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Sample records for cholinergic receptor binding

  1. Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors.

    PubMed

    Aráoz, Rómulo; Vilariño, Natalia; Botana, Luis M; Molgó, Jordi

    2010-07-01

    Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and cholinergic synapses, including the neuromuscular junction. Anatoxin-a and its methylene homologue homoanatoxin-a are potent agonists of nicotinic acetylcholine receptors. Since the structural determination of anatoxin-a, several mass spectrometry-based methods have been developed for detection of anatoxin-a and, later, homoanatoxin-a. Mass spectrometry-based techniques provide accuracy, precision, selectivity, sensitivity, reproducibility, adequate limit of detection, and structural and quantitative information for analyses of cyanobacterial anatoxins from cultured and environmental cyanobacterial samples. However, these physicochemical techniques will only detect known toxins for which toxin standards are commercially available, and they require highly specialized laboratory personnel and expensive equipment. Receptor-based assays are functional methods that are based on the mechanism of action of a class of toxins and are thus, suitable tools for survey of freshwater reservoirs for cyanobacterial anatoxins. The competition between cyanobacterial anatoxins and a labelled ligand for binding to nicotinic acetylcholine receptors is measured radioactively or non-radioactively providing high-throughput screening formats for routine detection of this class of neurotoxins. The mouse bioassay is the method of choice for marine toxin monitoring, but has to be replaced by fully validated functional methods. In this paper we review the ligand-binding assays developed for detection of cyanobacterial and algal neurotoxins targeting the nicotinic acetylcholine receptors and for high-throughput screening of novel nicotinic agents.

  2. Binding of /sup 3/H-acetylcholine to cholinergic receptors in bovine cerebral arteries

    SciTech Connect

    Shimohama, S.; Tsukahara, T.; Taniguchi, T.; Fujiwara, M.

    1985-11-18

    Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, /sup 3/H-acetylcholine (ACh), as the ligand. Specific binding of /sup 3/H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (K/sub D/) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of /sup 3/H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC/sub 50/ values of cholinergic drugs for /sup 3/H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC/sub 50/ values of nicotinic cholinergic agents such as nicotine, cytisine and ..cap alpha..-bungarotoxin exceeded 50 ..mu..M. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic reporters in these arteries. 18 references, 2 figures, 2 tables.

  3. Nicotinic Cholinergic Receptor Binding Sites in the Brain: Regulation in vivo

    NASA Astrophysics Data System (ADS)

    Schwartz, Rochelle D.; Kellar, Kenneth J.

    1983-04-01

    Tritiated acetylcholine was used to measure binding sites with characteristics of nicotinic cholinergic receptors in rat brain. Regulation of the binding sites in vivo was examined by administering two drugs that stimulate nicotinic receptors directly or indirectly. After 10 days of exposure to the cholinesterase inhibitor diisopropyl fluorophosphate, binding of tritiated acetylcholine in the cerebral cortex was decreased. However, after repeated administration of nicotine for 10 days, binding of tritiated acetylcholine in the cortex was increased. Saturation analysis of tritiated acetylcholine binding in the cortices of rats treated with diisopropyl fluorophosphate or nicotine indicated that the number of binding sites decreased and increased, respectively, while the affinity of the sites was unaltered.

  4. ( sup 3 H)cytisine binding to nicotinic cholinergic receptors in brain

    SciTech Connect

    Pabreza, L.A.; Dhawan, S.; Kellar, K.J. )

    1991-01-01

    Cytisine, a ganglionic agonist, competes with high affinity for brain nicotinic cholinergic receptors labeled by any of several nicotinic {sup 3}H-agonist ligands. Here we have examined the binding of ({sup 3}H)cytisine in rat brain homogenates. ({sup 3}H)Cytisine binds with high affinity (Kd less than 1 nM), and specific binding represented 60-90% of total binding at all concentrations examined up to 15 nM. The nicotinic cholinergic agonists nicotine, acetylcholine, and carbachol compete with high affinity for ({sup 3}H)cytisine binding sites, whereas among nicotinic receptor antagonists only dihydro-beta-erythroidine competes with high affinity (in the nanomolar range). Comparison of binding in several brain regions showed that ({sup 3}H)cytisine binding is higher in the thalamus, striatum, and cortex than in the hippocampus, cerebellum, or hypothalamus. The pharmacology and brain regional distribution of ({sup 3}H)cytisine binding sites are those predicted for neuronal nicotinic receptor agonist recognition sites. The high affinity and low nonspecific binding of ({sup 3}H)cytisine should make it a very useful ligand for studying neuronal nicotinic receptors.

