVA cells and also promoted dendritic cell (DC) activation toward a DC1 phenotype, in vitro. Moreover, incubation of DCs, NK cells and HN-expressing B16-OVA cells further enhanced NK cell activation through the NK-DC crosstalk, in a cell-to-cell contact- and IL-12-dependent fashion. Immunization of mice with HN-expressing B16-OVA cells resulted in > 85% survival rate after subsequent challenge with parental B16 or B16.OVA tumor cells. Tumor rejection was dependent on both NK and CD8+ T cells but not on CD4+ T cells, demonstrating induction of an effective adaptive immune response through innate immune cell activation. Our data indicate the potential of using robust NK cell activation, which through the NK-DC crosstalk stimulates effective antitumor responses, providing an alternate vaccine strategy. PMID:23162755

Neurokinin-1 receptor: functional significance in the immune system in reference to selected infections and inflammation

PubMed Central

Douglas, Steven D.; Leeman, Susan E.

2010-01-01

The G-protein coupled receptor (GPCR), Neurokinin-1 Receptor (NK1R), and its preferred ligand, substance P (SP), are reviewed in relationship to the immune system and selected infections. NK1R and substance P are ubiquitous throughout the animal kingdom. This important pathway has unique functions in numerous cells and tissues. The interaction of SP with its preferred receptor, NK1R, leads to the activation of nuclear factor-kappa-b (NF-κb) and proinflammatory cytokines. NK1R has two isoforms, both a full-length and a truncated form. These isoforms have different functional significances and differ in cell signaling capability. The proinflammatory signals modulated by substance P are important in bacterial, viral, fungal, and parasitic diseases, as well as in immune system function. The SP-NK1R system is a major Class 1, rhodopsin-like GPCR ligand-receptor interaction. PMID:21091716

Upregulating substance P levels to treat obstructive sleep apnea.

PubMed

Ursavas, Ahmet

2008-05-01

The neuropeptide (tachykinin) substance P is widely distributed in the central and peripheral nervous systems. Substance P has been suggested to function as a neurotransmitter, cotransmitter, or neuromodulator in the central and peripheral nervous system. substance P also influences sleep physiology. Neurokinin 1 (NK-1) receptors may also be implicated in the control of sleep/wake behavior. Obstructive sleep apnea syndrome (OSAS) is defined as repeated episodes of upper airway occlusion during sleep with subsequent excessive daytime sleepiness (EDS). Substance P levels are found to be significantly lowered in patients with OSAS. The aim of this review was to investigate the relationship between substance P, EDS and other OSAS complications. The literature was searched using standard methods. Medline and Embase were searched systematically from 1974 to the end of February 2008 for relevant articles published in English. EDS seen in some OSAS patients may be associated with various pathophysiological mechanisms including changes in substance P levels. Intravenous substance P administration in OSAS patients can be effective in the treatment of EDS. Further studies on the possible relationship between low serum substance P and hypertension, reproductive function disorders, memory and learning problems in OSAS cases is required.

Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

PubMed Central

Firor, Amelia E.; Pinz, Kevin G.; Jares, Alexander; Liu, Hua; Salman, Huda; Golightly, Marc; Lan, Fengshuo; Jiang, Xun; Ma, Yupo

2016-01-01

Peripheral T-cell lymphomas (PTCLS) comprise a diverse group of difficult to treat, very aggressive non-Hodgkin's lymphomas (NHLS) with poor prognoses and dismal patient outlook. Despite the fact that PTCLs comprise the majority of T-cell malignancies, the standard of care is poorly established. Chimeric antigen receptor (CAR) immunotherapy has shown in B-cell malignancies to be an effective curative option and this extends promise into treating T-cell malignancies. Because PTCLS frequently develop from mature T-cells, CD3 is similarly strongly and uniformly expressed in many PTCL malignancies, with expression specific to the hematological compartment thus making it an attractive target for CAR design. We engineered a robust 3rd generation anti-CD3 CAR construct (CD3CAR) into an NK cell line (NK-92). We found that CD3CAR NK-92 cells specifically and potently lysed diverse CD3+ human PTCL primary samples as well as T-cell leukemia cells lines ex vivo. Furthermore, CD3CAR NK-92 cells effectively controlled and suppressed Jurkat tumor cell growth in vivo and significantly prolonged survival. In this study, we present the CAR directed targeting of a novel target - CD3 using CAR modified NK-92 cells with an emphasis on efficacy, specificity, and potential for new therapeutic approaches that could improve the current standard of care for PTCLs. PMID:27494836

An N-terminal fragment of substance P, substance P(1-7), down-regulates neurokinin-1 binding in the mouse spinal cord.

PubMed

Yukhananov RYu; Larson, A A

1994-08-29

Injected intrathecally, substance P (SP) down-regulates neurokinin-1 (NK-1) binding in the spinal cord and desensitizes rats to the behavioral effect of SP. N-terminal fragments of SP, such as SP(1-7), induce antinociception and play a role in desensitization to SP in mice. The goal of this study was to assess the abilities of N- and C-terminal fragments of SP to down-regulate NK-1 binding. Binding of [3H]SP to mouse spinal cord membranes was inhibited by SP, CP-96,345, and to a lesser extent by SP(5-11), but not SP(1-7), consistent with these binding sites being NK-1 receptors. Injection of SP(5-11) intrathecally did not affect the affinity (Kd) or concentration (Bmax) of [3H]SP binding. However, injection of 1 nmol of SP(1-7) decreased the Bmax of [3H]SP binding in the spinal cord at 6 h after its injection just as this dose of SP decreased the Bmax at 24 h. These data suggest that the N-terminus of SP is responsible for down-regulation of NK-1 binding. As SP(5-11) did not down-regulate NK-1 binding, activation of NK-1 sites does not appear necessary or sufficient for down-regulation of SP binding. In contrast, SP(1-7), in spite of its inability to interact with NK-1 sites, did down-regulate SP binding, suggesting an indirect mechanism dissociated from NK-1 receptors.

Synergistic inhibition of natural killer cells by the nonsignaling molecule CD94

PubMed Central

Cheent, Kuldeep S.; Jamil, Khaleel M.; Cassidy, Sorcha; Liu, Mengya; Mbiribindi, Berenice; Mulder, Arend; Claas, Frans H. J.; Purbhoo, Marco A.; Khakoo, Salim I.

2013-01-01

Peptide selectivity is a feature of inhibitory receptors for MHC class I expressed by natural killer (NK) cells. CD94–NKG2A operates in tandem with the polymorphic killer cell Ig-like receptors (KIR) and Ly49 systems to inhibit NK cells. However, the benefits of having two distinct inhibitory receptor–ligand systems are not clear. We show that noninhibitory peptides presented by HLA-E can augment the inhibition of NKG2A+ NK cells mediated by MHC class I signal peptides through the engagement of CD94 without a signaling partner. Thus, CD94 is a peptide-selective NK cell receptor, and NK cells can be regulated by nonsignaling interactions. We also show that KIR+ and NKG2A+ NK cells respond with differing stoichiometries to MHC class I down-regulation. MHC-I–bound peptide functions as a molecular rheostat controlling NK cell function. Selected peptides which in isolation do not inhibit NK cells can have different effects on KIR and NKG2A receptors. Thus, these two inhibitory systems may complement each other by having distinct responses to bound peptide and surface levels of MHC class I. PMID:24082146

Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity.

PubMed

Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S

2017-01-01

Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application.

Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity

PubMed Central

Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S.

2017-01-01

Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application. PMID:28572802

Slp-76 is a critical determinant of NK-cell mediated recognition of missing-self targets.

PubMed

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-07-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele-while no major defects were observed in conventional T-cell development/function, a marked defect in NK cell mediated elimination of β2-microglobulin (β2M) deficient target cells was observed. Further studies revealed Slp-76 to control NK-cell receptor expression and maturation; however, activation of Slp-76(ace/ace) NK cells through ITAM-containing NK-cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M(-/-) target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76(ace/ace) NK cells. Overall these studies establish Slp-76 as a critical determinant of NK-cell development and NK cell mediated elimination of missing-self target cells in mice. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Slp-76 is a critical determinant of NK cell-mediated recognition of missing-self targets

PubMed Central

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-01-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying “missing-self” recognition, including the involvement of activating receptors, remain poorly understood. Using ENU mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell-mediated recognition and elimination of “missing-self” targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation [Thr428Ile] in the SH2 domain of Slp-76—a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele—while no major defects were observed in conventional T cell development/function, a marked defect in NK cell-mediated elimination of β2-Microglobulin (β2M)-deficient target cells was observed. Further studies revealed Slp-76 to control NK cell receptor expression and maturation, however, activation of Slp-76ace/ace NK cells through ITAM-containing NK cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M−/− target cell synapse, revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76ace/ace NK cells. Overall these studies establish Slp-76 as a critical determinant of NK cell development and NK cell-mediated elimination of missing-self target cells. PMID:25929249

High expression of NKG2A/CD94 and low expression of granzyme B are associated with reduced cord blood NK cell activity.

PubMed

Wang, Yanyan; Xu, Han; Zheng, Xiaodong; Wei, Haiming; Sun, Rui; Tian, Zhigang

2007-10-01

Human umbilical cord blood (CB) has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). It was reported that the activity of CB NK cells was lower than that of adult peripheral blood (PB) NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-gamma, TNF-alpha and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.

Kinome Analysis of Receptor-Induced Phosphorylation in Human Natural Killer Cells

PubMed Central

König, Sebastian; Nimtz, Manfred; Scheiter, Maxi; Ljunggren, Hans-Gustaf; Bryceson, Yenan T.; Jänsch, Lothar

2012-01-01

Background Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation. Results A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. Conclusions The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses. PMID:22238634

Blockade of neurokinin-1 receptors in the ventral respiratory column does not affect breathing but alters neurochemical release

PubMed Central

Muere, Clarissa; Neumueller, Suzanne; Olesiak, Samantha; Miller, Justin; Hodges, Matthew R.; Pan, Lawrence

2015-01-01

Substance P (SP) and its receptor, neurokinin-1 (NK1R), have been shown to be excitatory modulators of respiratory frequency and to stabilize breathing regularity. Studies in anesthetized mice suggest that tonic activation of NK1Rs is particularly important when other excitatory inputs to the pre-Bötzinger complex in the ventral respiratory column (VRC) are attenuated. Consistent with these findings, muscarinic receptor blockade in the VRC of intact goats elicits an increase in breathing frequency associated with increases in SP and serotonin concentrations, suggesting an involvement of these substances in neuromodulator compensation. To gain insight on the contribution to breathing of endogenous SP and NK1R activation, and how NK1R modulates the release of other neurochemicals, we individually dialyzed antagonists to NK1R (133, 267, 500 μM Spantide; 3 mM RP67580) throughout the VRC of awake and sleeping goats. We found that NK1R blockade with either Spantide at any dose or RP67580 had no effect on breathing or regularity. Both antagonists significantly (P < 0.001) increased SP, while RP67580 also increased serotonin and glycine and decreased thyrotropin-releasing hormone concentrations in the dialysate. Taken together, these data support the concept of neuromodulator interdependence, and we believe that the loss of excitatory input from NK1Rs was locally compensated by changes in other neurochemicals. PMID:25635003

Blockade of neurokinin-1 receptors in the ventral respiratory column does not affect breathing but alters neurochemical release.

PubMed

Muere, Clarissa; Neumueller, Suzanne; Olesiak, Samantha; Miller, Justin; Hodges, Matthew R; Pan, Lawrence; Forster, Hubert V

2015-03-15

Substance P (SP) and its receptor, neurokinin-1 (NK1R), have been shown to be excitatory modulators of respiratory frequency and to stabilize breathing regularity. Studies in anesthetized mice suggest that tonic activation of NK1Rs is particularly important when other excitatory inputs to the pre-Bötzinger complex in the ventral respiratory column (VRC) are attenuated. Consistent with these findings, muscarinic receptor blockade in the VRC of intact goats elicits an increase in breathing frequency associated with increases in SP and serotonin concentrations, suggesting an involvement of these substances in neuromodulator compensation. To gain insight on the contribution to breathing of endogenous SP and NK1R activation, and how NK1R modulates the release of other neurochemicals, we individually dialyzed antagonists to NK1R (133, 267, 500 μM Spantide; 3 mM RP67580) throughout the VRC of awake and sleeping goats. We found that NK1R blockade with either Spantide at any dose or RP67580 had no effect on breathing or regularity. Both antagonists significantly (P < 0.001) increased SP, while RP67580 also increased serotonin and glycine and decreased thyrotropin-releasing hormone concentrations in the dialysate. Taken together, these data support the concept of neuromodulator interdependence, and we believe that the loss of excitatory input from NK1Rs was locally compensated by changes in other neurochemicals. Copyright © 2015 the American Physiological Society.

CX3CR1-dependent recruitment of mature NK cells into the central nervous system contributes to control autoimmune neuroinflammation.

PubMed

Hertwig, Laura; Hamann, Isabell; Romero-Suarez, Silvina; Millward, Jason M; Pietrek, Rebekka; Chanvillard, Coralie; Stuis, Hanna; Pollok, Karolin; Ransohoff, Richard M; Cardona, Astrid E; Infante-Duarte, Carmen

2016-08-01

Fractalkine receptor (CX3CR1)-deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild-type and CX3CR1-deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1-deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1-deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b(+) NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4(+) T cells and kill these autoreactive cells more efficiently than the immature CD11b(-) fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Estradiol alters body temperature regulation in the female mouse.

PubMed

Krajewski-Hall, Sally J; Blackmore, Elise M; McMinn, Jessi R; Rance, Naomi E

2018-01-01

Hot flushes are due to estrogen withdrawal and characterized by the episodic activation of heat dissipation effectors. Recent studies (in humans and rats) have implicated neurokinin 3 (NK 3 ) receptor signaling in the genesis of hot flushes. Although transgenic mice are increasingly used for biomedical research, there is limited information on how 17β-estradiol and NK 3 receptor signaling alters thermoregulation in the mouse. In this study, a method was developed to measure tail skin temperature (T SKIN ) using a small data-logger attached to the surface of the tail, which, when combined with a telemetry probe for core temperature (T CORE ), allowed us to monitor thermoregulation in freely-moving mice over long durations. We report that estradiol treatment of ovariectomized mice reduced T CORE during the light phase (but not the dark phase) while having no effect on T SKIN or activity. Estradiol also lowered T CORE in mice exposed to ambient temperatures ranging from 20 to 36°C. Unlike previous studies in the rat, estradiol treatment of ovariectomized mice did not reduce T SKIN during the dark phase. Subcutaneous injections of an NK 3 receptor agonist (senktide) in ovariectomized mice caused an acute increase in T SKIN and a reduction in T CORE , consistent with the activation of heat dissipation effectors. These changes were reduced by estradiol, suggesting that estradiol lowers the sensitivity of central thermoregulatory pathways to NK 3 receptor activation. Overall, we show that estradiol treatment of ovariectomized mice decreases T CORE during the light phase, reduces the thermoregulatory effects of senktide and modulates thermoregulation differently than previously described in the rat.

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

PubMed

Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

2002-07-01

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

Influence of human cytomegalovirus infection on the NK cell receptor repertoire in children.

PubMed

Monsiváis-Urenda, Adriana; Noyola-Cherpitel, Daniel; Hernández-Salinas, Alba; García-Sepúlveda, Christian; Romo, Neus; Baranda, Lourdes; López-Botet, Miguel; González-Amaro, Roberto

2010-05-01

Human cytomegalovirus (hCMV) infection is usually asymptomatic but may cause disease in immunocompromised hosts. It has been reported that hCMV infection may shape the NK cell receptor (NKR) repertoire in adult individuals, promoting a variable expansion of the CD94/NKG2C+ NK cell subset. We explored the possible relationship between this viral infection and the expression pattern of different NKR including CD94/NKG2C, CD94/NKG2A, immunoglobulin-like transcript 2 (ILT2, CD85j), KIR2DL1/2DS1, KIR3DL1, and CD161 in peripheral blood lymphocytes from healthy children, seropositive (n=21) and seronegative (n=20) for hCMV. Consistent with previous observations in adults, a positive serology for hCMV was associated with increased numbers of NKG2C+ NK and T cells as well as with ILT2+ T lymphocytes. Moreover, the proportions of CD161+ and NKG2C+CD56-CD3- NK cells also tended to be increased in hCMV+ individuals. Excretion of the virus was associated with higher proportions of NKG2C+ NK cells. Altogether, these data reveal that hCMV may have a profound influence on the NKR repertoire in early childhood.

Inflammation-induced abnormalities in the subcellular localization and trafficking of the neurokinin 1 receptor in the enteric nervous system

PubMed Central

Lieu, TinaMarie; Pelayo, Juan Carlos; Eriksson, Emily M.; Veldhuis, Nicholas A.; Bunnett, Nigel W.

2015-01-01

Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK1R) in enteric neurons. We studied NK1R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10−/−-piroxicam and trinitrobenzenesulfonic acid colitis models. NK1R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK1R endocytosis and recycling. Deletion of β-arrestin2, which associates with the activated NK1R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK1R endocytosis in myenteric but not submucosal neurons. Whereas the NK1R in uninflamed neurons recycled within 60 min, NK1R recycling in inflamed neurons was delayed for >120 min, suggesting defective recycling machinery. Inflammation was associated with β-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK1R recycling. We conclude that inflammation evokes redistribution of NK1R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK1R recycling. Defective recycling may be secondary to upregulation of β-arrestin2 and downregulation of ECE-1. Internalized NK1R may generate sustained proinflammatory signals that disrupt normal neuronal functions. PMID:26138465

Balance between activating NKG2D, DNAM-1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts.

PubMed

Nielsen, Natasja; Pascal, Veronique; Fasth, Andreas E R; Sundström, Yvonne; Galsgaard, Elisabeth D; Ahern, David; Andersen, Martin; Baslund, Bo; Bartels, Else M; Bliddal, Henning; Feldmann, Marc; Malmström, Vivianne; Berg, Louise; Spee, Pieter; Söderström, Kalle

2014-08-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast-like synoviocytes (FLS) are central components of the aggressive, tumour-like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA-FLS and NK cells. We used cultured RA-FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA-FLS/NK cell cross-talk. We show that RA-FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM-1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA-FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA-E expressed on RA-FLS further enhanced Nishi cell degranulation in co-culture with RA-FLS. Using cultured RA-FLS and the human NK cell line Nishi as an in vitro model system of RA-FLS/NK cell cross-talk, our results suggest that cell-mediated cytotoxicity of RA-FLS may be one mechanism by which NK cells influence local joint inflammation in RA. © 2014 John Wiley & Sons Ltd.

Balance between activating NKG2D, DNAM-1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

PubMed Central

Nielsen, Natasja; Pascal, Veronique; Fasth, Andreas E R; Sundström, Yvonne; Galsgaard, Elisabeth D; Ahern, David; Andersen, Martin; Baslund, Bo; Bartels, Else M; Bliddal, Henning; Feldmann, Marc; Malmström, Vivianne; Berg, Louise; Spee, Pieter; Söderström, Kalle

2014-01-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast-like synoviocytes (FLS) are central components of the aggressive, tumour-like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA-FLS and NK cells. We used cultured RA-FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA-FLS/NK cell cross-talk. We show that RA-FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM-1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA-FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA-E expressed on RA-FLS further enhanced Nishi cell degranulation in co-culture with RA-FLS. Using cultured RA-FLS and the human NK cell line Nishi as an in vitro model system of RA-FLS/NK cell cross-talk, our results suggest that cell-mediated cytotoxicity of RA-FLS may be one mechanism by which NK cells influence local joint inflammation in RA. PMID:24673109

The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection.

PubMed

Rajalingam, Raja

2016-01-01

Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

Neurokinin-1 receptor antagonists CP-96,345 and L-733,060 protect mice from cytokine-mediated liver injury.

