Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor

PubMed Central

Liedtke, Wolfgang; Choe, Yong; Martí-Renom, Marc A.; Bell, Andrea M.; Denis, Charlotte S.; Šali, Andrej; Hudspeth, A. J.; Friedman, Jeffrey M.; Heller, Stefan

2008-01-01

SUMMARY The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nevous system, the channel is expressed neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells. PMID:11081638

An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors

PubMed Central

Huang, Susan M.; Bisogno, Tiziana; Trevisani, Marcello; Al-Hayani, Abdulmonem; De Petrocellis, Luciano; Fezza, Filomena; Tognetto, Michele; Petros, Timothy J.; Krey, Jocelyn F.; Chu, Constance J.; Miller, Jeffrey D.; Davies, Stephen N.; Geppetti, Pierangelo; Walker, J. Michael; Di Marzo, Vincenzo

2002-01-01

The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and the pungent ingredient of “hot” chili peppers, capsaicin. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. Here we examined the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous “capsaicin-like” substance in mammalian nervous tissues. We found that NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. We also gained evidence for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency (EC50 ≈ 50 nM) and efficacy similar to those of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia (EC50 = 1.5 ± 0.3 μg). Our data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors. PMID:12060783

Opioid receptor subtypes: fact or artifact?

PubMed

Dietis, N; Rowbotham, D J; Lambert, D G

2011-07-01

There is a vast amount of pharmacological evidence favouring the existence of multiple subtypes of opioid receptors. In addition to the primary classification of µ (mu: MOP), δ (delta: DOP), κ (kappa: KOP) receptors, and the nociceptin/orphanin FQ peptide receptor (NOP), various groups have further classified the pharmacological µ into µ(1-3), the δ into δ(1-2)/δ(complexed/non-complexed), and the κ into κ(1-3). From an anaesthetic perspective, the suggestions that µ(1) produced analgesia and µ(2) produced respiratory depression are particularly important. However, subsequent to the formal identification of the primary opioid receptors (MOP/DOP/KOP/NOP) by cloning and the use of this information to produce knockout animals, evidence for these additional subtypes is lacking. Indeed, knockout of a single gene (and hence receptor) results in a loss of all function associated with that receptor. In the case of MOP knockout, analgesia and respiratory depression is lost. This suggests that further sub-classification of the primary types is unwise. So how can the wealth of pharmacological data be reconciled with new molecular information? In addition to some simple misclassification (κ(3) is probably NOP), there are several possibilities which include: (i) alternate splicing of a common gene product, (ii) receptor dimerization, (iii) interaction of a common gene product with other receptors/signalling molecules, or (iv) a combination of (i)-(iii). Assigning variations in ligand activity (pharmacological subtypes) to one or more of these molecular suggestions represents an interesting challenge for future opioid research.

Sensitization of transient receptor potential vanilloid 1 by the prokineticin receptor agonist Bv8.

PubMed

Vellani, Vittorio; Colucci, Mariantonella; Lattanzi, Roberta; Giannini, Elisa; Negri, Lucia; Melchiorri, Pietro; McNaughton, Peter A

2006-05-10

Small mammalian proteins called the prokineticins [prokineticin 1 (PK1) and PK2] and two corresponding G-protein-coupled receptors [prokineticin receptor 1 (PKR1) and PKR2] have been identified recently, but the physiological role of the PK/PKR system remains mostly unexplored. Bv8, a protein extracted from frog skin, is a convenient and potent agonist for both PKR1 and PKR2, and injection of Bv8 in vivo causes a potent and long-lasting hyperalgesia. Here, we investigate the cellular basis of hyperalgesia caused by activation of PKRs. Bv8 caused increases in [Ca]i in a population of isolated dorsal root ganglion (DRG) neurons, which we identified as nociceptors, or sensors for painful stimuli, from their responses to capsaicin, bradykinin, mustard oil, or proteases. Bv8 enhanced the inward current carried by the heat and capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) via a pathway involving activation of protein kinase Cepsilon (PKCepsilon), because Bv8 caused translocation of PKCepsilon to the neuronal membrane and because PKC antagonists reduced both the enhancement of current carried by TRPV1 and behavioral hyperalgesia in rodents. The neuronal population expressing PKRs consisted partly of small peptidergic neurons and partly of neurons expressing the N52 marker for myelinated fibers. Using single-cell reverse transcriptase-PCR, we found that mRNA for PKR1 was mainly expressed in small DRG neurons. Exposure to GDNF (glial cell line-derived neurotrophic factor) induced de novo expression of functional receptors for Bv8 in a nonpeptidergic population of neurons. These results show that prokineticin receptors are expressed in nociceptors and cause heat hyperalgesia by sensitizing TRPV1 through activation of PKCepsilon. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.

Sensing of blood pressure increase by transient receptor potential vanilloid 1 receptors on baroreceptors.

PubMed

Sun, Hao; Li, De-Pei; Chen, Shao-Rui; Hittelman, Walter N; Pan, Hui-Lin

2009-12-01

The arterial baroreceptor is critically involved in the autonomic regulation of homoeostasis. The transient receptor potential vanilloid 1 (TRPV1) receptor is expressed on both somatic and visceral sensory neurons. Here, we examined the TRPV1 innervation of baroreceptive pathways and its functional significance in the baroreflex. Resiniferatoxin (RTX), an ultrapotent analog of capsaicin, was used to ablate TRPV1-expressing afferent neurons and fibers in adult rats. Immunofluorescence labeling revealed that TRPV1 immunoreactivity was present on nerve fibers and terminals in the adventitia of the ascending aorta and aortic arch, the nodose ganglion neurons, and afferent fibers in the solitary tract of the brainstem. RTX treatment eliminated TRPV1 immunoreactivities in the aorta, nodose ganglion, and solitary tract. Renal sympathetic nerve activity, blood pressure, and heart rate were recorded in anesthetized rats. The baroreflex was triggered by lowering and raising blood pressure through intravenous infusion of sodium nitroprusside and phenylephrine, respectively. Inhibition of sympathetic nerve activity and heart rate by the phenylephrine-induced increase in blood pressure was largely impaired in RTX-treated rats. The maximum gain of the baroreflex function was significantly lower in RTX-treated than vehicle-treated rats. Furthermore, blocking of TRPV1 receptors significantly blunted the baroreflex and decreased the maximum gain of baroreflex function in the high blood pressure range. Our findings provide important new information that TRPV1 is expressed along the entire baroreceptive afferent pathway. TRPV1 receptors expressed on baroreceptive nerve endings can function as mechanoreceptors to detect the increase in blood pressure and maintain the homoeostasis.

Sensing of Blood Pressure Increase by Transient Receptor Potential Vanilloid 1 Receptors on Baroreceptors

PubMed Central

Sun, Hao; Li, De-Pei; Chen, Shao-Rui; Hittelman, Walter N.

2009-01-01

The arterial baroreceptor is critically involved in the autonomic regulation of homoeostasis. The transient receptor potential vanilloid 1 (TRPV1) receptor is expressed on both somatic and visceral sensory neurons. Here, we examined the TRPV1 innervation of baroreceptive pathways and its functional significance in the baroreflex. Resiniferatoxin (RTX), an ultrapotent analog of capsaicin, was used to ablate TRPV1-expressing afferent neurons and fibers in adult rats. Immunofluorescence labeling revealed that TRPV1 immunoreactivity was present on nerve fibers and terminals in the adventitia of the ascending aorta and aortic arch, the nodose ganglion neurons, and afferent fibers in the solitary tract of the brainstem. RTX treatment eliminated TRPV1 immunoreactivities in the aorta, nodose ganglion, and solitary tract. Renal sympathetic nerve activity, blood pressure, and heart rate were recorded in anesthetized rats. The baroreflex was triggered by lowering and raising blood pressure through intravenous infusion of sodium nitroprusside and phenylephrine, respectively. Inhibition of sympathetic nerve activity and heart rate by the phenylephrine-induced increase in blood pressure was largely impaired in RTX-treated rats. The maximum gain of the baroreflex function was significantly lower in RTX-treated than vehicle-treated rats. Furthermore, blocking of TRPV1 receptors significantly blunted the baroreflex and decreased the maximum gain of baroreflex function in the high blood pressure range. Our findings provide important new information that TRPV1 is expressed along the entire baroreceptive afferent pathway. TRPV1 receptors expressed on baroreceptive nerve endings can function as mechanoreceptors to detect the increase in blood pressure and maintain the homoeostasis. PMID:19726694

Identifying the receptor subtype selectivity of retinoid X and retinoic acid receptors via quantum mechanics.

PubMed

Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

2017-03-01

Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

Capsaicin and N-arachidonoyl-dopamine (NADA) decrease tension by activating both cannabinoid and vanilloid receptors in fast skeletal muscle fibers of the frog.

PubMed

Trujillo, Xóchitl; Ortiz-Mesina, Mónica; Uribe, Tannia; Castro, Elena; Montoya-Pérez, Rocío; Urzúa, Zorayda; Feria-Velasco, Alfredo; Huerta, Miguel

2015-02-01

Previous studies have indicated that vanilloid receptor (VR1) mRNA is expressed in muscle fibers. In this study, we evaluated the functional effects of VR1 activation. We measured caffeine-induced contractions in bundles of the extensor digitorum longus muscle of Rana pipiens. Isometric tension measurements showed that two VR1 agonists, capsaicin (CAP) and N-arachidonoyl-dopamine (NADA), reduced muscle peak tension to 57 ± 4 % and 71 ± 3% of control, respectively. The effect of CAP was partially blocked by a VR1 blocker, capsazepine (CPZ), but the effect of NADA was not changed by CPZ. Because NADA is able to act on cannabinoid receptors, which are also present in muscle fibers, we tested the cannabinoid antagonist AM281. We found that AM281 antagonized both CAP and NADA effects. AM281 alone reduced peak tension to 80 ± 6 % of control. With both antagonists, the CAP effect was completely blocked, and the NADA effect was partially blocked. These results provide pharmacological evidence of the functional presence of the VR1 receptor in fast skeletal muscle fibers of the frog and suggest that capsaicin and NADA reduce tension by activating both cannabinoid and vanilloid receptors.

Single-residue molecular switch for high-temperature dependence of vanilloid receptor TRPV3

PubMed Central

Liu, Beiying; Qin, Feng

2017-01-01

Thermal transient receptor potential (TRP) channels, a group of ion channels from the transient receptor potential family, play important functions in pain and thermal sensation. These channels are directly activated by temperature and possess strong temperature dependence. Furthermore, their temperature sensitivity can be highly dynamic and use-dependent. For example, the vanilloid receptor transient receptor potential 3 (TRPV3), which has been implicated as a warmth detector, becomes responsive to warm temperatures only after intensive stimulation. Upon initial activation, the channel exhibits a high-temperature threshold in the noxious temperature range above 50 °C. This use dependence of heat sensitivity thus provides a mechanism for sensitization of thermal channels. However, how the channels acquire the use dependence remains unknown. Here, by comparative studies of chimeric channels between use-dependent and use-independent homologs, we have determined the molecular basis that underlies the use dependence of temperature sensitivity of TRPV3. Remarkably, the restoration of a single residue that is apparently missing in the use-dependent homologs could largely eliminate the use dependence of heat sensitivity of TRPV3. The location of the region suggests a mechanism of temperature-dependent gating of thermal TRP channels involving an intracellular region assembled around the TRP domain. PMID:28154143

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

PubMed

Bianchi, B R; Lynch, K J; Touma, E; Niforatos, W; Burgard, E C; Alexander, K M; Park, H S; Yu, H; Metzger, R; Kowaluk, E; Jarvis, M F; van Biesen, T

1999-07-02

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Dopamine modulation of transient receptor potential vanilloid type 1 (TRPV1) receptor in dorsal root ganglia neurons.

PubMed

Chakraborty, Saikat; Rebecchi, Mario; Kaczocha, Martin; Puopolo, Michelino

2016-03-15

The transient receptor potential vanilloid type 1 (TRPV1) receptor plays a key role in the modulation of nociceptor excitability. To address whether dopamine can modulate the activity of TRPV1 channels in nociceptive neurons, the effects of dopamine and dopamine receptor agonists were tested on the capsaicin-activated current recorded from acutely dissociated small diameter (<27 μm) dorsal root ganglia (DRG) neurons. Dopamine or SKF 81297 (an agonist at D1/D5 receptors), caused inhibition of both inward and outward currents by ∼60% and ∼48%, respectively. The effect of SKF 81297 was reversed by SCH 23390 (an antagonist at D1/D5 receptors), confirming that it was mediated by activation of D1/D5 dopamine receptors. In contrast, quinpirole (an agonist at D2 receptors) had no significant effect on the capsaicin-activated current. Inhibition of the capsaicin-activated current by SKF 81297 was mediated by G protein coupled receptors (GPCRs), and highly dependent on external calcium. The inhibitory effect of SKF 81297 on the capsaicin-activated current was not affected when the protein kinase A (PKA) activity was blocked with H89, or when the protein kinase C (PKC) activity was blocked with bisindolylmaleimide II (BIM). In contrast, when the calcium-calmodulin-dependent protein kinase II (CaMKII) was blocked with KN-93, the inhibitory effect of SKF 81297 on the capsaicin-activated current was greatly reduced, suggesting that activation of D1/D5 dopamine receptors may be preferentially linked to CaMKII activity. We suggest that modulation of TRPV1 channels by dopamine in nociceptive neurons may represent a way for dopamine to modulate incoming noxious stimuli. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

The sympathetic nervous system is controlled by transient receptor potential vanilloid 1 in the regulation of body temperature

PubMed Central

Alawi, Khadija M.; Aubdool, Aisah A.; Liang, Lihuan; Wilde, Elena; Vepa, Abhinav; Psefteli, Maria-Paraskevi; Brain, Susan D.; Keeble, Julie E.

2015-01-01

Transient receptor potential vanilloid 1 (TRPV1) is involved in sensory nerve nociceptive signaling. Recently, it has been discovered that TRPV1 receptors also regulate basal body temperature in multiple species from mice to humans. In the present study, we investigated whether TRPV1 modulates basal sympathetic nervous system (SNS) activity. C57BL6/J wild-type (WT) mice and TRPV1 knockout (KO) mice were implanted with radiotelemetry probes for measurement of core body temperature. AMG9810 (50 mg/kg) or vehicle (2% DMSO/5% Tween 80/10 ml/kg saline) was injected intraperitoneally. Adrenoceptor antagonists or vehicle (5 ml/kg saline) was injected subcutaneously. In WT mice, the TRPV1 antagonist, AMG9810, caused significant hyperthermia, associated with increased noradrenaline concentrations in brown adipose tissue. The hyperthermia was significantly attenuated by the β-adrenoceptor antagonist propranolol, the mixed α-/β-adrenoceptor antagonist labetalol, and the α1-adrenoceptor antagonist prazosin. TRPV1 KO mice have a normal basal body temperature, indicative of developmental compensation. d-Amphetamine (potent sympathomimetic) caused hyperthermia in WT mice, which was reduced in TRPV1 KO mice, suggesting a decreased sympathetic drive in KOs. This study provides new evidence that TRPV1 controls thermoregulation upstream of the SNS, providing a potential therapeutic target for sympathetic hyperactivity thermoregulatory disorders.—Alawi, K. M., Aubdool, A. A., Liang, L., Wilde, E., Vepa, A., Psefteli, M.-P., Brain, S. D., Keeble, J. E. The sympathetic nervous system is controlled by transient receptor potential vanilloid 1 in the regulation of body temperature. PMID:26136480

Involvement of Transient Receptor Potential Vanilloid (TRPV) 4 in mouse sperm thermotaxis.

PubMed

Hamano, Koh-Ichi; Kawanishi, Tae; Mizuno, Atsuko; Suzuki, Makoto; Takagi, Yuji

2016-08-25

Transient Receptor Potential Vanilloid (TRPV) 4 is one of the temperature-sensitive ion channels involved in temperature receptors, and it is known to be activated from 35 to 40ºC. Here we analyzed sperm motility function of Trpv4 knockout (KO) mouse in temperature-gradient conditions to elucidate the thermotaxis of mouse sperm and the involvement of TRPV4 in thermotaxis. The sperm were introduced at the vertical column end of a T-shaped chamber filled with medium in a plastic dish, and we measured the number of sperm that arrived at both ends of the wide column where we had established a temperature gradient of approx. 2ºC, and we evaluated the sperm's thermotaxis. Large numbers of wild-type (WT) mouse sperm migrated into the high level of the temperature gradient that was set in the wide column, and thermotaxis was confirmed. The ratio of migrated sperm at the high temperature level of the T-shaped chamber was decreased in the KO sperm and Ruthenium red (a TRPV antagonist) treated sperm compared with the WT sperm. The thermotaxis of the mouse sperm was confirmed, and the involvement of TRPV4 in this thermotaxis was suggested.

Lorcaserin and pimavanserin: emerging selectivity of serotonin receptor subtype-targeted drugs.

PubMed

Meltzer, Herbert Y; Roth, Bryan L

2013-12-01

Serotonin (5-hydroxytryptamine, or 5-HT) receptors mediate a plethora of physiological phenomena in the brain and the periphery. Additionally, serotonergic dysfunction has been implicated in nearly every neuropsychiatric disorder. The effects of serotonin are mediated by fourteen GPCRs. Both the therapeutic actions and side effects of commonly prescribed drugs are frequently due to nonspecific actions on various 5-HT receptor subtypes. For more than 20 years, the search for clinically efficacious drugs that selectively target 5-HT receptor subtypes has been only occasionally successful. This review provides an overview of 5-HT receptor pharmacology and discusses two recent 5-HT receptor subtype-selective drugs, lorcaserin and pimavanserin, which target the 5HT2C and 5HT2A receptors and provide new treatments for obesity and Parkinson's disease psychosis, respectively.

Use Dependence of Heat Sensitivity of Vanilloid Receptor TRPV2

PubMed Central

Liu, Beiying; Qin, Feng

2016-01-01

Thermal TRP channels mediate temperature transduction and pain sensation. The vanilloid receptor TRPV2 is involved in detection of noxious heat in a subpopulation of high-threshold nociceptors. It also plays a critical role in development of thermal hyperalgesia, but the underlying mechanism remains uncertain. Here we analyze the heat sensitivity of the TRPV2 channel. Heat activation of the channel exhibits strong use dependence. Prior heat activation can profoundly alter its subsequent temperature responsiveness, causing decreases in both temperature activation threshold and slope sensitivity of temperature dependence while accelerating activation time courses. Notably, heat and agonist activations differ in cross use-dependence. Prior heat stimulation can dramatically sensitize agonist responses, but not conversely. Quantitative analyses indicate that the use dependence in heat sensitivity is pertinent to the process of temperature sensing by the channel. The use dependence of TRPV2 reveals that the channel can have a dynamic temperature sensitivity. The temperature sensing structures within the channel have multiple conformations and the temperature activation pathway is separate from the agonist activation pathway. Physiologically, the use dependence of TRPV2 confers nociceptors with a hypersensitivity to heat and thus provides a mechanism for peripheral thermal hyperalgesia. PMID:27074678

Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

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

Baumgold, J.

1986-05-01

Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and themore » remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.« less

Use Dependence of Heat Sensitivity of Vanilloid Receptor TRPV2.

PubMed

Liu, Beiying; Qin, Feng

2016-04-12

Thermal TRP channels mediate temperature transduction and pain sensation. The vanilloid receptor TRPV2 is involved in detection of noxious heat in a subpopulation of high-threshold nociceptors. It also plays a critical role in development of thermal hyperalgesia, but the underlying mechanism remains uncertain. Here we analyze the heat sensitivity of the TRPV2 channel. Heat activation of the channel exhibits strong use dependence. Prior heat activation can profoundly alter its subsequent temperature responsiveness, causing decreases in both temperature activation threshold and slope sensitivity of temperature dependence while accelerating activation time courses. Notably, heat and agonist activations differ in cross use-dependence. Prior heat stimulation can dramatically sensitize agonist responses, but not conversely. Quantitative analyses indicate that the use dependence in heat sensitivity is pertinent to the process of temperature sensing by the channel. The use dependence of TRPV2 reveals that the channel can have a dynamic temperature sensitivity. The temperature sensing structures within the channel have multiple conformations and the temperature activation pathway is separate from the agonist activation pathway. Physiologically, the use dependence of TRPV2 confers nociceptors with a hypersensitivity to heat and thus provides a mechanism for peripheral thermal hyperalgesia. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

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

Baumgold, J.; Cohen, V.I.; Paek, R.

1991-01-01

In an effort at synthesizing centrally-active subtype-selective antimuscarinic agents, the authors derivatized QNB (quinuclidinyl benzilate), a potent muscarinic antagonist, by replacing one of the phenyl groups with less lipophilic heterocyclic moieties. The displacement of ({sup 3}H)-N-methyl scopolamine binding by these novel compounds to membranes from cells expressing ml - m4 receptor subtypes was determined. Most of the novel 4-bromo-QNB analogues were potent and slightly selective for ml receptors. The 2-thienyl derivative was the most potent, exhibiting a 2-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potencymore » at m2 receptors. This compound was also considerably less lipophilic than BrQNB as determined from its retention time on C18 reverse phase HPLC. This compound may therefore be useful both for pharmacological studies and as a candidate for a radioiodinated SPECT imaging agent for ml muscarinic receptors in human brain.« less

Selective targeting of G-protein-coupled receptor subtypes with venom peptides.

PubMed

Näreoja, K; Näsman, J

2012-02-01

The G-protein-coupled receptor (GPCR) family is one of the largest gene superfamilies with approx. 370 members responding to endogenous ligands in humans and a roughly equal amount of receptors sensitive to external stimuli from the surrounding. A number of receptors from this superfamily are well recognized targets for medical treatment of various disease conditions, whereas for many others the potential medical benefit of interference is still obscure. A general problem associated with GPCR research and therapeutics is the insufficient specificity of available ligands to differentiate between closely homologous receptor subtypes. In this context, venom peptides could make a significant contribution to the development of more specific drugs. Venoms from certain animals specialized in biochemical hunting contain a mixture of molecules that are directed towards a variety of membrane proteins. Peptide toxins isolated from these mixtures usually exhibit high specificity for their targets. Muscarinic toxins found from mamba snakes attracted much attention during the 1990s. These are 65-66 amino acid long peptides with a structural three-finger folding similar to the α-neurotoxins and they target the muscarinic acetylcholine receptors in a subtype-selective manner. Recently, several members of the three-finger toxins from mamba snakes as well as conotoxins from marine cone snails have been shown to selectively interact with subtypes of adrenergic receptors. In this review, we will discuss the GPCR-directed peptide toxins found from different venoms and how some of these can be useful in exploring specific roles of receptor subtypes. © 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.

Pharmacological and molecular characterization of muscarinic receptor subtypes in human esophageal smooth muscle.

PubMed

Preiksaitis, H G; Krysiak, P S; Chrones, T; Rajgopal, V; Laurier, L G

2000-12-01

Esophageal peristalsis is dependent on activation of muscarinic receptors, but little is known about the roles of specific receptor subtypes in the human esophagus. We examined muscarinic receptor expression and function in human esophageal smooth muscle obtained from patients undergoing resection for cancer. [(3)H]Quinuclidinyl benzylate (QNB)-specific binding was similar in longitudinal muscle (B(max) = 106 +/- 22 fmol/mg of protein, K(d) = 68 +/- 9 pM) and circular muscle (B(max) = 81 +/- 16 fmol/mg of protein, K(d) = 79 +/- 15 pM). Subtype-selective antagonists inhibited [(3)H]QNB similarly in muscle from both layers. Further analysis of antagonist inhibition of [(3)H]QNB binding showed a major site (60-70%) with antagonist affinity profile consistent with the M2 subtype and a second site that could not be classified. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated the presence of all five known muscarinic receptor subtypes, and immunocytochemistry on acutely isolated smooth muscle cells confirmed the expression of each subtype on the muscle cells. Subtype-selective antagonists had similar inhibitory effects on carbachol-evoked contractions in longitudinal muscle and circular muscle strips with pA(2) values of 9.5 +/- 0.1 and 9.6 +/- 0.2 for 4-diphenylacetoxy-N-methylpiperidine methiodide, 7.1 +/- 0.1 and 7.0 +/- 0.2 for pirenzepine, and 6.2 +/- 0.2 and 6.4 +/- 0.2 for methoctramine, respectively. We conclude that human esophageal smooth muscle expresses muscarinic receptor subtypes M1 through M5. The antagonist sensitivity profile for muscle contraction is consistent with activation of the M3 subtype.

Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol

PubMed Central

Blednov, Y.A.; Harris, R.A.

2009-01-01

The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol. PMID:19705551

Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*

PubMed Central

Dalrymple, Matthew B.; Jaeger, Werner C.; Eidne, Karin A.; Pfleger, Kevin D. G.

2011-01-01

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors. PMID:21378163

Temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes.

PubMed

Dalrymple, Matthew B; Jaeger, Werner C; Eidne, Karin A; Pfleger, Kevin D G

2011-05-13

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors.

The adrenergic receptor subtypes present in frog (Rana esculenta) skin.

PubMed

Bellantuono, Vito; Cassano, Giuseppe; Lippe, Claudio

2008-08-01

Frog skin transports ions and water under hormonal control. In spite of the fundamental role played by adrenergic stimulation in maintaining the water balance of the organism, the receptor subtype(s) present in the skin have not been identified yet. We measured the increase in short-circuit current (ISC, an estimate of ion transport) induced by cirazoline, clonidine, xamoterol, formoterol, or BRL 37344, in order to verify the presence of alpha1, alpha2, beta1, beta2, or beta3 receptor subtypes, respectively. Only after treatment with formoterol, BRL 37344 and, to a lesser extent, cirazoline was measured a significant increase in ISC (57%, 33.2%, and 4.7%, respectively). The formoterol and BRL 37344 concentrations producing half-maximal effect (EC50) were 1.12 and 70.1 nM, respectively. Moreover, the formoterol effect was inhibited by treatment with ICI 118551 (antagonist of beta2 receptors) while SR 59230A (antagonist of beta3 receptors) had no effect; opposite findings were obtained when the BRL 37344 stimulation was investigated. Finally, by measuring the transepithelial fluxes of 22Na+ and 36Cl-, we demonstrated that Na+ absorption is increased by activation of beta2 and beta3 and is cAMP-sensitive, whereas the Cl- secretion is only increased by activation of beta2 receptors and is cAMP- and calmodulin-sensitive.

Periaqueductal gray glutamatergic, cannabinoid and vanilloid receptor interplay in defensive behavior and aversive memory formation.

PubMed

Back, Franklin P; Carobrez, Antonio P

2018-06-01

Stimulation of the midbrain periaqueductal gray matter (PAG) in humans elicits sensations of fear and impending terror, and mediates predator defensive responses in rodents. In rats, pharmacological stimulation of the dorsolateral portion of the PAG (dlPAG) with N-Methyl-d-Aspartate (NMDA) induces aversive conditioning that acts as an unconditioned stimulus (US). In the present work, we investigated the interplay between the vanilloid TRPV1 and cannabinoid CB1 receptors in the NMDA-dlPAG defensive response and in subsequent aversive learning. Rats were subjected to dlPAG NMDA infusion in an olfactory conditioned stimulus (CS) task allowing the evaluation of immediate and long-term defensive behavioral responses during CS presentation. The results indicated that an intermediate dose of NMDA (50 pmol) induced both immediate and long-term effects. A sub-effective dose of NMDA (25 pmol) was potentiated by the TRPV1 receptor agonist capsaicin (CAP, 1 nmol) and the CB1 receptor antagonist, AM251 (200 pmol). CAP (10 nmol) or the combination of CAP (1 nmol) and AM251 (200 pmol) induced long-term effects without increasing immediate defensive responses. The glutamate release inhibitor riluzole (2 or 4 nmol) and the AMPA/kainate receptor antagonist DNQX (2 or 4 nmol) potentiated the immediate effects but blocked the long-term effects. The results showed that immediate defensive responses rely on NMDA receptors, and aversive learning on the fine-tuning of TRPV1, CB1, metabotropic glutamate and AMPA receptors located in pre- and postsynaptic membranes. In conclusion, the activity of the dlPAG determines core affective aspects of aversive memory formation controlled by local TRPV1/CB1 balance. Copyright © 2018 Elsevier Ltd. All rights reserved.

Bidirectional modulation of visual plasticity by cholinergic receptor subtypes in the frog optic tectum

PubMed Central

Yu, Chuan-Jiang; Butt, Christopher M.; Debski, Elizabeth A.

2008-01-01

Cholinergic input to the optic tectum is necessary for visual map maintenance. To understand why, we examined the effects of activation of the different cholinergic receptor subtypes in tectal brain slices and determined whether the retinotectal map was affected by manipulations of their activity in vivo. Both α-bungarotoxin sensitive and insensitive nicotinic receptor agonists increased spontaneous postsynaptic currents (sPSCs) in a subpopulation of patch-clamped tectal cells; application of subtype selective receptor antagonists reduced nicotine-induced increases in sPSCs. Activation of α-bungarotoxin insensitive nicotinic receptors also induced substantial inward current in some cells. Muscarinic receptor mediated outward current responses were blocked by the M2-like muscarinic receptor antagonists himbacine or AF-DX 384 and mimicked by application of the M2-like agonist oxotremorine. A less frequently observed muscarinic response involving a change in sPSC frequency appeared to be mediated by M1-like muscarinic receptors. In separate experiments, pharmacological manipulation of cholinergic receptor subtype activation led to changes in the activity-dependent visual map created in the tectum by retinal ganglion cell terminals. Chronic exposure of the tectum to either α-bungarotoxin insensitive, α-bungarotoxin sensitive or M1-like receptor antagonists resulted in map disruption. However, treatment with the M2-like receptor antagonist, AF-DX 384, compressed the map. We conclude that nicotinic or M1-like muscarinic receptors control input to tectal cells while α-bungarotoxin insensitive nicotinic receptors and M2-like muscarinic receptors change tectal cell responses to that input. Blockade of the different cholinergic receptor subtypes can have opposing effects on map topography that are consistent with expected effects on tectal cell activity levels. PMID:12670313

Ontogenetic expression of the vanilloid receptors TRPV1 and TRPV2 in the rat retina.

PubMed

Leonelli, Mauro; Martins, Daniel O; Kihara, Alexandre H; Britto, Luiz R G

2009-11-01

The present study aimed to analyze the gene and protein expression and the pattern of distribution of the vanilloid receptors TRPV1 and TRPV2 in the developing rat retina. During the early phases of development, TRPV1 was found mainly in the neuroblastic layer of the retina and in the pigmented epithelium. In the adult, TRPV1 was found in microglial cells, blood vessels, astrocytes and in neuronal structures, namely synaptic boutons of both retinal plexiform layers, as well as in cell bodies of the inner nuclear layer and the ganglion cell layer. The pattern of distribution of TRPV1 was mainly punctate, and there was higher TRPV1 labeling in the peripheral retina than in central regions. TRPV2 expression was quite distinct. Its expression was virtually undetectable by immunoblotting before P1, and that receptor was found by immunohistochemistry only by postnatal day 15 (P15). RNA and protein analysis showed that the adult levels are only reached by P60, which includes small processes in the retinal plexiform layers, and labeled cellular bodies in the inner nuclear layer and the ganglion cell layer. There was no overlapping between the signal observed for both receptors. In conclusion, our results showed that the patterns of distribution of TRPV1 and TRPV2 are different during the development of the rat retina, suggesting that they have specific roles in both visual processing and in providing specific cues to neural development.

Dense transient receptor potential cation channel, vanilloid family, type 2 (TRPV2) immunoreactivity defines a subset of motoneurons in the dorsal lateral nucleus of the spinal cord, the nucleus ambiguus and the trigeminal motor nucleus in rat.

PubMed

Lewinter, R D; Scherrer, G; Basbaum, A I

2008-01-02

The transient receptor potential cation channel, vanilloid family, type 2 (TRPV2) is a member of the TRPV family of proteins and is a homologue of the capsaicin/vanilloid receptor (transient receptor potential cation channel, vanilloid family, type 1, TRPV1). Like TRPV1, TRPV2 is expressed in a subset of dorsal root ganglia (DRG) neurons that project to superficial laminae of the spinal cord dorsal horn. Because noxious heat (>52 degrees C) activates TRPV2 in transfected cells this channel has been implicated in the processing of high intensity thermal pain messages in vivo. In contrast to TRPV1, however, which is restricted to small diameter DRG neurons, there is significant TRPV2 immunoreactivity in a variety of CNS regions. The present report focuses on a subset of neurons in the brainstem and spinal cord of the rat including the dorsal lateral nucleus (DLN) of the spinal cord, the nucleus ambiguus, and the motor trigeminal nucleus. Double label immunocytochemistry with markers of motoneurons, combined with retrograde labeling, established that these cells are, in fact, motoneurons. With the exception of their smaller diameter, these cells did not differ from other motoneurons, which are only lightly TRPV2-immunoreactive. As for the majority of DLN neurons, the densely-labeled populations co-express androgen receptor and follow normal DLN ontogeny. The functional significance of the very intense TRPV2 expression in these three distinct spinal cord and brainstem motoneurons groups remains to be determined.

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

PubMed

Seguin, L; Widdowson, P S; Giesen-Crouse, E

1992-12-01

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of transient receptor potential vanilloid 1 activation and sensitization by allyl isothiocyanate.

PubMed

Gees, Maarten; Alpizar, Yeranddy A; Boonen, Brett; Sanchez, Alicia; Everaerts, Wouter; Segal, Andrei; Xue, Fenqin; Janssens, Annelies; Owsianik, Grzegorz; Nilius, Bernd; Voets, Thomas; Talavera, Karel

2013-09-01

Allyl isothiocyanate (AITC; aka, mustard oil) is a powerful irritant produced by Brassica plants as a defensive trait against herbivores and confers pungency to mustard and wasabi. AITC is widely used experimentally as an inducer of acute pain and neurogenic inflammation, which are largely mediated by the activation of nociceptive cation channels transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 (TRPV1). Although it is generally accepted that electrophilic agents activate these channels through covalent modification of cytosolic cysteine residues, the mechanism underlying TRPV1 activation by AITC remains unknown. Here we show that, surprisingly, AITC-induced activation of TRPV1 does not require interaction with cysteine residues, but is largely dependent on S513, a residue that is involved in capsaicin binding. Furthermore, AITC acts in a membrane-delimited manner and induces a shift of the voltage dependence of activation toward negative voltages, which is reminiscent of capsaicin effects. These data indicate that AITC acts through reversible interactions with the capsaicin binding site. In addition, we show that TRPV1 is a locus for cross-sensitization between AITC and acidosis in nociceptive neurons. Furthermore, we show that residue F660, which is known to determine the stimulation by low pH in human TRPV1, is also essential for the cross-sensitization of the effects of AITC and low pH. Taken together, these findings demonstrate that not all reactive electrophiles stimulate TRPV1 via cysteine modification and help understanding the molecular bases underlying the surprisingly large role of this channel as mediator of the algesic properties of AITC.

Cockroach GABAB receptor subtypes: molecular characterization, pharmacological properties and tissue distribution.

PubMed

Blankenburg, S; Balfanz, S; Hayashi, Y; Shigenobu, S; Miura, T; Baumann, O; Baumann, A; Blenau, W

2015-01-01

γ-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system (CNS). Its effects are mediated by either ionotropic GABAA receptors or metabotropic GABAB receptors. GABAB receptors regulate, via Gi/o G-proteins, ion channels, and adenylyl cyclases. In humans, GABAB receptor subtypes are involved in the etiology of neurologic and psychiatric disorders. In arthropods, however, these members of the G-protein-coupled receptor family are only inadequately characterized. Interestingly, physiological data have revealed important functions of GABAB receptors in the American cockroach, Periplaneta americana. We have cloned cDNAs coding for putative GABAB receptor subtypes 1 and 2 of P. americana (PeaGB1 and PeaGB2). When both receptor proteins are co-expressed in mammalian cells, activation of the receptor heteromer with GABA leads to a dose-dependent decrease in cAMP production. The pharmacological profile differs from that of mammalian and Drosophila GABAB receptors. Western blot analyses with polyclonal antibodies have revealed the expression of PeaGB1 and PeaGB2 in the CNS of the American cockroach. In addition to the widespread distribution in the brain, PeaGB1 is expressed in salivary glands and male accessory glands. Notably, PeaGB1-like immunoreactivity has been detected in the GABAergic salivary neuron 2, suggesting that GABAB receptors act as autoreceptors in this neuron. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Transient Receptor Potential Vanilloid 1 Antagonist Capsazepine Improves the Impaired Lung Mechanics during Endotoxemia.

PubMed

Cabral, Layla D M; Giusti-Paiva, Alexandre

2016-11-01

Acute lung injury (ALI) caused by systemic inflammatory response remains a leading cause of morbidity and mortality in critically ill patients. Management of patients with sepsis is largely limited to supportive therapies, reflecting an incomplete understanding of the underlying pathophysiology. Furthermore, there have been limited advances in the treatments for ALI. In this study, lung function and a histological analysis were performed to evaluate the impact of transient receptor potential vanilloid-1 receptor (TRPV1) antagonist (capsazepine; CPZ) on the lipopolysaccharide (LPS)-induced lung injury in mice. For this, adult mice pre-treated with CPZ or vehicle received intraperitoneal injections of LPS or saline and 24 hr after, the mice were anaesthetized, and lung mechanics was evaluated. The LPS-challenged mice exhibited substantial mechanical impairment, characterized by increases in respiratory system resistance, respiratory system elastance, tissue damping and tissue elastance. The pre-treatment with CPZ prevented the increase in respiratory system resistance and decreased the increase in tissue damping during endotoxemia. In addition, mice pre-treated with CPZ had an attenuated lung injury evidenced by reduction on collapsed area of the lung parenchyma induced by LPS. This suggests that the TRPV1 antagonist capsazepine has a protective effect on lung mechanics in ALI during endotoxemia and that it may be a target for enhanced therapeutic efficacy in ALI. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Effects of piperine, the pungent component of black pepper, at the human vanilloid receptor (TRPV1)

PubMed Central

McNamara, Fergal N; Randall, Andrew; Gunthorpe, Martin J

2005-01-01

We have characterised the effects of piperine, a pungent alkaloid found in black pepper, on the human vanilloid receptor TRPV1 using whole-cell patch-clamp electrophysiology. Piperine produced a clear agonist activity at the human TRPV1 receptor yielding rapidly activating whole-cell currents that were antagonised by the competitive TRPV1 antagonist capsazepine and the non-competitive TRPV1 blocker ruthenium red. The current–voltage relationship of piperine-activated currents showed pronounced outward rectification (25±4-fold between −70 and +70 mV) and a reversal potential of 0.0±0.4 mV, which was indistinguishable from that of the prototypical TRPV1 agonist capsaicin. Although piperine was a less potent agonist (EC50=37.9±1.9 μM) than capsaicin (EC50=0.29±0.05 μM), it demonstrated a much greater efficacy (approximately two-fold) at TRPV1. This difference in efficacy did not appear to be related to the proton-mediated regulation of the receptor since a similar degree of potentiation was observed for responses evoked by piperine (230±20%, n=11) or capsaicin (284±32%, n=8) upon acidification to pH 6.5. The effects of piperine upon receptor desensitisation were also unable to explain this effect since piperine resulted in more pronounced macroscopic desensitisation (t1/2=9.9±0.7 s) than capsaicin (t1/2>20 s) and also caused greater tachyphylaxis in response to repetitive agonist applications. Overall, our data suggest that the effects of piperine at human TRPV1 are similar to those of capsaicin except for its propensity to induce greater receptor desensitisation and, rather remarkably, exhibit a greater efficacy than capsaicin itself. These results may provide insight into the TRPV1-mediated effects of piperine on gastrointestinal function. PMID:15685214

Histamine receptors in human detrusor smooth muscle cells: physiological properties and immunohistochemical representation of subtypes.

PubMed

Neuhaus, Jochen; Weimann, Annett; Stolzenburg, Jens-Uwe; Dawood, Waled; Schwalenberg, Thilo; Dorschner, Wolfgang

2006-06-01

The potent inflammatory mediator histamine is released from activated mast cells in interstitial cystitis (IC). Here, we report on the histamine receptor subtypes involved in the intracellular calcium response of cultured smooth muscle cells (cSMC). Fura-2 was used to monitor the calcium response in cSMC, cultured from human detrusor biopsies. The distribution of histamine receptor subtypes was addressed by immunocytochemistry in situ and in vitro. Histamine stimulated a maximum of 92% of the cells (n=335), being more effective than carbachol (70%, n=920). HTMT (H1R-agonist), dimaprit (H2R) and MTH (H3R) lead to significant lower numbers of reacting cells (60, 48 and 54%). Histamine receptor immunoreactivity (H1R, H2R, H3R, H4R) was found in situ and in vitro. Histamine-induced calcium increase is mediated by distinct histamine receptors. Thus, pre-therapeutic evaluation of histamine receptor expression in IC patients may help to optimize therapy by using a patient-specific cocktail of subtype-specific histamine receptor antagonists.

Nociceptin inhibits vanilloid TRPV-1-mediated neurosensitization induced by fenoterol in human isolated bronchi.

PubMed

Faisy, Christophe; Naline, Emmanuel; Rouget, Céline; Risse, Paul-André; Guerot, Emmanuel; Fagon, Jean-Yves; Chinet, Thierry; Roche, Nicolas; Advenier, Charles

2004-09-01

Chronic exposure to beta(2)-adrenoceptor agonists, especially fenoterol, has been shown to increase smooth muscle contraction to endothelin-1 in human bronchi partly through tachykinin-mediated pathways. The purpose of this work was to further investigate the role of sensory nerves in fenoterol-induced sensitization of human airways and the effect of nociceptin, a nociceptin/orphanin FQ (NOP) receptor agonist, on the increase in contraction after fenoterol exposure. Human bronchi from 62 patients were sensitized to endothelin-1 by prolonged incubation with fenoterol (0.1 microM, 15 h). The sensitizing effect of fenoterol was inhibited by high concentration of capsaicin (10 microM, 30 min before fenoterol sensitization), which induces depletion of mediators from sensory nerves, or co-incubation of fenoterol and capsazepine (1 microM), a vanilloid TRPV-1 receptor antagonist. Moreover, short pretreatment of bronchi with capsaicin (10 microM) or capsazepine (1 microM) after sensitization by fenoterol decreased the rise in smooth muscle contraction to endothelin-1. Nociceptin (1 microM) also inhibited the increased contraction in fenoterol-sensitized bronchi. Tertiapin (10 microM), an inhibitor of the inward-rectifier K(+) channels, but not naloxone (0.1 microM), a DOP/KOP/MOP receptor antagonist, prevented the inhibitory effect of nociceptin. In conclusion, fenoterol induces sensitization of human isolated bronchi to endothelin-1 in part through the stimulation of the vanilloid TRPV-1 receptor on tachykininergic sensory nerves. Nociceptin inhibits airway hyperresponsiveness via NOP receptor activation. This effect involves inward-rectifier K(+) channels.

Phosphoinositide system-linked serotonin receptor subtypes and their pharmacological properties and clinical correlates.

PubMed Central

Pandey, S C; Davis, J M; Pandey, G N

1995-01-01

Serotonergic neurotransmission represents a complex mechanism involving pre- and post-synaptic events and distinct 5-HT receptor subtypes. Serotonin (5-HT) receptors have been classified into several categories, and they are termed as 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 type receptors. 5-HT1 receptors have been further subdivided into 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E and 5-HT1F. 5-HT2 receptors have been divided into 5-HT2A, 5-HT2B and 5-HT2C receptors. All 5-HT2 receptor subtypes are linked to the multifunctional phosphoinositide (PI) signalling system. 5-HT3 receptors are considered ion-gated receptors and are also linked to the PI signalling system by an unknown mechanism. The 5-HT2A receptor subtype is the most widely studied of the 5-HT receptors in psychiatric disorders (for example, suicide, depression and schizophrenia) as well as in relation to the mechanism of action of antidepressant drugs. The roles of 5-HT2C and 5-HT3 receptors in psychiatric disorders are less clear. These 5-HT receptors also play an important role in alcoholism. It has been shown that 5-HT2A, 5-HT2C and 5-HT3 antagonists cause attenuation of alcohol intake in animals and humans. However, the exact mechanisms are unknown. The recent cloning of the cDNAs for 5-HT2A, 5-HT2C and 5-HT3 receptors provides the opportunity to explore the molecular mechanisms responsible for the alterations in these receptors during illness as well as pharmacotherapy. This review article will focus on the current research into the pharmacological properties, molecular biology, and clinical correlates of 5-HT2A, 5-HT2C and 5-HT3 receptors. PMID:7786883

The role of transient receptor potential vanilloid type-2 ion channels in innate and adaptive immune responses

PubMed Central

Santoni, Giorgio; Farfariello, Valerio; Liberati, Sonia; Morelli, Maria B.; Nabissi, Massimo; Santoni, Matteo; Amantini, Consuelo

2013-01-01

The transient receptor potential vanilloid type-2 (TRPV2), belonging to the transient receptor potential channel family, is a specialized ion channel expressed in human and other mammalian immune cells. This channel has been found to be expressed in CD34+ hematopoietic stem cells, where its cytosolic Ca2+ activity is crucial for stem/progenitor cell cycle progression, growth, and differentiation. In innate immune cells, TRPV2 is expressed in granulocytes, macrophages, and monocytes where it stimulates fMet-Leu-Phe migration, zymosan-, immunoglobulin G-, and complement-mediated phagocytosis, and lipopolysaccharide-induced tumor necrosis factor-alpha and interleukin-6 production. In mast cells, activation of TRPV2 allows intracellular Ca2+ ions flux, thus stimulating protein kinase A-dependent degranulation. In addition, TRPV2 is highly expressed in CD56+ natural killer cells. TRPV2 orchestrates Ca2+ signal in T cell activation, proliferation, and effector functions. Moreover, messenger RNA for TRPV2 are expressed in CD4+ and CD8+ T lymphocytes. Finally, TRPV2 is expressed in CD19+ B lymphocytes where it regulates Ca2+ release during B cell development and activation. Overall, the specific expression of TRPV2 in immune cells suggests a role in immune-mediated diseases and offers new potential targets for immunomodulation. PMID:23420671

Transient receptor potential vanilloid-type 2 targeting on stemness in liver cancer.

PubMed

Hu, Zecheng; Cao, Xiaocheng; Fang, Yu; Liu, Guoxing; Xie, Chengzhi; Qian, Ke; Lei, Xiaohua; Cao, Zhenyu; Du, Huihui; Cheng, Xiangding; Xu, Xundi

2018-06-12

The malignant phenotype of the cells resulting from human liver cancer is driven by liver cancer stem-like cells (LCSLCs). Transient Receptor Potential Vanilloid-type 2 channel (TRPV2) contributes to the progression of different tumor types, including liver cancer. In the current study, the TRPV2 expression levels give rise to the effect on stemness in liver cancer cell lines. TRPV2 knockdown in HepG2 cells enhanced spheroid and colony formation, and expression levels of CD133, CD44 and ALDH1 whereas the opposite effects were observed in TRPV2 enforced expression in SMMC-7721 cells. Furthermore, TRPV2 overexpression restored inhibition of spheroid and colony formation, and stem cell markers expression in HepG2 cells with TRPV2 silencing. The addition of the TRPV2 agonist probenecid and the TRPV2 antagonist tranilast suppressed and/or increased in vitro spheroid and colony formation, and stem cell marker expression of LCSLCs and/or liver cancer cell lines, respectively. Notably, probenecid and tranilast significantly inhibited or promoted tumor growth of HepG2 xenografts in the severe combined immunodeficiency (SCID) mouse model, respectively. TRPV2 expression at protein levels revealed converse correlation with those of CD133 and CD44 in human hepatocellular carcinoma (HCC) tissue. Collectively, the data demonstrate that TRPV2 exert effects on stemness of liver cancer and is a potential target in the treatment of human liver cancer patients. Copyright © 2018. Published by Elsevier Masson SAS.

Involvement of transient receptor potential vanilloid 2 in intra-oral incisional pain.

PubMed

Urata, K; Shinoda, M; Ikutame, D; Iinuma, T; Iwata, K

2018-03-05

To examine whether transient receptor potential vanilloid 2 (TRPV2) contributes to the changes in intra-oral thermal and mechanical sensitivity following the incision of buccal mucosa. Buccal mucosal pain threshold was measured after the incision. Changes in the number of TRPV2-immunoreactive (IR) trigeminal ganglion (TG) neurons which innervate the whisker pad skin and buccal mucosa, changes in the number of isolectin B4-negative/isolectin B4-positive TRPV2-IR TG neurons which innervate the whisker pad skin and the buccal mucosa, and the effect of peripheral TRPV2 antagonism on the pain threshold of incisional whisker pad skin and buccal mucosa were examined after these injuries. Buccal mucosal pain hypersensitivities were induced on day 3 following the incision. The total number of TRPV2-IR TG neurons and the number of isolectin B4-negative TRPV2-IR TG neurons which innervate the whisker pad skin and buccal mucosa were increased. Buccal mucosal TRPV2 antagonism completely suppressed the heat and mechanical hypersensitivities, but not cold hypersensitivity. TRPV2 antagonist administration to the incisional whisker pad skin only partially suppressed pain hypersensitivities. The increased expression of TRPV2 in peptidergic TG neurons innervating the incisional buccal mucosa is predominantly involved in buccal mucosal heat hyperalgesia and mechanical allodynia following buccal mucosal incision. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

Subtype-dependent postnatal development of taste receptor cells in mouse fungiform taste buds.

PubMed

Ohtubo, Yoshitaka; Iwamoto, Masafumi; Yoshii, Kiyonori

2012-06-01

Taste buds contain two types of taste receptor cells, inositol 1,4,5-triphosphate receptor type 3-immunoreactive cells (type II cells) and synaptosomal-associating protein-25-immunoreactive cells (type III cells). We investigated their postnatal development in mouse fungiform taste buds immunohistochemically and electrophysiologically. The cell density, i.e. the number of cells per taste bud divided by the maximal area of the horizontal cross-section of the taste bud, of type II cells increased by postnatal day (PD)49, where as that of type III cells was unchanged throughout the postnatal observation period and was equal to that of the adult cells at PD1. The immunoreactivity of taste bud cell subtypes was the same as that of their respective subtypes in adult mice throughout the postnatal observation period. Almost all type II cells were immunoreactive to gustducin at PD1, and then the ratio of gustducin-immunoreactive type II cells to all type II cells decreased to a saturation level, ∼60% of all type II cells, by PD15. Type II and III cells generated voltage-gated currents similar to their respective adult cells even at PD3. These results show that infant taste receptor cells are as excitable as those of adults and propagate in a subtype-dependent manner. The relationship between the ratio of each taste receptor cell subtype to all cells and taste nerve responses are discussed. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

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.

Transient Receptor Potential Vanilloid 1 Expression Mediates Capsaicin-Induced Cell Death.

PubMed

Ramírez-Barrantes, Ricardo; Córdova, Claudio; Gatica, Sebastian; Rodriguez, Belén; Lozano, Carlo; Marchant, Ivanny; Echeverria, Cesar; Simon, Felipe; Olivero, Pablo

2018-01-01

The transient receptor potential (TRP) ion channel family consists of a broad variety of non-selective cation channels that integrate environmental physicochemical signals for dynamic homeostatic control. Involved in a variety of cellular physiological processes, TRP channels are fundamental to the control of the cell life cycle. TRP channels from the vanilloid (TRPV) family have been directly implicated in cell death. TRPV1 is activated by pain-inducing stimuli, including inflammatory endovanilloids and pungent exovanilloids, such as capsaicin (CAP). TRPV1 activation by high doses of CAP (>10 μM) leads to necrosis, but also exhibits apoptotic characteristics. However, CAP dose-response studies are lacking in order to determine whether CAP-induced cell death occurs preferentially via necrosis or apoptosis. In addition, it is not known whether cytosolic Ca 2+ and mitochondrial dysfunction participates in CAP-induced TRPV1-mediated cell death. By using TRPV1-transfected HeLa cells, we investigated the underlying mechanisms involved in CAP-induced TRPV1-mediated cell death, the dependence of CAP dose, and the participation of mitochondrial dysfunction and cytosolic Ca 2+ increase. Together, our results contribute to elucidate the pathophysiological steps that follow after TRPV1 stimulation with CAP. Low concentrations of CAP (1 μM) induce cell death by a mechanism involving a TRPV1-mediated rapid and transient intracellular Ca 2+ increase that stimulates plasma membrane depolarization, thereby compromising plasma membrane integrity and ultimately leading to cell death. Meanwhile, higher doses of CAP induce cell death via a TRPV1-independent mechanism, involving a slow and persistent intracellular Ca 2+ increase that induces mitochondrial dysfunction, plasma membrane depolarization, plasma membrane loss of integrity, and ultimately, cell death.

Protease-activated receptor 2-mediated protection of myocardial ischemia-reperfusion injury: role of transient receptor potential vanilloid receptors

PubMed Central

Zhong, Beihua

2009-01-01

Activation of the protease-activated receptor 2 (PAR2) or the transient receptor potential vanilloid type 1 (TRPV1) channels expressed in cardiac sensory afferents containing calcitonin gene-related peptide (CGRP) and/or substance P (SP) has been proposed to play a protective role in myocardial ischemia-reperfusion (I/R) injury. However, the interaction between PAR2 and TRPV1 is largely unknown. Using gene-targeted TRPV1-null mutant (TRPV1−/−) or wild-type (WT) mice, we test the hypothesis that TRPV1 contributes to PAR2-mediated cardiac protection via increasing the release of CGRP and SP. Immunofluorescence labeling showed that TRPV1 coexpressed with PAR2, PKC-ε, or PKAc in cardiomyocytes, cardiac blood vessels, and perivascular nerves in WT but not TRPV1−/− hearts. WT or TRPV1−/− hearts were Langendorff perfused with the selective PAR2 agonist, SLIGRL, in the presence or absence of various antagonists, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). The recovery rate of coronary flow, the maximum rate of left ventricular pressure development, left ventricular end-diastolic pressure, and left ventricular developed pressure were evaluated after I/R. SLIGRL improved the recovery of hemodynamic parameters, decreased lactate dehydrogenase release, and reduced the infarct size in both WT and TRPV1−/− hearts (P < 0.05). The protection of SLIGRL was significantly surpassed for WT compared with TRPV1−/− hearts (P < 0.05). CGRP8–37, a selective CGRP receptor antagonist, RP67580, a selective neurokinin-1 receptor antagonist, PKC-ε V1–2, a selective PKC-ε inhibitor, or H-89, a selective PKA inhibitor, abolished SLIGRL protection by inhibiting the recovery of the rate of coronary flow, maximum rate of left ventricular pressure development, and left ventricular developed pressure, and increasing left ventricular end-diastolic pressure in WT but not TRPV1−/− hearts. Radioimmunoassay showed that SLIGRL increased the release

Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists.

PubMed

Katz, Jonathan L; Hiranita, Takato; Kopajtic, Theresa A; Rice, Kenner C; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H; McCurdy, Christopher R

2016-07-01

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. U.S. Government work not protected by U

Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists

PubMed Central

Hiranita, Takato; Kopajtic, Theresa A.; Rice, Kenner C.; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H.; McCurdy, Christopher R.

2016-01-01

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

A Molecular Determinant of Subtype-Specific Desensitization in Ionotropic Glutamate Receptors.

PubMed

Alsaloum, Matthew; Kazi, Rashek; Gan, Quan; Amin, Johansen; Wollmuth, Lonnie P

2016-03-02

AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the "hydrophobic box," located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPAR-like. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process. Rapid communication between cells in the nervous system depends on ion channels that are directly activated by neurotransmitter molecules. Here, we studied ionotropic glutamate receptors (iGluRs), which are ion channels activated by the neurotransmitter glutamate. By comparing the sequences of a vast number of iGluR proteins from diverse animal species, assisted by available structural information, we identified a highly conserved motif. We showed that a single amino acid difference in this motif between mammalian iGluR subtypes has dramatic effects on receptor function. These results have implications in both the evolution of synaptic function, as well as the role of iGluRs in health and disease

An Inotropic Action Caused by Muscarinic Receptor Subtype 3 in Canine Cardiac Purkinje Fibers

PubMed Central

Urushidani, Tetsuro; Tachibana, Shigehiro

2013-01-01

Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers. PMID:24260719

Engineering vanilloid-sensitivity into the rat TRPV2 channel.

PubMed

Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

2016-05-13

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1.

Reversal of pathological pain through specific spinal GABAA receptor subtypes.

PubMed

Knabl, Julia; Witschi, Robert; Hösl, Katharina; Reinold, Heiko; Zeilhofer, Ulrike B; Ahmadi, Seifollah; Brockhaus, Johannes; Sergejeva, Marina; Hess, Andreas; Brune, Kay; Fritschy, Jean-Marc; Rudolph, Uwe; Möhler, Hanns; Zeilhofer, Hanns Ulrich

2008-01-17

Inflammatory diseases and neuropathic insults are frequently accompanied by severe and debilitating pain, which can become chronic and often unresponsive to conventional analgesic treatment. A loss of synaptic inhibition in the spinal dorsal horn is considered to contribute significantly to this pain pathology. Facilitation of spinal gamma-aminobutyric acid (GABA)ergic neurotransmission through modulation of GABA(A) receptors should be able to compensate for this loss. With the use of GABA(A)-receptor point-mutated knock-in mice in which specific GABA(A) receptor subtypes have been selectively rendered insensitive to benzodiazepine-site ligands, we show here that pronounced analgesia can be achieved by specifically targeting spinal GABA(A) receptors containing the alpha2 and/or alpha3 subunits. We show that their selective activation by the non-sedative ('alpha1-sparing') benzodiazepine-site ligand L-838,417 (ref. 13) is highly effective against inflammatory and neuropathic pain yet devoid of unwanted sedation, motor impairment and tolerance development. L-838,417 not only diminished the nociceptive input to the brain but also reduced the activity of brain areas related to the associative-emotional components of pain, as shown by functional magnetic resonance imaging in rats. These results provide a rational basis for the development of subtype-selective GABAergic drugs for the treatment of chronic pain, which is often refractory to classical analgesics.

Iron overload causes osteoporosis in thalassemia major patients through interaction with transient receptor potential vanilloid type 1 (TRPV1) channels

PubMed Central

Rossi, Francesca; Perrotta, Silverio; Bellini, Giulia; Luongo, Livio; Tortora, Chiara; Siniscalco, Dario; Francese, Matteo; Torella, Marco; Nobili, Bruno; Di Marzo, Vincenzo; Maione, Sabatino

2014-01-01

The pathogenesis of bone resorption in β-thalassemia major is multifactorial and our understanding of the underlying molecular and cellular mechanisms remains incomplete. Considering the emerging importance of the endocannabinoid/endovanilloid system in bone metabolism, it may be instructive to examine a potential role for this system in the development of osteoporosis in patients with β-thalassemia major and its relationship with iron overload and iron chelation therapy. This study demonstrates that, in thalassemic-derived osteoclasts, tartrate-resistant acid phosphatase expression inversely correlates with femoral and lumbar bone mineral density, and directly correlates with ferritin levels and liver iron concentration. The vanilloid agonist resiniferatoxin dramatically reduces cathepsin K levels and osteoclast numbers in vitro, without affecting tartrate-resistant acid phosphatase expression. The iron chelators deferoxamine, deferiprone and deferasirox decrease both tartrate-resistant acid phosphatase and cathepsin K expression, as well as osteoclast activity. Taken together, these data show that transient receptor potential vanilloid type 1 activation/desensitization influences tartrate-resistant acid phosphatase expression and activity, and this effect is dependent on iron, suggesting a pivotal role for iron overload in the dysregulation of bone metabolism in patients with thalassemia major. Our applied pharmacology provides evidence for the potential of iron chelators to abrogate these effects by reducing osteoclast activity. Whether iron chelation therapy is capable of restoring bone health in humans requires further study, but the potential to provide dual benefits for patients with β-thalassemia major –preventing iron-overload and alleviating associated osteoporotic changes – is exciting. PMID:25216685

Effects of body temperature on neural activity in the hippocampus: regulation of resting membrane potentials by transient receptor potential vanilloid 4.

PubMed

Shibasaki, Koji; Suzuki, Makoto; Mizuno, Atsuko; Tominaga, Makoto

2007-02-14

Physiological body temperature is an important determinant for neural functions, and it is well established that changes in temperature have dynamic influences on hippocampal neural activities. However, the detailed molecular mechanisms have never been clarified. Here, we show that hippocampal neurons express functional transient receptor potential vanilloid 4 (TRPV4), one of the thermosensitive TRP (transient receptor potential) channels, and that TRPV4 is constitutively active at physiological temperature. Activation of TRPV4 at 37 degrees C depolarized the resting membrane potential in hippocampal neurons by allowing cation influx, which was observed in wild-type (WT) neurons, but not in TRPV4-deficient (TRPV4KO) cells, although dendritic morphology, synaptic marker clustering, and synaptic currents were indistinguishable between the two genotypes. Furthermore, current injection studies revealed that TRPV4KO neurons required larger depolarization to evoke firing, equivalent to WT neurons, indicating that TRPV4 is a key regulator for hippocampal neural excitabilities. We conclude that TRPV4 is activated by physiological temperature in hippocampal neurons and thereby controls their excitability.

Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

PubMed

Gonzalez-Cabrera, Pedro J; Shi, Ting; Yun, June; McCune, Dan F; Rorabaugh, Boyd R; Perez, Dianne M

2004-11-01

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis

PubMed Central

Bohonyi, Noémi; Pohóczky, Krisztina; Szalontai, Bálint; Perkecz, Anikó; Kovács, Krisztina; Kajtár, Béla; Orbán, Lajos; Varga, Tamás; Szegedi, Sarolta; Bódis, József; Koppán, Miklós

2017-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis (n = 15), uterine fibroid-induced moderate dysmenorrhoea (n = 7) and tubal infertility with no pain (n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis.

PubMed

Bohonyi, Noémi; Pohóczky, Krisztina; Szalontai, Bálint; Perkecz, Anikó; Kovács, Krisztina; Kajtár, Béla; Orbán, Lajos; Varga, Tamás; Szegedi, Sarolta; Bódis, József; Helyes, Zsuzsanna; Koppán, Miklós

2017-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis ( n = 15), uterine fibroid-induced moderate dysmenorrhoea ( n = 7) and tubal infertility with no pain ( n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first

G protein γ (Gγ) subtype dependent targeting of GRK2 to M3 receptor by Gβγ.

PubMed

Samaradivakara, Saroopa; Kankanamge, Dinesh; Senarath, Kanishka; Ratnayake, Kasun; Karunarathne, Ajith

2018-06-11

Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gβγ. Since we have shown that signaling efficacy of Gβγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gβγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gβγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization. Copyright © 2018 Elsevier Inc. All rights reserved.

Modulation of the adaptive response to stress by brain activation of selective somatostatin receptor subtypes.

PubMed

Stengel, Andreas; Rivier, Jean; Taché, Yvette

2013-04-01

Somatostatin-14 was discovered in 1973 in the hypothalamus as a peptide inhibiting growth hormone release. Somatostatin interacts with five receptor subtypes (sst(1-5)) which are widely distributed in the brain with a distinct, but overlapping, expression pattern. During the last few years, the development of highly selective peptide agonists and antagonists provided new insight to characterize the role of somatostatin receptor subtypes in the pleiotropic actions of somatostatin. Recent evidence in rodents indicates that the activation of selective somatostatin receptor subtypes in the brain blunts stress-corticotropin-releasing factor (CRF) related ACTH release (sst2/5), sympathetic-adrenal activaton (sst5), stimulation of colonic motility (sst1), delayed gastric emptying (sst5), suppression of food intake (sst2) and the anxiogenic-like (sst2) response. These findings suggest that brain somatostatin signaling pathways may play an important role in dampening CRF-mediated endocrine, sympathetic, behavioral and visceral responses to stress. Published by Elsevier Inc.

Engineering vanilloid-sensitivity into the rat TRPV2 channel

PubMed Central

Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

2016-01-01

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1. DOI: http://dx.doi.org/10.7554/eLife.16409.001 PMID:27177419

Effect of neonatal handling on serotonin 1A sub-type receptors in the rat hippocampus.

PubMed

Stamatakis, A; Mantelas, A; Papaioannou, A; Pondiki, S; Fameli, M; Stylianopoulou, F

2006-06-19

Serotonin 1A sub-type receptors play an important role in the etiopathogenesis of depression, which is known to occur more often in females than males. Early experiences can be a predisposing factor for depression; however, the underlying cellular processes remain unknown. In an effort to address such issues, we employed neonatal handling, an experimental model of early experience, which has been previously shown to render females more vulnerable to display enhanced depression-like behavior in response to chronic stress, while it increases the ability of males to cope. In rat pre-pubertal (30 days of age) and adult (90 days) hippocampus, of both males and females, the effect of neonatal handling on serotonin 1A sub-type receptor mRNA and protein levels was determined by in situ hybridization and immunohistochemistry, respectively, while the number of binding sites was determined by in vitro autoradiography using [(3)H]8-hydroxy-2(di-n-propylamino)tetralin as the ligand. Our results revealed a significant sex difference in serotonin 1A sub-type receptor mRNA, protein and binding sites, with females having higher levels than males. Handling resulted in statistically significant decreased numbers of cells positive for serotonin 1A sub-type receptor mRNA or protein, as well as [(3)H]8-hydroxy-2(di-n-propylamino)tetralin binding sites in the area 4 of Ammon's horn and dentate gyrus of both pre-pubertal males and females. In adult animals the number of serotonin 1A sub-type receptor mRNA positive cells was increased as a result of handling in the area 1 of Ammon's horn, area 4 of Ammon's horn and dentate gyrus of males, while it was decreased only in the area 4 of Ammon's horn of females. Furthermore, the number of serotonin sub-type 1A receptor immunopositive cells, as well as [(3)H]8-hydroxy-2(di-n-propylamino)tetralin binding sites was increased in the area 1 of Ammon's horn, area 4 of Ammon's horn and dentate gyrus of handled males, whereas it was decreased in these

Pharmacological identification of cholinergic receptor subtypes on Drosophila melanogaster larval heart.

PubMed

Malloy, Cole A; Ritter, Kyle; Robinson, Jonathan; English, Connor; Cooper, Robin L

2016-01-01

The Drosophila melanogaster heart is a popular model in which to study cardiac physiology and development. Progress has been made in understanding the role of endogenous compounds in regulating cardiac function in this model. It is well characterized that common neurotransmitters act on many peripheral and non-neuronal tissues as they flow through the hemolymph of insects. Many of these neuromodulators, including acetylcholine (ACh), have been shown to act directly on the D. melanogaster larval heart. ACh is a primary neurotransmitter in the central nervous system (CNS) of vertebrates and at the neuromuscular junctions on skeletal and cardiac tissue. In insects, ACh is the primary excitatory neurotransmitter of sensory neurons and is also prominent in the CNS. A full understanding regarding the regulation of the Drosophila cardiac physiology by the cholinergic system remains poorly understood. Here we use semi-intact D. melanogaster larvae to study the pharmacological profile of cholinergic receptor subtypes, nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), in modulating heart rate (HR). Cholinergic receptor agonists, nicotine and muscarine both increase HR, while nAChR agonist clothianidin exhibits no significant effect when exposed to an open preparation at concentrations as low as 100 nM. In addition, both nAChR and mAChR antagonists increase HR as well but also display capabilities of blocking agonist actions. These results provide evidence that both of these receptor subtypes display functional significance in regulating the larval heart's pacemaker activity.

Berberine via suppression of transient receptor potential vanilloid 4 channel improves vascular stiffness in mice

PubMed Central

Wang, Jie; Guo, Tao; Peng, Qi-Sheng; Yue, Shou-Wei; Wang, Shuang-Xi

2015-01-01

Berberine, as an alkaloid found in many Chinese herbs, improves vascular functions in patients with cardiovascular diseases. We determined the effects of berberine in hypertension and vascular ageing, and elucidated the underlying mechanisms. In isolated aortas, berberine dose-dependently elicited aortic relaxation. In cultured cells, berberine induced the relaxation of vascular smooth muscle cells (VSMCs). Overexpression of transient receptor potential vanilloid 4 (TRPV4) channel by genetic approaches abolished the berberine-induced reduction in intracellular Ca2+ concentration in VSMCs and attenuated berberine-elicited vessel dilation in mice aortas. In deoxycorticosterone acetate (DOCA)-induced hypertensive model, treatment of mice with berberine or RN-1734, a pharmacological inhibitor of TRPV4, significantly decreased systemic blood pressure (BP) in control mice or mice infected with an adenovirus vector. However, berberine-induced effects of lowering BP were reversed by overexpressing TRPV4 in mice by infecting with adenovirus. Furthermore, long-term administration of berberine decreased mean BP and pulse BP, increased artery response to vasodilator and reduced vascular collagen content in aged mice deficient in apolipoprotein E (Apoe-KO), but not in Apoe-KO old mice with lentivirus-mediated overexpression of TRPV4 channel. In conclusion, berberine induces direct vasorelaxation to lower BP and reduces vascular stiffness in aged mice through suppression of TRPV4. PMID:26177349

Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity

PubMed Central

Yin, Shijin; Luo, Jialie; Qian, Aihua; Du, Junhui; Yang, Qing; Zhou, Shentai; Yu, Weihua; Du, Guangwei; Clark, Richard B.; Walters, Edgar T.; Carlton, Susan M.; Hu, Hongzhen

2013-01-01

Retinoids are structurally related derivatives of vitamin A and are required for normal vision as well as cell proliferation and differentiation. Clinically, retinoids are effective in treating many skin disorders and cancers. Application of retinoids evokes substantial irritating side effects, including pain and inflammation; however, the precise mechanisms accounting for the sensory hypersensitivity are not understood. Here we show that both naturally occurring and synthetic retinoids activate recombinant or native transient receptor potential channel vanilloid subtype 1 (TRPV1), an irritant receptor for capsaicin, the pungent ingredient of chili peppers. In vivo, retinoids produced pain-related behaviors that were either eliminated or significantly reduced by genetic or pharmacological inhibition of TRPV1 function. These findings identify TRPV1 as an ionotropic receptor for retinoids and provide cellular and molecular insights into retinoid-evoked hypersensitivity. These findings also suggest that selective TRPV1 antagonists are potential therapeutic drugs for treating retinoid-induced sensory hypersensitivity. PMID:23925292

US incidence of breast cancer subtypes defined by joint hormone receptor and HER2 status.

PubMed

Howlader, Nadia; Altekruse, Sean F; Li, Christopher I; Chen, Vivien W; Clarke, Christina A; Ries, Lynn A G; Cronin, Kathleen A

2014-04-28

In 2010, Surveillance, Epidemiology, and End Results (SEER) registries began collecting human epidermal growth factor 2 (HER2) receptor status for breast cancer cases. Breast cancer subtypes defined by joint hormone receptor (HR; estrogen receptor [ER] and progesterone receptor [PR]) and HER2 status were assessed across the 28% of the US population that is covered by SEER registries. Age-specific incidence rates by subtype were calculated for non-Hispanic (NH) white, NH black, NH Asian Pacific Islander (API), and Hispanic women. Joint HR/HER2 status distributions by age, race/ethnicity, county-level poverty, registry, stage, Bloom-Richardson grade, tumor size, and nodal status were evaluated using multivariable adjusted polytomous logistic regression. All statistical tests were two-sided. Among case patients with known HR/HER2 status, 36810 (72.7%) were found to be HR(+)/HER2(-), 6193 (12.2%) were triple-negative (HR(-)/HER2(-)), 5240 (10.3%) were HR(+)/HER2(+), and 2328 (4.6%) were HR(-)/HER2(+); 6912 (12%) had unknown HR/HER2 status. NH white women had the highest incidence rate of the HR(+)/HER2(-) subtype, and NH black women had the highest rate of the triple-negative subtype. Compared with women with the HR(+)/HER2(-) subtype, triple-negative patients were more likely to be NH black and Hispanic; HR(+)/HER2(+) patients were more likely to be NH API; and HR(-)/HER2(+) patients were more likely to be NH black, NH API, and Hispanic. Patients with triple-negative, HR(+)/HER2(+), and HR(-)/HER2(+) breast cancer were 10% to 30% less likely to be diagnosed at older ages compared with HR(+)/HER2(-) patients and 6.4-fold to 20.0-fold more likely to present with high-grade disease. In the future, SEER data can be used to monitor clinical outcomes in women diagnosed with different molecular subtypes of breast cancer for a large portion (approximately 28%) of the US population. Published by Oxford University Press 2014.

The sympathetic nervous system is controlled by transient receptor potential vanilloid 1 in the regulation of body temperature.

PubMed

Alawi, Khadija M; Aubdool, Aisah A; Liang, Lihuan; Wilde, Elena; Vepa, Abhinav; Psefteli, Maria-Paraskevi; Brain, Susan D; Keeble, Julie E

2015-10-01

Transient receptor potential vanilloid 1 (TRPV1) is involved in sensory nerve nociceptive signaling. Recently, it has been discovered that TRPV1 receptors also regulate basal body temperature in multiple species from mice to humans. In the present study, we investigated whether TRPV1 modulates basal sympathetic nervous system (SNS) activity. C57BL6/J wild-type (WT) mice and TRPV1 knockout (KO) mice were implanted with radiotelemetry probes for measurement of core body temperature. AMG9810 (50 mg/kg) or vehicle (2% DMSO/5% Tween 80/10 ml/kg saline) was injected intraperitoneally. Adrenoceptor antagonists or vehicle (5 ml/kg saline) was injected subcutaneously. In WT mice, the TRPV1 antagonist, AMG9810, caused significant hyperthermia, associated with increased noradrenaline concentrations in brown adipose tissue. The hyperthermia was significantly attenuated by the β-adrenoceptor antagonist propranolol, the mixed α-/β-adrenoceptor antagonist labetalol, and the α1-adrenoceptor antagonist prazosin. TRPV1 KO mice have a normal basal body temperature, indicative of developmental compensation. d-Amphetamine (potent sympathomimetic) caused hyperthermia in WT mice, which was reduced in TRPV1 KO mice, suggesting a decreased sympathetic drive in KOs. This study provides new evidence that TRPV1 controls thermoregulation upstream of the SNS, providing a potential therapeutic target for sympathetic hyperactivity thermoregulatory disorders. © FASEB.

Novel oxotremorine-related heterocyclic derivatives: Synthesis and in vitro pharmacology at the muscarinic receptor subtypes.

PubMed

Dallanoce, Clelia; De Amici, Marco; Barocelli, Elisabetta; Bertoni, Simona; Roth, Bryan L; Ernsberger, Paul; De Micheli, Carlo

2007-12-15

A set of novel heterocyclic ligands (6-27) structurally related to Oxotremorine 2 was designed, synthesized and tested at muscarinic receptor subtypes (mAChRs). In the binding experiments at cloned human receptors (hm1-5), compounds 7 and 15 evidenced a remarkable affinity and selectivity for the hm2 subtype. The in vitro functional assays, performed on a selected group of derivatives at M(1), M(2), and M(3) tissue preparations, singled out the 3-butynyloxy-5-methylisoxazole trimethylammonium salt 7 as a potent unselective muscarinic agonist [pEC(50): 7.40 (M(1)), 8.18 (M(2)), and 8.14 (M(3))], whereas its 5-phenyl analogue 12 behaved as a muscarinic antagonist, slightly selective for the M(1) subtype [pK(B): 6.88 (M(1)), 5.95 (M(2)), 5.53 (M(3))]. Moreover, the functional data put in evidence that the presence of the piperidine ring may generate a functional selectivity, e.g., an M(1) antagonist/M(2) partial agonist/M(3) full agonist profile (compound 21), at variance with the corresponding quaternary ammonium salt (compound 22) which behaved as a muscarinic agonist at all M(1-3) receptors, with an appreciable selectivity for the cardiac M(2) receptors.

Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel – an overview of the current mutational data

PubMed Central

2013-01-01

This review aims to create an overview of the currently available results of site-directed mutagenesis studies on transient receptor potential vanilloid type 1 (TRPV1) receptor. Systematization of the vast number of data on the functionally important amino acid mutations of TRPV1 may provide a clearer picture of this field, and may promote a better understanding of the relationship between the structure and function of TRPV1. The review summarizes information on 112 unique mutated sites along the TRPV1, exchanged to multiple different residues in many cases. These mutations influence the effect or binding of different agonists, antagonists, and channel blockers, alter the responsiveness to heat, acid, and voltage dependence, affect the channel pore characteristics, and influence the regulation of the receptor function by phosphorylation, glycosylation, calmodulin, PIP2, ATP, and lipid binding. The main goal of this paper is to publish the above mentioned data in a form that facilitates in silico molecular modelling of the receptor by promoting easier establishment of boundary conditions. The better understanding of the structure-function relationship of TRPV1 may promote discovery of new, promising, more effective and safe drugs for treatment of neurogenic inflammation and pain-related diseases and may offer new opportunities for therapeutic interventions. PMID:23800232

The three subtypes of atrial natriuretic peptide (ANP) receptors are expressed in the rat adrenal gland.

PubMed

Grandclément, B; Ronsin, B; Morel, G

1997-03-01

Atrial natriuretic peptide (ANP) actions are mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANP receptor-A (or GC-A), -B (or GC-B) and -C (the so-called clearance receptor). In rat adrenal gland, the mRNA for each subtype was detected using 35S-dUTP or digoxigenin-11-dUTP specific labeled probes, and in situ hybridization at light and electron microscopic levels respectively. The three subtypes were expressed the most abundantly in the zona glomerulosa. The amount of GC-A mRNA expression, quantified using macro-autoradiography and densitometry, was higher than the amounts of GC-B mRNA and ANPR-C mRNA both in zona glomerulosa and medullary of adrenal gland. At electron microscopic level, the three subtypes of ANPR were revealed in glomerulosa cells. A noticeable signal was also present in the medullary area, especially for GC-A mRNA, in adrenaline-containing chromaffin cells. No signal was detected in noradrenaline-containing chromaffin cells. The subcellular localization of the three mRNAs is similar: in the cytoplasmic matrix and in the euchromatin of the nucleus in each cell of glomerulosa, and in the same compartments of the adrenaline-containing chromaffin cells. These data indicate that the adrenal gland is an important target tissue for ANP action both in glomerulosa cells and adrenaline-containing chromaffin cells. The mRNA expression levels were different for each ANPR subtype.

Transient Receptor Potential Vanilloid 4 Activation-Induced Increase in Glycine-Activated Current in Mouse Hippocampal Pyramidal Neurons.

PubMed

Qi, Mengwen; Wu, Chunfeng; Wang, Zhouqing; Zhou, Li; Men, Chen; Du, Yimei; Huang, Songming; Chen, Lei; Chen, Ling

2018-01-01

Glycine plays an important role in regulating hippocampal inhibitory/ excitatory neurotransmission through activating glycine receptors (GlyRs) and acting as a co-agonist of N-methyl-d-aspartate-type glutamate receptors. Activation of transient receptor potential vanilloid 4 (TRPV4) is reported to inhibit hippocampal A-type γ-aminobutyric acid receptor, a ligand-gated chloride ion channel. GlyRs are also ligand-gated chloride ion channels and this paper aimed to explore whether activation of TRPV4 could modulate GlyRs. Whole-cell patch clamp recording was employed to record glycine-activated current (IGly) and Western blot was conducted to assess GlyRs subunits protein expression. Application of TRPV4 agonist (GSK1016790A or 5,6-EET) increased IGly in mouse hippocampal CA1 pyramidal neurons. This action was blocked by specific antagonists of TRPV4 (RN-1734 or HC-067047) and GlyR (strychnine), indicating that activation of TRPV4 increases strychnine-sensitive GlyR function in mouse hippocampal pyramidal neurons. GSK1016790A-induced increase in IGly was significantly attenuated by protein kinase C (PKC) (BIM II or D-sphingosine) or calcium/calmodulin-dependent protein kinase II (CaMKII) (KN-62 or KN-93) antagonists but was unaffected by protein kinase A or protein tyrosine kinase antagonists. Finally, hippocampal protein levels of GlyR α1 α2, α3 and β subunits were not changed by treatment with GSK1016790A for 30 min or 1 h, but GlyR α2, α3 and β subunits protein levels increased in mice that were intracerebroventricularly (icv.) injected with GSK1016790A for 5 d. Activation of TRPV4 increases GlyR function and expression, and PKC and CaMKII signaling pathways are involved in TRPV4 activation-induced increase in IGly. This study indicates that GlyRs may be effective targets for TRPV4-induced modulation of hippocampal inhibitory neurotransmission. © 2018 The Author(s). Published by S. Karger AG, Basel.

Targeting the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Assembly Domain Attenuates Inflammation-induced Hypersensitivity*

PubMed Central

Flynn, Robyn; Chapman, Kevin; Iftinca, Mircea; Aboushousha, Reem; Varela, Diego; Altier, Christophe

2014-01-01

The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief. PMID:24808184

Expression and diagnosis of transient receptor potential vanilloid1 in urothelium of patients with overactive bladder.

PubMed

Zhang, H Y; Chu, J F; Li, P; Li, N; Lv, Z H

2015-01-01

This study was carried out to test expression of transient receptor potential vanilloid1 (TRPV1) in urothelium of female patients with overactive bladder (OAB) and explore clinical significance of TRPV1 in diagnosing female OAB. TRPV1 expression in urothelium of female OAB patients (n=21) and healthy females (n=9) was detected using Strept Avidin-Biotin Complex (SABC), an immunohistochemical method and image analysis system. Relative content of TRPV1 was expressed by average optical density (AOD) and was analyzed through data of urodynamics. Compared to TRPV1 expression in urothelium of healthy females (AOD 0.3658 ± 0.1009), TRPV1 expression in OAB patients was much higher (AOD 0.4834 ± 0.1252) and the difference was significant P less than 0.05. Observation and comparison in clinic of urodynamic parameters of female patients and healthy females revealed that the former had lower indexes with remarkable differences (P less than 0.05) such as Qmax, first desire volume (FDV), strong desire volume (SDV), maximum cyst capacity (MCC) and bladder compliance (BC). Thus high expression of TRPV1 in urothelium of female OAB patients is closely correlated to OAB occurrence, showing great importance of improved bladder sensitivity in female OAB occurrence mechanism.

Targeting the transient receptor potential vanilloid type 1 (TRPV1) assembly domain attenuates inflammation-induced hypersensitivity.

PubMed

Flynn, Robyn; Chapman, Kevin; Iftinca, Mircea; Aboushousha, Reem; Varela, Diego; Altier, Christophe

2014-06-13

The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of two H3-histamine receptor subtypes

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

West, R.E. Jr.; Zweig, A.; Shih, N.Y.

The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-(3H)methylhistamine ((3H)NAMHA). The results of (3H)NAMHA saturation binding and NAMHA inhibition of (3H)NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of (3H)NAMHA binding by the specific high affinity H3 antagonist thioperamide revealedmore » two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for (3H) NAMHA was reduced less than 2-fold, whereas (3H)NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data.« less

Differentiation of muscarinic cholinergic receptor subtypes in human cortex and pons - Implications for anti-motion sickness therapy

NASA Technical Reports Server (NTRS)

Mccarthy, Bruce G.; Peroutka, Stephen J.

1988-01-01

Radioligand binding studies were used to analyze muscarinic cholinergic receptor subtypes in human cortex and pons. Muscarinic cholinergic receptors were labeled by H-3-quinuclidinyl benzilate (H-3-QNB). Scopolamine was equipotent in both brain regions and did not discriminate subtypes of H-3-QNB binding. By contrast, the M1 selective antagonist pirenzepine was approximately 33-fold more potent in human cortex than pons. Carbachol, a putative M2 selective agonist, was more than 100-fold more potent in human pons than cortex. These results demonstrate that the human pons contains a relatively large proportion of carbachol-sensitive muscarinic cholinergic receptors. Drugs targeted to this subpopulation of muscarinic cholinergic receptors may prove to be effective anti-motion sickness agents with less side effects than scopolamine.

Activation of transient receptor potential vanilloid-1 (TRPV1) influences how retinal ganglion cell neurons respond to pressure-related stress

PubMed Central

Sappington, Rebecca M; Sidorova, Tatiana; Ward, Nicholas J; Chakravarthy, Rohini; Ho, Karen W; Calkins, David J

2015-01-01

Our recent studies implicate the transient receptor potential vanilloid-1 (TRPV1) channel as a mediator of retinal ganglion cell (RGC) function and survival. With elevated pressure in the eye, TRPV1 increases in RGCs, supporting enhanced excitability, while Trpv1 -/- accelerates RGC degeneration in mice. Here we find TRPV1 localized in monkey and human RGCs, similar to rodents. Expression increases in RGCs exposed to acute changes in pressure. In retinal explants, contrary to our animal studies, both Trpv1 -/- and pharmacological antagonism of the channel prevented pressure-induced RGC apoptosis, as did chelation of extracellular Ca2+. Finally, while TRPV1 and TRPV4 co-localize in some RGC bodies and form a protein complex in the retina, expression of their mRNA is inversely related with increasing ocular pressure. We propose that TRPV1 activation by pressure-related insult in the eye initiates changes in expression that contribute to a Ca2+-dependent adaptive response to maintain excitatory signaling in RGCs. PMID:25713995

Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery

PubMed Central

Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G.; Mandrup, Susanne

2006-01-01

Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARγ2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARγ2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (α, γ, and β/δ), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci. PMID:16847324

Anxiety and Depression: Mouse Genetics and Pharmacological Approaches to the Role of GABAA Receptor Subtypes

PubMed Central

Smith, Kiersten S.; Rudolph, Uwe

2012-01-01

GABAA receptors mediate fast synaptic inhibitory neurotransmission throughout the central nervous system. Recent work indicates a role for GABAA receptors in physiologically modulating anxiety and depression levels. In this review, we summarize research that led to the identification of the essential role of GABAA receptors in counteracting trait anxiety and depression-related behaviors, and research aimed at identifying individual GABAA receptor subtypes involved in physiological and pharmacological modulation of emotions. PMID:21810433

Targeting Transient Receptor Potential Vanilloid 1 (TRPV1) Channel Softly: The Discovery of Passerini Adducts as a Topical Treatment for Inflammatory Skin Disorders.

PubMed

Serafini, Marta; Griglio, Alessia; Aprile, Silvio; Seiti, Fabio; Travelli, Cristina; Pattarino, Franco; Grosa, Giorgio; Sorba, Giovanni; Genazzani, Armando A; Gonzalez-Rodriguez, Sara; Butron, Laura; Devesa, Isabel; Fernandez-Carvajal, Asia; Pirali, Tracey; Ferrer-Montiel, Antonio

2018-05-24

Despite being an old molecule, capsaicin is still a hot topic in the scientific community, and the development of new capsaicinoids is a promising pharmacological approach in the management of skin disorders related to inflammation and pruritus. Here we report the synthesis and the evaluation of capsaicin soft drugs that undergo deactivation by the hydrolyzing activity of skin esterases. The implanting of an ester group in the lipophilic moiety of capsaicinoids by the Passerini multicomponent reaction affords both agonists and antagonists that retain transient receptor potential vanilloid 1 channel (TRPV1) modulating activity and, at the same time, are susceptible to hydrolysis. The most promising antagonist identified shows in vivo anti-nociceptive activity on pruritus and hyperalgesia without producing hyperthermia, thus validating it as novel treatment for dermatological conditions that implicate TRPV1 channel dysfunction.

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.

Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes.

PubMed

Forman, Stuart A; Miller, Keith W

2016-11-01

IV general anesthetics, including propofol, etomidate, alphaxalone, and barbiturates, produce important actions by enhancing γ-aminobutyric acid type A (GABAA) receptor activation. In this article, we review scientific studies that have located and mapped IV anesthetic sites using photoaffinity labeling and substituted cysteine modification protection. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABAA receptors, and drugs that display stereoselectivity also show remarkably selective interactions with distinct interfacial sites. These results suggest strategies for developing new drugs that selectively modulate distinct GABAA receptor subtypes.

Sensory fibers containing vanilloid receptor-1 (VR-1) mediate spinal cord stimulation-induced vasodilation.

PubMed

Wu, Mingyuan; Komori, Naoka; Qin, Chao; Farber, Jay P; Linderoth, Bengt; Foreman, Robert D

2006-08-30

Spinal cord stimulation (SCS) is used to improve peripheral blood flow in selected populations of patients with ischemia of the extremities. Previous studies show that antidromic activation of sensory fibers is an important mechanism that contributes to SCS-induced vasodilation. However, the characteristics of sensory fibers involved in vasodilation are not fully known. This study investigated the contribution of vanilloid receptor type 1 (VR-1) containing fibers to SCS-induced vasodilation. A unipolar ball electrode was placed on the left dorsal column at the lumbar 2-3 spinal cord segments (L2-L3) in sodium pentobarbital anesthetized, paralyzed and ventilated rats. Cutaneous blood flows from both ipsilateral (left) and contralateral (right) hind foot pads were recorded with laser Doppler flow perfusion monitors. SCS (50 Hz; 0.2 ms) was applied through the ball electrode at 30%, 60%, 90% and 300% of motor threshold (MT). Resiniferatoxin (RTX), an ultra potent analog of capsaicin and VR-1 receptor agonist, was used to suppress the activities of VR-1 containing sensory fibers. SCS at 30%, 60%, 90% and also at 300% of MT significantly increased cutaneous blood flow in the ipsilateral foot pad compared to that in the contralateral side. RTX (2 microg/kg, i.v.) significantly attenuated SCS-induced vasodilation of the ipsilateral side (P<0.05, n=7) compared with responses prior to RTX administration. A pledget of cotton soaked with RTX (2 microg/ml) placed on L2-L3 spinal cord significantly decreased SCS-induced vasodilation of the ipsilateral side at 30%, 60%, 90% and 300% of MT (P<0.05, n=7) compared with responses prior to RTX administration. Additionally, topical application of a pledget of cotton soaked with RTX (2 microg/ml) on the sciatic nerve at the middle level of the thigh or on the tibial nerve at the lower level of the lower hindlimb also decreased SCS-induced vasodilation (n=5). SCS-induced vasodilation is predominantly mediated via VR-1 containing sensory

The effects of cannabinoid CB1, CB2 and vanilloid TRPV1 receptor antagonists on cocaine addictive behavior in rats.

PubMed

Adamczyk, Przemysław; Miszkiel, Joanna; McCreary, Andrew C; Filip, Małgorzata; Papp, Mariusz; Przegaliński, Edmund

2012-03-20

There is evidence that indicates that tonic activation of cannabinoid CB1 receptors plays a role in extinction/reinstatement of cocaine seeking-behavior but is not involved in the maintenance of cocaine self-administration. To further explore the importance of other endocannabinoid-related receptors in an animal model of cocaine addiction, the present paper examines cannabinoid CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) and the transient receptor potential vanilloid type-1 (TRPV1) receptor antagonist N-(3-methoxyphenyl)-4-chlorocinnamide (SB366791) on intravenous (i.v.) cocaine self-administration and extinction/reinstatement of cocaine-seeking behavior in rats. For comparison and reference purposes, the effect of the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) was also examined. Moreover, for comparison effects of those drugs on operant lever responding for artificial (cocaine) vs. natural (food) reward, food self-administration was also evaluated. Our findings show that AM251 (1-3mg/kg), SR144528 (0.1-1mg/kg) and SB366791 (0.3-1mg/kg) did not affect cocaine self-administration. However, AM251 (0.1-1mg/kg), SR144528 (0.1-1mg/kg) and SB366791 (0.1-1mg/kg) decreased cocaine-induced reinstatement of cocaine-seeking behavior, and AM251 (0.3-1mg/kg) decreased cue-induced reinstatement. Moreover, AM251 (3mg/kg), SR144528 (0.1-1mg/kg) and SB366791 (0.1-1mg/kg) slightly decreased food self-administration behavior, but only AM251 (3mg/kg) reduced food reward. In conclusion, our results indicate for the first time, that tonic activation of CB2 or TRPV1 receptors is involved in cocaine-induced reinstatement of cocaine-seeking behavior, but their activity is not necessary for the rewarding effect of this psychostimulant. In contrast to CB1 receptors, neither CB2 nor

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+.

PubMed

Li, Mufeng; Silberberg, Shai D; Swartz, Kenton J

2013-09-03

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg(2+). Here we investigated the active forms of ATP and found that the action of MgATP(2-) and ATP(4-) differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP(2-) promotes opening with very low efficacy. In contrast, both free ATP and MgATP(2-) robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg(2+) to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP(2-) and weak regulation by Mg(2+). These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP(2-) and regulation by Mg(2+), and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

PubMed Central

Li, Mufeng; Silberberg, Shai D.; Swartz, Kenton J.

2013-01-01

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons. PMID:23959888

Mechanisms Underlying Tolerance after Long-Term Benzodiazepine Use: A Future for Subtype-Selective GABAA Receptor Modulators?

PubMed Central

Vinkers, Christiaan H.; Olivier, Berend

2012-01-01

Despite decades of basic and clinical research, our understanding of how benzodiazepines tend to lose their efficacy over time (tolerance) is at least incomplete. In appears that tolerance develops relatively quickly for the sedative and anticonvulsant actions of benzodiazepines, whereas tolerance to anxiolytic and amnesic effects probably does not develop at all. In light of this evidence, we review the current evidence for the neuroadaptive mechanisms underlying benzodiazepine tolerance, including changes of (i) the GABAA receptor (subunit expression and receptor coupling), (ii) intracellular changes stemming from transcriptional and neurotrophic factors, (iii) ionotropic glutamate receptors, (iv) other neurotransmitters (serotonin, dopamine, and acetylcholine systems), and (v) the neurosteroid system. From the large variance in the studies, it appears that either different (simultaneous) tolerance mechanisms occur depending on the benzodiazepine effect, or that the tolerance-inducing mechanism depends on the activated GABAA receptor subtypes. Importantly, there is no convincing evidence that tolerance occurs with α subunit subtype-selective compounds acting at the benzodiazepine site. PMID:22536226

Pharmacology of modality-specific transient receptor potential vanilloid-1 antagonists that do not alter body temperature.

PubMed

Reilly, Regina M; McDonald, Heath A; Puttfarcken, Pamela S; Joshi, Shailen K; Lewis, LaGeisha; Pai, Madhavi; Franklin, Pamela H; Segreti, Jason A; Neelands, Torben R; Han, Ping; Chen, Jun; Mantyh, Patrick W; Ghilardi, Joseph R; Turner, Teresa M; Voight, Eric A; Daanen, Jerome F; Schmidt, Robert G; Gomtsyan, Arthur; Kort, Michael E; Faltynek, Connie R; Kym, Philip R

2012-08-01

The transient receptor potential vanilloid-1 (TRPV1) channel is involved in the development and maintenance of pain and participates in the regulation of temperature. The channel is activated by diverse agents, including capsaicin, noxious heat (≥ 43°C), acidic pH (< 6), and endogenous lipids including N-arachidonoyl dopamine (NADA). Antagonists that block all modes of TRPV1 activation elicit hyperthermia. To identify efficacious TRPV1 antagonists that do not affect temperature antagonists representing multiple TRPV1 pharmacophores were evaluated at recombinant rat and human TRPV1 channels with Ca(2+) flux assays, and two classes of antagonists were identified based on their differential ability to inhibit acid activation. Although both classes of antagonists completely blocked capsaicin- and NADA-induced activation of TRPV1, select compounds only partially inhibited activation of the channel by protons. Electrophysiology and calcitonin gene-related peptide release studies confirmed the differential pharmacology of these antagonists at native TRPV1 channels in the rat. Comparison of the in vitro pharmacological properties of these TRPV1 antagonists with their in vivo effects on core body temperature confirms and expands earlier observations that acid-sparing TRPV1 antagonists do not significantly increase core body temperature. Although both classes of compounds elicit equivalent analgesia in a rat model of knee joint pain, the acid-sparing antagonist tested is not effective in a mouse model of bone cancer pain.

Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates*

PubMed Central

Ohkita, Masashi; Saito, Shigeru; Imagawa, Toshiaki; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

2012-01-01

The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca2+]i). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca2+]i increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function. PMID:22130664

Control of glycinergic input to spinal dorsal horn neurons by distinct muscarinic receptor subtypes revealed using knockout mice.

PubMed

Zhang, Hong-Mei; Zhou, Hong-Yi; Chen, Shao-Rui; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

2007-12-01

Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA(B) receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABA(B) receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn 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.

Neuroendocrine mediators up-regulate alpha1b- and alpha1d-adrenergic receptor subtypes in human monocytes.

PubMed

Rouppe van der Voort, C; Kavelaars, A; van de Pol, M; Heijnen, C J

1999-03-01

Beta2- and alpha2-adrenergic receptors (AR) are thought to be the main AR subtypes to exert the effects of catecholamines on the immune system. However, in the present study, we demonstrate that another subtype of AR can be induced in human monocytes. Expression of alpha1b- and alpha1d-AR mRNA can be obtained by culturing freshly isolated human peripheral blood monocytes with the neuroendocrine mediators dexamethasone or the beta2-AR agonist terbutaline. Using the human monocytic cell line THP-1, we demonstrate that increased levels of alpha1b- and alpha1d-mRNA are accompanied by increased levels of receptor protein as determined by Western blot analysis and radioligand binding assays. This study describes for the first time regulated expression of alpha1-AR subtypes in human monocytes.

Preclinical characterization of three transient receptor potential vanilloid receptor 1 antagonists for early use in human intradermal microdose analgesic studies.

PubMed

Sjögren, E; Halldin, M M; Stålberg, O; Sundgren-Andersson, A K

2018-05-01

The transient receptor potential vanilloid receptor 1 (TRPV1) is a nonselective cation channel involved in the mediation of peripheral pain to the central nervous system. As such, the TRPV1 is an accessible molecular target that lends itself well to the understanding of nociceptive signalling. This study encompasses preclinical investigations of three molecules with the prospect to establish them as suitable analgesic model compounds in human intradermal pain relief studies. The inhibitory effectiveness was evaluated by means of in vitro assays, TRPV1 expressing Chinese hamster ovary cells (CHO-K1) and rat dorsal root ganglion cultures in fluorescent imaging plate reader and whole cell patch clamp systems, as well as in vivo by capsaicin-evoked pain-related behavioural response studies in rat. Secondary pharmacology, pharmacokinetics and preclinical safety were also assessed. In vitro, all three compounds were effective at inhibiting capsaicin-activated TRPV1. The concentration producing 50% inhibition (IC 50 ) determined was in the range of 3-32 nmol/L and 10-501 nmol/L using CHO-K1 and dorsal root ganglion cultures, respectively. In vivo, all compounds showed dose-dependent reduction in capsaicin-evoked pain-related behavioural responses in rat. None of the three compounds displayed any significant activity on any of the secondary targets tested. The compounds were also shown to be safe from a toxicological, drug metabolism and pharmacokinetic perspective, for usage in microgram doses in the human skin. The investigated model compounds displayed ideal compound characteristics as pharmacological and translational tools to address efficacy on the human native TRPV1 target in human skin in situ. This work details the pharmaceutical work-up of three TRPV1-active investigational compounds, to obtain regulatory approval, for subsequent use in humans. This fast and cost-effective preclinical development path may impact research beyond the pain management area, as

Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

PubMed Central

Sniatecki, Jan J.; Goloborodko, Evgeny; Steege, Andreas; Zavaritskaya, Olga; Vetter, Jan M.; Grus, Franz H.; Patzak, Andreas; Wess, Jürgen; Pfeiffer, Norbert

2011-01-01

Purpose. To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. Methods. Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor–deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. Results. Only mRNA for the M3 receptor was detected in retinal arterioles. Thus, M3 receptor–deficient mice (M3R−/−) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R−/− mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R−/−, mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non–subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor Nω-nitro-l-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Conclusions. These findings provide evidence that endothelial M3 receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase. PMID:21873683

Dynamic control of glutamatergic synaptic input in the spinal cord by muscarinic receptor subtypes defined using knockout mice.

PubMed

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2010-12-24

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M(2), M(3), and M(4)) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M(3)-single KO and M(1)/M(3) double-KO mice. In addition, the M(3) antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M(5)-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M(2)/M(4) double-KO mice, but not M(2)- or M(4)-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M(2)/M(4) antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M(2) and M(4) receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M(5) is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord.

Dynamic Control of Glutamatergic Synaptic Input in the Spinal Cord by Muscarinic Receptor Subtypes Defined Using Knockout Mice*

PubMed Central

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2010-01-01

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M2, M3, and M4) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M3-single KO and M1/M3 double-KO mice. In addition, the M3 antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M5-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M2/M4 double-KO mice, but not M2- or M4-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M2/M4 antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M2 and M4 receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M5 is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord. PMID:20940295

Transient receptor potential vanilloid 2 (TRPV2), a potential novel biomarker in childhood asthma.

PubMed

Cai, Xin; Yang, Yong-chang; Wang, Jing-feng; Wang, Qiang; Gao, Jie; Fu, Wen-liang; Zhu, Ze-yi; Wang, Yuan-yuan; Zou, Min-ji; Wang, Jia-xi; Xu, Dong-qun; Xu, Dong-gang

2013-03-01

The presence of transient receptor potential vanilloid 2 (TRPV2) in human peripheral blood cells may suggest a role under pathological conditions. The aim of this study was to explore the relationship between the expression profile of TRPV2 gene and childhood asthma in the north of China. The effects of allergens exposure on the expression of TRPV2 gene were also investigated. Sixty asthmatics children confirmed by physician diagnosis and 60 healthy children as a control group were recruited. Serum total IgE and specific IgE were measured. Using quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), TRPV2 was detected in total RNA extracted from peripheral blood lymphocytes. Student's t-test and chi-square test were used to analyze the relationship between TRPV2 transcript and different parameter variables on susceptibility of childhood asthma. Multiple logistic regression was used to analyze the associations between TRPV2 gene and allergens. The expression level of TRPV2 gene was increased 2.6 times in asthmatic children compared with controls (p < .01). The up-regulation of TRPV2 gene and sensitization to one of three the allergens-spring pollen, dust mite, and dog and cat hair-were correlated with childhood asthma. In addition, the hypersensitivity to spring pollen, cockroach, and dust mite and up-regulation of TRPV2 gene expression may be the risk factors for the childhood asthma in Beijing. The increased expression of TRPV2 gene in peripheral lymphocytes is closely correlated with childhood asthma in the north of China. This study provides a potential new biomarker of childhood asthma and lays the basis for further clarification of the pathogenesis underlying asthma.

The N-terminal domain of GluR6-subtype glutamate receptor ion channels

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

Kumar, Janesh; Schuck, Peter; Jin, Rongsheng

2009-09-25

The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs.more » This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.« less

A possible participation of transient receptor potential vanilloid type 1 channels in the antidepressant effect of fluoxetine.

PubMed

Manna, Shyamshree S S; Umathe, Sudhir N

2012-06-15

The present study investigated the influence of transient receptor vanilloid type 1 (TRPV1) channel agonist (capsaicin) and antagonist (capsazepine) either alone or in combination with traditional antidepressant drug, fluoxetine; or a serotonin hydroxylase inhibitor, para-chlorophenylalanine; or a glutamate N-methyl-D-aspartate (NMDA) receptor agonist, NMDA on the forced swim test and tail suspension test using male Swiss mice. Results revealed that intracerebroventricular injections of capsaicin (200 and 300 μg/mouse) and capsazepine (100 and 200 μg/mouse) reduced the immobility time, exhibiting antidepressant-like activity that was comparable to the effects of fluoxetine (2.5-10 μg/mouse) in both the tests. However, in the presence of inactive dose (10 μg/mouse) of capsazepine, capsaicin (300 μg/mouse) had no influence on the indices of both tests, signifying that the effects are TRPV1-mediated. Further, the antidepressant-like effects of both the TRPV1 ligands were neutralized in mice-pretreated with NMDA (0.1 μg/mouse), suggestive of the fact that decreased glutamatergic transmission might contribute to the antidepressant-like activity. In addition, co-administration of sub-threshold dose of capsazepine (10 μg/mouse) and fluoxetine (1.75 μg/mouse) produced a synergistic effect in both the tests. In contrast, inactive doses of capsaicin (10 and 100 μg/mouse) partially abolished the antidepressant effect of fluoxetine (10 μg/mouse), while its effect was potentiated by active dose of capsaicin (200 μg/mouse). Moreover, pretreatment of mice with para-chlorophenylalanine (300 mg/kg/day × 3 days, i.p.) attenuated the effects of capsaicin and capsazepine, demonstrating a probable interplay between serotonin and TRPV1, at least in parts. Thus, our data indicate a possible role of TRPV1 in depressive-like symptoms. Copyright © 2012 Elsevier B.V. All rights reserved.

Participation of transient receptor potential vanilloid 1 in paclitaxel-induced acute visceral and peripheral nociception in rodents.

PubMed

Rossato, Mateus Fortes; Rigo, Flavia Karine; Oliveira, Sara Marchesan; Guerra, Gustavo Petri; Silva, Cássia Regina; Cunha, Thiago Mattar; Gomez, Marcus Vinícius; Ferreira, Juliano; Trevisan, Gabriela

2018-06-05

The clinical use of paclitaxel as a chemotherapeutic agent is limited by the severe acute and chronic hypersensitivity caused when it is administered via intraperitoneal or intravenous routes. Thus far, evidence has suggested that transient receptor potential vanilloid-1 (TRPV1) has a key role in the chronic neuropathy induced by paclitaxel. Despite this, the role of TRPV1 in paclitaxel -related acute nociception, especially the development of visceral nociception, has not been evaluated. Thus, the goal of this study was to evaluate the participation of TRPV1 in a model of acute nociception induced by paclitaxel in rats and mice. A single intraperitoneal (i.p.) paclitaxel administration (1 mg/kg, i.p.) produced an immediate visceral nociception response 1 h after administration, caused mechanical and heat hypersensitivity, and diminished burrowing behaviour 24 h after administration. These nociceptive responses were reduced by SB-366791 treatment (0.5 mg/kg, i.p., a TRPV1 antagonist). In addition, TRPV1-positive sensory fibre ablation (using resiniferatoxin, 200 µg/kg, s.c.) reduced visceral nociception and mechanical or heat hypersensitivity caused by paclitaxel injection. Similarly, TRPV1 deficient mice showed a pronounced reduction in mechanical allodynia to paclitaxel acute injection and did not develop heat hypersensitivity. Moreover, 24 h after its injection, paclitaxel induced chemical hypersensitivity to capsaicin (a TRPV1 agonist, 0.01 nmol/site) and increased TRPV1 immunoreactivity in the dorsal root ganglion and sciatic nerve. In conclusion, TRPV1 is involved in mechanical and heat hypersensitivity and spontaneous-pain behaviour induced 24 h after a single paclitaxel injection. This receptor is also involved in visceral nociception induced immediately after paclitaxel administration. Copyright © 2018 Elsevier B.V. All rights reserved.

Pore helix domain is critical to camphor sensitivity of transient receptor potential vanilloid 1 channel.

PubMed

Marsakova, Lenka; Touska, Filip; Krusek, Jan; Vlachova, Viktorie

2012-04-01

The recent discovery that camphor activates and strongly desensitizes the capsaicin-sensitive and noxious heat-sensitive channel transient receptor potential vanilloid subfamily member 1 (TRPV1) has provided new insights and opened up new research paths toward understanding why this naturally occurring monoterpene is widely used in human medicine for its local counter-irritant, antipruritic, and anesthetic properties. However, the molecular basis for camphor sensitivity remains mostly unknown. The authors attempt to explore the nature of the activation pathways evoked by camphor and narrow down a putative interaction site at TRPV1. The authors transiently expressed wild-type or specifically mutated recombinant TRPV1 channels in human embryonic kidney cells HEK293T and recorded cation currents with the whole cell, patch clamp technique. To monitor changes in the spatial distribution of phosphatidylinositol 4,5-bisphosphate, they used fluorescence resonance energy transfer measurements from cells transfected with the fluorescent protein-tagged pleckstrin homology domains of phospholipase C. The results revealed that camphor modulates TRPV1 channel through the outer pore helix domain by affecting its overall gating equilibrium. In addition, camphor, which generally is known to decrease the fluidity of cell plasma membranes, may also regulate the activity of TRPV1 by inducing changes in the spatial distribution of phosphatidylinositol-4,5-bisphosphate on the inner leaflet of the plasma membrane. The findings of this study provide novel insights into the structural basis for the modulation of TRPV1 channel by camphor and may provide an explanation for the mechanism by which camphor modulates thermal sensation in vivo.

Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice

PubMed Central

Smith, Kiersten S.; Engin, Elif; Meloni, Edward G.; Rudolph, Uwe

2012-01-01

GABAA receptor modulating drugs such as benzodiazepines (BZs) have been used to treat anxiety disorders for over five decades. In order to determine whether the same or different GABAA receptor subtypes are necessary for the anxiolytic-like action of BZs in unconditioned anxiety and conditioned fear models, we investigated the role of different GABAA receptor subtypes by challenging wild type, α1(H101R), α2(H101R) and α3(H126R) mice bred on the C57BL/6J background with diazepam or chlordiazepoxide in the elevated plus maze and the fear-potentiated startle paradigms. Both drugs significantly increased open arm exploration in the elevated plus maze in wild type, α1(H101R) and α3(H126R), but this effect was abolished in α2(H101R) mice; these were expected results based on previous published results. In contrast, while administration of diazepam and chlordiazepoxide significantly attenuated fear-potentiated startle (FPS) in wild type mice and α3(H126R) mice, the fear-reducing effects of these drugs were absent in both α1(H101R) and α2(H101R) point mutants, indicating that both α1- and α2-containing GABAA receptors are necessary for BZs to exert their effects on conditioned fear responses.. Our findings illustrate both an overlap and a divergence between the GABAA receptor subtype requirements for the impact of BZs, specifically that both α1- and α2-containing GABAA receptors are necessary for BZs to reduce conditioned fear whereas only α2-containing GABAA receptors are needed for BZ-induced anxiolysis in unconditioned tests of anxiety. This raises the possibility that GABAergic pharmacological interventions for specific anxiety disorders can be differentially tailored. PMID:22465203

The potentiating effect of calcitonin gene-related peptide on transient receptor potential vanilloid-1 activity and the electrophysiological responses of rat trigeminal neurons to nociceptive stimuli.

PubMed

Chatchaisak, Duangthip; Connor, Mark; Srikiatkhachorn, Anan; Chetsawang, Banthit

2018-05-01

Growing evidence suggests that calcitonin gene-related peptide (CGRP) participates in trigeminal nociceptive responses. However, the role of CGRP in sensitization or desensitization of nociceptive transduction remains poorly understood. In this study, we sought to further investigate the CGRP-induced up-regulation of transient receptor potential vanilloid-1 (TRPV1) and the responses of trigeminal neurons to nociceptive stimuli. Rat trigeminal ganglion (TG) organ cultures and isolated trigeminal neurons were incubated with CGRP. An increase in TRPV1 levels was observed in CGRP-incubated TG organ cultures. CGRP potentiated capsaicin-induced increase in phosphorylated CaMKII levels in the TG organ cultures. The incubation of the trigeminal neurons with CGRP significantly increased the inward currents in response to capsaicin challenge, and this effect was inhibited by co-incubation with the CGRP receptor antagonist, BIBN4068BS or the inhibitor of protein kinase A, H-89. These findings reveal that CGRP acting on trigeminal neurons may play a significant role in facilitating cellular events that contribute to the peripheral sensitization of the TG in nociceptive transmission.

Distribution profiles of transient receptor potential melastatin- and vanilloid-related channels in rat spermatogenic cells and sperm.

PubMed

Li, Shilin; Wang, Xinghuan; Ye, Haixia; Gao, Weicheng; Pu, Xiaoyong; Yang, Zhonghua

2010-03-01

In the present study, we aimed to investigate the expression and distribution of transient receptor potential melastatin (TRPM)- and vanilloid (TRPV)- related channels in rat spermatogenic cells and spermatozoa. Spermatogenic cells and spermatozoa were obtained from male Sprague-Dawley rats. Reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of all TRPM and TRPV channel members with specific primers. Western blot analysis was applied for detecting the expression of TRPM and TRPV channel proteins. Immunohistochemistry staining for TRPM4, TRPM7 and TRPV5 was also performed in rat testis. The mRNAs of TRPM3, TRPM4, TRPM7 and TRPV5 were detected in the spermatogenic cells and spermatozoa in rat. Western blot analysis verified the expression of TRPM4, TRPM7 and TRPV5 in the rat spermatogenic cells and spermatozoa. Immunocytochemistry staining for TRPM and TRPV channel families indicated that TRPM4 and TRPM7 proteins were highly expressed in different stages of spermatogenic cells and spermatozoa, while TRPV5 protein was lowly expressed in these cells. Our results demonstrate that mRNAs or proteins for TRPM3, TRPM4, TRPM7 and TRPV5 exist in rat spermatogenic cells and spermatozoa. These data presented here may assist in elucidating the possible physiological function of TRPM and TRPV channels in spermatogenic cells and spermatozoa.

Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling.

PubMed

Alea, Mileidys Perez; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Fuxe, Kjell; Garriga, Pere

2011-08-15

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function. Copyright © 2011 Elsevier B.V. All rights reserved.

Kappa2 opioid receptor subtype binding requires the presence of the DOR-1 gene.

PubMed

Ansonoff, Michael A; Wen, Ting; Pintar, John E

2010-01-01

Over the past several years substantial evidence has documented that opioid receptor homo- and heterodimers form in cell lines expressing one or more of the opioid receptors. We used opioid receptor knockout mice to determine whether in vivo pharmacological characteristics of kappa1 and kappa2 opioid receptors changed following knockout of specific opioid receptors. Using displacement of the general opioid ligand diprenorphine, we observed that occupancy or knockout of the DOR-1 gene increases the binding density of kappa1 receptors and eliminates kappa2 receptors in crude membrane preparations while the total density of kappa opioid binding sites is unchanged. Further, the analgesic potency of U69,593 in cumulative dose response curves is enhanced in mice lacking the DOR-1 gene. These results demonstrate that the DOR-1 gene is required for the expression of the kappa2 opioid receptor subtype and are consistent with the possibility that a KOR-1/DOR-1 heterodimer mediates kappa2 pharmacology.

The role of endogenous molecules in modulating pain through transient receptor potential vanilloid 1 (TRPV1)

PubMed Central

Morales-Lázaro, Sara L; Simon, Sidney A; Rosenbaum, Tamara

2013-01-01

Pain is a physiological response to a noxious stimulus that decreases the quality of life of those sufferring from it. Research aimed at finding new therapeutic targets for the treatment of several maladies, including pain, has led to the discovery of numerous molecular regulators of ion channels in primary afferent nociceptive neurons. Among these receptors is TRPV1 (transient receptor potential vanilloid 1), a member of the TRP family of ion channels. TRPV1 is a calcium-permeable channel, which is activated or modulated by diverse exogenous noxious stimuli such as high temperatures, changes in pH, and irritant and pungent compounds, and by selected molecules released during tissue damage and inflammatory processes. During the last decade the number of endogenous regulators of TRPV1's activity has increased to include lipids that can negatively regulate TRPV1, as is the case for cholesterol and PIP2 (phosphatidylinositol 4,5-biphosphate) while, in contrast, other lipids produced in response to tissue injury and ischaemic processes are known to positively regulate TRPV1. Among the latter, lysophosphatidic acid activates TRPV1 while amines such as N-acyl-ethanolamines and N-acyl-dopamines can sensitize or directly activate TRPV1. It has also been found that nucleotides such as ATP act as mediators of chemically induced nociception and pain and gases, such as hydrogen sulphide and nitric oxide, lead to TRPV1 activation. Finally, the products of lipoxygenases and omega-3 fatty acids among other molecules, such as divalent cations, have also been shown to endogenously regulate TRPV1 activity. Here we provide a comprehensive review of endogenous small molecules that regulate the function of TRPV1. Acting through mechanisms that lead to sensitization and desensitization of TRPV1, these molecules regulate pathways involved in pain and nociception. Understanding how these compounds modify TRPV1 activity will allow us to comprehend how some pathologies are associated with

The role of endogenous molecules in modulating pain through transient receptor potential vanilloid 1 (TRPV1).

PubMed

Morales-Lázaro, Sara L; Simon, Sidney A; Rosenbaum, Tamara

2013-07-01

Pain is a physiological response to a noxious stimulus that decreases the quality of life of those sufferring from it. Research aimed at finding new therapeutic targets for the treatment of several maladies, including pain, has led to the discovery of numerous molecular regulators of ion channels in primary afferent nociceptive neurons. Among these receptors is TRPV1 (transient receptor potential vanilloid 1), a member of the TRP family of ion channels. TRPV1 is a calcium-permeable channel, which is activated or modulated by diverse exogenous noxious stimuli such as high temperatures, changes in pH, and irritant and pungent compounds, and by selected molecules released during tissue damage and inflammatory processes. During the last decade the number of endogenous regulators of TRPV1's activity has increased to include lipids that can negatively regulate TRPV1, as is the case for cholesterol and PIP2 (phosphatidylinositol 4,5-biphosphate) while, in contrast, other lipids produced in response to tissue injury and ischaemic processes are known to positively regulate TRPV1. Among the latter, lysophosphatidic acid activates TRPV1 while amines such as N-acyl-ethanolamines and N-acyl-dopamines can sensitize or directly activate TRPV1. It has also been found that nucleotides such as ATP act as mediators of chemically induced nociception and pain and gases, such as hydrogen sulphide and nitric oxide, lead to TRPV1 activation. Finally, the products of lipoxygenases and omega-3 fatty acids among other molecules, such as divalent cations, have also been shown to endogenously regulate TRPV1 activity. Here we provide a comprehensive review of endogenous small molecules that regulate the function of TRPV1. Acting through mechanisms that lead to sensitization and desensitization of TRPV1, these molecules regulate pathways involved in pain and nociception. Understanding how these compounds modify TRPV1 activity will allow us to comprehend how some pathologies are associated with

Receptor mimicry by antibody F045–092 facilitates universal binding to the H3 subtype of influenza virus

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

Lee, Peter S.; Ohshima, Nobuko; Stanfield, Robyn L.

Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012–2013. Here we describe an antibody, F045–092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045–092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045–092 extends its recognition to divergent subtypes, including H1, H2 and H13, using the enhancedmore » avidity of its IgG to overcome lower-affinity Fab binding, as observed with other antibodies that target the receptor-binding site. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small-molecule therapeutics.« less

Racial Variations in Prostate Cancer Molecular Subtypes and Androgen Receptor Signaling Reflect Anatomic Tumor Location.

PubMed

Faisal, Farzana A; Sundi, Debasish; Tosoian, Jeffrey J; Choeurng, Voleak; Alshalalfa, Mohammed; Ross, Ashley E; Klein, Eric; Den, Robert; Dicker, Adam; Erho, Nicholas; Davicioni, Elai; Lotan, Tamara L; Schaeffer, Edward M

2016-07-01

Prostate cancer (PCa) subtypes based on ETS gene expression have been described. Recent studies suggest there are racial differences in tumor location, with PCa located anteriorly more often among African-American (AA) compared to Caucasian-American (CA) men. In this retrospective analysis of a multi-institutional cohort treated by radical prostatectomy (179 CA, 121 AA), we evaluated associations among molecular subtype, race, anatomic tumor location, and androgen receptor (AR) signaling. Subtype (m-ERG(+), m-ETS(+), m-SPINK1(+), or triple-negative) was determined using distribution-based outlier analysis. AR signaling was investigated using gene expression profiling of canonical AR targets. m-ERG(+) was more common in CA than AA men (47% vs 22%, p<0.001). AA men were more likely to be m-SPINK1(+) (13% vs 7%; p=0.069) and triple-negative (50% vs 37%; p=0.043). Racial differences in molecular subtypes did not persist when tumors were analyzed by location, suggesting a biologically important relationship between tumor location and subtype. Accordingly, anterior tumor location was associated with higher Decipher scores and lower global AR signaling. This study demonstrates associations among patient race, prostate cancer molecular subtypes, and tumor location. Location-specific differences in androgen regulation may further underlie these relationships. Copyright © 2015. Published by Elsevier B.V.

Pharmacological characterization of the cloned kappa opioid receptor as a kappa 1b subtype.

PubMed

Lai, J; Ma, S W; Zhu, R H; Rothman, R B; Lentes, K U; Porreca, F

1994-10-27

Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.

Receptor-Defined Subtypes of Breast Cancer in Indigenous Populations in Africa: A Systematic Review and Meta-Analysis

PubMed Central

Eng, Amanda; McCormack, Valerie; dos-Santos-Silva, Isabel

2014-01-01

Background Breast cancer is the most common female cancer in Africa. Receptor-defined subtypes are a major determinant of treatment options and disease outcomes but there is considerable uncertainty regarding the frequency of poor prognosis estrogen receptor (ER) negative subtypes in Africa. We systematically reviewed publications reporting on the frequency of breast cancer receptor-defined subtypes in indigenous populations in Africa. Methods and Findings Medline, Embase, and Global Health were searched for studies published between 1st January 1980 and 15th April 2014. Reported proportions of ER positive (ER+), progesterone receptor positive (PR+), and human epidermal growth factor receptor-2 positive (HER2+) disease were extracted and 95% CI calculated. Random effects meta-analyses were used to pool estimates. Fifty-four studies from North Africa (n = 12,284 women with breast cancer) and 26 from sub-Saharan Africa (n = 4,737) were eligible. There was marked between-study heterogeneity in the ER+ estimates in both regions (I2>90%), with the majority reporting proportions between 0.40 and 0.80 in North Africa and between 0.20 and 0.70 in sub-Saharan Africa. Similarly, large between-study heterogeneity was observed for PR+ and HER2+ estimates (I2>80%, in all instances). Meta-regression analyses showed that the proportion of ER+ disease was 10% (4%–17%) lower for studies based on archived tumor blocks rather than prospectively collected specimens, and 9% (2%–17%) lower for those with ≥40% versus those with <40% grade 3 tumors. For prospectively collected samples, the pooled proportions for ER+ and triple negative tumors were 0.59 (0.56–0.62) and 0.21 (0.17–0.25), respectively, regardless of region. Limitations of the study include the lack of standardized procedures across the various studies; the low methodological quality of many studies in terms of the representativeness of their case series and the quality of the procedures for collection

CO-LOCALIZATION OF THE VANILLOID CAPSAICIN RECEPTOR AND SUBSTANCE P IN SENSORY NERVE FIBERS INNERVATING COCHLEAR AND VERTEBRO-BASILAR ARTERIES

PubMed Central

VASS, Z.; DAI, C. F.; STEYGER, P. S.; JANCSÓ, G.; TRUNE, D. R.; NUTTALL, A. L.