  5. Alterations in alpha-adrenergic and muscarinic cholinergic receptor binding in rat brain following nonionizing radiation

    SciTech Connect

    Gandhi, V.C.; Ross, D.H.

    1987-01-01

    Microwave radiation produces hyperthermia. The mammalian thermoregulatory system defends against changes in temperature by mobilizing diverse control mechanisms. Neurotransmitters play a major role in eliciting thermoregulatory responses. The involvement of adrenergic and muscarinic cholinergic receptors was investigated in radiation-induced hyperthermia. Rats were subjected to radiation at 700 MHz frequency and 15 mW/cm/sup 2/ power density and the body temperature was raised by 2.5 degrees C. Of six brain regions investigated only the hypothalamus showed significant changes in receptor states, confirming its pivotal role in thermoregulation. Adrenergic receptors, studied by (/sup 3/H)clonidine binding, showed a 36% decrease in binding following radiation after a 2.5 degrees C increase in body temperature, suggesting a mechanism to facilitate norepinephrine release. Norepinephrine may be speculated to maintain thermal homeostasis by activating heat dissipation. Muscarinic cholinergic receptors, studied by (3H)quinuclidinyl benzilate binding, showed a 65% increase in binding at the onset of radiation. This may be attributed to the release of acetylcholine in the hypothalamus in response to heat cumulation. The continued elevated binding during the period of cooling after radiation was shut off may suggest the existence of an extra-hypothalamic heat-loss pathway.

  6. Cholinergic nicotinic receptors in the vestibular epithelia.

    PubMed

    Thornhill, R A

    1991-10-01

    Receptor binding studies specific for nicotinic cholinergic receptors have been carried out on isolated vestibular epithelia of the frogs Rana catesbiana and Rana temporaria. Evidence is presented for the presence of nicotinic-like cholinergic receptors specifically associated with the sensory areas. PMID:1797345

  7. Kinetics of in vivo binding of antagonist to muscarinic cholinergic receptor in the human heart studied by Positron Emission Tomography

    SciTech Connect

    Syrota, A.; Paillotin, G.; Davy, J.M.; Aumont, M.C.

    1984-08-27

    Positron Emission Tomography (PET) was used to analyze in vivo antagonist binding to human myocardial muscarinic cholinergic receptor. The methiodide salt of the muscarinic antagonist, quinuclidinyl benzilate (MQNB), was labeled with the positron emitter, Carbon-11, and injected intravenously to 8 normal subjects. /sup 11/C-MQNB concentration was determined in vivo in the ventricular septum from 40 cross-sectional images acquired at the same transverse level over a period of 70 minutes. In 4 subjects, various amounts of unlabeled atropine were rapidly injected at 20 minutes to study whether atropine competitively inhibited MQNB. The kinetics of binding of /sup 11/C-MQNB were not the same in vivo and in vitro. The apparent dissociation rate of /sup 11/C-MQNB in vivo was much slower (by 1 to 2 orders of magnitude) than that observed in vitro with /sup 3/H-QNB. After atropine injection, /sup 11/C-MQNB dissociated from its binding sites at a rate that apparently depended on the amount of atropine present. /sup 11/C-MQNB kinetics were analyzed with a mathematical model which assumes the existence of a boundary layer containing free ligand in the vicinity of the binding sites. The dissociation rate of the radioligand depends on the probability of its rebinding to a free receptor site. 11 references, 1 table.

  8. Low-level exposure to methylmercury modifies muscarinic cholinergic receptor binding characteristics in rat brain and lymphocytes: physiologic implications and new opportunities in biologic monitoring.