PubMed

Bang, Renate; Sass, Gabriele; Kiemer, Alexandra K; Vollmar, Angelika M; Neuhuber, Winfried L; Tiegs, Gisa

2003-04-01

Previously, we have shown that primary afferent sensory neurons are necessary for disease activity in T cell-mediated immune hepatitis in mice. In the present study, we analyzed the possible role of substance P (SP), an important proinflammatory neuropeptide of these nerve fibers, in an in vivo mouse model of liver inflammation. Liver injury was induced by bacterial lipopolysaccharide (LPS) in D-galactosamine (GalN)-sensitized mice. Depletion of primary afferent nerve fibers by neonatal capsaicin treatment down-regulated circulating levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) and protected mice from GalN/LPS-induced liver injury. Likewise, pretreatment of mice with antagonists of the SP-specific neurokinin-1 receptor (NK-1R), i.e., (2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine (CP-96,345) and (2S,3S)3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine (L-733,060), dose dependently protected mice from GalN/LPS-induced liver injury. The presence of the NK-1R in the murine liver was demonstrated by reverse transcription-polymerase chain reaction, sequence analysis, and immunocytochemistry. NK-1R blockade reduced inflammatory liver damage, i.e., edema formation, neutrophil infiltration, hepatocyte apoptosis, and necrosis. To get further insight into the mechanism by which receptor blockade attenuated GalN/LPS-induced liver damage, we analyzed plasma levels and intrahepatic expression of TNFalpha, IFNgamma, interleukin (IL)-6, and IL-10. NK-1R blockade clearly inhibited GalN/LPS-induced production of TNFalpha and IFNgamma, whereas synthesis of the hepatoprotective cytokines IL-6 and IL-10 was increased. NK-1 receptor antagonists might be potent drugs for treatment of inflammatory liver disease, most likely by inhibiting SP effects.

Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ-opioid receptors and GABA(B) receptors, but not α2 adrenergic receptors.

PubMed

Zhang, Guohua; Chen, Wenling; Marvizón, Juan Carlos G

2010-09-01

GABA(B) , μ-opioid and adrenergic α(2) receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ-opioid and GABA(B) receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage-gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high-frequency dorsal root stimulation was inhibited by the μ-opioid agonist DAMGO and the GABA(B) agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low-frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α(2) adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low-frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ-opioid and GABA(B) receptors, but not by α(2) receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd. No claim to original US government works.

TGF-β1 Downregulates the Expression of CX3CR1 by Inducing miR-27a-5p in Primary Human NK Cells.

PubMed

Regis, Stefano; Caliendo, Fabio; Dondero, Alessandra; Casu, Beatrice; Romano, Filomena; Loiacono, Fabrizio; Moretta, Alessandro; Bottino, Cristina; Castriconi, Roberta

2017-01-01

Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX 3 CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX 3 CR1. We demonstrated the functional interaction of miR-27a-5p with the 3' untranslated region (3'UTR) of CX 3 CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX 3 CR1 3'UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX 3 CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX 3 CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX 3 CR1 expression.

Ly49H Engagement Compensates for the Absence of Type I Interferon Signaling in Stimulating NK Cell Proliferation during MCMV Infection

PubMed Central

Geurs, Theresa L.; Zhao, Yun M.; Hill, Elaise B.; French, Anthony R.

2009-01-01

NK cells vigorously proliferate during viral infections, resulting in an expanded pool of innate lymphocytes that are able to participate in early host defense. The relative contributions of cytokines and activation receptors in stimulating NK cell proliferation during viral infections are not well characterized. In this study, we demonstrated that signaling through the NK cell activation receptor Ly49H was able to compensate for the absence of cytokine stimulation in the preferential phase of viral-induced proliferation during MCMV infection. In the absence of type I interferon stimulation, NK cell proliferation was strongly biased toward cells expressing the Ly49H receptor, even at early time points when minimal preferential Ly49H-mediated proliferation was observed in wild-type mice. In the absence of effective Ly49H signaling or following infection with virus that did not express the ligand for Ly49H, no difference was observed in the proliferation of subsets of NK cells that either express or lack expression of Ly49H, although the overall proliferation of NK cells in IFNαβR−/− mice was substantially reduced. These results highlight the contribution of NK cell activation receptors in stimulating proliferation and subsequent expansion of NK cells that are able to recognize virally infected cells. PMID:19828630

Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

PubMed Central

Holmes, Tim D.; Wilson, Erica B.; Black, Emma V. I.; Benest, Andrew V.; Vaz, Candida; Tan, Betty; Tanavde, Vivek M.; Cook, Graham P.

2014-01-01

Interactions between natural killer (NK) cells and dendritic cells (DCs) aid DC maturation and promote T-cell responses. Here, we have analyzed the response of human NK cells to tumor cells, and we identify a pathway by which NK–DC interactions occur. Gene expression profiling of tumor-responsive NK cells identified the very rapid induction of TNF superfamily member 14 [TNFSF14; also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT)], a cytokine implicated in the enhancement of antitumor responses. TNFSF14 protein expression was induced by three primary mechanisms of NK cell activation, namely, via the engagement of CD16, by the synergistic activity of multiple target cell-sensing NK-cell activation receptors, and by the cytokines IL-2 and IL-15. For antitumor responses, TNFSF14 was preferentially produced by the licensed NK-cell population, defined by the expression of inhibitory receptors specific for self-MHC class I molecules. In contrast, IL-2 and IL-15 treatment induced TNFSF14 production by both licensed and unlicensed NK cells, reflecting the ability of proinflammatory conditions to override the licensing mechanism. Importantly, both tumor- and cytokine-activated NK cells induced DC maturation in a TNFSF14-dependent manner. The coupling of TNFSF14 production to tumor-sensing NK-cell activation receptors links the tumor immune surveillance function of NK cells to DC maturation and adaptive immunity. Furthermore, regulation by NK cell licensing helps to safeguard against TNFSF14 production in response to healthy tissues. PMID:25512551

Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor

PubMed Central

Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

2015-01-01

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

PubMed

Paust, Silke; Gill, Harvinder S; Wang, Bao-Zhong; Flynn, Michael P; Moseman, E Ashley; Senman, Balimkiz; Szczepanik, Marian; Telenti, Amalio; Askenase, Philip W; Compans, Richard W; von Andrian, Ulrich H

2010-12-01

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells.

PubMed

Ziegler, Sabrina; Weiss, Esther; Schmitt, Anna-Lena; Schlegel, Jan; Burgert, Anne; Terpitz, Ulrich; Sauer, Markus; Moretta, Lorenzo; Sivori, Simona; Leonhardt, Ines; Kurzai, Oliver; Einsele, Hermann; Loeffler, Juergen

2017-07-21

Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.

Recent developments in the clinical pharmacology of rolapitant: subanalyses in specific populations

PubMed Central

Rapoport, Bernardo Leon; Aapro, Matti; Chasen, Martin R; Jordan, Karin; Navari, Rudolph M; Schnadig, Ian; Schwartzberg, Lee

2017-01-01

Knowledge of the involvement of the neurokinin substance P in emesis has led to the development of the neurokinin-1 receptor antagonists (NK-1 RAs) for control of chemotherapy-induced nausea and vomiting (CINV), in combination with serotonin type 3 receptor antagonists and corticosteroids. The NK-1 RA rolapitant, recently approved in oral formulation, has nanomolar affinity for the NK-1 receptor, as do the other commercially available NK-1 RAs, aprepitant and netupitant. Rolapitant is rapidly absorbed and has a long half-life in comparison to aprepitant and netupitant. All three NK-1 RAs undergo metabolism by cytochrome P450 (CYP) 3A4, necessitating caution with the concomitant use of CYP3A4 inhibitors, but in contrast to aprepitant and netupitant, rolapitant does not inhibit or induce CYP3A4. However, rolapitant is a moderate inhibitor of CYP2D6, and concomitant use with CYP2D6 substrates with narrow therapeutic indices should be avoided. Aprepitant, netupitant, and rolapitant have all demonstrated efficacy in the control of delayed CINV in patients receiving moderately and highly emetogenic chemotherapy in randomized controlled trials, including over multiple cycles of chemotherapy. We reviewed recent post hoc analyses of clinical trial data demonstrating that rolapitant is efficacious in the control of CINV in patient populations with specific tumor types, namely, breast cancers, gastrointestinal/colorectal cancers, and lung cancers. In addition, we show that rolapitant has efficacy in the control of CINV in specific age groups of patients receiving chemotherapy (<65 and ≥65 years of age). Overall, the safety profile of rolapitant in these specific patient populations was consistent with that observed in primary analyses of phase 3 trials. PMID:28919712

Neural contractions in colonic strips from patients with diverticular disease: role of endocannabinoids and substance P.

PubMed

Guagnini, F; Valenti, M; Mukenge, S; Matias, I; Bianchetti, A; Di Palo, S; Ferla, G; Di Marzo, V; Croci, T

2006-07-01

Diverticulosis is a common disease of not completely defined pathogenesis. Motor abnormalities of the intestinal wall have been frequently described but very little is known about their mechanisms. We investigated in vitro the neural response of colonic longitudinal muscle strips from patients undergoing surgery for complicated diverticular disease (diverticulitis). The neural contractile response to electrical field stimulation of longitudinal muscle strips from the colon of patients undergoing surgery for colonic cancer or diverticulitis was challenged by different receptor agonists and antagonists. Contractions of colonic strips from healthy controls and diverticulitis specimens were abolished by atropine. The beta adrenergic agonist (-) isoprenaline and the tachykinin NK1 receptor antagonist SR140333 had similar potency in reducing the electrical twitch response in controls and diseased tissues, while the cannabinoid receptor agonist (+)WIN 55,212-2 was 100 times more potent in inhibiting contractions in controls (IC50 42 nmol/l) than in diverticulitis strips. SR141716, a selective antagonist of the cannabinoid CB1 receptor, had no intrinsic activity in control preparations but potentiated the neural twitch in diseased tissues by up to 196% in a concentration dependent manner. SR141716 inhibited (+)WIN 55,212-2 induced relaxation in control strips but had no efficacy on (+)WIN 55,212-2 responses in strips from diverticular disease patients. Colonic levels of the endogenous ligand of cannabinoid and vanilloid TRPV1 receptors anandamide were more than twice those of control tissues (54 v 27 pmol/g tissue). The axonal conduction blocker tetrodotoxin had opposite effects in the two preparations, completely inhibiting the contractions of control strips but potentiating those in diverticular preparations, an effect selectively inhibited by SR140333. Neural control of colon motility is profoundly altered in patients with diverticulitis. Their raised levels of anandamide, apparent desensitisation of the presynaptic neural cannabinoid CB1 receptor, and the SR141716 induced intrinsic response, suggest that endocannabinoids may be involved in the pathophysiology of complications of colonic diverticular disease.

Nociceptin effects in the airways.

PubMed

Peiser, C; Undem, B J; Fischer, A

2000-07-01

The opioid-like heptadecapeptide nociceptin (NC) has the following effects in the airways (investigated in isolated tracheae and bronchi from guinea pig or rat): the electric field stimulation (EFS)-induces release of acetylcholine (ACh), the tachykinin substance P (SP) and calcitonin gene-related peptide (CGRP) is reduced after pretreatment with NC, and EFS-induced tachykinergic nonadrenergic-noncholinergic (NANC) bronchoconstriction is inhibited by NC. Both the NC-mediated inhibition of neurotransmission and of smooth muscle contraction occurred in a concentration-dependent manner. Because these effects were naloxone-insensitive, were blocked by the NC receptor antagonist [F/G]NC(1-13)NH(2), and could be mimicked by the NC analogs, NCNH(2) and NC(1-13)NH(2), it is thought that they are distinct from the classic opioid receptors. That these pharmacological actions of NC are of relevance for airway physiology is highly probable given the presence of NC-immunoreactivity in the nerve fibers of the airways and of opioid-like receptor (ORL-1) transcripts in the jugular ganglia, from where the tachykinin-containing afferents arise.

Copy Number Variation of KIR Genes Influences HIV-1 Control

PubMed Central

Shianna, Kevin V.; Feng, Sheng; Urban, Thomas J.; Ge, Dongliang; De Luca, Andrea; Martinez-Picado, Javier; Wolinsky, Steven M.; Martinson, Jeremy J.; Jamieson, Beth D.; Bream, Jay H.; Martin, Maureen P.; Borrow, Persephone; Letvin, Norman L.; McMichael, Andrew J.; Haynes, Barton F.; Telenti, Amalio; Carrington, Mary; Goldstein, David B.; Alter, Galit

2011-01-01

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection. PMID:22140359

The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway

PubMed Central

WANG, DONGDONG; SAGA, YASUSHI; SATO, NAOTO; NAKAMURA, TOSHIKAZU; TAKIKAWA, OSAMU; MIZUKAMI, HIROAKI; MATSUBARA, SHIGEKI; FUJIWARA, HIROYUKI

2016-01-01

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway. PMID:27082119

Spinal N-methyl-D-aspartate receptors and nociception-evoked release of primary afferent substance P.

PubMed

Nazarian, A; Gu, G; Gracias, N G; Wilkinson, K; Hua, X Y; Vasko, M R; Yaksh, T L

2008-03-03

Dorsal horn N-methyl-D-aspartate (NMDA) receptors contribute significantly to spinal nociceptive processing through an effect postsynaptic to non-primary glutamatergic axons, and perhaps presynaptic to the primary afferent terminals. The present study sought to examine the regulatory effects of NMDA receptors on primary afferent release of substance P (SP), as measured by neurokinin 1 receptor (NK1r) internalization in the spinal dorsal horn of rats. The effects of intrathecal NMDA alone or in combination with D-serine (a glycine site agonist) were initially examined on basal levels of NK1r internalization. NMDA alone or when co-administered with D-serine failed to induce NK1r internalization, whereas activation of spinal TRPV1 receptors by capsaicin resulted in a notable NK1r internalization. To determine whether NMDA receptor activation could potentiate NK1r internalization or pain behavior induced by a peripheral noxious stimulus, intrathecal NMDA was given prior to an intraplantar injection of formalin. NMDA did not alter the formalin-induced NK1r internalization nor did it enhance the formalin paw flinching behavior. To further characterize the effects of presynaptic NMDA receptors, the NMDA antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and MK-801 were intrathecally administered to assess their regulatory effects on formalin-induced NK1r internalization and pain behavior. AP-5 had no effect on formalin-induced NK1r internalization, whereas MK-801 produced only a modest reduction. Both antagonists, however, reduced the formalin paw flinching behavior. In subsequent in vitro experiments, perfusion of NMDA in spinal cord slice preparations did not evoke basal release of SP or calcitonin gene-related peptide (CGRP). Likewise, perfusion of NMDA did not enhance capsaicin-evoked release of the two peptides. These results suggest that presynaptic NMDA receptors in the spinal cord play little if any role on the primary afferent release of SP.

Cellular and behavioural profile of the novel, selective neurokinin1 receptor antagonist, vestipitant: a comparison to other agents.

PubMed

Brocco, Mauricette; Dekeyne, Anne; Mannoury la Cour, Clotilde; Touzard, Manuelle; Girardon, Sylvie; Veiga, Sylvie; de Nanteuil, Guillaume; deJong, Trynke R; Olivier, Berend; Millan, Mark J

2008-10-01

This study characterized the novel neurokinin (NK)(1) antagonist, vestipitant, under clinical evaluation for treatment of anxiety and depression. Vestipitant possessed high affinity for human NK(1) receptors (pK(i), 9.4), and potently blocked Substance P-mediated phosphorylation of Extracellular-Regulated-Kinase. In vivo, it occupied central NK(1) receptors in gerbils (Inhibitory Dose(50), 0.11 mg/kg). At similar doses, it abrogated nociception elicited by formalin in gerbils, and blocked foot-tapping and locomotion elicited by the NK(1) agonist, GR73632, in gerbils and guinea pigs, respectively. Further, vestipitant attenuated fear-induced foot-tapping in gerbils, separation-induced distress-vocalizations in guinea pigs, marble-burying behaviour in mice, and displayed anxiolytic actions in Vogel conflict and fear-induced ultrasonic vocalization procedures in rats. These actions were mimicked by CP99,994, L733,060 and GR205,171 which acted stereoselectively vs its less active isomer, GR226,206. In conclusion, vestipitant is a potent NK(1) receptor antagonist: its actions support the utility of NK(1) receptor blockade in the alleviation of anxiety and, possibly, depression.

Activation-specific metabolic requirements for NK cell IFN-γ production1

PubMed Central

Keppel, Molly P.; Topcagic, Nermina; Mah, Annelise Y.; Vogel, Tiphanie P.; Cooper, Megan A.

2014-01-01

There has been increasing recognition of the importance of cellular metabolism and metabolic substrates for the function and differentiation of immune cells. Here, for the first time, we investigate the metabolic requirements for production of IFN-γ by freshly isolated NK cells. Primary murine NK cells mainly utilize mitochondrial oxidative phosphorylation at rest and with short-term activation. Remarkably, we discovered significant differences in the metabolic requirements of murine NK cell IFN-γ production depending upon the activation signal. Stimulation of NK cell IFN-γ production was independent of glycolysis or mitochondrial oxidative phosphorylation when cells were activated with IL-12+IL-18. By contrast, stimulation via activating NK receptors required glucose-driven oxidative phosphorylation. Prolonged treatment with high-dose, but not low dose, IL-15 eliminated the metabolic requirement for receptor stimulation. In summary, this study demonstrates that metabolism provides an essential second signal for induction of IFN-γ production by activating NK cell receptors that can be reversed with prolonged high-dose IL-15 treatment. PMID:25595780

Neurokinin-3 Receptor Activation in the Retrochiasmatic Area is Essential for the Full Preovulatory LH Surge in Ewes

PubMed Central

Porter, Katrina L.; Hileman, Stanley M.; Hardy, Steven L.; Nestor, Casey C; Lehman, Michael N.; Goodman, Robert L.

2014-01-01

Neurokinin B (NKB) is essential for human reproduction and has been shown to stimulate LH secretion in several species, including sheep. Ewes express the neurokinin-3 receptor (NK3R) in the retrochiasmatic area (RCh) and there is one report that placement of senktide, an NK3R agonist, therein stimulates LH secretion that resembles an LH surge in ewes. In this study, we first confirmed that local administration of senktide to the RCh produced a surge-like increase in LH secretion, and then tested the effects of this agonist in two other areas implicated in the control of LH secretion and where NK3R is found in high abundance: the preoptic area (POA) and arcuate nucleus (ARC). Bilateral microimplants containing senktide induced a dramatic surge-like increase in LH when given in the POA similar to that seen with RCh treatment. In contrast, senktide treatment in the ARC resulted in a much smaller, but significant, increase in LH concentrations suggestive of an effect on tonic secretion. The possible role of POA and RCh NK3R activation in the LH surge was next tested by treating ewes with SB222200, an NK3R antagonist, in each area during an E2-induced LH surge. SB222200 in the RCh, but not in the POA, reduced LH surge amplitude by about 40% compared to controls, indicating that NK3R activation in the former region is essential for full expression of the preovulatory LH surge. Based on these data, we propose that NKB actions in the RCh are an important component of the preovulatory LH surge in ewes. PMID:25040132

The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection

PubMed Central

Rajalingam, Raja

2016-01-01

Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR–HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants. PMID:28066408

Impaired plasmacytoid dendritic cell (PDC)-NK cell activity in viremic human immunodeficiency virus infection attributable to impairments in both PDC and NK cell function.

PubMed

Conry, Sara J; Milkovich, Kimberly A; Yonkers, Nicole L; Rodriguez, Benigno; Bernstein, Helene B; Asaad, Robert; Heinzel, Frederick P; Tary-Lehmann, Magdalena; Lederman, Michael M; Anthony, Donald D

2009-11-01

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections impair plasmacytoid dendritic cell (PDC) and natural killer (NK) cell subset numbers and functions, though little is known about PDC-NK cell interactions during these infections. We evaluated PDC-dependent NK cell killing and gamma interferon (IFN-gamma) and granzyme B production, using peripheral blood mononuclear cell (PBMC)-based and purified cell assays of samples from HCV- and HIV-infected subjects. CpG-enhanced PBMC killing and IFN-gamma and granzyme B activity (dependent on PDC and NK cells) were impaired in viremic HIV infection. In purified PDC-NK cell culture experiments, CpG-enhanced, PDC-dependent NK cell activity was cell contact and IFN-alpha dependent, and this activity was impaired in viremic HIV infection but not in HCV infection. In heterologous PDC-NK cell assays, impaired PDC-NK cell killing activity was largely attributable to an NK cell defect, while impaired PDC-NK cell IFN-gamma-producing activity was attributable to both PDC and NK cell defects. Additionally, the response of NK cells to direct IFN-alpha stimulation was defective in viremic HIV infection, and this defect was not attributable to diminished IFN-alpha receptor expression, though IFN-alpha receptor and NKP30 expression was closely associated with killer activity in viremic HIV infection but not in healthy controls. These data indicate that during uncontrolled HIV infection, PDC-dependent NK cell function is impaired, which is in large part attributable to defective IFN-alpha-induced NK cell activity and not to altered IFN-alpha receptor, NKP30, NKP44, NKP46, or NKG2D expression.