2014-01-01

Evidence suggests that capsaicin-sensitive substance P (SP)-containing trigeminal ganglion neurons innervate the spiral modiolar artery (SMA), radiating arterioles, and the stria vascularis of the cochlea. Antidromic electrical or chemical stimulation of trigeminal sensory nerves results in neurogenic plasma extravasation in inner ear tissues. The primary aim of this study was to reveal the possible morphological basis of cochlear vascular changes mediated by capsaicin-sensitive sensory nerves. Therefore, the distribution of SP and capsaicin receptor (transient receptor potential vanilloid type 1—TRPV1) was investigated by double immunolabeling to demonstrate the anatomical relationships between the cochlear and vertebro-basilar blood vessels and the trigeminal sensory fiber system. Extensive TRPV1 and SP expression and co-localization were observed in axons within the adventitial layer of the basilar artery, the anterior inferior cerebellar artery, the SMA, and the radiating arterioles of the cochlea. There appears to be a functional relationship between the trigeminal ganglion and the cochlear blood vessels since electrical stimulation of the trigeminal ganglion induced significant plasma extravasation from the SMA and the radiating arterioles. The findings suggest that stimulation of paravascular afferent nerves may result in permeability changes in the basilar and cochlear vascular bed and may contribute to the mechanisms of vertebro-basilar type of headache through the release of SP and stimulation of TPVR1, respectively. We propose that vertigo, tinnitus, and hearing deficits associated with migraine may arise from perturbations of capsaicin-sensitive trigeminal sensory ganglion neurons projecting to the cochlea. PMID:15026132

Effects of nucleotides on [3H]bradykinin binding in guinea pig: further evidence for multiple B2 receptor subtypes.

PubMed

Seguin, L; Widdowson, P S

1993-02-01

We have suggested recently the existence of three subtypes of B2 bradykinin receptors in tissues of guinea pigs. We have classified these B2 bradykinin receptors into B2a, B2b, and B2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [3H]bradykinin to the B2 subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B2a and B2b subtypes, specific [3H]bradykinin binding was reduced with GDP (100 microM) and the nonmetabolized analogue of GTP, guanyl-5'-yl-imidodiphosphate (GppNHp; 100 microM). Competition studies with bradykinin and with [Hyp3]bradykinin, which shows approximately 20-fold greater selectivity for the B2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 microM). The competition experiments demonstrated that binding to the B2a subtype, which has higher affinity for [Hyp3]bradykinin and bradykinin than the B2b subtype, was lost in the presence of GppNHp, whereas binding to the B2b subtype was unaffected. In contrast, GppNHp (100 microM) and GDP (100 microM) failed to alter specific [3H]bradykinin binding to B2b and B2c subtypes in lung. [3H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 microM each). Based on this evidence, we suggest that the B2a bradykinin subtype is coupled to G proteins. The B2b and B2c subtypes are either not coupled to G proteins, or may be coupled to the Go-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular basis of urgency: regional difference of vanilloid receptor expression in the human urinary bladder.

PubMed

Liu, Lu; Mansfield, Kylie J; Kristiana, Ika; Vaux, Kenneth J; Millard, Richard J; Burcher, Elizabeth

2007-01-01

Treatments targeting vanilloid receptor TRPV1 are effective in some bladder disorders. Our aim was to determine the expression profiles of TRPV1 in regions of human bladder and test the hypothesis that there would be an upregulation of TRPV1 in mucosa of patients with bladder hypersensitivity but not idiopathic detrusor overactivity (IDO). Women with sensory urgency (SU), interstitial cystitis (IC), and IDO were investigated by videourodynamics and cystoscopy. Control biopsies were used for comparison. Biopsies were dissected into mucosa and muscle, and evaluated for TRPV1 mRNA expression using quantitative competitive RT-PCR (QC-RT-PCR). TRPV1 mRNA from SU trigonal mucosa was significantly higher than control trigonal mucosa or SU bladder body mucosa. In contrast, in IDO patients, there was no difference between trigonal mucosa and body mucosa. In IC biopsies, RNA quality was substandard and unable to be used for analysis. The most striking finding was that TRPV1 mRNA expressed in SU trigonal mucosa was significantly inversely correlated with the bladder volume at first sensation of filling during cystometry. No such relationship was seen for IDO trigonal mucosa. No difference was seen in bladder body mucosa from any disease groups compared with age-matched control. The symptoms of SU were associated with the increased expression of TRPV1 mRNA in the trigonal mucosa. No upregulation or regional differences of TRPV1 mRNA were seen in IDO patients. TRPV1 may play a role in SU and premature first bladder sensation on filling.

Quantitative autoradiographic analysis of muscarinic receptor subtypes and their role in representational memory

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

Messer, W.S.

1986-01-01

Autoradiographic techniques were used to examine the distribution of muscarinic receptors in rat brain slices. Agonist and selective antagonist binding were examined by measuring the ability for unlabeled ligands to inhibit (/sup 3/H)-1-QNB labeling of muscarinic receptors. The distribution of high affinity pirenzepine binding sites (M/sub 1/ subtype) was distinct from the distribution of high affinity carbamylcholine sites, which corresponded to the M/sub 2/ subtype. In a separate assay, the binding profile for pirenzepine was shown to differ from the profile for scopolamine, a classical muscarinic antagonist. Muscarinic antagonists, when injected into the Hippocampus, impaired performance of a representational memorymore » task. Pirenzepine, the M/sub 1/ selective antagonist, produced representational memory deficits. Scopolamine, a less selective muscarinic antagonist, caused increases in running times in some animals which prevented a definitive interpretation of the nature of the impairment. Pirenzepine displayed a higher affinity for the hippocampus and was more effective in producing a selective impairment of representational memory than scopolamine. The data indicated that cholinergic activity in the hippocampus was necessary for representation memory function.« less

Functional acetylcholine muscarinic receptor subtypes in human brain microcirculation: identification and cellular localization.

PubMed

Elhusseiny, A; Cohen, Z; Olivier, A; Stanimirović, D B; Hamel, E

1999-07-01

Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P<0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P<0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AFDX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood-brain barrier permeability, functions that could become

Structure-based prediction of subtype selectivity of histamine H3 receptor selective antagonists in clinical trials.

PubMed

Kim, Soo-Kyung; Fristrup, Peter; Abrol, Ravinder; Goddard, William A

2011-12-27

Histamine receptors (HRs) are excellent drug targets for the treatment of diseases, such as schizophrenia, psychosis, depression, migraine, allergies, asthma, ulcers, and hypertension. Among them, the human H(3) histamine receptor (hH(3)HR) antagonists have been proposed for specific therapeutic applications, including treatment of Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), epilepsy, and obesity. However, many of these drug candidates cause undesired side effects through the cross-reactivity with other histamine receptor subtypes. In order to develop improved selectivity and activity for such treatments, it would be useful to have the three-dimensional structures for all four HRs. We report here the predicted structures of four HR subtypes (H(1), H(2), H(3), and H(4)) using the GEnSeMBLE (GPCR ensemble of structures in membrane bilayer environment) Monte Carlo protocol, sampling ∼35 million combinations of helix packings to predict the 10 most stable packings for each of the four subtypes. Then we used these 10 best protein structures with the DarwinDock Monte Carlo protocol to sample ∼50 000 × 10(20) poses to predict the optimum ligand-protein structures for various agonists and antagonists. We find that E206(5.46) contributes most in binding H(3) selective agonists (5, 6, 7) in agreement with experimental mutation studies. We also find that conserved E5.46/S5.43 in both of hH(3)HR and hH(4)HR are involved in H(3)/ H(4) subtype selectivity. In addition, we find that M378(6.55) in hH(3)HR provides additional hydrophobic interactions different from hH(4)HR (the corresponding amino acid of T323(6.55) in hH(4)HR) to provide additional subtype bias. From these studies, we developed a pharmacophore model based on our predictions for known hH(3)HR selective antagonists in clinical study [ABT-239 1, GSK-189,254 2, PF-3654746 3, and BF2.649 (tiprolisant) 4] that suggests critical selectivity directing elements are: the basic proton

Metabotropic glutamate receptor subtype 5: molecular pharmacology, allosteric modulation and stimulus bias

PubMed Central

Sengmany, K

2015-01-01

The metabotropic glutamate receptor subtype 5 (mGlu5) is a family C GPCR that has been implicated in various neuronal processes and, consequently, in several CNS disorders. Over the past few decades, GPCR‐based drug discovery, including that for mGlu5 receptors, has turned considerable attention to targeting allosteric binding sites. Modulation of endogenous agonists by allosteric ligands offers the advantages of spatial and temporal fine‐tuning of receptor activity, increased selectivity and reduced adverse effects with the potential to elicit improved clinical outcomes. Further, with greater appreciation of the multifaceted nature of the transduction of mGlu5 receptor signalling, it is increasingly apparent that drug discovery must take into consideration unique receptor conformations and the potential for stimulus‐bias. This novel paradigm proposes that different ligands may differentially modulate distinct signalling pathways arising from the same receptor. We review our current understanding of the complexities of mGlu5 receptor signalling and regulation, and how these relate to allosteric ligands. Ultimately, a deeper appreciation of these relationships will provide the foundation for targeted drug design of compounds with increased selectivity, not only for the desired receptor but also for the desired signalling outcome from the receptor. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein‐Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc PMID:26276909

Reduced Serotonin Receptor Subtypes in a Limbic and a Neocortical Region in Autism

PubMed Central

Oblak, Adrian; Gibbs, Terrell T.; Blatt, Gene J.

2013-01-01

Autism is a behaviorally defined, neurological disorder with symptom onset before the age of three. Abnormalities in social-emotional behaviors are a core deficit in autism and are characterized by impaired reciprocal social interaction, lack of facial expressions, and the inability to recognize familiar faces. The posterior cingulate cortex (PCC) and fusiform gyrus (FG) are two regions within an extensive limbic-cortical network that contribute to social-emotional behaviors. Evidence indicates that changes in brains of individuals with autism begin prenatally. Serotonin (5HT) is one of the earliest expressed neurotransmitters, and plays an important role in synaptogenesis, neurite outgrowth, and neuronal migration. Abnormalities in 5HT systems have been implicated in several psychiatric disorders including autism, as evidenced by immunology, imaging, genetics, pharmacotherapy, and neuropathology. Although information is known regarding peripheral 5HT in autism, there is emerging evidence that 5HT systems in the CNS, including various 5HT receptor subtypes and transporters, are affected in autism. The present study demonstrated significant reductions in 5HT1A receptor binding density in superficial and deep layers of the PCC and FG, and in the density of 5HT2A receptors in superficial layers of the PCC and FG. Significant reduction in the density of serotonin transporters (5-HTT) was also found in the deep layers of the FG, but normal levels were demonstrated in both layers of the PCC and superficial layers of the FG. These studies provide potential substrates for decreased 5-HT modulation/innervation in the autism brain, and implicate two 5-HT receptor subtypes as potential neuromarkers for novel or existing pharmacotherapies. PMID:23894004

Immunohistochemical Localization of AT1a, AT1b, and AT2 Angiotensin II Receptor Subtypes in the Rat Adrenal, Pituitary, and Brain with a Perspective Commentary

PubMed Central

Premer, Courtney; Lamondin, Courtney; Mitzey, Ann; Speth, Robert C.; Brownfield, Mark S.

2013-01-01

Angiotensin II increases blood pressure and stimulates thirst and sodium appetite in the brain. It also stimulates secretion of aldosterone from the adrenal zona glomerulosa and epinephrine from the adrenal medulla. The rat has 3 subtypes of angiotensin II receptors: AT1a, AT1b, and AT2. mRNAs for all three subtypes occur in the adrenal and brain. To immunohistochemically differentiate these receptor subtypes, rabbits were immunized with C-terminal fragments of these subtypes to generate receptor subtype-specific antibodies. Immunofluorescence revealed AT1a and AT2 receptors in adrenal zona glomerulosa and medulla. AT1b immunofluorescence was present in the zona glomerulosa, but not the medulla. Ultrastructural immunogold labeling for the AT1a receptor in glomerulosa and medullary cells localized it to plasma membrane, endocytic vesicles, multivesicular bodies, and the nucleus. AT1b and AT2, but not AT1a, immunofluorescence was observed in the anterior pituitary. Stellate cells were AT1b positive while ovoid cells were AT2 positive. In the brain, neurons were AT1a, AT1b, and AT2 positive, but glia was only AT1b positive. Highest levels of AT1a, AT1b, and AT2 receptor immunofluorescence were in the subfornical organ, median eminence, area postrema, paraventricular nucleus, and solitary tract nucleus. These studies complement those employing different techniques to characterize Ang II receptors. PMID:23573410

Expression Profiling of the Transient Receptor Potential Vanilloid (TRPV) Channels 1, 2, 3 and 4 in Mucosal Epithelium of Human Ulcerative Colitis.

PubMed

Rizopoulos, Theodoros; Papadaki-Petrou, Helen; Assimakopoulou, Martha

2018-06-15

The Transient Receptor Potential (TRP) family of selective and non-selective ion channels is well represented throughout the mammalian gastrointestinal track. Several members of the Transient Receptor Potential Vanilloid (TRPV) subfamily have been identified in contributing to modulation of mobility, secretion and sensitivity of the human intestine. Previous studies have focused on the detection of TRPV mRNA levels in colon tissue of patients with inflammatory bowel disease (IBD) whereas little information exists regarding TRPV channel expression in the colonic epithelium. The aim of this study was to evaluate the expression levels of TRPV1, TRPV2, TRPV3 and TRPV4 in mucosa epithelial cells of colonic biopsies from patients with ulcerative colitis (UC) in comparison to colonic resections from non-IBD patients (control group). Immunohistochemistry, using specific antibodies and quantitative analyses of TRPV-immunostained epithelial cells, was performed in semi-serial sections of the samples. TRPV1 expression was significantly decreased whereas TRPV4 expression was significantly increased in the colonic epithelium of UC patients compared to patients in the control group ( p < 0.05). No significant difference for TRPV2 and TRPV3 expression levels between UC and control specimens was detected ( p > 0.05). There was no correlation between TRPV channel expression and the clinical features of the disease ( p > 0.05). Further investigation is needed to clarify the role of TRPV channels in human bowel inflammatory response.

Randomised clinical trial: the efficacy of a transient receptor potential vanilloid 1 antagonist AZD1386 in human oesophageal pain.

PubMed

Krarup, A L; Ny, L; Astrand, M; Bajor, A; Hvid-Jensen, F; Hansen, M B; Simrén, M; Funch-Jensen, P; Drewes, A M

2011-05-01

Many patients with gastro-oesophageal reflux disease (GERD) are hypersensitive to heat and acid and may respond insufficiently to standard treatment. Antagonists of the heat and acid receptor 'transient receptor potential vanilloid 1'(TRPV1) are a potential drug class for GERD treatment. To investigate the effect of a TRPV1 antagonist (AZD1386) on experimentally induced oesophageal pain. Twenty-two healthy men (20-31 years) participated in this randomised, placebo-controlled, double-blinded, crossover study examining the effects of a single-dose oral AZD1386 (30 and 95 mg). Subjects were block-randomised. On treatment days, participants were stimulated with painful heat, distension, electrical current and acid in the oesophagus. Heat and pressure pain on the forearm were somatic control stimuli. intention-to-treat. A total of 21 participants completed the protocol and 1 voluntarily discontinued. In the oesophagus, both 30 and 95 mg of AZD1386 increased pain thresholds to heat stimuli 23% [95% confidence interval (CI): 10-38%] and 28%, respectively (CI: 14-43%). The skin heat tolerance was increased 2.1 °C (CI: 1.1-3.2 °C) after 30 mg AZD1386 and 4.0 °C (CI: 3.0-5.0 °C) after 95 mg. Heat analgesia persisted for 2.5 h. Pain thresholds to the other stimuli were unaffected by AZD1386. 50% reported 'feeling cold' and body temperature increased in all subjects exposed to 30 and 95 mg AZD1386 (mean increase 0.4±0.3 °C and 0.7±0.3 °C, respectively, P<0.05). AZD1386 increased oesophageal and skin heat pain thresholds and had a safe adverse-event profile. This drug class may have a potential for treatment of GERD. © 2011 Blackwell Publishing Ltd.

Reduced serotonin receptor subtypes in a limbic and a neocortical region in autism.

PubMed

Oblak, Adrian; Gibbs, Terrell T; Blatt, Gene J

2013-12-01

Autism is a behaviorally defined, neurological disorder with symptom onset before the age of 3. Abnormalities in social-emotional behaviors are a core deficit in autism, and are characterized by impaired reciprocal-social interaction, lack of facial expressions, and the inability to recognize familiar faces. The posterior cingulate cortex (PCC) and fusiform gyrus (FG) are two regions within an extensive limbic-cortical network that contribute to social-emotional behaviors. Evidence indicates that changes in brains of individuals with autism begin prenatally. Serotonin (5-HT) is one of the earliest expressed neurotransmitters, and plays an important role in synaptogenesis, neurite outgrowth, and neuronal migration. Abnormalities in 5-HT systems have been implicated in several psychiatric disorders, including autism, as evidenced by immunology, imaging, genetics, pharmacotherapy, and neuropathology. Although information is known regarding peripheral 5-HT in autism, there is emerging evidence that 5-HT systems in the central nervous system, including various 5-HT receptor subtypes and transporters, are affected in autism. The present study demonstrated significant reductions in 5-HT1A receptor-binding density in superficial and deep layers of the PCC and FG, and in the density of 5-HT(2A) receptors in superficial layers of the PCC and FG. A significant reduction in the density of serotonin transporters (5-HTT) was also found in the deep layers of the FG, but normal levels were demonstrated in both layers of the PCC and superficial layers of the FG. This study provides potential substrates for decreased 5-HT modulation/innervation in the autism brain, and implicate two 5-HT receptor subtypes as potential neuromarkers for novel or existing pharmacotherapies. © 2013 International Society for Autism Research, Wiley Periodicals, Inc.

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease

PubMed Central

Karmouty-Quintana, Harry; Cicko, Sanja; Ayata, Korcan; Zissel, Gernot; Goldmann, Torsten; Lungarella, Giuseppe; Ferrari, Davide; Di Virgilio, Francesco; Robaye, Bernard; Boeynaems, Jean-Marie; Blackburn, Michael R.; Idzko, Marco

2017-01-01

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases. The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms. Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes. Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF. PMID:28415591

Short-term increases in transient receptor potential vanilloid-1 mediate stress-induced enhancement of neuronal excitation.

PubMed

Weitlauf, Carl; Ward, Nicholas J; Lambert, Wendi S; Sidorova, Tatiana N; Ho, Karen W; Sappington, Rebecca M; Calkins, David J

2014-11-12

Progression of neurodegeneration in disease and injury is influenced by the response of individual neurons to stressful stimuli and whether this response includes mechanisms to counter declining function. Transient receptor potential (TRP) cation channels transduce a variety of disease-relevant stimuli and can mediate diverse stress-dependent changes in physiology, both presynaptic and postsynaptic. Recently, we demonstrated that knock-out or pharmacological inhibition of the TRP vanilloid-1 (TRPV1) capsaicin-sensitive subunit accelerates degeneration of retinal ganglion cell neurons and their axons with elevated ocular pressure, the critical stressor in the most common optic neuropathy, glaucoma. Here we probed the mechanism of the influence of TRPV1 on ganglion cell survival in mouse models of glaucoma. We found that induced elevations of ocular pressure increased TRPV1 in ganglion cells and its colocalization at excitatory synapses to their dendrites, whereas chronic elevation progressively increased ganglion cell Trpv1 mRNA. Enhanced TRPV1 expression in ganglion cells was transient and supported a reversal of the effect of TRPV1 on ganglion cells from hyperpolarizing to depolarizing, which was also transient. Short-term enhancement of TRPV1-mediated activity led to a delayed increase in axonal spontaneous excitation that was absent in ganglion cells from Trpv1(-/-) retina. In isolated ganglion cells, pharmacologically activated TRPV1 mobilized to discrete nodes along ganglion cell dendrites that corresponded to sites of elevated Ca(2+). These results suggest that TRPV1 may promote retinal ganglion cell survival through transient enhancement of local excitation and axonal activity in response to ocular stress. Copyright © 2014 the authors 0270-6474/14/3415369-13$15.00/0.

Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide opiate receptor subtype.

PubMed

Mantione, K; Zhu, W; Rialas, C; Casares, F; Cadet, P; Franklin, A L; Tonnesen, J; Stefano, G B

2002-03-01

We have previously demonstrated that Mytilus edulis pedal ganglia contain opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu opiate receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu opiate receptor was present and selective toward M6G. In examining a receptor displacement analysis, both opiate alkaloids displaced [3H]-dihydromorphine binding to the mu opiate receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu opiate receptor may be present.

Somatostatin, acting at receptor subtype 1, inhibits Rho activity, the assembly of actin stress fibers, and cell migration.

PubMed

Buchan, Alison M J; Lin, Chin-Yu; Choi, Jimmy; Barber, Diane L

2002-08-09

Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin- and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.

Molecular cloning and characterization of chicken prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4).

PubMed

Kwok, Amy Ho Yan; Wang, Yajun; Wang, Crystal Ying; Leung, Frederick C

2008-06-01

Prostaglandin E(2) (PGE(2)) is an important chemical mediator responsible for regulation of many vital physiological processes. Four receptor subtypes have been identified to mediate its biological actions. Among these subtypes, prostaglandin E receptor subtypes 2 and 4 (EP(2) and EP(4)), both coupled to cAMP-protein kinase A (cAMP-PKA) signaling pathway, are proposed to play crucial roles under both physiological and pathological conditions. Though both receptors were extensively studied in mammals, little is known about their functionality and expression in non-mammalian species including chicken. In present study, the full-length cDNAs for chicken EP(2) and EP(4) receptors were first cloned from adult chicken ovary and testis, respectively. Chicken EP(2) is 356 amino acids in length and shows high amino acid identity to that of human (61%), mouse (63%), and rat (61%). On the other hand, the full-length cDNA of EP(4) gene encodes a precursor of 475 amino acids with a high degree of amino acid identity to that of mammals, including human (87%), mouse (86%), rat (84%), dog (85%), and cattle (83%), and a comparatively lower sequence identity to zebrafish (52%). RT-PCR assays revealed that EP(2) mRNA was expressed in all tissues examined including the oviduct, while EP(4) expression was detected only in a few tissues. Using the pGL3-CRE-luciferase reporter system, we also demonstrated that PGE(2) could induce luciferase activity in DF-1 cells expressing EP(2) and EP(4) in dose-dependent manners (EC(50): <1 nM), confirming that both receptors could be activated by PGE(2) and functionally coupled to the cAMP-PKA signaling pathway. Together, our study establishes a molecular basis to understand the physiological roles of PGE(2) in target tissues of chicken.

Forsythoside A exerts antipyretic effect on yeast-induced pyrexia mice via inhibiting transient receptor potential vanilloid 1 function

PubMed Central

Liu, Cuiling; Su, Hongchang; Wan, Hongye; Qin, Qingxia; Wu, Xuan; Kong, Xiangying; Lin, Na

2017-01-01

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel gated by noxious heat, playing major roles in thermoregulation. Forsythoside A (FT-A) is the most abundant phenylethanoid glycosides in Fructus Forsythiae, which has been prescribed as a medicinal herb for treating fever in China for a long history. However, how FT-A affects pyrexia and what is the underlying molecular mechanism remain largely unknown. Here we found that FT-A exerted apparent antipyretic effect through decreasing the levels of prostaglandin E2 (PGE2) and interleukin 8 (IL-8) in a dose-dependent fashion on the yeast induced pyrexia mice. Interestingly, FT-A significantly downregulated TRPV1 expression in the hypothalamus and dorsal root ganglion (DRG) of the yeast induced pyrexia mice. Moreover, FT-A inhibited IL-8 and PGE2 secretions, and calcium influx in the HEK 293T-TRPV1 cells after stimulated with capsaicin, the specific TRPV1 agonist. Further investigation of the molecular mechanisms revealed that FT-A treatment rapidly inhibited phosphorylation of extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 in both yeast induced pyrexia mice and HEK 293T-TRPV1 cells. These results suggest that FT-A may serve as a potential antipyretic agent and the therapeutic action of Fructus Forsythiae on pyretic related disease is, in part, due to the FT-A activities. PMID:28123347

4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes.

PubMed

Madsen, Ulf; Pickering, Darryl S; Nielsen, Birgitte; Bräuner-Osborne, Hans

2005-01-01

4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies, and the series of 4-alkyl-HIBO analogues have been extended in this paper in the search for versatile agents. Pharmacological characterization of five new analogues, branched and unbranched 4-alkyl-HIBO analogues, have been carried out. The present compounds are all weak antagonists at Group I mGluRs (mGluR1 and 5) presenting only small differences in potencies (Ki values ranging from 89 to 670 microM). Affinities were studied at native and cloned iGluRs, and the compounds described show preference for the AMPA receptor subtypes GluR1 and 2 over GluR3 and 4. However, compared to previous 4-alkyl-HIBO analogues, these compounds show a remarkably high affinity for the Kain preferring subtype GluR5. The observed GluR5 affinities were either similar or higher compared to their GluR1 and 2 affinity. Isopropyl-HIBO showed the highest affinity for GluR5 (Ki=0.16 microM), and represents a unique compound with high affinity towards the three subtypes GluR1, 2 and 5. In general, these compounds represent new selectivity profiles compared to previously reported Glu receptor analogues.

Muscarinic acetylcholine receptor subtype expression in avian vestibular hair cells, nerve terminals and ganglion cells.

PubMed

Li, G Q; Kevetter, G A; Leonard, R B; Prusak, D J; Wood, T G; Correia, M J

2007-04-25

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and peripheral nervous system and play an important role in modulating the cell activity and function. We have shown that the cholinergic agonist carbachol reduces the pigeon's inwardly rectifying potassium channel (pKir2.1) ionic currents in native vestibular hair cells. We have cloned and sequenced pigeon mAChR subtypes M2-M5 and we have studied the expression of all five mAChR subtypes (M1-M5) in the pigeon vestibular end organs (semicircular canal ampullary cristae and utricular maculae), vestibular nerve fibers and the vestibular (Scarpa's) ganglion using tissue immunohistochemistry (IH), dissociated single cell immunocytochemistry (IC) and Western blotting (WB). We found that vestibular hair cells, nerve fibers and ganglion cells each expressed all five (M1-M5) mAChR subtypes. Two of the three odd-numbered mAChRs (M1, M5) were present on the hair cell cilia, supporting cells and nerve terminals. And all three odd numbered mAChRs (M1, M3 and M5) were expressed on cuticular plates, myelin sheaths and Schwann cells. Even-numbered mAChRs were seen on the nerve terminals. M2 was also shown on the cuticular plates and supporting cells. Vestibular efferent fibers and terminals were not identified in our studies. Results from WB of the dissociated vestibular epithelia, nerve fibers and vestibular ganglia were consistent with the results from IH and IC. Our findings suggest that there is considerable co-expression of the subtypes on the neural elements of the labyrinth. Further electrophysiological and pharmacological studies should delineate the mechanisms of action of muscarinic acetylcholine receptors on structures in the labyrinth.

Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons

PubMed Central

Cohen, Matthew R.; Johnson, William M.; Pilat, Jennifer M.; Kiselar, Janna; DeFrancesco-Lisowitz, Alicia; Zigmond, Richard E.

2015-01-01

Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca2+-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca2+-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca2+ signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca2+ signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function. PMID:26416880

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

PubMed

Costa, M Adelina; Barbosa, A; Neto, E; Sá-e-Sousa, A; Freitas, R; Neves, J M; Magalhães-Cardoso, T; Ferreirinha, F; Correia-de-Sá, P

2011-05-01

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation. Copyright © 2010 Wiley-Liss, Inc.

Differential Regulation of Cell Proliferation and Apoptosis by Melatonin Receptor Subtype-Signaling in the Adult Murine Brain.

PubMed

Fredrich, Michaela; Christ, Elmar; Korf, Horst-Werner

2018-06-27

Background/Aims: Zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis are regulated by melatonin receptor (MT)-mediated signaling in the adult hippocampus and hypothalamic-hypophyseal system. There are two G-protein-coupled MT-subtypes, MT1 and MT2. Therefore, the present study examined which MT-subtype is required for regulation of ZT-dependent changes in cell proliferation and/or apoptosis in the adult murine brain and pituitary. Adult melatonin-proficient (C3H) mice with targeted deletion of MT1 (MT1 KO) or MT2 (MT2 KO) were adapted to a 12-hour light, 12-hour dark photoperiod and sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 or activated caspase-3 served to quantify proliferating and apoptotic cells in the hippocampal subgranular zone (SGZ) and granule cell layer, the hypothalamic median eminence (ME), and the hypophyseal pars tuberalis. ZT-dependent changes in cell proliferation were found exclusively in the SGZ and ME of MT1 KO mice, while apoptosis showed no ZT-dependent changes in the regions analyzed, neither in MT1 nor in MT2 KO mice. Comparison with our previous studies in C3H mice with functional MTs and MT1/2 KO mice revealed that MT2-mediated signaling is required and sufficient for ZT-dependent changes in cell proliferation in the SGZ and ME, while ZT-dependent changes in apoptosis require signaling from both MT-subtypes. Our results indicate that generation and timing of ZT-dependent changes in cell proliferation and apoptosis by melatonin require different MT-subtype-constellations and emphasize the importance to shed light on the specific function of each receptor-subtype in different tissues and physiological conditions.
. ©2018S. Karger AG, Basel.

Muscarinic receptor subtypes involved in urothelium-derived relaxatory effects in the inflamed rat urinary bladder.

PubMed

Andersson, M; Aronsson, P; Doufish, D; Lampert, A; Tobin, G

2012-09-25

Functional studies have shown altered cholinergic mechanisms in the inflamed bladder, which partly depend on muscarinic receptor-induced release of nitric oxide (NO). The current study aimed to characterize which muscarinic receptor subtypes that are involved in the regulation of the nitrergic effects in the bladder cholinergic response during cystitis. For this purpose, in vitro examinations of carbachol-evoked contractions of inflamed and normal bladder preparations were performed. The effects of antagonists with different selectivity for the receptor subtypes were assessed on intact and urothelium-denuded bladder preparations. In preparations from cyclophosphamide (CYP; in order to induce cystitis) pre-treated rats, the response to carbachol was about 75% of that of normal preparations. Removal of the urothelium or administration of a nitric oxide synthase inhibitor re-established the responses in the inflamed preparations. Administration of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the carbachol-induced contractile responses of preparations from CYP pre-treated rats less potently than controls. Pirenzepine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) affected the carbachol-induced contractile responses to similar extents in preparations of CYP pre-treated and control rats. However, the Schild slopes for the three antagonists were all significantly different from unity in the preparations from CYP pre-treated rats. Again, L-NNA or removal of the urothelium eliminated any difference compared to normal preparations. This study confirms that muscarinic receptor stimulation in the inflamed rat urinary bladder induces urothelial release of NO, which counteracts detrusor contraction. Copyright © 2012 Elsevier B.V. All rights reserved.

Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

PubMed

Ilyaskina, Olga S; Lemoine, Horst; Bünemann, Moritz

2018-05-08

G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of G i/o , G q/11 , G s , and G 12/13 proteins to muscarinic M 1 , M 2 , and M 3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M 3 -R interactions but rather the affinities of G q and G o proteins to M 3 -Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

What Can Crystal Structures of Aminergic Receptors Tell Us about Designing Subtype-Selective Ligands?

PubMed Central

Michino, Mayako; Beuming, Thijs; Donthamsetti, Prashant; Newman, Amy Hauck; Javitch, Jonathan A.

2015-01-01

G protein–coupled receptors (GPCRs) are integral membrane proteins that represent an important class of drug targets. In particular, aminergic GPCRs interact with a significant portion of drugs currently on the market. However, most drugs that target these receptors are associated with undesirable side effects, which are due in part to promiscuous interactions with close homologs of the intended target receptors. Here, based on a systematic analysis of all 37 of the currently available high-resolution crystal structures of aminergic GPCRs, we review structural elements that contribute to and can be exploited for designing subtype-selective compounds. We describe the roles of secondary binding pockets (SBPs), as well as differences in ligand entry pathways to the orthosteric binding site, in determining selectivity. In addition, using the available crystal structures, we have identified conformational changes in the SBPs that are associated with receptor activation and explore the implications of these changes for the rational development of selective ligands with tailored efficacy. PMID:25527701

Characterization of Transient Receptor Potential Vanilloid-1 (TRPV1) Variant Activation by Coal Fly Ash Particles and Associations with Altered Transient Receptor Potential Ankyrin-1 (TRPA1) Expression and Asthma*

PubMed Central

Stockmann, Chris; Romero, Erin G.; Lu, Zhenyu; Shapiro, Darien; Stone, Bryan L.; Fassl, Bernhard; Nkoy, Flory; Uchida, Derek A.; Ward, Robert M.; Veranth, John M.; Reilly, Christopher A.

2016-01-01

Transient receptor potential (TRP) channels are activated by environmental particulate materials. We hypothesized that polymorphic variants of transient receptor potential vanilloid-1 (TRPV1) would be uniquely responsive to insoluble coal fly ash compared with the prototypical soluble agonist capsaicin. Furthermore, these changes would manifest as differences in lung cell responses to these agonists and perhaps correlate with changes in asthma symptom control. The TRPV1-I315M and -T469I variants were more responsive to capsaicin and coal fly ash. The I585V variant was less responsive to coal fly ash particles due to reduced translation of protein and an apparent role for Ile-585 in activation by particles. In HEK-293 cells, I585V had an inhibitory effect on wild-type TRPV1 expression, activation, and internalization/agonist-induced desensitization. In normal human bronchial epithelial cells, IL-8 secretion in response to coal fly ash treatment was reduced for cells heterozygous for TRPV1-I585V. Finally, both the I315M and I585V variants were associated with worse asthma symptom control with the effects of I315M manifesting in mild asthma and those of the I585V variant manifesting in severe, steroid-insensitive individuals. This effect may be due in part to increased transient receptor potential ankyrin-1 (TRPA1) expression by lung epithelial cells expressing the TRPV1-I585V variant. These findings suggest that specific molecular interactions control TRPV1 activation by particles, differential activation, and desensitization of TRPV1 by particles and/or other agonists, and cellular changes in the expression of TRPA1 as a result of I585V expression could contribute to variations in asthma symptom control. PMID:27758864

Characterization of Transient Receptor Potential Vanilloid-1 (TRPV1) Variant Activation by Coal Fly Ash Particles and Associations with Altered Transient Receptor Potential Ankyrin-1 (TRPA1) Expression and Asthma.

PubMed

Deering-Rice, Cassandra E; Stockmann, Chris; Romero, Erin G; Lu, Zhenyu; Shapiro, Darien; Stone, Bryan L; Fassl, Bernhard; Nkoy, Flory; Uchida, Derek A; Ward, Robert M; Veranth, John M; Reilly, Christopher A

2016-11-25

Transient receptor potential (TRP) channels are activated by environmental particulate materials. We hypothesized that polymorphic variants of transient receptor potential vanilloid-1 (TRPV1) would be uniquely responsive to insoluble coal fly ash compared with the prototypical soluble agonist capsaicin. Furthermore, these changes would manifest as differences in lung cell responses to these agonists and perhaps correlate with changes in asthma symptom control. The TRPV1-I315M and -T469I variants were more responsive to capsaicin and coal fly ash. The I585V variant was less responsive to coal fly ash particles due to reduced translation of protein and an apparent role for Ile-585 in activation by particles. In HEK-293 cells, I585V had an inhibitory effect on wild-type TRPV1 expression, activation, and internalization/agonist-induced desensitization. In normal human bronchial epithelial cells, IL-8 secretion in response to coal fly ash treatment was reduced for cells heterozygous for TRPV1-I585V. Finally, both the I315M and I585V variants were associated with worse asthma symptom control with the effects of I315M manifesting in mild asthma and those of the I585V variant manifesting in severe, steroid-insensitive individuals. This effect may be due in part to increased transient receptor potential ankyrin-1 (TRPA1) expression by lung epithelial cells expressing the TRPV1-I585V variant. These findings suggest that specific molecular interactions control TRPV1 activation by particles, differential activation, and desensitization of TRPV1 by particles and/or other agonists, and cellular changes in the expression of TRPA1 as a result of I585V expression could contribute to variations in asthma symptom control. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Muscarinic receptor M1 and M2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro autoradiography.

PubMed Central

Gupta, N; McAllister, R; Drance, S M; Rootman, J; Cynader, M S

1994-01-01

Muscarinic cholinergic agents are used to lower intraocular pressure in the medical management of glaucoma and subtypes of muscarinic receptors have now been recognised in many tissues including the eye. To localise muscarinic receptors and their M1 and M2 subtypes in the human eye, in vitro ligand binding and autoradiographic techniques with densitometric quantitation on postmortem eye sections were used. As ligands, [3H] quinuclydinyl benzylate (QNB) (non-subtype specific muscarinic antagonist), [3H]pirenzipine (M1 antagonist), [3H]oxotremorine (M2 muscarinic agonist), [3H]AFDX-116(11[(2[diethylaminomethyl]1-piperidinyl)acetyl]5 , 11dihydro-6H-pyrido [2,3b][1,4]benzodiazepine-6-one) (M2 antagonist) were studied. Specific binding sites for QNB, pirenzipine, and AFDX-116 were localised in the entire ciliary muscle, the iris, and ciliary epithelium. [3H]oxotremorine localised only in the longitudinal portion of the ciliary muscle, and additionally, was not localised in the iris or ciliary epithelium. These results suggest that oxotremorine, by binding selectively to receptors on the longitudinal ciliary muscle and inducing its contraction, may modulate outflow facility independently from accommodation and miosis. Images PMID:7918268

Improvements in the Methodology for Analyzing Receptor Subtypes and Neuronal Populations Affected by Anticholinesterase Exposure.

DTIC Science & Technology

1984-11-14

Slide-mounted tissue sections can be treated with [ H]forskolin (a diterpene plant derivative which is a potent activator of adenylate cyclase) to...protein activities are altered in response to the chronic presence of anticholinesterase agents. Significant progress and improvement has been made in...359 FILE COPY IMPROVEMENTS IN THE METHODOLOGY FOR ANALYZING RECEPTOR SUBTYPES AND NEURONAL POPULATIONS AFFECTED BY ANTICHOLINESTERASE EXPOSURE Annual

Modulation of transient receptor potential vanilloid 4-mediated membrane currents and synaptic transmission by protein kinase C

PubMed Central

Cao, De-Shou; Yu, Shuang-Quan; Premkumar, Louis S

2009-01-01

Background Transient receptor potential Vanilloid (TRPV) receptors are involved in nociception and are expressed predominantly in sensory neurons. TRPV1, a non-selective cation channel has been extensively studied and is responsible for inflammatory thermal hypersensitivity. In this study, the expression and function of TRPV4 have been characterized and compared with those of TRPV1. Results Immunohistochemical studies revealed that both TRPV1 and TRPV4 were co-expressed in dorsal root ganglion (DRG) neuronal cell bodies and in the central terminals of laminae I and II of the spinal dorsal horn (DH). In Ca2+ fluorescence imaging and whole-cell patch-clamp experiments, TRPV1- and TRPV4-mediated responses were observed in a population of the same DRG neurons. Sensitization of TRPV1 has been shown to be involved in inflammatory pain conditions. Incubation with phorbol 12, 13-dibutyrate (PDBu), a PKC activator, resulted in a significant potentiation of TRPV4 currents in DRG neurons. In TRPV4 expressing HEK 293T cells, PDBu increased 4α-phorbol 12, 13-didecanoate (4α-PDD)-induced single-channel activity in cell-attached patches, which was abrogated by bisindolylmaleimide (BIM), a selective PKC inhibitor. TRPV4 is also expressed at the central terminals of sensory neurons. Activation of TRPV4 by 4α-PDD increased the frequency of miniature excitatory post synaptic currents (mEPSCs) in DRG-DH neuronal co-cultures. 4α-PDD-induced increase in the frequency of mEPSCs was further enhanced by PDBu. The expression of TRP channels has been shown in other areas of the CNS; application of 4α-PDD significantly increased the mEPSC frequency in cultured hippocampal neurons, which was further potentiated by PDBu, whereas, TRPV1 agonist capsaicin did not modulate synaptic transmission. Conclusion These results indicate that TRPV4 and TRPV1 are co-expressed in certain DRG neurons and TRPV4 can be sensitized by PKC not only in DRG neuronal cell bodies, but also in the central sensory

Expression of transient receptor potential vanilloid 1 (TRPV1) and 2 (TRPV2) in human peripheral blood.

PubMed

Saunders, Cassandra Im; Kunde, Dale A; Crawford, Amanda; Geraghty, Dominic P

2007-02-01

The vanilloid receptor family of cation channels includes the capsaicin-sensitive, proton- and heat-activated TRPV1 and noxious heat-activated TRPV2. The present study demonstrates both gene and protein expression of TRPV1 and TRPV2 in human peripheral blood cells (PBCs) using molecular and immunocytochemical techniques. Using reverse-transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR), TRPV1 and TRPV2 mRNA was detected in mRNA isolated from human whole peripheral blood. Using qRT-PCR, TRPV2 mRNA was highly expressed in human whole blood isolates (9.33+/-1.19 x 10(4)copies per 10(6)copies of the housekeeping gene GAPDH), whereas TRPV1 message was detected at approximately 150-fold lower levels (638+/-121 copies per 10(6)copies GAPDH). At the protein level, TRPV1 and TRPV2 activity was determined immunocytochemically in a lymphocyte-enriched mononuclear cell preparation (83+/-2% lymphocytes). Cells were labelled with rabbit anti-TRPV1 or goat anti-TRPV2 (1:500) and subsequently labelled with goat Texas red- (TRPV1) or FITC-(TRPV2) conjugated secondary antibodies (1:1000). All cells demonstrated punctate TRPV1-immunoreactivity, which appeared to be on the plasma membrane and in the cytoplasm. In contrast, cells within subjects appeared to express the TRPV1 protein at varying intensities. TRPV2-immunoreactivity appeared diffuse. This is the first study to demonstrate the presence of both TRPV1 and TRPV2 in human peripheral lymphocytes. Further studies need to be undertaken in order to determine the role of TRPV channels in these cells.

Transient Receptor Potential Vanilloid Type 1–Dependent Regulation of Liver-Related Neurons in the Paraventricular Nucleus of the Hypothalamus Diminished in the Type 1 Diabetic Mouse

PubMed Central

Gao, Hong; Miyata, Kayoko; Bhaskaran, Muthu D.; Derbenev, Andrei V.; Zsombok, Andrea

2012-01-01

The paraventricular nucleus (PVN) of the hypothalamus controls the autonomic neural output to the liver, thereby participating in the regulation of hepatic glucose production (HGP); nevertheless, mechanisms controlling the activity of liver-related PVN neurons are not known. Transient receptor potential vanilloid type 1 (TRPV1) is involved in glucose homeostasis and colocalizes with liver-related PVN neurons; however, the functional role of TRPV1 regarding liver-related PVN neurons has to be elucidated. A retrograde viral tracer was used to identify liver-related neurons within the brain-liver circuit in control, type 1 diabetic, and insulin-treated mice. Our data indicate that TRPV1 regulates liver-related PVN neurons. This TRPV1-dependent excitation diminished in type 1 diabetic mice. In vivo and in vitro insulin restored TRPV1 activity in a phosphatidylinositol 3-kinase/protein kinase C–dependent manner and stimulated TRPV1 receptor trafficking to the plasma membrane. There was no difference in total TRPV1 protein expression; however, increased phosphorylation of TRPV1 receptors was observed in type 1 diabetic mice. Our data demonstrate that TRPV1 plays a pivotal role in the regulation of liver-related PVN neurons. Moreover, TRPV1-dependent excitation of liver-related PVN neurons diminishes in type 1 diabetes, thus indicating that the brain-liver autonomic circuitry is altered in type 1 diabetes and may contribute to the autonomic dysfunction of HGP. PMID:22492526

The relaxant 5-HT receptor in the dog coronary artery smooth muscle: pharmacological resemblance to the cloned 5-ht7 receptor subtype.

PubMed Central

Terrón, J. A.

1996-01-01

1. The relaxant effect of 5-hydroxytryptamine (5-HT) in the dog isolated coronary artery deprived of endothelium is mediated by a receptor unrelated to the 5-HT1, 5-HT2, 5-HT3 or 5-HT4 types. Based upon the pharmacological characteristics of this relaxant 5-HT receptor and those reported for the new members of the 5-HT receptor family, the present study explored the possibility that the relaxant 5-HT receptor referred to above, corresponds to the cloned 5-ht7 subtype. Thus, the relaxing and/or blocking effects of several 5-HT receptor drugs as well as some typical and atypical antipsychotic drugs with high affinity for the cloned 5-ht7 receptor in precontracted ring segments were analyzed. 2. 5-HT, 5-carboxamidotryptamine (5-CT) and 5-methoxytryptamine, but not 8-OH-DPAT or sumatriptan, produced concentration-dependent relaxations in endothelium-denuded canine coronary artery rings precontracted with prostaglandin F2a (2 microM). Clozapine (1 microM) produced in some cases a small relaxing effect and antagonized 5-HT- and 5-CT-induced relaxation suggesting a partial agonist effect. In the presence of the 5-HT1D receptor antagonist, GR127935 (100 nM), the rank order of agonist potency was 5-CT > 5-HT > clozapine > or = 5-methoxytryptamine. 8-OH-DPAT and sumatriptan remained inactive as agonists. 3. In GR127935-treated preparations, methiothepin (3 nM) and mianserin (1 microM), as well as the antipsychotics, clozapine (1 microM), pimozide (300 nM), risperidone (3 nM) and spiperone (1 microM), failed to induce a significant relaxation in prostaglandin F2x-precontracted vessels, but produced significant rightward displacements of the concentration-response curves to 5-HT and 5-CT without significantly reducing the Emax. In a final set of experiments with 5-CT, metergoline (100 nM) and mesulergine (300 nM) behaved as competitive antagonists. In contrast, lisuride (3 nM) noncompetitively antagonized 5-CT-induced relaxation. The estimated affinity (apparent pKa values) of

Discrimination of putative M1 and M2 muscarinic receptor subtypes in rat brain by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)

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

Norman, A.B.; Creese, I.

1986-03-01

The EC/sub 50/ of EEDQ for the inhibition of (/sup 3/H)(-)QNB binding in vitro was approximately 3 fold lower for homogenates of hippocampus than brainstem (containing predominantly putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes respectively). Furthermore, the time-dependent loss of (/sup 3/H)(-)QNB binding produced by 100 ..mu..M EEDQ was faster in homogenates of hippocampus than brainstem. Administration of EEDQ (20 mg/kg i.p.) irreversibly reduced the Bmax of (/sup 3/H)(-)QNB binding by 56% and 34% in hippocampus and brainstem respectively. Pirenzepine competition for the remaining (/sup 3/H)(-)QNB binding sites following in vitro and in vivo treatment with EEDQ revealedmore » a significant increase in the proportion of (/sup 3/H)(-)QNB binding sites having low affinity for pirenzepine (M/sub 2/ receptors), indicating that the high affinity pirenzepine binding sites (M/sub 1/ receptors) were selectively and irreversibly lost. Thus, EEDQ discriminates the same putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes that are discriminated by pirenzepine. The reduction of (/sup 3/H)(-)QNB binding could be prevented both in vitro and in vivo by atropine or scopolamine. These data may indicate differences in the accessibility of these putative receptor subtypes to EEDQ or, alternatively, differences in the availability of carboxyl groups able to interact with EEDQ at the ligand recognition site of M/sub 1/ and M/sub 2/ muscarinic receptors.« less

The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies.

PubMed

Muldoon, P P; Jackson, K J; Perez, E; Harenza, J L; Molas, S; Rais, B; Anwar, H; Zaveri, N T; Maldonado, R; Maskos, U; McIntosh, J M; Dierssen, M; Miles, M F; Chen, X; De Biasi, M; Damaj, M I

2014-08-01

Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. © 2014 The British Pharmacological Society.

The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies

PubMed Central

Muldoon, P P; Jackson, K J; Perez, E; Harenza, J L; Molas, S; Rais, B; Anwar, H; Zaveri, N T; Maldonado, R; Maskos, U; McIntosh, J M; Dierssen, M; Miles, M F; Chen, X; De Biasi, M; Damaj, M I

2014-01-01

BACKGROUND AND PURPOSE Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. EXPERIMENTAL APPROACHES To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. KEY RESULTS BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. CONCLUSION AND IMPLICATIONS Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. PMID:24750073

Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

PubMed

Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

2006-10-01

To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

Regulation of ocular surface inflammation by prostaglandin E receptor subtype EP3.

PubMed

Ueta, Mayumi

2010-11-01

We first investigated whether the prostaglandin (PG) E2-PGE receptor subtype EP3 axis regulates the development of murine experimental allergic conjunctivitis because it has been reported that this pathway negatively regulates allergic reactions in a murine allergic asthma model. We observed that EP3 is constitutively expressed in mice conjunctival epithelium. EP3 knockout mice demonstrated significantly increased eosinophil infiltration in conjunctiva after ragweed challenge compared with wild-type mice. Consistently, significantly higher expression of eotaxin-1 messenger RNA was observed in Ptger3-/- mice. Conversely, treatment of wild-type mice with an EP3-selective agonist significantly decreased eosinophil infiltration, which was blunted in Ptger3-/- mice. Expression of cyclooxygenase-2 and PGE synthases was upregulated and PGE2 content increased in the eyelids after ragweed challenge. These data suggest that PGE2 acts on EP3 in the conjunctival epithelium and downregulates the progression of experimental allergic conjunctivitis. We next examined and compared the expression of EP3 in human conjunctival epithelium in various ocular surface diseases. Human conjunctival epithelium expressed EP3-specific messenger RNA and EP3 protein. Although we could clearly find positive signals in the conjunctival epithelium from patients with noninflammatory ocular surface diseases such as conjunctivochalasis and pterygium, we could not find positive signals in that from those with inflammatory disorders such as Stevens-Johnson syndrome and ocular cicatricial pemphigoid. Likewise, expression of the PGE receptor subtype EP4 was clearly found in the conjunctival epithelium from patients with conjunctivochalasis and pterygium but not from patients with Stevens-Johnson syndrome and ocular cicatricial pemphigoid.

Valium without dependence? Individual GABAA receptor subtype contribution toward benzodiazepine addiction, tolerance, and therapeutic effects.

PubMed

Cheng, Tianze; Wallace, Dominique Marie; Ponteri, Benjamin; Tuli, Mahir

2018-01-01

Benzodiazepines are one of the most prescribed medications as first-line treatment of anxiety, insomnia, and epilepsy around the world. Over the past two decades, advances in the neuropharmacological understanding of gamma aminobutyric acid (GABA) A receptors revealed distinct contributions from each subtype and produced effects. Recent findings have highlighted the importance of α 1 containing GABA A receptors in the mechanisms of addiction and tolerance in benzodiazepine treatments. This has shown promise in the development of tranquilizers with minimal side effects such as cognitive impairment, dependence, and tolerance. A valium-like drug without its side effects, as repeatedly demonstrated in animals, is achievable.

Learning and Memory Impairments in a Congenic C57BL/6 Strain of Mice That Lacks the M2 Muscarinic Acetylcholine Receptor Subtype

PubMed Central

Bainbridge, Natalie K.; Koselke, Lisa R.; Jeon, Jongrye; Bailey, Kathleen R.; Wess, Jürgen; Crawley, Jacqueline N.; Wrenn, Craige C.

2009-01-01

The neurotransmitter acetylcholine is an important modulator of cognitive functions including attention, learning, and memory. The actions of acetylcholine are mediated by five distinct muscarinic acetylcholine receptor subtypes (M1-M5). The lack of drugs with a high degree of selectivity for these subtypes has impeded the determination of which subtypes mediate which components of cholinergic neurotransmission relevant to cognitive abilities. The present study examined the behavioral functions of the M2 muscarinic receptor subtype by utilizing congenic C57BL/6 mice possessing a null-mutation in the M2 muscarinic receptor gene (M2−/− mice). Comprehensive assessment of general health and neurological function found no major differences between M2−/− and wild-type (M2+/+) mice. In tests of learning and memory, M2−/− mice were impaired in the acquisition (trials to criterion), but not the retention (72 hr) of a passive avoidance task. In a novel open field, M2−/− mice were impaired in between-sessions, but not within-session habituation. In a holeboard test of spatial memory, M2−/− mice committed more errors in working memory than M2+/+ mice. Reference memory did not differ between the genotypes. M2−/− mice showed no impairments in either cued or contextual fear conditioning. These findings replicate and extend earlier findings in a hybrid strain and solidify the interpretation that the M2 receptor plays a critical role in specific components of cognitive abilities. PMID:18346798

Analysis of opioid receptor subtype antagonist effects upon mu opioid agonist-induced feeding elicited from the ventral tegmental area of rats.

PubMed

Lamonte, Nicole; Echo, Joyce A; Ackerman, Tsippa F; Christian, Garrison; Bodnar, Richard J

2002-03-01

The present study examined opioid receptor(s) mediation of feeding elicited by mu opioid agonists in the ventral tegmental area using general or selective opioid antagonist pretreatment. Naltrexone as well as equimolar doses of selective mu and kappa, but not delta opioid antagonists in the ventral tegmental area significantly reduced mu agonist-induced feeding, indicating a pivotal role for these receptor subtypes in the full expression of this response.

Angiotensin II and its different receptor subtypes in placenta and fetal membranes.

PubMed

Kalenga, M K; de Gasparo, M; Thomas, K; de Hertogh, R

1996-01-01

The recent discovery of a local renin-angiotensin system in trophoblastic tissues has raised many questions regarding its role in the physiology of normal gestation and its implications in the pathophysiology of hypertension during pregnancy. In this article, the authors first review the most interesting aspects of the chorioplacental renin-angiotensin system, dwelling on the tissue distribution of angiotensin II and its receptor subtypes in the placenta and fetal membranes of different species. The relationship between angiotensin II and other locally synthesized chorioplacental substances is also analysed and the therapeutic implications of phenomena observed in pregnancy-associated hypertension are discussed.

Association Between Imaging Characteristics and Different Molecular Subtypes of Breast Cancer.

PubMed

Wu, Mingxiang; Ma, Jie

2017-04-01

Breast cancer can be divided into four major molecular subtypes based on the expression of hormone receptor (estrogen receptor and progesterone receptor), human epidermal growth factor receptor 2, HER2 status, and molecular proliferation rate (Ki67). In this study, we sought to investigate the association between breast cancer subtype and radiological findings in the Chinese population. Medical records of 300 consecutive invasive breast cancer patients were reviewed from the database: the Breast Imaging Reporting and Data System. The imaging characteristics of the lesions were evaluated. The molecular subtypes of breast cancer were classified into four types: luminal A, luminal B, HER2 overexpressed (HER2), and basal-like breast cancer (BLBC). Univariate and multivariate logistic regression analyses were performed to assess the association between the subtype (dependent variable) and mammography or 15 magnetic resonance imaging (MRI) indicators (independent variables). Luminal A and B subtypes were commonly associated with "clustered calcification distribution," "nipple invasion," or "skin invasion" (P <0.05). The BLBC subtype was more commonly associated with "rim enhancement" and persistent inflow type enhancement in delayed phase (P <0.05). HER2 overexpressed cancers showed association with persistent enhancement in the delayed phase on MRI and "clustered calcification distribution" on mammography (P <0.05). Certain radiological features are strongly associated with the molecular subtype and hormone receptor status of breast tumor, which are potentially useful tools in the diagnosis and subtyping of breast cancer. Copyright © 2017 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.

Treatment and prognosis of breast cancer patients with brain metastases according to intrinsic subtype.

PubMed

Kuba, Sayaka; Ishida, Mayumi; Nakamura, Yoshiaki; Yamanouchi, Kosho; Minami, Shigeki; Taguchi, Kenichi; Eguchi, Susumu; Ohno, Shinji

2014-11-01

How breast cancer subtypes should affect treatment decisions for breast cancer patients with brain metastases is unclear. We analyzed local brain metastases treatments and their outcomes according to subtype in patients with breast cancer and brain metastases. We reviewed records and database information for women treated at the National Kyushu Cancer Center between 2001 and 2010. Patients were divided into three breast cancer subtype groups: Luminal (estrogen receptor positive and/or progesterone receptor positive, but human epidermal growth factor receptor 2 negative); human epidermal growth factor receptor 2 positive and triple negative (estrogen receptor negative, progesterone receptor negative and human epidermal growth factor receptor 2 negative). Of 524 advanced breast cancer patients, we reviewed 65 (12%) with brain metastases and records showing estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 status, as well as outcome data; there were 26 (40%) Luminal, 26 (40%) had human epidermal growth factor receptor 2 and 13 (20%) had triple negative subtypes. There was no statistical difference in the number of brain metastases among subtypes; however, rates of stereotactic radiosurgery or surgery for brain metastases differed significantly by subtype (human epidermal growth factor receptor 2: 81%, Luminal: 42% and triple negative: 47%; P = 0.03). Patients having the human epidermal growth factor receptor 2 subtype, a performance status of ≤1 and ≤4 brain metastases, who underwent systemic therapy after brain metastases and underwent stereotactic radiosurgery or surgery, were predicted to have longer overall survival after brain metastases. Multivariate analysis demonstrated that not having systemic therapy and not having the human epidermal growth factor receptor 2 subtype were independent factors associated with an increased risk of death (hazard ratio 2.4, 95% confidence interval 1.01-5.6; P = 0.05 and hazard ratio 2.9, 95

Dose-dependent EEG effects of zolpidem provide evidence for GABA(A) receptor subtype selectivity in vivo.

PubMed

Visser, S A G; Wolters, F L C; van der Graaf, P H; Peletier, L A; Danhof, M

2003-03-01

Zolpidem is a nonbenzodiazepine GABA(A) receptor modulator that binds in vitro with high affinity to GABA(A) receptors expressing alpha(1) subunits but with relatively low affinity to receptors expressing alpha(2), alpha(3), and alpha(5) subunits. In the present study, it was investigated whether this subtype selectivity could be detected and quantified in vivo. Three doses (1.25, 5, and 25 mg) of zolpidem were administered to rats in an intravenous infusion over 5 min. The time course of the plasma concentrations was determined in conjunction with the change in the beta-frequency range of the EEG as pharmacodynamic endpoint. The concentration-effect relationship of the three doses showed a dose-dependent maximum effect and a dose-dependent potency. The data were analyzed for one- or two-site binding using two pharmacodynamic models based on 1) the descriptive model and 2) a novel mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for GABA(A) receptor modulators that aims to separates drug- and system-specific properties, thereby allowing the estimation of in vivo affinity and efficacy. The application of two-site models significantly improved the fits compared with one-site models. Furthermore, in contrast to the descriptive model, the mechanism-based PK/PD model yielded dose-independent estimates for affinity (97 +/- 40 and 33,100 +/- 14,800 ng x ml(-1)). In conclusion, the mechanism-based PK/PD model is able to describe and explain the observed dose-dependent EEG effects of zolpidem and suggests the subtype selectivity of zolpidem in vivo.

The distribution of the orphan bombesin receptor subtype-3 in the rat CNS.

PubMed

Jennings, C A; Harrison, D C; Maycox, P R; Crook, B; Smart, D; Hervieu, G J

2003-01-01

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor that shares between 47 and 51% homology with other known bombesin receptors. The natural ligand for BRS-3 is currently unknown and little is known about the mechanisms regulating BRS-3 gene expression. Unlike other mammalian bombesin receptors that have been shown to be predominantly expressed in the CNS and gastrointestinal tract, expression of the BRS-3 receptor in the rat brain has previously not been observed. To gain further understanding of the biology of BRS-3, we have studied the distribution of BRS-3 mRNA and protein in the rat CNS. The mRNA expression pattern was studied using reverse transcription followed by quantitative polymerase chain reaction. Using immunohistological techniques, the distribution of BRS-3 protein in the rat brain was investigated using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The BRS-3 receptor was found to be widely expressed in the rat brain at both mRNA and protein levels. Particularly strong immunosignals were observed in the cerebral cortex, hippocampal formation, hypothalamus and thalamus. Other regions of the brain such as the basal ganglia, midbrain and reticular formation were also immunopositive for BRS-3. In conclusion, our neuroanatomical data provide evidence that BRS-3 is as widely expressed in the rat brain as other bombesin-like peptide receptors and suggest that this receptor may also have important roles in the CNS, mediating the functions of a so far unidentified ligand.

Pharmacological profiles of cloned mammalian P2Y-receptor subtypes.

PubMed

von Kügelgen, Ivar

2006-06-01

Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling. P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors (GPCRs). So far, the P2Y family is composed out of 8 human subtypes that have been cloned and functionally defined; species orthologues have been found in many vertebrates. P2Y1-, P2Y2-, P2Y4-, P2Y6-, and P2Y11-receptors all couple to stimulation of phospholipase C. The P2Y11-receptor mediates in addition a stimulation of adenylate cyclase. In contrast, activation of the P2Y12-, P2Y13-, and P2Y14-receptors causes an inhibition of adenylate cyclase activity. The expression of P2Y1-receptors is widespread. The receptor is involved in blood platelet aggregation, vasodilatation and neuromodulation. It is activated by ADP and ADP analogues including 2-methylthio-ADP (2-MeSADP). 2'-Deoxy-N6-methyladenosine-3',5'-bisphosphate (MRS2179) and 2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate (MRS2279) are potent and selective antagonists. P2Y2 transcripts are abundantly distributed. One important example for its functional role is the control of chloride ion fluxes in airway epithelia. The P2Y2-receptor is activated by UTP and ATP and blocked by suramin. The P2Y2-agonist diquafosol is used for the treatment of the dry eye disease. P2Y4-receptors are expressed in the placenta and in epithelia. The human P2Y4-receptor has a strong preference for UTP as agonist, whereas the rat P2Y4-receptor is activated about equally by UTP and ATP. The P2Y4-receptor is not blocked by suramin. The P2Y6-receptor has a widespread distribution including heart, blood vessels, and brain. The receptor prefers UDP as agonist and is selectively blocked by 1,2-di-(4-isothiocyanatophenyl)ethane (MRS2567). The P2Y11-receptor may play a role in the differentiation of immunocytes. The human P2Y11-receptor is activated by ATP as naturally occurring agonist and

Transient receptor potential vanilloid-3 (TRPV3) activation plays a central role in cardiac fibrosis induced by pressure overload in rats via TGF-β1 pathway.