    PubMed Central

    Coccini, T; Randine, G; Candura, S M; Nappi, R E; Prockop, L D; Manzo, L

    2000-01-01

    Methylmercury (MeHg) affects several parameters of cholinergic function. These alterations are thought to play a role in MeHg neurotoxicity. In vitro experiments have indicated that MeHg acts as a strong competitive inhibitor of radioligand binding to muscarinic cholinergic receptors (mAChRs) in rat brain. Furthermore, rat brain mAChRs share several pharmacologic characteristics of similar receptors present on lymphocytes. Using the muscarinic antagonist [(3)H]quinuclidinyl benzilate (QNB) to label receptors, we investigated the in vivo interactions of MeHg with rat brain mAChRs. We also investigated whether MeHg-induced central mAChR changes are reflected by similar alterations in splenic lymphocytes. Exposure to low doses of MeHg--0.5 or 2 mg/kg/day in drinking water--for 16 days significantly increased (20-44% of control) mAChRs density (B(max)) in the hippocampus and cerebellum without affecting receptor affinity (K(d)). The effect of MeHg did not occur immediately; it was not apparent until 2 weeks after the termination of treatment. No significant changes in [(3)H]QNB binding were observed in the cerebral cortex. In splenic lymphocytes, mAChR density was remarkably increased (95-198% of control) by day 14 of MeHg exposure and remained enhanced 14 days after the cessation of treatment. These results suggest up-regulation of mAChRs in selected brain regions (hippocampus and cerebellum) after prolonged low-level ingestion of MeHg in rats. These cerebral effects are delayed in onset and are preceded by a marked increase in density of mAChRs on lymphocytes. In chronic MeHg exposure, peripheral lymphocytes may represent a sensitive target for the interaction of MeHg with mAChRs and, therefore, may be predictive indicators of later adaptive response involving cerebral mAChRs. Additionally, the effect of MeHg on lymphocyte mAChRs in vivo indicates that this receptor system should be investigated further as a possible target for MeHg immunotoxicity. Images Figure 1

  9. In vitro and in vivo characterization of 4-[125I]iododexetimide binding to muscarinic cholinergic receptors in the rat heart.

    PubMed

    Matsumura, K; Uno, Y; Scheffel, U; Wilson, A A; Dannals, R F; Wagner, H N

    1991-01-01

    4-[125I]iododexetimide binding to muscarinic cholinergic receptors (mAChR) was evaluated in the rat heart. 4-[125I]iododexetimide displayed high in vitro affinity (Kd = 14.0 nM) for rat myocardial mAChR. In vivo, there was high accumulation of 4-[125I]iododexetimide in the rat atrium and ventricle which could be blocked by approximately 60% by preinjection of atropine. In contrast, accumulation of the radiolabeled stereoisomer, 4-[125I]iodolevetimide, was 63% lower than 4-[125I]iodolevetimide and was not blocked by atropine. The blood clearance of 4-[125I]iododexetimide was rapid, providing heart-to-blood ratios of up to 14:1; however, heart-to-lung and heart-to-liver ratios were below unity. The data indicate that 4-[125I]iododexetimide binds potently to rat mAChR. However, since nonspecific binding is relatively high, it is not clear whether iododexetimide labeled with 123I will be useful in SPECT imaging studies of myocardial mAChR. Further studies in humans are indicated. PMID:1988640

  10. Muscarinic cholinergic receptors in pancreatic acinar carcinoma of rat.

    PubMed

    Taton, G; Delhaye, M; Swillens, S; Morisset, J; Larose, L; Longnecker, D S; Poirier, G G

    1985-04-15

    The active enantiomer of tritiated quinuclidinyl benzilate (3H(-)QNB) was used as a ligand to evaluate the muscarinic receptors. The 3H(-)QNB binding characteristics of muscarinic cholinergic receptors obtained from normal and neoplastic tissues were studied to determine changes in receptor properties during neoplastic transformation. Saturable and stereospecific binding sites for 3H(-)QNB are present in homogenates of rat pancreatic adenocarcinoma. The proportions of high- and low-affinity agonist binding sites are similar for neoplastic and normal tissues. The density of muscarinic receptors is higher in neoplastic (200 femtomoles/mg protein) than in normal pancreatic homogenates (80 femtomoles/mg protein). The muscarinic binding sites of the neoplastic and fetal pancreas show similar KD values which are higher than those observed for normal pancreas. PMID:2580801