Subpopulations of neurokinin 1 receptor-expressing neurons in the rat lateral amygdala display a differential pattern of innervation from distinct glutamatergic afferents.

PubMed

Sreepathi, H K; Ferraguti, F

2012-02-17

Substance P by acting on its preferred receptor neurokinin 1 (NK1) in the amygdala appears to be critically involved in the modulation of fear and anxiety. The present study was undertaken to identify neurochemically specific subpopulations of neuron expressing NK1 receptors in the lateral amygdaloid nucleus (LA), a key site for regulating these behaviors. We also analyzed the sources of glutamatergic inputs to these neurons. Immunofluorescence analysis of the co-expression of NK1 with calcium binding proteins in LA revealed that ~35% of NK1-containing neurons co-expressed parvalbumin (PV), whereas no co-localization was detected in the basal amygdaloid nucleus. We also show that neurons expressing NK1 receptors in LA did not contain detectable levels of calcium/calmodulin kinase IIα, thus suggesting that NK1 receptors are expressed by interneurons. By using a dual immunoperoxidase/immunogold-silver procedure at the ultrastructural level, we found that in LA ~75% of glutamatergic synapses onto NK1-expressing neurons were labeled for the vesicular glutamate transporter 1 indicating that they most likely are of cortical, hippocampal, or intrinsic origin. The remaining ~25% were immunoreactive for the vesicular glutamate transporter 2 (VGluT2), and may then originate from subcortical areas. On the other hand, we could not detect VGluT2-containing inputs onto NK1/PV immunopositive neurons. Our data add to previous localization studies by describing an unexpected variation between LA and basal nucleus of the amygdala (BA) in the neurochemical phenotype of NK1-expressing neurons and reveal the relative source of glutamatergic inputs that may activate these neurons, which in turn regulate fear and anxiety responses. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Subpopulations of neurokinin 1 receptor-expressing neurons in the rat lateral amygdala display a differential pattern of innervation from distinct glutamatergic afferents

PubMed Central

Sreepathi, H.K.; Ferraguti, F.

2012-01-01

Substance P by acting on its preferred receptor neurokinin 1 (NK1) in the amygdala appears to be critically involved in the modulation of fear and anxiety. The present study was undertaken to identify neurochemically specific subpopulations of neuron expressing NK1 receptors in the lateral amygdaloid nucleus (LA), a key site for regulating these behaviors. We also analyzed the sources of glutamatergic inputs to these neurons. Immunofluorescence analysis of the co-expression of NK1 with calcium binding proteins in LA revealed that ∼35% of NK1-containing neurons co-expressed parvalbumin (PV), whereas no co-localization was detected in the basal amygdaloid nucleus. We also show that neurons expressing NK1 receptors in LA did not contain detectable levels of calcium/calmodulin kinase IIα, thus suggesting that NK1 receptors are expressed by interneurons. By using a dual immunoperoxidase/immunogold-silver procedure at the ultrastructural level, we found that in LA ∼75% of glutamatergic synapses onto NK1-expressing neurons were labeled for the vesicular glutamate transporter 1 indicating that they most likely are of cortical, hippocampal, or intrinsic origin. The remaining ∼25% were immunoreactive for the vesicular glutamate transporter 2 (VGluT2), and may then originate from subcortical areas. On the other hand, we could not detect VGluT2-containing inputs onto NK1/PV immunopositive neurons. Our data add to previous localization studies by describing an unexpected variation between LA and basal nucleus of the amygdala (BA) in the neurochemical phenotype of NK1-expressing neurons and reveal the relative source of glutamatergic inputs that may activate these neurons, which in turn regulate fear and anxiety responses. PMID:22210508

Immunolocalization of Substance P and NK-1 Receptor in ADIPOSE Stem Cells.

PubMed

Muñoz, Miguel; Muñoz, Mario F; Ayala, Antonio

2017-12-01

Substance P (SP) is a neuropeptide belonging to the thachykinin peptide family. SP, after binding to its receptor, the neurokinin 1 receptor (NK1R), controls several transcription factors such as NF-κB, hypoxia inducible factor (HIF-1α), c-myc, c-fos, c-jun, and AP-1. SP and NK1R have a widespread distribution in both the central and peripheral nervous systems. They are also present in cells not belonging to the nervous system (immune cells, placenta, etc.). SP is located in all body fluids, that is, blood, cerebrospinal fluid, etc., making it ubiquitous throughout the human body. SP and NK1R genes are expressed in the stem cell line TF-1 and in primary stem cells derived from human placental cord blood. However, to our knowledge, the presence of SP and the NK1R receptor in adipose stem cells (ADSC) is unknown. We demonstrated by immunofluorescence the localization of SP and NK1R in human and rat ADSC. SP and NK1R are located in both the cytoplasm and the nucleus of these cells. The NK1R is higher in the nucleus than in the cytoplasm of ADSCs. By Western blot we demonstrated the presence of different isoforms of NK1R that have different subcellular locations in the ADSC. SP induces proliferation and mitogenesis through NK1R in ADSCs. These findings reported here for the first time suggest an important role for a SP/NK1R system, either as genetic and/or epigenetic factor, in both the cytoplasm and nucleus functions of the ADSCs. J. Cell. Biochem. 118: 4686-4696, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The expression of human natural killer cell receptors in early life.

PubMed

Sundström, Y; Nilsson, C; Lilja, G; Kärre, K; Troye-Blomberg, M; Berg, L

2007-01-01

Natural killer (NK) cells play an important role in tumour immunosurveillance and the early defence against viral infections. Recognition of altered cells (i.e. infected- or tumour-cells) is achieved through a multiple receptor recognition strategy which gives the NK cells inhibitory or activating signals depending on the ligands present on the target cell. NK cells originate from the bone marrow where they develop and proliferate. However, further maturation processes and homeostasis of NK cells in peripheral blood are not well understood. To determine the proportions of cells and the expression of NK cell receptors, mononuclear cells from children at three time points during early childhood were compared, i.e. cord blood (CB), 2 and 5 years of age. The proportion of NK cells was high in CB, but the interferon-gamma (IFN-gamma) production low compared to later in life. In contrast, the proportion of T cells was low in CB. This may indicate a deviation of the regulatory function of NK cells in CB compared to later in life, implying an importance of innate immunity in early life before the adaptive immune system matures. Additionally, we found that the proportion of LIR-1(+) NK cells increased with increasing age while CD94(+)NKG2C(-) (NKG2A(+)) NK cells and the level of expression of NKG2D, NKp30 and NKp46 decreased with age. These age related changes in NK cell populations defined by the expression of activating and inhibitory receptors may be the result of pathogen exposure and/or a continuation of the maturation process that begins in the bone marrow.

Substance P-induced trafficking of beta-arrestins. The role of beta-arrestins in endocytosis of the neurokinin-1 receptor.

PubMed

McConalogue, K; Déry, O; Lovett, M; Wong, H; Walsh, J H; Grady, E F; Bunnett, N W

1999-06-04

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and beta-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of beta-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or beta-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of beta-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and beta-arrestin1 into the same endosomes containing SP and the transferrin receptor (2-10 min); (c) prolonged colocalization of the NK1R and beta-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and beta-arrestin1 in the cytosol (4-6 h). SP stimulated a similar redistribution of immunoreactive beta-arrestin1 and beta-arrestin2. In contrast, SP did not affect Galphaq/11 distribution, which remained at the plasma membrane. Expression of the dominant negative beta-arrestin319-418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of beta-arrestins to the plasma membrane, where they participate in NK1R endocytosis. beta-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and beta-arrestins return to the cytosol.

From the Cover: Prenatal Nicotinic Exposure Attenuates Respiratory Chemoreflexes Associated With Downregulation of Tyrosine Hydroxylase and Neurokinin 1 Receptor in Rat Pup Carotid Body.

PubMed

Zhao, Lei; Zhuang, Jianguo; Gao, Xiuping; Ye, Chunyan; Lee, Lu-Yuan; Xu, Fadi

2016-09-01

Maternal cigarette smoke is the major risk of sudden infant death syndrome (SIDS). A depressed ventilatory response to hypoxia (HVR) and hypercapnia (HCVR) is thought to be responsible for the pathogenesis of SIDS and the carotid body is critically involved in these responses. We have recently reported that prenatal nicotinic exposure (PNE) over the full gestation induces depressed HVR in rat pups. Here, we asked whether PNE (1) depressed not only HVR but also HCVR that were dependent on the carotid body, (2) affected some important receptors and neurochemicals expressed in the carotid body, such as tyrosine hydroxylase (TH), neurokinin-1 receptor (NK1R), and α7 nicotinic acetylcholine receptor (α7nAChR), and (3) blunted the ventilatory responses to activation of these receptors. To this end, HVR and HCVR in Ctrl and PNE pups were measured with plethysmography before and after carotid body ablation (Series I), mRNA expression and/or immunoreactivity (IR) of TH, NK1R, and α7nAChR in the carotid body were examined by RT-PCR and immunohistochemistry (Series II), and the ventilatory responses were tested before and after intracarotid injection of substance P (NK1R agonist) and AR-R17779 (α7nAChR agonist) (Series III). Our results showed that PNE (1) significantly depressed both HVR and HCVR and these depressions were abolished by carotid body ablation, (2) reduced the relative population of glomus cells, mRNA NK1R, and α7nAChR and IR of NK1R and TH in the carotid body, and (3) decreased ventilatory responses to intracarotid injection of substance P or AR-R17779. These results suggest that PNE acting via the carotid body could strikingly blunt HVR and HCVR, likely through downregulating TH and NK1R. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Up-regulation of NKG2A inhibitory receptor on circulating NK cells contributes to transfusion-induced immunodepression in patients with β-thalassemia major.

PubMed

Zou, Yong; Song, Zhi-Xing; Lu, Ying; Liang, Xiao-Li; Yuan, Qing; Liao, Si-Hong; Bao, Jun-Jie

2016-08-01

Accumulating evidence has shown that allogeneic blood transfusions can induce significant immunosuppression in recipients, and thereby increase the risk of postoperative infection and/or tumor relapse. Although it is well known that natural killer (NK) cells are responsible for the immunodepression effects of transfusion, the underlying mechanisms remain obscure. In this study, we investigated the role of NK cells in transfusion-induced immunodepression in β-thalassemia major. The proportion of circulating NK cells and the expression of NK receptors (NKG2A, CD158a, NKP30, NKP46 and NKG2D) as well as CD107a were detected by multicolor flow cytometry. IFN-γ production by circulating NK cells was detected by intracellular cytokine staining. Our results showed that the proportion and cytotoxicity (CD107a expression) of circulating NK cells in transfusion-dependent β-thalassemia major patients were remarkably lower than those of β-thalassemia minor patients or healthy volunteers. Expression of NKG2A inhibitory receptor on circulating NK cells in patients with β-thalassemia major was remarkably up-regulated, but there were no significant differences in the expression levels of NKP30, NKP46, NKG2D, CD158a and IFN-γ. These results indicate NKG2A inhibitory receptor may play a key role in transfusion-induced immunodepression of NK cells in patients with β-thalassemia major.

Maternal uterine NK cell–activating receptor KIR2DS1 enhances placentation

PubMed Central

Xiong, Shiqiu; Sharkey, Andrew M.; Kennedy, Philippa R.; Gardner, Lucy; Farrell, Lydia E.; Chazara, Olympe; Bauer, Julien; Hiby, Susan E.; Colucci, Francesco; Moffett, Ashley

2013-01-01

Reduced trophoblast invasion and vascular conversion in decidua are thought to be the primary defect of common pregnancy disorders including preeclampsia and fetal growth restriction. Genetic studies suggest these conditions are linked to combinations of polymorphic killer cell Ig-like receptor (KIR) genes expressed by maternal decidual NK cells (dNK) and HLA-C genes expressed by fetal trophoblast. Inhibitory KIR2DL1 and activating KIR2DS1 both bind HLA-C2, but confer increased risk or protection from pregnancy disorders, respectively. The mechanisms underlying these genetic associations with opposing outcomes are unknown. We show that KIR2DS1 is highly expressed in dNK, stimulating strong activation of KIR2DS1+ dNK. We used microarrays to identify additional responses triggered by binding of KIR2DS1 or KIR2DL1 to HLA-C2 and found different responses in dNK coexpressing KIR2DS1 with KIR2DL1 compared with dNK only expressing KIR2DL1. Activation of KIR2DS1+ dNK by HLA-C2 stimulated production of soluble products including GM-CSF, detected by intracellular FACS and ELISA. We demonstrated that GM-CSF enhanced migration of primary trophoblast and JEG-3 trophoblast cells in vitro. These findings provide a molecular mechanism explaining how recognition of HLA class I molecules on fetal trophoblast by an activating KIR on maternal dNK may be beneficial for placentation. PMID:24091323

Irradiation of breast cancer cells enhances CXCL16 ligand expression and induces the migration of natural killer cells expressing the CXCR6 receptor.

PubMed

Yoon, Mee Sun; Pham, Chanh Tin; Phan, Minh-Trang Thi; Shin, Dong-Jun; Jang, Youn-Young; Park, Min-Ho; Kim, Sang-Ki; Kim, Seokho; Cho, Duck

2016-12-01

Few studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells. The expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96 h after irradiation of breast cancer cell lines for migration and blocking assays. The activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R 2  = 0.91; P <0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (P = 0.043) and SKBR3 (P = 0.043) cells, but not in MDA-MB231 (P = 0.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines. Our data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Endocytosis as a mechanism of regulating natural killer cell function: unique endocytic and trafficking pathway for CD94/NKG2A.

PubMed

Peruzzi, Giovanna; Masilamani, Madhan; Borrego, Francisco; Coligan, John E

2009-01-01

Natural killer (NK) cells are lymphocytes generally recognized as sentinels of the innate immune system due to their inherent capacity to deal with diseased (stressed) cells, including malignant and infected. This ability to recognize many potentially pathogenic situations is due to the expression of a diverse panel of activation receptors. Because NK cell activation triggers an aggressive inflammatory response, it is important to have a means of throttling this response. Hence, NK cells also express a panel of inhibitory receptors that recognize ligands expressed by "normal" cells. Little or nothing is known about the endocytosis and trafficking of NK cell receptors, which are of great relevance to understanding how NK cells maintain the appropriate balance of activating and inhibitory receptors on their cell surface. In this review, we focus on the ITIM-containing inhibitory receptor CD94/NKG2A showing that it is endocytosed by a previously undescribed macropinocytic-like process that may be related to the maintenance of its surface expression.

PD-1 mediates functional exhaustion of activated NK cells in patients with Kaposi sarcoma

PubMed Central

Beldi-Ferchiou, Asma; Lambert, Marion; Dogniaux, Stéphanie; Vély, Frédéric; Vivier, Eric; Olive, Daniel; Dupuy, Stéphanie; Levasseur, Frank; Zucman, David; Lebbé, Céleste; Sène, Damien; Hivroz, Claire; Caillat-Zucman, Sophie

2016-01-01

Programmed Death-1 (PD-1), an inhibitory receptor expressed by activated lymphocytes, is involved in regulating T- and B-cell responses. PD-1 and its ligands are exploited by a variety of cancers to facilitate tumor escape through PD-1-mediated functional exhaustion of effector T cells. Here, we report that PD-1 is upregulated on Natural Killer (NK) cells from patients with Kaposi sarcoma (KS). PD-1 was expressed in a sub-population of activated, mature CD56dimCD16pos NK cells with otherwise normal expression of NK surface receptors. PD-1pos NK cells from KS patients were hyporesponsive ex vivo following direct triggering of NKp30, NKp46 or CD16 activating receptors, or short stimulation with NK cell targets. PD-1pos NK cells failed to degranulate and release IFNγ, but exogenous IL-2 or IL-15 restored this defect. That PD-1 contributed to NK cell functional impairment and was not simply a marker of dysfunctional NK cells was confirmed in PD-1-transduced NKL cells. In vitro, PD-1 was induced at the surface of healthy control NK cells upon prolonged contact with cells expressing activating ligands, i.e. a condition mimicking persistent stimulation by tumor cells. Thus, PD-1 appears to plays a critical role in mediating NK cell exhaustion. The existence of this negative checkpoint fine-tuning NK activation highlights the possibility that manipulation of the PD-1 pathway may be a strategy for circumventing tumor escape not only from the T cell-, but also the NK-cell mediated immune surveillance. PMID:27662664

Variable NK cell receptors and their MHC class I ligands in immunity, reproduction and human evolution.

PubMed

Parham, Peter; Moffett, Ashley

2013-02-01

Natural killer (NK) cells have roles in immunity and reproduction that are controlled by variable receptors that recognize MHC class I molecules. The variable NK cell receptors found in humans are specific to simian primates, in which they have progressively co-evolved with MHC class I molecules. The emergence of the MHC-C gene in hominids drove the evolution of a system of NK cell receptors for MHC-C molecules that is most elaborate in chimpanzees. By contrast, the human system of MHC-C receptors seems to have been subject to different selection pressures that have acted in competition on the immunological and reproductive functions of MHC class I molecules. We suggest that this compromise facilitated the development of the bigger brains that enabled archaic and modern humans to migrate out of Africa and populate other continents.

Synthesis and Evaluation of a Novel 64Cu- and 67Ga-Labeled Neurokinin 1 Receptor Antagonist for in Vivo Targeting of NK1R-Positive Tumor Xenografts.

PubMed

Zhang, Hanwen; Kanduluru, Ananda Kumar; Desai, Pooja; Ahad, Afruja; Carlin, Sean; Tandon, Nidhi; Weber, Wolfgang A; Low, Philip S

2018-04-18

Neurokinin 1 receptor (NK1R) is expressed in gliomas and neuroendocrine malignancies and represents a promising target for molecular imaging and targeted radionuclide therapy. The goal of this study was to synthesize and evaluate a novel NK1R ligand (NK1R-NOTA) for targeting NK1R-expressing tumors. Using a carboxymethyl moiety linked to L-733060 as a starting reagent, NK1R-NOTA was synthesized in a three-step reaction and then labeled with 64 Cu (or 67 Ga for in vitro studies) in the presence of CH 3 COONH 4 buffer. The radioligand affinity and cellular uptake were evaluated with NK1R-transduced HEK293 cells (HEK293-NK1R) and NK1R nontransduced HEK293 cells (HEK293-WT) and their xenografts. Radiolabeled NK1R-NOTA was obtained with a radiochemical purity of >95% and specific activities of >7.0 GBq/μmol for 64 Cu and >5.0 GBq/μmol for 67 Ga. Both 64 Cu- and 67 Ga-labeled NK1R-NOTA demonstrated high levels of uptake in HEK293-NK1R cells, whereas co-incubation with an excess of NK1R ligand L-733060 reduced the level of uptake by 90%. Positron emission tomography (PET) imaging showed that [ 64 Cu]NK1R-NOTA had a accumulated rapidly in HEK293-NK1R xenografts and a 10-fold lower level of uptake in HEK293-WT xenografts. Radioactivity was cleared by gastrointestinal tract and urinary systems. Biodistribution studies confirmed that the tumor-to-organ ratios were ≥5 for all studied organs at 1 h p.i., except kidneys, liver, and intestine, and that the tumor-to-intestine and tumor-to-kidney ratios were also improved 4 and 20 h post-injection. [ 64 Cu]NK1R-NOTA is a promising ligand for PET imaging of NK1R-expressing tumor xenografts. Delayed imaging with [ 64 Cu]NK1R-NOTA improves image contrast because of the continuous clearance of radioactivity from normal organs.

Neuropeptides activate human mast cell degranulation and chemokine production

PubMed Central

Kulka, Marianna; Sheen, Cecilia H; Tancowny, Brian P; Grammer, Leslie C; Schleimer, Robert P

2008-01-01

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcεRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34+ progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-γ (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte–macrophage colony-stimulating factor, but not IL-4, interferon-γ or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases. PMID:17922833

Decreased Brain Neurokinin-1 Receptor Availability in Chronic Tennis Elbow.