PubMed

Liu, Yan; Qi, Hanping; E, Mingyao; Shi, Pilong; Zhang, Qianhui; Li, Shuzhi; Wang, Ye; Cao, Yonggang; Chen, Yunping; Ba, Lina; Gao, Jingquan; Huang, Wei; Sun, Hongli

2018-02-01

Cardiac fibrosis is a common pathologic change along with pressure overload. Recent studies indicated that transient receptor potential (TRP) channels played multiple roles in heart. However, the functional role of transient receptor potential vanilloid-3 (TRPV3) in cardiac fibrosis remained unclear. The present study was designed to investigate the relationship between TRPV3 activation and pressure overload-induced cardiac fibrosis. Pressure overload rats were successfully established by abdominal aortic constriction (AAC), and cardiac fibrosis was simulated by 100 nM angiotensin II (Ang II) in neonatal cardiac fibroblasts. Echocardiographic parameters, cardiac fibroblast proliferation, cell cycle, intracellular calcium concentration ([Ca 2+ ] i ), and the protein expressions of collagen I, collagen III, transforming growth factor beta 1 (TGF-β 1 ), cyclin E, and cyclin-dependent kinase 2 (CDK2) were measured. Echocardiographic and histological measurements suggested that the activation of TRPV3 exacerbated the cardiac dysfunction and increased interstitial fibrosis in pressure overload rats. Further results showed that TRPV3 activation upregulated the expressions of collagen I, collagen III, TGF-β 1 , cyclin E, and CDK2 in vivo and in vitro. At the same time, blocking TGF-β 1 pathway could partially reverse the effect of TRPV3 activation. These results suggested that TRPV3 activation exacerbated cardiac fibrosis by promoting cardiac fibroblast proliferation through TGF-β 1 /CDK2/cyclin E pathway in the pressure-overloaded rat hearts.

Stereocontrolled dopamine receptor binding and subtype selectivity of clebopride analogues synthesized from aspartic acid.

PubMed

Einsiedel, Jürgen; Weber, Klaus; Thomas, Christoph; Lehmann, Thomas; Hübner, Harald; Gmeiner, Peter

2003-10-06

Employing the achiral 4-aminopiperidine derivative clebopride as a lead compound, chiral analogues were developed displaying dopamine receptor binding profiles that proved to be strongly dependent on the stereochemistry. Compared to the D1 receptor, the test compounds showed high selectivity for the D2-like subtypes including D2(long), D2(short), D3 and D4. The highest D4 and D3 affinities were observed for the cis-3-amino-4-methylpyrrolidines 3e and the enantiomer ent3e resulting in K(i) values of 0.23 and 1.8 nM, respectively. The benzamides of type 3 and 5 were synthesized in enantiopure form starting from (S)-aspartic acid and its unnatural optical antipode.

Ca2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1.

PubMed

Wang, Sheng; Wang, Sen; Asgar, Jamila; Joseph, John; Ro, Jin Y; Wei, Feng; Campbell, James N; Chung, Man-Kyo

2017-05-19

Capsaicin is an ingredient in spicy peppers that produces burning pain by activating transient receptor potential vanilloid 1 (TRPV1), a Ca 2+ -permeable ion channel in nociceptors. Capsaicin has also been used as an analgesic, and its topical administration is approved for the treatment of certain pain conditions. The mechanisms underlying capsaicin-induced analgesia likely involve reversible ablation of nociceptor terminals. However, the mechanisms underlying these effects are not well understood. To visualize TRPV1-lineage axons, a genetically engineered mouse model was used in which a fluorophore is expressed under the TRPV1 promoter. Using a combination of these TRPV1-lineage reporter mice and primary afferent cultures, we monitored capsaicin-induced effects on afferent terminals in real time. We found that Ca 2+ influx through TRPV1 is necessary for capsaicin-induced ablation of nociceptive terminals. Although capsaicin-induced mitochondrial Ca 2+ uptake was TRPV1-dependent, dissipation of the mitochondrial membrane potential, inhibition of the mitochondrial transition permeability pore, and scavengers of reactive oxygen species did not attenuate capsaicin-induced ablation. In contrast, MDL28170, an inhibitor of the Ca 2+ -dependent protease calpain, diminished ablation. Furthermore, overexpression of calpastatin, an endogenous inhibitor of calpain, or knockdown of calpain 2 also decreased ablation. Quantitative assessment of TRPV1-lineage afferents in the epidermis of the hind paws of the reporter mice showed that EGTA and MDL28170 diminished capsaicin-induced ablation. Moreover, MDL28170 prevented capsaicin-induced thermal hypoalgesia. These results suggest that TRPV1/Ca 2+ /calpain-dependent signaling plays a dominant role in capsaicin-induced ablation of nociceptive terminals and further our understanding of the molecular mechanisms underlying the effects of capsaicin on nociceptors. © 2017 by The American Society for Biochemistry and Molecular Biology

Elevated expression of transient receptor potential vanilloid type 1 in dorsal root ganglia of rats with endometriosis

PubMed Central

Lian, Yu-Ling; Cheng, Ming-Jun; Zhang, Xian-Xia; Wang, Li

2017-01-01

Pain is the most pronounced complaint of women with endometriosis, however the underlying mechanism is still poorly understood. In the present study, the authors evaluate the effect of transient receptor potential vanilloid type 1 (TRPV1) of dorsal root ganglia (DRG) on endometriosis-associated pain. A total of 36 SD rats were randomly divided into a sham group (n=9) and a Model group (n=27), accepted auto-transplanted pieces of fat or uterus to the pelvic cavity. At 4 weeks, the Model group was randomly subdivided into the following groups: ENDO group (no treatment, n=9), BCTC group (Model + BCTC, an antagonist of TRPV1, n=9), Vehicle group (Model + cyclodextrin, the vehicle of BCTC, n=9). Tail-flick test was performed prior to surgery, 1 h prior to and following treatment of BCTC or cyclodextrin. The expression of TRPV1, substance P (SP), calcitonin gene-related peptide (CGRP) in L1-L6 DRG was measured via immunohistochemistry, western blotting and RT-qPCR. The results indicated that the Model group exhibited a significant decrease in tail flick latency compared to pre-surgical baseline, and the expression of TRPV1, SP, CGRP protein and mRNA in L1-L6 DRG significantly increased compared to the sham group. BCTC significantly improved tail flick latency, and downregulated the expression of TRPV1, SP and CGRP protein and mRNA levels in L1-L6 DRG compared to ENDO group. However, there were no significant differences of those in Vehicle group compared with the ENDO group. Taken together, the current study provides evidence that TRPV1 expressed in DRG may serve an important role in endometriosis-associated pain. PMID:28627595

Low-Level Blast Exposure Increases Transient Receptor Potential Vanilloid 1 (TRPV1) Expression in the Rat Cornea

PubMed Central

Por, Elaine D.; Choi, Jae-Hyek; Lund, Brian J.

2016-01-01

ABSTRACT Background: Blast-related ocular injuries sustained by military personnel have led to rigorous efforts to elucidate the effects of blast exposure on neurosensory function. Recent studies have provided some insight into cognitive and visual deficits sustained following blast exposure; however, limited data are available on the effects of blast on pain and inflammatory processes. Investigation of these secondary effects of blast exposure is necessary to fully comprehend the complex pathophysiology of blast-related injuries. The overall purpose of this study is to determine the effects of single and repeated blast exposure on pain and inflammatory mediators in ocular tissues. Methods: A compressed air shock tube was used to deliver a single or repeated blast (68.0 ± 2.7 kPa) to anesthetized rats daily for 5 days. Immunohistochemistry was performed on ocular tissues to determine the expression of the transient receptor potential vanilloid 1 (TRPV1) channel, calcitonin gene-related peptide (CGRP), substance P (SP), and endothelin-1 (ET-1) following single and repeated blast exposure. Neutrophil infiltration and myeloperoxidase (MPO) expression were also assessed in blast tissues via immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis, respectively. Results: TRPV1 expression was increased in rat corneas exposed to both single and repeated blast. Increased secretion of CGRP, SP, and ET-1 was also detected in rat corneas as compared to control. Moreover, repeated blast exposure resulted in neutrophil infiltration in the cornea and stromal layer as compared to control animals. Conclusion: Single and repeated blast exposure resulted in increased expression of TRPV1, CGRP, SP, and ET-1 as well as neutrophil infiltration. Collectively, these findings provide novel insight into the activation of pain and inflammation signaling mediators following blast exposure. PMID:27049881

Role of the Outer Pore Domain in Transient Receptor Potential Vanilloid 1 Dynamic Permeability to Large Cations*

PubMed Central

Munns, Clare H.; Chung, Man-Kyo; Sanchez, Yuly E.; Amzel, L. Mario; Caterina, Michael J.

2015-01-01

Transient receptor potential vanilloid 1 (TRPV1) has been shown to alter its ionic selectivity profile in a time- and agonist-dependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-d-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-of-function substitutions within the TRPV1 pore turret (N628P and S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered capsaicin-induced changes in NMDG permeability, the loss-of-function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin-evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity. PMID:25568328

Role of the outer pore domain in transient receptor potential vanilloid 1 dynamic permeability to large cations.

PubMed

Munns, Clare H; Chung, Man-Kyo; Sanchez, Yuly E; Amzel, L Mario; Caterina, Michael J

2015-02-27

Transient receptor potential vanilloid 1 (TRPV1) has been shown to alter its ionic selectivity profile in a time- and agonist-dependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-D-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-of-function substitutions within the TRPV1 pore turret (N628P and S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered capsaicin-induced changes in NMDG permeability, the loss-of-function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin-evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Drug-induced mild therapeutic hypothermia obtained by administration of a transient receptor potential vanilloid type 1 agonist.

PubMed

Fosgerau, Keld; Weber, Uno J; Gotfredsen, Jacob W; Jayatissa, Magdalena; Buus, Carsten; Kristensen, Niels B; Vestergaard, Mogens; Teschendorf, Peter; Schneider, Andreas; Hansen, Philip; Raunsø, Jakob; Køber, Lars; Torp-Pedersen, Christian; Videbaek, Charlotte

2010-10-09

The use of mechanical/physical devices for applying mild therapeutic hypothermia is the only proven neuroprotective treatment for survivors of out of hospital cardiac arrest. However, this type of therapy is cumbersome and associated with several side-effects. We investigated the feasibility of using a transient receptor potential vanilloid type 1 (TRPV1) agonist for obtaining drug-induced sustainable mild hypothermia. First, we screened a heterogeneous group of TRPV1 agonists and secondly we tested the hypothermic properties of a selected candidate by dose-response studies. Finally we tested the hypothermic properties in a large animal. The screening was in conscious rats, the dose-response experiments in conscious rats and in cynomologus monkeys, and the finally we tested the hypothermic properties in conscious young cattle (calves with a body weight as an adult human). The investigated TRPV1 agonists were administered by continuous intravenous infusion. Screening: Dihydrocapsaicin (DHC), a component of chili pepper, displayed a desirable hypothermic profile with regards to the duration, depth and control in conscious rats. Dose-response experiments: In both rats and cynomologus monkeys DHC caused a dose-dependent and immediate decrease in body temperature. Thus in rats, infusion of DHC at doses of 0.125, 0.25, 0.50, and 0.75 mg/kg/h caused a maximal ΔT (°C) as compared to vehicle control of -0.9, -1.5, -2.0, and -4.2 within approximately 1 hour until the 6 hour infusion was stopped. Finally, in calves the intravenous infusion of DHC was able to maintain mild hypothermia with ΔT > -3°C for more than 12 hours. Our data support the hypothesis that infusion of dihydrocapsaicin is a candidate for testing as a primary or adjunct method of inducing and maintaining therapeutic hypothermia.

Cancer cachexia causes skeletal muscle damage via transient receptor potential vanilloid 2‐independent mechanisms, unlike muscular dystrophy

PubMed Central

Suzuki, Nobuyuki; Ohtake, Hitomi; Kamauchi, Shinya; Hashimoto, Naohiro; Kiyono, Tohru; Wakabayashi, Shigeo

2015-01-01

Abstract Background Muscle wasting during cancer cachexia contributes to patient morbidity. Cachexia‐induced muscle damage may be understood by comparing its symptoms with those of other skeletal muscle diseases, but currently available data are limited. Methods We modelled cancer cachexia in mice bearing Lewis lung carcinoma/colon adenocarcinoma and compared the associated muscle damage with that in a murine muscular dystrophy model (mdx mice). We measured biochemical and immunochemical parameters: amounts/localization of cytoskeletal proteins and/or Ca2+ signalling proteins related to muscle function and abnormality. We analysed intracellular Ca2+ mobilization and compared results between the two models. Involvement of Ca2+‐permeable channel transient receptor potential vanilloid 2 (TRPV2) was examined by inoculating Lewis lung carcinoma cells into transgenic mice expressing dominant‐negative TRPV2. Results Tumourigenesis caused loss of body and skeletal muscle weight and reduced muscle force and locomotor activity. Similar to mdx mice, cachexia muscles exhibited myolysis, reduced sarcolemmal sialic acid content, and enhanced lysosomal exocytosis and sarcolemmal localization of phosphorylated Ca2+/CaMKII. Abnormal autophagy and degradation of dystrophin also occurred. Unlike mdx muscles, cachexia muscles did not exhibit regeneration markers (centrally nucleated fibres), and levels of autophagic proteolytic pathway markers increased. While a slight accumulation of TRPV2 was observed in cachexia muscles, Ca2+ influx via TRPV2 was not elevated in cachexia‐associated myotubes, and the course of cachexia pathology was not ameliorated by dominant‐negative inhibition of TRPV2. Conclusions Thus, cancer cachexia may induce muscle damage through TRPV2‐independent mechanisms distinct from those in muscular dystrophy; this may help treat patients with tumour‐induced muscle wasting. PMID:27239414

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice*

PubMed Central

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2014-01-01

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. PMID:24695732

A new class of pyrazolo[5,1-c][1,2,4]triazines as γ-aminobutyric type A (GABAA) receptor subtype ligand: synthesis and pharmacological evaluation.

PubMed

Guerrini, Gabriella; Ciciani, Giovanna; Daniele, Simona; Martini, Claudia; Costagli, Camilla; Guarino, Chiara; Selleri, Silvia

2018-05-15

A comparison between compounds with pyrazolo[1,5-a]pyrimidine structure (series 4-6) and pyrazolo[5,1-c][1,2,4]triazine core (series 9) as ligands at GABA A -receptor subtype, was evaluated. Moreover, for pyrazolotriazine derivatives having binding recognition, the interaction on recombinant rat α(1-3,5) GABA A receptor subtypes, was performed. Among these latter, emerge compounds 9c, 9k, 9l, 9m and 9n as α1-selective and 9h as α2-selective ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.

Roles of Hippocampal Somatostatin Receptor Subtypes in Stress Response and Emotionality.

PubMed

Prévôt, Thomas D; Gastambide, François; Viollet, Cécile; Henkous, Nadia; Martel, Guillaume; Epelbaum, Jacques; Béracochéa, Daniel; Guillou, Jean-Louis

2017-07-01

Altered brain somatostatin functions recently appeared as key elements for the pathogenesis of stress-related neuropsychiatric disorders. The hippocampus exerts an inhibitory feedback on stress but the mechanisms involved remain unclear. We investigated herein the role of hippocampal somatostatin receptor subtypes in both stress response and behavioral emotionality using C57BL/6, wild type and sst 2 or sst 4 knockout mice. Inhibitory effects of hippocampal infusions of somatostatin agonists on stress-induced hypothalamo-pituitary-adrenal axis (HPA) activity were tested by monitoring peripheral blood and local hippocampus corticosterone levels, the latter by using microdialysis. Anxiolytic and antidepressant-like effects were determined in the elevated-plus maze, open field, forced swimming, and stress-sensitive beam walking tests. Hippocampal injections of somatostatin analogs and sst 2 or sst 4, but not sst 1 or sst 3 receptor agonists produced rapid and sustained inhibition of HPA axis. sst 2 agonists selectively produced anxiolytic-like behaviors whereas both sst 2 and sst 4 agonists had antidepressant-like effects. Consistent with these findings, high corticosterone levels and anxiety were found in sst 2 KO mice and depressive-like behaviors observed in both sst 2 KO and sst 4 KO strains. Both hippocampal sst 2 and sst 4 receptors selectively inhibit stress-induced HPA axis activation but mediate anxiolytic and antidepressive effects through distinct mechanisms. Such results are to be accounted for in development of pathway-specific somatostatin receptor agents in the treatment of hypercortisolism (Cushing's disease) and stress-related neuropsychiatric disorders.

New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes.

PubMed

Barocelli, E; Ballabeni, V; Bertoni, S; Dallanoce, C; De Amici, M; De Micheli, C; Impicciatore, M

2000-06-30

Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel.

PubMed

Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren; Altier, Christophe

2016-01-01

Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel.

PubMed

Yang, Fan; Vu, Simon; Yarov-Yarovoy, Vladimir; Zheng, Jie

2016-06-28

Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S-S498F-L505T-Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular "glue" that bridges the S4-S5 linker to the S1-S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor.

Multiple receptor subtypes mediate the effects of serotonin on rat subfornical organ neurons

NASA Technical Reports Server (NTRS)

Scrogin, K. E.; Johnson, A. K.; Schmid, H. A.

1998-01-01

The subfornical organ (SFO) receives significant serotonergic innervation. However, few reports have examined the functional effects of serotonin on SFO neurons. This study characterized the effects of serotonin on spontaneously firing SFO neurons in the rat brain slice. Of 31 neurons tested, 80% responded to serotonin (1-100 microM) with either an increase (n = 15) or decrease (n = 10) in spontaneous activity. Responses to serotonin were dose dependent and persisted after synaptic blockade. Excitatory responses could also be mimicked by the 5-hydroxytryptamine (5-HT)2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI; 1-10 microM) and could be blocked by the 5-HT2A/2C-receptor antagonist LY-53,857 (10 microM). LY-53,857 unmasked inhibitory responses to serotonin in 56% of serotonin-excited cells tested. Serotonin-inhibited cells were also inhibited by the 5-HT1A-receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT; 1-10 microM; n = 7). The data indicate that SFO neurons are responsive to serotonin via postsynaptic activation of multiple receptor subtypes. The results suggest that excitatory responses to serotonin are mediated by 5-HT2A or 5-HT2C receptors and that inhibitory responses may be mediated by 5-HT1A receptors. In addition, similar percentages of serotonin-excited and -inhibited cells were also sensitive to ANG II. As such the functional relationship between serotonin and ANG II in the SFO remains unclear.

Multiple receptor subtypes mediate the effects of serotonin on rat subfornical organ neurons.

PubMed

Scrogin, K E; Johnson, A K; Schmid, H A

1998-12-01

The subfornical organ (SFO) receives significant serotonergic innervation. However, few reports have examined the functional effects of serotonin on SFO neurons. This study characterized the effects of serotonin on spontaneously firing SFO neurons in the rat brain slice. Of 31 neurons tested, 80% responded to serotonin (1-100 microM) with either an increase (n = 15) or decrease (n = 10) in spontaneous activity. Responses to serotonin were dose dependent and persisted after synaptic blockade. Excitatory responses could also be mimicked by the 5-hydroxytryptamine (5-HT)2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI; 1-10 microM) and could be blocked by the 5-HT2A/2C-receptor antagonist LY-53,857 (10 microM). LY-53,857 unmasked inhibitory responses to serotonin in 56% of serotonin-excited cells tested. Serotonin-inhibited cells were also inhibited by the 5-HT1A-receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT; 1-10 microM; n = 7). The data indicate that SFO neurons are responsive to serotonin via postsynaptic activation of multiple receptor subtypes. The results suggest that excitatory responses to serotonin are mediated by 5-HT2A or 5-HT2C receptors and that inhibitory responses may be mediated by 5-HT1A receptors. In addition, similar percentages of serotonin-excited and -inhibited cells were also sensitive to ANG II. As such the functional relationship between serotonin and ANG II in the SFO remains unclear.

[6]-Gingerol induces bone loss in ovary intact adult mice and augments osteoclast function via the transient receptor potential vanilloid 1 channel.

PubMed

Khan, Kainat; Singh, Akanksha; Mittal, Monika; Sharan, Kunal; Singh, Nidhi; Dixit, Preety; Sanyal, Sabyasachi; Maurya, Rakesh; Chattopadhyay, Naibedya

2012-12-01

[6]-Gingerol, a major constituent of ginger, is considered to have several health beneficial effects. The effect of 6-gingerol on bone cells and skeleton of mice was investigated. The effects of 6-gingerol on mouse bone marrow macrophages and osteoblasts were studied. 6-Gingerol-stimulated osteoclast differentiation of bone marrow macrophages but had no effect on osteoblasts. Capsazepine, an inhibitor of TRPV1 (transient receptor potential vanilloid 1) channel, attenuated the pro-osteoclastogenic effect of 6-gingerol or capsaicin (an agonist of TRPV1). Similar to capsaicin, 6-gingerol stimulated Ca(2) + influx in osteoclasts. The effect of daily feeding of 6-gingerol for 5 wk on the skeleton of adult female Balb/cByJ mice was investigated. Mice treated with capsaicin and ovariectomized (OVx) mice served as controls for osteopenia. 6-Gingerol caused increase in trabecular osteoclast number, microarchitectural erosion at all trabecular sites and loss of vertebral stiffness, and these effects were comparable to capsaicin or OVx group. Osteoclast-specific serum and gene markers of 6-gingerol-treated mice were higher than the OVx group. Bone formation was unaffected by 6-gingerol. Daily feeding of 6-gingerol to skeletally mature female mice caused trabecular osteopenia, and the mechanism appeared to be activation of osteoclast formation via the TRPV1 channel. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes.

PubMed

Harizi, Hedi; Grosset, Christophe; Gualde, Norbert

2003-06-01

We have reported previously that PGE(2) inhibits dendritic cells (DC) functions. Because E prostanoid receptor (EPR) subtypes involved in this action are unknown, expression and functions of these receptors were examined in DC. Western blot and flow cytometry analyses showed that all EPRs were coexpressed in DC. In a dose-dependent manner, lipopolysaccharide (LPS) enhanced EP(2)R/EP(4)R but not EP(1)R/EP(3)R expressions. NS-398, a cyclooxygenase (COX)-2-selective inhibitor, suppressed LPS-enhanced EP(2)R/EP(4)R expression, suggesting that COX-2-issued prostaglandin E(2) (PGE(2)) modulates DC function through stimulation of specific EPR subtypes. Using selective agonists, we found that butaprost, an EP(2)R agonist, and PGE(1) alcohol, an EP(2)R and EP(2)R/EP(4)R agonist, inhibited major histocompatibility complex class II expression and enhanced interleukin-10 production from DC. However, no effect was observed with sulprostone and 17-phenyl-omega-trinor-PGE(2), selective agonists for EP(1)R and EP(1)R/EP(3)R, respectively. Treatment of DC with dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, mimics PGE(2)-induced, inhibitory effects. Taken together, our data demonstrate that EP(2)R/EP(4)R are efficient for mediating PGE(2)-induced modulation of DC functions.

A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio).

PubMed

McPartland, J M; Glass, Michelle; Matias, Isabel; Norris, Ryan W; Kilpatrick, C William

2007-05-01

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.

The SOL-2/Neto Auxiliary Protein Modulates the Function of AMPA-Subtype Ionotropic Glutamate Receptors

PubMed Central

Wang, Rui; Mellem, Jerry E.; Jensen, Michael; Brockie, Penelope J.; Walker, Craig S.; Hoerndli, Frédéric J.; Madsen, David M.; Maricq, Andres V.

2012-01-01

Summary The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents. PMID:22958824

The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors.

PubMed

Wang, Rui; Mellem, Jerry E; Jensen, Michael; Brockie, Penelope J; Walker, Craig S; Hoerndli, Frédéric J; Hauth, Linda; Madsen, David M; Maricq, Andres V

2012-09-06

The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents. Copyright © 2012 Elsevier Inc. All rights reserved.

Elevated expression of transient receptor potential vanilloid type 1 in dorsal root ganglia of rats with endometriosis.

PubMed

Lian, Yu-Ling; Cheng, Ming-Jun; Zhang, Xian-Xia; Wang, Li

2017-08-01

Pain is the most pronounced complaint of women with endometriosis, however the underlying mechanism is still poorly understood. In the present study, the authors evaluate the effect of transient receptor potential vanilloid type 1 (TRPV1) of dorsal root ganglia (DRG) on endometriosis-associated pain. A total of 36 SD rats were randomly divided into a sham group (n=9) and a Model group (n=27), accepted auto‑transplanted pieces of fat or uterus to the pelvic cavity. At 4 weeks, the Model group was randomly subdivided into the following groups: ENDO group (no treatment, n=9), BCTC group (Model + BCTC, an antagonist of TRPV1, n=9), Vehicle group (Model + cyclodextrin, the vehicle of BCTC, n=9). Tail‑flick test was performed prior to surgery, 1 h prior to and following treatment of BCTC or cyclodextrin. The expression of TRPV1, substance P (SP), calcitonin gene‑related peptide (CGRP) in L1‑L6 DRG was measured via immunohistochemistry, western blotting and RT‑qPCR. The results indicated that the Model group exhibited a significant decrease in tail flick latency compared to pre‑surgical baseline, and the expression of TRPV1, SP, CGRP protein and mRNA in L1‑L6 DRG significantly increased compared to the sham group. BCTC significantly improved tail flick latency, and downregulated the expression of TRPV1, SP and CGRP protein and mRNA levels in L1‑L6 DRG compared to ENDO group. However, there were no significant differences of those in Vehicle group compared with the ENDO group. Taken together, the current study provides evidence that TRPV1 expressed in DRG may serve an important role in endometriosis-associated pain.

Analgesic effects of the novel semicarbazide-sensitive amine oxidase inhibitor SZV 1287 in mouse pain models with neuropathic mechanisms: Involvement of transient receptor potential vanilloid 1 and ankyrin 1 receptors.

PubMed

Horváth, Ádám; Tékus, Valéria; Bencze, Noémi; Szentes, Nikolett; Scheich, Bálint; Bölcskei, Kata; Szőke, Éva; Mócsai, Attila; Tóth-Sarudy, Éva; Mátyus, Péter; Pintér, Erika; Helyes, Zsuzsanna

2018-05-01

Semicarbazide-sensitive amine oxidase (SSAO) produces tissue irritants by deamination of primary amines, which activate transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors expressed predominantly on nociceptors. Since there are no data about its functions in pain, we studied the effects and mechanisms of action of our novel SSAO inhibitor and dual TRPA1/TRPV1 antagonist multi-target drug SZV 1287 in different pain models. Acute chemonociception was induced by TRPV1 and TRPA1 activation (resiniferatoxin and formalin, respectively), chronic arthritis by K/BxN serum transfer, traumatic mononeuropathy by sciatic nerve ligation. SZV 1287 (20 mg/kg i.p.) was investigated in C57BL/6J wildtype (WT), TRPA1- (TRPA1 -/- ) and TRPV1-deficient (TRPV1 -/- ) mice. Paw mechanonociception was measured by aesthesiometry, thermonociception by hot plate, nocifensive behavior by licking duration, volume by plethysmometry, myeloperoxidase activity by luminescence and plasma extravasation by fluorescence imaging, glia activation in pain-related brain regions by immunohistochemistry. SZV 1287 significantly inhibited both TRPA1 and TRPV1 activation-induced acute chemonociception and hyperalgesia. In K/BxN arthritis, daily SZV 1287 injections significantly decreased hyperalgesia, L4-L6 spinal dorsal horn microgliosis, edema and myeloperoxidase activity. SZV 1287-evoked antihyperalgesic and anti-edema effects were absent in TRPV1 -/- , and remarkably reduced in TRPA1 -/- mice. In contrast, myeloperoxidase-inhibitory effect was absent in TRPA1 -/-, but not in TRPV1 -/- animals. Acute SZV 1287 administration resulted in approximately 50% significant reduction of neuropathic hyperalgesia 7 days after nerve ligation, which was not observed in either TRPA1 -/- or TRPV1 -/- mice. SZV 1287 inhibits chronic inflammatory and neuropathic pain via TRPV1 and TRPA1/TRPV1 activation, respectively, highlighting its drug developmental potential. Copyright © 2018 Elsevier

Lysophosphatidylcholine-induced cytotoxicity in osteoblast-like MG-63 cells: involvement of transient receptor potential vanilloid 2 (TRPV2) channels.

PubMed

Fallah, Abdallah; Pierre, Rachel; Abed, Elie; Moreau, Robert

2013-01-01

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Accordingly, atherogenic determinants such as oxidized low density lipoprotein (OxLDL) particles have been shown to alter bone cell functions. In this work, we investigated the cytotoxicity of lysophosphatidylcholine (lysoPC), a major phospholipid component generated upon LDL oxidation, on bone-forming MG-63 osteoblast-like cells. Cell viability was reduced by lysoPC in a concentration-dependent manner with a LC50 of 18.7±0.7 μM. LysoPC-induced cell death was attributed to induction of both apoptosis and necrosis. Since impairment of intracellular calcium homeostasis is often involved in mechanism of cell death, we determined the involvement of calcium in lysoPC-induced cytotoxicity. LysoPC promoted a rapid and transient increase in intracellular calcium attributed to mobilization from calcium stores, followed by a sustained influx. Intracellular calcium mobilization was associated to phospholipase C (PLC)-dependent mobilization of calcium from the endoplasmic reticulum since inhibition of PLC or calcium depletion of reticulum endoplasmic with thapsigargin prevented the calcium mobilization. The calcium influx induced by lysoPC was abolished by inhibition of transient receptor potential vanilloid (TRPV) channels with ruthenium red whereas gadolinium, which inhibits canonical TRP (TRPC) channels, was without effect. Accordingly, expression of TRPV2 and TRPV4 were shown in MG-63 cells. The addition of TRPV2 inhibitor Tranilast in the incubation medium prevent the calcium influx triggered by lysoPC and reduced lysoPC-induced cytotoxicity whereas TRPV4 inhibitor RN 1734 was without effect, which confirms the involvement of TRPV2 activation in lysoPC-induced cell death.

PKCepsilon-dependent potentiation of TTX-resistant Nav1.8 current by neurokinin-1 receptor activation in rat dorsal root ganglion neurons.

PubMed

Cang, Chun-Lei; Zhang, Hua; Zhang, Yu-Qiu; Zhao, Zhi-Qi

2009-06-30

Substance P (SP), which mainly exists in a subtype of small-diameter dorsal root ganglion (DRG) neurons, is an important signal molecule in pain processing in the spinal cord. Our previous results have proved the expression of SP receptor neurokinin-1 (NK-1) on DRG neurons and its interaction with transient receptor potential vanilloid 1 (TRPV1) receptor. In this study we investigated the effect of NK-1 receptor agonist on Na(v)1.8, a tetrodotoxin (TTX)-resistant sodium channel, in rat small-diameter DRG neurons employing whole-cell patch clamp recordings. NK-1 agonist [Sar(9), Met(O2)(11)]-substance P (Sar-SP) significantly enhanced the Na(v)1.8 currents in a subgroup of small-diameter DRG neurons under both the normal and inflammatory situation, and the enhancement was blocked by NK-1 antagonist Win51708 and protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), but not the protein kinase A (PKA) inhibitor H89. In particular, the inhibitor of PKCepsilon, a PKC isoform, completely blocked this effect. Under current clamp model, Sar-SP reduced the amount of current required to evoke action potentials and increased the firing rate in a subgroup of DRG neurons. These data suggest that activation of NK-1 receptor potentiates Na(v)1.8 sodium current via PKCepsilon-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.

Characterisation of the contribution of the GABA-benzodiazepine α1 receptor subtype to [11C]Ro15-4513 PET images

PubMed Central

Myers, James FM; Rosso, Lula; Watson, Ben J; Wilson, Sue J; Kalk, Nicola J; Clementi, Nicoletta; Brooks, David J; Nutt, David J; Turkheimer, Federico E; Lingford-Hughes, Anne R

2012-01-01

This positron emission tomography (PET) study aimed to further define selectivity of [11C]Ro15-4513 binding to the GABARα5 relative to the GABARα1 benzodiazepine receptor subtype. The impact of zolpidem, a GABARα1-selective agonist, on [11C]Ro15-4513, which shows selectivity for GABARα5, and the nonselective benzodiazepine ligand [11C]flumazenil binding was assessed in humans. Compartmental modelling of the kinetics of [11C]Ro15-4513 time-activity curves was used to describe distribution volume (VT) differences in regions populated by different GABA receptor subtypes. Those with low α5 were best fitted by one-tissue compartment models; and those with high α5 required a more complex model. The heterogeneity between brain regions suggested spectral analysis as a more appropriate method to quantify binding as it does not a priori specify compartments. Spectral analysis revealed that zolpidem caused a significant VT decrease (∼10%) in [11C]flumazenil, but no decrease in [11C]Ro15-4513 binding. Further analysis of [11C]Ro15-4513 kinetics revealed additional frequency components present in regions containing both α1 and α5 subtypes compared with those containing only α1. Zolpidem reduced one component (mean±s.d.: 71%±41%), presumed to reflect α1-subtype binding, but not another (13%±22%), presumed to reflect α5. The proposed method for [11C]Ro15-4513 analysis may allow more accurate selective binding assays and estimation of drug occupancy for other nonselective ligands. PMID:22214903

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

PubMed Central

Yang, Fan; Vu, Simon; Yarov-Yarovoy, Vladimir; Zheng, Jie

2016-01-01

Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S–S498F–L505T–Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular “glue” that bridges the S4–S5 linker to the S1–S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor. PMID:27298359

Differential regulation of primary afferent input to spinal cord by muscarinic receptor subtypes delineated using knockout mice.

PubMed

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2014-05-16

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Selective regulation of nuclear orphan receptors 4A by adenosine receptor subtypes in human mast cells

PubMed Central

Zhang, Li; Paine, Catherine

2010-01-01

Nuclear orphan receptors 4A (NR4A) are early responsive genes that belong to the superfamily of hormone receptors and comprise NR4A1, NR4A2 and NR4A3. They have been associated to transcriptional activation of multiple genes involved in inflammation, apoptosis and cell cycle control. Here, we establish a link between NR4As and adenosine, a paradoxical inflammatory molecule that can contribute to persistence of inflammation or mediate inflammatory shutdown. Transcriptomics screening of the human mast cell-line HMC-1 revealed a sharp induction of transcriptionally active NR4A2 and NR4A3 by the adenosine analogue NECA. The concomitant treatment of NECA and the adenosine receptor A2A (A2AAR) selective antagonist SCH-58261 exaggerated this effect, suggesting that upregulation of these factors in mast cells is mediated by other AR subtypes (A2B and A3) and that A2AAR activation counteracts NR4A2 and NR4A3 induction. In agreement with this, A2AAR-silencing amplified NR4A induction by NECA. Interestingly, a similar A2AAR modulatory effect was observed on ERK1/2 phosphorylation because A2AAR blockage exacerbated NECA-mediated phosphorylation of ERK1/2. In addition, PKC or MEK1/2 inhibition prevented ERK1/2 phosphorylation and antagonized AR-mediated induction of NR4A2 and NR4A3, suggesting the involvement of these kinases in AR to NR4A signaling. Finally, we observed that selective A2AAR activation with CGS-21680 blocked PMA-induced ERK1/2 phosphorylation and modulated the overexpression of functional nuclear orphan receptors 4A. Taken together, these results establish a novel PKC/ERK/nuclear orphan receptors 4A axis for adenosinergic signaling in mast cells, which can be modulated by A2AAR activation, not only in the context of adenosine but of other mast cell activating stimuli as well. PMID:21234122

Receptor-Binding Profiles of H7 Subtype Influenza Viruses in Different Host Species

PubMed Central

Gambaryan, Alexandra S.; Matrosovich, Tatyana Y.; Philipp, Jennifer; Munster, Vincent J.; Fouchier, Ron A. M.; Cattoli, Giovanni; Capua, Ilaria; Krauss, Scott L.; Webster, Robert G.; Banks, Jill; Bovin, Nicolai V.; Klenk, Hans-Dieter

2012-01-01

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4(6-O-HSO3)GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO3)GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry. PMID:22345462

Muscarinic acetylcholine receptor M1 and M3 subtypes mediate acetylcholine-induced endothelium-independent vasodilatation in rat mesenteric arteries.

PubMed

Tangsucharit, Panot; Takatori, Shingo; Zamami, Yoshito; Goda, Mitsuhiro; Pakdeechote, Poungrat; Kawasaki, Hiromu; Takayama, Fusako

2016-01-01

The present study investigated pharmacological characterizations of muscarinic acetylcholine receptor (AChR) subtypes involving ACh-induced endothelium-independent vasodilatation in rat mesenteric arteries. Changes in perfusion pressure to periarterial nerve stimulation and ACh were measured before and after the perfusion of Krebs solution containing muscarinic receptor antagonists. Distributions of muscarinic AChR subtypes in mesenteric arteries with an intact endothelium were studied using Western blotting. The expression level of M1 and M3 was significantly greater than that of M2. Endothelium removal significantly decreased expression levels of M2 and M3, but not M1. In perfused mesenteric vascular beds with intact endothelium and active tone, exogenous ACh (1, 10, and 100 nmol) produced concentration-dependent and long-lasting vasodilatations. In endothelium-denuded preparations, relaxation to ACh (1 nmol) disappeared, but ACh at 10 and 100 nmol caused long-lasting vasodilatations, which were markedly blocked by the treatment of pirenzepine (M1 antagonist) or 4-DAMP (M1 and M3 antagonist) plus hexamethonium (nicotinic AChR antagonist), but not methoctramine (M2 and M4 antagonist). These results suggest that muscarinic AChR subtypes, mainly M1, distribute throughout the rat mesenteric arteries, and that activation of M1 and/or M3 which may be located on CGRPergic nerves releases CGRP, causing an endothelium-independent vasodilatation. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

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.

Pharmacological Studies of NOP Receptor Agonists as Novel Analgesics

DTIC Science & Technology

2009-05-01

found in hot-chili peppers that evokes pain sensation by activating at the TRPV1 . TRPV1 and the up-regulation of its expression have been strongly...via nociceptin/orphanin FQ receptors. Br J Pharmacol 137:1355-1361. Knotkova H, Pappagallo M, Szallasi A (2008) Capsaicin ( TRPV1 Agonist) therapy...Szallasi A, Cortright DN, Blum CA, Eid SR (2007). The vanilloid receptor TRPV1 : 10 years from channel cloning to antagonist proof of concept. Nat Rev Drug

Actions of subtype-specific purinergic ligands on rat spiral ganglion neurons.

PubMed

Ito, Ken; Iwasaki, Shinichi; Kondo, Kenji; Dulon, Didier; Kaga, Kimitaka

2004-08-01

In a previous study we showed that, in rat spiral ganglion neurons (SGNs), the adenosine 5'-triphosphate (ATP)-evoked currents were a combination of the activation of ionotropic receptors (the first fast current) and the activation of metabotropic receptors which secondarily opened non-selective cation channels. These two conductances imply the involvement of different receptor subtypes. In the present study, we tested three subtype-specific purinergic ligands: alpha,beta-methylene ATP (a;pha,beta-meATP) for P2X receptors, uridine 5'-triphosphate (UTP) for P2Y receptors and 2'-3'-O-(4-benzoylbenzoyl) ATP (Bz-ATP) for P2Z (P2X(7)) receptors. Application of 100 microM alpha,beta-meATP did not trigger any significant change in membrane conductance, while the SGNs were responsive to ATP. Pressure application of UTP (100 microM, 1 s) evoked an inward current averaging 344+/-169 pA at a holding potential of -50 mV. The conductance developed after a latency averaging 1.5+/-0.6 s, took 4-6 s to peak and reversed slowly within 15-30 s. The current-voltage curve reversed near 0 mV, suggesting a non-selective cation conductance, like the second component of the ATP conductance. Bz-ATP evoked an inward current which developed without latency, was sustained during ligand application and was rapidly inactivated at the end of application: the same characteristics as the first component of the ATP-evoked current. The Bz-ATP conductance reversed around -10 mV, indicating also a non-selective cation conductance. These results suggest that, in SGNs, ATP acts via two different receptor subtypes, ionotropic P2Z receptors and metabotropic P2Y receptors, and that these two receptor subtypes can assume different physiological roles.

Transient Receptor Potential Vanilloid-1 (TRPV1) Is a Mediator of Lung Toxicity for Coal Fly Ash Particulate Material

PubMed Central

Deering-Rice, Cassandra E.; Johansen, Mark E.; Roberts, Jessica K.; Thomas, Karen C.; Romero, Erin G.; Lee, Jeewoo; Yost, Garold S.; Veranth, John M.

2012-01-01

Environmental particulate matter (PM) pollutants adversely affect human health, but the molecular basis is poorly understood. The ion channel transient receptor potential vanilloid-1 (TRPV1) has been implicated as a sensor for environmental PM and a mediator of adverse events in the respiratory tract. The objectives of this study were to determine whether TRPV1 can distinguish chemically and physically unique PM that represents important sources of air pollution; to elucidate the molecular basis of TRPV1 activation by PM; and to ascertain the contributions of TRPV1 to human lung cell and mouse lung tissue responses exposed to an insoluble PM agonist, coal fly ash (CFA1). The major findings of this study are that TRPV1 is activated by some, but not all of the prototype PM materials evaluated, with rank-ordered responses of CFA1 > diesel exhaust PM > crystalline silica; TRP melastatin-8 is also robustly activated by CFA1, whereas other TRP channels expressed by airway sensory neurons and lung epithelial cells that may also be activated by CFA1, including TRPs ankyrin 1 (A1), canonical 4α (C4α), M2, V2, V3, and V4, were either slightly (TRPA1) or not activated by CFA1; activation of TRPV1 by CFA1 occurs via cell surface interactions between the solid components of CFA1 and specific amino acid residues of TRPV1 that are localized in the putative pore-loop region; and activation of TRPV1 by CFA1 is not exclusive in mouse lungs but represents a pathway by which CFA1 affects the expression of selected genes in lung epithelial cells and airway tissue. PMID:22155782

Human pDCs display sex-specific differences in type I interferon subtypes and interferon α/β receptor expression.

PubMed

Ziegler, Susanne M; Beisel, Claudia; Sutter, Kathrin; Griesbeck, Morgane; Hildebrandt, Heike; Hagen, Sven H; Dittmer, Ulf; Altfeld, Marcus

2017-02-01

The outcomes of many diseases differ between women and men, with women experiencing a higher incidence and more severe pathogenesis of autoimmune and some infectious diseases. It has been suggested that this is partially due to activation of plasmacytoid dendritic cells (pDCs), the main producers of interferon (IFN)-α, in response to toll-like receptor (TLR)7 stimulation. We investigated the induction of type I IFN (IFN-I) subtypes upon TLR7 stimulation on isolated pDCs. Our data revealed a sex-specific differential expression of IFN-Is, with pDCs from females showing a significantly higher mRNA expression of all 13 IFN-α subtypes. In addition, pDCs from females had higher levels of IFN-β mRNA after stimulation, indicating that sex differences in IFN-I production by pDCs were mediated by a signaling event upstream of the first loop of IFN-I mRNA transcription. Furthermore, the surface expression levels of the common IFN-α/β receptor subunit 2 were significantly higher on pDCs from females in comparison to males. These data indicate that higher IFN-α production is already established at the mRNA level and propose a contribution of higher IFN-α/β receptor 2 expression on pDCs to the immunological differences in IFN-I production observed between females and males. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Yangambin, a lignan obtained from Ocotea duckei, differentiates putative PAF receptor subtypes in the gastrointestinal tract of rats.

PubMed

Jesus-Morais, C M; Assis, E F; Cordeiro, R S; Barbosa-Filho, J M; Lima, W T; Silva, Z L; Bozza, P T; Castro-Faria-Neto, H C

2000-04-01

We investigated the presence of PAF receptor subtypes in the tissues of the gastrointestinal tract, airways, blood vessels and in murine macrophages. For this purpose we have used a competitive PAF receptor antagonist, yangambin (YAN), extracted from the Brazilian plant "louro de cheiro" (Ocotea duckei Vattimo). Rat duodenum, jejunum, ileum, colon, stomach fundus, trachea and bronchia were removed and 1.5-2 cm muscle segments from those regions were mounted in a 10 ml organ bath with aerated physiological solution at 37 degrees C. PAF evoked a contraction of the rat jejunum, ileum, colon and stomach fundus. The contraction was slow and resistant to wash and was followed by desensitization to further doses of PAF. Contractions induced by PAF (10(-6) M) were inhibited by YAN (10(-7) to M-2 x 10(-5) M) and WEB 2086 (10(-6) m to M-5 M) in rat jejunum, ileum and colon but not in the stomach fundus. In the rat stomach fundus only WEB 2086 (5 x 10(-6) M) was able to block PAF-induced contraction. The contractions induced by acetylcholine, histamine, 5-hydroxytryptamine and vasopressin were not inhibited by prior administration of YAN. Yangambin also significantly inhibited PAF-induced vascular permeability in rat duodenum, jejunum, ileum, colon, and mesentery. Yangambin significantly inhibited PAF-induced lipid body formation in mice peritoneal macrophages. We suggest that YAN is a selective PAF antagonist which is able to discriminate putative PAF receptors subtypes present in the stomach fundus.

Carboxyl-terminal Domain of Transient Receptor Potential Vanilloid 1 Contains Distinct Segments Differentially Involved in Capsaicin- and Heat-induced Desensitization*

PubMed Central

Joseph, John; Wang, Sen; Lee, Jongseok; Ro, Jin Y.; Chung, Man-Kyo

2013-01-01

Multiple Ca2+-dependent processes are involved in capsaicin-induced desensitization of transient receptor potential vanilloid 1 (TRPV1), but desensitization of TRPV1 by heat occurs even in the absence of extracellular Ca2+, although the mechanisms are unknown. In this study, we tested the hypothesis that capsaicin and heat desensitize TRPV1 through distinct mechanisms involving distinct structural segments of TRPV1. In HEK293 cells that heterologously express TRPV1, we found that heat-induced desensitization was not affected by the inclusion of intracellular ATP or alanine mutation of Lys155, both of which attenuate capsaicin-induced desensitization, suggesting that heat-induced desensitization occurs through mechanisms distinct from capsaicin-induced desensitization. To determine protein domains involved in heat-induced desensitization, we generated chimeric proteins between TRPV1 and TRPV3, a heat-gated channel lacking heat-induced desensitization. We found that TRPV1 with the carboxyl-terminal domain (CTD) of TRPV3 retained heat activation but was impaired in heat-induced desensitization. Further experiments using chimeric or deletion mutants within TRPV1 CTD indicated that the distal half of CTD regulates the activation and desensitization of TRPV1 in modality-specific manners. Within the distal CTD, we identified two segments that distinctly regulated capsaicin- and heat-induced desensitization. The results suggest that the activation and desensitization of TRPV1 by capsaicin and heat can be modulated differentially and disproportionally through different regions of TRPV1 CTD. Identifying the domains involved in thermal regulation of TRPV1 may facilitate the development of novel anti-hyperalgesic approaches aimed at attenuating activation and enhancing desensitization of TRPV1 by thermal stimuli. PMID:24174527

Immunohistochemical localization and functional characterization of somatostatin receptor subtypes in a corticotropin releasing hormone-secreting adrenal phaeochromocytoma: review of the literature and report of a case

PubMed Central

Ruggeri, R.M.; Ferraù, F.; Campennì, A.; Simone, A.; Barresi, V.; Giuffrè, G.; Tuccari, G.; Baldari, S.; Trimarchi, F.

2009-01-01

Somastostatin receptors are frequently expressed in phaeochromocytoma but data on somatostatin receptor subtyping are scanty and the functional response to the somatostatin analogue octretide is still debated.We report an unusual case of pheochromocytoma, causing ectopic Cushing’s syndrome due to CRH production by the tumour cells, in a 50-yr-old woman. Abdominal computed tomography revealed an inhomogeneous, 9-cm mass in the right adrenal gland, and [111In-DTPA0] octreotide scintigraphy showed an abnormal uptake of the radiotracer in the right perirenal region, corresponding to the adrenal mass. The patient underwent laparoscopic surgery and formalin-fixed and paraffin-embedded samples were studied. The tumour was extensively characterized by immunohistochemistry and somatostatin receptor (SSTRs) subtypes expression was analyzed. Histological and immunohistochemical examination of the surgical specimens displayed a typical pheochromocytoma, which was found to be immunoreative to S-100, chromogranin A and neurofilaments. Immunostaining for SSTR subtypes showed a positive reaction for SSTR1, SSTR2A, SSTR2B, antisera on tumour cells. The intense and diffuse immunostaining for corticotropin releasing hormone (CRH) antiserum indicated that Cushing’s disease was dependent on CRH overproduction by the pheochromocytoma, in which no immunostaining for adrenocorticotropic hormone was found. Our report confirms the heterogeneity of the pattern of SSTR expression in pheochromocytomas, and provide further evidence for functional SSTR subtype SSTR2a in a subgroup of pheochromocytomas, suggesting that these tumours may represent potential target for octreotide treatment.

Immunohistochemical localization and functional characterization of somatostatin receptor subtypes in a corticotropin releasing hormone- secreting adrenal phaeochromocytoma: review of the literature and report of a case.

PubMed

Ruggeri, Rosaria M; Ferraù, F; Campennì, A; Simone, A; Barresi, V; Giuffrè, G; Tuccari, G; Baldari, S; Trimarchi, F

2009-01-01

Somastostatin receptors are frequently expressed in phaeochromocytoma but data on somatostatin receptor subtyping are scanty and the functional response to the somatostatin analogue octretide is still debated.We report an unusual case of pheochro-mocytoma,causing ectopic Cushing's syndrome due to CRH production by the tumour cells, in a 50-yr-old woman. Abdominal computed tomography revealed an inhomogeneous,9-cm mass in the right adrenal gland,and [111In-DTPA0] octreotide scintigraphy showed an abnormal uptake of the radiotracer in the right perirenal region,corresponding to the adrenal mass.The patient underwent laparoscopic surgery and formalin-fixed and paraffin embedded samples were studied. The tumour was extensively characterized by immunohistochemistry and somatostatin receptor (SSTRs) subtypes expression was analyzed.Histological and immunohistochemical examination of the surgical specimens displayed a typical pheochromocytoma,which was found to be immunoreative to S-100, chromogranin A and neurofilaments. Immunostaining for SSTR subtypes showed a positive reaction for SSTR1, SSTR2A, SSTR2B, antisera on tumour cells. The intense and diffuse immunostaining for corticotropin releasing hormone (CRH) antiserum indicated that Cushing's disease was dependent on CRH overproduction by the pheochromocytoma,in which no immunostaining for adrenocorticotropic hormone was found. Our report confirms the heterogeneity of the pattern of SSTR expression in pheochromocytomas,and provide further evidence for functional SSTR subtype SSTR2a in a subgroup of pheochromocytomas,suggesting that these tumours may represent potential target for octreotide treatment.

TRPV1 recapitulates native capsaicin receptor in sensory neurons in association with Fas-associated factor 1.

PubMed

Kim, Sangsung; Kang, Changjoong; Shin, Chan Young; Hwang, Sun Wook; Yang, Young Duk; Shim, Won Sik; Park, Min-Young; Kim, Eunhee; Kim, Misook; Kim, Byung-Moon; Cho, Hawon; Shin, Youngki; Oh, Uhtaek

2006-03-01

TRPV1, a cloned capsaicin receptor, is a molecular sensor for detecting adverse stimuli and a key element for inflammatory nociception and represents biophysical properties of native channel. However, there seems to be a marked difference between TRPV1 and native capsaicin receptors in the pharmacological response profiles to vanilloids or acid. One plausible explanation for this overt discrepancy is the presence of regulatory proteins associated with TRPV1. Here, we identify Fas-associated factor 1 (FAF1) as a regulatory factor, which is coexpressed with and binds to TRPV1 in sensory neurons. When expressed heterologously, FAF1 reduces the responses of TRPV1 to capsaicin, acid, and heat, to the pharmacological level of native capsaicin receptor in sensory neurons. Furthermore, silencing FAF1 by RNA interference augments capsaicin-sensitive current in native sensory neurons. We therefore conclude that FAF1 forms an integral component of the vanilloid receptor complex and that it constitutively modulates the sensitivity of TRPV1 to various noxious stimuli in sensory neurons.

Expression Analysis of Dopamine Receptor Subtypes in Normal Human Pituitaries, Nonfunctioning Pituitary Adenomas and Somatotropinomas, and the Association between Dopamine and Somatostatin Receptors with Clinical Response to Octreotide-LAR in Acromegaly

PubMed Central

Neto, Leonardo Vieira; Machado, Evelyn de O.; Luque, Raul M.; Taboada, Giselle F.; Marcondes, Jorge B.; Chimelli, Leila M. C.; Quintella, Leonardo Pereira; Niemeyer, Paulo; de Carvalho, Denise P.; Kineman, Rhonda D.; Gadelha, Mônica R.

2009-01-01

Context: Dopamine receptor (DR) and somatostatin receptor subtype expression in pituitary adenomas may predict the response to postsurgical therapies. Objectives: Our objectives were to assess and compare the mRNA levels of DR1-5 and somatostatin receptors 1–5 in normal pituitaries (NPs), nonfunctioning pituitary adenomas (NFPAs), and somatotropinomas. In addition, we determined whether the level of DR expression correlates with the in vivo response to octreotide-LAR in acromegalic patients. Design and Patients: Eight NPs, 30 NFPAs, and 39 somatotropinomas were analyzed for receptor mRNA levels by real-time RT-PCR. The DR2 short variant was estimated as the DR2 long/DR2 total (DR2T). The relationship between DR expression and the postsurgical response to octreotide-LAR was assessed in 19 of the acromegalic patients. Results: DR3 was not detected. The relationship between expression levels of DR subtypes in NPs and somatotropinomas was DR2T⋙DR4≫DR5>DR1, whereas in NFPAs, DR2T⋙DR4≫DR1>DR5. The DR2 short variant was the predominant DR2 variant in the majority of samples. In acromegalics treated with octreotide-LAR, DR1 was negatively correlated with percent GH reduction (3 months: r = −0.67, P = 0.002; and 6 months: r = −0.58, P = 0.009), and DR5 was positively correlated with percent IGF-I reduction (3 months: r = 0.55, P = 0.01; and 6 months: r = 0.47, P = 0.04). Conclusions: DR2 is the predominant DR subtype in NPs, NFPAs, and somatotropinomas. The fact that DR1, DR4, and DR5 are also expressed in many adenomas tested suggests that these receptors might also play a role in the therapeutic impact of postsurgical medical therapies in patients with NFPA and acromegaly. This was supported by the finding that the in vivo response to octreotide-LAR was negatively associated with DR1 and positively associated with DR5. PMID:19293270

Serotonin homeostasis and serotonin receptors as actors of cortical construction: special attention to the 5-HT3A and 5-HT6 receptor subtypes

PubMed Central

Vitalis, Tania; Ansorge, Mark S.; Dayer, Alexandre G.

2013-01-01

Cortical circuits control higher-order cognitive processes and their function is highly dependent on their structure that emerges during development. The construction of cortical circuits involves the coordinated interplay between different types of cellular processes such as proliferation, migration, and differentiation of neural and glial cell subtypes. Among the multiple factors that regulate the assembly of cortical circuits, 5-HT is an important developmental signal that impacts on a broad diversity of cellular processes. 5-HT is detected at the onset of embryonic telencephalic formation and a variety of serotonergic receptors are dynamically expressed in the embryonic developing cortex in a region and cell-type specific manner. Among these receptors, the ionotropic 5-HT3A receptor and the metabotropic 5-HT6 receptor have recently been identified as novel serotonergic targets regulating different aspects of cortical construction including neuronal migration and dendritic differentiation. In this review, we focus on the developmental impact of serotonergic systems on the construction of cortical circuits and discuss their potential role in programming risk for human psychiatric disorders. PMID:23801939

Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion

USGS Publications Warehouse

Jett, David A.; Hill, E.F.; Fernando, J.C.; Eldefrawi, M.E.; Eldefrawi, A.T.

1993-01-01

The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs

Transient receptor potential vanilloid type 2 (TRPV2) expression in normal urothelium and in urothelial carcinoma of human bladder: correlation with the pathologic stage.

PubMed

Caprodossi, Sara; Lucciarini, Roberta; Amantini, Consuelo; Nabissi, Massimo; Canesin, Giacomo; Ballarini, Patrizia; Di Spilimbergo, Adriana; Cardarelli, Marco Andrea; Servi, Lucilla; Mammana, Gabriele; Santoni, Giorgio

2008-09-01

To evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. Bladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal (n=6) and cancer bladder tissues (n=58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. Enhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. Normal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.

Breast Cancer Subtype is Associated With Axillary Lymph Node Metastasis

PubMed Central

He, Zhen-Yu; Wu, San-Gang; Yang, Qi; Sun, Jia-Yuan; Li, Feng-Yan; Lin, Qin; Lin, Huan-Xin

2015-01-01

Abstract The purpose of this study was to assess whether breast cancer subtype (BCS) as determined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 can predict the axillary lymph node metastasis in breast cancer. Patients who received breast conserving surgery or mastectomy and axillary lymph node dissection were identified from 2 cancer centers. The associations between clinicopathological variables and axillary lymph node involvement were evaluated in univariate and multivariate regression analyses. A total of 3471 patients met the inclusion criteria, and 53.0% had axillary lymph node metastases at diagnosis. Patients with hormone receptor (HR)−/human epidermal growth factor receptor 2 (HER2)− subtype had a higher grade disease and the lowest rate of lymphovascular invasion. Univariate and multivariable logistic regression analyses showed that BCS was significantly associated with lymph node involvement. Patients with the HR−/HER2− subtype had the lowest odds of having nodal positivity than those with other BCSs. HR+/HER2− (odds ratio [OR] 1.651, 95% confidence interval [CI]: 1.349–2.021, P < 0.001), HR+/HER2+ (OR 1.958, 95%CI 1.542–2.486, P < 0.001), and HR−/HER2+ (OR 1.525, 95%CI 1.181–1.970, P < 0.001) tumors had higher risk of nodal positivity than the HR−/HER2− subtype. The other independent predictors of nodal metastases included tumor size, tumor grade, and lymphovascular invasion. Breast cancer subtype can predict the presence of axillary lymph node metastasis in breast cancer. HR−/HER2− is associated with a reduced risk of axillary lymph node metastasis compared to other BCSs. Our findings may play an important role in guiding axillary treatment considerations if further confirmed in larger sample size studies. PMID:26632910

Dynamic regulation of glycinergic input to spinal dorsal horn neurones by muscarinic receptor subtypes in rats.

PubMed

Wang, Xiu-Li; Zhang, Hong-Mei; Li, De-Pei; Chen, Shao-Rui; Pan, Hui-Lin

2006-03-01

Activation of spinal muscarinic acetylcholine receptors (mAChRs) inhibits nociception. However, the cellular mechanisms of this action are not fully known. In this study, we determined the role of mAChR subtypes in regulation of synaptic glycine release in the spinal cord. Whole-cell voltage-clamp recordings were performed on lamina II neurones in the rat spinal cord slices. The mAChR agonist oxotremorine-M significantly increased the frequency of glycinergic sIPSCs but not mIPSCs. Surprisingly, the effect of oxotremorine-M on sIPSCs was largely attenuated at a higher concentration. On the other hand, 1-10 microm oxotremorine-M dose-dependently increased the frequency of sIPSCs in rats pretreated with intrathecal pertussis toxin. Furthermore, oxotremorine-M also dose-dependently increased the frequency of sIPSCs in the presence of himbacine (an M2/M4 mAChR antagonist) or AF-DX116 (an M2 mAChR antagonist). The M3 mAChR antagonist 4-DAMP abolished the stimulatory effect of oxotremorine-M on sIPSCs. Interestingly, the GABA(B) receptor antagonist CGP55845 potentiated the stimulatory effect of oxotremorine-M on sIPSCs. In the presence of CGP55845, both himbacine and AF-DX116 similarly reduced the potentiating effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that the M3 subtype is present on the somatodendritic site of glycinergic neurones and is mainly responsible for muscarinic potentiation of glycinergic input to spinal dorsal horn neurones. Concurrent stimulation of mAChRs on adjacent GABAergic interneurones attenuates synaptic glycine release through presynaptic GABA(B) receptors on glycinergic interneurones. This study illustrates a complex dynamic interaction between GABAergic and glycinergic synapses in the spinal cord dorsal horn.

Allosteric modulation of nicotinic and GABAA receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model.