  11. Cholinergic receptors in the upper respiratory system of the rat.

    PubMed

    Klaassen, A B; Kuijpers, W; Scheres, H M; Rodrigues de Miranda, J F; Beld, A J

    1986-04-01

    Radioligand receptor binding might give more detailed information on the innervation pattern of the nasal mucosa and the character of the various neuroreceptors involved. With respect to the cholinergic receptors, this technique reveals that specific binding of tritiated I-quinuclidinyl benzilate to rat nasal mucosa homogenates occurs to a homogeneous class of binding sites, with a dissociation constant of 0.06 +/- 0.02 nM and a receptor density of 8 +/- 2 pmole/g of tissue. Binding is stereoselectively inhibited by benzetimide hydrochloride enantiomers. Pirenzepine displacement (inhibition constant = 0.5 X 10(-6) M) classifies tritiated I-quinuclidinyl benzilate binding sites as M2-muscarinic receptors. Methylfurthrethonium inhibits tritiated I-quinuclidinyl benzilate binding at high concentrations, pointing to the presence of low-affinity agonist binding sites, probably admixed with a small proportion of high-affinity agonist binding sites. These data obtained in the rat open new perspectives for studying muscarinic receptors in the human nose to elucidate the supposed disturbance of autonomic nerve regulation in nasal hyperreactivity. PMID:3511926

  12. Cholinergic innervation and receptors in the cerebellum.

    PubMed

    Jaarsma, D; Ruigrok, T J; Caffé, R; Cozzari, C; Levey, A I; Mugnaini, E; Voogd, J

    1997-01-01

    We have studied the source and ultrastructural characteristics of ChAT-immunoreactive fibers in the cerebellum of the rat, and the distribution of muscarinic and nicotinic receptors in the cerebellum of the rat, rabbit, cat and monkey, in order to define which of the cerebellar afferents may use ACh as a neurotransmitter, what target structures are they, and which cholinergic receptor mediate the actions of these pathways. Our data confirm and extend previous observations that cholinergic markers occur at relatively low density in the cerebellum and show not only interspecies variability, but also heterogeneity between cerebellar lobules in the same species. As previously demonstrated by Barmack et al. (1992a,b), the predominant fiber system in the cerebellum that might use ACh as a transmitter or a co-transmitter is formed by mossy fibers originating in the vestibular nuclei and innervating the nodulus and ventral uvula. Our results show that these fibers innervate both granule cells and unipolar brush cells, and that the presumed cholinergic action of these fibers most likely is mediated by nicotinic receptors. In addition to cholinergic mossy fibers, the rat cerebellum is innervated by beaded ChAT-immunoreactive fibers. We have demonstrated that these fibers originate in the pedunculopontine tegmental nucleus (PPTg), the lateral paragigantocellular nucleus (LPGi), and to a lesser extent in various raphe nuclei. In both the cerebellar cortex and the cerebellar nuclei these fibers make asymmetric synaptic junctions with small and medium-sized dendritic profiles. Both muscarinic and nicotinic receptor could mediate the action of these diffuse beaded fibers. In the cerebellar nuclei the beaded cholinergic fibers form a moderately dense network, and could in principle have a significant effect on neuronal activity. For instance, the cholinergic fibers arising in the PPTg may modulate the excitability of the cerebellonuclear neurons in relation to sleep and arousal (e

  13. Neurotransmitter Receptor Binding in Bovine Cerebral Microvessels

    NASA Astrophysics Data System (ADS)

    Peroutka, Stephen J.; Moskowitz, Michael A.; Reinhard, John F.; Synder, Solomon H.

    1980-05-01

    Purified preparations of microvessels from bovine cerebral cortex contain substantial levels of alpha-adrenergic, beta-adrenergic, and histamine 1 receptor binding sites but only negligible serotonin, muscarinic cholinergic, opiate, and benzodiazepine receptor binding. Norepinephrine and histamine may be endogenous regulators of the cerebral microcirculation at the observed receptors.