PubMed

Linnman, Clas; Catana, Ciprian; Svärdsudd, Kurt; Appel, Lieuwe; Engler, Henry; Långström, Bengt; Sörensen, Jens; Furmark, Tomas; Fredrikson, Mats; Borsook, David; Peterson, Magnus

Substance P is released in painful and inflammatory conditions, affecting both peripheral processes and the central nervous system neurokinin 1 (NK1) receptor. There is a paucity of data on human brain alterations in NK1 expression, how this system may be affected by treatment, and interactions between central and peripheral tissue alterations. Ten subjects with chronic tennis elbow (lateral epicondylosis) were selected out of a larger (n = 120) randomized controlled trial evaluating graded exercise as a treatment for chronic tennis elbow (lateral epicondylosis). These ten subjects were examined by positron emission tomography (PET) with the NK1-specific radioligand 11C-GR205171 before, and eight patients were followed up after treatment with graded exercise. Brain binding in the ten patients before treatment, reflecting NK1-receptor availability (NK1-RA), was compared to that of 18 healthy subjects and, longitudinally, to the eight of the original ten patients that agreed to a second PET examination after treatment. Before treatment, patients had significantly lower NK1-RA in the insula, vmPFC, postcentral gyrus, anterior cingulate, caudate, putamen, amygdala and the midbrain but not the thalamus and cerebellum, with the largest difference in the insula contralateral to the injured elbow. No significant correlations between brain NK1-RA and pain, functional severity, or peripheral NK1-RA in the affected limb were observed. In the eight patients examined after treatment, pain ratings decreased in everyone, but there were no significant changes in NK1-RA. These findings indicate a role for the substance P (SP) / NK1 receptor system in musculoskeletal pain and tissue healing. As neither clinical parameters nor successful treatment response was reflected in brain NK1-RA after treatment, this may reflect the diverse function of the SP/NK1 system in CNS and peripheral tissue, or a change too small or slow to capture over the three-month treatment.

Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation

PubMed Central

Sivori, Simona; Falco, Michela; Marcenaro, Emanuela; Parolini, Silvia; Biassoni, Roberto; Bottino, Cristina; Moretta, Lorenzo; Moretta, Alessandro

2002-01-01

In this study we analyzed the progression of cell surface receptor expression during the in vitro-induced human natural killer (NK) cell maturation from CD34+ Lin− cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor. PMID:11917118

Identification of an elaborate NK-specific system regulating HLA-C expression

PubMed Central

Ivarsson, Martin A.; Walker-Sperling, Victoria E.; Subleski, Jeff; Johnson, Jenna K.; Wright, Paul W.; Carrington, Mary; McVicar, Daniel W.

2018-01-01

The HLA-C gene appears to have evolved in higher primates to serve as a dominant source of ligands for the KIR2D family of inhibitory MHC class I receptors. The expression of NK cell-intrinsic MHC class I has been shown to regulate the murine Ly49 family of MHC class I receptors due to the interaction of these receptors with NK cell MHC in cis. However, cis interactions have not been demonstrated for the human KIR and HLA proteins. We report the discovery of an elaborate NK cell-specific system regulating HLA-C expression, indicating an important role for HLA-C in the development and function of NK cells. A large array of alternative transcripts with differences in intron/exon content are generated from an upstream NK-specific HLA-C promoter, and exon content varies between HLA-C alleles due to SNPs in splice donor/acceptor sites. Skipping of the first coding exon of HLA-C generates a subset of untranslatable mRNAs, and the proportion of untranslatable HLA-C mRNA decreases as NK cells mature, correlating with increased protein expression by mature NK cells. Polymorphism in a key Ets-binding site of the NK promoter has generated HLA-C alleles that lack significant promoter activity, resulting in reduced HLA-C expression and increased functional activity. The NK-intrinsic regulation of HLA-C thus represents a novel mechanism controlling the lytic activity of NK cells during development. PMID:29329284

Imatinib and Nilotinib Off-Target Effects on Human NK Cells, Monocytes, and M2 Macrophages.

PubMed

Bellora, Francesca; Dondero, Alessandra; Corrias, Maria Valeria; Casu, Beatrice; Regis, Stefano; Caliendo, Fabio; Moretta, Alessandro; Cazzola, Mario; Elena, Chiara; Vinti, Luciana; Locatelli, Franco; Bottino, Cristina; Castriconi, Roberta

2017-08-15

Tyrosine kinase inhibitors (TKIs) are used in the clinical management of hematological neoplasms. Moreover, in solid tumors such as stage 4 neuroblastomas (NB), imatinib showed benefits that might depend on both on-target and immunological off-target effects. We investigated the effects of imatinib and nilotinib on human NK cells, monocytes, and macrophages. High numbers of monocytes died upon exposure to TKI concentrations similar to those achieved in patients. Conversely, NK cells were highly resistant to the TKI cytotoxic effect, were properly activated by immunostimulatory cytokines, and degranulated in the presence of NB cells. In NB, neither drug reduced the expression of ligands for activating NK receptors or upregulated that of HLA class I, B7-H3, PD-L1, and PD-L2, molecules that might limit NK cell function. Interestingly, TKIs modulated the chemokine receptor repertoire of immune cells. Acting at the transcriptional level, they increased the surface expression of CXCR4, an effect observed also in NK cells and monocytes of patients receiving imatinib for chronic myeloid leukemia. Moreover, TKIs reduced the expression of CXCR3 (in NK cells) and CCR1 (in monocytes). Monocytes also decreased the expression of M-CSFR, and low numbers of cells underwent differentiation toward macrophages. M0 and M2 macrophages were highly resistant to TKIs and maintained their phenotypic and functional characteristics. Importantly, also in the presence of TKIs, the M2 immunosuppressive polarization was reverted by TLR engagement, and M1-oriented macrophages fully activated autologous NK cells. Our results contribute to better interpreting the off-target efficacy of TKIs in tumors and to envisaging strategies aimed at facilitating antitumor immune responses. Copyright © 2017 by The American Association of Immunologists, Inc.

Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases.

PubMed

Roosterman, Dirk; Cottrell, Graeme S; Schmidlin, Fabien; Steinhoff, Martin; Bunnett, Nigel W

2004-07-16

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.

Production of proinflammatory cytokines without invocation of cytotoxic effects by an Epstein-Barr virus-infected natural killer cell line established from a patient with hypersensitivity to mosquito bites.

PubMed

Suzuki, Daisuke; Tsuji, Kazuhide; Yamamoto, Takenobu; Fujii, Kazuyasu; Iwatsuki, Keiji

2010-10-01

Cumulative evidence supports that Epstein-Barr virus (EBV)-infected natural killer (NK) cells induce severe systemic and cutaneous inflammation in patients with hypersensitivity to mosquito bites (HMB). In order to understand the pathogenesis of HMB, we established an EBV-infected cell line and characterized the cytological profiles. A novel EBV-infected NK-cell line, designated NKED, was established from a patient with HMB and used for the present study along with two other NK-cell lines, KAI3 and KHYG-1. NKED expressed the latency II-related transcripts. NKED cells were positive for CD2 and CD161 antigens, and negative for CD3, CD16, CD34, CD56, and T-cell receptor α/β and γ/δ antigens. Although NKED cells contained several cytotoxic molecules, the cells had an extremely poor cytotoxic activity. The majority of NKED cells were negative for perforin, major histocompatibility complex class I-restricted NK-cell receptors, CD94 and KIR2D, and an activating receptor, NKG2D. NKED cells, however, secreted higher levels of tumor necrosis factor-α. Stimulation with phorbol 12-myristate 13-acetate or tumor necrosis factor-α induced expression of BZLF1 messenger RNA in the NKED and KAI3 cells, indicating the transition from the latent- to the lytic-cycle infection. These data suggested that NKED cells revealed a very low cytotoxic effect probably because of the low expression levels of perforin, but had the ability to release proinflammatory cytokines. NKED cells did not reflect the characteristics of HMB, as they were different from pathogenic NK cells proliferating in the HMB patient, but the difference indicated that pathogenic NK cells could change their character in the presence of interleukin-2. Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

KHYG-1 and NK-92 represent different subtypes of LFA-1-mediated NK cell adhesiveness.

PubMed

Suck, Garnet; Tan, Suet-Mien; Chu, Sixian; Niam, Madelaine; Vararattanavech, Ardcharaporn; Lim, Tsyr Jong; Koh, Mickey B C

2011-01-01

Novel cancer cellular therapy approaches involving long-term ex vivo IL-2 stimulated highly cytotoxic natural killer (NK) cells are emerging. However, adhesion properties of such NK cells are not very well understood. Herein, we describe the novel observation of permanently activated alphaLbeta2 integrin leukocyte function-associated antigen (LFA)-1 adhesion receptor in long-term IL-2 activated NK cells and the permanent NK cell lines KHYG-1 and NK-92. We show that such cytokine activated NK effectors constitutively adhered to the LFA-1-ligand ICAM-1, whereas binding to the lower affinity ligand ICAM-3 required additional exogenous activating conditions. The results demonstrate an extended conformation and an intermediate affinity state for the LFA-1 population expressed by the NK cells. Interestingly, adhesion to ICAM-1 or K562 induced pronounced cell spreading in KHYG-1, but not in NK-92, and partially in long-term IL-2 stimulated primary NK cells. It is conceivable that such differential adhesion characteristics may impact motility potential of such NK effectors with relevance to clinical tumor targeting. KHYG-1 could be a useful model in planning future targeted therapeutic approaches involving NK effectors with augmented functions.

Role of tachykinins in ozone-induced acute lung injury in guinea pigs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tepper, J.S.; Costa, D.L.; Fitzgerald, S.

To examine the hypothesis that the acute reversible changes caused by ozone (O3) exposure are mediated by tachykinin release, guinea pigs were depleted of tachykinins by use of repeated capsaicin (CAP) injections before O3 exposure in an attempt to prevent O3-induced functional changes. Unexpectedly, CAP pretreatment caused divergent results in the functional responses to O3. Ventilatory measurements obtained from CAP-pretreated O3-exposed (CAP-O3) animals were exacerbated rather than diminished compared with the effects of O3 alone. Similarly, lavage fluid protein accumulation was enhanced in the CAP-O3 group compared with the O3-exposed group. In better agreement with our initial hypothesis, the CAP-O3more » group was less responsive than the O3-exposed animals to histamine aerosol challenge. Additionally, Evans blue dye accumulation, a hallmark of tachykinin release, was increased in O3-exposed animals and was partially blocked in the CAP-O3 group. These data suggest that tachykinin-containing sensory fibers are unlikely to mediate the acute effects of O3 exposure on tidal breathing and lavage fluid protein accumulation but may play a role in causing post-O3 airway hyperreactivity and protein extravasation into the trachea.« less

Cytokine-Induced Memory-Like Differentiation Enhances Unlicensed Natural Killer Cell Antileukemia and FcγRIIIa-Triggered Responses.

PubMed

Wagner, Julia A; Berrien-Elliott, Melissa M; Rosario, Maximillian; Leong, Jeffrey W; Jewell, Brea A; Schappe, Timothy; Abdel-Latif, Sara; Fehniger, Todd A

2017-03-01

Cytokine-induced memory-like natural killer (NK) cells differentiate after short-term preactivation with IL-12, IL-15, and IL-18 and display enhanced effector function in response to cytokines or tumor targets for weeks after the initial preactivation. Conventional NK cell function depends on a licensing signal, classically delivered by an inhibitory receptor engaging its cognate MHC class I ligand. How licensing status integrates with cytokine-induced memory-like NK cell responses is unknown. We investigated this interaction using killer cell immunoglobulin-like receptor- and HLA-genotyped primary human NK cells. Memory-like differentiation resulted in enhanced IFN-γ production triggered by leukemia targets or FcγRIIIa ligation within licensed NK cells, which exhibited the highest functionality of the NK cell subsets interrogated. IFN-γ production by unlicensed memory-like NK cells was also enhanced to a level comparable with that of licensed control NK cells. Mechanistically, differences in responses to FcγRIIIa-based triggering were not explained by alterations in key signaling intermediates, indicating that the underlying biology of memory-like NK cells is distinct from that of adaptive NK cells in human cytomegalovirus-positive individuals. Additionally, memory-like NK cells responded robustly to cytokine receptor restimulation with no impact of licensing status. These results demonstrate that both licensed and unlicensed memory-like NK cell populations have enhanced functionality, which may be translated to improve leukemia immunotherapy. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Effect of peptidases on the ability of exogenous and endogenous neurokinins to produce neurokinin 1 receptor internalization in the rat spinal cord.

PubMed

Marvizon, Juan Carlos G; Wang, Xueren; Lao, Li-Jun; Song, Bingbing

2003-12-01

The ability of peptidases to restrict neurokinin 1 receptor (NK1R) activation by exogenously applied or endogenously released neurokinins was investigated by measuring NK1R internalization in rat spinal cord slices. Concentration-response curves for substance P and neurokinin A were obtained in the presence and absence of 10 microm thiorphan, an inhibitor of neutral endopeptidase (EC 3.4.24.11), plus 10 microm captopril, an inhibitor of dipeptidyl carboxypeptidase (EC 3.4.15.1). These inhibitors significantly decreased the EC50 of substance P to produce NK1R internalization from 32 to 9 nm, and the EC50 of neurokinin A from 170 to 60 nm. Substance P was significantly more potent than neurokinin A, both with and without these peptidase inhibitors. In the presence of peptidase inhibitors, neurokinin B was 10 times less potent than neurokinin A and 64 times less potent than substance P (EC50=573 nm). Several aminopeptidase inhibitors (actinonin, amastatin, bacitracin, bestatin and puromycin) failed to further increase the effect of thiorphan plus captopril on the NK1R internalization produced by 10 nm substance P. Electrical stimulation of the dorsal root produced NK1R internalization by releasing endogenous neurokinins. Thiorphan plus captopril increased NK1R internalization produced by 1 Hz stimulation, but not by 30 Hz stimulation. Therefore, NEN and DCP restrict NK1R activation by endogenous neurokinins when they are gradually released by low-frequency firing of primary afferents, but become saturated or inhibited when primary afferents fire at a high frequency.

NK-92 cell, another ideal carrier for chimeric antigen receptor.

PubMed

Wang, Wan-Ning; Zhou, Guang-Yu; Zhang, Wen-Long

2017-08-01

The remarkable clinical outcomes of the treatment for B-cell malignancies through the application of CD19 chimeric antigen receptor T (CAR-T) cells have made adoptive immunotherapy with genetically modified immune effector cells a hotspot in the field of antitumor. However, numerous toxicities of CAR-T cells have been identified. Thus, some studies have resorted to another cytotoxic cell, NK-92 cell, to reach for better efficacy with minimal toxicity. Preclinical studies have confirmed the safety and feasibility of the genetically modified NK-92 cells with highly specific cytotoxicity in vitro and in vivo. Therefore, it is expected that NK-92 cell becomes another ideal carrier for CAR for its unique advantages over primary NK cells, parental NK-92 cells and autologous T cells.

Adaptive reconfiguration of the human NK-cell compartment in response to cytomegalovirus: a different perspective of the host-pathogen interaction.

PubMed

Muntasell, Aura; Vilches, Carlos; Angulo, Ana; López-Botet, Miguel

2013-05-01

As discussed in this review, human cytomegalovirus (HCMV) infection in healthy individuals is associated with a variable and persistent increase of NK cells expressing the CD94/NKG2C activating receptor. The expansion of NKG2C(+) NK cells reported in other infectious diseases is systematically associated with HCMV co-infection. The functionally mature NKG2C(bright) NK-cell subset expanding in HCMV(+) individuals displays inhibitory Ig-like receptors (KIR and LILRB1) specific for self HLA class I, and low levels of NKp46 and NKp30 activating receptors. Such reconfiguration of the NK-cell compartment appears particularly marked in immunocompromised patients and in children with symptomatic congenital infection, thus suggesting that its magnitude may be inversely related with the efficiency of the T-cell-mediated response. This effect of HCMV infection is reminiscent of the pattern of response of murine Ly49H(+) NK cells against murine CMV (MCMV), and it has been hypothesized that a cognate interaction of the CD94/NKG2C receptor with HCMV-infected cells may drive the expansion of the corresponding NK-cell subset. Yet, the precise role of NKG2C(+) cells in the control of HCMV infection, the molecular mechanisms underlying the NK-cell compartment redistribution, as well as its putative influence in the response to other pathogens and tumors remain open issues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tyrosine Kinase Btk Is Required for NK Cell Activation

PubMed Central

Bao, Yan; Zheng, Jian; Han, Chaofeng; Jin, Jing; Han, Huanxing; Liu, Yinping; Lau, Yu-Lung; Tu, Wenwei; Cao, Xuetao

2012-01-01

Bruton tyrosine kinase (Btk) is not only critical for B cell development and differentiation but is also involved in the regulation of Toll-like receptor-triggered innate response of macrophages. However, whether Btk is involved in the regulation of natural killer (NK) cell innate function remains unknown. Here, we show that Btk expression is up-regulated during maturation and activation of mouse NK cells. Murine Btk−/− NK cells have decreased innate immune responses to the TLR3 ligand, with reduced expressions of IFN-γ, perforin, and granzyme-B and decreased cytotoxic activity. Furthermore, Btk is found to promote TLR3-triggered NK cell activation mainly by activating the NF-κB pathway. Poly(I:C)-induced NK cell-mediated acute hepatitis was observed to be attenuated in Btk−/− mice or the mice with in vivo administration of the Btk inhibitor. Correspondingly, liver damage was aggravated in Btk−/− mice after the adoptive transfer of Btk+/+ NK cells, further indicating that Btk-mediated NK cell activation contributes to TLR3-triggered acute liver injury. Importantly, reduced TLR3-triggered activation of human NK cells was observed in Btk-deficient patients with X-linked agammaglobulinemia, as evidenced by the reduced IFN-γ, CD69, and CD107a expression and cytotoxic activity. These results indicate that Btk is required for activation of NK cells, thus providing insight into the physiological significance of Btk in the regulation of immune cell functions and innate inflammatory response. PMID:22589540

Tyrosine kinase Btk is required for NK cell activation.

PubMed

Bao, Yan; Zheng, Jian; Han, Chaofeng; Jin, Jing; Han, Huanxing; Liu, Yinping; Lau, Yu-Lung; Tu, Wenwei; Cao, Xuetao

2012-07-06

Bruton tyrosine kinase (Btk) is not only critical for B cell development and differentiation but is also involved in the regulation of Toll-like receptor-triggered innate response of macrophages. However, whether Btk is involved in the regulation of natural killer (NK) cell innate function remains unknown. Here, we show that Btk expression is up-regulated during maturation and activation of mouse NK cells. Murine Btk(-/-) NK cells have decreased innate immune responses to the TLR3 ligand, with reduced expressions of IFN-γ, perforin, and granzyme-B and decreased cytotoxic activity. Furthermore, Btk is found to promote TLR3-triggered NK cell activation mainly by activating the NF-κB pathway. Poly(I:C)-induced NK cell-mediated acute hepatitis was observed to be attenuated in Btk(-/-) mice or the mice with in vivo administration of the Btk inhibitor. Correspondingly, liver damage was aggravated in Btk(-/-) mice after the adoptive transfer of Btk(+/+) NK cells, further indicating that Btk-mediated NK cell activation contributes to TLR3-triggered acute liver injury. Importantly, reduced TLR3-triggered activation of human NK cells was observed in Btk-deficient patients with X-linked agammaglobulinemia, as evidenced by the reduced IFN-γ, CD69, and CD107a expression and cytotoxic activity. These results indicate that Btk is required for activation of NK cells, thus providing insight into the physiological significance of Btk in the regulation of immune cell functions and innate inflammatory response.

Boosting Natural Killer Cell-Based Immunotherapy with Anticancer Drugs: a Perspective.

PubMed

Cifaldi, Loredana; Locatelli, Franco; Marasco, Emiliano; Moretta, Lorenzo; Pistoia, Vito

2017-12-01

Natural killer (NK) cells efficiently recognize and kill tumor cells through several mechanisms including the expression of ligands for NK cell-activating receptors on target cells. Different clinical trials indicate that NK cell-based immunotherapy represents a promising antitumor treatment. However, tumors develop immune-evasion strategies, including downregulation of ligands for NK cell-activating receptors, that can negatively affect antitumor activity of NK cells, which either reside endogenously, or are adoptively transferred. Thus, restoration of the expression of NK cell-activating ligands on tumor cells represents a strategic therapeutic goal. As discussed here, various anticancer drugs can fulfill this task via different mechanisms. We envision that the combination of selected chemotherapeutic agents with NK cell adoptive transfer may represent a novel strategy for cancer immunotherapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Signalling through NK1.1 triggers NK cells to die but induces NK T cells to produce interleukin-4.