PubMed

Yoshimura, Ryan F; Tran, Minhtam B; Hogenkamp, Derk J; Ayala, Narielle L; Johnstone, Timothy; Dunnigan, Andrew J; Gee, Timothy K; Gee, Kelvin W

2017-11-01

Autism spectrum disorder (ASD) is associated with two core symptoms (social communication deficits and stereotyped repetitive behaviors) in addition to a number of comorbidities. There are no FDA-approved drugs for the core symptoms and the changes that underlie these behaviors are not fully understood. One hypothesis is an imbalance of the excitation (E)/inhibition (I) ratio with excessive E and diminished I occurring in specific neuronal circuits. Data suggests that both gamma-aminobutyric acid A (GABA A ) and α7 nicotinic acetylcholine receptors (nAChRs) significantly impact E/I. BTBR T + tf/J (BTBR) mice are a model that display an autism-like phenotype with impaired social interaction and stereotyped behavior. A β2/3-subunit containing GABA A receptor (GABA A R) subtype selective positive allosteric modulator (PAM), 2-261, and an α7 nAChR subtype selective PAM, AVL-3288, were tested in social approach and repetitive self-grooming paradigms. 2-261 was active in the social approach but not the self-grooming paradigm, whereas AVL-3288 was active in both. Neither compound impaired locomotor activity. Modulating α7 nAChRs alone may be sufficient to correct these behavioral and cognitive deficits. GABAergic and nicotinic compounds are already in various stages of clinical testing for treatment of the core symptoms and comorbidities associated with ASD. Our findings and those of others suggest that compounds that have selective activities at GABA A R subtypes and the α7 nAChR may address not only the core symptoms, but many of the associated comorbidities as well and warrant further investigation in other models of ASD. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Discovery and Characterization of Novel Subtype-Selective Allosteric Agonists for the Investigation of M1 Receptor Function in the Central Nervous System

PubMed Central

2009-01-01

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M1−M5. Of the mAChR subtypes, M1 is among the most heavily expressed in regions that are critical for learning and memory and has been viewed as the most critical mAChR subtype for memory and attention mechanisms. Unfortunately, it has been difficult to develop selective activators of M1 and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M1. Using a functional high-throughput screening and subsequent diversity-oriented synthesis approach, we have discovered a novel series of highly selective M1 allosteric agonists. These compounds activate M1 with EC50 values in the 150−500 nM range and have unprecedented, clean ancillary pharmacology (no substantial activity at 10 μM across a large panel of targets). Targeted mutagenesis revealed a potentially novel allosteric binding site in the third extracellular loop of the M1 receptor for these allosteric agonists. Optimized compounds, such as VU0357017, provide excellent brain exposure after systemic dosing and have robust in vivo efficacy in reversing scopolamine-induced deficits in a rodent model of contextual fear conditioning. This series of selective M1 allosteric agonists provides critical research tools to allow dissection of M1-mediated effects in the CNS and potential leads for novel treatments for Alzheimer’s disease and schizophrenia. PMID:21961051

Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Adenophostin A and Its Analogues

PubMed Central

Saleem, Huma; Tovey, Stephen C.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

2013-01-01

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca2+ channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca2+ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca2+ signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes. PMID:23469136

Moclobemide attenuates anoxia and glutamate-induced neuronal damage in vitro independently of interaction with glutamate receptor subtypes.

PubMed

Verleye, Marc; Steinschneider, Remy; Bernard, François Xavier; Gillardin, Jean-Marie

2007-03-23

Recent data suggested the existence of a bidirectional relation between depression and neurodegenerative diseases resulting from cerebral ischemia injury. Glutamate, a major excitatory neurotransmitter, has long been recognised to play a key role in the pathophysiology of anoxia or ischemia, due to its excessive accumulation in the extracellular space and the subsequent activation of its receptors. A characteristic response to glutamate is the increase in cytosolic Na(+) and Ca(2+) levels which is due mainly to influx from the extracellular space, with a consequent cell swelling and oxidative metabolism dysfunction. The present study examined the in vitro effects of the antidepressant and type-A monoamine oxidase inhibitor, moclobemide, in neuronal-astroglial cultures from rat cerebral cortex exposed to anoxia (for 5 and 7 h) or to glutamate (2 mM for 6 h), two in vitro models of brain ischemia. In addition, the affinity of moclobemide for the different glutamate receptor subtypes and an interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and K(+) excess, respectively, were evaluated. Moclobemide (10-100 microM) included in the culture medium during anoxia or with glutamate significantly increased in a concentration-dependent manner the amount of surviving neurons compared to controls. Moclobemide displayed no binding affinity for the different glutamate receptor subtypes (IC(50)>100 microM) and did not block up to 300 microM the entry of Na(+) and of Ca(2+) activated by veratridine and K(+), respectively. These results suggest that the neuroprotective properties of moclobemide imply neither the glutamate neurotransmission nor the Na(+) and Ca(2+) channels.

Occurrence of breast cancer subtypes in adolescent and young adult women

PubMed Central

2012-01-01

Introduction Breast cancers are increasingly recognized as heterogeneous based on expression of receptors for estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2). Triple-negative tumors (ER-/PR-/HER2-) have been reported to be more common among younger women, but occurrence of the spectrum of breast cancer subtypes in adolescent and young adult (AYA) women aged between 15 and 39 years is otherwise poorly understood. Methods Data regarding all 5,605 AYA breast cancers diagnosed in California during the period 2005 to 2009, including ER and PR status (referred to jointly as hormone receptor (HR) status) and HER2 status, was obtained from the population-based California Cancer Registry. Incidence rates were calculated by subtype (triple-negative; HR+/HER2-; HR+/HER2+; HR-/HER2+), and logistic regression was used to evaluate differences in subtype characteristics by age group. Results AYAs had higher proportions of HR+/HER2+, triple-negative and HR-/HER2+ breast cancer subtypes and higher proportions of patients of non-White race/ethnicity than did older women. AYAs also were more likely to be diagnosed with stage III/IV disease and high-grade tumors than were older women. Rates of HR+/HER2- and triple-negative subtypes in AYAs varied substantially by race/ethnicity. Conclusions The distribution of breast cancer subtypes among AYAs varies from that observed in older women, and varies further by race/ethnicity. Observed subtype distributions may explain the poorer breast cancer survival previously observed among AYAs. PMID:22452927

Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide

PubMed Central

Bisogno, Tiziana; Hanuš, Lumír; De Petrocellis, Luciano; Tchilibon, Susanna; Ponde, Datta E; Brandi, Ines; Moriello, Aniello Schiano; Davis, John B; Mechoulam, Raphael; Di Marzo, Vincenzo

2001-01-01

(−)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA).CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5′ pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca2+ concentrations in cells over-expressing human VR1; (b) [14C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [14C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase.Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC50=3.2 – 3.5 μM, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67 – 70% of the effect obtained with ionomycin (4 μM). CBD (10 μM) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive.(+)-5′-DMH-CBD and (+)-7-hydroxy-5′-DMH-CBD inhibited [14C]-AEA uptake (IC50=10.0 and 7.0 μM); the (−)-enantiomers were slightly less active (IC50=14.0 and 12.5 μM). CBD and (+)-CBD were also active (IC50=22.0 and 17.0 μM).CBD (IC50=27.5 μM), (+)-CBD (IC50=63.5 μM) and (−)-7-hydroxy-CBD (IC50=34 μM), but not the other analogues (IC50>100 μM), weakly inhibited [14C]-AEA hydrolysis.Only the (+)-isomers exhibited high affinity for CB1 and/or CB2 cannabinoid receptors.These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield

Function of brain α2B-adrenergic receptor characterized with subtype-selective α2B antagonist and KO mice.

PubMed

Luhrs, Lauren; Manlapaz, Cynthia; Kedzie, Karen; Rao, Sandhya; Cabrera-Ghayouri, Sara; Donello, John; Gil, Daniel

2016-12-17

Noradrenergic signaling, through the α 2A and α 2C adrenergic receptors modulates the cognitive and behavioral symptoms of disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction. However, it is unknown whether the α 2B receptor has any significant role in CNS function. The present study elucidates the potential role of the α 2B receptor in CNS function via the discovery and use of the first subtype-selective α 2B antagonist (AGN-209419), and behavioral analyses of α-receptor knockout (KO) mice. Using AGN-209419 as radioligand, α 2B receptor binding sites were identified within the olfactory bulb, cortex, thalamus, cerebellum, and striatum. Based on the observed expression patterns of α 2 subtypes in the brain, we compared α 2B KO, α 2A KO and α 2C KO mice behavioral phenotypes with their respective wild-type lines in anxiety (plus maze), compulsive (marble burying), and sensorimotor (prepulse inhibition) tasks. α 2B KO mice exhibited increased marble burying and α 2C KO mice exhibited an increased startle response to a pulse stimulus, but otherwise intact prepulse inhibition. To further explore compulsive behavior, we evaluated novelty-induced locomotor hyperactivity and found that α 2B KO and α 2C KO mice exhibited increased locomotion in the open field. Interestingly, when challenged with amphetamine, α 2C KO mice increased activity at lower doses relative to either α 2A KO or WT mice. However, α 2B KO mice exhibited stereotypy at doses of amphetamine that were only locomotor stimulatory to all other genotypes. Following co-administration of AGN-209419 with low-dose amphetamine in WT mice, stereotypy was observed, mimicking the α 2B KO phenotype. These findings suggest that the α 2B receptor is involved in CNS behaviors associated with sensorimotor gating and compulsivity, and may be therapeutically relevant for disorders such as schizophrenia, ADHD, post-traumatic stress disorder, addiction, and

Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.

PubMed

Sulk, Mathias; Seeliger, Stephan; Aubert, Jerome; Schwab, Verena D; Cevikbas, Ferda; Rivier, Michel; Nowak, Pawel; Voegel, Johannes J; Buddenkotte, Jörg; Steinhoff, Martin

2012-04-01

Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea.

Distribution and Expression of Non-Neuronal Transient Receptor Potential (TRPV) Ion Channels in Rosacea

PubMed Central

Sulk, Mathias; Seeliger, Stephan; Aubert, Jerome; Schwab, Verena D.; Cevikbas, Ferda; Rivier, Michel; Nowak, Pawel; Voegel, Johannes J.; Buddenkotte, Jörg; Steinhoff, Martin

2011-01-01

Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea. PMID:22189789

ROLES OF OPIOID RECEPTOR SUBTYPES IN MEDIATING ALCOHOL SEEKING INDUCED BY DISCRETE CUES AND CONTEXT

PubMed Central

Marinelli, Peter W.; Funk, Douglas; Harding, Stephen; Li, Zhaoxia; Juzytsch, Walter; Lê, A.D.

2009-01-01

The aim of this study was to assess the effects of selective blockade of the delta (DOP) or mu opioid (MOP) receptors on alcohol seeking induced by discrete cues and context. In Experiment 1, rats were trained to self-administer alcohol in an environment with distinct sensory properties. After extinction in a different context with separate sensory properties, rats were tested for context-induced renewal in the original context following treatment with the DOP receptor antagonist naltrindole (0 – 15-mg/kg, IP) or the MOP receptor antagonist CTOP (0 – 3-µg/kg ICV). In a separate set of experiments, reinstatement was tested with the presentation of a discrete light+tone cue previously associated with alcohol delivery, following extinction without the cue. In Experiment 2, the effects of naltrindole (0 – 5-mg/kg, IP) or CTOP (0 – 3-µg/kg µg ICV) were assessed. For context-induced renewal, 7.5-mg/kg naltrindole reduced responding without affecting locomotor activity. Both doses of CTOP attenuated responding in the first 15 min of the renewal test session; however, total responses did not differ at the end of the session. For discrete cue-induced reinstatement, 1 and 5-mg/kg naltrindole attenuated responding, but CTOP had no effect. We conclude that while DOP receptors mediate alcohol seeking induced by discrete cues and context, MOP receptors may play a modest role only in context-induced renewal. These findings point to a differential involvement of opioid receptor subtypes in the effects of different kinds of conditioned stimuli on alcohol seeking, and support a more prominent role for DOP receptors. PMID:19686472

Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

PubMed

Lai, H; Carino, M A

1992-07-01

Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

Opposing functions of spinal M2, M3, and M4 receptor subtypes in regulation of GABAergic inputs to dorsal horn neurons revealed by muscarinic receptor knockout mice.

PubMed

Zhang, Hong-Mei; Chen, Shao-Rui; Matsui, Minoru; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

2006-03-01

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.

Clinical Overestimation of HER2 Positivity in Early Estrogen and Progesterone Receptor-Positive Breast Cancer and the Value of Molecular Subtyping Using BluePrint.

PubMed

Myburgh, Ettienne J; Langenhoven, Lizanne; Grant, Kathleen A; van der Merwe, Lize; Kotze, Maritha J

2017-08-01

Human epidermal growth factor receptor 2 (HER2) positivity is an important prognostic and predictive indicator in breast cancer. HER2 status is determined by immunohistochemistry and fluorescent in situ hybridization (FISH), which are potentially inaccurate techniques as a result of several technical factors, polysomy of chromosome 17, and amplification or overexpression of CEP17 (centromeric probe for chromosome 17) and/or HER2. In South Africa, HER2-positive tumors are excluded from a MammaPrint (MP; Agendia BV, Amsterdam, Netherlands) pretest algorithm. Clinical HER2 status has been reported to correlate poorly with molecular subtype. The aim of this study was to investigate the correlation of clinical HER2 status with BluePrint (BP) molecular subtyping. Clinico-pathologic and genomic information was extracted from a prospectively collected central MP database containing records of 256 estrogen receptor-positive and/or progesterone receptor-positive tumors. Twenty-one tumors considered HER2 positive on immunohistochemistry or FISH were identified for this study. The median age of patients was 56 years (range, 34 to 77 years), with a median tumor size of 16 mm (3 to 27 mm). Four (19%) tumors were confirmed HER2-enriched subtype, six (29%) were luminal A, and 11 (52%) were luminal B. The positive predictive values of HER2/CEP17 ratio ≥ 2 and HER2 copy number ≥ 6 were only 29% and 40%, respectively. The differences in means for HER2/CEP17 ratio were significant between BP HER2-enriched versus luminal ( P = .0249; 95% CI, 0.12 to 1.21) and MP high-risk versus low-risk tumors ( P = .0002; 95% CI, 0.40 to 1.06). Of the 21 tumors considered clinically HER2 positive, only four were HER2-enriched subtype with BP, indicating an overestimation of HER2 positivity. FISH testing has a poor positive predictive value.

Tonic suppression of spontaneous baroreceptor reflex by endogenous angiotensins via AT(2) subtype receptors at nucleus reticularis ventrolateralis in the rat.

PubMed

Lin, K; Chan, S H; Chan, J Y

2001-04-01

We evaluated the role of endogenous angiotensins at the rostral nucleus reticularis ventrolateralis (NRVL) in the modulation of spontaneous baroreceptor reflex (BRR) response and the subtype of angiotensin receptors involved using rats anesthetized and maintained with pentobarbital sodium. Bilateral microinjection of angiotensin II (ANG II) or its active metabolite angiotensin III (ANG III) (5, 10, or 20 pmol) into the NRVL significantly suppressed the spontaneous BRR response, as represented by the magnitude of transfer function between systemic arterial pressure and heart rate signals. The inhibitory effect of ANG III (20 pmol) was discernibly reversed by coadministration with its peptide antagonist, [Ile(7)]ANG III (1.6 nmol), or the nonpeptide AT(2) receptor antagonist, PD-123319 (1.6 nmol), but not by the nonpeptide AT(1) receptor antagonist, losartan (1.6 nmol). On the other hand, the peptide antagonist, [Sar(1), Ile(8)]ANG II (1.6 nmol) or both non-peptide antagonists appreciably reversed the suppressive action of ANG II (20 pmol). Whereas losartan produced minimal effect, blocking the endogenous activity of the angiotensins by microinjection into the bilateral NRVL of PD-123319, [Sar(1), Ile(8)]ANG II or [Ile(7)]ANG III elicited significant enhancement of the spontaneous BRR response. We conclude that under physiologic conditions both endogenous ANG II and ANG III may exert a tonic inhibitory modulation on the spontaneous BRR response by acting selectively on the AT(2) subtype receptors at the NRVL. Copyright 2001 Wiley-Liss, Inc.

[Effect of high-fat diet on expression of transient receptor potential vanilloid 1 in respiratory tract and dorsal root ganglion of mice].

PubMed

Zhu, Lian; Xu, Zhi-Liang

2017-07-01

To investigate the effect of high-fat diet on the expression of transient receptor potential vanilloid 1 (TRPV1) in the respiratory system and the dorsal root ganglion (DRG) of mice, as well as its effect on the excitability of sensory neurons. A total of 20 C57BL/6 mice were randomly divided into normal-diet (ND) group and high-fat diet (HFD) group, with 10 mice in each group. The mice were given corresponding diets and body weights were monitored. After 7 weeks of feeding, lung tissue, bronchial tissue, and DRG at thoracic segments 3-4 were collected and immunohistochemical staining was performed. A patch clamp was used to measure the number of action potentials and TRPV1 current intensity in the DRG. After 7 weeks of feeding, the HFD group had significantly greater mean weight gain than the ND group (6.4±2.6 g vs 2.3±0.5 g; P<0.001). The HFD group had significantly higher expression of TRPV1 in the bronchus, pulmonary alveoli, and DRG than the ND group (P<0.05). Compared with the ND group, the HFD group had significant increases in the TRPV1 current intensity and number of action potentials in the DRG (P<0.05). High-fat diet induces a significant increase in body weight and leads to high expression of TRPV1 and high excitability in the respiratory system and the peripheral sensory neurons. This suggests that TRPV1 may be an important factor in the physiopathological mechanisms of bronchial hyperresponsiveness.

Age-Specific Incidence of Breast Cancer Subtypes: Understanding the Black–White Crossover

PubMed Central

2012-01-01

Background Breast cancer incidence is higher among black women than white women before age 40 years, but higher among white women than black women after age 40 years (black–white crossover). We used newly available population-based data to examine whether the age-specific incidences of breast cancer subtypes vary by race and ethnicity. Methods We classified 91908 invasive breast cancers diagnosed in California between January 1, 2006, and December 31, 2009, by subtype based on tumor expression of estrogen receptor (ER) and progesterone receptor (PR)—together referred to as hormone receptor (HR)—and human epidermal growth factor receptor 2 (HER2). Breast cancer subtypes were classified as ER or PR positive and HER2 negative (HR+/HER2−), ER or PR positive and HER2 positive (HR+/HER2+), ER and PR negative and HER2 positive (HR−/HER2+), and ER, PR, and HER2 negative (triple-negative). We calculated and compared age-specific incidence rates, incidence rate ratios, and 95% confidence intervals by subtype and race (black, white, Hispanic, and Asian). All P values are two-sided. Results We did not observe an age-related black–white crossover in incidence for any molecular subtype of breast cancer. Compared with white women, black women had statistically significantly higher rates of triple-negative breast cancer at all ages but statistically significantly lower rates of HR+/HER2− breast cancers after age 35 years (all P < .05). The age-specific incidence of HR+/HER2+ and HR−/HER2+ subtypes did not vary markedly between white and black women. Conclusions The black–white crossover in breast cancer incidence occurs only when all breast cancer subtypes are combined and relates largely to higher rates of triple-negative breast cancers and lower rates of HR+/HER2− breast cancers in black vs white women. PMID:22773826

Ameliorating Endothelial Mitochondrial Dysfunction Restores Coronary Function via Transient Receptor Potential Vanilloid 1-Mediated Protein Kinase A/Uncoupling Protein 2 Pathway.

PubMed

Xiong, Shiqiang; Wang, Peijian; Ma, Liqun; Gao, Peng; Gong, Liuping; Li, Li; Li, Qiang; Sun, Fang; Zhou, Xunmei; He, Hongbo; Chen, Jing; Yan, Zhencheng; Liu, Daoyan; Zhu, Zhiming

2016-02-01

Coronary heart disease arising from atherosclerosis is a leading cause of cardiogenic death worldwide. Mitochondria are the principal source of reactive oxygen species (ROS), and defective oxidative phosphorylation by the mitochondrial respiratory chain contributes to ROS generation. Uncoupling protein 2 (UCP2), an adaptive antioxidant defense factor, protects against mitochondrial ROS-induced endothelial dysfunction in atherosclerosis. The activation of transient receptor potential vanilloid 1 (TRPV1) attenuates vascular dysfunction. Therefore, whether TRPV1 activation antagonizes coronary lesions by alleviating endothelial mitochondrial dysfunction and enhancing the activity of the protein kinase A/UCP2 pathway warrants examination. ApoE(-/-), ApoE(-/-)/TRPV1(-/-), and ApoE(-/-)/UCP2(-/-) mice were fed standard chow, a high-fat diet (HFD), or the HFD plus 0.01% capsaicin. HFD intake profoundly impaired coronary vasodilatation and myocardial perfusion and shortened the survival duration of ApoE(-/-) mice. TRPV1 or UCP2 deficiency exacerbated HFD-induced coronary dysfunction and was associated with increased ROS generation and reduced nitric oxide production in the endothelium. The activation of TRPV1 by capsaicin upregulated UCP2 expression via protein kinase A phosphorylation, thereby alleviating endothelial mitochondrial dysfunction and inhibiting mitochondrial ROS generation. In vivo, dietary capsaicin supplementation enhanced coronary relaxation and prolonged the survival duration of HFD-fed ApoE(-/-) mice. These effects were not observed in ApoE(-/-) mice lacking the TRPV1 or UCP2 gene. The upregulation of protein kinase A /UCP2 via TRPV1 activation ameliorates coronary dysfunction and prolongs the lifespan of atherosclerotic mice by ameliorating endothelial mitochondrial dysfunction. Dietary capsaicin supplementation may represent a promising intervention for the primary prevention of coronary heart disease. © 2015 American Heart Association, Inc.

Possible involvement of the transient receptor potential vanilloid type 1 channel in postoperative adhesive obstruction and its prevention by a kampo (traditional Japanese) medicine, daikenchuto.

PubMed

Tokita, Yohei; Yamamoto, Masahiro; Satoh, Kazuko; Nishiyama, Mitsue; Iizuka, Seiichi; Imamura, Sachiko; Kase, Yoshio

2011-01-01

This study focused on the localization of transient receptor potential vanilloid type 1 (TRPV1) in the intestines in postoperative adhesion model rats and investigated the underlying mechanism for the anti-adhesion action of daikenchuto (DKT), especially in relation to TRPV1. Postoperative intestinal adhesion was induced by sprinkling talc in the small intestine. The expression of TRPV1 mRNA was examined by in situ hybridization and real-time RT-PCR. The effects of DKT and its major ingredient, hydroxy sanshool, with or without ruthenium red, a TRP-channel antagonist, on talc-induced intestinal adhesions were evaluated. The level of TRPV1 mRNA was higher in the adhesion regions of talc-treated rats than in normal small intestine of sham-operated rats. Localization of TRPV1 mRNA expression was identified in the submucosal plexus of both sham-operated and talc-treated rats; and in talc-treated rats, it was observed also in the myenteric plexus and regions of adhesion. Capsaicin, DKT, and hydroxy sanshool significantly prevented formation of intestinal adhesions. The effects of DKT and hydroxy sanshool were abrogated by subcutaneous injection of ruthenium red. These results suggest that pharmacological modulation of TRPV1 might be a possible therapeutic option in postoperative intestinal adhesion, which might be relevant to the prevention of postoperative adhesive obstruction by DKT.

Crystal structure of arrestin-3 reveals the basis of the difference in receptor binding between two non-visual subtypes

PubMed Central

Zhan, Xuanzhi; Gimenez, Luis E.; Gurevich, Vsevolod V.; Spiller, Benjamin W.

2011-01-01

Arrestins are multi-functional proteins that regulate signaling and trafficking of the majority of G protein-coupled receptors (GPCRs), as well as sub-cellular localization and activity of many other signaling proteins. Here we report the first crystal structure of arrestin-3, solved at 3.0Å. Arrestin-3 is an elongated two-domain molecule with the overall fold and key inter-domain interactions that hold free protein in the basal conformation similar to the other subtypes. Arrestin-3 is the least selective member of the family, binding wide variety of GPCRs with high affinity and demonstrating lower preference for active phosphorylated forms of the receptors. In contrast to the other three arrestins, part of the receptor-binding surface in the arrestin-3 C-domain does not form a contiguous β-sheet, consistent with increased flexibility. By swapping the corresponding elements between arrestin-2 and -3 we show that the presence of this loose structure correlates with reduced arrestin selectivity for activated receptor, consistent with a conformational change in this β-sheet upon receptor binding. PMID:21215759

Identification of N-methyl-D-aspartic acid (NMDA) receptor subtype-specific binding sites that mediate direct interactions with scaffold protein PSD-95.

PubMed

Cousins, Sarah L; Stephenson, F Anne

2012-04-13

N-methyl-D-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149-1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins.

The 5-HT1-like receptor mediating the increase in canine external carotid blood flow: close resemblance to the 5-HT1D subtype.

PubMed Central

Villalón, C M; Terrón, J A

1994-01-01

1. It has recently been shown that the increase in external carotid blood flow induced by 5-hydroxy-tryptamine (5-HT) in the anaesthetized dog, being mimicked by 5-carboxamidotryptamine (5-CT), inhibited by methiothepin, vagosympathectomy and sympatho-inhibitory drugs, and resistant to blockade by ritanserin and MDL 72222, is mediated by stimulation of prejunctional 5-HT1-like receptors leading to an inhibitory action on carotid sympathetic nerves; these 5-HT1-like receptors are unrelated to either the 5-HT1A, 5-HT1B or 5-HT1C (now 5-HT2C) receptor subtypes. Inasmuch as 5-CT, 5-methoxytryptamine, sumatriptan and metergoline display high affinity, amongst other 5-HT binding sites, for the 5-HT1D subtype, in the present study we have used these drugs in an attempt to determine whether the above inhibitory prejunctional 5-HT1-like receptors correlate with the 5-HT1D subtype. 2. One-minute intracarotid (i.c.) infusions of 5-HT (0.3, 1, 3 and 10 micrograms), 5-CT (0.01, 0.03, 0.1 and 0.3 micrograms), 5-methoxytryptamine (1, 3, 10 and 30 micrograms) and sumatriptan (1, 3, 10, 30 and 100 micrograms) resulted in dose-dependent increases in external carotid blood flow (without changes in mean arterial blood pressure or heart rate) with the following rank order of agonist potency: 5-CT >> 5-HT > 5-methoxytryptamine > or = sumatriptan. Interestingly, sumatriptan-induced vasodilatation was followed by a more pronounced vasoconstriction. 3. The external carotid vasodilator effects of 5-HT, 5-CT, 5-methoxytryptamine and sumatriptan were dose-dependently and specifically antagonized by metergoline (10, 30 and/or 100 micrograms kg-1, i.v.).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7812603

Analysis of transient receptor potential ankyrin 1 (TRPA1) in frogs and lizards illuminates both nociceptive heat and chemical sensitivities and coexpression with TRP vanilloid 1 (TRPV1) in ancestral vertebrates.

PubMed

Saito, Shigeru; Nakatsuka, Kazumasa; Takahashi, Kenji; Fukuta, Naomi; Imagawa, Toshiaki; Ohta, Toshio; Tominaga, Makoto

2012-08-31

Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1.

Analysis of Transient Receptor Potential Ankyrin 1 (TRPA1) in Frogs and Lizards Illuminates Both Nociceptive Heat and Chemical Sensitivities and Coexpression with TRP Vanilloid 1 (TRPV1) in Ancestral Vertebrates*

PubMed Central

Saito, Shigeru; Nakatsuka, Kazumasa; Takahashi, Kenji; Fukuta, Naomi; Imagawa, Toshiaki; Ohta, Toshio; Tominaga, Makoto

2012-01-01

Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1. PMID:22791718

FAS Death Receptor: A Breast Cancer Subtype-Specific Radiation Response Biomarker and Potential Therapeutic Target

PubMed Central

Horton, Janet K.; Siamakpour-Reihani, Sharareh; Lee, Chen-Ting; Zhou, Ying; Chen, Wei; Geradts, Joseph; Fels, Diane R.; Hoang, Peter; Ashcraft, Kathleen A.; Groth, Jeff; Kung, Hsiu-Ni; Dewhirst, Mark W.; Chi, Jen-Tsan A.

2015-01-01

Although a standardized approach to radiotherapy has been used to treat breast cancer, regardless of subtype (e.g., luminal, basal), recent clinical data suggest that radiation response may vary significantly among subtypes. We hypothesized that this clinical variability may be due, in part, to differences in cellular radiation response. In this study, we utilized RNA samples for microarray analysis from two sources: 1. Paired pre- and postirradiation breast tumor tissue from 32 early-stage breast cancer patients treated in our unique preoperative radiation Phase I trial; and 2. Sixteen biologically diverse breast tumor cell lines exposed to 0 and 5 Gy irradiation. The transcriptome response to radiation exposure was derived by comparing gene expression in samples before and after irradiation. Genes with the highest coefficient of variation were selected for further evaluation and validated at the RNA and protein level. Gene editing and agonistic antibody treatment were performed to assess the impact of gene modulation on radiation response. Gene expression in our cohort of luminal breast cancer patients was distinctly different before and after irradiation. Further, two distinct patterns of gene expression were observed in our biologically diverse group of breast cancer cell lines pre- versus postirradiation. Cell lines that showed significant change after irradiation were largely luminal subtype, while gene expression in the basal and HER2+ cell lines was minimally impacted. The 100 genes with the most significant response to radiation in patients were identified and analyzed for differential patterns of expression in the radiation-responsive versus nonresponsive cell lines. Fourteen genes were identified as significant, including FAS, a member of the tumor necrosis factor receptor family known to play a critical role in programed cell death. Modulation of FAS in breast cancer cell lines altered radiation response phenotype and enhanced radiation sensitivity in

Repeat low-level blast exposure increases transient receptor potential vanilloid 1 (TRPV1) and endothelin-1 (ET-1) expression in the trigeminal ganglion

PubMed Central

Burke, Teresa A.; Doyle Brackley, Allison; Jeske, Nathaniel A.; Cleland, Jeffery M.; Lund, Brian J.

2017-01-01

Blast-associated sensory and cognitive trauma sustained by military service members is an area of extensively studied research. Recent studies in our laboratory have revealed that low-level blast exposure increased expression of transient receptor potential vanilloid 1 (TRPV1) and endothelin-1 (ET-1), proteins well characterized for their role in mediating pain transmission, in the cornea. Determining the functional consequences of these alterations in protein expression is critical to understanding blast-related sensory trauma. Thus, the purpose of this study was to examine TRPV1 and ET-1 expression in ocular associated sensory tissues following primary and tertiary blast. A rodent model of blast injury was used in which anesthetized animals, unrestrained or restrained, received a single or repeat blast (73.8 ± 5.5 kPa) from a compressed air shock tube once or daily for five consecutive days, respectively. Behavioral and functional analyses were conducted to assess blast effects on nocifensive behavior and TRPV1 activity. Immunohistochemistry and Western Blot were also performed with trigeminal ganglia (TG) to determine TRPV1, ET-1 and glial fibrillary associated protein (GFAP) expression following blast. Increased TRPV1, ET-1 and GFAP were detected in the TG of animals exposed to repeat blast. Increased nocifensive responses were also observed in animals exposed to repeat, tertiary blast as compared to single blast and control. Moreover, decreased TRPV1 desensitization was observed in TG neurons exposed to repeat blast. Repeat, tertiary blast resulted in increased TRPV1, ET-1 and GFAP expression in the TG, enhanced nociception and decreased TRPV1 desensitization. PMID:28797041

Allosteric Modulation of Metabotropic Glutamate Receptors

PubMed Central

Sheffler, Douglas J.; Gregory, Karen J.; Rook, Jerri M.; Conn, P. Jeffrey

2013-01-01

The development of receptor subtype-selective ligands by targeting allosteric sites of G protein-coupled receptors (GPCRs) has proven highly successful in recent years. One GPCR family that has greatly benefited from this approach is the metabotropic glutamate receptors (mGlus). These family C GPCRs participate in the neuromodulatory actions of glutamate throughout the CNS, where they play a number of key roles in regulating synaptic transmission and neuronal excitability. A large number of mGlu subtype-selective allosteric modulators have been identified, the majority of which are thought to bind within the transmembrane regions of the receptor. These modulators can either enhance or inhibit mGlu functional responses and, together with mGlu knockout mice, have furthered the establishment of the physiologic roles of many mGlu subtypes. Numerous pharmacological and receptor mutagenesis studies have been aimed at providing a greater mechanistic understanding of the interaction of mGlu allosteric modulators with the receptor, which have revealed evidence for common allosteric binding sites across multiple mGlu subtypes and the presence for multiple allosteric sites within a single mGlu subtype. Recent data have also revealed that mGlu allosteric modulators can display functional selectivity toward particular signal transduction cascades downstream of an individual mGlu subtype. Studies continue to validate the therapeutic utility of mGlu allosteric modulators as a potential therapeutic approach for a number of disorders including anxiety, schizophrenia, Parkinson’s disease, and Fragile X syndrome. PMID:21907906

Identification of N-Methyl-d-aspartic Acid (NMDA) Receptor Subtype-specific Binding Sites That Mediate Direct Interactions with Scaffold Protein PSD-95*

PubMed Central

Cousins, Sarah L.; Stephenson, F. Anne

2012-01-01

N-methyl-d-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149–1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins. PMID:22375001

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1.

PubMed

Lee, Yi-Chen; Lu, Shui-Chin; Hsieh, Yu-Lin

2018-02-13

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 µg/kg, respectively) and concluded that a critical dose of RTX (50 µg/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established

Identification of Critical Residues Involved in Ligand Binding and G Protein Signaling in Human Somatostatin Receptor Subtype 2

PubMed Central

Parry, Jesse J.; Chen, Ronald; Andrews, Rebecca; Lears, Kimberly A.

2012-01-01

G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling. PMID:22495673

Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5′-O-(1-boranotriphosphate) derivatives

PubMed Central

Tulapurkar, M E; Laubinger, W; Nahum, V; Fischer, B; Reiser, G

2004-01-01

P2Y-nucleotide receptors represent important targets for drug development. The lack of stable and receptor specific agonists, however, has prevented successful therapeutic applications. A novel series of P-boronated ATP derivatives (ATP-α-B) were synthesized by substitution of a nonbridging O at Pα with a BH3 group. This introduces a chiral center, thus resulting in diastereoisomers. In addition, at C2 of the adenine ring a further substitution was made (Cl- or methylthio-). The pairs of diastereoisomers were denoted here as A and B isomers. Here, we tested the receptor subtype specificity of these analogs on HEK 293 cells stably expressing rat P2Y1 and rat P2Y2 receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y1-GFP, rP2Y2-GFP). We investigated agonist-induced receptor endocytosis, [Ca2+]i rise and arachidonic acid (AA) release. Agonist-induced endocytosis of rP2Y1-GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP-α-B analogs. Both 2-MeS-substituted diastereoisomers induced a greater degree of agonist-induced receptor endocytosis as compared to the 2-Cl-substituted derivatives. Endocytosis results are in accordance with the potency to induce Ca2+ release by these compounds in HEK 293 cells stably transfected with rP2Y1. In case of rP2Y2-GFP, the borano-nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca2+ release, AA release and in inducing receptor endocytosis. The different ATP-α-B derivatives and also the diastereoisomers were equally ineffective. Thus, the new agonists may be considered as potent and highly specific agonist drug candidates for P2Y1 receptors. The difference in activity of the ATP analogs at P2Y receptors could be used as a tool to investigate structural differences between P2Y receptor subtypes. PMID:15197109

Structure of the full-length TRPV2 channel by cryo-EM

NASA Astrophysics Data System (ADS)

Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

2016-03-01

Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a `minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ~5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

Structure of the full-length TRPV2 channel by cryo-EM.

PubMed

Huynh, Kevin W; Cohen, Matthew R; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T; Zhou, Z Hong; Moiseenkova-Bell, Vera Y

2016-03-29

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

miR-155 functions downstream of angiotensin II receptor subtype 1 and calcineurin to regulate cardiac hypertrophy.

PubMed

Yang, Yong; Zhou, Yong; Cao, Zheng; Tong, Xin Zhu; Xie, Hua Qiang; Luo, Tao; Hua, Xian Ping; Wang, Han Qin

2016-09-01

Cardiac hypertrophy is characterized by maladaptive tissue remodeling that may lead to heart failure or sudden death. MicroRNAs (miRs) are negative regulators of angiotensin II and the angiotensin II receptor subtype 1 (AGTR 1 ), which are two components involved in cardiac hypertrophy. In the present study, the interaction between angiotensin II receptor subtype 1 (AGTR 1 ) signaling and miR-155 was investigated. Rat H9C2 (2-1) cardiomyocytes were transfected with miR-155 analogues or inhibitors, then stimulated with angiotensin II to induce cardiac hypertrophy. miR-155 expression was revealed to be altered following transfection with chemically-modified miR-155 analogues and inhibitors in rat cardiomyocytes. In cell cardiac hypertrophy models, the cell surface area, AGTR 1 , atrial natriuretic peptide and myosin heavy chain-β mRNA expression levels were revealed to be lower in cells stimulated with miR-155 analogue-transfected cells treated with angiotensin II compared with cells stimulated with angiotensin alone (P<0.05), as determined using reverse transcription-polymerase chain reaction (PCR), quantitative PCR and western blot analyses. Furthermore, calcineurin mRNA and protein, intracellular free calcium and nuclear factor of activated T-cells-4 proteins were downregulated in miR-155 analogue-transfected cells treated with angiotensin II, as compared with cells stimulated with angiotensin II alone (P<0.05). In conclusion, the current study indicates that miR-155 may improve cardiac hypertrophy by downregulating AGTR 1 and suppressing the calcium signaling pathways activated by AGTR 1 .

Cannabinoid hyperemesis syndrome: potential mechanisms for the benefit of capsaicin and hot water hydrotherapy in treatment.

PubMed

Richards, John R; Lapoint, Jeff M; Burillo-Putze, Guillermo

2018-01-01

Cannabinoid hyperemesis syndrome is a clinical disorder that has become more prevalent with increasing use of cannabis and synthetic cannabinoids, and which is difficult to treat. Standard antiemetics commonly fail to alleviate the severe nausea and vomiting characteristic of the syndrome. Curiously, cannabinoid hyperemesis syndrome patients often report dramatic relief of symptoms with hot showers and baths, and topical capsaicin. In this review, we detail the pharmacokinetics and pharmacodynamics of capsaicin and explore possible mechanisms for its beneficial effect, including activation of transient receptor potential vanilloid 1 and neurohumoral regulation. Putative mechanisms responsible for the benefit of hot water hydrotherapy are also investigated. An extensive search of PubMed, OpenGrey, and Google Scholar from inception to April 2017 was performed to identify known and theoretical thermoregulatory mechanisms associated with the endocannabinoid system. The searches resulted in 2417 articles. These articles were screened for relevant mechanisms behind capsaicin and heat activation having potential antiemetic effects. References from the selected articles were also hand-searched. A total of 137 articles were considered relevant and included. Capsaicin: Topical capsaicin is primarily used for treatment of neuropathic pain, but it has also been used successfully in some 20 cases of cannabinoid hyperemesis syndrome. The pharmacokinetics and pharmacodynamics of capsaicin as a transient receptor potential vanilloid 1 agonist may explain this effect. Topical capsaicin has a longer half-life than oral administration, thus its potential duration of benefit is longer. Capsaicin and transient receptor potential vanilloid 1: Topical capsaicin binds and activates the transient receptor potential vanilloid 1 receptor, triggering influx of calcium and sodium, as well as release of inflammatory neuropeptides leading to transient burning, stinging, and itching. This elicits

Effects of norepinephrine on alpha-subtype receptors in the feline pulmonary vascular bed.

PubMed

Kaye, Alan D; Hoover, Jason M; Baber, Syed R; Ibrahim, Ikhlass N; Fields, Aaron M

2004-11-01

To test the hypothesis that norepinephrine induces a pressor response in the pulmonary vascular bed of the cat and identify the alpha-(1)adrenoceptor subtypes involved in the mediation or modulation of these effects. Prospective vehicle controlled study. University research laboratory. Intact chest preparation, adult mongrel cats. In separate experiments, the effects of 5-methyl-urapidil, a selective alpha-(1)A-subtype adrenoceptor antagonist, chloroethylclonidine, an alpha-(1)B-subtype and -(1)D-subtype adrenoceptor antagonist, and BMY 7378, the selective alpha-(1)D-subtype adrenoceptor antagonist, were investigated on pulmonary arterial responses to norepinephrine and other agonists in the pulmonary vascular bed of the cat. The systemic pressure and lobar arterial perfusion pressure were continuously monitored, electronically averaged, and permanently recorded. In the feline pulmonary vascular bed of the isolated left lower lobe, norepinephrine induced a dose-dependent vasoconstrictor response that was not significantly altered after administration of BMY 7378. However, the responses to norepinephrine were significantly attenuated following administration of 5-methyl-urapidil and chloroethylclonidine. The results of the present study suggest that norepinephrine has potent vasopressor activity in the pulmonary vascular bed of the cat and that this response may be mediated or modulated by both alpha-(1)A-subtype and -(1)B-subtype adrenoceptor sensitive pathways.

Dietary fat intake and development of specific breast cancer subtypes.

PubMed

Sieri, Sabina; Chiodini, Paolo; Agnoli, Claudia; Pala, Valeria; Berrino, Franco; Trichopoulou, Antonia; Benetou, Vassiliki; Vasilopoulou, Effie; Sánchez, María-José; Chirlaque, Maria-Dolores; Amiano, Pilar; Quirós, J Ramón; Ardanaz, Eva; Buckland, Genevieve; Masala, Giovanna; Panico, Salvatore; Grioni, Sara; Sacerdote, Carlotta; Tumino, Rosario; Boutron-Ruault, Marie-Christine; Clavel-Chapelon, Françoise; Fagherazzi, Guy; Peeters, Petra H M; van Gils, Carla H; Bueno-de-Mesquita, H Bas; van Kranen, Henk J; Key, Timothy J; Travis, Ruth C; Khaw, Kay Tee; Wareham, Nicholas J; Kaaks, Rudolf; Lukanova, Annekatrin; Boeing, Heiner; Schütze, Madlen; Sonestedt, Emily; Wirfält, Elisabeth; Sund, Malin; Andersson, Anne; Chajes, Veronique; Rinaldi, Sabina; Romieu, Isabelle; Weiderpass, Elisabete; Skeie, Guri; Dagrun, Engeset; Tjønneland, Anne; Halkjær, Jytte; Overvard, Kim; Merritt, Melissa A; Cox, David; Riboli, Elio; Krogh, Vittorio

2014-04-09

We prospectively evaluated fat intake as predictor of developing breast cancer (BC) subtypes defined by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor (HER2), in a large (n = 337327) heterogeneous cohort of women, with 10062 BC case patients after 11.5 years, estimating BC hazard ratios (HRs) by Cox proportional hazard modeling. High total and saturated fat were associated with greater risk of ER(+)PR(+) disease (HR = 1.20, 95% confidence interval [CI] = 1.00 to 1.45; HR = 1.28, 95% CI = 1.09 to 1.52; highest vs lowest quintiles) but not ER(-)PR(-) disease. High saturated fat was statistically significantly associated with greater risk of HER2(-) disease. High saturated fat intake particularly increases risk of receptor-positive disease, suggesting saturated fat involvement in the etiology of this BC subtype. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Greater absolute risk for all subtypes of breast cancer in the US than Malaysia.

PubMed

Horne, Hisani N; Beena Devi, C R; Sung, Hyuna; Tang, Tieng Swee; Rosenberg, Philip S; Hewitt, Stephen M; Sherman, Mark E; Anderson, William F; Yang, Xiaohong R

2015-01-01

Hormone receptor (HR) negative breast cancers are relatively more common in low-risk than high-risk countries and/or populations. However, the absolute variations between these different populations are not well established given the limited number of cancer registries with incidence rate data by breast cancer subtype. We, therefore, used two unique population-based resources with molecular data to compare incidence rates for the 'intrinsic' breast cancer subtypes between a low-risk Asian population in Malaysia and high-risk non-Hispanic white population in the National Cancer Institute's surveillance, epidemiology, and end results 18 registries database (SEER 18). The intrinsic breast cancer subtypes were recapitulated with the joint expression of the HRs (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor-2 (HER2). Invasive breast cancer incidence rates overall were fivefold greater in SEER 18 than in Malaysia. The majority of breast cancers were HR-positive in SEER 18 and HR-negative in Malaysia. Notwithstanding the greater relative distribution for HR-negative cancers in Malaysia, there was a greater absolute risk for all subtypes in SEER 18; incidence rates were nearly 7-fold higher for HR-positive and 2-fold higher for HR-negative cancers in SEER 18. Despite the well-established relative breast cancer differences between low-risk and high-risk countries and/or populations, there was a greater absolute risk for HR-positive and HR-negative subtypes in the US than Malaysia. Additional analytical studies are sorely needed to determine the factors responsible for the elevated risk of all subtypes of breast cancer in high-risk countries like the United States.

Memory Effects of Benzodiazepines: Memory Stages and Types Versus Binding-Site Subtypes

PubMed Central

Savić, Miroslav M.; Obradović, Dragan I.; Ugrešić, Nenad D.; Bokonjić, Dubravko R.

2005-01-01

Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the α1 and α1 subunits, whereas the effects on procedural memory can be mainly mediated by the α1 subunit. The pervading involvement of the α1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of α5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states. PMID:16444900

Epidemiological risk factors associated with inflammatory breast cancer subtypes.

PubMed

Atkinson, Rachel L; El-Zein, Randa; Valero, Vicente; Lucci, Anthony; Bevers, Therese B; Fouad, Tamer; Liao, Weiqin; Ueno, Naoto T; Woodward, Wendy A; Brewster, Abenaa M

2016-03-01

In this single-institution case-control study, we identified risk factors associated with inflammatory breast cancer (IBC) subtypes based on staining of estrogen receptor (ER), progesterone receptor (PR) and expression of human epidermal growth factor 2 (HER2neu) to determine distinct etiologic pathways. We identified 224 women with IBC and 396 cancer-free women seen at the MD Anderson Cancer Center. Multinomial logistic regression was used to estimate odds ratios (ORs) and 95 % confidence intervals (CIs) for associations between breast cancer risk factors and the IBC tumor subtypes: luminal (ER+ and/or PR+/HER2neu-), HER2neu+ (any ER and PR, HER2neu+), and triple-negative (ER-/PR-/HER2neu-). In multivariable analysis, compared with women age ≥26 at first pregnancy, women age <26 had a higher risk of triple-negative IBC (OR 3.32, 95% CI 1.37-8.05). Women with a history of breast-feeding had a lower risk of triple-negative (OR 0.30; 95% CI 0.15-0.62) and luminal IBC (OR 0.35, 95% CI 0.18-0.68). A history of smoking was associated with an increased risk of luminal IBC (OR 2.37; 95% CI 1.24-4.52). Compared with normal-weight women, those who were overweight or obese (body mass index ≥25 kg/m(2)) had a higher risk of all three tumor subtypes (p < 0.01 for all subtypes). Overweight or obese status is important modifiable risk factor for IBC of any subtype. Modifiable risk factors, age at first pregnancy (≥26), breast-feeding, and smoking may be associated with specific IBC subtypes. These results highlight the importance of evaluating epidemiologic risk factors for IBC for the identification of subtype-specific prevention strategies.

XEN-D0501, a Novel Transient Receptor Potential Vanilloid 1 Antagonist, Does Not Reduce Cough in Patients with Refractory Cough.

PubMed

Belvisi, Maria G; Birrell, Mark A; Wortley, Michael A; Maher, Sarah A; Satia, Imran; Badri, Huda; Holt, Kimberley; Round, Patrick; McGarvey, Lorcan; Ford, John; Smith, Jaclyn A

2017-11-15

Heightened cough responses to inhaled capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, are characteristic of patients with chronic cough. However, previously, a TRPV1 antagonist (SB-705498) failed to improve spontaneous cough frequency in these patients, despite small reductions in capsaicin-evoked cough. XEN-D0501 (a potent TRPV1 antagonist) was compared with SB-705498 in preclinical studies to establish whether an improved efficacy profile would support a further clinical trial of XEN-D0501 in refractory chronic cough. XEN-D0501 and SB-705498 were profiled against capsaicin in a sensory nerve activation assay and in vivo potency established against capsaicin-induced cough in the guinea pig. Twenty patients with refractory chronic cough participated in a double-blind, randomized, placebo-controlled crossover study evaluating the effect of 14 days of XEN-D0501 (oral, 4 mg twice daily) versus placebo on awake cough frequency (primary outcome), capsaicin-evoked cough, and patient-reported outcomes. XEN-D0501 was more efficacious and 1,000-fold more potent than SB-705498 at inhibiting capsaicin-induced depolarization of guinea pig and human isolated vagus nerve. In vivo XEN-D0501 completely inhibited capsaicin-induced cough, whereas 100 times more SB-705498 was required to achieve the same effect. In patients, XEN-D0501 substantially reduced maximal cough responses to capsaicin (mean change from baseline, XEN-D0501, -19.3 ± 16.4) coughs; placebo, -1.8 ± 5.8 coughs; P < 0.0001), but not spontaneous awake cough frequency (mean change from baseline, XEN-D0501, 6.7  ± 16.9 coughs/h; placebo, 0.4 ± 13.7 coughs/h; P = 0.41). XEN-D0501 demonstrated superior efficacy and potency in preclinical and clinical capsaicin challenge studies; despite this improved pharmacodynamic profile, spontaneous cough frequency did not improve, ruling out TRPV1 as an effective therapeutic target for refractory cough. Clinical trial registered

Fatty acid metabolism in breast cancer subtypes

PubMed Central

Monaco, Marie E.

2017-01-01

Dysregulation of fatty acid metabolism is recognized as a component of malignant transformation in many different cancers, including breast; yet the potential for targeting this pathway for prevention and/or treatment of cancer remains unrealized. Evidence indicates that proteins involved in both synthesis and oxidation of fatty acids play a pivotal role in the proliferation, migration and invasion of breast cancer cells. The following essay summarizes data implicating specific fatty acid metabolic enzymes in the genesis and progression of breast cancer, and further categorizes the relevance of specific metabolic pathways to individual intrinsic molecular subtypes of breast cancer. Based on mRNA expression data, the less aggressive luminal subtypes appear to rely on a balance between de novo fatty acid synthesis and oxidation as sources for both biomass and energy requirements, while basal-like, receptor negative subtypes overexpress genes involved in the utilization of exogenous fatty acids. With these differences in mind, treatments may need to be tailored to individual subtypes. PMID:28412757

Expression changes of serotonin receptor gene subtype 5HT3a in peripheral blood mononuclear cells from schizophrenic patients treated with haloperidol and Olanzapin.

PubMed

Shariati, Gholam Reza; Ahangari, Ghasem; Hossein-nezhad, Arash; Asadi, Seyed Mohammad; Pooyafard, Farzaneh; Ahmadkhaniha, Hamid Reza

2009-09-01

Serotonin receptors are involved in pathophysiology of schizophrenia and may mediate other neurotransmitter effects. We investigated serotonin receptors gene expression in peripheral blood mononuclear cells (PBMC) of naïve schizophrenic patients, before and after treatment. Also serotonin receptor gene expression was compared in two treatment groups including Haloperidol and Olanzapine. The PBMC was separated from whole blood by Ficoll-hypaque. The total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for 5HT(3a) serotonin receptor mRNA and beta-actin as internal control. The results showed the presence of subtype of serotonin receptor in lymphocytes. Serotonin gene expression showed significant changes in Olanzapine treatment group which correlated with Clinical Global Impression (CGI) score improvement. In conclusion, the present study has shown that human PBMC express serotonin receptors 5HT(3a). Moreover, clinical symptom improvement of Olanzapin may be demonstrated by a change in serotonin receptor gene expression.

Efficacy of ABT-116, an antagonist of transient receptor potential vanilloid type 1, in providing analgesia for dogs with chemically induced synovitis.

PubMed

Cathcart, Curtis J; Johnston, Spencer A; Reynolds, Lisa R; Al-Nadaf, Sami; Budsberg, Steven C

2012-01-01

To investigate the ability of ABT-116 (a proprietary antagonist of transient receptor potential vanilloid type 1) administered at 2 doses to attenuate lameness in dogs with experimentally induced urate synovitis. 8 purpose-bred mixed-breed dogs. In a 4-way crossover study, dogs orally received each of low-dose ABT-116 treatment (LDA; 10 mg/kg), high-dose ABT-116 treatment (HDA; 30 mg/kg), firocoxib (5 mg/kg), and no treatment (nontreatment) once a day for 2 days, in a randomly assigned order. Synovitis was induced on the second day of each treatment period by intra-articular injection of either stifle joint with sodium urate, alternating between joints for each treatment period, beginning with the left stifle joint. Ground reaction forces, clinical lameness scores, and rectal temperature were assessed before the injection (baseline) and at various points afterward. Lameness scores at the 2-, 6-, and 12-hour assessment points were higher than baseline scores for HDA and nontreatment, whereas scores at the 2- and 6-hour points were higher than baseline scores for LDA. For firocoxib, there was no difference from baseline scores in lameness scores at any point. Compared with baseline values, peak vertical force and vertical impulse were lower at 2 and 6 hours for HDA and nontreatment and at 2 hours for LDA. No changes in these values were evident for firocoxib. The HDA or LDA resulted in higher rectal temperatures than did treatment with firocoxib or nothing, but those temperatures did not differ among treatments. HDA had no apparent effect on sodium urate-induced lameness; LDA did attenuate the lameness but not as completely as firocoxib treatment. High rectal temperature is an adverse effect of oral ABT-116 administration that may be of clinical concern.

Genetic variability of prostaglandin E2 receptor subtype EP4 gene in aspirin-intolerant chronic urticaria.

PubMed

Palikhe, Nami Shrestha; Sin, Hye Jung; Kim, Seung Hyun; Sin, Hyun Jung; Hwang, Eui Kyung; Ye, Young Min; Park, Hae-Sim

2012-08-01

Prostaglandin E2 receptor subtype EP4 (PTGER4) is one of the four subtypes of receptors for prostaglandin E2 (PGE2). Overproduction of cysteinyl leukotriene in mast cells may be related with suppression of PGE2 in patients with aspirin hypersensitivity. Considering the association of PTGER4 in mast cells, urticaria- and aspirin-related disease, we hypothesized the genetic variability of PTGER4 may be associated with aspirin-intolerant chronic urticaria (AICU). The case-control study was performed in 141 with AICU, 153 with aspirin-tolerant chronic urticaria (ATCU) and 174 with normal controls (NCs). PTGER4 promoter single-nucleotide polymorphism was genotyped using a primer extension method with the SNAPshot ddNTP primer extension kit. The functional variability of PTGER4 promoter polymorphism was carried out by dual-luciferase system and electrophoretic mobility shift assay (EMSA) in human mast cells (HMC-1). Furthermore, the effect of aspirin was performed for PTGER4 mRNA expression using real-time PCR, and PGE2 production was checked in HMC-1 cells using ELISA. AICU patients carrying GG genotype at -1254 G>A showed significantly higher frequency compared with NC (P=0.032). Similarly, the minor allele frequency, G allele was significantly higher in AICU compared with NC (P=0.031). In vitro functional study demonstrated that the -1254 G allele had lower luciferase activity (P<0.001) in HMC-1 cells. EMSA finding showed that PTGER4 -1254 G produced a specific band. Significantly decreased PTGER4 expression (P=0.008) and PGE2 production by aspirin exposure was confirmed in in vitro HMC cell line model (P=0.001). The PTGER4 -1254 G allele demonstrated a higher frequency in AICU patients and lower promoter activity with decreased expression of PTGER4 and contributes to the development of AICU.

Moxibustion relieves visceral hyperalgesia via inhibition of transient receptor potential vanilloid 1 (TRPV1) and heat shock protein (HSP) 70 expression in rat bone marrow cells.

PubMed

Zou, Weiying; Lin, Hua; Liu, Wenwen; Yang, Bei; Wu, Lei; Duan, Limin; Ling, Ping; Zhu, Lingyan; Dai, Qun; Zhao, Lintong; Zou, Ting; Zhang, Dalei

2016-04-01

To investigate the effects of moxibustion on visceral hyperalgesia (VH) and bone marrow cell transient receptor potential vanilloid type 1 (TRPV1) and heat shock protein (HSP) 70 expression in a rat model of VH. Mechanical colorectal distension was performed to induce VH in neonatal Sprague-Dawley rats. Eight-week-old VH rats were treated with moxibustion at acupuncture point BL25 or an ipsilateral non-acupuncture point. Abdominal withdrawal reflex (AWR) scoring and pain threshold pressure assessment were performed before and after moxibustion treatment for 7 consecutive days. The expression of TRPV1 and HSP70 in bone marrow cells was quantified by real-time quantitative PCR. The expression of TRPV1 and HSP70 in bone marrow cells was increased in rats with VH. Moxibustion at BL25 significantly decreased AWR scores and increased pain threshold pressure in rats with VH. Furthermore, moxibustion at BL25 significantly inhibited the VH-induced increase in the expression of TRPV1 and HSP70 in bone marrow cells. The up-regulation of TRPV1 and HSP70 expression in bone marrow cells may be involved in visceral pain development and the analgesic effect of moxibustion on VH may be mediated through down-regulation of TRPV1 and HSP70 expression in bone marrow cells. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Evidence for Alpha Receptors in the Human Ureter

NASA Astrophysics Data System (ADS)

Madeb, Ralph; Knopf, Joy; Golijanin, Dragan; Bourne, Patricia; Erturk, Erdal

2007-04-01

An immunohistochemical and western blot expression analysis of human ureters was performed in order to characterize the alpha-1-adrenergic receptor distribution along the length of the human ureteral wall. Mapping the distribution will assist in understanding the potential role alpha -1-adrenergic receptors and their subtype density might have in the pathophysiology of ureteral colic and stone passage. Patients diagnosed with renal cancer or bladder cancer undergoing nephrectomy, nephroureterectomy, or cystectomy had ureteral specimens taken from the proximal, mid, distal and tunneled ureter. Tissues were processed for fresh frozen examination and fixed in formalin. None of the ureteral specimens were involved with cancer. Serial histologic sections and immunohistochemical studies were performed using antibodies specific for alpha-1-adrenergic receptor subtypes (alpha 1a, alpha 1b, alpha 1d). The sections were examined under a light microscope and scored as positive or negative. In order to validate and quantify the alpha receptor subtypes along the human ureter. Western blotting techniques were applied. Human ureter stained positively for alpha -1-adrenergic receptors. Immunostaining appeared red, with intense reaction in the smooth muscle of the ureter and endothelium of the neighboring blood vessels. There was differential expression between all the receptors with the highest staining for alpha-1D subtype. The highest protein expression for all three subtypes was in the renal pelvis and decreased with advancement along the ureter to the distal ureter. At the distal ureter, there was marked increase in expression as one progressed towards the ureteral orifice. The same pattern of protein expression was exhibited for all three alpha -1-adrenergic receptor subtypes. We provide preliminary evidence for the ability to detect and quantify the alpha-1-receptor subtypes along the human ureter which to the best of our knowledge has never been done with

Adenosine receptor desensitization and trafficking.

PubMed

Mundell, Stuart; Kelly, Eamonn

2011-05-01

As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. Copyright © 2010 Elsevier B.V. All rights reserved.

Transient receptor potential channel superfamily: Role in lower urinary tract function.

PubMed

Ogawa, Teruyuki; Imamura, Tetsuya; Nakazawa, Masaki; Hiragata, Shiro; Nagai, Takashi; Minagawa, Tomonori; Yokoyama, Hitoshi; Ishikawa, Masakuni; Domen, Takahisa; Ishizuka, Osamu

2015-11-01

Lower urinary tract symptoms associated with neurogenic bladder and overactive bladder syndrome are mediated in part by members of the transient receptor potential channel superfamily. The best studied member of this superfamily is the vanilloid receptor. Other transient receptor potential channels, such as the melastatin receptor and the ankyrin receptor, are also active in the pathogenesis of lower urinary tract dysfunction. However, the detailed mechanisms by which the transient receptor potential channels contribute to lower urinary tract symptoms are still not clear, and the therapeutic benefits of modulating transient receptor potential channel activity have not been proved in the clinical setting. In the present review, to better understand the pathophysiology and therapeutic potential for lower urinary tract symptoms, we summarize the presence and role of different members of the transient receptor potential channel superfamily in the lower urinary tract. © 2015 The Japanese Urological Association.

Glucocorticoid-induced fetal programming alters the functional complement of angiotensin receptor subtypes within the kidney.

PubMed

Gwathmey, TanYa M; Shaltout, Hossam A; Rose, James C; Diz, Debra I; Chappell, Mark C

2011-03-01

We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80 to 81 days gestation with full-term delivery. Renal nuclear and plasma membrane fractions were isolated from sheep age 1.0 to 1.5 years for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were significantly higher in the BMX-exposed adult offspring versus CON sheep. The proportion of nuclear AT(1) receptors sensitive to losartan was 2-fold higher (67 ± 6% vs 27 ± 9%; P<0.01) in BMX compared with CON. In contrast, the proportion of AT(2) sites was only one third that of controls (BMX, 25 ± 11% vs CON, 78 ± 4%; P<0.01), with a similar reduction in sites sensitive to the Ang-(1-7) antagonist D-Ala7-Ang-(1-7) with BMX exposure. Functional studies revealed that Ang II stimulated ROS to a greater extent in BMX than in CON sheep (16 ± 3% vs 6 ± 4%; P<0.05); however, NO production to Ang II was attenuated in BMX (26 ± 7% vs 82 ± 14%; P<0.05). BMX exposure was also associated with a reduction in the Ang-(1-7) NO response (75 ± 8% vs 131 ± 26%; P<0.05). We conclude that altered expression of angiotensin receptor subtypes may be one mechanism whereby functional changes in NO- and ROS-dependent signaling pathways may favor the sustained increase in blood pressure evident in fetal programming.