  14. Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors

    SciTech Connect

    Pan, G.Z.; Lu, L.; Qian, J.; Xue, B.G.

    1987-03-01

    In dispersed acini from rat pancreas, it was found that bovine pancreatic polypeptide (BPP) and its C-fragment hexapeptide amide (PP-6), at concentrations of 0.1 and 30 ..mu..M, respectively, could significantly inhibit amylase secretion stimulated by carbachol, and this inhibition by BPP was dose dependent. /sup 45/Ca outflux induced by carbachol was also inhibited by BPP or PP-6, but they had no effect on cholecystokinin octapeptide- (CCK-8) or A23187-stimulated /sup 45/Ca outflux. BPP was also capable of displacing the specific binding of (/sup 3/H)-quinuclidinyl benzilate to its receptors, and it possessed a higher affinity (K/sub i/35nM) than carbachol (K/sub i/ 1.8 ..mu..M) in binding with M-receptors. It is concluded from this study that BPP acts as an antagonist of muscarinic cholinergic receptors in rat pancreatic acini. In addition, BPP inhibited the potentiation of amylase secretion caused by the combination of carbachol plus secretin or vasoactive intestinal peptide. This may be a possible explanation of the inhibitory effect of BPP on secretin-induced pancreatic enzyme secretion shown in vivo, since pancreatic enzyme secretion stimulated by secretin under experimental conditions may be the result of potentiation of enzyme release produced by the peptide in combination with a cholinergic stimulant.

  15. Cholinergic muscarinic receptors in rat cochlea.

    PubMed

    van Megen, Y J; Klaassen, A B; Rodrigues de Miranda, J F; Kuijpers, W

    1988-11-22

    Specific 3H-1-quinuclidinylbenzilate (3H-1-QNB) binding to rat cochlea homogenates occurs to a homogeneous class of binding sites with Kd = 0.13 +/- 0.01 nM and Bmax = 0.57 +/- 0.07 fmol per cochlea. Binding is stereoselectively inhibited by benzetimide enantiomers. Dexetimide was more effective than levetimide in displacing 3H-1-QNB from its binding sites (Ki = 4 x 10(-10) M and 6.5 x 10(-6) M, respectively). Pirenzepine inhibits 3H-1-QNB binding with low affinity (Ki = 2 x 10(-6) M), classifying the binding sites as muscarinic M2 receptors. PMID:3214711

  16. Basic and modern concepts on cholinergic receptor: A review

    PubMed Central

    Tiwari, Prashant; Dwivedi, Shubhangi; Singh, Mukesh Pratap; Mishra, Rahul; Chandy, Anish

    2013-01-01

    Cholinergic system is an important system and a branch of the autonomic nervous system which plays an important role in memory, digestion, control of heart beat, blood pressure, movement and many other functions. This article serves as both structural and functional sources of information regarding cholinergic receptors and provides a detailed understanding of the determinants governing specificity of muscarinic and nicotinic receptor to researchers. The study helps to give overall information about the fundamentals of the cholinergic system, its receptors and ongoing research in this field.

  17. Decreased hippocampal muscarinic cholinergic receptor binding measured by 123I-iododexetimide and single-photon emission computed tomography in epilepsy.

    PubMed

    Müller-Gärtner, H W; Mayberg, H S; Fisher, R S; Lesser, R P; Wilson, A A; Ravert, H T; Dannals, R F; Wagner, H N; Uematsu, S; Frost, J J

    1993-08-01

    Regional binding of 123I-iododexetimide, a muscarinic acetylcholine receptor antagonist, was measured in vivo in the temporal lobes of 4 patients with complex partial seizures using single-photon emission computed tomography. In the anterior hippocampus ipsilateral to the electrical focus, 123I-iododexetimide binding was decreased by 40 +/- 9% (mean +/- SD, p < 0.01) compared with the contralateral hippocampus; 123I-iododexetimide binding in other temporal lobe regions was symmetrical. The data indicate a regionally specific change of muscarinic acetylcholine receptor in anterior hippocampus in complex partial seizures of temporal lobe origin. PMID:8338348