PubMed

Asea, A; Stein-Streilein, J

1998-02-01

In vivo inoculation of specific antibody is an accepted protocol for elimination of specific cell populations. Except for anti-CD3 and anti-CD4, it is not known if the depleted cells are eliminated by signalling through the target molecule or through a more non-specific mechanism. C57BL/6 mice were inoculated with anti-natural killer (NK1.1) monoclonal antibody (mAb). Thereafter spleen cells were harvested, stained for both surface and intracellular markers, and analysed by flow cytometry. As early as 2 hr post inoculation, NK cells were signalled to become apoptotic while signalling through the NK1.1 molecule activated NK1.1+ T-cell receptor (TCR)+ (NK T) cells to increase in number, and produce interleukin-4 (IL-4). Anti NK1.1 mAb was less efficient at signalling apoptosis in NK cells when NK T-cell deficient [beta 2-microglobulin beta 2m-deficient] mice were used compared with wild type mice. Efficient apoptotic signalling was restored when beta 2m-deficient mice were reconstituted with NK T cells. NK-specific antibody best signals the apoptotic process in susceptible NK cells when resistant NK T cells are present, activated, and secrete IL-4.

TGF-β1 Downregulates the Expression of CX3CR1 by Inducing miR-27a-5p in Primary Human NK Cells

PubMed Central

Regis, Stefano; Caliendo, Fabio; Dondero, Alessandra; Casu, Beatrice; Romano, Filomena; Loiacono, Fabrizio; Moretta, Alessandro; Bottino, Cristina; Castriconi, Roberta

2017-01-01

Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX3CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX3CR1. We demonstrated the functional interaction of miR-27a-5p with the 3′ untranslated region (3′UTR) of CX3CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX3CR1 3′UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX3CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX3CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX3CR1 expression. PMID:28791023

Haemagglutinin-neuraminidase from HPIV3 mediates human NK regulation of T cell proliferation via NKp44 and NKp46.

PubMed

McQuaid, Samantha; Loughran, Sinead; Power, Patrick; Maguire, Paula; Walls, Dermot; Grazia Cusi, Maria; Orvell, Claes; Johnson, Patricia

2018-06-01

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

Spatial association of prolyl oligopeptidase, inositol 1,4,5-triphosphate type 1 receptor, substance P and its neurokinin-1 receptor in the rat brain: an immunohistochemical colocalization study.

PubMed

Myöhänen, T T; Venäläinen, J I; Garcia-Horsman, J A; Männistö, P T

2008-06-02

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyzes proline-containing peptides shorter than 30 amino acids. It has been suggested that POP is associated with cognitive functions, possibly via the cleavage of neuropeptides such as substance P (SP). Recently, several studies have also linked POP to the inositol 1,4,5-triphosphate (IP(3)) signaling. However, the neuroanatomical interactions between these substances are not known. We used double-labeled immunofluorescence to determine the POP colocalization with SP, SP receptor (neurokinin-1 receptor, NK-1R) and IP(3) type 1 receptor (IP(3)R1) in the rat brain. Furthermore, since striatal and cortical GABAergic neurons are involved in SP neurotransmission, we studied the coexpression of POP, SP and GABA by triple-labeled immunofluorescence. POP was moderately present in IP(3)R1-containing cells in cortex; the colocalization was particularly high in the thalamus, hippocampal CA1 field and cerebellar Purkinje cells. Colocalization of POP with SP and NK1-receptor was infrequent throughout the brain, though some POP and SP coexpression was observed in cerebellar Purkinje cells. We also found that POP partially colocalized with SP-containing GABAergic neurons in striatum and cortex. Our findings support the view that POP is at least spatially associated with the IP(3)-signaling in the thalamus, hippocampus and cerebellar Purkinje cells. This might point to a role for POP in the regulation of long-term potentiation and/or depression. Moreover, the low degree of colocalization of POP, SP and its NK-1R suggests that a transport system is needed either for POP or SP to make hydrolysis possible and that POP may act both intra- and extracellularly.

Complementary Phosphorylation Sites in the Adaptor Protein SLP-76 Promote Synergistic Activation of Natural Killer Cells

PubMed Central

Kim, Hun Sik; Long, Eric O.

2013-01-01

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require the induction of synergistic signals from co-activation receptors, such as CD314 (NKG2D) and CD244 (2B4), which bind to ligands expressed on target cells. Synergy is required to overcome inhibition of the guanine nucleotide exchange factor (GEF) Vav1, a central regulator of NK cell activation, by the E3 ubiquitin ligase c-Cbl. However, the molecular basis for this synergy is unknown. Here, we showed that the adaptor protein Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) was required for this synergy, and that distinct tyrosine residues in SLP-76 were phosphorylated by each receptor of a synergistic pair. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76, each of which enables binding of SLP-76 to Vav1, was unique to receptors that stimulate ligand-dependent target cell killing, because antibody-dependent stimulation by Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that complementation of separate phospho-tyrosine targets in SLP-76 forms the basis of synergistic NK cell activation. PMID:22786724

Complementary phosphorylation sites in the adaptor protein SLP-76 promote synergistic activation of natural killer cells.

PubMed

Kim, Hun Sik; Long, Eric O

2012-07-10

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require synergistic signals from specific pairs of co-activation receptors, such as CD314 (also known as NKG2D) and CD244 (2B4), which bind to distinct ligands present on target cells. These signals are required to overcome inhibition mediated by the E3 ubiquitin ligase c-Cbl of the guanine nucleotide exchange factor Vav1, which promotes activation of NK cells. Here, we showed that the adaptor protein SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons) was required for this synergy and that distinct tyrosine residues in SLP-76 were phosphorylated by each member of a pair of synergistic receptors. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76 enabled binding of SLP-76 to Vav1. Selective phosphorylation of SLP-76 at these residues was restricted to receptors that stimulated ligand-dependent target cell killing; antibody-dependent stimulation of the Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 mutant proteins showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that combined phosphorylation of separate tyrosine residues in SLP-76 forms the basis of synergistic NK cell activation.

The Hypothalamic-Pituitary-Adrenal Axis and Serotonin Metabolism in Individual Brain Nuclei of Mice with Genetic Disruption of the NK1 Receptor Exposed to Acute Stress.

PubMed

Culman, Juraj; Mühlenhoff, Stephan; Blume, Annegret; Hedderich, Jürgen; Lützen, Ulf; Hunt, Stephen P; Rupniak, Nadia M J; Zhao, Yi

2018-06-15

Mice lacking the substance P (SP) neurokinin-1 (NK1) receptor (NK1R-/-mice) were used to investigate whether SP affects serotonin (5-HT) function in the brain and to assess the effects of acute immobilisation stress on the hypothalamic-pituitary-adrenocortical (HPA) axis and 5-HT turnover in individual brain nuclei. Basal HPA activity and the expression of hypothalamic corticotropin-releasing hormone (CRH) in wild-type (WT)- and NK1R-/- mice were identical. Stress-induced increases in plasma ACTH concentration were considerably higher in NK1R-/- mice than in WT mice while corticosterone concentrations were equally elevated in both mouse lines. Acute stress did not alter the expression of CRH. In the dorsal raphe nucleus (DRN), basal 5-HT turnover was increased in NK1R-/- mice and a 15 min stress further magnified 5-HT utilisation in this region. In the frontoparietal cortex, medial prefrontal cortex, central nucleus of amygdala, and the hippocampal CA1 region, stress increased 5-HT and/or 5-hydroxyindoleacetic acid (5-HIAA) concentrations to a similar extent in WT and NK1R-/- mice. 5-HT turnover in the hypothalamic paraventricular nucleus was not affected by stress, but stress induced similar increases in 5-HT and 5-HIAA in the ventromedial and dorsomedial hypothalamic nuclei in WT and NK1R-/- mice. Our findings indicate that NK1 receptor activation suppresses ACTH release during acute stress but does not exert sustained inhibition of the HPA axis. Genetic deletion of the NK1 receptor accelerates 5-HT turnover in DRN under basal and stress conditions. No differences between the responses of serotonergic system to acute stress in WT and NK1R-/- mice occur in forebrain nuclei linked to the regulation of anxiety and neuroendocrine stress responses.

The Activating NKG2C Receptor Is Significantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation in Patients with Severe Graft-versus-Host Disease.

PubMed

Kordelas, Lambros; Steckel, Nina-Kristin; Horn, Peter A; Beelen, Dietrich W; Rebmann, Vera

2016-10-27

Natural killer (NK) cells play a central role in the innate immune system. In allogeneic stem cell transplantation (alloSCT), alloreactive NK cells derived by the graft are discussed to mediate the elimination of leukemic cells and dendritic cells in the patient and thereby to reduce the risk for leukemic relapses and graft-versus-host reactions. The alloreactivity of NK cells is determined by various receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors, which both recognize the non-classical human leukocyte antigen E (HLA-E). Here we analyze the contribution of these receptors to NK cell alloreactivity in 26 patients over the course of the first year after alloSCT due to acute myeloid leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results show that NK cells expressing the activating CD94/NKG2C receptor are significantly reduced in patients after alloSCT with severe acute and chronic graft-versus-host disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in patients with severe acute and chronic GvHD after receiving an HLA-mismatched graft. Collectively, these results provide evidence for the first time that CD94/NKG2C is involved in GvHD prevention.

The Activating NKG2C Receptor Is Significantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation in Patients with Severe Graft-versus-Host Disease

PubMed Central

Kordelas, Lambros; Steckel, Nina-Kristin; Horn, Peter A.; Beelen, Dietrich W.; Rebmann, Vera

2016-01-01

Natural killer (NK) cells play a central role in the innate immune system. In allogeneic stem cell transplantation (alloSCT), alloreactive NK cells derived by the graft are discussed to mediate the elimination of leukemic cells and dendritic cells in the patient and thereby to reduce the risk for leukemic relapses and graft-versus-host reactions. The alloreactivity of NK cells is determined by various receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors, which both recognize the non-classical human leukocyte antigen E (HLA-E). Here we analyze the contribution of these receptors to NK cell alloreactivity in 26 patients over the course of the first year after alloSCT due to acute myeloid leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results show that NK cells expressing the activating CD94/NKG2C receptor are significantly reduced in patients after alloSCT with severe acute and chronic graft-versus-host disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in patients with severe acute and chronic GvHD after receiving an HLA-mismatched graft. Collectively, these results provide evidence for the first time that CD94/NKG2C is involved in GvHD prevention. PMID:27801784

Divergent β-Arrestin-dependent Signaling Events Are Dependent upon Sequences within G-protein-coupled Receptor C Termini*

PubMed Central

Pal, Kasturi; Mathur, Maneesh; Kumar, Puneet; DeFea, Kathryn

2013-01-01

β-Arrestins are multifunctional adaptor proteins that, upon recruitment to an activated G-protein-coupled receptor, can promote desensitization of G-protein signaling and receptor internalization while simultaneously eliciting an independent signal. The result of β-arrestin signaling depends upon the activating receptor. For example, activation of two Gαq-coupled receptors, protease-activated receptor-2 (PAR2) and neurokinin-1 receptor (NK1R), results in drastically different signaling events. PAR2 promotes β-arrestin-dependent membrane-sequestered extracellular signal-regulated kinase (ERK1/2) activation, cofilin activation, and cell migration, whereas NK1R promotes nuclear ERK1/2 activation and proliferation. Using bioluminescence resonance energy transfer to monitor receptor/β-arrestin interactions in real time, we observe that PAR2 has a higher apparent affinity for both β-arrestins than does NK1R, recruits them at a faster rate, and exhibits more rapid desensitization of the G-protein signal. Furthermore, recruitment of β-arrestins to PAR2 does not require prior Gαq signaling events, whereas inhibition of Gαq signaling intermediates inhibits recruitment of β-arrestins to NK1R. Using chimeric receptors in which the C terminus of PAR2 is fused to the N terminus of NK1R and vice versa and a critical Ser/Thr mutant of PAR2, we demonstrate that interactions between β-arrestins and specific phosphoresidues in the C termini of each receptor are crucial for determining the rate and magnitude of β-arrestin recruitment as well as the ultimate signaling outcome. PMID:23235155

Prenatal nicotinic exposure upregulates pulmonary C-fiber NK1R expression to prolong pulmonary C-fiber-mediated apneic response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Lei; Zhuang, Jianguo; Zang, Na

Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA{sub 1} receptor, ADA{sub 1}R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA{sub 1}R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to changemore » BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChR or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin. - Highlights: • PNE upregulated NK1R and TRPV1 gene and protein expression in the N/J ganglia. • PNE only elevated NK1R mRNA in vagal pulmonary C-neurons. • Blockage of peripheral NK1R reduced the PNE-induced PCF sensitization. • PNE induced gene and protein changes in NK1R and TRPV1 dependent on action of α7nAChR.« less

Targeting the Epidermal Growth Factor Receptor Can Counteract the Inhibition of Natural Killer Cell Function Exerted by Colorectal Tumor-Associated Fibroblasts

PubMed Central

Costa, Delfina; Venè, Roberta; Benelli, Roberto; Romairone, Emanuele; Scabini, Stefano; Catellani, Silvia; Rebesco, Barbara; Mastracci, Luca; Grillo, Federica; Minghelli, Simona; Loiacono, Fabrizio; Zocchi, Maria Raffaella; Poggi, Alessandro

2018-01-01

Mesenchymal stromal cells (MSC) present in the tumor microenvironment [usually named tumor-associated fibroblasts (TAF)] can exert immunosuppressive effects on T and natural killer (NK) lymphocytes, favoring tumor immune escape. We have analyzed this mechanism in colorectal carcinoma (CRC) and found that co-culture of NK cells with TAF can prevent the IL-2-mediated NKG2D upregulation. This leads to the impairment of NKG2D-mediated recognition of CRC cells, sparing the NK cell activation through DNAM1 or FcγRIIIA (CD16). In situ, TAF express detectable levels of epidermal growth factor receptor (EGFR); thus, the therapeutic anti-EGFR humanized antibody cetuximab can trigger the antibody-dependent cellular cytotoxicity of TAF, through the engagement of FcγRIIIA on NK cells. Importantly, in the tumor, we found a lymphoid infiltrate containing NKp46+CD3− NK cells, enriched in CD16+ cells. This population, sorted and cultured with IL-2, could be triggered via CD16 and via NKG2D. Of note, ex vivo NKp46+CD3− cells were able to kill autologous TAF; in vivo, this might represent a control mechanism to reduce TAF-mediated regulatory effect on NK cell function. Altogether, these findings suggest that MSC from the neoplastic mucosa (TAF) of CRC patients can downregulate the immune cell recognition of CRC tumor cells. This immunosuppression can be relieved by the anti-EGFR antibody used in CRC immunotherapy. PMID:29910806

The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT).

PubMed

Foley, Bree; Felices, Martin; Cichocki, Frank; Cooley, Sarah; Verneris, Michael R; Miller, Jeffrey S

2014-03-01

Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed 'the missing self hypothesis' to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Impaired natural killer cell self-education and "missing-self" responses in Ly49-deficient mice.

PubMed

Bélanger, Simon; Tu, Megan M; Rahim, Mir Munir Ahmed; Mahmoud, Ahmad B; Patel, Rajen; Tai, Lee-Hwa; Troke, Angela D; Wilhelm, Brian T; Landry, Josette-Renée; Zhu, Qinzhang; Tung, Kenneth S; Raulet, David H; Makrigiannis, Andrew P

2012-07-19

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.

Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection.

PubMed

Lee, Seung-Hwan; Kim, Kwang-Sin; Fodil-Cornu, Nassima; Vidal, Silvia M; Biron, Christine A

2009-09-28

Natural killer (NK) cells have the potential to deliver both direct antimicrobial effects and regulate adaptive immune responses, but NK cell yields have been reported to vary greatly during different viral infections. Activating receptors, including the Ly49H molecule recognizing mouse cytomegalovirus (MCMV), can stimulate NK cell expansion. To define Ly49H's role in supporting NK cell proliferation and maintenance under conditions of uncontrolled viral infection, experiments were performed in Ly49h(-/-), perforin 1 (Prf1)(-/-), and wild-type (wt) B6 mice. NK cell numbers were similar in uninfected mice, but relative to responses in MCMV-infected wt mice, NK cell yields declined in the absence of Ly49h and increased in the absence of Prf1, with high rates of proliferation and Ly49H expression on nearly all cells. The expansion was abolished in mice deficient for both Ly49h and Prf1 (Ly49h(-/-)Prf1(-/-)), and negative consequences for survival were revealed. The Ly49H-dependent protection mechanism delivered in the absence of Prf1 was a result of interleukin 10 production, by the sustained NK cells, to regulate the magnitude of CD8 T cell responses. Thus, the studies demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection to regulate adaptive immune responses.

Localization of NK1 receptors and roles of substance-P in subepithelial fibroblasts of rat intestinal villi.

PubMed

Furuya, Sonoko; Furuya, Kishio; Shigemoto, Ryuichi; Sokabe, Masahiro

2010-11-01

Subepithelial fibroblasts of the intestinal villi, which form a contractile cellular network beneath the epithelium, are in close contact with epithelial cells, nerve varicosities, capillaries, smooth muscles and immune cells, and secrete extracellular matrix molecules, growth factors and cytokines, etc. Cultured subepithelial fibroblasts of the rat duodenal villi display various receptors such as endothelins, ATP, substance-P and bradykinin, and release ATP in response to mechanical stimulation. In this study, the presence of functional NK1 receptors (NK1R) was pharmacologically confirmed in primary culture by Ca(2+) measurement, and the effects of substance-P were measured in an acute preparation of epithelium-free duodenal villi from 2- to 3-week-old rats using a two-photon laser microscope. Substance-P elicited an increase in the intracellular Ca(2+) concentration and contraction of the subepithelial fibroblasts in culture and the isolated villi. The localization of NK1R and substance-P in the villi was examined by light and electron microscopic immunohistochemistry. NK1R-like immunoreactivity was intensely localized on the plasma membrane of villous subepithelial fibroblasts in 10-day- to 4-week-old rats and mice and was decreased or absent in adulthood. The pericryptal fibroblasts of the small and large intestine were NK1R immuno-negative. These villous subepithelial fibroblasts form synapse-like structures with both substance-P-immunopositive and -immunonegative nerve varicosities. Here, we propose that the mutual interaction between villous subepithelial fibroblasts and afferent neurons via substance-P and ATP plays important roles in the maturation of the structure and function of the small intestine.

Improving efficacy of cancer immunotherapy by genetic modification of natural killer cells.

PubMed

Burga, Rachel A; Nguyen, Tuongvan; Zulovich, Jane; Madonna, Sarah; Ylisastigui, Loyda; Fernandes, Rohan; Yvon, Eric

2016-11-01

Natural killer (NK) cells are members of the innate immune system that recognize target cells via activating and inhibitory signals received through cell receptors. Derived from the lymphoid lineage, NK cells are able to produce cytokines and exert a cytotoxic effect on viral infected and malignant cells. It is their unique ability to lyse target cells rapidly and without prior education that renders NK cells a promising effector cell for adoptive cell therapy. However, both viruses and tumors employ evasion strategies to avoid attack by NK cells, which represent biological challenges that need to be harnessed to fully exploit the cytolytic potential of NK cells. Using genetic modification, the function of NK cells can be enhanced to improve their homing, cytolytic activity, in vivo persistence and safety. Examples include gene modification to express chemokine, high-affinity Fc receptor and chimeric antigen receptors, suicide genes and the forced expression of cytokines such as interleukin (IL)-2 and IL-15. Preclinical studies have clearly demonstrated that such approaches are effective in improving NK-cell function, homing and safety. In this review, we summarize the recent advances in the genetic manipulations of NK cells and their application for cellular immunotherapeutic strategies. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Clinical Grade Purification and Expansion of NK Cell Products for an Optimized Manufacturing Protocol

PubMed Central

Koehl, Ulrike; Brehm, Claudia; Huenecke, Sabine; Zimmermann, Stefanie-Yvonne; Kloess, Stephan; Bremm, Melanie; Ullrich, Evelyn; Soerensen, Jan; Quaiser, Andrea; Erben, Stephanie; Wunram, Claudia; Gardlowski, Tanja; Auth, Eileen; Tonn, Torsten; Seidl, Christian; Meyer-Monard, Sandrine; Stern, Martin; Passweg, Jakob; Klingebiel, Thomas; Bader, Peter; Schwabe, Dirk; Esser, Ruth

2013-01-01

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized. PMID:23730623

High-efficiency lysis of cervical cancer by allogeneic NK cells derived from umbilical cord progenitors is independent of HLA status.