Structure of the full-length TRPV2 channel by cryo-EM

PubMed Central

Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

2016-01-01

Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a ‘minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2–6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels. PMID:27021073

Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs.

PubMed

Smith, N; Broadley, K J

2008-09-01

Endogenous adenosine levels are raised in the lungs during asthma attacks. 5'-adenosine monophosphate (5'-AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea-pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper-reactivity (AHR) and inflammatory cell recruitment to the lungs. The aim of this study was to investigate the roles of A(1), A(2A), A(2B) and A(3) adenosine receptors in these responses to inhaled 5'-AMP in sensitized guinea-pigs. Comparisons were made with the effect of dexamethasone treatment on 5'-AMP-induced responses. Functional airways responses to inhaled 5'-AMP (3 and 300 mM) of actively sensitized, conscious guinea-pigs were determined by whole-body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mM) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. 5'-AMP at 3 mM caused an immediate bronchoconstriction (EAR), whereas 300 mM caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A(2A) receptor antagonist, ZM241385, further enhanced cell influx after 5'-AMP inhalation (3 and 300 mM), and blocked the immediate bronchodilator response to 300 mM 5'-AMP, exposing an EAR. The A(2B) receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5'-AMP (300 mM). The A(3) receptor antagonist, MRS1220, inhibited 5'-AMP-induced inflammatory cell influx. The A(1) receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5'-AMP inhalation (300 mM). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5'-AMP (300 mM). All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5'-AMP in sensitized guinea-pigs.

Identification and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype

USDA-ARS?s Scientific Manuscript database

Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and Seven Stx2 subtypes have been described in E. coli, which were found to differ in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated...

Specificity of arrestin subtypes in regulating airway smooth muscle G protein-coupled receptor signaling and function.

PubMed

Pera, Tonio; Hegde, Akhil; Deshpande, Deepak A; Morgan, Sarah J; Tiegs, Brian C; Theriot, Barbara S; Choi, Yeon H; Walker, Julia K L; Penn, Raymond B

2015-10-01

Arrestins have been shown to regulate numerous G protein-coupled receptors (GPCRs) in studies employing receptor/arrestin overexpression in artificial cell systems. Which arrestin isoforms regulate which GPCRs in primary cell types is poorly understood. We sought to determine the effect of β-arrestin-1 or β-arrestin-2 inhibition or gene ablation on signaling and function of multiple GPCRs endogenously expressed in airway smooth muscle (ASM). In vitro [second messenger (calcium, cAMP generation)], ex vivo (ASM tension generation in suspended airway), and in vivo (invasive airway resistance) analyses were performed on human ASM cells and murine airways/whole animal subject to β-arrestin-1 or -2 knockdown or knockout (KO). In both human and murine model systems, knockdown or KO of β-arrestin-2 relative to control missense small interfering RNA or wild-type mice selectively increased (40-60%) β2-adrenoceptor signaling and function. β-arrestin-1 knockdown or KO had no effect on signaling and function of β2-adrenoceptor or numerous procontractile GPCRs, but selectively inhibited M3 muscarinic acetylcholine receptor signaling (∼50%) and function (∼25% ex vivo, >50% in vivo) without affecting EC50 values. Arrestin subtypes differentially regulate ASM GPCRs and β-arrestin-1 inhibition represents a novel approach to managing bronchospasm in obstructive lung diseases. © FASEB.

Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide

PubMed Central

Vellani, Vittorio; Mapplebeck, Sarah; Moriondo, Andrea; Davis, John B; McNaughton, Peter A

2001-01-01

The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium

Advanced oxidation protein products sensitized the transient receptor potential vanilloid 1 via NADPH oxidase 1 and 4 to cause mechanical hyperalgesia.

PubMed

Ding, Ruoting; Jiang, Hui; Sun, Baihui; Wu, Xiaoliang; Li, Wei; Zhu, Siyuan; Liao, Congrui; Zhong, Zhaoming; Chen, Jianting

2016-12-01

Oxidative stress is a possible pathogenesis of hyperalgesia. Advanced oxidation protein products (AOPPs), a new family of oxidized protein compounds, have been considered as a novel marker of oxidative stress. However, the role of AOPPs in the mechanism of hyperalgesia remains unknown. Our study aims to investigate whether AOPPs have an effect on hyperalgesia and the possible underlying mechanisms. To identify the AOPPs involved, we induced hyperalgesia in rats by injecting complete Freund's adjuvant (CFA) in hindpaw. The level of plasma AOPPs in CFA-induced rats was 1.6-fold in comparison with what in normal rats (P<0.05). After intravenous injection of AOPPs-modified rat serum albumin (AOPPs-RSA) in Sprague-Dawley rats, the paw mechanical thresholds, measured by the electronic von Frey system, significantly declined. Immunofluorescence staining indicated that AOPPs increased expressions of NADPH oxidase 1 (Nox1), NADPH oxidase 4 (Nox4), transient receptor potential vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP) in the dorsal root ganglia (DRG) tissues. In-vitro studies were performed on primary DRG neurons which were obtained from both thoracic and lumbar DRG of rats. Results indicated that AOPPs triggered reactive oxygen species (ROS) production in DRG neurons, which were significantly abolished by ROS scavenger N-acetyl-l-cysteine (NAC) and small-interfering RNA (siRNA) silencing of Nox1 or Nox4. The expressions of Nox1, Nox4, TRPV1 and CGRP were significantly increased in AOPPs-induced DRG neurons. And relevant siRNA or inhibitors notably suppressed the expressions of these proteins and the calcium influxes in AOPPs-induced DRG neurons. In conclusion, AOPPs increased significantly in CFA-induced hyperalgesia rats and they activated Nox1/Nox4-ROS to sensitize TRPV1-dependent Ca2+ influx and CGRP release which led to inducing mechanical hyperalgesia. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Inhibition of transient receptor potential vanilloid-1 confers neuroprotection, reduces tumor necrosis factor-alpha, and increases IL-10 in a rat stroke model.

PubMed

Hakimizadeh, Elham; Shamsizadeh, Ali; Roohbakhsh, Ali; Arababadi, Mohammad Kazemi; Hajizadeh, Mohammad R; Shariati, Mehdi; Rahmani, Mohammad R; Allahtavakoli, Mohammad

2017-08-01

Stroke is a major cause of mortality and long-term disability in adults. Transient receptor potential vanilloid-1 (TRPV1) plays a crucial role in neuroinflammation. In this study, the effects of TRPV1 agonist (capsaicin) and antagonist (AMG9810) on cerebral ischemia were investigated. Forty male Wistar rats were assigned to the following experimental groups: sham, vehicle) ischemic), AMG9810 (selective TRPV1 antagonist, 0.5 mg/kg; 3 h after stroke), and capsaicin (1 mg/kg; 3 h after stroke). Stroke was induced by permanent middle cerebral artery occlusion and neurological deficits were evaluated 1, 3, and 7 days after stroke. Then, infarct volume, brain edema, body temperature, mRNA expression of TRPV1, and serum concentrations of tumor necrosis factor-alpha (TNF-α) and IL-10 were measured. Compared to the vehicle group, AMG9810 significantly decreased the infarct volume (P < 0.01). Latency for the removal of sticky labels from the forepaw and the hanging time were significantly decreased and increased, respectively, following administration of AMG9810 (P < 0.01 and P < 0.001 vs. vehicle) 3 and 7 days after stroke. Compared to the sham group, the mRNA expression of TRPV1 was significantly increased in vehicle group (P < 0.01). Administration of AMG9810 significantly increased the anti-inflammatory cytokine IL-10 and decreased the inflammatory cytokine TNF-α (P < 0.05). Moreover, our results indicate that AMG9810 might a promising candidate for the hypothermic treatment of stroke. The findings also suggest a key role for AMG9810 in reducing inflammation after stroke and imply that TRPV1 could be a potential target for the treatment of ischemic stroke. © 2017 Société Française de Pharmacologie et de Thérapeutique.

Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice

PubMed Central

Mulak, Agata; Larauche, Muriel; Biraud, Mandy; Million, Mulugeta; Rivier, Jean; Taché, Yvette

2014-01-01

Somatostatin interacts with 5 G-protein-coupled receptor (sst1–5). Octreotide, a stable sst2≫3≥5 agonist, octreotide, exerts a visceral anti-hyperalgesic effect in experimental and clinical studies. Little is known on the receptor subtypes involved. We investigated the influence of the stable sst1–5 agonist, ODT8-SST and selective receptor subtype peptide agonists (3 or 10 μg/mouse) injected intraperitoneally (ip) on visceral hypersensitivity in mice induced by repeated noxious colorectal distensions (4 sets of 3 CRD, each at 55 mmHg) or corticotropin-releasing factor receptor 1 agonist, cortagine given between 2 sets of graded CRD (15, 30, 45, and 60 mmHg, 3 times each pressure). The mean visceromotor response (VMR) was assessed using a non-invasive manometry method and values were expressed as percentage of the VMR to the 1st set of CRD baseline or to the 60 mmHg CRD, respectively. ODT8-SST (10 μg) and the sst2 agonist, S-346-011 (3 and 10 μg) prevented mechanically-induced visceral hypersensitivity in the 3 sets of CRD, the sst1 agonist (10 μg) blocked only the 2nd set and showed a trend at 3 μg while the sst4 agonist had no effect. The selective sst2 antagonist, S-406-028 blocked the sst2 agonist but not the sst1 agonist effect. The sst1 agonist (3 and 10 μg) prevented cortagine-induced hypersensitivity to CRD at each pressure while the sst2 agonist at 10 μg reduced it. These data indicate that in addition to sst2, the sst1 agonist may provide a novel promising target to alleviate visceral hypersensitivity induced by mechanoreceptor sensitization and more prominently, stress-related visceral nociceptive sensitization. PMID:25451334

Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice.

PubMed

Mulak, Agata; Larauche, Muriel; Biraud, Mandy; Million, Mulugeta; Rivier, Jean; Taché, Yvette

2015-01-01

Somatostatin interacts with five G-protein-coupled receptor (sst1-5). Octreotide, a stable sst2≫3≥5 agonist, exerts a visceral anti-hyperalgesic effect in experimental and clinical studies. Little is known on the receptor subtypes involved. We investigated the influence of the stable sst1-5 agonist, ODT8-SST and selective receptor subtype peptide agonists (3 or 10μg/mouse) injected intraperitoneally (ip) on visceral hypersensitivity in mice induced by repeated noxious colorectal distensions (four sets of three CRD, each at 55mmHg) or corticotropin-releasing factor receptor 1 agonist, cortagine given between two sets of graded CRD (15, 30, 45, and 60mmHg, three times each pressure). The mean visceromotor response (VMR) was assessed using a non-invasive manometry method and values were expressed as percentage of the VMR to the 1st set of CRD baseline or to the 60mmHg CRD, respectively. ODT8-SST (10μg) and the sst2 agonist, S-346-011 (3 and 10μg) prevented mechanically induced visceral hypersensitivity in the three sets of CRD, the sst1 agonist (10μg) blocked only the 2nd set and showed a trend at 3μg while the sst4 agonist had no effect. The selective sst2 antagonist, S-406-028 blocked the sst2 agonist but not the sst1 agonist effect. The sst1 agonist (3 and 10μg) prevented cortagine-induced hypersensitivity to CRD at each pressure while the sst2 agonist at 10μg reduced it. These data indicate that in addition to sst2, the sst1 agonist may provide a novel promising target to alleviate visceral hypersensitivity induced by mechanoreceptor sensitization and more prominently, stress-related visceral nociceptive sensitization. Copyright © 2014 Elsevier Inc. All rights reserved.

Asian ethnicity and breast cancer subtypes: a study from the California Cancer Registry.

PubMed

Telli, Melinda L; Chang, Ellen T; Kurian, Allison W; Keegan, Theresa H M; McClure, Laura A; Lichtensztajn, Daphne; Ford, James M; Gomez, Scarlett L

2011-06-01

The distribution of breast cancer molecular subtypes has been shown to vary by race/ethnicity, highlighting the importance of host factors in breast tumor biology. We undertook the current analysis to determine population-based distributions of breast cancer subtypes among six ethnic Asian groups in California. We defined immunohistochemical (IHC) surrogates for each breast cancer subtype among Chinese, Japanese, Filipina, Korean, Vietnamese, and South Asian patients diagnosed with incident, primary, invasive breast cancer between 2002 and 2007 in the California Cancer Registry as: hormone receptor-positive (HR+)/HER2- [estrogen receptor-positive (ER+) and/or progesterone receptor-positive (PR+), human epidermal growth factor receptor 2-negative (HER2-)], triple-negative (ER-, PR-, and HER2-), and HER2-positive (ER±, PR±, and HER2+). We calculated frequencies of breast cancer subtypes among Asian ethnic groups and evaluated their associations with clinical and demographic factors. Complete IHC data were available for 8,140 Asian women. Compared to non-Hispanic White women, Korean [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.5-2.2], Filipina (OR = 1.3, 95% CI = 1.2-1.5), Vietnamese (OR = 1.3, 95% CI = 1.1-1.6), and Chinese (OR = 1.1, 95% CI = 1.0-1.3) women had a significantly increased risk of being diagnosed with HER2-positive breast cancer subtypes after adjusting for age, stage, grade, socioeconomic status, histology, diagnosis year, nativity, and hospital ownership status. We report a significant ethnic disparity in HER2-positive breast cancer in a large population-based cohort enriched for Asian-Americans. Given the poor prognosis and high treatment costs of HER2-positive breast cancer, our results have implications for healthcare resource utilization, cancer biology, and clinical care.

Pharmacological interference with metabotropic glutamate receptor subtype 7 but not subtype 5 differentially affects within- and between-session extinction of Pavlovian conditioned fear.

PubMed

Toth, Iulia; Dietz, Monika; Peterlik, Daniel; Huber, Sabine E; Fendt, Markus; Neumann, Inga D; Flor, Peter J; Slattery, David A

2012-03-01

Fear extinction is defined as the attenuation of a conditioned-fear memory by re-exposing animals to the conditioned stimulus without the aversive stimulus. This process is known to be effectively enhanced via administration of D-cycloserine (DCS), a partial NMDA-receptor agonist. However, other glutamatergic mechanisms, such as interference with metabotropic glutamate receptor (mGluR) subtypes 5 and 7 in the extinction of aversive memories are insufficiently understood. Using the allosteric mGluR5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), the mGluR7 allosteric agonist N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082), and DCS for comparison, we aimed to study how pharmacological blockade of mGluR5 and activation of mGluR7 influenced within- and between-session conditioned-fear extinction training and extinction retention in rats. We show that when injected before extinction training, mGluR7 activation with AMN082 enhanced freezing and thereby attenuated within-session fear extinction, whereas both DCS and the mGluR5 receptor antagonist MPEP had no effect on this process. However, these differential drug effects were not long lasting, as no difference in extinction retention were observed 24 h later. Therefore, we assessed whether the compounds affect 24 h consolidation of extinction training following incomplete extinction training (between-session extinction). Similar to DCS, AMN082- but not MPEP-treated rats showed facilitated extinction retention, as exhibited by decreased freezing. Finally, using fluoxetine, we provide evidence that the effect of AMN082 on between-session extinction retention is most likely not via increasing 5-HT transmission. These findings demonstrate that mGluR7 activation differentially modulates conditioned-fear extinction, in dependence on the protocol employed, and suggests drugs with AMN082-like mechanisms as potential add-on drugs following exposure-based psychotherapy for fear-related human

Ionotropic and metabotropic receptor mediated airway sensory nerve activation.

PubMed

Lee, Min-Goo; Kollarik, Marian; Chuaychoo, Benjamas; Undem, Bradley J

2004-01-01

There are several receptors capable of inducing activating generator potentials in cough-associated afferent terminals in the airways. The chemical receptors leading to generator potentials can be subclassified into ionotropic and metabotropic types. An ionotropic receptor has an agonist-binding domain, and also serves directly as an ion channel that is opened upon binding of the agonist. Examples of ionotropic receptors found in airway sensory nerve terminals include receptors for serotonin (5-HT3 receptors), ATP (P2X receptors), acetylcholine (nicotinic receptors), receptors for capsaicin and related vanilloids (TRPV1 receptors), and acid receptors (acid sensing ion channels). Afferent nerve terminals can also be depolarized via activation of metabotropic or G-protein coupled receptors (GPCRs). Among the GPCRs that can lead to activation of airway afferent fibers include bradykinin B2 and adenosine A1 receptors. The signaling events leading to GPCR-mediated membrane depolarization are more complex than that seen with ionotropic receptors. The GPCR-mediated effects are thought to occur through classical second messenger systems such as activation of phospholipase C. This may lead to membrane depolarization through interaction with specific ionotropic receptors (such as TRPV1) and/or various types of calcium activated channels.

Functional characterization of the beta-adrenergic receptor subtypes expressed by CA1 pyramidal cells in the rat hippocampus.

PubMed

Hillman, Kristin L; Doze, Van A; Porter, James E

2005-08-01

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists

Identification of the dopamine autoreceptor in the guinea-pig retina as D2 receptor using novel subtype-selective antagonists

PubMed Central

Weber, Bernd; Schlicker, Eberhard; Sokoloff, Pierre; Stark, Holger

2001-01-01

Dopamine release in the retina is subject to modulation via autoreceptors, which belong to the D2 receptor family (encompassing the D2, D3 and D4 receptors). The aim of the present study was to determine the receptor subtype (D2 vs D3) involved in the inhibition of dopamine release in guinea-pig retinal discs, using established (haloperidol, (S)-nafadotride) and novel dopamine receptor antagonists (ST-148, ST-198). hD2L and hD3 receptors were expressed in CHO cells and the pKi values determined in binding studies with [125I]-iodosulpride were: haloperidol 9.22 vs 8.54; ST-148 7.85 vs 6.60; (S)-nafadotride 8.52 vs 9.51; ST-198 6.14 vs 7.92. The electrically evoked tritium overflow from retinal discs preincubated with [3H]-noradrenaline (which represents quasi-physiological dopamine release) was inhibited by the dopamine receptor agonists B-HT 920 (talipexole) and quinpirole (maximally by 82 and 71%; pEC50 5.80 and 5.83). The concentration-response curves of these agonists were shifted to the right by haloperidol (apparent pA2 8.69 and 8.23) and ST-148 (7.52 and 7.66). (S)-Nafadotride 0.01 μM and ST-198 0.32 μM did not affect the concentration-response curve of B-HT 920. The dopamine autoreceptor in the guinea-pig retina can be classified as a D2 receptor. ST-148 and ST-198 show an improved selectivity for D2 and D3 receptors when compared to haloperidol and (S)-nafadotride, respectively. PMID:11498509

Opioid-receptor subtype agonist-induced enhancements of sucrose intake are dependent upon sucrose concentration.

PubMed

Ruegg, H; Yu, W Z; Bodnar, R J

1997-07-01

Selective mu ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO)), delta1 ([D-Pen2, D-Pen5]-enkephalin (DPDPE)), delta2 ([D-Ala2, Glu4]-Deltorphin (Delt II)), kappa1 (U50488H) and kappa3 (naloxone benzoylhydrazone (NalBzOH)) opioid agonists each stimulate food intake in rats. Whereas studies with selective opioid antagonists implicate mu and kappa1 receptors in the mediation of sucrose intake, studies with selective opioid agonists implicate mu and delta receptors in the mediation of saccharin intake. The present study determined if specific delta1, delta2, kappa1, kappa3 and mu opioid-receptor subtype agonists produced similar alterations in sucrose intake as a function of sucrose concentration (0.5%, 2.5%, 10%) across a 1-h time-course. Each of these agonists significantly increased sucrose intake with variations in pattern, magnitude, and consistency as a function of sucrose concentration. Whereas the mu opioid agonist, DAMGO, and the delta1 opioid agonist, DPDPE, each enhanced sucrose intake at higher (2.5%, 10%), but not lower (0.5%), concentrations, the delta2 opioid agonist, Delt II, increased sucrose intake at lower (0.5%, 2.5%), but not higher (10%), concentrations. Kappa opioid agonists produced less consistent effects. The kappa1 opioid agonist, U50488H, increased sucrose intake at high (10%) concentrations and decreased sucrose intake at low (0.5%) concentrations, and the kappa3 opioid agonist, NalBzOH, inconsistently increased sucrose intake at the 0.5% (20 microg) and 10% (1 microg) concentrations. Thus, these data further implicate mu, delta1, and delta2 opioid mediation of palatable intake, particularly of its orosensory characteristics.

Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats

PubMed Central

Ting, K N; Blaylock, N A; Sugden, D; Delagrange, P; Scalbert, E; Wilson, V G

1999-01-01

In this study reverse transcriptase-polymerase chain reaction (RT–PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4-phenyl-2-propionamidotetraline (4-P-PDOT) and 2-benzyl-N-pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated.Using RT–PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR.At low concentrations of the MT2 selective ligands, neither 10 nM 4-P-PDOT (pEC50=8.70±0.31 (control) vs 8.73±0.16, n=6) nor 60 nM 2-benzyl-N-pentanoyltryptamine (pEC50=8.53±0.20 (control) vs 8.83±0.38, n=6) significantly altered the potency of melatonin in the rat tail artery.At concentrations non-selective for mt1 and MT2 receptors, 4-P-PDOT (3 μM) and 2-benzyl-N-pentanoyltryptamine (5 μM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4-P-PDOT, the estimated pKB (6.17±0.16, n=8) is similar to the binding affinity for mt1 receptor.Pre-incubation with 1 μM melatonin did not affect the conversion of [3H]-adenine to [3H]-cyclic AMP under basal condition (0.95±0.19% conversion (control) vs 0.92±0.19%, n=4) or following exposure to 30 μM forskolin (5.20±1.30% conversion (control) vs 5.35±0.90%, n=4).Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway. PMID:10433507

Down-regulation of angiotensin II receptor subtypes and desensitization of cyclic GMP production in neuroblastoma N1E-115 cells.

PubMed

Reagan, L P; Ye, X; Maretzski, C H; Fluharty, S J

1993-01-01

Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either 125I-AngII or the high-affinity antagonist 125I-[Sarc1,Ile8]-AngII. This apparent down-regulation was dose related with an ED50 of 1 nM, and maximal decreases of approximately 90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37 degrees C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1 receptor subtype during the first 5 min, followed by a decline in both AT1 and AT2 receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc1,Thr8]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface AngII receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

EEG sleep activities react topographically different to GABAergic sleep modulation by flunitrazepam: relationship to regional distribution of benzodiazepine receptor subtypes?

PubMed

Scheuler, W

Spectral analysis was performed to study the response of various EEG sleep activities to a modification of GABAergic sleep regulation by flunitrazepam. We observed sleep stage- and sleep cycle-dependent differences in the topographic distribution of the reactions. An increase in power density was found in the frontal regions for the alpha 2 and sigma 1 frequency band whereas a decrease in power density was emphasized in the posterior regions for the delta and alpha 1 frequency band. These topographic differences might be related to the regional distribution of benzodiazepine receptor subtypes.

Ultradian oscillation in expression of four melatonin receptor subtype genes in the pineal gland of the grass puffer, a semilunar-synchronized spawner, under constant darkness.

PubMed

Ikegami, Taro; Maruyama, Yusuke; Doi, Hiroyuki; Hattori, Atsuhiko; Ando, Hironori

2015-01-01

Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel 1a 1.4, mel 1a 1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0-15.4 h, suggesting the presence of a circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer.

Ultradian oscillation in expression of four melatonin receptor subtype genes in the pineal gland of the grass puffer, a semilunar-synchronized spawner, under constant darkness

PubMed Central

Ikegami, Taro; Maruyama, Yusuke; Doi, Hiroyuki; Hattori, Atsuhiko; Ando, Hironori

2015-01-01

Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel1a1.4, mel1a1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0–15.4 h, suggesting the presence of a circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer. PMID:25688184

The analgesic effect of clonixine is not mediated by 5-HT3 subtype receptors.

PubMed

Paeile, C; Bustamante, S E; Sierralta, F; Bustamante, D; Miranda, H F

1995-10-01

1. The analgesic effect of clonixinate of L-lysine (Clx) in the nociceptive C-fiber reflex in rat and in the writhing test in mice is reported. 2. Clx was administered by three routes, i.v., i.t. and i.c.v., inducing a dose-dependent antinociception. 3. The antinociceptive effect of Clx was 40-45% with respect to the control integration values in the nociceptive C-fiber reflex method. 4. The writhing test yielded ED50 values (mg/kg) of 12.0 +/- 1.3 (i.p.), 1.8 +/- 0.2 (i.t.) and 0.9 +/- 0.1 (i.c.v.) for Clx administration. 5. Ondansetron was not able to antagonize the antinociception response of Clx in the algesiometric tests used. 6. Chlorophenilbiguanide did not produce any significative change in the analgesic effect of Clx in the nociceptive C-fiber reflex method. 7. It is suggested that the mechanism of action of the central analgesia of Clx is not mediated by 5-HT3 subtype receptors.

(3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

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

Norman, A.B.; Battaglia, G.; Creese, I.

1985-12-01

In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5more » mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.« less

A coupling of homology modeling with multiple molecular dynamics simulation for identifying representative conformation of GPCR structures: a case study on human bombesin receptor subtype-3.

PubMed

Nowroozi, Amin; Shahlaei, Mohsen

2017-02-01

In this study, a computational pipeline was therefore devised to overcome homology modeling (HM) bottlenecks. The coupling of HM with molecular dynamics (MD) simulation is useful in that it tackles the sampling deficiency of dynamics simulations by providing good-quality initial guesses for the native structure. Indeed, HM also relaxes the severe requirement of force fields to explore the huge conformational space of protein structures. In this study, the interaction between the human bombesin receptor subtype-3 and MK-5046 was investigated integrating HM, molecular docking, and MD simulations. To improve conformational sampling in typical MD simulations of GPCRs, as in other biomolecules, multiple trajectories with different initial conditions can be employed rather than a single long trajectory. Multiple MD simulations of human bombesin receptor subtype-3 with different initial atomic velocities are applied to sample conformations in the vicinity of the structure generated by HM. The backbone atom conformational space distribution of replicates is analyzed employing principal components analysis. As a result, the averages of structural and dynamic properties over the twenty-one trajectories differ significantly from those obtained from individual trajectories.

Subtypes of attention deficit-hyperactivity disorder (ADHD) and cannabis use.

PubMed

Loflin, Mallory; Earleywine, Mitch; De Leo, Joseph; Hobkirk, Andrea

2014-03-01

The current study examined the association between subtypes of attention-deficit/hyperactivity disorder (ADHD) and cannabis use within a sample of 2811 current users. Data were collected in 2012 from a national U.S. survey of cannabis users. A series of logistic regression equations and chi-squares were assessed for proportional differences between users. When asked about the ADHD symptoms they have experienced when not using cannabis, a higher proportion of daily users met symptom criteria for an ADHD diagnoses of the subtypes that include hyperactive-impulsive symptoms than the inattentive subtype. For nondaily users, the proportions of users meeting symptom criteria did not differ by subtype. These results have implications for identifying which individuals with ADHD might be more likely to self-medicate using cannabis. Furthermore, these findings indirectly support research linking relevant cannabinoid receptors to regulatory control.

In breast cancer subtypes steroid sulfatase (STS) is associated with less aggressive tumour characteristics.

PubMed

McNamara, Keely M; Guestini, Fouzia; Sauer, Torill; Touma, Joel; Bukholm, Ida Rashida; Lindstrøm, Jonas C; Sasano, Hironobu; Geisler, Jürgen

2018-05-01

The majority of breast cancer cases are steroid dependent neoplasms, with hormonal manipulation of either CYP19/aromatase or oestrogen receptor alpha axis being the most common therapy. Alternate pathways of steroid actions are documented, but their interconnections and correlations to BC subtypes and clinical outcome could be further explored. We evaluated selected steroid receptors (Androgen Receptor, Oestrogen Receptor alpha and Beta, Glucocorticoid Receptor) and oestrogen pathways (steroid sulfatase (STS), 17β-hydroxysteroid dehydrogenase 2 (17βHSD2) and aromatase) in a cohort of 139 BC cases from Norway. Using logistic and cox regression analysis, we examined interactions between these and clinical outcomes such as distant metastasis, local relapse and survival. Our principal finding is an impact of STS expression on the risk for distant metastasis (p<0.001) and local relapses (p <0.001), HER2 subtype (p<0.015), and survival (p<0.001). The suggestion of a beneficial effect of alternative oestrogen synthesis pathways was strengthened by inverted, but non-significant findings for 17βHSD2. Increased intratumoural metabolism of oestrogens through STS is associated with significantly lower incidence of relapse and/or distant metastasis and correspondingly improved prognosis. The enrichment of STS in the HER2 overexpressing subtype is intriguing, especially given the possible role of HER-2 over-expression in endocrine resistance.

Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist {alpha}-conotoxin OmIA that discriminates {alpha}3 vs. {alpha}6 nAChR subtypes

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

Chi, Seung-Wook; Kim, Do-Hyoung; Olivera, Baldomero M.

2006-06-23

{alpha}-Conotoxin OmIA from Conus omaria is the only {alpha}-conotoxin that shows a {approx}20-fold higher affinity to the {alpha}3{beta}2 over the {alpha}6{beta}2 subtype of nicotinic acetylcholine receptor. We have determined a three-dimensional structure of {alpha}-conotoxin OmIA by nuclear magnetic resonance spectroscopy. {alpha}-Conotoxin OmIA has an '{omega}-shaped' overall topology with His{sup 5}-Asn{sup 12} forming an {alpha}-helix. Structural features of {alpha}-conotoxin OmIA responsible for its selectivity are suggested by comparing its surface characteristics with other functionally related {alpha}4/7 subfamily conotoxins. Reduced size of the hydrophilic area in {alpha}-conotoxin OmIA seems to be associated with the reduced affinity towards the {alpha}6{beta}2 nAChR subtype.

Somatostatin receptor subtype-4 agonist NNC 26-9100 decreases extracellular and intracellular Aβ₁₋₄₂ trimers.

PubMed

Sandoval, Karin E; Farr, Susan A; Banks, William A; Crider, Albert M; Morley, John E; Witt, Ken A

2012-05-15

Soluble amyloid β-protein (Aβ) oligomers are primary mediators of synaptic dysfunction associated with the progression of Alzheimer's disease. Such Aβ oligomers exist dependent on their rates of aggregation and metabolism. Use of selective somatostatin receptor-subtype agonists have been identified as a potential means to mitigate Aβ accumulation in the brain, via regulation of the enzyme neprilysin. Herein, we first evaluated the impact of the somatostatin receptor subtype-4 agonist 1-[3-[N-(5-Bromopyridin-2-yl)-N-(3,4-dichlorobenzyl)amino]propyl]-3-[3-(1H-imidazol-4-yl)propyl]thiourea (NNC 26-9100) on learning and memory in 12-month SAMP8 mice (i.c.v. injection). NNC 26-9100 (0.2 μg-dose) was shown to enhance both learning (T-maze) and memory (object recognition) compared to vehicle controls. Cortical and hippocampal tissues were evaluated subsequent to NNC 26-9100 (0.2 μg) or vehicle administration for changes in neprilysin activity, along with protein expression of amyloid-precursor protein (APP), neprilysin, and Aβ₁₋₄₂ oligomers within respective cellular fractions (extracellular, intracellular and membrane). NNC 26-9100 increased neprilysin activity in cortical tissue, with an associated protein expression increase in the extracellular fraction and decreased in the intracellular fraction. A decrease in intracellular APP expression was found with treatment in both cortical and hippocampal tissues. NNC 26-9100 also significantly decreased expression of Aβ₁₋₄₂ trimers within both the extracellular and intracellular cortical fractions. No expression changes were found in membrane fractions for any protein. These finding suggest the potential use of selective SSTR4 agonists to mitigate toxic oligomeric forms of Aβ₁₋₄₂ in critical regions of the brain identified with learning and memory decline. Copyright © 2012 Elsevier B.V. All rights reserved.

Impaired Bone Resorption by Lipopolysaccharide In Vivo in Mice Deficient in the Prostaglandin E Receptor EP4 Subtype

PubMed Central

Sakuma, Yoko; Tanaka, Kiyoshi; Suda, Michio; Komatsu, Yasato; Yasoda, Akihiro; Miura, Masako; Ozasa, Ami; Narumiya, Shuh; Sugimoto, Yukihiko; Ichikawa, Atsushi; Ushikubi, Fumitaka; Nakao, Kazuwa

2000-01-01

In a previous study we showed that the involvement of EP4 subtype of the prostaglandin E (PGE) receptor is crucial for lipopolysaccharide (LPS)-induced osteoclast formation in vitro. The present study was undertaken to test whether EP4 is actually associated with LPS-induced bone resorption in vivo. In wild-type (WT) mice, osteoclast formation in vertebrae and tibiae increased 5 days after systemic LPS injection, and urinary excretion of deoxypyridinoline, a sensitive marker for bone resorption, statistically increased 10 days after injection. In EP4 knockout (KO) mice, however, LPS injection caused no significant changes in these parameters throughout the experiment. LPS exposure for 4 h strongly induced osteoclast differentiation factor (ODF) mRNA expression in primary osteoblastic cells (POB) both from WT and EP4 KO mice, and this expression was not inhibited by indomethacin, suggesting prostaglandin (PG) independence. LPS exposure for 24 h further induced ODF expression in WT POB, but not in EP4 KO POB. Indomethacin partially inhibited ODF expression in WT POB, but not in EP4 KO POB. These data suggest that ODF is induced both PG dependently and PG independently. LPS exposure for 24 h induced slightly greater osteoclastgenesis inhibitory factor (OCIF) mRNA expression in EP4 KO than in WT POB. These findings suggest that the reduced ODF expression and apparently increased OCIF expression also are responsible for the markedly reduced LPS-induced osteoclast formation in EP4 KO mice. Our results show that the EP4 subtype of the PGE receptor is involved in LPS-induced bone resorption in vivo also. Since LPS is considered to be largely involved in bacterially induced bone loss, such as in periodontitis and osteomyelitis, our study is expected to help broaden our understanding of the pathophysiology of these conditions. PMID:11083800

Systematic Analysis of Primary Sequence Domain Segments for the Discrimination Between Class C GPCR Subtypes.

PubMed

König, Caroline; Alquézar, René; Vellido, Alfredo; Giraldo, Jesús

2018-03-01

G-protein-coupled receptors (GPCRs) are a large and diverse super-family of eukaryotic cell membrane proteins that play an important physiological role as transmitters of extracellular signal. In this paper, we investigate Class C, a member of this super-family that has attracted much attention in pharmacology. The limited knowledge about the complete 3D crystal structure of Class C receptors makes necessary the use of their primary amino acid sequences for analytical purposes. Here, we provide a systematic analysis of distinct receptor sequence segments with regard to their ability to differentiate between seven class C GPCR subtypes according to their topological location in the extracellular, transmembrane, or intracellular domains. We build on the results from the previous research that provided preliminary evidence of the potential use of separated domains of complete class C GPCR sequences as the basis for subtype classification. The use of the extracellular N-terminus domain alone was shown to result in a minor decrease in subtype discrimination in comparison with the complete sequence, despite discarding much of the sequence information. In this paper, we describe the use of Support Vector Machine-based classification models to evaluate the subtype-discriminating capacity of the specific topological sequence segments.

Role of gamma-aminobutyric acid type A (GABAA) receptor subtypes in acute benzodiazepine physical dependence-like effects: evidence from squirrel monkeys responding under a schedule of food presentation

PubMed Central

Fischer, Bradford D.; Teixeira, Laura P.; van Linn, Michael L.; Namjoshi, Ojas A.; Cook, James M.; Rowlett, James K.

2013-01-01

Rationale Assays of schedule-controlled responding can be used to characterize the pharmacology of benzodiazepines and other GABAA receptor modulators, and are sensitive to changes in drug effects that are related to physical dependence. Objective The present study used this approach to investigate the role of GABAA receptor subtypes in mediating dependence-like effects following benzodiazepine administration. Methods Squirrel monkeys (n=6) were trained on a fixed-ratio schedule of food reinforcement. Initially, the response rate-decreasing effects of chlordiazepoxide (0.1–10 mg/kg; nonselective GABAA receptor agonist), zolpidem (0.032–1.0 mg/kg; α1 subunit-containing GABAA subtype-preferring agonist) and HZ-166 (0.1–10 mg/kg; functionally selective α2 and α3 subunit-containing GABAA receptor agonist) were assessed. Next, acute dependence-like effects following single injections of chlordiazepoxide, zolpidem and HZ-166 were assessed with flumazenil (0.1–3.2 mg/kg; nonselective GABAA receptor antagonist). Finally, acute dependence-like effects following zolpidem administration were assessed with βCCt and 3-PBC (0.1–3.2 mg/kg and 0.32–10 mg/kg, respectively; α1 subunit-containing GABAA receptor antagonists). Results Chlordiazepoxide, zolpidem and HZ-166 produced dose- and time-dependent decreases in response rates, whereas flumazenil, βCCt and 3-PBC were ineffective. After the drug effects waned, flumazenil produced dose-dependent decreases in response rates following administration of 10 mg/kg chlordiazepoxide and 1.0 mg/kg zolpidem, but not following any dose of HZ-166. Further, both βCCt and 3-PBC produced dose-dependent decreases in response rates when administered after 1.0 mg/kg zolpidem. Conclusions These data raise the possibility that α1 subunit-containing GABAA receptors play a major role in physical dependence-related behaviors following a single injection of a benzodiazepine. PMID:23354533

Role of gamma-aminobutyric acid type A (GABAA) receptor subtypes in acute benzodiazepine physical dependence-like effects: evidence from squirrel monkeys responding under a schedule of food presentation.

PubMed

Fischer, Bradford D; Teixeira, Laura P; van Linn, Michael L; Namjoshi, Ojas A; Cook, James M; Rowlett, James K

2013-05-01

Assays of schedule-controlled responding can be used to characterize the pharmacology of benzodiazepines and other GABAA receptor modulators, and are sensitive to changes in drug effects that are related to physical dependence. The present study used this approach to investigate the role of GABAA receptor subtypes in mediating dependence-like effects following benzodiazepine administration. Squirrel monkeys (n = 6) were trained on a fixed-ratio schedule of food reinforcement. Initially, the response rate-decreasing effects of chlordiazepoxide (0.1-10 mg/kg; nonselective GABAA receptor agonist), zolpidem (0.032-1.0 mg/kg; α1 subunit-containing GABAA subtype-preferring agonist), and HZ-166 (0.1-10 mg/kg; functionally selective α2 and α3 subunit-containing GABAA receptor agonist) were assessed. Next, acute dependence-like effects following single injections of chlordiazepoxide, zolpidem, and HZ-166 were assessed with flumazenil (0.1-3.2 mg/kg; nonselective GABAA receptor antagonist). Finally, acute dependence-like effects following zolpidem administration were assessed with βCCt and 3-PBC (0.1-3.2 mg/kg and 0.32-10 mg/kg, respectively; α1 subunit-containing GABAA receptor antagonists). Chlordiazepoxide, zolpidem, and HZ-166 produced dose- and time-dependent decreases in response rates, whereas flumazenil, βCCT, and 3-PBC were ineffective. After the drug effects waned, flumazenil produced dose-dependent decreases in response rates following administration of 10 mg/kg chlordiazepoxide and 1.0 mg/kg zolpidem, but not following any dose of HZ-166. Further, both βCCT and 3-PBC produced dose-dependent decreases in response rates when administered after 1.0 mg/kg zolpidem. These data raise the possibility that α1 subunit-containing GABAA receptors play a major role in physical dependence-related behaviors following a single injection of a benzodiazepine.

Somatostatin receptor subtypes SSTR2 and SSTR5 couple negatively to an L-type Ca2+ current in the pituitary cell line AtT-20.

PubMed

Tallent, M; Liapakis, G; O'Carroll, A M; Lolait, S J; Dichter, M; Reisine, T

1996-04-01

The somatostatin receptor subtypes SSTR2 and SSTR5 mediate distinct endocrine and exocrine functions of somatostatin and may also be involved in mediating the neuromodulatory actions of somatostatin in the brain. To investigate whether these receptors couple to voltage-sensitive Ca2+ channels, SSTR2 and SSTR5 selective agonists were tested for their effects on AtT-20 cells using whole cell patch clamp techniques. The SSTR2 selective agonist MK 678 inhibited Ca2+ currents in AtT-20 cells. The effects of MK 678 were reversible and blocked by pertussis toxin pretreatment, suggesting that SSTR2 couples to the L-type Ca2+ channels via G proteins. Other SSTR2-selective agonists, including BIM 23027 and NC8-12, were able to inhibit the Ca2+ currents in these cells. The SSTR5 selective agonist BIM 23052 also inhibited the Ca2+ currents in these cells and this effect was reversible and blocked by pertussis toxin treatment. The ability of SSTR5 to mediate inhibition of the Ca2+ current was greatly attenuated by pretreatment with the SSTR5-selective agonist BIM 23052, whereas SSTR2-mediated inhibition of the Ca2+ current was not altered by pretreatment with the SSTR2-selective agonist MK 678. Thus, the SSTR2 and SSTR5 couplings to the Ca2+ current are differentially regulated. The peptide L362,855, which we previously have shown to have high affinity for the cloned SSTR5, had minimal effects on Ca2+ currents in AtT-20 cells at concentrations up to 100 nM and did not alter the ability of MK 678 to inhibit Ca2+ currents. However, it completely antagonized the effects of the SSTR5-selective agonist BIM 23052 on the Ca2+ currents. L362,855 is an antagonist/partial agonist at SSTR5 since it can reduce Ca2+ currents in these cells at concentrations above 100 nM. L362,855 is also an antagonist/partial agonist at the cloned rat SSTR5 expressed in CHO cells since it is able to block the inhibition of cAMP accumulation induced by somatostatin at concentrations below 100 nM but at

Role of the nicotinic acetylcholine receptor α3 subtype in vascular inflammation.

PubMed

Yang, Cui; Li, Zhengtao; Yan, Saimei; He, Yonghui; Dai, Rong; Leung, George Pek-Heng; Pan, Shitian; Yang, Jinyan; Yan, Rong; Du, Guanhua

2016-11-01

Vascular inflammation is a major factor contributing to the development of vascular diseases. The aim of this study was to investigate the role of the nicotinic acetylcholine receptor α3 subtype (α3-nAChR) in vascular inflammation. Vascular inflammation was studied in apolipoprotein E knockout (ApoE -/- ) mice fed a high-fat diet. Inflammatory markers were measured in mouse aortic endothelial cells (MAECs) and macrophages after α3-nAChRs were antagonized pharmacologically, or after the gene of α3-nAChRs was silenced. Treatment with α-conotoxin MII (MII; an α3-nAChR antagonist) increased the number of inflammatory cells infiltrating the aortic walls and further impaired the endothelium-dependent vasodilatations in the aorta of ApoE -/- mice. MII also increased the plasma levels of inflammatory cytokines. Furthermore, the infiltration of classical activated macrophages into the arterial wall of ApoE -/- mice was markedly elevated by MII but that of alternative activated macrophages was reduced. In MAECs, the lipopolysaccharide-stimulated secretion of adhesion molecules and inflammatory cytokines was enhanced by MII, or by silencing the gene of α3-nAChRs. This effect was reversed by inhibitors of the PI3K-Akt-IκKα/β-IκBα-NFκB pathways. In macrophages, the classical activation was enhanced, but the alternative activation was reduced when the gene of α3-nACh receptors was silenced. These effects were prevented by inhibitors of the IκKα/β-IκBα-NFκB and JAK2-STAT6-PPARγ pathways respectively. α3-nAChRs play a pivotal role in regulating the inflammatory responses in endothelial cells and macrophages. The mechanisms involve the modulations of multiple cell signalling pathways. © 2016 The British Pharmacological Society.

Design and Synthesis of a Series of l-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid.

PubMed

Krogsgaard-Larsen, Niels; Delgar, Claudia G; Koch, Karina; Brown, Patricia M G E; Møller, Charlotte; Han, Liwei; Huynh, Tri H V; Hansen, Stinne W; Nielsen, Birgitte; Bowie, Derek; Pickering, Darryl S; Kastrup, Jette Sandholm; Frydenvang, Karla; Bunch, Lennart

2017-01-12

Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (K i = 4 μM). In a functional assay, 1b displayed full antagonist activity with IC 50 = 6 ± 2 μM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

Supervised Risk Predictor of Breast Cancer Based on Intrinsic Subtypes

PubMed Central

Parker, Joel S.; Mullins, Michael; Cheang, Maggie C.U.; Leung, Samuel; Voduc, David; Vickery, Tammi; Davies, Sherri; Fauron, Christiane; He, Xiaping; Hu, Zhiyuan; Quackenbush, John F.; Stijleman, Inge J.; Palazzo, Juan; Marron, J.S.; Nobel, Andrew B.; Mardis, Elaine; Nielsen, Torsten O.; Ellis, Matthew J.; Perou, Charles M.; Bernard, Philip S.

2009-01-01

Purpose To improve on current standards for breast cancer prognosis and prediction of chemotherapy benefit by developing a risk model that incorporates the gene expression–based “intrinsic” subtypes luminal A, luminal B, HER2-enriched, and basal-like. Methods A 50-gene subtype predictor was developed using microarray and quantitative reverse transcriptase polymerase chain reaction data from 189 prototype samples. Test sets from 761 patients (no systemic therapy) were evaluated for prognosis, and 133 patients were evaluated for prediction of pathologic complete response (pCR) to a taxane and anthracycline regimen. Results The intrinsic subtypes as discrete entities showed prognostic significance (P = 2.26E-12) and remained significant in multivariable analyses that incorporated standard parameters (estrogen receptor status, histologic grade, tumor size, and node status). A prognostic model for node-negative breast cancer was built using intrinsic subtype and clinical information. The C-index estimate for the combined model (subtype and tumor size) was a significant improvement on either the clinicopathologic model or subtype model alone. The intrinsic subtype model predicted neoadjuvant chemotherapy efficacy with a negative predictive value for pCR of 97%. Conclusion Diagnosis by intrinsic subtype adds significant prognostic and predictive information to standard parameters for patients with breast cancer. The prognostic properties of the continuous risk score will be of value for the management of node-negative breast cancers. The subtypes and risk score can also be used to assess the likelihood of efficacy from neoadjuvant chemotherapy. PMID:19204204

Receptor subtypes involved in callosally-induced postsynaptic potentials in rat frontal agranular cortex in vitro.

PubMed

Kawaguchi, Y

1992-01-01

A slice preparation of rat frontal agranular cortex preserving commissural inputs has been used for intracellular recording from layer V pyramidal cells, in order to characterize the synaptic potentials induced by stimulation of the corpus callosum and to reveal the subtypes of amino acid receptors involved. Stimulation of the corpus callosum induced EPSPs followed by early IPSPs with a peak latency of 30 +/- 2 ms and late IPSPs with a peak latency of 185 +/- 18 ms. Reversal potentials for early and late IPSPs were -75 +/- 5 mV (early) and -96 +/- 5 mV (late). Late IPSPs were more dependent on extracellular K+ concentration. The early IPSPs were blocked by GABAA antagonists, bicuculline and picrotoxin, whereas the late IPSPs were reduced by the GABAB antagonist, phaclofen. CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), an antagonist of non-NMDA (N-methyl-D-aspartate) receptors, suppressed both EPSPs and late IPSPs at 5 microM. Early IPSPs remained at this concentration but were suppressed by 20 microM CNQX. In Mg(2+)-free solution, EPSPs were larger and more prolonged than in control solution. These enhanced EPSPs persisted after 5 to 20 microM CNQX, but were reduced in amplitude, and their onset was delayed by 3.6 +/- 0.8 ms. The remaining EPSPs were suppressed by 50 microM APV (DL-2-amino-5-phosphono-valeric acid), an antagonist of NMDA receptors. In Mg(2+)-free solution containing 5 to 20 microM CNQX, the late IPSPs were not diminished. The remaining late IPSPs were suppressed by APV or by phaclofen.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of age and biological subtype on the risk and timing of brain metastasis in breast cancer patients.

PubMed

Hung, Man-Hsin; Liu, Chun-Yu; Shiau, Cheng-Ying; Hsu, Chin-Yi; Tsai, Yi-Fang; Wang, Yu-Ling; Tai, Ling-Chen; King, Kuang-Liang; Chao, Ta-Chung; Chiu, Jen-Hwey; Su, Cheng-Hsi; Lo, Su-Shun; Tzeng, Cheng-Hwai; Shyr, Yi-Ming; Tseng, Ling-Ming

2014-01-01

Brain metastasis is a major complication of breast cancer. This study aimed to analyze the effect of age and biological subtype on the risk and timing of brain metastasis in breast cancer patients. We identified subtypes of invasive ductal carcinoma of the breast by determining estrogen receptor, progesterone receptor and HER2 status. Time to brain metastasis according to age and cancer subtype was analyzed by Cox proportional hazard analysis. Of the 2248 eligible patients, 164 (7.3%) developed brain metastasis over a median follow-up of 54.2 months. Age 35 or younger, HER2-enriched subtype, and triple-negative breast cancer were significant risk factors of brain metastasis. Among patients aged 35 or younger, the risk of brain metastasis was independent of biological subtype (P = 0.507). Among patients aged 36-59 or >60 years, those with triple-negative or HER2-enriched subtypes had consistently increased risk of brain metastasis, as compared with those with luminal A tumors. Patients with luminal B tumors had higher risk of brain metastasis than luminal A only in patients >60 years. Breast cancer subtypes are associated with differing risks of brain metastasis among different age groups. Patients age 35 or younger are particularly at risk of brain metastasis independent of biological subtype.

Racial Differences in PAM50 Subtypes in the Carolina Breast Cancer Study.

PubMed

Troester, Melissa A; Sun, Xuezheng; Allott, Emma H; Geradts, Joseph; Cohen, Stephanie M; Tse, Chiu-Kit; Kirk, Erin L; Thorne, Leigh B; Mathews, Michelle; Li, Yan; Hu, Zhiyuan; Robinson, Whitney R; Hoadley, Katherine A; Olopade, Olufunmilayo I; Reeder-Hayes, Katherine E; Earp, H Shelton; Olshan, Andrew F; Carey, Lisa A; Perou, Charles M

2018-02-01

African American breast cancer patients have lower frequency of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-negative disease and higher subtype-specific mortality. Racial differences in molecular subtype within clinically defined subgroups are not well understood. Using data and biospecimens from the population-based Carolina Breast Cancer Study (CBCS) Phase 3 (2008-2013), we classified 980 invasive breast cancers using RNA expression-based PAM50 subtype and recurrence (ROR) score that reflects proliferation and tumor size. Molecular subtypes (Luminal A, Luminal B, HER2-enriched, and Basal-like) and ROR scores (high vs low/medium) were compared by race (blacks vs whites) and age (≤50 years vs > 50 years) using chi-square tests and analysis of variance tests. Black women of all ages had a statistically significantly lower frequency of Luminal A breast cancer (25.4% and 33.6% in blacks vs 42.8% and 52.1% in whites; younger and older, respectively). All other subtype frequencies were higher in black women (case-only odds ratio [OR] = 3.11, 95% confidence interval [CI] = 2.22 to 4.37, for Basal-like; OR = 1.45, 95% CI = 1.02 to 2.06, for Luminal B; OR = 2.04, 95% CI = 1.33 to 3.13, for HER2-enriched). Among clinically HR+/HER2- cases, Luminal A subtype was less common and ROR scores were statistically significantly higher among black women. Multigene assays highlight racial disparities in tumor subtype distribution that persist even in clinically defined subgroups. Differences in tumor biology (eg, HER2-enriched status) may be targetable to reduce disparities among clinically ER+/HER2- cases. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

[Effect of hydrostatic pressure on intracellular free calcium concentration and transient receptor potential vanilloid expression in human bladder smooth muscle cells].

PubMed

Han, Zhenwei; Wang, Kunjie; Chen, Lin; Wei, Tangqiang; Luo, Deyi; Li, Shengfu

2012-04-01

To explore the effect of hydrostatic pressure on intracellular free calcium concentration ([Ca2+]i) and the gene expression of transient receptor potential vanilloid (TRPV) in cultured human bladder smooth muscle cells (hb-SMCs), and to preliminarily probe into the possible molecular mechanism of hb-SMCs proliferation stimulated by hydrostatic pressure. The passage 6-7 hb-SMCs were loaded with Ca2+ indicator Fluo-3/AM. When the hb-SMCs were under 0 cm H2O (1cm H2O = 0.098 kPa) (group A) or 200 cm H2O hydrostatic pressure for 30 minutes (group B) and then removing the 200 cm H2O hydrostatic pressure (group C), the [Ca2+]i was measured respectively by inverted laser scanning confocal microscope. When the hb-SMCs were given the 200 cm H2O hydrostatic pressure for 0 hour, 2 hours, 6 hours, 12 hours, and 24 hours, the mRNA expressions of TRPV1, TRPV2, and TRPV4 were detected by RT-PCR technique. The [Ca2+]i of group A, group B, and group C were (100.808 +/- 1.724), (122.008 +/- 1.575), and (99.918 +/- 0.887) U, respectively; group B was significantly higher than groups A and C (P < 0.001). The [Ca2+]i of group C decreased to the base line level of group A after removing the pressure (t = 0.919, P = 0.394). The TRPV1, TRPV2, and TRPV4 genes expressed in hb-SMCs under 200 cm H2O hydrostatic pressure at 0 hour, 2 hours, 6 hours, 12 hours, and 24 hours, but the expressions had no obvious changes with time. There was no significant difference in the expressions of TRPV1, TRPV2, and TRPV4 among 3 groups (P > 0.05). The [Ca2+]i of hb-SMCs increases significantly under high hydrostatic pressure. As possible genes in stretch-activated cation channel, the TRPV1, TRPV2, and TRPV4 express in hb-SMCs under 200 cm H2O hydrostatic pressure. It is possible that the mechanical pressure regulates the [Ca2+]i of hb-SMCs by opening the stretch-activated cation channel rather than up-regulating its expression.

Geographic differences in the distribution of molecular subtypes of breast cancer in Brazil

PubMed Central

2014-01-01

Background To compare the distribution of the intrinsic molecular subtypes of breast cancer based on immunohistochemical profile in the five major geographic regions of Brazil, a country of continental dimension, with a wide racial variation of people. Methods The study was retrospective observational. We classified 5,687 invasive breast cancers by molecular subtype based on immunohistochemical expression of estrogen-receptor (ER), progesterone-receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 proliferation index. Cases were classified as luminal A (ER and/or PR positive and HER2 negative, Ki-67 < 14%), luminal B (ER and/or PR positive, HER2 negative, and Ki-67 > 14%), triple-positive (ER and/or PR positive and HER2 positive), HER2-enriched (ER and PR negative, and HER2- positive), and triple-negative (TN) (ER negative, PR negative, and HER2- negative). Comparisons of the ages of patients and molecular subtypes between different geographic regions were performed. Results South and Southeast regions with a higher percentage of European ancestry and higher socioeconomic status presented with the highest proportion of luminal tumors. The North region presented with more aggressive subtypes (HER2-enriched and triple-negative), while the Central-West region predominated triple-positive carcinomas. The Northeast—a region with a high African influence—presented intermediate frequency of the different molecular subtypes. The differences persisted in subgroups of patients under and over 50 years. Conclusions The geographic regions differ according to the distribution of molecular subtypes of breast cancer. However, other differences, beside those related to African ancestry, such as socioeconomic, climatic, nutritional, and geographic, have to be considered to explain our results. The knowledge of the differences in breast cancer characteristics among the geographic regions may help to organize healthcare programs in large countries

Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. III. Agonist and antagonist properties at serotonin, 5-HT(1) and 5-HT(2), receptor subtypes.

PubMed

Newman-Tancredi, Adrian; Cussac, Didier; Quentric, Yann; Touzard, Manuelle; Verrièle, Laurence; Carpentier, Nathalie; Millan, Mark J

2002-11-01

Although certain antiparkinson agents interact with serotonin (5-HT) receptors, little information is available concerning functional actions. Herein, we characterized efficacies of apomorphine, bromocriptine, cabergoline, lisuride, piribedil, pergolide, roxindole, and terguride at human (h)5-HT(1A), h5-HT(1B), and h5-HT(1D) receptors [guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding], and at h5-HT(2A), h5-HT(2B), and h5-HT(2C) receptors (depletion of membrane-bound [(3)H]phosphatydilinositol). All drugs stimulated h5-HT(1A) receptors with efficacies (compared with 5-HT, 100%) ranging from modest (apomorphine, 35%) to high (cabergoline, 93%). At h5-HT(1B) receptors, efficacies varied from mild (terguride, 37%) to marked (cabergoline, 102%) and potencies were modest (pEC(50) values of 5.8-7.6): h5-HT(1D) sites were activated with a similar range of efficacies and greater potency (7.1-8.5). Piribedil and apomorphine were inactive at h5-HT(1B) and h5-HT(1D) receptors. At h5-HT(2A) receptors, terguride, lisuride, bromocriptine, cabergoline, and pergolide displayed potent (7.6-8.8) agonist properties (49-103%), whereas apomorphine and roxindole were antagonists and piribedil was inactive. Only pergolide (113%/8.2) and cabergoline (123%/8.6) displayed pronounced agonist properties at h5-HT(2B) receptors. At 5-HT(2C) receptors, lisuride, bromocriptine, pergolide, and cabergoline were efficacious (75-96%) agonists, apomorphine and terguride were antagonists, and piribedil was inactive. MDL100,907 and SB242,084, selective antagonists at 5-HT(2A) and 5-HT(2C) receptors, respectively, abolished these actions of pergolide, cabergoline, and bromocriptine. In conclusion, antiparkinson agents display markedly different patterns of agonist and antagonist properties at multiple 5-HT receptor subtypes. Although all show modest (agonist) activity at 5-HT(1A) sites, their contrasting actions at 5-HT(2A) and 5-HT(2C) sites may be of particular significance to their

Molecular cloning, molecular evolution and gene expression of cDNAs encoding thyrotropin-releasing hormone receptor subtypes in a teleost, the sockeye salmon (Oncorhynchus nerka).

PubMed

Saito, Yuichi; Mekuchi, Miyuki; Kobayashi, Noriaki; Kimura, Makoto; Aoki, Yasuhiro; Masuda, Tomohiro; Azuma, Teruo; Fukami, Motohiro; Iigo, Masayuki; Yanagisawa, Tadashi

2011-11-01

Molecular cloning of thyrotropin-releasing hormone receptors (TRHR) was performed in a teleost, the sockeye salmon (Oncorhynchus nerka). Four different TRHR cDNAs were cloned and named TRHR1, TRHR2a, TRHR2b and TRHR3 based on their similarity to known TRHR subtypes in vertebrates. Important residues for TRH binding were conserved in deduced amino acid sequences of the three TRHR subtypes except for the TRHR2b. Seven transmembrane domains were predicted for TRHR1, TRHR2a and TRHR3 proteins but only five for TRHR2b which appears to be truncated. In silico database analysis identified putative TRHR sequences including invertebrate TRHR and reptilian, avian and mammalian TRHR3. Phylogenetic analyses predicted the molecular evolution of TRHR in vertebrates: from the common ancestral TRHR (i.e. invertebrate TRHR), the TRHR2 subtype diverged first and then TRHR1 and TRHR3 diverged. Reverse transcription-polymerase chain reaction analyses revealed TRHR1 transcripts in the brain (hypothalamus), retina, pituitary gland and large intestine; TRHR2a in the brain (telencephalon and hypothalamus); and TRHR3 in the brain (olfactory bulbs) and retina. Copyright © 2011 Elsevier Inc. All rights reserved.

Neuromedin B receptor in esophagus: evidence for subtypes of bombesin receptors

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

Von Schrenck, T.; Heinz-Erian, P.; Moran, T.