  18. An autoradiographic analysis of cholinergic receptors in mouse brain after chronic nicotine treatment

    SciTech Connect

    Pauly, J.R.; Marks, M.J.; Gross, S.D.; Collins, A.C. )

    1991-09-01

    Quantitative autoradiographic procedures were used to examine the effects of chronic nicotine infusion on the number of central nervous system nicotinic cholinergic receptors. Female DBA mice were implanted with jugular cannulas and infused with saline or various doses of nicotine (0.25, 0.5, 1.0 or 2.0 mg/kg/hr) for 10 days. The animals were then sacrificed and the brains were removed and frozen in isopentane. Cryostat sections were collected and prepared for autoradiographic procedures as previously described. Nicotinic cholinergic receptors were labeled with L-(3H)nicotine or alpha-(125I)bungarotoxin; (3H)quinuclidinyl benzilate was used to measure muscarinic cholinergic receptor binding. Chronic nicotine infusion increased the number of sites labeled by (3H)nicotine in most brain areas. However, the extent of the increase in binding as well as the dose-response curves for the increase were widely different among brain regions. After the highest treatment dose, binding was increased in 67 of 86 regions measured. Septal and thalamic regions were most resistant to change. Nicotinic binding measured by alpha-(125I)bungarotoxin also increased after chronic treatment, but in a less robust fashion. At the highest treatment dose, only 26 of 80 regions were significantly changes. Muscarinic binding was not altered after chronic nicotine treatment. These data suggest that brain regions are not equivalent in the mechanisms that regulate alterations in nicotinic cholinergic receptor binding after chronic nicotine treatment.

  19. 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.

  20. Characterization of muscarinic cholinergic receptors on rat pancreatic acini by N-[3H]methylscopolamine binding. Their relationship with calcium 45 efflux and amylase secretion.

    PubMed

    Dehaye, J P; Winand, J; Poloczek, P; Christophe, J

    1984-01-10

    N-[3H]Methylscopolamine (NMS) binding, amylase secretion, and 45Ca efflux from dispersed rat pancreatic acini were investigated in parallel, in the presence or absence of 4 muscarinic agonists and 3 muscarinic antagonists. Scatchard analysis of [3H]NMS saturation isotherms gave a KD of 0.9 nM and an average binding capacity of 24,000 sites per cell. Binding competition curves with the antagonists atropine, dexetimide, and NMS gave KD values of 3.5, 3.5, and 0.5 nM, respectively. With the 3 full agonists oxotremorine, muscarine, and carbamylcholine, the receptor population could be divided into two classes of binding sites: a minor one (15%) with high affinity (KD = 20-35 nM) and a major one (85%) with low affinity (KD = 3-65 microM). There was a receptor reserve of about 50% with respect to carbamylcholine-stimulated amylase secretion. Further analysis of dose-effect curves suggests that low affinity binding sites were involved in the secretory response to muscarinic stimulation. Pilocarpine, like muscarinic antagonists, recognized all binding sites with the same affinity but acted as a partial agonist on amylase secretion and 45Ca efflux. PMID:6200472

  1. Synthesis and biological evaluation of [125I]- and [123I]-4-iododexetimide, a potent muscarinic cholinergic receptor antagonist.

    PubMed

    Wilson, A A; Dannals, R F; Ravert, H T; Frost, J J; Wagner, H N

    1989-05-01

    A series of halogenated racemic analogues of dexetimide (1) was synthesized and their affinity for the muscarinic cholinergic receptor measured. One analogue, 4-iododexetimide (21), was efficiently labeled with 125I and 123I at high specific activity. In vitro binding studies and in vivo biodistribution studies suggest that 123I-labeled 21 may be useful for imaging muscarinic cholinergic receptors in the living human brain with single photon emission computed tomography. PMID:2785211

  2. Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and. alpha. -bungarotoxin

    SciTech Connect

    Conti-Tronconi, B.M.; Tang, Fen; Diethelm, B.M.; Spencer, S.R. ); Reinhardt-Maelicke, S.; Maelicke, A. )

    1990-07-03

    Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) {alpha} subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, the authors used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind {alpha}-bungarotoxin ({alpha}-BTX) and several {alpha}-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the {alpha}-subunit, and bind to AChR in a mutually exclusive fashion with {alpha}-BTX. The binding of WF5 and W2 to Torpedo AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. Peptides {alpha}181-200 and {alpha}55-74 both inhibited binding of {sup 125}I-{alpha}-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound {alpha}-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR {alpha} subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region.