PubMed

Veluchamy, John P; Heeren, A Marijne; Spanholtz, Jan; van Eendenburg, Jaap D H; Heideman, Daniëlle A M; Kenter, Gemma G; Verheul, Henk M; van der Vliet, Hans J; Jordanova, Ekaterina S; de Gruijl, Tanja D

2017-01-01

Down-regulation of HLA in tumor cells, low numbers and dysfunctionality of NK cells are commonly observed in patients with end-stage cervical cancer. Adoptive transfer of high numbers of cytotoxic NK cells might be a promising treatment approach in this setting. Here, we explored the cytotoxic efficacy on ten cervical cancer cell lines of activated allogeneic NK cells from two sources, i.e., peripheral blood (PBNK) with and without cetuximab (CET), a tumor-specific monoclonal antibody directed against EGFR, or derived from umbilical cord blood (UCB-NK). Whereas CET monotherapy was ineffective against the panel of cervical cancer cell lines, irrespective of their EGFR expression levels and despite their RAS wt status, it significantly enhanced the in vitro cytotoxic efficacy of activated PBNK (P = 0.002). Equally superior cytotoxicity over activated PBNK alone was achieved by UCB-NK (P < 0.001). Both PBNK- and UCB-NK-mediated cytotoxic activity was dependent on the NK-activating receptors natural killer group 2, member D receptor (NKG2D) and DNAX accessory molecule-1 (DNAM-1) (P < 0.05) and unrelated to expression levels of the inhibitory receptors HLA-E and/or HLA-G. Most strikingly, whereas the PBNK's cytotoxic activity was inversely correlated with HLA-ABC levels (P = 0.036), PBNK + CET and UCB-NK cytotoxicity were entirely independent of HLA-ABC expression. In conclusion, this study provides a rationale to initiate a clinical trial for cervical cancer with adoptively transferred allogeneic NK cells, employing either UCB-NK or PBNK + CET for EGFR-expressing tumors. Adoptive transfer of UCB-NK might serve as a generally applicable treatment for cervical cancer, enabled by HLA-, histology- and HPV-independent killing mechanisms.

Ex vivo expansion of highly cytotoxic human NK cells by cocultivation with irradiated tumor cells for adoptive immunotherapy.

PubMed

Lim, Seon Ah; Kim, Tae-Jin; Lee, Jung Eun; Sonn, Chung Hee; Kim, Kwanghee; Kim, Jiyoung; Choi, Jong Gwon; Choi, Il-Kyu; Yun, Chae-Ok; Kim, Jae-Hong; Yee, Cassian; Kumar, Vinay; Lee, Kyung-Mi

2013-04-15

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I-negative or -mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1-mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1-lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation. ©2012 AACR.

A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

PubMed

Fujii, Rika; Schlom, Jeffrey; Hodge, James W

2018-05-01

OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for chordoma.

Modulation of hypoglossal motoneuron excitability by NK1 receptor activation in neonatal mice in vitro

PubMed Central

Yasuda, Kouichi; Robinson, Dean M; Selvaratnam, Subramaniam R; Walsh, Carmen W; McMorland, Angus J C; Funk, Gregory D

2001-01-01

The effects of substance P (SP), acting at NK1 receptors, on the excitability and inspiratory activity of hypoglossal (XII) motoneurons (MNs) were investigated using rhythmically active medullary-slice preparations from neonatal mice (postnatal day 0–3). Local application of the NK1 agonist [SAR9,Met (O2)11]-SP (SPNK1) produced a dose-dependent, spantide- (a non-specific NK receptor antagonist) and GR82334-(an NK1 antagonist) sensitive increase in inspiratory burst amplitude recorded from XII nerves. Under current clamp, SPNK1 significantly depolarized XII MNs, potentiated repetitive firing responses to injected currents and produced a leftward shift in the firing frequency-current relationships without affecting slope. Under voltage clamp, SPNK1 evoked an inward current and increased input resistance, but had no effect on inspiratory synaptic currents. SPNK1 currents persisted in the presence of TTX, were GR82334 sensitive, were reduced with hyperpolarization and reversed near the expected EK. Effects of the α1-noradrenergic receptor agonist phenylephrine (PE) on repetitive firing behaviour were virtually identical to those of SPNK1. Moreover, SPNK1 currents were completely occluded by PE, suggesting that common intracellular pathways mediate the actions of NK1 and α1-noradrenergic receptors. In spite of the similar actions of SPNK1 and PE on XII MN responses to somally injected current, α1-noradrenergic receptor activation potentiated inspiratory synaptic currents and was more than twice as effective in potentiating XII nerve inspiratory burst amplitude. GR82334 reduced XII nerve inspiratory burst amplitude and generated a small outward current in XII MNs. These observations, together with the first immunohistochemical evidence in the newborn for SP immunopositive terminals in the vicinity of SPNK1-sensitive inspiratory XII MNs, support the endogenous modulation of XII MN excitability by SP. In contrast to phrenic MNs (Ptak et al. 2000), blocking NMDA receptors with AP5 had no effect on the modulation of XII nerve activity by SPNK1. In conclusion, SPNK1 modulates XII motoneuron responses to inspiratory drive primarily through inhibition of a resting, postsynaptic K+ leak conductance. The results establish the functional significance of SP in controlling upper airway tone during early postnatal life and indicate differential modulation of motoneurons controlling airway and pump muscles by SP. PMID:11454963

NKG2C zygosity influences CD94/NKG2C receptor function and the NK-cell compartment redistribution in response to human cytomegalovirus.

PubMed

Muntasell, Aura; López-Montañés, María; Vera, Andrea; Heredia, Gemma; Romo, Neus; Peñafiel, Judith; Moraru, Manuela; Vila, Joan; Vilches, Carlos; López-Botet, Miguel

2013-12-01

Human cytomegalovirus (HCMV) infection promotes a persistent expansion of a functionally competent NK-cell subset expressing the activating CD94/NKG2C receptor. Factors underlying the wide variability of this effect observed in HCMV-seropositive healthy individuals and exacerbated in immunocompromized patients are uncertain. A deletion of the NKG2C gene has been reported, and an apparent relation of NKG2C genotype with circulating NKG2C(+) NK-cell numbers was observed in HCMV(+) children. We have assessed the influence of NKG2C gene dose on the NK-cell repertoire in a cohort of young healthy adults (N = 130, median age 19 years). Our results revealed a relation of NKG2C copy number with surface receptor levels and with NKG2C(+) NK-cell numbers in HCMV(+) subjects, independently of HLA-E dimorphism. Functional studies showed quantitative differences in signaling (i.e. iCa(2+) influx), degranulation, and IL-15-dependent proliferation, in response to NKG2C engagement, between NK cells from NKG2C(+/+) and hemizygous subjects. These observations provide a mechanistic interpretation on the way the NKG2C genotype influences steady-state NKG2C(+) NK-cell numbers, further supporting an active involvement of the receptor in the HCMV-induced reconfiguration of the NK-cell compartment. The putative implications of NKG2C zygosity over viral control and other clinical variables deserve attention. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Requirement and Redundancy of the Src Family Kinases Fyn and Lyn in Perforin-Dependent Killing of Cryptococcus neoformans by NK Cells

PubMed Central

Oykhman, Paul; Timm-McCann, Martina; Xiang, Richard F.; Islam, Anowara; Li, Shu Shun; Stack, Danuta; Huston, Shaunna M.; Ma, Ling Ling

2013-01-01

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase–extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse. PMID:23918783

Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma.

PubMed

Oelsner, Sarah; Friede, Miriam E; Zhang, Congcong; Wagner, Juliane; Badura, Susanne; Bader, Peter; Ullrich, Evelyn; Ottmann, Oliver G; Klingemann, Hans; Tonn, Torsten; Wels, Winfried S

2017-02-01

Natural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications. To enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3ζ (CAR 63.z), composite CD28-CD3ζ or CD137-CD3ζ signaling domains (CARs 63.28.z and 63.137.z). Exposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R γ null mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo. Our data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Expression of retinoid-related orphan receptor (ROR)γt on NK22 cells in the peripheral blood and uterine endometrium of women with unexplained recurrent pregnancy loss and unexplained infertility.

PubMed

Fuchinoue, Kohei; Fukui, Atsushi; Chiba, Hitomi; Kamoi, Mai; Funamizu, Ayano; Taima, Ayako; Fukuhara, Rie; Mizunuma, Hideki

2016-11-01

Recently, NK22 cells, a subset of interleukin (IL)-22-producing natural killer (NK) cells, were identified. We have previously reported the higher percentage of NK22 cells in women suffering recurrent pregnancy loss (RPL). Moreover, we have also reported lower expression of NKp46, a kind of natural cytotoxicity receptor (NCR), on NK cells and the changes of NK cell producing cytokines in women who experience RPL. NK22 cells express NCRs, such as NKp44 or NKp46. Retinoid-related orphan receptor γt (RORγt) is known as a regulator of NK22 cells; however, in NK22 cells of peripheral blood (PB) and the uterine endometrium (UE), the relationship between NCRs and RORγt is unclear. We investigate RORγt expression NK22 cells in the PB and UE of women with unexplained infertility (uI) or unexplained RPL (uRPL). Lymphocytes were extracted from PB and UE, derived from women with uI or uRPL. Expression of RORγt and NCRs in NK cells and NK cell-produced cytokines were analyzed by flow cytometry. CD56 + /NKp46 + /RORγt + cells were positively correlated with CD56 + /IL-22 + cells in both PB and UE. CD56 bright /NKp46 bright /RORγt + cells were significantly higher in uRPL than in uI, and endometrial CD56 bright /NKp46 bright /RORγt + cells were positively correlated with PB. In UE, CD56 bright /RORγt + cells were negatively correlated with CD56 bright /interferon-γ + and CD56 bright /tumor necrosis factor-α + cells of uRPL. RORγt may be associated with NK22 cells in reproduction. Particularly, higher expression of RORγt may be associated with elevated NK22 cells in uRPL. © 2016 Japan Society of Obstetrics and Gynecology.

Peripheral substance P and neurokinin-1 receptors have a role in inflammatory and neuropathic orofacial pain models.

PubMed

Teodoro, Fernanda C; Tronco Júnior, Marcos F; Zampronio, Aleksander R; Martini, Alessandra C; Rae, Giles A; Chichorro, Juliana G

2013-06-01

There is accumulating evidence that substance P released from peripheral sensory neurons participates in inflammatory and neuropathic pain. In this study it was investigated the ability of substance P to induce orofacial nociception and thermal and mechanical hyperalgesia, as well as the role of NK1 receptors on models of orofacial inflammatory and neuropathic pain. Substance P injected into the upper lip at 1, 10 and 100 μg/50 μL failed to induce nociceptive behavior. Also, substance P (0.1-10 μg/50 μL) injected into the upper lip did not evoke orofacial cold hyperalgesia and when injected at 1 μg/50 μL did not induce mechanical hyperalgesia. However, substance P at this latter dose induced orofacial heat hyperalgesia, which was reduced by the pre-treatment of rats with a non-peptide NK1 receptor antagonist (SR140333B, 3mg/kg). Systemic treatment with SR140333B (3 mg/kg) also reduced carrageenan-induced heat hyperalgesia, but did not exert any influence on carrageenan-induced cold hyperalgesia. Blockade of NK1 receptors with SR140333B also reduced by about 50% both phases of the formalin response evaluated in the orofacial region. Moreover, heat, but not cold or mechanical, hyperalgesia induced by constriction of the infraorbital nerve, a model of trigeminal neuropathic pain, was abolished by pretreatment with SR140333B. Considering that substance P was peripherally injected (i.e. upper lip) and the NK1 antagonist used lacks the ability to cross the blood-brain-barrier, our results demonstrate that the peripheral SP/NK1 system participates in the heat hyperalgesia associated with inflammation or nerve injury and in the persistent pain evoked by formalin in the orofacial region. Copyright © 2012 Elsevier Ltd. All rights reserved.

The correlation between NK cell and liver function in patients with primary hepatocellular carcinoma.

PubMed

Sha, Wei Hong; Zeng, Xiao Hui; Min, Lu

2014-05-01

This study aimed to detect the expression of natural killer (NK) cell receptor natural killer group 2D (NKG2D) in the peripheral blood of patients with primary hepatocellular carcinoma and to discuss the correlation between NK cell cytotoxicity and liver function. The number of NK cells and the expression of NK cell receptor NKG2D in peripheral blood were determined by flow cytometry in patients with primary hepatocellular carcinoma, hepatitis B cirrhosis, chronic hepatitis B, and healthy controls. When compared with patients in the healthy and the chronic hepatitis B groups, the primary hepatocellular carcinoma group showed significant decreases in all parameters, including the cytotoxicity of NK cells on K562 cells, expression rate of NKG2D in NK cells, number of NKG2D(+) NK cells, expression level of NKG2D, and number of NK cells (p<0.05). The activity of NK cells showed a positive correlation, whereas the Child-Pugh scores in the primary hepatocellular carcinoma and the hepatitis B cirrhosis groups showed a negative correlation with all parameters detected above. The decrease of NK cell activity in patients with primary hepatocellular carcinoma is closely related to their lower expression of NKG2D. Liver function affects the expression of NKG2D and the activity of NK cells.

Effect of peptidases on the ability of exogenous and endogenous neurokinins to produce neurokinin 1 receptor internalization in the rat spinal cord

PubMed Central

Marvizón, Juan Carlos G; Wang, Xueren; Lao, Li-Jun; Song, Bingbing

2003-01-01

The ability of peptidases to restrict neurokinin 1 receptor (NK1R) activation by exogenously applied or endogenously released neurokinins was investigated by measuring NK1R internalization in rat spinal cord slices. Concentration–response curves for substance P and neurokinin A were obtained in the presence and absence of 10 μM thiorphan, an inhibitor of neutral endopeptidase (EC 3.4.24.11), plus 10 μM captopril, an inhibitor of dipeptidyl carboxypeptidase (EC 3.4.15.1). These inhibitors significantly decreased the EC50 of substance P to produce NK1R internalization from 32 to 9 nM, and the EC50 of neurokinin A from 170 to 60 nM. Substance P was significantly more potent than neurokinin A, both with and without these peptidase inhibitors. In the presence of peptidase inhibitors, neurokinin B was 10 times less potent than neurokinin A and 64 times less potent than substance P (EC50=573 nM). Several aminopeptidase inhibitors (actinonin, amastatin, bacitracin, bestatin and puromycin) failed to further increase the effect of thiorphan plus captopril on the NK1R internalization produced by 10 nM substance P. Electrical stimulation of the dorsal root produced NK1R internalization by releasing endogenous neurokinins. Thiorphan plus captopril increased NK1R internalization produced by 1 Hz stimulation, but not by 30 Hz stimulation. Therefore, NEN and DCP restrict NK1R activation by endogenous neurokinins when they are gradually released by low-frequency firing of primary afferents, but become saturated or inhibited when primary afferents fire at a high frequency. PMID:14623771

Thiorphan-induced survival and proliferation of rat thymocytes by activation of Akt/survivin pathway and inhibition of caspase-3 activity.

PubMed

Amantini, Consuelo; Mosca, Michela; Lucciarini, Roberta; Perfumi, Marina Cecilia; Santoni, Giorgio

2008-10-01

The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK(1)R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5(+) thymocytes, namely on the CD4(+)CD8(+) (double positive; DP) and CD4(+) subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK(1)R mRNA expression as well as SP and NK(1)R protein levels in an NK(1)R-dependent manner. Thiorphan increased CD10(+)CD4(+) and CD10(+)DP thymocyte numbers, and an NK(1)R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor alpha chain expression, and concanavalin A-induced proliferation of CD5(+) thymocytes, and it inhibited spontaneous and NK(1)R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.

IFN-γ Stimulated Human Umbilical-Tissue-Derived Cells Potently Suppress NK Activation and Resist NK-Mediated Cytotoxicity In Vitro

PubMed Central

Noone, Cariosa; Kihm, Anthony; O'Dea, Shirley; Mahon, Bernard P.

2013-01-01

Umbilical cord tissue represents a unique source of cells with potential for cell therapy applications for multiple diseases. Human umbilical tissue-derived cells (hUTC) are a developmentally early stage, homogenous population of cells that are HLA-ABC dim, HLA-DR negative, and lack expression of co-stimulatory molecules in the unactivated state. The lack of HLA-DR and co-stimulatory molecule expression on unactivated hUTC may account for their reduced immunogenicity, facilitating their use in allogeneic settings. However, such approaches could be confounded by host innate cells such as natural killer (NK) cells. Here, we evaluate in vitro NK cell interactions with hUTC and compare them with human mesenchymal stem cells (MSC). Our investigations show that hUTC suppress NK activation, through prostaglandin-E2 secretion in a contact-independent manner. Prestimulation of hUTC or human MSC with interferon gamma (IFN-γ) induced expression of the tryptophan degrading enzyme indoleamine 2, 3 dioxygenase, facilitating enhanced suppression. However, resting NK cells of different killer immunoglobulin-like receptor haplotypes did not kill hUTC or MSC; only activated NK cells had the ability to kill nonstimulated hUTC and, to a lesser extent, MSC. The cell killing process involved signaling through the NKG2D receptor and the perforin/granzyme pathway; this was supported by CD54 (ICAM-1) expression by hUTC. IFN-γ-stimulated hUTC or hMSC were less susceptible to NK killing; in this case, protection was associated with elevated HLA-ABC expression. These data delineate the different mechanisms in a two-way interaction between NK cells and two distinct cell therapies, hUTC or hMSC, and how these interactions may influence their clinical applications. PMID:23795941

Human natural killer cell receptors for HLA-class I molecules. Evidence that the Kp43 (CD94) molecule functions as receptor for HLA-B alleles

PubMed Central

1994-01-01

GL183 or EB6 (p58) molecules have been shown to function as receptors for different HLA-C alleles and to deliver an inhibitory signal to natural killer (NK) cells, thus preventing lysis of target cells. In this study, we analyzed a subset of NK cells characterized by a p58- negative surface phenotype. We show that p58-negative clones, although specific for class I molecules do not recognize HLA-C alleles. In addition, by the use of appropriate target cells transfected with different HLA-class I alleles we identified HLA-B7 as the protective element recognized by a fraction of p58-negative clones. In an attempt to identify the receptor molecules expressed by HLA-B7-specific clones, monoclonal antibodies (mAbs) were selected after mice immunization with such clones. Two of these mAbs, termed XA-88 and XA-185, and their F(ab')2 fragments, were found to reconstitute lysis of B7+ target cells by B7-specific NK clones. Both mAbs were shown to be directed against the recently clustered Kp43 molecule (CD94). Thus, mAb-mediated masking of Kp43 molecules interferes with recognition of HLA-B7 and results in target cell lysis. Moreover, in a redirected killing assay, the cross- linking of Kp43 molecules mediated by the XA185 mAb strongly inhibited the cytolytic activity of HLA-B7-specific NK clones, thus mimicking the functional effect of B7 molecules. Taken together, these data strongly suggest that Kp43 molecules function as receptors for HLA-B7 and that this receptor/ligand interaction results in inhibition of the NK- mediated cytolytic activity. Indirect immunofluorescence and FACS analysis of a large number of random NK clones showed that Kp43 molecules (a) were brightly expressed on a subset of p58-negative clones, corresponding to those specific for HLA-B7; (b) displayed a medium/low fluorescence in the p58-negative clones that are not B7- specific as well as in most p58+ NK clones; and (c) were brightly expressed as in the p58+ clone ET34 (GL183-/EB6+, Cw4-specific). Functional analysis revealed that Kp43 functioned as an inhibitory receptor only in NK clones displaying bright fluorescence. These studies also indicate that some NK clones (e.g., the ET34) can coexpress two distinct receptors (p58 and Kp43) for different class I alleles (Cw4 and B7). Finally, we show that Kp43 molecules function as receptors only for some HLA-B alleles and that still undefined receptor(s) must exist for other HLA-B alleles including B27. PMID:8046333

Interleukin-1beta-induced hyperresponsiveness to [Sar9,Met(O2)11]substance P in isolated human bronchi.