1989-04-01

To identify receptors for bombesin-related peptides in the rat esophagus, we measured binding of 125I-Bolton-Hunter neuromedin B (125I-BH-neuromedin B) and 125I-(Tyr4)bombesin to tissue sections from the rat esophagus and compared the results with those for rat pancreas. Esophagus bound both tracers, whereas pancreas bound only 125I-(Tyr4)bombesin. In each tissue binding was saturable, dependent on pH, on time, and on temperature, reversible, and specific. Autoradiography demonstrated binding of both tracers only to the muscularis mucosae of the esophagus and binding of 125I-(Tyr4)bombesin diffusely over pancreatic acini. In the esophagus, the relative potencies for inhibition of binding of both tracers were asmore » follows: neuromedin B greater than bombesin greater than GRP = neuromedin C; similar relative potencies were found for causing contraction of muscle strips from whole esophagus and from the isolated muscularis mucosae. In pancreas tissue sections and dispersed acini, the relative potencies for inhibition of binding of 125I-(Tyr4)bombesin were as follows: bombesin greater than GRP = neuromedin C much greater than neuromedin B. Similar relative potencies were found for stimulation of enzyme secretion from dispersed pancreatic acini. Computer analysis in both tissues demonstrated only a single binding site. The present study demonstrates that rat esophagus muscle possesses specific receptors for bombesin-related peptides. Furthermore, this study shows that the esophageal bombesin receptors represent a previously unidentified class of bombesin receptors in that they have a higher affinity for neuromedin B than for bombesin. In contrast, the pancreatic bombesin receptors have, like all other bombesin receptors described to date, a high affinity for bombesin, but low affinity for neuromedin B.« less

The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund's adjuvant

PubMed Central

Yu, Lu; Yang, Fei; Luo, Hao; Liu, Feng-Yu; Han, Ji-Sheng; Xing, Guo-Gang; Wan, You

2008-01-01

Background The present study aims to investigate the role of transient receptor potential vanilloid 1 (TRPV1) in dorsal root ganglion (DRG) neurons in chronic pain including thermal hyperalgesia and mechanical allodynia. Chronic inflammatory nociception of rats was produced by intraplantar injection of complete Freund's adjuvant (CFA) and data was collected until day 28 following injection. Results Thermal hyperalgesia was evident from day 1 to day 28 with peak at day 7, while mechanical allodynia persisted from day 1 to day 14 and was greatest at day 7. Intrathecal administration of AMG 9810 at day 7, a selective TRPV1 antagonist, significantly reduced thermal hyperalgesia and mechanical allodynia. TRPV1 expression in DRG detected by Western blotting was increased relative to baseline throughout the observation period. Double labeling of TRPV1 with neuronal marker neurofilament 200 (NF200), calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4) was used to distinguish different subtypes of DRG neurons. TRPV1 expression was increased in the medium-sized myelinated A fiber (NF200 positive) neurons and in small non-peptidergic (IB4 positive) neurons from day 1 to day 14 and was increased in small peptidergic (CGRP positive) neurons from day 1 to day 28. Conclusion TRPV1 expression increases in all three types of DRG neurons after CFA injection and plays a role in CFA-induced chronic inflammatory pain including thermal hyperalgesia and mechanical allodynia. PMID:19055783

Male breast cancer according to tumor subtype and race: a population-based study.

PubMed

Chavez-Macgregor, Mariana; Clarke, Christina A; Lichtensztajn, Daphne; Hortobagyi, Gabriel N; Giordano, Sharon H

2013-05-01

Breast cancer occurs rarely in men. To the authors' knowledge, no population-based estimates of the incidence of human epidermal growth factor receptor 2 (HER2)-positive breast cancer or of the distribution of breast cancer subtypes among male breast cancer patients have been published to date. Therefore, the objective of the current study was to explore breast tumor subtype distribution by race/ethnicity among men in the large, ethnically diverse population of California. This study included men who were diagnosed with invasive breast cancer between 2005 and 2009 with known estrogen receptor (ER) and progesterone receptor (PR) (together, hormone receptor [HR]) status and HER2 status reported to the California Cancer Registry. Among the men with HR-positive tumors, survival probabilities between groups were compared using log-rank tests. Six hundred six patients were included. The median age at diagnosis was 68 years. Four hundred ninety-four men (81.5%) had HR-positive tumors (defined as ER-positive and/or PR-positive and HER2-negative). Ninety men (14.9%) had HER2-positive tumors, and 22 (3.6%) had triple receptor-negative (TN) tumors. Among the patients with HR-positive tumors, non-Hispanic black men and Hispanic men were more likely to have PR-negative tumors than non-Hispanic white men. No statistically significant differences in survival were observed according to tumor subtype (P = .08). Differences in survival according to race/ethnicity were observed among all patients (P = .087) and among those with HR-positive tumors (P = .0170), and non-Hispanic black men had poorer outcomes. In this large, representative cohort of men with breast cancer, the distribution of tumor subtypes was different from that reported for women and varied by patient race/ethnicity. Non-Hispanic black men were more likely to have TN tumors and ER-positive/PR-negative tumors than white men. Copyright © 2013 American Cancer Society.

Structure-activity studies of RFamide peptides reveal subtype-selective activation of neuropeptide FF1 and FF2 receptors.

PubMed

Findeisen, Maria; Rathmann, Daniel; Beck-Sickinger, Annette G

2011-06-06

Selectivity is a major issue in closely related multiligand/multireceptor systems. In this study we investigated the RFamide systems of hNPFF₁R and hNPFF₂R that bind the endogenous peptide hormones NPFF, NPAF, NPVF, and NPSF. By use of a systematic approach, we characterized the role of the C-terminal dipeptide with respect to agonistic properties using synthesized [Xaa 7]NPFF and [Xaa 8]NPFF analogues. We were able to identify only slight differences in potency upon changing the position of Arg 7, as all modifications resulted in identical behavior at the NPFF₁R and NPFF₂R. However, the C-terminal Phe 8 was able to be replaced by Trp or His with only a minor loss in potency at the NPFF₂R relative to the NPFF₁R. Analogues with shorter side chains, such as α-amino-4-guanidino butyric acid ([Agb 7]NPFF) or phenylglycine ([Phg 8]NPFF), decreased efficacy for the NPFF₁ R to 25-31 % of the maximal response, suggesting that these agonist-receptor complexes are more susceptible to structural modifications. In contrast, mutations to the conserved Asp 6.59 residue in the third extracellular loop of both receptors revealed a higher sensitivity toward the hNPFF₂R receptor than toward hNPFF₁R. These data provide new insight into the subtype-specific agonistic activation of the NPFF₁ and NPFF(2) receptors that are necessary for the development of selective agonists. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhancement effects of nicotine on neurogenic relaxation responses in the corpus cavernosum in rabbits: the role of nicotinic acetylcholine receptor subtypes.

PubMed

Ozturk Fincan, Gokce Sevim; Vural, Ismail Mert; Ercan, Zeynep Sevim; Sarioglu, Yusuf

2010-02-10

Nicotine acts as an agonist of nicotinic acetylcholine receptors, which belong to a superfamily of neurotransmitter-gated ion channels. We previously demonstrated that nicotine increases the electrical field stimulation (EFS)-evoked nitrergic relaxation responses via activation of nicotinic acetylcholine receptors. The aim of the present study is to investigate the subtypes of nicotinic acetylcholine receptors in rabbit corpus cavernosum. EFS-evoked relaxation responses were recorded from corpus cavernosum strips obtained from rabbits with an isometric force displacement transducers. Effects of nicotine on EFS-evoked relaxations were examined in pre-contracted tissues. Then the effect of nicotine on the EFS-evoked relaxations was examined in the presence of hexamethonium, dihydro-beta-erythroidine, mecamylamine or alpha-bungarotoxin. In our study, nicotine (3 x 10(-5), 10(-4)) transiently increased nitrergic relaxations induced by EFS in the rabbit isolated corpus cavernosum. While hexamethonium and mecamylamine near totally inhibited or abolished the neurorelaxation response to nicotine (3 x 10(-5)) on EFS, dihydro-beta-erythroidine and alpha-bungarotoxin partially inhibited these responses. These findings demonstrated that the alpha3-beta4, alpha4-beta2 and alpha7 subunits of nicotinic acetylcholine receptors play role on the nicotine-induced augmentation in EFS-evoked relaxation responses in rabbit corpus cavernosum. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Species differences in the relative densities of D1- and D2-like dopamine receptor subtypes in the Japanese quail and rats: an in vitro quantitative receptor autoradiography study.

PubMed

Kleitz, Hayley K; Cornil, Charlotte A; Balthazart, Jacques; Ball, Gregory F

2009-01-01

Evidence has accumulated that the regulation of male sexual behavior by dopamine might not be the same in Japanese quail (and perhaps all birds) as it is in mammals. For example, the non-selective dopamine receptor agonist, apomorphine (APO), facilitates male sexual behavior in rats but inhibits it in quail. Although the general organization of the dopamine system is similar in birds and mammals, it is possible that the relative distribution and/or density of binding sites are different. We therefore compared the relative densities of D1-like and D2-like receptor subtypes in Japanese quail and rats, with the use of in vitro quantitative receptor autoradiography. Brain sections from 8 male rats and 8 male quail were labeled with [(3)H]SCH-23390 and [(3)H]Spiperone. In general we found a systematic species difference in the relative density of D1- vs. D2-like receptors such that the D2/D1 ratio is higher in quail than in rats in areas, known to be important target sites for dopamine action such as striatal regions or the preoptic area, which is also associated with activation of sexual behavior. This difference might explain the variation in the behavioral effectiveness of APO in rats as compared to quail; with a higher relative density of D2-like receptors in quail, a similar dose of APO would be more likely to activate inhibitory processes in quail than in rats. (c) 2009 S. Karger AG, Basel.

Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.

PubMed

Khan, Samir A; Rossi, Ana M; Riley, Andrew M; Potter, Barry V L; Taylor, Colin W

2013-04-15

IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3. Activation of IP(3)R is initiated by IP(3) binding to the IBC (IP(3)-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 μM) stimulated IP(3) binding to the isolated NT (N-terminal; residues 1-604) of IP(3)R1 and IP(3)R2, but not to that of IP(3)R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP(3)) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP(3)R activation. IP(3) binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP(3)R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP(3) binding to the chimaeric NT and IP(3)-evoked Ca(2+) release from the chimaeric IP(3)R. This is the first systematic analysis of the effects of a thiol reagent on each IP(3)R subtype. We conclude that thimerosal selectively sensitizes IP(3)R1 and IP(3)R2 to IP(3) by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

Subtype-selective regulation of IP3 receptors by thimerosal via cysteine residues within the IP3-binding core and suppressor domain

PubMed Central

Khan, Samir A.; Rossi, Ana M.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

2013-01-01

IP3R (IP3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP3R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP3-evoked Ca2+ release via IP3R1 and IP3R2, but inhibited IP3R3. Activation of IP3R is initiated by IP3 binding to the IBC (IP3-binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP3 binding to the isolated NT (N-terminal; residues 1–604) of IP3R1 and IP3R2, but not to that of IP3R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP3) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP3R activation. IP3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP3R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP3 binding to the chimaeric NT and IP3-evoked Ca2+ release from the chimaeric IP3R. This is the first systematic analysis of the effects of a thiol reagent on each IP3R subtype. We conclude that thimerosal selectively sensitizes IP3R1 and IP3R2 to IP3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor. PMID:23282150

Label noise in subtype discrimination of class C G protein-coupled receptors: A systematic approach to the analysis of classification errors.

PubMed

König, Caroline; Cárdenas, Martha I; Giraldo, Jesús; Alquézar, René; Vellido, Alfredo

2015-09-29

The characterization of proteins in families and subfamilies, at different levels, entails the definition and use of class labels. When the adscription of a protein to a family is uncertain, or even wrong, this becomes an instance of what has come to be known as a label noise problem. Label noise has a potentially negative effect on any quantitative analysis of proteins that depends on label information. This study investigates class C of G protein-coupled receptors, which are cell membrane proteins of relevance both to biology in general and pharmacology in particular. Their supervised classification into different known subtypes, based on primary sequence data, is hampered by label noise. The latter may stem from a combination of expert knowledge limitations and the lack of a clear correspondence between labels that mostly reflect GPCR functionality and the different representations of the protein primary sequences. In this study, we describe a systematic approach, using Support Vector Machine classifiers, to the analysis of G protein-coupled receptor misclassifications. As a proof of concept, this approach is used to assist the discovery of labeling quality problems in a curated, publicly accessible database of this type of proteins. We also investigate the extent to which physico-chemical transformations of the protein sequences reflect G protein-coupled receptor subtype labeling. The candidate mislabeled cases detected with this approach are externally validated with phylogenetic trees and against further trusted sources such as the National Center for Biotechnology Information, Universal Protein Resource, European Bioinformatics Institute and Ensembl Genome Browser information repositories. In quantitative classification problems, class labels are often by default assumed to be correct. Label noise, though, is bound to be a pervasive problem in bioinformatics, where labels may be obtained indirectly through complex, many-step similarity modelling processes

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

PubMed

Lee, Jonathan K; Garbe, James C; Vrba, Lukas; Miyano, Masaru; Futscher, Bernard W; Stampfer, Martha R; LaBarge, Mark A

2015-01-01

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

PubMed Central

Trujillo, Cleber A.; Sathler, Luciana B.; Juliano, Maria A.; Juliano, Luiz; Ulrich, Henning; Ferreira, Sergio T.

2013-01-01

Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD. PMID:23894286

Molecular recognition at adenine nucleotide (P2) receptors in platelets.

PubMed

Jacobson, Kenneth A; Mamedova, Liaman; Joshi, Bhalchandra V; Besada, Pedro; Costanzi, Stefano

2005-04-01

Transmembrane signaling through P2Y receptors for extracellular nucleotides controls a diverse array of cellular processes, including thrombosis. Selective agonists and antagonists of the two P2Y receptors present on the platelet surface-the G (q)-coupled P2Y (1) subtype and the G (i)-coupled P2Y (12) subtype-are now known. High-affinity antagonists of each have been developed from nucleotide structures. The (N)-methanocarba bisphosphate derivatives MRS2279 and MRS2500 are potent and selective P2Y (1) receptor antagonists. The carbocyclic nucleoside AZD6140 is an uncharged, orally active P2Y (12) receptor antagonist of nM affinity. Another nucleotide receptor on the platelet surface, the P2X (1) receptor, the activation of which may also be proaggregatory, especially under conditions of high shear stress, has high-affinity ligands, although high selectivity has not yet been achieved. Although alpha,beta-methylene-adenosine triphosphate (ATP) is the classic agonist for the P2X (1) receptor, where it causes rapid desensitization, the agonist BzATP is among the most potent in activating this subtype. The aromatic sulfonates NF279 and NF449 are potent antagonists of the P2X (1) receptor. The structures of the two platelet P2Y receptors have been modeled, based on a rhodopsin template, to explain the basis for nucleotide recognition within the putative transmembrane binding sites. The P2Y (1) receptor model, especially, has been exploited in the design and optimization of antagonists targeted to interact selectively with that subtype.

Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies

PubMed Central

Lehmann, Brian D.; Bauer, Joshua A.; Chen, Xi; Sanders, Melinda E.; Chakravarthy, A. Bapsi; Shyr, Yu; Pietenpol, Jennifer A.

2011-01-01

Triple-negative breast cancer (TNBC) is a highly diverse group of cancers, and subtyping is necessary to better identify molecular-based therapies. In this study, we analyzed gene expression (GE) profiles from 21 breast cancer data sets and identified 587 TNBC cases. Cluster analysis identified 6 TNBC subtypes displaying unique GE and ontologies, including 2 basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal (M), a mesenchymal stem–like (MSL), and a luminal androgen receptor (LAR) subtype. Further, GE analysis allowed us to identify TNBC cell line models representative of these subtypes. Predicted “driver” signaling pathways were pharmacologically targeted in these cell line models as proof of concept that analysis of distinct GE signatures can inform therapy selection. BL1 and BL2 subtypes had higher expression of cell cycle and DNA damage response genes, and representative cell lines preferentially responded to cisplatin. M and MSL subtypes were enriched in GE for epithelial-mesenchymal transition, and growth factor pathways and cell models responded to NVP-BEZ235 (a PI3K/mTOR inhibitor) and dasatinib (an abl/src inhibitor). The LAR subtype includes patients with decreased relapse-free survival and was characterized by androgen receptor (AR) signaling. LAR cell lines were uniquely sensitive to bicalutamide (an AR antagonist). These data may be useful in biomarker selection, drug discovery, and clinical trial design that will enable alignment of TNBC patients to appropriate targeted therapies. PMID:21633166

Predicting Subtype Selectivity for Adenosine Receptor Ligands with Three-Dimensional Biologically Relevant Spectrum (BRS-3D)

NASA Astrophysics Data System (ADS)

He, Song-Bing; Ben Hu; Kuang, Zheng-Kun; Wang, Dong; Kong, De-Xin

2016-11-01

Adenosine receptors (ARs) are potential therapeutic targets for Parkinson’s disease, diabetes, pain, stroke and cancers. Prediction of subtype selectivity is therefore important from both therapeutic and mechanistic perspectives. In this paper, we introduced a shape similarity profile as molecular descriptor, namely three-dimensional biologically relevant spectrum (BRS-3D), for AR selectivity prediction. Pairwise regression and discrimination models were built with the support vector machine methods. The average determination coefficient (r2) of the regression models was 0.664 (for test sets). The 2B-3 (A2B vs A3) model performed best with q2 = 0.769 for training sets (10-fold cross-validation), and r2 = 0.766, RMSE = 0.828 for test sets. The models’ robustness and stability were validated with 100 times resampling and 500 times Y-randomization. We compared the performance of BRS-3D with 3D descriptors calculated by MOE. BRS-3D performed as good as, or better than, MOE 3D descriptors. The performances of the discrimination models were also encouraging, with average accuracy (ACC) 0.912 and MCC 0.792 (test set). The 2A-3 (A2A vs A3) selectivity discrimination model (ACC = 0.882 and MCC = 0.715 for test set) outperformed an earlier reported one (ACC = 0.784). These results demonstrated that, through multiple conformation encoding, BRS-3D can be used as an effective molecular descriptor for AR subtype selectivity prediction.

Mechanism of Activation of Enteric Nociceptive Neurons via Interaction of TLR4 and TRPV1 Receptors.

PubMed

Filippova, L V; Fedorova, A V; Nozdrachev, A D

2018-03-01

Evidence obtained by immunohistochemical double labeling and confocal laser scanning microscopy suggests that capsaicin, a ligand of the TRPV1 nociceptive vanilloid receptor, increases the number of TLR4-positive neurons in the rat colon myenteric plexus. In colitis caused by trinitrobenzene sulfonate, an increase in TRPV1 expression was more significant in both plexuses. Specific inhibitor of the TLR4 (C34) pattern-recognition receptor reduces TRPV1 expression in enteric neurons of both intact rats and rats with induced acute colitis. Thus, stimulation of nociceptive neurons by means of direct activation of their receptors of innate immunity (TLR4) is one of the possible mechanisms underlying the visceral pain in bacterial invasion and inflammatory bowel diseases.

ROLE OF ESTROGEN RECEPTOR-α ON FOOD DEMAND ELASTICITY

PubMed Central

Minervini, Vanessa; Rowland, Neil E.; Robertson, Kimberly L.; Foster, Thomas C.

2016-01-01

Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions. PMID:25869426

Role of estrogen receptor-α on food demand elasticity.

PubMed

Minervini, Vanessa; Rowland, Neil E; Robertson, Kimberly L; Foster, Thomas C

2015-05-01

Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions. © Society for the Experimental Analysis of Behavior.

PAM50 Breast Cancer Subtyping by RT-qPCR and Concordance with Standard Clinical Molecular Markers

PubMed Central

2012-01-01

Background Many methodologies have been used in research to identify the “intrinsic” subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions. Methods We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and “intrinsic” subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments. Results ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a

Rapid Antidepressant Actions of Scopolamine: Role of Medial Prefrontal Cortex and M1-subtype Muscarinic Acetylcholine Receptors

PubMed Central

Navarria, Andrea; Wohleb, Eric S.; Voleti, Bhavya; Ota, Kristie T.; Dutheil, Sophie; Lepack, Ashley E.; Dwyer, Jason M.; Fuchikami, Manabu; Becker, Astrid; Drago, Filippo; Duman, Ronald S.

2015-01-01

Clinical studies demonstrate that scopolamine, a nonselective muscarinic acetycholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1) in the medial prefrontal cortex (mPFC). The present study extends these findings to determine the role of the mPFC and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. Administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that systemic administration of a selective M1-AChR antagonist, VU0255035 produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions, and found that scopolamine or VU0255035 administration blocked the anhedonic response caused by CUS, an effect that requires chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine, and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants. PMID:26102021

Rapid antidepressant actions of scopolamine: Role of medial prefrontal cortex and M1-subtype muscarinic acetylcholine receptors.

PubMed

Navarria, Andrea; Wohleb, Eric S; Voleti, Bhavya; Ota, Kristie T; Dutheil, Sophie; Lepack, Ashley E; Dwyer, Jason M; Fuchikami, Manabu; Becker, Astrid; Drago, Filippo; Duman, Ronald S

2015-10-01

Clinical studies demonstrate that scopolamine, a non-selective muscarinic acetylcholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1). The present study extends these findings to determine the role of the medial prefrontal cortex (mPFC) and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. The administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or the PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that the systemic administration of a selective M1-AChR antagonist, VU0255035, produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions and found that a single dose of scopolamine or VU0255035 blocked the anhedonic response caused by CUS, an effect that requires the chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants. Copyright © 2015 Elsevier Inc. All rights reserved.

Bombesin-like peptide receptors in human bronchial epithelial cells.

PubMed

Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A

1996-01-01

Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

Spontaneous calcium transients in human neural progenitor cells mediated by transient receptor potential channels.

PubMed

Morgan, Peter J; Hübner, Rayk; Rolfs, Arndt; Frech, Moritz J

2013-09-15

Calcium signals affect many developmental processes, including proliferation, migration, survival, and apoptosis, processes that are of particular importance in stem cells intended for cell replacement therapies. The mechanisms underlying Ca(2+) signals, therefore, have a role in determining how stem cells respond to their environment, and how these responses might be controlled in vitro. In this study, we examined the spontaneous Ca(2+) activity in human neural progenitor cells during proliferation and differentiation. Pharmacological characterization indicates that in proliferating cells, most activity is the result of transient receptor potential (TRP) channels that are sensitive to Gd(3+) and La(3+), with the more subtype selective antagonist Ruthenium red also reducing activity, suggesting the involvement of transient receptor potential vanilloid (TRPV) channels. In differentiating cells, Gd(3+) and La(3+)-sensitive TRP channels also appear to underlie the spontaneous activity; however, no sub-type-specific antagonists had any effect. Protein levels of TRPV2 and TRPV3 decreased in differentiated cells, which is demonstrated by western blot. Thus, it appears that TRP channels represent the main route of Ca(2+) entry in human neural progenitor cells (hNPCs), but the responsible channel types are subject to substitution under differentiating conditions. The level of spontaneous activity could be increased and decreased by lowering and raising the extracellular K(+) concentration. Proliferating cells in low K(+) slowed the cell cycle, with a disproportionate increased percentage of cells in G1 phase and a reduction in S phase. Taken together, these results suggest a link between external K(+) concentration, spontaneous Ca(2+) transients, and cell cycle distribution, which is able to influence the fate of stem and progenitor cells.

Clinical Characteristics and Prognosis of Pregnancy-Associated Breast Cancer: Poor Survival of Luminal B Subtype.

PubMed

Bae, Soo Youn; Jung, Seung Pil; Jung, Eun Sung; Park, Sung Min; Lee, Se Kyung; Yu, Jong Han; Lee, Jeong Eon; Kim, Seok Won; Nam, Seok Jin

2018-06-18

Pregnancy-associated breast cancer (PABC) is rare and is generally defined as breast cancer diagnosed during pregnancy or within 1 year of delivery. The average ages of marriage and childbearing are increasing, and PABC is expected to also increase. This study is intended to increase understanding of the characteristics of PABC. A database of 2,810 patients with breast cancer diagnosed when they were less than 40 years of age was reviewed. The clinicopathological factors and survival of PABC (40 patients) were compared to those of patients with young breast cancer (YBC, non-pregnant or over 12 months after delivery; 2,770 patients). PABC had significantly lower estrogen receptor (ER) and progesterone receptor (PR) expression (ER-positive 50.0%, PR-positive 45.0%) and higher HER2 overexpression (38.5%) than YBC. The most common subtype of PABC was triple-negative breast cancer (TNBC; 35.9%), and luminal A subtype represented only 7.7% of cases. In univariate analysis, PABC had significantly worse disease-free survival (DFS) and breast cancer-specific survival (BCSS) compared to YBC. In multivariate analysis, PABC was associated with worse BCSS (HR 4.0, 95% CI 1.2-12.9, p = 0.019) and survival, but there was no difference in DFS between PABC and YBC. In subgroup analysis by subtype, luminal B subtype of PABC showed worse DFS (HR 3.5; 95% CI 1.1-11.2, p = 0.039) and BCSS (HR 10.2, 95% CI 1.2-87.1, p = 0.035), especially with high Ki67. However, no differences were demonstrated in other subtypes. In this study, PABC showed lower expression of ER/PR, higher overexpression of HER2, fewer luminal A subtype, and more TNBC subtype compared to YBC. PABC had worse BCSS, especially luminal B subtype, compared to YBC. © 2018 S. Karger AG, Basel.

Role of Dopamine Receptors Subtypes, D1-Like and D2-Like, within the Nucleus Accumbens Subregions, Core and Shell, on Memory Consolidation in the One-Trial Inhibitory Avoidance Task

ERIC Educational Resources Information Center

Manago, Francesca; Castellano, Claudio; Oliverio, Alberto; Mele, Andrea; De Leonibus, Elvira

2009-01-01

Recent evidence demonstrated that dopamine within the nucleus accumbens mediates consolidation of both associative and nonassociative memories. However, the specific contribution of the nucleus accumbens subregions, core and shell, and of D1 and D2 receptors subtypes has not been yet clarified. The aim of this study was, therefore, to directly…

8-(2-Furyl)adenine derivatives as A₂A adenosine receptor ligands.

PubMed

Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Thomas, Ajiroghene; Klotz, Karl-Norbert; Federico, Stephanie; Cacciari, Barbara; Spalluto, Giampiero; Volpini, Rosaria

2013-01-01

Selective adenosine receptor modulators are potential tools for numerous therapeutic applications, including cardiovascular, inflammatory, and neurodegenerative diseases. In this work, the synthesis and biological evaluation at the four human adenosine receptor subtypes of a series of 9-substituted 8-(2-furyl)adenine derivatives are reported. Results show that 8-(2-furyl)-9-methyladenine is endowed with high affinity at the A₂A subtype. Further modification of this compound with introduction of arylacetyl or arylcarbamoyl groups in N(6)-position takes to different effects on the A₂A affinity and in particular on the selectivity versus the other three adenosine receptor subtypes. A molecular modelling analysis at three different A₂A receptor crystal structures provides an interpretation of the obtained biological results. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

High prevalence of luminal B breast cancer intrinsic subtype in Colombian women

PubMed Central

Serrano-Gomez, Silvia Juliana; Sanabria-Salas, Maria Carolina; Hernández-Suarez, Gustavo; García, Oscar; Silva, Camilo; Romero, Alejandro; Mejía, Juan Carlos; Miele, Lucio; Fejerman, Laura; Zabaleta, Jovanny

2016-01-01

Breast cancer is the most frequent malignancy in women worldwide. Distinct intrinsic subtypes of breast cancer have different prognoses, and their relative prevalence varies significantly among ethnic groups. Little is known about the prevalence of breast cancer intrinsic subtypes and their association with clinicopathological data and genetic ancestry in Latin Americans. Immunohistochemistry surrogates from the 2013 St. Gallen International Expert Consensus were used to classify breast cancers in 301 patients from Colombia into intrinsic subtypes. We analyzed the distribution of subtypes by clinicopathological variables. Genetic ancestry was estimated from a panel of 80 ancestry informative markers. Luminal B breast cancer subtype was the most prevalent in our population (37.2%) followed by luminal A (26.3%), non-basal triple negative (NBTN) (11.6%), basal like (9%), human epidermal growth factor receptor 2 (HER2) enriched (8.6%) and unknown (7.3%). We found statistical significant differences in distribution between Colombian region (P = 0.007), age at diagnosis (P = 0.0139), grade (P < 0.001) and recurrence (P < 0.001) according to intrinsic subtype. Patients diagnosed with HER2-enriched, basal-like and NBTN breast cancer had the highest African ancestry. Future studies analyzing the molecular profiles of breast cancer in Colombian women will help us understand the molecular basis of this subtype distribution and compare the molecular characteristics of the different intrinsic subtypes in Colombian patients. PMID:27207651

Regulation of IP 3 Receptors by IP 3 and Ca 2+

NASA Astrophysics Data System (ADS)

Taylor, Colin W.; Swatton, Jane E.

Inositol 1,4,5-trisphosphate ( IP 3) receptors are intracellular Ca 2+ channels that mediate release of Ca 2+ from intracellular stores. The channels are oligomeric assemblies of four subunits, each of which has an N-terminal IP 3-binding domain and each of which contributes to formation of the Ca 2+ channel. In mammals, three different genes encode IP 3 receptors subunits and the type 1 receptor (and perhaps the type 2 receptor) is also expressed as splice variants. Further diversity arises from assembly of the receptor in hetero- and homo-tetrameric channels. The subtypes differ in their expression and regulation, but they share the key property of being regulated by both IP3 and cytosolic Ca 2+. All three mammalian IP 3 subtypes, and probably also the IP 3 receptors expressed in invertebrates, are biphasically regulated by cytosolic Ca2+, although the underlying mechanisms appear to differ between subtypes. The interactions between IP 3 and Ca 2+ in controlling IP 3 receptor gating, and the physiological significance of such regulation will be reviewed.

Tamoxifen therapy improves overall survival in luminal A subtype of ductal carcinoma in situ: a study based on nationwide Korean Breast Cancer Registry database.

PubMed

Hwang, Ki-Tae; Kim, Eun-Kyu; Jung, Sung Hoo; Lee, Eun Sook; Kim, Seung Il; Lee, Seokwon; Park, Heung Kyu; Kim, Jongjin; Oh, Sohee; Kim, Young A

2018-06-01

To determine the prognostic role of tamoxifen therapy for patients with ductal carcinoma in situ (DCIS) according to molecular subtypes. Data of 14,944 patients with DCIS were analyzed. Molecular subtypes were classified into four categories based on expression of estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Kaplan-Meier estimator was used for overall survival analysis while Cox proportional hazards model was used for univariate and multivariate analyses. Luminal A subtype (ER/PR+, HER2-) showed higher (P = .009) survival rate than triple-negative (TN) subtype. Tamoxifen therapy group showed superior (P < .001) survival than no-tamoxifen therapy group. It had survival benefit only for luminal A subtype (P = .001). Tamoxifen therapy resulted in higher survival rate in subgroups with positive ER (P = .006), positive PR (P = .009), and negative HER2 (P < .001). In luminal A subtype, tamoxifen therapy showed lower hazard ratio (HR) compared to no-tamoxifen therapy (HR, 0.420; 95% CI 0.250-0.705; P = .001). Tamoxifen therapy was a significant independent factor by multivariate analysis (HR, 0.538; 95% CI 0.306-0.946; P = .031) as well as univariate analysis. Tamoxifen therapy group showed superior prognosis than the no-tamoxifen therapy group. Its prognostic influence was only effective for luminal A subtype. Patients with luminal A subtype showed higher survival rate than those with TN subtype. Active tamoxifen therapy is recommended for DCIS patients with luminal A subtype, and routine tests for ER, PR, and HER2 should be considered for DCIS.

Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.

PubMed

Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S

2015-06-01

We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors.

Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

PubMed

Joseph, Lauren; Thomsen, Morgane

2017-06-30

Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

Feeding condition and the relative contribution of different dopamine receptor subtypes to the discriminative stimulus effects of cocaine in rats.

PubMed

Baladi, Michelle G; Newman, Amy H; France, Charles P

2014-02-01

The contribution of dopamine receptor subtypes in mediating the discriminative stimulus effects of cocaine is not fully established. Many drug discrimination studies use food to maintain responding, necessitating food restriction, which can alter drug effects. This study established stimulus control with cocaine (10 mg/kg) in free-feeding and food-restricted rats responding under a schedule of stimulus shock termination (SST) and in food-restricted rats responding under a schedule of food presentation to examine whether feeding condition or the reinforcer used to maintain responding impacts the effects of cocaine. Dopamine receptor agonists and antagonists were examined for their ability to mimic or attenuate, respectively, the effects of cocaine. Apomorphine, quinpirole, and lisuride occasioned >90 % responding on the cocaine-associated lever in free-feeding rats responding under a schedule of SST; apomorphine, but not quinpirole or lisuride, occasioned >90 % responding on the cocaine lever in food-restricted rats responding under a schedule of SST. In food-restricted rats responding for food these drugs occasioned little cocaine lever responding and were comparatively more potent in decreasing responding. In free-feeding rats, the effects of cocaine were attenuated by the D2/D3 receptor antagonist raclopride and the D3 receptor-selective antagonist PG01037. In food-restricted rats, raclopride and the D2 receptor-selective antagonist L-741,626 attenuated the effects of cocaine. Raclopride antagonized quinpirole in all groups while PG01037 antagonized quinpirole only in free-feeding rats. These results demonstrate significant differences in the discriminative stimulus of cocaine that are due to feeding conditions and not to the use of different reinforcers across procedures.

Feeding condition and the relative contribution of different dopamine receptor subtypes to the discriminative stimulus effects of cocaine in rats

PubMed Central

Baladi, Michelle G; Newman, Amy H; France, Charles P

2013-01-01

Rationale The contribution of dopamine receptor subtypes in mediating the discriminative stimulus effects of cocaine is not fully established. Many drug discrimination studies use food to maintain responding, necessitating food restriction, which can alter drug effects. Objective This study established stimulus control with cocaine (10 mg/kg) in free-feeding and food-restricted rats responding under a schedule of stimulus shock termination (SST) and in food-restricted rats responding under a schedule of food presentation to examine whether feeding condition or the reinforcer used to maintain responding impacts the effects of cocaine. Method Dopamine receptor agonists and antagonists were examined for their ability to mimic or attenuate, respectively, the effects of cocaine. Result Apomorphine, quinpirole, and lisuride occasioned >90% responding on the cocaine-associated lever in free-feeding rats responding under a schedule of SST; apomorphine, but not quinpirole or lisuride, occasioned >90% responding on the cocaine lever in food-restricted rats responding under a schedule of SST. In food-restricted rats responding for food these drugs occasioned little cocaine lever responding and were comparatively more potent in decreasing responding. In free-feeding rats, the effects of cocaine were attenuated by the D2/D3 receptor antagonist raclopride and the D3 receptor-selective antagonist PG01037. In food-restricted rats, raclopride and the D2 receptor-selective antagonist L-741,626 attenuated the effects of cocaine. Raclopride antagonized quinpirole in all groups while PG01037 antagonized quinpirole only in free-feeding rats. Conclusion These results demonstrate significant differences in the discriminative stimulus of cocaine that are due to feeding conditions and not to the use of different reinforcers across procedures. PMID:24030470

Association of Hormone-Related Characteristics and Breast Cancer Risk by Estrogen Receptor/Progesterone Receptor Status in the Shanghai Breast Cancer Study

PubMed Central

Bao, Ping-Ping; Shu, Xiao Ou; Gao, Yu-Tang; Zheng, Ying; Cai, Hui; Deming, Sandra L.; Ruan, Zhi-Xian; Gu, Kai; Lu, Wei; Zheng, Wei

2011-01-01

Etiologic differences between subtypes of breast cancer defined by estrogen receptor (ER) and progesterone receptor (PR) status are not well understood. The authors evaluated associations of hormone-related factors with breast cancer subtypes in a population-based case-control study involving 1,409 ER-positive (ER+)/PR-positive (PR+) cases, 712 ER-negative (ER−)/PR-negative (PR−) cases, 301 ER+/PR− cases, 254 ER−/PR+ cases, and 3,474 controls aged 20–70 years in Shanghai, China (phase I, 1996–1998; phase II, 2002–2005). Polytomous logistic regression and Wald tests for heterogeneity across subtypes were conducted. Breast cancer risks associated with age at menarche, age at menopause, breastfeeding, age at first livebirth, waist-to-hip ratio, and oral contraceptive use did not differ by hormone receptor status. Among postmenopausal women, higher parity (≥2 children vs. 1) was associated with reduced risk (odds ratio (OR) = 0.69, 95% confidence interval (CI): 0.52, 0.91) and higher body mass index (BMI; weight (kg)/height (m)2) with increased risk (highest quartile: OR = 2.40, 95% CI: 1.65, 3.47) of the ER+/PR+ subtype but was unrelated to the ER−/PR− subtype (for parity, Pheterogeneity = 0.02; for BMI, Pheterogeneity < 0.01). Hormone replacement therapy (OR = 2.25, 95% CI: 1.40, 3.62) and alcohol consumption (OR = 1.59, 95% CI: 1.01, 2.51) appeared to be preferentially associated with the ER+/PR− subtype. These findings indicate that BMI, parity, hormone replacement therapy, and alcohol consumption may play different roles in subtypes of breast cancer. More research is needed to better understand the etiology of 2 relatively rare subtypes, ER+/PR− tumors and ER−/PR+ tumors. PMID:21768404

Nicotinic Acetylcholine Receptors in Sensory Cortex

ERIC Educational Resources Information Center

Metherate, Raju

2004-01-01

Acetylcholine release in sensory neocortex contributes to higher-order sensory function, in part by activating nicotinic acetylcholine receptors (nAChRs). Molecular studies have revealed a bewildering array of nAChR subtypes and cellular actions; however, there is some consensus emerging about the major nAChR subtypes and their functions in…

Important roles of P2Y receptors in the inflammation and cancer of digestive system.

PubMed

Wan, Han-Xing; Hu, Jian-Hong; Xie, Rei; Yang, Shi-Ming; Dong, Hui

2016-05-10

Purinergic signaling is important for many biological processes in humans. Purinoceptors P2Y are widely distributed in human digestive system and different subtypes of P2Y receptors mediate different physiological functions from metabolism, proliferation, differentiation to apoptosis etc. The P2Y receptors are essential in many gastrointestinal functions and also involve in the occurrence of some digestive diseases. Since different subtypes of P2Y receptors are present on the same cell of digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic functions on the same cell. Recently, growing lines of evidence strongly suggest the involvement of P2Y receptors in the pathogenesis of several digestive diseases. In this review, we will focus on their important roles in the development of digestive inflammation and cancer. We anticipate that as the special subtypes of P2Y receptors are studied in depth, specific modulators for them will have good potentials to become promising new drugs to treat human digestive diseases in the near future.

Insulin oversecretion in MSG-obese rats is related to alterations in cholinergic muscarinic receptor subtypes in pancreatic islets.

PubMed

Miranda, Rosiane A; Agostinho, Aryane R; Trevenzoli, Isis H; Barella, Luiz F; Franco, Claudinéia C S; Trombini, Amanda B; Malta, Ananda; Gravena, Clarice; Torrezan, Rosana; Mathias, Paulo C F; de Oliveira, Júlio C

2014-01-01

Impaired pancreatic beta cell function and insulin secretion/action are a link between obesity and type 2 diabetes, which are worldwide public health burdens. We aimed to characterize the muscarinic acetylcholine receptor (mAChR) M1-M4 subtypes in isolated pancreatic islets from pre-diabetic obese rats that had been treated neonatally with monosodium L-glutamate (MSG). At 90 days of age, both the MSG and the control groups underwent biometric and biochemical evaluation. Anti-muscarinic drugs were used to study mAChR function either in vivo or in vitro. The results demonstrated that atropine treatment reduced insulin secretion in the MSG-treated and control groups, whereas treatment with an M2mAChR-selective antagonist increased secretion. Moreover, the insulinostatic effect of an M3mAChR-selective antagonist was significantly higher in the MSG-treated group. M1mAChR and M3mAChR expression was increased in the MSG-obese group by 55% and 73%, respectively. In contrast, M2mAChR expression decreased by 25% in the MSG group, whereas M4mAChR expression was unchanged. Functional changes in and altered content of the mAChR (M1-M4) subtypes are pivotal to the demand for high pancreatic beta cell insulin secretion in MSG-obese rats, which is directly associated with vagal hyperactivity and peripheral insulin resistance. © 2014 S. Karger AG, Basel.

Structure-Function of α1-Adrenergic Receptors

PubMed Central

Perez, Dianne M.

2007-01-01

Summary The Easson-Stedman hypothesis provided the rationale for the first studies of drug design for the α1-adrenergic receptor. Through chemical modifications of the catecholamine core structure, the need was established for a protonated amine, a β-hydroxyl on a chiral center, and an aromatic ring with substitutions capable of hydrogen bonding. After the receptors were cloned and three α1-adrenergic receptor subtypes were discovered, drug design became focused on the analysis of receptor structure and new interactions were uncovered. It became clear that α1 and β-adrenergic receptors did not share stringent homology in the ligand-binding pocket but this difference has allowed for more selective drug design. Novel discoveries on allosterism and agonist trafficking may be used in the future design of therapeutics with fewer side effects. This review will explore past and current knowledge of the structure-function of the α1-adrenergic receptor subtypes. PMID:17052695

Localization of P2X receptor subtypes 2, 3 and 7 in human urinary bladder.

PubMed

Svennersten, Karl; Hallén-Grufman, Katarina; de Verdier, Petra J; Wiklund, N Peter; Poljakovic, Mirjana

2015-08-08

Voiding dysfunctions are a common problem that has a severe negative impact on the quality of life. Today there is a need for new drug targets for these conditions. The role of ATP receptors in bladder physiology has been studied for some time, primarily in animal models. The aim of this work is to investigate the localization of the ATP receptors P2X2, P2X3 and P2X7 and their colocalization with vimentin and actin in the human urinary bladder. Immunohistochemical analysis was conducted on full-thickness bladder tissues from fundus and trigonum collected from 15 patients undergoing open radical cystectomy due to chronic cystitis, bladder cancer or locally advanced prostate cancer. Colocalization analyses were performed between the three different P2X subtypes and the structural proteins vimentin and actin. Specimens were examined using epifluorescence microscopy and correlation coefficients were calculated for each costaining as well as the mean distance from the laminin positive basal side of the urothelium to the vimentin positive cells located in the suburothelium. P2X2 was expressed in vimentin positive cells located in the suburothelium. Less distinct labelling of P2X2 was also observed in actin positive smooth muscle cells and in the urothelium. P2X3 was expressed in vimentin positive cells surrounding the smooth muscle, and in vimentin positive cells located in the suburothelium. Weaker P2X3 labelling was seen in the urothelium. P2X7 was expressed in the smooth muscle cells and the urothelium. In the suburothelium, cells double positive for P2X2 and vimentin where located closer to the urothelium while cells double positive for P2X3 and vimentin where located further from the urothelium. The results from this study demonstrate that there is a significant difference in the expression of the purinergic P2X2, P2X3 and P2X7 receptors in the different histological layers of the human urinary bladder.

High prevalence of luminal B breast cancer intrinsic subtype in Colombian women.

PubMed

Serrano-Gomez, Silvia Juliana; Sanabria-Salas, Maria Carolina; Hernández-Suarez, Gustavo; García, Oscar; Silva, Camilo; Romero, Alejandro; Mejía, Juan Carlos; Miele, Lucio; Fejerman, Laura; Zabaleta, Jovanny

2016-07-01

Breast cancer is the most frequent malignancy in women worldwide. Distinct intrinsic subtypes of breast cancer have different prognoses, and their relative prevalence varies significantly among ethnic groups. Little is known about the prevalence of breast cancer intrinsic subtypes and their association with clinicopathological data and genetic ancestry in Latin Americans. Immunohistochemistry surrogates from the 2013 St. Gallen International Expert Consensus were used to classify breast cancers in 301 patients from Colombia into intrinsic subtypes. We analyzed the distribution of subtypes by clinicopathological variables. Genetic ancestry was estimated from a panel of 80 ancestry informative markers. Luminal B breast cancer subtype was the most prevalent in our population (37.2%) followed by luminal A (26.3%), non-basal triple negative (NBTN) (11.6%), basal like (9%), human epidermal growth factor receptor 2 (HER2) enriched (8.6%) and unknown (7.3%). We found statistical significant differences in distribution between Colombian region (P = 0.007), age at diagnosis (P = 0.0139), grade (P < 0.001) and recurrence (P < 0.001) according to intrinsic subtype. Patients diagnosed with HER2-enriched, basal-like and NBTN breast cancer had the highest African ancestry. Future studies analyzing the molecular profiles of breast cancer in Colombian women will help us understand the molecular basis of this subtype distribution and compare the molecular characteristics of the different intrinsic subtypes in Colombian patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Incidence and risk factors for breast cancer subtypes in three distinct South-East Asian ethnic groups: Chinese, Malay and natives of Sarawak, Malaysia.

PubMed

Devi, C R Beena; Tang, Tieng Swee; Corbex, Marilys

2012-12-15

We determined the incidences of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) subtypes among breast cancer cases in Sarawak, Malaysia and their correlation with various risk factors in the three ethnic groups: Chinese, Malay and native. Subtype status was ascertained for 1,034 cases of female breast cancer (93% of all cases diagnosed since 2003), and the age-standardized incidence rates (ASRs) of each subtype were inferred. Case-case comparisons across subtypes were performed for reproductive risk factors. We found 48% luminal A (ER+/PR+/HER2-), 29% triple-negative (ER-/PR-/HER2-), 12% triple-positive (ER+/PR+/HER2+) and 11% HER2-overexpressing (ER-/PR-/HER2+) subtypes, with ASRs of 10.6, 6.0, 2.8 and 2.8 per 100,000, respectively. The proportions of subtypes and ASRs differed significantly by ethnic groups: HER2-positive cases were more frequent in Malays (29%; 95% CI [23;35]) than Chinese (22%; [19;26] and natives (21%; [16;26]); triple-negative cases were less frequent among Chinese (23%; [20;27]) than Malays (33%; [27;39]) and natives (37%; [31;43]). The results of the case-case comparison were in accordance with those observed in western case series. Some uncommon associations, such as between triple-negative subtype and older age at menopause (OR, 1.59; p < 0.05), were found. The triple-negative and HER2+ subtypes predominate in our region, with significant differences among ethnic groups. Our results support the idea that the risk factors for different subtypes vary markedly. Westernized populations are more likely to have factors that increase the risk for the luminal A type, while risk factors for the triple-negative type are more frequent in local populations. Copyright © 2012 UICC.

Molecular basis for subtype-specificity and high-affinity zinc inhibition in the GluN1-GluN2A NMDA receptor amino terminal domain

PubMed Central

Romero-Hernandez, Annabel; Simorowski, Noriko; Karakas, Erkan

2016-01-01

Summary Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete due to lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc binding site and reveals distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability supporting the general model where the bi-lobe motion in ATD regulates the channel activity in NMDA receptors. PMID:27916457

Adrenergic receptors inhibit TRPV1 activity in the dorsal root ganglion neurons of rats.

PubMed

Matsushita, Yumi; Manabe, Miki; Kitamura, Naoki; Shibuya, Izumi

2018-01-01

Transient receptor potential vanilloid type 1 (TRPV1) is a polymodal receptor channel that responds to multiple types of stimuli, such as heat, acid, mechanical pressure and some vanilloids. Capsaicin is the most commonly used vanilloid to stimulate TRPV1. TRPV1 channels are expressed in dorsal root ganglion neurons that extend to Aδ- and C-fibers and have a role in the transduction of noxious inputs to the skin into the electrical signals of the sensory nerve. Although noradrenergic nervous systems, including the descending antinociceptive system and the sympathetic nervous system, are known to modulate pain sensation, the functional association between TRPV1 and noradrenaline in primary sensory neurons has rarely been examined. In the present study, we examined the effects of noradrenaline on capsaicin-evoked currents in cultured dorsal root ganglion neurons of the rat by the whole-cell voltage clamp method. Noradrenaline at concentrations higher than 0.1 pM significantly reduced the amplitudes of the inward capsaicin currents recorded at -60 mV holding potential. This inhibitory action was reversed by either yohimbine (an α2 antagonist, 10 nM) or propranolol (a β antagonist, 10 nM). The α2 agonists, clonidine (1 pM) and dexmedetomidine (1 pM) inhibited capsaicin currents, and yohimbine (1 nM) reversed the effects of clonidine. The inhibitory action of noradrenaline was not seen in the neurons pretreated with pertussis toxin (100 μg/ml for 24 h) and the neurons dialyzed intracellularly with guanosine 5'- [β-thio] diphosphate (GDPβS, 200 μM), the catalytic subunit of protein kinase A (250 U/ml) or okadaic acid (1 μM). These results suggest that noradrenaline directly acts on dorsal root ganglion neurons to inhibit the activity of TRPV1 depending on the activation of α2-adrenoceptors followed by the inhibition of the adenylate cyclase/cAMP/protein kinase A pathway.

Risk Differences Between Prediabetes And Diabetes According To Breast Cancer Molecular Subtypes.

PubMed

Crispo, A; Augustin, L S A; Grimaldi, M; Nocerino, F; Giudice, A; Cavalcanti, E; Di Bonito, M; Botti, G; De Laurentiis, M; Rinaldo, M; Esposito, E; Riccardi, G; Amore, A; Libra, M; Ciliberto, G; Jenkins, D J A; Montella, M

2017-05-01

Hyperglycemia and hyperinsulinemia may play a role in breast carcinogenesis and prediabetes and diabetes have been associated with increased breast cancer (BC) risk. However, whether BC molecular subtypes may modify these associations is less clear. We therefore investigated these associations in all cases and by BC molecular subtypes among women living in Southern Italy. Cases were 557 patients with non-metastatic incident BC and controls were 592 outpatients enrolled during the same period as cases and in the same hospital for skin-related non-malignant conditions. Adjusted multivariate logistic regression models were built to assess the risks of developing BC in the presence of prediabetes or diabetes. The analyses were repeated by strata of BC molecular subtypes: Luminal A, Luminal B, HER2+, and Triple Negative (TN). Prediabetes and diabetes were significantly associated with higher BC incidence after controlling for known risk factors (OR = 1.94, 95% CI 1.32-2.87 and OR = 2.46, 95% CI 1.38-4.37, respectively). Similar results were seen in Luminal A and B while in the TN subtype only prediabetes was associated with BC (OR = 2.43, 95% CI 1.11-5.32). Among HER2+ patients, only diabetes was significantly associated with BC risk (OR = 3.04, 95% CI 1.24-7.47). Furthermore, when postmenopausal HER2+ was split into hormone receptor positive versus negative, the association with diabetes remained significant only in the former (OR = 5.13, 95% CI 1.53-17.22). These results suggest that prediabetes and diabetes are strongly associated with BC incidence and that these metabolic conditions may be more relevant in the presence of breast cancer molecular subtypes with positive hormone receptors. J. Cell. Physiol. 232: 1144-1150, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Bladder pain induced by prolonged peripheral alpha 1A adrenoceptor stimulation involves the enhancement of transient receptor potential vanilloid 1 activity and an increase of urothelial adenosine triphosphate release.

PubMed

Matos, R; Cordeiro, J M; Coelho, A; Ferreira, S; Silva, C; Igawa, Y; Cruz, F; Charrua, A

2016-12-01

Pathophysiological mechanisms of chronic visceral pain (CVP) are unknown. This study explores the association between the sympathetic system and bladder nociceptors activity by testing the effect of a prolonged adrenergic stimulation on transient receptor potential vanilloid 1 (TRPV1) activity and on urothelial adenosine triphosphate (ATP) release. Female Wistar rats received saline, phenylephrine (PHE), PHE + silodosin, PHE + naftopidil or PHE + prazosin. TRPV1 knockout and wild-type mice received saline or PHE. Visceral pain behaviour tests were performed before and after treatment. Cystometry was performed, during saline and capsaicin infusion. Fos immunoreactivity was assessed in L6 spinal cord segment. Human urothelial ATP release induced by mechanical and thermal stimulation was evaluated. Subcutaneous, but not intrathecal, PHE administration induced pain, which was reversed by silodosin, a selective alpha 1A adrenoceptor antagonist, but not by naftopidil, a relatively selective antagonist for alpha 1D adrenoceptor. Silodosin also reversed PHE-induced bladder hyperactivity and L6 spinal cord Fos expression. Thus, in subsequent experiments, only silodosin was used. Wild-type, but not TRPV1 knockout, mice exhibited phenylephrine-induced pain. Capsaicin induced a greater increase in voiding contractions in PHE-treated rats than in control animals, and silodosin reversed this effect. When treated with PHE, ATP release from human urothelial cells was enhanced either by mechanical stimulation or by lowering the thermal threshold of urothelial TRPV1, which becomes abnormally responsive at body temperature. This study suggests that the activation of peripheral alpha 1A adrenoceptors induces CVP, probably through its interaction with TRPV1 and ATP release. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Regulation and Functional Implications of Opioid Receptor Splicing in Opioid Pharmacology and HIV Pathogenesis

PubMed Central

Regan, Patrick M.; Langford, T. Dianne; Khalili, Kamel

2015-01-01

Despite the identification and characterization of four opioid receptor subtypes and the genes from which they are encoded, pharmacological data does not conform to the predications of a four opioid receptor model. Instead, current studies of opioid pharmacology suggest the existence of additional receptor subtypes; however, no additional opioid receptor subtype has been identified to date. It is now understood that this discrepancy is due to the generation of multiple isoforms of opioid receptor subtypes. While several mechanisms are utilized to generate these isoforms, the primary mechanism involves alternative splicing of the pre-mRNA transcript. Extensive alternative splicing patterns for opioid receptors have since been identified and discrepancies in opioid pharmacology are now partially attributed to variable expression of these isoforms. Recent studies have been successful in characterizing the localization of these isoforms as well as their specificity in ligand binding; however, the regulation of opioid receptor splicing specificity is poorly characterized. Furthermore, the functional significance of individual receptor isoforms and the extent to which opioid- and/or HIV-mediated changes in the opioid receptor isoform profile contributes to altered opioid pharmacology or the well-known physiological role of opioids in the exacerbation of HIV neurocognitive dysfunction is unknown. As such, the current review details constitutive splicing mechanisms as well as the specific architecture of opioid receptor genes, transcripts, and receptors in order to highlight the current understanding of opioid receptor isoforms, potential mechanisms of their regulation and signaling, and their functional significance in both opioid pharmacology and HIV-associated neuropathology. PMID:26529364

Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx*

PubMed Central

Schindl, Rainer; Fritsch, Reinhard; Jardin, Isaac; Frischauf, Irene; Kahr, Heike; Muik, Martin; Riedl, Maria Christine; Groschner, Klaus; Romanin, Christoph

2012-01-01

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca2+ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca2+ and Na+ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca2+ influx in HEK293 cells. PMID:22932896

The Search for a Subtype-Selective PET Imaging Agent for the GABAA Receptor Complex: Evaluation of the Radiotracer [11C]ADO in Nonhuman Primates.

PubMed

Lin, Shu-Fei; Bois, Frederic; Holden, Daniel; Nabulsi, Nabeel; Pracitto, Richard; Gao, Hong; Kapinos, Michael; Teng, Jo-Ku; Shirali, Anupama; Ropchan, Jim; Carson, Richard E; Elmore, Charles S; Vasdev, Neil; Huang, Yiyun

2017-01-01

The myriad physiological functions of γ-amino butyric acid (GABA) are mediated by the GABA-benzodiazepine receptor complex comprising of the GABA A , GABA B , and GABA C groups. The various GABA A subunits with region-specific distributions in the brain subserve different functional and physiological roles. For example, the sedative and anticonvulsive effects of classical benzodiazepines are attributed to the α 1 subunit, and the α 2 and α 3 subunits mediate the anxiolytic effect. To optimize pharmacotherapies with improved efficacy and devoid of undesirable side effects for the treatment of anxiety disorders, subtype-selective imaging radiotracers are required to assess target engagement at GABA sites and determine the dose-receptor occupancy relationships. The goal of this work was to characterize, in nonhuman primates, the in vivo binding profile of a novel positron emission tomography (PET) radiotracer, [ 11 C]ADO, which has been indicated to have functional selectivity for the GABA A α 2 /α 3 subunits. High specific activity [ 11 C]ADO was administrated to 3 rhesus monkeys, and PET scans of 120-minute duration were performed on the Focus-220 scanner. In the blood, [ 11 C]ADO metabolized at a fairly rapid rate, with ∼36% of the parent tracer remaining at 30 minutes postinjection. Uptake levels of [ 11 C]ADO in the brain were high (peak standardized uptake value of ∼3.0) and consistent with GABA A distribution, with highest activity levels in cortical areas, intermediate levels in cerebellum and thalamus, and lowest uptake in striatal regions and amygdala. Tissue kinetics was fast, with peak uptake in all brain regions within 20 minutes of tracer injection. The one-tissue compartment model provided good fits to regional time-activity curves and reliable measurement of kinetic parameters. The absolute test-retest variability of regional distribution volumes ( V T ) was low, ranging from 4.5% to 8.7%. Pretreatment with flumazenil (a subtype nonselective

Resolution of Novel Pancreatic Ductal Adenocarcinoma Subtypes by Global Phosphotyrosine Profiling*

PubMed Central

Humphrey, Emily S.; Su, Shih-Ping; Nagrial, Adnan M.; Hochgräfe, Falko; Pajic, Marina; Lehrbach, Gillian M.; Parton, Robert G.; Yap, Alpha S.; Horvath, Lisa G.; Chang, David K.; Biankin, Andrew V.; Wu, Jianmin; Daly, Roger J.

2016-01-01

Comprehensive characterization of signaling in pancreatic ductal adenocarcinoma (PDAC) promises to enhance our understanding of the molecular aberrations driving this devastating disease, and may identify novel therapeutic targets as well as biomarkers that enable stratification of patients for optimal therapy. Here, we use immunoaffinity-coupled high-resolution mass spectrometry to characterize global tyrosine phosphorylation patterns across two large panels of human PDAC cell lines: the ATCC series (19 cell lines) and TKCC series (17 cell lines). This resulted in the identification and quantification of over 1800 class 1 tyrosine phosphorylation sites and the consistent segregation of both PDAC cell line series into three subtypes with distinct tyrosine phosphorylation profiles. Subtype-selective signaling networks were characterized by identification of subtype-enriched phosphosites together with pathway and network analyses. This revealed that the three subtypes characteristic of the ATCC series were associated with perturbations in signaling networks associated with cell-cell adhesion and epithelial-mesenchyme transition, mRNA metabolism, and receptor tyrosine kinase (RTK) signaling, respectively. Specifically, the third subtype exhibited enhanced tyrosine phosphorylation of multiple RTKs including the EGFR, ERBB3 and MET. Interestingly, a similar RTK-enriched subtype was identified in the TKCC series, and 'classifier' sites for each series identified using Random Forest models were able to predict the subtypes of the alternate series with high accuracy, highlighting the conservation of the three subtypes across the two series. Finally, RTK-enriched cell lines from both series exhibited enhanced sensitivity to the small molecule EGFR inhibitor erlotinib, indicating that their phosphosignature may provide a predictive biomarker for response to this targeted therapy. These studies highlight how resolution of subtype-selective signaling networks can provide a

Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response.

PubMed

Che, Pulin; Chen, Yunjia; Lu, Roujian; Peng, Ning; Gannon, Mary; Wyss, J Michael; Jiao, Kai; Wang, Qin

2015-01-01

The α2 adrenergic receptor (AR) subtypes are important for blood pressure control. When activated, the α2A subtype elicits a hypotensive response whereas the α2B subtype mediates a hypertensive effect that counteracts the hypotensive response by the α2A subtype. We have previously shown that spinophilin attenuates the α2AAR-dependent hypotensive response; in spinophilin null mice, this response is highly potentiated. In this study, we demonstrate that spinophilin impedes arrestin-dependent phosphorylation and desensitization of the α2BAR subtype by competing against arrestin binding to this receptor subtype. The Del301-303 α2BAR, a human variation that shows impaired phosphorylation and desensitization and is linked to hypertension in certain populations, exhibits preferential interaction with spinophilin over arrestin. Furthermore, Del301-303 α2BAR-induced ERK signaling is quickly desensitized in cells without spinophilin expression, showing a profile similar to that induced by the wild type receptor in these cells. Together, these data suggest a critical role of spinophilin in sustaining α2BAR signaling. Consistent with this notion, our in vivo study reveals that the α2BAR-elicited hypertensive response is diminished in spinophilin deficient mice. In arrestin 3 deficient mice, where the receptor has a stronger binding to spinophilin, the same hypertensive response is enhanced. These data suggest that interaction with spinophilin is indispensable for the α2BAR to elicit the hypertensive response. This is opposite of the negative role of spinophilin in regulating α2AAR-mediated hypotensive response, suggesting that spinophilin regulation of these closely related receptor subtypes can result in distinct functional outcomes in vivo. Thus, spinophilin may represent a useful therapeutic target for treatment of hypertension.

Therapeutic potential of metabotropic glutamate receptor modulators.

PubMed

Hovelsø, N; Sotty, F; Montezinho, L P; Pinheiro, P S; Herrik, K F; Mørk, A

2012-03-01

Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and is a major player in complex brain functions. Glutamatergic transmission is primarily mediated by ionotropic glutamate receptors, which include NMDA, AMPA and kainate receptors. However, glutamate exerts modulatory actions through a family of metabotropic G-protein-coupled glutamate receptors (mGluRs). Dysfunctions of glutamatergic neurotransmission have been implicated in the etiology of several diseases. Therefore, pharmacological modulation of ionotropic glutamate receptors has been widely investigated as a potential therapeutic strategy for the treatment of several disorders associated with glutamatergic dysfunction. However, blockade of ionotropic glutamate receptors might be accompanied by severe side effects due to their vital role in many important physiological functions. A different strategy aimed at pharmacologically interfering with mGluR function has recently gained interest. Many subtype selective agonists and antagonists have been identified and widely used in preclinical studies as an attempt to elucidate the role of specific mGluRs subtypes in glutamatergic transmission. These studies have allowed linkage between specific subtypes and various physiological functions and more importantly to pathological states. This article reviews the currently available knowledge regarding the therapeutic potential of targeting mGluRs in the treatment of several CNS disorders, including schizophrenia, addiction, major depressive disorder and anxiety, Fragile X Syndrome, Parkinson's disease, Alzheimer's disease and pain.