PubMed

Barchasz, E; Naline, E; Molimard, M; Moreau, J; Georges, O; Emonds-Alt, X; Advenier, C

1999-08-20

Interleukin-1beta has been reported to induce airway hyperresponsiveness in several animal models. In this study, we have investigated whether interleukin-1beta was able to potentiate the contractions of human isolated small bronchi (internal diameter < or = 1 mm) provoked by a specific tachykinin NK1 receptor agonist, [Sar9,Met(O2)11]substance P. Pre-incubation of human isolated small bronchi with interleukin-1beta (10 ng/ml, in Krebs-Henseleit solution, at 21 degrees C for 15 h) potentiated the contractile response to [Sar9,Met(O2)11]substance P. It also increased the [Sar9,Met(O2)11]substance P-induced release of thromboxane B2, the stable metabolite of thromboxane A2. Indomethacin (10(-6) M), a non-specific cyclooxygenase inhibitor, or GR 32191 ((1R-(1alpha(Z)),2beta,3beta,5alpha))-(+)-7-(5-(((1,1' -biphenyl)-4-yl)-methoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl)-4-hept enoic acid, hydrochloride) (10(-6) M), a prostanoid TP-receptor antagonist, blocked the contractions induced by [Sar9,Met(O2)11]substance P both in control experiments and after interleukin-1beta pre-treatment, indicating that prostanoids and thromboxane receptors are directly implicated in the [Sar9,Met(O2)11]substance P-induced contractile response. The thromboxane mimetic U-46619 (10(-8)-10(-6) M) (9,11-dideoxy-11alpha,9alpha-epoxymethano-prostaglandin F2alpha)-induced contractions of human isolated small bronchi were not enhanced by interleukin-1beta pre-treatment, suggesting that no up-regulation of thromboxane receptors occurred. Furthermore, the cyclooxygenase-2 inhibitor CGP 28238 (6-(2,4-difluorophenoxy)-5-methyl-sulfonylamino-1-indanon e) (10(-6) M) had no direct effect on [Sar9,Met(O2)11]substance P-provoked contractions, but inhibited the interleukin-1beta-induced potentiation of [Sar9,Met(O2)11]substance P response. In conclusion, our results show that interleukin-1beta pre-treatment is able to potentiate the contractions of isolated human small bronchi provoked by [Sar9,Met(O2)11]substance P both by increasing prostanoid synthesis and by inducing a cyclooxygenase-2 pathway.

Cancer Immunology at the Crossroads: Killer immunoglobulin-like receptors and tumor immunity

PubMed Central

Benson, Don M; Caligiuri, Michael A

2014-01-01

Natural killer (NK) cells, large granular lymphocytes comprising a key cellular subset of innate immunity, were originally named for their capacity to elicit potent cytotoxicity against tumor cells independent of prior sensitization or gene rearrangement. This process is facilitated through the expression of activating and inhibitory receptors that provide for NK cell “education” and a subsequent ability to survey, recognize and lyse infected or transformed cells, especially those lacking or possessing mutated major histocompatibility complex (MHC) class I expression. Since these original observations were made, how NK cells recognize candidate target cells continues to be the topic of ongoing investigation. It is now appreciated that NK cells express a diverse repertoire of activating and inhibitory receptors of which killer immunoglobulin-like receptors (KIR) appear to play a critical role in mediating self-tolerance as well as facilitating cytotoxicity against infected or transformed cells. Additionally, in the presence of an activating signal, the absence or mismatch of MHC class I molecules on such targets (which serve as inhibitory KIR ligands) promotes NK cell-mediated lysis. An increasing understanding of the complexities of KIR biology has provided recent opportunities to leverage the NK cell versus tumor effect as a novel avenue of therapeutic immunotherapy for cancer. The present review seeks to summarize the current understanding of KIR expression and function and highlight ongoing efforts to translate these discoveries into novel NK cell-mediated immunotherapies for cancer. PMID:24592397

Peptide-specific recognition of human cytomegalovirus strains controls adaptive natural killer cells.

PubMed

Hammer, Quirin; Rückert, Timo; Borst, Eva Maria; Dunst, Josefine; Haubner, André; Durek, Pawel; Heinrich, Frederik; Gasparoni, Gilles; Babic, Marina; Tomic, Adriana; Pietra, Gabriella; Nienen, Mikalai; Blau, Igor Wolfgang; Hofmann, Jörg; Na, Il-Kang; Prinz, Immo; Koenecke, Christian; Hemmati, Philipp; Babel, Nina; Arnold, Renate; Walter, Jörn; Thurley, Kevin; Mashreghi, Mir-Farzin; Messerle, Martin; Romagnani, Chiara

2018-05-01

Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C + NK cells has remained unclear. Here we found that adaptive NKG2C + NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C + NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C + NK cell populations among HCMV-seropositive people.

Histaminergic regulation of NK-cells: protection against monocyte-induced apoptosis.

PubMed

Hansson, M; Asea, A; Hermodsson, S; Hellstrand, K

1996-08-01

Human natural killer (NK) cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology and degradation of DNA into oligonucleosomal fragments. The monocyte-induced apoptosis in NK-cells was prevented by the biogenic amine histamine at concentrations exceeding 0.1 microM. The protective effect of histamine was blocked by the H2-receptor (H2R) antagonist ranitidine but not by AH202399 A, a chemical control to ranitidine devoid of H2R affinity. It is concluded that histaminergic mechanisms may serve to protect NK cells from damage inflicted by products of the oxidative metabolism of monocytes.

Activation of natural killer cells by hepatitis C virus particles in vitro

PubMed Central

Farag, M M S; Weigand, K; Encke, J; Momburg, F

2011-01-01

Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection. PMID:21682720

Truncated neurokinin-1 receptor is increased in colonic epithelial cells from patients with colitis-associated cancer

PubMed Central

Gillespie, Earl; Leeman, Susan E.; Watts, Luisa A.; Coukos, Jennifer A.; O'Brien, Michael J.; Cerda, Sandra R.; Farraye, Francis A.; Stucchi, Arthur F.; Becker, James M.

2011-01-01

Patients with chronic ulcerative colitis (UC) are at high risk for developing colorectal cancer. In this study, archival formalin-fixed paraffin-embedded colonic tissue from patients with UC who developed carcinoma (CA) or high-grade dysplasia (HGD) was examined for changes in expression of the proinflammatory and mitogenic neurokinin-1 receptor (NK-1R). Laser capture microscopy was used to microdissect epithelia from areas of colons that showed histologic evidence of CA, HGD, and epithelia that were not dysplastic or cancerous but did contain evidence of prior inflammation (quiescent colitis). mRNA was extracted from the dissected tissue, and PCR array analysis was performed on extracted mRNA. Two antibodies were necessary to separately estimate the protein levels of the truncated (tr-NK-1R) and full-length (fl-NK-1R) receptors by immunohistochemistry. mRNA expression of tr-NK-1R increased 14-fold (P = 0.02) when comparing the HGD and CA groups. In contrast, the fl-NK-1R transcript showed no significant differences among groups. The protein levels of the total NK-1R increased by 40% (P = 0.02) in HGD and 80% (P = 0.0007) in CA compared with quiescent colitis. There were no significant changes in protein levels of the fl-NK-1R. We conclude that the increase in total NK-1R protein in HGD and CA is attributable to an increase in tr-NK-1R, suggesting there may be a functional role for tr-NK-1R in malignant transformation in colitis-associated cancer. The tr-NK-1R could prove useful as a diagnostic marker to identify patients at risk for neoplasia and may serve as a useful therapeutic target in the treatment of colitis-associated cancer. PMID:21969570

Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

PubMed Central

Gross, Catharina C.; Schulte-Mecklenbeck, Andreas; Rünzi, Anna; Kuhlmann, Tanja; Posevitz-Fejfár, Anita; Schwab, Nicholas; Schneider-Hohendorf, Tilman; Herich, Sebastian; Held, Kathrin; Konjević, Matea; Hartwig, Marvin; Dornmair, Klaus; Hohlfeld, Reinhard; Ziemssen, Tjalf; Klotz, Luisa; Meuth, Sven G.; Wiendl, Heinz

2016-01-01

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4β1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4+ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4+ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4+ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4+ T cells and the cytolytic activity of NK cells. PMID:27162345

The aryl hydrocarbon receptor is required for the maintenance of liver-resident natural killer cells

PubMed Central

2016-01-01

A tissue-resident population of natural killer cells (NK cells) in the liver has recently been described to have the unique capacity to confer immunological memory in the form of hapten-specific contact hypersensitivity independent of T and B cells. Factors regulating the development and maintenance of these liver-resident NK cells are poorly understood. The aryl hydrocarbon receptor (AhR) is a transcription factor modulated by exogenous and endogenous ligands that is important in the homeostasis of immune cells at barrier sites, such as the skin and gut. In this study, we show that liver-resident NK (NK1.1+CD3−) cells, defined as CD49a+TRAIL+CXCR6+DX5− cells in the mouse liver, constitutively express AhR. In AhR−/− mice, there is a significant reduction in the proportion and absolute number of these cells, which results from a cell-intrinsic dependence on AhR. This deficiency in liver-resident NK cells appears to be the result of higher turnover and increased susceptibility to cytokine-induced cell death. Finally, we show that this deficiency has functional implications in vivo. Upon hapten exposure, AhR−/− mice are not able to mount an NK cell memory response to hapten rechallenge. Together, these data demonstrate the requirement of AhR for the maintenance of CD49a+TRAIL+CXCR6+DX5− liver-resident NK cells and their hapten memory function. PMID:27670593

Continuous infusion of substance P into rat striatum alleviates nociceptive behavior via phosphorylation of extracellular signal-regulated kinase 1/2.

PubMed

Nakamura, Yoki; Izumi, Hiroki; Fukushige, Ryo; Shimizu, Takumi; Watanabe, Kyohei; Morioka, Norimitsu; Hama, Aldric; Takamatsu, Hiroyuki; Nakata, Yoshihiro

2014-12-01

Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an 'acute' phase (0-15 min) and 'tonic' phase (16-120 min), which is accompanied by significant phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post-formalin injection. To uncover a possible relationship between the slow-onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 μg/mL in microdialysis fiber, 1 μL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of 'tonic' phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre-treatment with NK1 receptor antagonist CP96345 (10 μM) blocked the effect of SP infusion. However, formalin-induced 'tonic' phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the 'tonic' phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP-NK1 receptor-ERK1/2 system. Noxious stimulation induces a slow-onset substance P (SP) release as a volume transmitter, activating extra-synaptic NK1 receptors, and evokes phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The SP-NK1-ERK1/2 system in the striatum decreases tonic nociception. © 2014 International Society for Neurochemistry.

Uterine Natural Killer cells regulate endometrial bleeding in women and are suppressed by the progesterone receptor modulator asoprisnil

PubMed Central

Wilkens, Julia; Male, Victoria; Ghazal, Peter; Forster, Thorsten; Gibson, Douglas A.; Williams, Alistair RW; Brito-Mutunayagam, Savita L; Craigon, Marie; Lourenco, Paula; Cameron, Iain T; Chwalisz, Kristof; Moffett, Ashley; Critchley, Hilary OD

2013-01-01

Uterine NK cells (uNK) play a role in the regulation of placentation but their functions in non-pregnant endometrium are not understood. We have previously reported suppression of endometrial bleeding and alteration of spiral artery morphology in women exposed to asoprisnil, a progesterone receptor modulator. We now compare global endometrial gene expression in asoprisnil-treated versus control women, and we demonstrate a statistically significant reduction of genes in the IL-15 pathway, known to play a key role in uNK development and function. Suppression of IL-15 by asoprisnil was also observed at mRNA level (p<0.05), and immunostaining for NK cell marker CD56 revealed a striking reduction of uNK in asoprisnil-treated endometrium (p<0.001). IL-15 levels in normal endometrium are progesterone-responsive. Progesterone receptor (PR) positive stromal cells transcribe both IL-15 and IL-15RA. Thus, the response of stromal cells to progesterone will be to increase IL-15 trans-presentation to uNK, supporting their expansion and differentiation. In asoprisnil-treated endometrium, there is a marked down-regulation of stromal PR expression and virtual absence of uNK. These novel findings indicate that the IL-15 pathway provides a missing link in the complex interplay between endometrial stromal cells, uNK and spiral arteries affecting physiological and pathological endometrial bleeding. PMID:23913972

Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality

PubMed Central

Bárcena, Paloma; Jara-Acevedo, María; Tabernero, María Dolores; López, Antonio; Sánchez, María Luz; García-Montero, Andrés C.; Muñoz-García, Noemí; Vidriales, María Belén; Paiva, Artur; Lecrevisse, Quentin; Lima, Margarida; Langerak, Anton W.; Böttcher, Sebastian; van Dongen, Jacques J.M.

2015-01-01

Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56low NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56low NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94hi/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality. PMID:26556869

Survival in acute myeloid leukemia is associated with NKp44 splice variants

PubMed Central

Hadad, Uzi; Teltsh, Omri; Edri, Avishay; Rubin, Eitan; Campbell, Kerry S.; Rosental, Benyamin; Porgador, Angel

2016-01-01

NKp44 is a receptor encoded by the NCR2 gene, which is expressed by cytokine-activated natural killer (NK) cells that are involved in anti-AML immunity. NKp44 has three splice variants corresponding to NKp44ITIM+ (NKp44-1) and NKp44ITIM− (NKp44-2, and NKp44-3) isoforms. RNAseq data of AML patients revealed similar survival of NKp46+NKp44+ and NKp46+NKp44− patients. However, if grouped according to the NKp44 splice variant profile, NKp44-1 expression was significantly associated with poor survival of AML patients. Moreover, activation of PBMC from healthy controls showed co-dominant expression of NKp44-1 and NKp44-3, while primary NK clones show more diverse NKp44 splice variant profiles. Cultured primary NK cells resulted in NKp44-1 dominance and impaired function associated with PCNA over-expression by target cells. This impaired functional phenotype could be rescued by blocking of NKp44 receptor. Human NK cell lines revealed co-dominant expression of NKp44-1 and NKp44-3 and showed a functional phenotype that was not inhibited by PCNA over-expression. Furthermore, transfection-based overexpression of NKp44-1, but not NKp44-2/NKp44-3, reversed the endogenous resistance of NK-92 cells to PCNA-mediated inhibition, and resulted in poor formation of stable lytic immune synapses. This research contributes to the understanding of AML prognosis by shedding new light on the functional implications of differential splicing of NKp44. PMID:27102296

Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

PubMed Central

Pelayo, Juan-Carlos; Poole, Daniel P; Steinhoff, Martin; Cottrell, Graeme S; Bunnett, Nigel W

2011-01-01

Abstract Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK1R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nm, 10 min) induced interaction of NK1R and β-arrestin at the plasma membrane, and the SP–NK1R–β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK1R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H+ATPase inhibitor bafilomycin A1, which prevent endosomal SP degradation, suppressed NK1R recycling by >50%. Preincubation of neurones with SP (10 nm, 5 min) desensitized Ca2+ transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK1R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP–NK1R–β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK1R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling. PMID:21878523

Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor.

PubMed

Schmidlin, F; Dery, O; DeFea, K O; Slice, L; Patierno, S; Sternini, C; Grady, E F; Bunnett, N W

2001-07-06

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

Prenatal Nicotinic Exposure Upregulates Pulmonary C-fiber NK1R Expression to Prolong Pulmonary C-fiber-Mediated Apneic Response

PubMed Central

Zhao, Lei; Zhuang, Jianguo; Zang, Na; Lin, Yong; Lee, Lu-Yuan; Xu, Fadi

2015-01-01

Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA1 receptor, ADA1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA1R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to change BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChRs or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin. PMID:26524655

Lipoic acid stimulates cAMP production via the EP2 and EP4 prostanoid receptors and inhibits IFN gamma synthesis and cellular cytotoxicity in NK cells

PubMed Central

Salinthone, Sonemany; Schillace, Robynn V.; Marracci, Gail H.; Bourdette, Dennis N.; Carr, Daniel W.

2008-01-01

The antioxidant lipoic acid (LA) treats and prevents the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In an effort to understand the therapeutic potential of LA in MS, we sought to define the cellular mechanisms that mediate the effects of LA on human natural killer (NK) cells, which are important in innate immunity as the first line of defense against invading pathogens and tumor cells. We discovered that LA stimulates cAMP production in NK cells in a dose-dependent manner. Studies using pharmacological inhibitors and receptor transfection experiments indicate that LA stimulates cAMP production via activation of the EP2 and EP4 prostanoid receptors and adenylyl cyclase. In addition, LA suppressed interleukin (IL)-12/IL-18 induced IFNγ secretion and cytotoxicity in NK cells. These novel findings suggest that LA may inhibit NK cell function via the cAMP signaling pathway. PMID:18562016

Polyclonal proliferation of activated suppressor/cytotoxic T cells with transient depression of natural killer cell function in acute infectious mononucleosis.

PubMed Central

Williams, M L; Loughran, T P; Kidd, P G; Starkebaum, G A

1989-01-01

In acute infectious mononucleosis large numbers of atypical lymphocytes proliferate in response to B cells infected with Epstein-Barr virus, generally resulting in a self-limited illness. Although both T-cells and NK cells are known to be involved, the precise origin of the large granular lymphocytes in this disorder is incompletely understood. Using two-colour immunofluorescent flow cytometry, we sequentially examined the phenotype of selected T cell and NK cell subsets from nine patients with infectious mononucleosis. In parallel, we determined whether these lymphocytes utilized a restricted repertoire of the T cell receptor gene and also measured their NK activity. Our results show that in acute infectious mononucleosis there was a greater than three-fold increase in T lymphocytes with the phenotype CD2+, CD3+, CD8+ and DR+. A modest increase in Leu7(HNK1)+ and CD4+ T cells was also seen. In addition, there was a three-fold increase in cells coexpressing CD3- and CD16+, the phenotype reported to represent most NK cells. In spite of this latter finding, however, a marked decrease in NK function was found at the time of diagnosis, gradually returning to normal by day 28. Finally, Southern blot analysis of DNA from patient lymphocytes showed polyclonal rearrangements of the T cell receptor beta chain gene. These studies indicate that the proliferation of activated suppressor/cytotoxic T lymphocytes in acute infectious mononucleosis is polyclonal and is associated with transient depression of NK function. Images Fig. 2 PMID:2527653

A synergistic interferon-γ production is induced by mouse hepatitis virus in interleukin-12 (IL-12)/IL-18-activated natural killer cells and modulated by carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1a receptor

PubMed Central

Jacques, Alexandre; Bleau, Christian; Turbide, Claire; Beauchemin, Nicole; Lamontagne, Lucie

2009-01-01

The production of interferon-γ (IFN-γ) by infiltrating natural killer (NK) cells in liver is involved in the control of mouse hepatitis virus (MHV) infection. The objectives of this study were to identify the mechanisms used by MHV type 3 to modulate the production of IFN-γ by NK cells during the acute hepatitis in susceptible C57BL/6 mice. Ex vivo and in vitro experiments revealed that NK cells, expressing carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1a (the MHV receptor), can produce a higher level of IFN-γ in the presence of both L2-MHV3 and interleukin-12 (IL-12)/IL-18. The synergistic production of IFN-γ by NK cells depends on viral replication rather than viral fixation only, because it is inhibited or not induced in cells infected with ultraviolet-inactivated viruses and in cells from Ceacam1a−/− mice infected with virulent viruses. The synergistic IFN-γ production involves the p38 mitogen-activated protein kinase (MAPK) rather than the extracellular signal-regulated kinase-1/2 MAPK signalling pathway. However, the signal triggered through the engagement of CEACAM1a decreases the production of IFN-γ, when these molecules are cross-linked using specific monoclonal antibodies. These results suggest that control of acute hepatitis by IFN-γ-producing NK cells may depend on both production of IL-12 and IL-18 in the liver environment and viral infection of NK cells. PMID:19740316

Shifting the Balance of Activating and Inhibitory Natural Killer Receptor Ligands on BRAFV600E Melanoma Lines with Vemurafenib.

PubMed

Frazao, Alexandra; Colombo, Marina; Fourmentraux-Neves, Emmanuelle; Messaoudene, Meriem; Rusakiewicz, Sylvie; Zitvogel, Laurence; Vivier, Eric; Vély, Frédéric; Faure, Florence; Dréno, Brigitte; Benlalam, Houssem; Bouquet, Fanny; Savina, Ariel; Pasmant, Eric; Toubert, Antoine; Avril, Marie-Françoise; Caignard, Anne

2017-07-01

Over 60% of human melanoma tumors bear a mutation in the BRAF gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib. However, these responses are transient, and relapses are frequent. Melanoma cells are efficiently lysed by activated natural killer (NK) cells. Melanoma cells express several stress-induced ligands that are recognized by activating NK-cell receptors. We have investigated the effect of vemurafenib on the immunogenicity of seven BRAF -mutated melanoma cells to NK cells and on their growth and sensitivity to NK-cell-mediated lysis. We showed that vemurafenib treatment modulated expression of ligands for two activating NK receptors, increasing expression of B7-H6, a ligand for NKp30, and decreasing expression of MICA and ULBP2, ligands for NKG2D. Vemurafenib also increased expression of HLA class I and HLA-E molecules, likely leading to higher engagement of inhibitory receptors (KIRs and NKG2A, respectively), and decreased lysis of vemurafenib-treated melanoma cell lines by cytokine-activated NK cells. Finally, we showed that whereas batimastat (a broad-spectrum matrix metalloprotease inhibitor) increased cell surface ULBP2 by reducing its shedding, vemurafenib lowered soluble ULBP2, indicating that BRAF signal inhibition diminished expression of both cell-surface and soluble forms of NKG2D ligands. Vemurafenib, inhibiting BRAF signaling, shifted the balance of activatory and inhibitory NK ligands on melanoma cells and displayed immunoregulatory effects on NK-cell functional activities. Cancer Immunol Res; 5(7); 582-93. ©2017 AACR . ©2017 American Association for Cancer Research.

Immunolocalization of a tachykinin-receptor-like protein in the central nervous system of Locusta migratoria migratorioides and neobellieria bullata.

PubMed

Veelaert, D; Oonk, H B; Vanden Eynde, G; Torfs, H; Meloen, R H; Schoofs, L; Parmentier, M; De Loof, A; Vanden Broeck, J

1999-05-10

Antisera raised against two distinct peptide regions of the Drosophila neurokinin-like receptor NKD were used to immunolocalize tachykinin-receptor-like proteins in the central nervous system of two insect species: the African migratory locust, Locusta migratoria, and the gray fleshfly, Neobellieria bullata. The resulting immunopositive staining patterns were identical for both antisera. Moreover, a very similar distribution of the immunoreactive material was observed in fleshflies and locusts. Immunoreactivity was found in nerve terminals of the retrocerebral complex, suggesting a presynaptic localization of the receptor in this part of the brain. Cell bodies were stained in the subesophageal ganglion: an anterior group of four larger cells and a posterior group of about 20 cells. These cells have axons projecting into the contralateral nervus corporis allati (NCA) II, bypassing the corpus allatum and projecting through the NCA I into the storage part of the corpus cardiacum. In the glandular part of the corpus cardiacum, the glandular adipokinetic hormone-producing cells did not show any immunopositive staining. In the locust, additional immunopositive staining was observed in internolaterally located neurons of the tritocerebrum and in important integrative parts of the neuropil such as the central body and the mushroom bodies.

Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3+CD5dimCD21- Cytotoxic Large Granular Lymphocytes.

PubMed

Lee, Soo-Hyeon; Shin, Dong-Jun; Kim, Yoseop; Kim, Cheol-Jung; Lee, Je-Jung; Yoon, Mee Sun; Uong, Tung Nguyen Thanh; Yu, Dohyeon; Jung, Ji-Youn; Cho, Duck; Jung, Bock-Gie; Kim, Sang-Ki; Suh, Guk-Hyun

2018-01-01

Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation, and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3 - CD21 - ) large granular lymphocytes. However, a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes, and that CD3 + CD5 dim CD21 - lymphocytes are putative canine NK cells. Based on our previous reports, we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study, we investigated the phenotypic and functional differences between CD3 + CD5 dim CD21 - (cytotoxic large granular lymphocytes) and CD3 - CD5 - CD21 - NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3 + CD5 dim CD21 - lymphocytes can be differentiated into non-B, non-T NK (CD3 - CD5 - CD21 - TCRαβ - TCRγδ - GranzymeB + ) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3 + CD5 dim CD21 - cells showed that CD3 - CD5 - CD21 - cells are derived from CD3 + CD5 dim CD21 - cells through phenotypic modulation. CD3 + CD5 dim CD21 - cells share more NK cell functional characteristics compared with CD3 - CD5 - CD21 - cells, including the expression of T-box transcription factors (Eomes, T-bet), the production of granzyme B and interferon-γ, and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion, the results of this study suggest that CD3 + CD5 dim CD21 - and CD3 - CD5 - CD21 - cells both contain a subset of putative NK cells, and the difference between the two populations may be due to the degree of maturation.

ROLE OF TACHYKININS IN OZONE-INDUCED ACUTE LUNG INJURY

EPA Science Inventory

To examine the hypothesis that the acute, reversible changes caused by O3 exposure are mediated by techykinin release, guinea pigs were depleted of tachykinins using repeated capsaicin (CAP) injections prior to O3 exposure, in an attempt to prevent O3-induced functional changes. ...

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.

PubMed

Woll, Petter S; Martin, Colin H; Miller, Jeffrey S; Kaufman, Dan S

2005-10-15

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

PubMed

Lugthart, Gertjan; Melsen, Janine E; Vervat, Carly; van Ostaijen-Ten Dam, Monique M; Corver, Willem E; Roelen, Dave L; van Bergen, Jeroen; van Tol, Maarten J D; Lankester, Arjan C; Schilham, Marco W

2016-07-01

Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

Involvement of enzymatic degradation in the inactivation of tachykinin neurotransmitters in neonatal rat spinal cord.

PubMed

Suzuki, H; Yoshioka, K; Yanagisawa, M; Urayama, O; Kurihara, T; Hosoki, R; Saito, K; Otsuka, M

1994-09-01

1. The possible involvement of enzymatic degradation in the inactivation of tachykinin neurotransmitters was examined in the spinal cord of the neonatal rat. 2. The magnitude of substance P (SP)- or neurokinin A (NKA)-evoked depolarization of a lumbar ventral root in the isolated spinal cord preparation was increased by a mixture of peptidase inhibitors, consisting of actinonin (6 microM), arphamenine B (6 microM), bestatin (10 microM), captopril (10 microM) and thiorphan (0.3 microM). The mixture augmented the response to NKA more markedly than that to SP. 3. In the isolated spinal cord-cutaneous nerve preparation, the saphenous nerve-evoked slow depolarization of the L3 ventral root was augmented by the mixture of peptidase inhibitors in the presence of naloxone (0.5 microM) but not in the presence of both naloxone and a tachykinin receptor antagonist, GR71251 (5 microM). 4. Application of capsaicin (0.5 microM) for 6 min to the spinal cord evoked an increase in the release of SP from the spinal cord. The amount of SP released was significantly augmented by the mixture of peptidase inhibitors. 5. Synaptic membrane fractions were prepared from neonatal rat spinal cords. These fractions showed degrading activities for SP and NKA and the activities were inhibited by the mixture of peptidase inhibitors. The degrading activity for NKA was higher than that for SP and the inhibitory effect of the mixture for NKA was more marked than that for SP. Although some other fractions obtained from homogenates of spinal cords showed higher degrading activities for SP, these activities were insensitive to the mixture of peptidase inhibitors. 6. Effects of individual peptidase inhibitors on the enzymatic degradation of SP and NKA by synaptic membrane fractions were examined. Thiorphan, actinonin and captopril inhibited SP degradation, while thiorphan and actinonin, but not captopril, inhibited NKA degradation. The potency of the inhibition of each peptidase inhibitor was lower than that of the mixture.7. The present results suggest that enzymatic degradation is involved in the inactivation of tachykinin neurotransmitters in the spinal cord of the neonatal rat.

Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma.

PubMed

Drexler, H G; Matsuo, Y

2000-05-01

Malignancies involving natural killer (NK) cells are rare disorders. The complexity of NK cell-involving disorders has only recently been appreciated. Modern classifications discern immature (precursor) from mature NK cell leukemias-lymphomas. Continuous NK leukemia-lymphoma cell lines represent important model systems to study these neoplasms. While there are a number of putative NK cell lines which are, however, either not characterized, not immortalized, non-malignant, non-NK, or plain false cell lines, six bona fide malignant NK cell lines have been established and are sufficiently well characterized: HANK1, KHYG-1, NK-92, NKL, NK-YS and YT. Except for YT which was derived from a not further defined acute lymphoblastic lymphoma, these cell lines were established from patients with various NK cell malignancies. Five of the six cell lines are constitutively interleukin-2-dependent. Their immunoprofile is remarkably similar: CD1-, CD2+, surface CD3 (but cytoplasmic CD3epsilon+), CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, TCRalphabeta-, TCRgammadelta-, negative for B cell and myelomonocytic markers. The immunoglobulin heavy chain and T cell receptor genes are all in germline configuration. All six lines show complex chromosomal alterations, with both numerical and structural aberrations, attesting to their malignant and monoclonal nature. Functionally, these cells which contain azurophilic granules in their cytoplasm are nearly universally positive in NK activity assays. Three of five cell lines are Epstein-Barr virus-positive (type II latency). The composite data on these six cell lines allow for the operational definition of a typical malignant NK cell line profile. NK leukemia-lymphoma cell lines will prove invaluable for studies of normal and malignant NK cell biology.

Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA, combined with soluble IL-21.

PubMed

Jiang, Bo; Wu, Xuan; Li, Xi-Ning; Yang, Xi; Zhou, Yulai; Yan, Haowei; Wei, An-Hui; Yan, Weiqun

2014-07-01

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56(bright)CD16(+) subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA demethylation and histone H3K9 acetylation determine the active transcription of the NKG2D gene in human CD8+ T and NK cells

PubMed Central

Fernández-Sánchez, Alba; Baragaño Raneros, Aroa; Carvajal Palao, Reyes; Sanz, Ana B.; Ortiz, Alberto; Ortega, Francisco; Suárez-Álvarez, Beatriz; López-Larrea, Carlos

2013-01-01

The human activating receptor NKG2D is mainly expressed by NK, NKT, γδ T and CD8+ T cells and, under certain conditions, by CD4+ T cells. This receptor recognizes a diverse family of ligands (MICA, MICB and ULBPs 1–6) leading to the activation of effector cells and triggering the lysis of target cells. The NKG2D receptor-ligand system plays an important role in the immune response to infections, tumors, transplanted graft and autoantigens. Elucidation of the regulatory mechanisms of NKG2D is therefore essential for therapeutic purposes. In this study, we speculate whether epigenetic mechanisms, such as DNA methylation and histone acetylation, participate in NKG2D gene regulation in T lymphocytes and NK cells. DNA methylation in the NKG2D gene was observed in CD4+ T lymphocytes and T cell lines (Jurkat and HUT78), while this gene was unmethylated in NKG2D-positive cells (CD8+ T lymphocytes, NK cells and NKL cell line) and associated with high levels of histone H3 lysine 9 acetylation (H3K9Ac). Treatment with the histone acetyltransferase (HAT) inhibitor curcumin reduces H3K9Ac levels in the NKG2D gene, downregulates NKG2D transcription and leads to a marked reduction in the lytic capacity of NKG2D-mediated NKL cells. These findings suggest that differential NKG2D expression in the different cell subsets is regulated by epigenetic mechanisms and that its modulation by epigenetic treatments might provide a new strategy for treating several pathologies. PMID:23235109

Altered respiratory response to substance P and reduced NK1 receptor binding in the nucleus of the solitary tract of aged rats.

PubMed

Mazzone, S B; Geraghty, D P

1999-04-24

The respiratory response to microinjection of substance P (SP) into the commissural nucleus of the solitary tract (cNTS) and binding of [125I]-Bolton-Hunter SP ([125I]-BHSP) to brain stem NK1 receptors were compared in young and aged rats. Injection of SP (750 pmol) into the cNTS of young rats (2 months) increased tidal volume (VT) but had no effect on respiratory rate (f). In aged rats (19-21 months), injection of SP had no significant effect on f or VT. The NTS of aged rats displayed significantly lower specific [125I]-BHSP binding than young rats, indicating a reduction in the number in NK1 receptors. These findings show that the respiratory response to microinjection of SP into the cNTS of aged rats is severely blunted and that this phenomenon may be due to a decrease in the number of NK1 receptors in the NTS. Copyright 1999 Elsevier Science B.V.

The absence of Grb2-associated binder 2 (Gab2) does not disrupt NK cell development and functions.

PubMed

Zompi, Simona; Gu, Hahiua; Colucci, Francesco

2004-10-01

Scaffolding molecules bind simultaneously and link together various components of signal-transduction pathways. Grb2-associated binder 2 (Gab2) is a scaffolding protein required for FcgammaR-initiated allergic responses in mast cells and FcgammaR-mediated phagocytosis in macrophages, where it links IgE and IgG receptors to the phosphatidylinositol-3 kinase (PI-3K) pathway. The FcgammaR expressed by natural killer (NK) cells triggers antibody-dependent cellular cytotoxicity (ADCC). We show here that mouse NK cells express Gab2 and that although PI-3K was required for ADCC, this FcgammaR-mediated function was normal in Gab2-/- NK cells. Moreover, NK cell development, spontaneous cytotoxicity, and responses to and production of cytokines were not perturbed in Gab2-/- mice. Considering the striking differences between the signaling requirements of FcgammaR in macrophages and NK cells, our findings suggest that the organization of signal transduction downstream of the same FcR can be cell type-specific. Conversely, Gab family members Gab1, Gab2, and Gab3 may play specific roles in different leukocytes. As pharmacological targeting of Gab2 in mast cells is a potential strategy to treat allergy, our results suggest prudence, as NK cells may participate in IgE-mediated anaphylaxis in a Gab2-independent manner.

Ca2+ signaling and emesis: Recent progress and new perspectives.

PubMed

Zhong, Weixia; Picca, Andrew J; Lee, Albert S; Darmani, Nissar A

2017-01-01

Cisplatin-like chemotherapeutics cause vomiting via calcium (Ca 2+ )-dependent release of multiple neurotransmitters (dopamine, serotonin, substance P, etc.) from the gastrointestinal enterochromaffin cells and/or the brainstem. Intracellular Ca 2+ signaling is triggered by activation of diverse emetic receptors (including tachykininergic NK 1 , serotonergic 5-HT 3 , dopaminergic D 2 , cholinergic M 1 , or histaminergic H 1 ) , whose activation in vomit-competent species can evoke emesis. Other emetogens such as cisplatin, rotavirus NSP4 protein and bacterial toxins can also induce intracellular Ca 2+ elevation. Netupitant is a highly selective neurokinin NK 1 receptor (NK 1 R) antagonist and palonosetron is a selective second-generation serotonin 5-HT 3 receptor (5-HT 3 R) antagonist with a distinct pharmacological profile. An oral fixed combination of netupitant/palonosetron (NEPA; Akynzeo(®)) with >85% antiemetic efficacy is available for use in the prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV). Cannabinoid CB 1 receptor agonists possess broad-spectrum antiemetic activity since they prevent vomiting caused by a variety of emetic stimuli including the chemotherapeutic agent cisplatin, 5-HT 3 R agonists, and D 2 R agonists. Our findings demonstrate that application of the L-type Ca 2+ channel (LTCC) agonist FPL 64176 and the intracellular Ca 2+ mobilizing agent thapsigargin (a sarco/endoplasmic reticulum Ca 2+ -ATPase inhibitor) cause vomiting in the least shrew. On the other hand, blockade of LTCCs by corresponding antagonists (nifedipine or amlodipine) not only provide broad-spectrum antiemetic efficacy against diverse agents that specifically activate emetogenic receptors such as 5-HT 3 , NK 1 , D 2 , and M 1 receptors, but can also potentiate the antiemetic efficacy of palonosetron against the non-specific emetogen, cisplatin. In this review, we will provide an overview of Ca 2+ involvement in the emetic process; discuss the relationship between Ca 2+ signaling and the prevailing therapeutics in control of vomiting; highlight the evidence for Ca 2+ -signaling blockers/inhibitors in suppressing emetic behavior in the least shrew model of emesis as well as in the clinical setting; and also draw attention to the clinical benefits of Ca 2+ -signaling blockers/inhibitors in the treatment of nausea and vomiting. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G

PubMed Central

Kailayangiri, Sareetha; Jamitzky, Silke; Schelhaas, Sonja; Jacobs, Andreas H.; Wiek, Constanze; Hanenberg, Helmut; Hartmann, Wolfgang; Wiendl, Heinz; Pankratz, Susann; Meltzer, Jutta; Farwick, Nicole; Greune, Lea; Fluegge, Maike; Rossig, Claudia

2017-01-01

ABSTRACT Activated and in vitro expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their in vivo efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen GD2 in activated NK cells increased their responses to GD2+ allogeneic EwS cells in vitro and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of GD2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate GD2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, in vitro co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells in vitro. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers. PMID:28197367

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells.

PubMed

Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

2012-07-01

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25-30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10(-6) M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

PubMed Central

Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

2012-01-01

Abstract Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10−6M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. PMID:22040127

Activation of natural killer cells by hepatitis C virus particles in vitro.

PubMed

Farag, M M S; Weigand, K; Encke, J; Momburg, F

2011-09-01

Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

Neurotrophins and tonsillar hypertrophy in children with obstructive sleep apnea.

PubMed

Goldbart, Aviv D; Mager, Edward; Veling, Maria C; Goldman, Julie L; Kheirandish-Gozal, Leila; Serpero, Laura D; Piedimonte, Giovanni; Gozal, David

2007-10-01

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p<0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p<0.01), and substance P concentrations in OSA patients were higher than in RI (p<0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA.

Neurotrophins and Tonsillar Hypertrophy in Children With Obstructive Sleep Apnea

PubMed Central

GOLDBART, AVIV D.; MAGER, EDWARD; VELING, MARIA C.; GOLDMAN, JULIE L.; KHEIRANDISH-GOZAL, LEILA; SERPERO, LAURA D.; PIEDIMONTE, GIOVANNI; GOZAL, DAVID

2013-01-01

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p < 0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p < 0.01), and substance P concentrations in OSA patients were higher than in RI (p < 0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA. PMID:17667845

Ubiquitylation of an internalized NK cell receptor by Triad3A disrupts sustained NF-κB signaling1

PubMed Central

Shahjahan Miah, S. M.; Purdy, Amanda K.; Rodin, Nicholas B.; MacFarlane, Alexander W.; Oshinsky, Jennifer; Alvarez-Arias, Diana A.; Campbell, Kerry S.

2011-01-01

KIR2DL4 (2DL4, CD158d) is a unique killer cell Ig-like receptor (KIR) expressed on human NK cells, which stimulates cytokine production, but mechanisms regulating its expression and function are poorly understood. By yeast two-hybrid screening, we identified the E3 ubiquitin ligase, Triad3A, as an interaction partner for the 2DL4 cytoplasmic domain. The protein interaction was confirmed in vivo, and Triad3A expression induced polyubiquitylation and degradation of 2DL4. Overexpression of Triad3A selectively abrogated cytokine-producing function of 2DL4, while Triad3A shRNA reversed ubiquitylation and restored cytokine production. Expression of Triad3A in an NK cell line did not affect receptor surface expression, internalization, or early signaling, but significantly reduced receptor turnover and suppressed sustained NF-κB activation. 2DL4 endocytosis was found to be vital to stimulate cytokine production, and Triad3A expression diminished localization of internalized receptor in early endosomes. Our results reveal a critical role for endocytosed 2DL4 receptor to generate sustained NF-κB signaling and drive cytokine production. We conclude that Triad3A is a key negative regulator of sustained 2DL4-mediated NF-κB signaling from internalized 2DL4, which functions by promoting ubiquitylation and degradation of endocytosed receptor from early endosomes. “This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.” PMID:21270397

Involvement of enzymatic degradation in the inactivation of tachykinin neurotransmitters in neonatal rat spinal cord.

PubMed Central

Suzuki, H; Yoshioka, K; Yanagisawa, M; Urayama, O; Kurihara, T; Hosoki, R; Saito, K; Otsuka, M

1994-01-01

1. The possible involvement of enzymatic degradation in the inactivation of tachykinin neurotransmitters was examined in the spinal cord of the neonatal rat. 2. The magnitude of substance P (SP)- or neurokinin A (NKA)-evoked depolarization of a lumbar ventral root in the isolated spinal cord preparation was increased by a mixture of peptidase inhibitors, consisting of actinonin (6 microM), arphamenine B (6 microM), bestatin (10 microM), captopril (10 microM) and thiorphan (0.3 microM). The mixture augmented the response to NKA more markedly than that to SP. 3. In the isolated spinal cord-cutaneous nerve preparation, the saphenous nerve-evoked slow depolarization of the L3 ventral root was augmented by the mixture of peptidase inhibitors in the presence of naloxone (0.5 microM) but not in the presence of both naloxone and a tachykinin receptor antagonist, GR71251 (5 microM). 4. Application of capsaicin (0.5 microM) for 6 min to the spinal cord evoked an increase in the release of SP from the spinal cord. The amount of SP released was significantly augmented by the mixture of peptidase inhibitors. 5. Synaptic membrane fractions were prepared from neonatal rat spinal cords. These fractions showed degrading activities for SP and NKA and the activities were inhibited by the mixture of peptidase inhibitors. The degrading activity for NKA was higher than that for SP and the inhibitory effect of the mixture for NKA was more marked than that for SP. Although some other fractions obtained from homogenates of spinal cords showed higher degrading activities for SP, these activities were insensitive to the mixture of peptidase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7529113