Therapeutic Potential of Metabotropic Glutamate Receptor Modulators

PubMed Central

Hovelsø, N; Sotty, F; Montezinho, L.P; Pinheiro, P.S; Herrik, K.F; Mørk, A

2012-01-01

Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and is a major player in complex brain functions. Glutamatergic transmission is primarily mediated by ionotropic glutamate receptors, which include NMDA, AMPA and kainate receptors. However, glutamate exerts modulatory actions through a family of metabotropic G-protein-coupled glutamate receptors (mGluRs). Dysfunctions of glutamatergic neurotransmission have been implicated in the etiology of several diseases. Therefore, pharmacological modulation of ionotropic glutamate receptors has been widely investigated as a potential therapeutic strategy for the treatment of several disorders associated with glutamatergic dysfunction. However, blockade of ionotropic glutamate receptors might be accompanied by severe side effects due to their vital role in many important physiological functions. A different strategy aimed at pharmacologically interfering with mGluR function has recently gained interest. Many subtype selective agonists and antagonists have been identified and widely used in preclinical studies as an attempt to elucidate the role of specific mGluRs subtypes in glutamatergic transmission. These studies have allowed linkage between specific subtypes and various physiological functions and more importantly to pathological states. This article reviews the currently available knowledge regarding the therapeutic potential of targeting mGluRs in the treatment of several CNS disorders, including schizophrenia, addiction, major depressive disorder and anxiety, Fragile X Syndrome, Parkinson’s disease, Alzheimer’s disease and pain. PMID:22942876

Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

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

Bahouth, S.W.; Malbon, C.C.

1987-05-01

Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in themore » control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.« less

Imaging features of breast cancers on digital breast tomosynthesis according to molecular subtype: association with breast cancer detection.

PubMed

Lee, Su Hyun; Chang, Jung Min; Shin, Sung Ui; Chu, A Jung; Yi, Ann; Cho, Nariya; Moon, Woo Kyung

2017-12-01

To evaluate imaging features of breast cancers on digital breast tomosynthesis (DBT) according to molecular subtype and to determine whether the molecular subtype affects breast cancer detection on DBT. This was an institutional review board--approved study with a waiver of informed consent. DBT findings of 288 invasive breast cancers were reviewed according to Breast Imaging Reporting and Data System lexicon. Detectability of breast cancer was quantified by the number of readers (0-3) who correctly detected the cancer in an independent blinded review. DBT features and the cancer detectability score according to molecular subtype were compared using Fisher's exact test and analysis of variance. Of 288 invasive cancers, 194 were hormone receptor (HR)-positive, 48 were human epidermal growth factor receptor 2 (HER2) positive and 46 were triple negative breast cancers. The most common DBT findings were irregular spiculated masses for HR-positive cancer, fine pleomorphic or linear branching calcifications for HER2 positive cancer and irregular masses with circumscribed margins for triple negative breast cancers (p < 0.001). Cancer detectability on DBT was not significantly different according to molecular subtype (p = 0.213) but rather affected by tumour size, breast density and presence of mass or calcifications. Breast cancers showed different imaging features according to molecular subtype; however, it did not affect the cancer detectability on DBT. Advances in knowledge: DBT showed characteristic imaging features of breast cancers according to molecular subtype. However, cancer detectability on DBT was not affected by molecular subtype of breast cancers.

Sigma receptors as potential therapeutic targets for neuroprotection.

PubMed

Nguyen, Linda; Kaushal, Nidhi; Robson, Matthew J; Matsumoto, Rae R

2014-11-15

Sigma receptors comprise a unique family of proteins that have been implicated in the pathophysiology and treatment of many central nervous system disorders, consistent with their high level of expression in the brain and spinal cord. Mounting evidence indicate that targeting sigma receptors may be particularly beneficial in a number of neurodegenerative conditions including Alzheimer׳s disease, Parkinson׳s disease, stroke, methamphetamine neurotoxicity, Huntington׳s disease, amyotrophic lateral sclerosis, and retinal degeneration. In this perspective, a brief overview is given on sigma receptors, followed by a focus on common mechanisms of neurodegeneration that appear amenable to modulation by sigma receptor ligands to convey neuroprotective effects and/or restorative functions. Within each of the major mechanisms discussed herein, the neuroprotective effects of sigma ligands are summarized, and when known, the specific sigma receptor subtype(s) involved are identified. Together, the literature suggests sigma receptors may provide a novel target for combatting neurodegenerative diseases through both neuronal and glial mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural basis for molecular recognition at serotonin receptors.

PubMed

Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D; Gao, Xiang; Zhou, X Edward; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L; Cherezov, Vadim; Stevens, Raymond C; Xu, H Eric

2013-05-03

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

Structure and dynamics of the M3 muscarinic acetylcholine receptor

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

Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.

2012-03-01

Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify themore » binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.« less

Quantification of Estrogen Receptor Expression in Normal Breast Tissue in Postmenopausal Women With Breast Cancer and Association With Tumor Subtypes.

PubMed

Gulbahce, H Evin; Blair, Cindy K; Sweeney, Carol; Salama, Mohamed E

2017-09-01

Estrogen exposure is important in the pathogenesis of breast cancer and is a contributing risk factor. In this study we quantified estrogen receptor (ER) alpha expression in normal breast epithelium (NBR) in women with breast cancer and correlated it with breast cancer subtypes. Tissue microarrays were constructed from 204 breast cancer patients for whom normal breast tissue away from tumor was available. Slides stained with ER were scanned and expression in normal terminal duct lobular epithelium was quantitated using computer-assisted image analysis. ER expression in normal terminal duct lobular epithelium of postmenopausal women with breast cancer was significantly associated with estrogen and triple (estrogen, progesterone receptors, and HER2) negative phenotypes. Also increased age at diagnosis was significantly associated with ER expression in NBR. ER positivity in normal epithelium did not vary by tumor size, lymph node status, tumor grade, or stage. On the basis of quantitative image analysis, we confirm that ER expression in NBR increases with age in women with breast cancer, and report for the first time, a significant association between ER expression in NBR with ER-negative and triple-negative cancers in postmenopausal women.

A family of photoswitchable NMDA receptors

PubMed Central

Berlin, Shai; Szobota, Stephanie; Reiner, Andreas; Carroll, Elizabeth C; Kienzler, Michael A; Guyon, Alice; Xiao, Tong; Trauner, Dirk; Isacoff, Ehud Y

2016-01-01

NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity. DOI: http://dx.doi.org/10.7554/eLife.12040.001 PMID:26929991

[Expression of epidermal growth factor receptor mutation specific antibodies in lung adenocarcinoma: evaluation of sensitivity, specificity and relationship to histologic subtypes].

PubMed

Lai, Y M; Feng, Q; Sun, Y; Wang, P; Shi, Y F; Zhao, M; Wu, Q; Li, X H

2016-09-08

To evaluate the expression of epidermal growth factor receptor (EGFR) mutation specific antibodies in invasive lung adenocarcinomas, and their sensitivity, specificity, as well as relationship to histological subtypes. Immunostaining with EGFR mutation-specific antibodies, del E746-A750 in exon 19 and L858R in exon 21, was performed in tissue microarrays of 884 cases of resection specimens to study the relationship between the immunophenotypes and morphologic subtypes. The sensitivity and specificity of the stains were compared with gene mutations detected by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR). Of the 884 cases, the expression of del E746-A750 in exon 19 was 3+ , 2+ , 1+ and 0 in 7 cases (0.79%), 38 cases (4.30%), 129 cases (14.59%) and 710 cases (80.32%), respectively. For L858R in exon 21, 3+ , 2+ , 1+ and 0 staining were seen in 82 cases (9.28%), 93 cases (10.52%), 82 cases (9.28%) and 627 cases (70.93%), respectively. For both antibodies, positive expression (1+ or more) was mainly observed in lepidic, acinar and papillary predominant subtypes, and rarely seen in solid subtype or invasive mucinous adenocarcinoma (P=0.014 and 0.016). If 1+ to 3+ expression was set as positive, the specificity of exon 19/exon 21 reached 98.59%/92.98%, while the sensitivity was relatively lower (62.86%/88.89%). If 2+ to 3+ expression was read as positive, the specificity and sensitivity were 99.30%/97.37% and 25.71%/74.60% for exon 19/exon 21. If only 3+ expression was considered positive, the specificity was 100.0% for both antibodies, with a low sensitivity (8.57% for exon 19 and 34.92% for exon 21). Of the 18 cases with E746-A750 del in exon 19 based on molecular detection, the sensitivity of immunohistochemistry for exon 19 was 88.89% if a positive cutoff value ≥1+ was used; in contrast, of the 8 cases harboring other deletions in exon 19, only two cases were positive as 1+ . Both the EGFR mutation specific antibodies del E746-A750 in

Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

PubMed Central

Gregory, Karen J.

2015-01-01

The metabotropic glutamate (mGlu) receptors are a group of eight family C G protein–coupled receptors that are expressed throughout the central nervous system (CNS) and periphery. Within the CNS the different subtypes are found in neurons, both pre- and/or postsynaptically, where they mediate modulatory roles and in glial cells. The mGlu receptor family provides attractive targets for numerous psychiatric and neurologic disorders, with the majority of discovery programs focused on targeting allosteric sites, with allosteric ligands now available for all mGlu receptor subtypes. However, the development of allosteric ligands remains challenging. Biased modulation, probe dependence, and molecular switches all contribute to the complex molecular pharmacology exhibited by mGlu receptor allosteric ligands. In recent years we have made significant progress in our understanding of this molecular complexity coupled with an increased understanding of the structural basis of mGlu allosteric modulation. PMID:25808929

Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons

PubMed Central

Pan, Y. Albert; Choy, Margaret; Prober, David A.; Schier, Alexander F.

2012-01-01

How neurons connect to form functional circuits is central to the understanding of the development and function of the nervous system. In the somatosensory system, perception of sensory stimuli to the head requires specific connections between trigeminal sensory neurons and their many target areas in the central nervous system. Different trigeminal subtypes have specialized functions and downstream circuits, but it has remained unclear how subtype-specific axonal projection patterns are formed. Using zebrafish as a model system, we followed the development of two trigeminal sensory neuron subtypes: one that expresses trpa1b, a nociceptive channel important for sensing environmental chemicals; and a distinct subtype labeled by an islet1 reporter (Isl1SS). We found that Trpa1b and Isl1SS neurons have overall similar axon trajectories but different branching morphologies and distributions of presynaptic sites. Compared with Trpa1b neurons, Isl1SS neurons display reduced branch growth and synaptogenesis at the hindbrain-spinal cord junction. The subtype-specific morphogenesis of Isl1SS neurons depends on the guidance receptor Robo2. robo2 is preferentially expressed in the Isl1SS subset and inhibits branch growth and synaptogenesis. In the absence of Robo2, Isl1SS afferents acquire many of the characteristics of Trpa1b afferents. These results reveal that subtype-specific activity of Robo2 regulates subcircuit morphogenesis in the trigeminal sensory system. PMID:22190641

Downregulation of Endothelial Transient Receptor Potential Vanilloid Type 4 Channel and Small-Conductance of Ca2+-Activated K+ Channels Underpins Impaired Endothelium-Dependent Hyperpolarization in Hypertension.

PubMed

Seki, Takunori; Goto, Kenichi; Kiyohara, Kanako; Kansui, Yasuo; Murakami, Noboru; Haga, Yoshie; Ohtsubo, Toshio; Matsumura, Kiyoshi; Kitazono, Takanari

2017-01-01

Endothelium-dependent hyperpolarization (EDH)-mediated responses are impaired in hypertension, but the underlying mechanisms have not yet been determined. The activation of small- and intermediate-conductance of Ca 2+ -activated K + channels (SK Ca and IK Ca ) underpins EDH-mediated responses. It was recently reported that Ca 2+ influx through endothelial transient receptor potential vanilloid type 4 channel (TRPV4) is a prerequisite for the activation of SK Ca /IK Ca in endothelial cells in specific beds. Here, we attempted to determine whether the impairment of EDH in hypertension is attributable to the dysfunction of TRPV4 and S/IK Ca , using isolated superior mesenteric arteries of 20-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wistar-Kyoto (WKY) rats. In the WKY arteries, EDH-mediated responses were reduced by a combination of SK Ca /IK Ca blockers (apamin plus TRAM-34; 1-[(2-chlorophenyl)diphenylmethl]-1H-pyrazole) and by the blockade of TRPV4 with the selective antagonist RN-1734 or HC-067047. In the SHRSP arteries, EDH-mediated hyperpolarization and relaxation were significantly impaired when compared with WKY. GSK1016790A, a selective TRPV4 activator, evoked robust hyperpolarization and relaxation in WKY arteries. In contrast, in SHRSP arteries, the GSK1016790A-evoked hyperpolarization was small and relaxation was absent. Hyperpolarization and relaxation to cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, a selective SK Ca activator, were marginally decreased in SHRSP arteries compared with WKY arteries. The expression of endothelial TRPV4 and SK Ca protein was significantly decreased in the SHRSP mesenteric arteries compared with those of WKY, whereas function and expression of IK Ca were preserved in SHRSP arteries. These findings suggest that EDH-mediated responses are impaired in superior mesenteric arteries of SHRSP because of a reduction in both TRPV4 and SK Ca input to EDH. © 2016 American

Serotonin Receptor Binding Characteristics of Geissoschizine Methyl Ether, an Indole Alkaloid in Uncaria Hook

PubMed Central

Ikarashi, Yasushi; Sekiguchi, Kyoji; Mizoguchi, Kazushige

2018-01-01

Background: Geissoschizine methyl ether (GM) is one of the indole alkaloids in Uncaria hook, and an active ingredient of yokukansan (YKS) that improves behavioral and psychological symp-toms of dementia (BPSD) in patients with several types of dementia. The pharmacological action of GM has been related to various serotonin (5-HT) receptor subtypes. Objective: The aim of this article is to review the binding characteristics of GM to the 5-HT receptor sub-types in the brains using our own data and previous findings. Methods: Competitive receptor-binding and agonist/antagonist activity assays for several 5-HT receptor subtypes were performed. Moreover, the articles describing pharmacokinetics and brain distribution of GM were searched in PubMed. Results: GM bound the following 5-HT receptor subtypes: 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT4, 5-HT5A, 5-HT6, and 5-HT7. Among these receptors, GM had partial agonistic activity for 5-HT1A receptors and antagonistic activity for 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT7 receptors. Also, GM was me-tabolized by various CYP isoforms, mainly CYP3A4. Parent/unchanged GM was detected in both the blood and brain of rats after oral administration of YKS. In the brains, GM was presumed to bind to 5-HT1A, 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT7 receptors on neuron-like large cells mainly in the frontal cor-tex. Conclusion: These results suggest that GM is a pharmacologically important alkaloid that regulates vari-ous serotonergic activities or functions by binding to multiple 5-HT receptor subtypes. Thus, this review provides recent 5-HT receptor-related evidence that GM is partly responsible for pharmacological effects of YKS. PMID:28322152

Synaptic muscarinic response types in hippocampal CA1 interneurons depend on different levels of presynaptic activity and different muscarinic receptor subtypes

PubMed Central

Bell, L. Andrew; Bell, Karen A.; McQuiston, A. Rory

2013-01-01

Depolarizing, hyperpolarizing and biphasic muscarinic responses have been described in hippocampal inhibitory interneurons, but the receptor subtypes and activity patterns required to synaptically activate muscarinic responses in interneurons have not been completely characterized. Using optogenetics combined with whole cell patch clamp recordings in acute slices, we measured muscarinic responses produced by endogenously released acetylcholine (ACh) from cholinergic medial septum/diagonal bands of Broca inputs in hippocampal CA1. We found that depolarizing responses required more cholinergic terminal stimulation than hyperpolarizing ones. Furthermore, elevating extracellular ACh with the acetylcholinesterase inhibitor physostigmine had a larger effect on depolarizing versus hyperpolarizing responses. Another subpopulation of interneurons responded biphasically, and periodic release of ACh entrained some of these interneurons to rhythmically burst. M4 receptors mediated hyperpolarizing responses by activating inwardly rectifying K+ channels, whereas the depolarizing responses were inhibited by the nonselective muscarinic antagonist atropine but were unaffected by M1, M4 or M5 receptor modulators. In addition, activation of M4 receptors significantly altered biphasic interneuron firing patterns. Anatomically, interneuron soma location appeared predictive of muscarinic response types but response types did not correlate with interneuron morphological subclasses. Together these observations suggest that the hippocampal CA1 interneuron network will be differentially affected by cholinergic input activity levels. Low levels of cholinergic activity will preferentially suppress some interneurons via hyperpolarization and increased activity will recruit other interneurons to depolarize, possibly because of elevated extracellular ACh concentrations. These data provide important information for understanding how cholinergic therapies will affect hippocampal network function

Cerebral Artery Alpha-1 AR Subtypes: High Altitude Long-Term Acclimatization Responses

PubMed Central

Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D.

2014-01-01

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10−5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function. PMID:25393740

Cerebral artery alpha-1 AR subtypes: high altitude long-term acclimatization responses.

PubMed

Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D

2014-01-01

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10-5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function.

Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

PubMed

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria Angel; Cilleros, Victor; Tomàs, Josep Maria

2016-01-01

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes. �

Characterization of the functional activity of botulinum neurotoxin subtype B6.

PubMed

Kohda, Tomoko; Nakamura, Keiji; Hosomi, Koji; Torii, Yasushi; Kozaki, Shunji; Mukamoto, Masafumi

2017-11-01

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A-G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C-terminal heavy chain (H C ) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously-characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. H C /B1 and H C /B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its H c .domain. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Lorcaserin and pimavanserin: emerging selectivity of serotonin receptor subtype–targeted drugs

PubMed Central

Meltzer, Herbert Y.; Roth, Bryan L.

2013-01-01

Serotonin (5-hydroxytryptamine, or 5-HT) receptors mediate a plethora of physiological phenomena in the brain and the periphery. Additionally, serotonergic dysfunction has been implicated in nearly every neuropsychiatric disorder. The effects of serotonin are mediated by fourteen GPCRs. Both the therapeutic actions and side effects of commonly prescribed drugs are frequently due to nonspecific actions on various 5-HT receptor subtypes. For more than 20 years, the search for clinically efficacious drugs that selectively target 5-HT receptor subtypes has been only occasionally successful. This review provides an overview of 5-HT receptor pharmacology and discusses two recent 5-HT receptor subtype–selective drugs, lorcaserin and pimavanserin, which target the 5HT2C and 5HT2A receptors and provide new treatments for obesity and Parkinson’s disease psychosis, respectively. PMID:24292660

Purinergic receptor immunoreactivity in the rostral ventromedial medulla.

PubMed

Close, L N; Cetas, J S; Heinricher, M M; Selden, N R

2009-01-23

The rostral ventromedial medulla (RVM) has long been recognized to play a pivotal role in nociceptive modulation. Pro-nociception within the RVM is associated with a distinct functional class of neurons, ON-cells that begin to discharge immediately before nocifensive reflexes. Anti-nociceptive function within the RVM, including the analgesic response to opiates, is associated with another distinct class, OFF-cells, which pause immediately prior to nocifensive reflexes. A third class of RVM neurons, NEUTRAL-cells, does not alter firing in association with nocifensive reflexes. ON-, OFF- and NEUTRAL-cells show differential responsiveness to various behaviorally relevant neuromodulators, including purinergic ligands. Iontophoresis of semi-selective P2X ligands, which are associated with nociceptive transmission in the spinal cord and dorsal root ganglia, preferentially activate ON-cells. By contrast, P2Y ligands activate OFF-cells and P1 ligands suppress the firing of NEUTRAL cells. The current study investigates the distribution of P2X, P2Y and P1 receptor immunoreactivity in RVM neurons of Sprague-Dawley rats. Co-localization with tryptophan hydroxylase (TPH), a well-established marker for serotonergic neurons was also studied. Immunoreactivity for the four purinergic receptor subtypes examined was abundant in all anatomical subdivisions of the RVM. By contrast, TPH-immunoreactivity was restricted to a relatively small subset of RVM neurons concentrated in the nucleus raphe magnus and pallidus, as expected. There was a significant degree of co-localization of each purinergic receptor subtype with TPH-immunoreactivity. This co-localization was most pronounced for P2Y1 receptor immunoreactivity, although this was the least abundant among the different purinergic receptor subtypes examined. Immunoreactivity for multiple purinergic receptor subtypes was often co-localized in single neurons. These results confirm the physiological finding that purinergic receptors are

Hippocampal 5-HT1A Receptor and Spatial Learning and Memory

PubMed Central

Glikmann-Johnston, Yifat; Saling, Michael M.; Reutens, David C.; Stout, Julie C.

2015-01-01

Spatial cognition is fundamental for survival in the topographically complex environments inhabited by humans and other animals. The hippocampus, which has a central role in spatial cognition, is characterized by high concentration of serotonin (5-hydroxytryptamine; 5-HT) receptor binding sites, particularly of the 1A receptor (5-HT1A) subtype. This review highlights converging evidence for the role of hippocampal 5-HT1A receptors in spatial learning and memory. We consider studies showing that activation or blockade of the 5-HT1A receptors using agonists or antagonists, respectively, lead to changes in spatial learning and memory. For example, pharmacological manipulation to induce 5-HT release, or to block 5-HT uptake, have indicated that increased extracellular 5-HT concentrations maintain or improve memory performance. In contrast, reduced levels of 5-HT have been shown to impair spatial memory. Furthermore, the lack of 5-HT1A receptor subtype in single gene knockout mice is specifically associated with spatial memory impairments. These findings, along with evidence from recent cognitive imaging studies using positron emission tomography (PET) with 5-HT1A receptor ligands, and studies of individual genetic variance in 5-HT1A receptor availability, strongly suggests that 5-HT, mediated by the 5-HT1A receptor subtype, plays a key role in spatial learning and memory. PMID:26696889

Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications.

PubMed

Lambrecht, G

2000-11-01

P2X receptors are nucleotide-gated cation channels composed of homomeric or heteromeric assemblies of three subunits. In the past 7 years, an extended series (P2X1-7) of P2X subunits has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2X receptor subtypes to the diverse physiological responses mediated by the native P2X receptors. However, the paucity of useful ligands, especially subtype-selective agonists and antagonists as well as radioligands, acts as a considerable impediment to progress. Most of the ligands available are highly limited in terms of their kinetics of action, receptor-affinity, subtype-selectivity and P2X receptor-specificity. Their suspected ability to be a substrate for ecto-nucleotidases or to inhibit these enzymes also complicates their use. A number of new antagonists at P2X receptors have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl 2',4'-disulfonate (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2X receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2X receptors. It then focuses on the pharmacological properties of a series of key P2 receptor agonists and antagonists and will finish with the discussion of some related therapeutic possibilities.

Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor) in experimental autoimmune encephalomyelitis models of multiple sclerosis.

PubMed

Sisay, Sofia; Pryce, Gareth; Jackson, Samuel J; Tanner, Carolyn; Ross, Ruth A; Michael, Gregory J; Selwood, David L; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some

Effects of volatile solvents on recombinant N-methyl-D-aspartate receptors expressed in Xenopus oocytes

PubMed Central

Cruz, Silvia L; Balster, Robert L; Woodward, John J

2000-01-01

We have previously shown that toluene dose-dependently inhibits recombinant N-methyl-D-aspartate (NMDA) receptors at micromolar concentrations. This inhibition was rapid, almost complete and reversible. The NR1/2B combination was the most sensitive receptor subtype tested with an IC50 value for toluene of 0.17 mM. We now report on the effects of other commonly abused solvents (benzene, m-xylene, ethylbenzene, propylbenzene, 1,1,1-trichlorethane (TCE) and those of a convulsive solvent, 2,2,2-trifluoroethyl ether (flurothyl), on NMDA-induced currents measured in Xenopus oocytes expressing NR1/2A or NR1/2B receptor subtypes. All of the alkylbenzenes and TCE produced a reversible inhibition of NMDA-induced currents that was dose- and subunit-dependent. The NR1/2B receptor subtype was several times more sensitive to these compounds than the NR1/2A subtype. The convulsant solvent flurothyl had no effect on NMDA responses in oocytes but potently inhibited ion flux through recombinant GABA receptors expressed in oocytes. Overall, these results suggest that abused solvents display pharmacological selectivity and that NR1/2B NMDA receptors may be an important target for the actions of these compounds on the brain. PMID:11090101

Species differences in the localization and number of CNS beta adrenergic receptors: Rat versus guinea pig

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

Booze, R.M.; Crisostomo, E.A.; Davis, J.N.

1989-06-01

The localization and number of beta adrenergic receptors were directly compared in the brains of rats and guinea pigs. The time course of association and saturability of (125I)cyanopindolol (CYP) binding to slide-mounted tissue sections was similar in rats (Kd = 17 pM) and guinea pigs (Kd = 20 pM). The beta-1 and beta-2 receptor subtypes were examined through the use of highly selective unlabeled receptor antagonists, ICI 118,551 (50 nM) and ICI 89,406 (70 nM). Dramatic species differences between rats and guinea pigs were observed in the neuroanatomical regional localization of the beta adrenergic receptor subtypes. For example, in themore » thalamus prominent beta-1 and beta-2 receptor populations were identified in the rat; however, the entire thalamus of the guinea pig had few, if any, beta adrenergic receptors of either subtype. Hippocampal area CA1 had high levels of beta-2 adrenergic receptors in both rats and guinea pigs but was accompanied by a widespread distribution of beta-2 adrenergic receptors only in rats. Quantitative autoradiographic analyses of 25 selected neuroanatomical regions (1) confirmed the qualitative differences in CNS beta adrenergic receptor localization, (2) determined that guinea pigs had significantly lower levels of beta adrenergic receptors than rats and (3) indicated a differential pattern of receptor subtypes between the two species. Knowledge of species differences in receptor patterns may be useful in designing effective experiments as well as in exploring the relationships between receptor and innervation patterns. Collectively, these data suggest caution be used in extrapolation of the relationships of neurotransmitters and receptors from studies of a single species.« less

Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor

PubMed Central

Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jurgen; Kobilka, Brian

2012-01-01

Acetylcholine (ACh), the first neurotransmitter to be identified1, exerts many of its physiological actions via activation of a family of G protein-coupled receptors (GPCRs) known as muscarinic ACh receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G protein coupling preference and the physiological responses they mediate.2–4 Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences.5–6 We describe here the structure of the Gq/11-coupled M3 mAChR bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor, offers new possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows the first structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and raise additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer new insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors. PMID:22358844

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

A network-based, integrative study to identify core biological pathways that drive breast cancer clinical subtypes

PubMed Central

Dutta, B; Pusztai, L; Qi, Y; André, F; Lazar, V; Bianchini, G; Ueno, N; Agarwal, R; Wang, B; Shiang, C Y; Hortobagyi, G N; Mills, G B; Symmans, W F; Balázsi, G

2012-01-01

Background: The rapid collection of diverse genome-scale data raises the urgent need to integrate and utilise these resources for biological discovery or biomedical applications. For example, diverse transcriptomic and gene copy number variation data are currently collected for various cancers, but relatively few current methods are capable to utilise the emerging information. Methods: We developed and tested a data-integration method to identify gene networks that drive the biology of breast cancer clinical subtypes. The method simultaneously overlays gene expression and gene copy number data on protein–protein interaction, transcriptional-regulatory and signalling networks by identifying coincident genomic and transcriptional disturbances in local network neighborhoods. Results: We identified distinct driver-networks for each of the three common clinical breast cancer subtypes: oestrogen receptor (ER)+, human epidermal growth factor receptor 2 (HER2)+, and triple receptor-negative breast cancers (TNBC) from patient and cell line data sets. Driver-networks inferred from independent datasets were significantly reproducible. We also confirmed the functional relevance of a subset of randomly selected driver-network members for TNBC in gene knockdown experiments in vitro. We found that TNBC driver-network members genes have increased functional specificity to TNBC cell lines and higher functional sensitivity compared with genes selected by differential expression alone. Conclusion: Clinical subtype-specific driver-networks identified through data integration are reproducible and functionally important. PMID:22343619

Serotonin Receptors in Hippocampus

PubMed Central

Berumen, Laura Cristina; Rodríguez, Angelina; Miledi, Ricardo; García-Alcocer, Guadalupe

2012-01-01

Serotonin is an ancient molecular signal and a recognized neurotransmitter brainwide distributed with particular presence in hippocampus. Almost all serotonin receptor subtypes are expressed in hippocampus, which implicates an intricate modulating system, considering that they can be localized as autosynaptic, presynaptic, and postsynaptic receptors, even colocalized within the same cell and being target of homo- and heterodimerization. Neurons and glia, including immune cells, integrate a functional network that uses several serotonin receptors to regulate their roles in this particular part of the limbic system. PMID:22629209

Distinct breast cancer subtypes in women with early-onset disease across races

PubMed Central

Singh, Mandeep; Ding, Yi; Zhang, Li-Ying; Song, Dong; Gong, Yun; Adams, Sylvia; Ross, Dara S; Wang, Jin-Hua; Grover, Shruti; Doval, Dinesh Chandra; Shao, Charles; He, Zi-Li; Chang, Victor; Chin, Warren W; Deng, Fang-Ming; Singh, Baljit; Zhang, David; Xu, Ru-Liang; Lee, Peng

2014-01-01

Background: Racial disparities among breast cancer (BCa) patients are known but not well studied in early-onset BCa. We analyzed molecular subtypes in early-onset BCa across five major races. Methods: A total of 2120 cases were included from non-Hispanic White (NHW), African American (AA) and Hispanic, Chinese and Indian. Based on ER, PR and HER-2 status, BCa was classified into 4 intrinsic subtypes as Luminal A, Luminal B, HER2/neu overexpression and Triple negative BCa (TNBC) subtypes. Data was stratified according to race and age as younger/early-onset group (40-years and younger) and older group (50-years and older). Results: In early-onset BCa, incidence of TNBC was significantly higher (p = 0.0369) in Indian women followed by AA, Hispanic, NHW and Chinese women. Incidence of Her2 over-expression subtype also was highest in Indian women, followed by Hispanic, Chinese, AA and NHW women. In contrast, Luminal B subtype was most significantly higher in AA women (p = 0.0000) followed by NHW (p = 0.0002), Chinese (p = 0.0003), Hispanic (0.0128) and Indian (p = 0.0468) women. Luminal A subtype was most significantly reduced in Indian women (p = 0.0113) followed by Hispanic, AA, NHW and Chinese women. These results were based on statistical analysis with the mean of older group populations. Conclusions: These results show significant disparities in receptor subtypes across races. This study will contribute in developing optimal clinical trial protocols and personalized management strategies for early-onset BCa patients. PMID:25057437

Structure and receptor binding preferences of recombinant hemagglutinins from avian and human H6 and H10 influenza A virus subtypes.

PubMed

Yang, Hua; Carney, Paul J; Chang, Jessie C; Villanueva, Julie M; Stevens, James

2015-04-01

During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections. Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Regulation of body temperature and brown adipose tissue thermogenesis by bombesin receptor subtype-3

PubMed Central

Lateef, Dalya M.; Abreu-Vieira, Gustavo; Xiao, Cuiying

2014-01-01

Bombesin receptor subtype-3 (BRS-3) regulates energy homeostasis, with Brs3 knockout (Brs3−/y) mice being hypometabolic, hypothermic, and hyperphagic and developing obesity. We now report that the reduced body temperature is more readily detected if body temperature is analyzed as a function of physical activity level and light/dark phase. Physical activity level correlated best with body temperature 4 min later. The Brs3−/y metabolic phenotype is not due to intrinsically impaired brown adipose tissue function or in the communication of sympathetic signals from the brain to brown adipose tissue, since Brs3−/y mice have intact thermogenic responses to stress, acute cold exposure, and β3-adrenergic activation, and Brs3−/y mice prefer a cooler environment. Treatment with the BRS-3 agonist MK-5046 increased brown adipose tissue temperature and body temperature in wild-type but not Brs3−/y mice. Intrahypothalamic infusion of MK-5046 increased body temperature. These data indicate that the BRS-3 regulation of body temperature is via a central mechanism, upstream of sympathetic efferents. The reduced body temperature in Brs3−/y mice is due to altered regulation of energy homeostasis affecting higher center regulation of body temperature, rather than an intrinsic defect in brown adipose tissue. PMID:24452453

Basal Subtype of Invasive Breast Cancer Is Associated With a Higher Risk of True Recurrence After Conventional Breast-Conserving Therapy

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

Hattangadi-Gluth, Jona A.; Wo, Jennifer Y.; Nguyen, Paul L.

2012-03-01

Purpose: To determine whether breast cancer subtype is associated with patterns of ipsilateral breast tumor recurrence (IBTR), either true recurrence (TR) or elsewhere local recurrence (ELR), among women with pT1-T2 invasive breast cancer (IBC) who receive breast-conserving therapy (BCT). Methods and Materials: From Jan 1998 to Dec 2003, 1,223 women with pT1-T2N0-3 IBC were treated with BCT (lumpectomy plus whole-breast radiation). Ninety percent of patients received adjuvant systemic therapy, but none received trastuzumab. Biologic cancer subtypes were approximated by determining estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), and human epidermal growth factor receptor-2-positive (HER-2+) expression, classified as luminal A (ER+ ormore » PR+ and HER-2 negative [HER-2-]), luminal B (ER+ or PR+ and HER-2+), HER-2 (ER- and PR- and HER-2+), and basal (ER- and PR- and HER-2- ) subtypes. Imaging, pathology, and operative reports were reviewed by two physicians independently, including an attending breast radiologist. Readers were blinded to subtype and outcome. TR was defined as IBTR within the same quadrant and within 3 cm of the primary tumor. All others were defined as ELR. Results: At a median follow-up of 70 months, 24 patients developed IBTR (5-year cumulative incidence of 1.6%), including 15 TR and 9 ELR patients. At 5 years, basal (4.4%) and HER-2 (9%) subtypes had a significantly higher incidence of TR than luminal B (1.2%) and luminal A (0.2%) subtypes (p < 0.0001). On multivariate analysis, basal subtype (hazard ratio [HR], 4.8, p = 0.01), younger age at diagnosis (HR, 0.97; p = 0.05), and increasing tumor size (HR, 2.1; p = 0.04) were independent predictors of TR. Only younger age (HR, 0.95; p = 0.01) significantly predicted for ELR. Conclusions: Basal and HER-2 subtypes are significantly associated with higher rates of TR among women with pT1-T2 IBC after BCT. Younger age predicts for both TR and ELR. Strategies to reduce TR in

Characterization of kinin receptors by bioassays.

PubMed

Gobeil, F; Regoli, D

1994-08-01

1. Using the classical pharmacological criteria recommended by Schild, namely the order of potency of selective agonists (e.g., bradykinin, desArg9-bradykinin, [Hyp3]BK and [Aib7]BK) and the apparent affinity of competitive antagonists (e.g., DArg[Hyp3,DPhe7,Leu8]BK and WIN 64338), we have attempted to characterize B2 receptor subtypes. It has been shown that vascular tissues (e.g., dog carotid and renal arteries, rabbit jugular vein and rabbit aorta) are very sensitive to kinin agonists and antagonists (pD2 and pA2 values for BK and HOE 140 on B2 receptors are 8.5-10.1 and 9.2-9.4, respectively, and for desArg9BK and desArg9[Leu8]BK on B1 receptors they are 7.3-8.6 and 7.3-7.8, respectively). Mechanisms of action of kinins differ between pharmacological preparations. Kinin may act directly on the smooth muscle (e.g., rabbit jugular vein and rabbit aorta) as well as indirectly through other endogenous mediators such as nitric oxide (EDRF) (e.g., dog carotid and renal arteries) and prostaglandins (e.g., dog renal artery). 2. Pharmacological analysis of rabbit jugular vein (RJV) and guinea pig ileum (GPI) has revealed different sensitivities to certain synthetic analogs of BK and to competitive B2 receptor antagonists between the two tissues. 3. Agonist order of potency ([Hyp3]BK > BK > [Aib7]BK) obtained for RJV differed from that obtained for GPI (BK > or = [Aib7]BK > [Hyp3]BK). Competitive antagonists such as DArg[Hyp3, DPhe7, Leu8]BK and WIN 64338 discriminate in favor of B2A (RJV) and B2B (GPI) receptor subtypes, respectively. These data demonstrate the existence of B2 receptor subtypes. Correlation between data obtained in the present study and those reported for binding to the human B2 receptor support the view that the human receptor is similar to that of the rabbit.

Integrating TRPV1 Receptor Function with Capsaicin Psychophysics

PubMed Central

Smutzer, Gregory; Devassy, Roni K.

2016-01-01

Capsaicin is a naturally occurring vanilloid that causes a hot, pungent sensation in the human oral cavity. This trigeminal stimulus activates TRPV1 receptors and stimulates an influx of cations into sensory cells. TRPV1 receptors function as homotetramers that also respond to heat, proinflammatory substances, lipoxygenase products, resiniferatoxin, endocannabinoids, protons, and peptide toxins. Kinase-mediated phosphorylation of TRPV1 leads to increased sensitivity to both chemical and thermal stimuli. In contrast, desensitization occurs via a calcium-dependent mechanism that results in receptor dephosphorylation. Human psychophysical studies have shown that capsaicin is detected at nanomole amounts and causes desensitization in the oral cavity. Psychophysical studies further indicate that desensitization can be temporarily reversed in the oral cavity if stimulation with capsaicin is resumed at short interstimulus intervals. Pretreatment of lingual epithelium with capsaicin modulates the perception of several primary taste qualities. Also, sweet taste stimuli may decrease the intensity of capsaicin perception in the oral cavity. In addition, capsaicin perception and hedonic responses may be modified by diet. Psychophysical studies with capsaicin are consistent with recent findings that have identified TRPV1 channel modulation by phosphorylation and interactions with membrane inositol phospholipids. Future studies will further clarify the importance of capsaicin and its receptor in human health and nutrition. PMID:26884754

Functional characterization of estrogen receptor subtypes, ER{alpha} and ER{beta}, mediating vitellogenin production in the liver of rainbow trout

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

Leanos-Castaneda, Olga; Kraak, Glen van der

2007-10-15

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ER{alpha} and ER{beta}, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ER{alpha} selective agonist, methyl-piperidino-pyrazole (MPP) an ER{alpha} selective antagonist, and diarylpropionitrile (DPN) an ER{beta} selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbowmore » trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [{sup 3}H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ER{alpha} could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ER{beta}. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ER{alpha}. On the other hand, once blocked ER{alpha} with MPP, the only manifestation of agonist activity of estradiol would be achieved via ER{beta}. In conclusion, the present results indicate that vitellogenin

Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP(8-37).

PubMed

Kawase, Tomoyuki; Okuda, Kazuhiro; Burns, Douglas M

2005-10-01

Calcitonin gene-related peptide (CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRPR) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRPR expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classic subtype 1 receptor (CGRPR1). The aim of the present study was to further characterize the MG63 CGRPR using a selective agonist of the putative CGRPR2, [Cys(Acm)(2,7)]CGRP, and a relatively specific antagonist of CGRPR1, CGRP(8-37). [Cys(Acm)(2,7)]CGRP acted as a significant agonist only upon ERK dephosphorylation, whereas this analog effectively antagonized CGRP-induced cAMP production and phosphorylation of cAMP response element-binding protein (CREB) and p38 MAPK. Although it had no agonistic action when used alone, CGRP(8-37) potently blocked CGRP actions on cAMP, CREB, and p38 MAPK but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA2 value for ERK is clearly distinguishable from those of the other three plots as judged using the 95% confidence intervals. Additional assays using 3-isobutyl-1-methylxanthine or the PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89) indicated that the cAMP-dependent pathway was predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38 MAPK phosphorylation. Considering previous data from Scatchard analysis of [125I]CGRP binding in connection with these results, these findings suggest that MG63 cells possess two functionally distinct CGRPR subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogs: one is best characterized as a variation of CGRPR1, and the second may be a novel variant of CGRPR2.

Genetic overlap between diagnostic subtypes of ischemic stroke.

PubMed

Holliday, Elizabeth G; Traylor, Matthew; Malik, Rainer; Bevan, Steve; Falcone, Guido; Hopewell, Jemma C; Cheng, Yu-Ching; Cotlarciuc, Ioana; Bis, Joshua C; Boerwinkle, Eric; Boncoraglio, Giorgio B; Clarke, Robert; Cole, John W; Fornage, Myriam; Furie, Karen L; Ikram, M Arfan; Jannes, Jim; Kittner, Steven J; Lincz, Lisa F; Maguire, Jane M; Meschia, James F; Mosley, Thomas H; Nalls, Mike A; Oldmeadow, Christopher; Parati, Eugenio A; Psaty, Bruce M; Rothwell, Peter M; Seshadri, Sudha; Scott, Rodney J; Sharma, Pankaj; Sudlow, Cathie; Wiggins, Kerri L; Worrall, Bradford B; Rosand, Jonathan; Mitchell, Braxton D; Dichgans, Martin; Markus, Hugh S; Levi, Christopher; Attia, John; Wray, Naomi R

2015-03-01

Despite moderate heritability, the phenotypic heterogeneity of ischemic stroke has hampered gene discovery, motivating analyses of diagnostic subtypes with reduced sample sizes. We assessed evidence for a shared genetic basis among the 3 major subtypes: large artery atherosclerosis (LAA), cardioembolism, and small vessel disease (SVD), to inform potential cross-subtype analyses. Analyses used genome-wide summary data for 12 389 ischemic stroke cases (including 2167 LAA, 2405 cardioembolism, and 1854 SVD) and 62 004 controls from the Metastroke consortium. For 4561 cases and 7094 controls, individual-level genotype data were also available. Genetic correlations between subtypes were estimated using linear mixed models and polygenic profile scores. Meta-analysis of a combined LAA-SVD phenotype (4021 cases and 51 976 controls) was performed to identify shared risk alleles. High genetic correlation was identified between LAA and SVD using linear mixed models (rg=0.96, SE=0.47, P=9×10(-4)) and profile scores (rg=0.72; 95% confidence interval, 0.52-0.93). Between LAA and cardioembolism and SVD and cardioembolism, correlation was moderate using linear mixed models but not significantly different from zero for profile scoring. Joint meta-analysis of LAA and SVD identified strong association (P=1×10(-7)) for single nucleotide polymorphisms near the opioid receptor μ1 (OPRM1) gene. Our results suggest that LAA and SVD, which have been hitherto treated as genetically distinct, may share a substantial genetic component. Combined analyses of LAA and SVD may increase power to identify small-effect alleles influencing shared pathophysiological processes. © 2015 American Heart Association, Inc.

[Functional selectivity of opioid receptors ligands].

PubMed

Audet, Nicolas; Archer-Lahlou, Elodie; Richard-Lalonde, Mélissa; Piñeyro-Filpo, Graciela

2010-01-01

Opiates are the most effective analgesics available for the treatment of severe pain. However, their clinical use is restricted by unwanted side effects such as tolerance, physical dependence and respiratory depression. The strategy to develop new opiates with reduced side effects has mainly focused on the study and production of ligands that specifically bind to different opiate receptors subtypes. However, this strategy has not allowed the production of novel therapeutic ligands with a better side effects profile. Thus, other research strategies need to be explored. One which is receiving increasing attention is the possibility of exploiting ligand ability to stabilize different receptor conformations with distinct signalling profiles. This newly described property, termed functional selectivity, provides a potential means of directing the stimulus generated by an activated receptor towards a specific cellular response. Here we summarize evidence supporting the existence of ligand-specific active conformations for two opioid receptors subtypes (delta and mu), and analyze how functional selectivity may contribute in the production of longer lasting, better tolerated opiate analgesics. double dagger.

Purinergic P2X receptors: structural models and analysis of ligand-target interaction.

PubMed

Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Marucci, Gabriella; Thomas, Ajiroghene; Volpini, Rosaria

2015-01-07

The purinergic P2X receptors are ligand-gated cation channels activated by the endogenous ligand ATP. They assemble as homo- or heterotrimers from seven cloned subtypes (P2X1-7) and all trimer subunits present a common topology consisting in intracellular N- and C- termini, two transmembrane domains and a large extracellular domain. These membrane proteins are present in virtually all mammalian tissues and regulate a large variety of responses in physio- and pathological conditions. The development of ligands that selectively activate or block specific P2X receptor subtypes hence represents a promising strategy to obtain novel pharmacological tools for the treatment of pain, cancer, inflammation, and neurological, cardiovascular, and endocrine diseases. The publication of the crystal structures of zebrafish P2X4 receptor in inactive and ATP-bound active forms provided structural data for the analysis of the receptor structure, the interpretation of mutagenesis data, and the depiction of ligand binding and receptor activation mechanism. In addition, the availability of ATP-competitive ligands presenting selectivity for P2X receptor subtypes supports the design of new potent and selective ligands with possibly improved pharmacokinetic profiles, with the final aim to obtain new drugs. This study describes molecular modelling studies performed to develop structural models of the human and rat P2X receptors in inactive and active states. These models allowed to analyse the role of some non-conserved residues at ATP binding site and to study the receptor interaction with some non-specific or subtype selective agonists and antagonists. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Low-level ultrahigh-frequency and ultrashort-pulse blue laser irradiation enhances osteoblast extracellular calcification by upregulating proliferation and differentiation via transient receptor potential vanilloid 1.

PubMed

Mikami, Risako; Mizutani, Koji; Aoki, Akira; Tamura, Yukihiko; Aoki, Kazuhiro; Izumi, Yuichi

2018-04-01

Low-level laser irradiation (LLLI) exerts various biostimulative effects, including promotion of wound healing and bone formation; however, few studies have examined biostimulation using blue lasers. The purpose of this study was to investigate the effects of low-level ultrahigh-frequency (UHF) and ultrashort-pulse (USP) blue laser irradiation on osteoblasts. The MC3T3-E1 osteoblast cell line was used in this study. Following LLLI with a 405 nm newly developed UHF-USP blue laser (80 MHz, 100 fs), osteoblast proliferation, and alkaline phosphatase (ALP) activity were assessed. In addition, mRNA levels of the osteoblast differentiation markers, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), and osteopontin (Opn) was evaluated, and extracellular calcification was quantified. To clarify the involvement of transient receptor potential (TRP) channels in LLLI-induced biostimulation, cells were treated prior to LLLI with capsazepine (CPZ), a selective inhibitor of TRP vanilloid 1 (TRPV1), and subsequent proliferation and ALP activity were measured. LLLI with the 405 nm UHF-USP blue laser significantly enhanced cell proliferation and ALP activity, compared with the non-irradiated control and LLLI using continuous-wave mode, without significant temperature elevation. LLLI promoted osteoblast proliferation in a dose-dependent manner up to 9.4 J/cm 2 and significantly accelerated cell proliferation in in vitro wound healing assay. ALP activity was significantly enhanced at doses up to 5.6 J/cm 2 , and expression of Osx and Alp mRNAs was significantly increased compared to that of the control on days 3 and 7 following LLLI at 5.6 J/cm 2 . The extent of extracellular calcification was also significantly higher as a result of LLLI 3 weeks after the treatment. Measurement of TRPV1 protein expression on 0, 3, and 7 days post-irradiation revealed no differences between the LLLI and control groups; however, promotion of cell

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

PubMed Central

ABBRACCHIO, MARIA P.; BURNSTOCK, GEOFFREY; BOEYNAEMS, JEAN-MARIE; BARNARD, ERIC A.; BOYER, JOSÉ L.; KENNEDY, CHARLES; KNIGHT, GILLIAN E.; FUMAGALLI, MARTA; GACHET, CHRISTIAN; JACOBSON, KENNETH A.; WEISMAN, GARY A.

2012-01-01

There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors deorphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review. PMID:16968944

Painful Pathways Induced by Toll-like Receptor Stimulation of Dorsal Root Ganglion Neurons

PubMed Central

Qi, Jia; Buzas, Krisztina; Fan, Huiting; Cohen, Jeffrey I.; Wang, Kening; Mont, Erik; Klinman, Dennis; Oppenheim, Joost J.; Howard, O.M. Zack

2011-01-01

We hypothesize that innate immune signals from infectious organisms and/or injured tissues may activate peripheral neuronal pain signals. In this study, we demonstrated that toll-like receptors 3/7/9 (TLRs) are expressed by human dorsal root ganglion neurons (DRGNs) and in cultures of primary mouse DRGNs. Stimulation of murine DRGNs with TLR ligands induced expression and production of proinflammatory chemokines and cytokines CCL5 (RANTES), CXCL10 (IP10), interleukin-1alpha, interleukin-1beta, and prostaglandin E2 (PGE2), which have previously been shown to augment pain. Further, TLR ligands up-regulated the expression of a nociceptive receptor transient receptor potential vanilloid type 1 (TRPV1), and enhanced calcium flux by TRPV1 expressing DRGNs. Using a tumor-induced temperature sensitivity model, we showed that in vivo administration of a TLR9 antagonist, known as a suppressive ODN, blocked tumor-induced temperature sensitivity. Taken together, these data indicate that stimulation of peripheral neurons by TLR ligands can induce nerve pain. PMID:21515789

Response to treatment of myasthenia gravis according to clinical subtype.

PubMed

Akaishi, Tetsuya; Suzuki, Yasushi; Imai, Tomihiro; Tsuda, Emiko; Minami, Naoya; Nagane, Yuriko; Uzawa, Akiyuki; Kawaguchi, Naoki; Masuda, Masayuki; Konno, Shingo; Suzuki, Hidekazu; Murai, Hiroyuki; Aoki, Masashi; Utsugisawa, Kimiaki

2016-11-17

We have previously reported using two-step cluster analysis to classify myasthenia gravis (MG) patients into the following five subtypes: ocular MG; thymoma-associated MG; MG with thymic hyperplasia; anti-acetylcholine receptor antibody (AChR-Ab)-negative MG; and AChR-Ab-positive MG without thymic abnormalities. The objectives of the present study were to examine the reproducibility of this five-subtype classification using a new data set of MG patients and to identify additional characteristics of these subtypes, particularly in regard to response to treatment. A total of 923 consecutive MG patients underwent two-step cluster analysis for the classification of subtypes. The variables used for classification were sex, age of onset, disease duration, presence of thymoma or thymic hyperplasia, positivity for AChR-Ab or anti-muscle-specific tyrosine kinase antibody, positivity for other concurrent autoantibodies, and disease condition at worst and current. The period from the start of treatment until the achievement of minimal manifestation status (early-stage response) was determined and then compared between subtypes using Kaplan-Meier analysis and the log-rank test. In addition, between subtypes, the rate of the number of patients who maintained minimal manifestations during the study period/that of patients who only achieved the status once (stability of improved status) was compared. As a result of two-step cluster analysis, 923 MG patients were classified into five subtypes as follows: ocular MG (AChR-Ab-positivity, 77%; histogram of onset age, skewed to older age); thymoma-associated MG (100%; normal distribution); MG with thymic hyperplasia (89%; skewed to younger age); AChR-Ab-negative MG (0%; normal distribution); and AChR-Ab-positive MG without thymic abnormalities (100%, skewed to older age). Furthermore, patients classified as ocular MG showed the best early-stage response to treatment and stability of improved status, followed by those classified as

First report on molecular breast cancer subtypes and their clinico-pathological characteristics in Eastern Morocco: series of 2260 cases.

PubMed

Elidrissi Errahhali, Manal; Elidrissi Errahhali, Mounia; Ouarzane, Meryem; El Harroudi, Tijani; Afqir, Said; Bellaoui, Mohammed

2017-01-09

Breast cancer is the most frequent malignancy among women in Eastern Morocco. In this paper, we provide the first report on molecular breast cancer subtypes in this region. This is the largest population-based study on breast cancer among Moroccan women. We analyzed 2260 breast cancer cases diagnosed at the Hassan II Regional Oncology Center between October 2005 and December 2012. Clinico-pathological and therapeutic features were studied. Molecular subtypes were determined and their associations with the clinico-pathological characteristics of the tumors were examined. The mean age at diagnosis was 48.7 years ±11.4. Invasive ductal carcinoma was the predominant histological type (77.1%), followed by lobular invasive carcinoma (15.3%). The mean size of breast tumors was 3.5 cm ± 1.96, and 84% of our patients are diagnosed with tumors of more than 2 cm. Histological grade II tumors were the most frequent (70.4%), followed by advanced histological grade (18%). Lymph node positive tumors were observed in 64.8% of cases and 29.3% of patients had distant metastasis. Most tumors were hormone receptor-positive (73%) and 28.6% were HER2 positive. 86.1% of patients with hormone receptor-positive breast cancer were given hormone therapy, while 68.9% of patients with HER2+ breast cancer received targeted therapy with Herceptin. Luminal A was the commonest molecular subtype, followed by Luminal B, Triple Negative and HER2. The highest prevalence of premenopausal patients was observed in Triple Negative subtype (72.2%), followed by HER2 (64.1%), Luminal B (62.2%), and Luminal A (55.1%). Luminal B subtype had a poorer prognosis than Luminal A. Compared with Triple Negative, HER2 subtype tend to spread more aggressively and is associated with poorer prognosis. Unlike Western countries, breast cancer occurs at an earlier age and is diagnosed at a more advanced stage in Eastern Morocco. In this region, hormone receptor-positive tumors are predominant and so the majority of

The Transient Receptor Potential Vanilloid 2 Cation Channel Is Abundant in Macrophages Accumulating at the Peri-Infarct Zone and May Enhance Their Migration Capacity towards Injured Cardiomyocytes following Myocardial Infarction

PubMed Central

Goryainov, Pavel; Landa, Natalie; Barshack, Iris; Avivi, Camila; Semo, Jonathan; Keren, Gad

2014-01-01

Purpose A novel family of transient receptor potential (TRP) channels, that may hold a role in calcium homeostasis, has recently been described. By employing a GeneChip array analysis we have demonstrated a clear and specific upregulation of the TRP vanilloid 2 (TRPV2) mRNA in the left ventricles (LV) 3–5 days post-acute myocardial infarction (MI) compared to sham-operated controls, both in rats and in mice. We sought to characterize the cardiac cellular subpopulations in which TRPV2 is overexpressed upon acute MI. Methods Lewis rats underwent an acute MI by ligation of the left anterior descending artery or chest opening only (sham). The animals were terminated at various time points and an immunohistochemical (IHC) and immunofloerescent (IFC) staining of the LV sections as well as a flow cytometry analysis of LV-derived cells were carried out, using anti-TRPV2 and anti-monocyte/macrophage antibodies. Rat alveolar macrophage cells, NR8383, transiently transfected with TRPV2 siRNA were allowed to migrate towards hypoxic conditioned media of the rat cardiac myoblast line H9C2 using a trans-well migration assay. The macrophage cells migrating to the bottom side of the inserts were counted. Results The IHC and IFC staining as well as the flow cytometry data demonstrated a substantial expression of TRPV2 in infiltrating macrophages in the peri-infarct region 3–5 days post-acute MI. The in vitro migration assay data demonstrated that following inhibition of the TRPV2 channel, the number of migrating macrophages towards conditioned medium of hypoxic cardiomyocytes was significantly reduced. Conclusions TRPV2 is highly expressed on the peri-infarct infiltrating macrophages and may play an important role in post-MI phagocytosis. Better characterization of this channel may pave the way for identifying a new target for modulating the dramatic post-MI immune reactions. PMID:25136832

Characterization of a Novel Small Molecule Subtype Specific Estrogen-Related Receptor α Antagonist in MCF-7 Breast Cancer Cells

PubMed Central

Chisamore, Michael J.; Cunningham, Michael E.; Flores, Osvaldo; Wilkinson, Hilary A.; Chen, J. Don

2009-01-01

Background The orphan nuclear receptor estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily. It was identified through a search for genes encoding proteins related to estrogen receptor α (ERα). An endogenous ligand has not been found. Novel ERRα antagonists that are highly specific for binding to the ligand binding domain (LBD) of ERRα have been recently reported. Research suggests that ERRα may be a novel drug target to treat breast cancer and/or metabolic disorders and this has led to an effort to characterize the mechanisms of action of N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine, a novel ERRα specific antagonist. Methodology/Principal Findings We demonstrate this ERRα ligand inhibits ERRα transcriptional activity in MCF-7 cells by luciferase assay but does not affect mRNA levels measured by real-time RT-PCR. Also, ERα (ESR1) mRNA levels were not affected upon treatment with the ERRα antagonist, but other ERRα (ESRRA) target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased. In vitro, the ERRα antagonist prevents the constitutive interaction between ERRα and nuclear receptor coactivators. Furthermore, we use Western blots to demonstrate ERRα protein degradation via the ubiquitin proteasome pathway is increased by the ERRα-subtype specific antagonist. We demonstrate by chromatin immunoprecipitation (ChIP) that the interaction between ACADM, ESRRA, and TFF1 endogenous gene promoters and ERRα protein is decreased when cells are treated with the ligand. Knocking-down ERRα (shRNA) led to similar genomic effects seen when MCF-7 cells were treated with our ERRα antagonist. Conclusions/Significance We report the mechanism of action of a novel ERRα specific antagonist that inhibits transcriptional activity of ERRα, disrupts the constitutive interaction between ERRα and nuclear coactivators, and induces proteasome

Medial prefrontal cortex TRPV1 and CB1 receptors modulate cardiac baroreflex activity by regulating the NMDA receptor/nitric oxide pathway.

PubMed

Lagatta, Davi C; Kuntze, Luciana B; Ferreira-Junior, Nilson C; Resstel, Leonardo B M

2018-05-29

The ventral medial prefrontal cortex (vMPFC) facilitates the cardiac baroreflex response through N-methyl-D-aspartate (NMDA) receptor activation and nitric oxide (NO) formation by neuronal NO synthase (nNOS) and soluble guanylate cyclase (sGC) triggering. Glutamatergic transmission is modulated by the cannabinoid receptor type 1 (CB 1 ) and transient receptor potential vanilloid type 1 (TRPV 1 ) receptors, which may inhibit or stimulate glutamate release in the brain, respectively. Interestingly, vMPFC CB 1 receptors decrease cardiac baroreflex responses, while TRPV 1 channels facilitate them. Therefore, the hypothesis of the present study is that the vMPFC NMDA/NO pathway is regulated by both CB 1 and TRPV 1 receptors in the modulation of cardiac baroreflex activity. In order to test this assumption, we used male Wistar rats that had stainless steel guide cannulae bilaterally implanted in the vMPFC. Subsequently, a catheter was inserted into the femoral artery, for cardiovascular recordings, and into the femoral vein for assessing baroreflex activation. The increase in tachycardic and bradycardic responses observed after the microinjection of a CB 1 receptors antagonist into the vMPFC was prevented by an NMDA antagonist as well as by the nNOS and sGC inhibition. NO extracellular scavenging also abolished these responses. These same pharmacological manipulations inhibited cardiac reflex enhancement induced by TRPV 1 agonist injection into the area. Based on these results, we conclude that vMPFC CB 1 and TRPV 1 receptors inhibit or facilitate the cardiac baroreflex activity by stimulating or blocking the NMDA activation and NO synthesis.

Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases the expression of prostaglandin E2 receptor subtype EP4. The roles of phosphatidylinositol 3-kinase and CCAAT/enhancer-binding protein beta.

PubMed

Han, ShouWei; Ritzenthaler, Jeffrey D; Wingerd, Byron; Roman, Jesse

2005-09-30

The prostaglandin E2 receptor subtype EP4 has been implicated in the growth and progression of human non-small cell lung carcinoma (NSCLC). However, the factors that control its expression have not been entirely elucidated. Our studies show that NSCLC cells express peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) protein and that treatment with a selective PPARbeta/delta agonist (GW501516) increases EP4 mRNA and protein levels. GW501516 induced NSCLC cell proliferation, and this effect was prevented by PPARbeta/delta antisense or EP4 short interfering RNA (siRNA). GW501516 increased the phosphorylation of Akt and decreased PTEN expression. The selective inhibitor of phosphatidylinositol 3-kinase (PI3-K), wortmannin, and PPARbeta/delta antisense, abrogated the effect of GW501516 on EP4 expression, whereas that of the inhibitor of Erk did not. GW501516 also increased EP4 promoter activity through effects on the region between -1555 and -992 bp in the EP4 promoter, and mutation of the CCAAT/enhancer-binding protein (C/EBP) site in this region abrogated the effect of GW501516. GW501516 increased not only the binding activity of C/EBP to the NF-IL6 site in the EP4 promoter, which was prevented by the inhibitor of PI3-K, but also increased C/EBPbeta protein in a dose- and PPARbeta/delta-dependent manner. The effect of GW501516 on EP4 protein was eliminated in the presence of C/EBPbeta siRNA. Finally, we showed that pretreatment of NSCLC with GW501516 further increased NSCLC cell proliferation in response to exogenous dimethyl-prostaglandin E2 (PGE2) that was diminished in the presence of PPARbeta/delta antisense and EP4 siRNA. Taken together, these findings suggest that activation of PPARbeta/delta induces PGE2 receptor subtype EP4 expression through PI3-K signals and increases human lung carcinoma cell proliferation in response to PGE2. The increase in transcription of the EP4 gene by PPARbeta/delta agonist was associated with increased C

GABAA receptor: Positive and negative allosteric modulators.

PubMed

Olsen, Richard W

2018-01-31

gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane transport of GABA, the GABA receptors Type A (GABA A R) and Type B (GABA B R) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABA B R is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABA A R pharmacology, the topic of this article. GABA A R are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABA A R the targets of agonist depressants and antagonist convulsants, but most GABA A R drugs act at other (allosteric) binding sites on the GABA A R proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABA A R subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABA A R subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABA A R subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABA A R subtype-dependent extracellular domain sites. Thus GABA A R subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of

Design of ligands for the nicotinic acetylcholine receptors: the quest for selectivity.