  3. Cholinergic ligand interactions with acetylcholine receptor proteins and solvent interactions with N,N-dialkylnicotinamides

    SciTech Connect

    Bean, J.W.

    1987-01-01

    A dual-chambered flow dialysis nuclear counting apparatus was used to monitor cholinergic ligand induced displacement of {sup 155}Eu{sup 3+} from acetylcholine receptor proteins. Acetylcholine, nicotine and carbamylcholine induced similar rates of displacement of {sup 155}Eu{sup 3+} probes of calcium binding sites in receptor proteins from wild type Drosophila melanogaster and Torpedo californica. The receptor isolated from a nicotine resistant strain of Drosophila melanogaster displayed an altered dependency of cholinergic ligand induced cation displacement with respect to the other two receptor proteins. Both Drosophila strains' solubilized receptor proteins migrated as three bands of molecular weights 68,000, 66,000, and 60,000 on denaturing polyacrylamide gels. Carbon-13 NMR techniques were employed to examine the effects of solvent environment on rotational energy barriers in a series of molecules related to the analeptic, nikethamide: N,N-dimethylnicotinamide, 1-nicotinoyl piperidine, and N,N-dipropylnicotinamide.

  4. Muscarinic cholinergic and alpha/sub 1/ adrenergic receptors in murine atria: phosphatidylinositol breakdown and receptor interaction

    SciTech Connect

    Scherer, R.W.

    1987-01-01

    Upon stimulation of muscarinic cholinergic receptors, there is a decrease in the force of contraction rate of firing in heart, while stimulation of ..cap alpha.. adrenergic receptors causes an increase in the force of contraction with no change in the heart rate. Yet both receptors stimulate the breakdown of phosphatidylinositol (PI). Therefore, the breakdown of PI was examined to determine how the process differed between the two receptor systems. Murine atria, prelabelled with (/sup 3/H)inositol, were stimulated with the muscarinic cholinergic agonists, carbamylcholine (CARB), and oxotremorine (OXO); and with the ..cap alpha.. adrenergic agonists, norepinephrine (NE) and phenylephrine (PE); either singly or in combination. Breakdown of PI was assessed by measurement of individual inositol phosphates by anion exchange chromatography. Binding of CARB to atrial muscarinic receptors was measured by competition with (/sup 3/H)quinuclidinyl benzilate.

  5. [Probable mechanism of recognition of cholinergic ligands by acetylcholine receptors].

    PubMed

    Demushkin, V P; Kotelevtsev, Iu V; Pliashkevich, Iu G; Khramtsov, N V

    1982-01-01

    Dryding's models were used for the conformational analysis of compounds affecting muscarin-specific acetylcholine receptor and nicotin-specific acetylcholine receptor. Ammonium group and ether oxygen (3.6 A apart from the ammonium group) specifically oriented to each other were shown to be necessary structural elements to reveal muscarin-type cholinergic activity. Ammonium group along with carbonyl oxygen or its substituent (5 A distance) are the necessary structural units providing nicotin-type cholinergic activity. The presence of two hydrophobic substituents (one in the ammonium area and the other neighbouring the second active grouping) is the additional factor. The developed principles were justified by the use of a series of synthetic samples. The compounds were obtained likely favouring affinitive modification of acetylcholine receptor (dissociation constants of acetylcholine receptor complexes equalling to 10(-4)--10(-7) M-1). PMID:7070378

  6. In vivo labeling of nicotinic cholinergic receptors in brain with [3H]cytisine.

    PubMed

    Flesher, J E; Scheffel, U; London, E D; Frost, J J

    1994-01-01

    [3H]Cytisine was evaluated as an in vivo ligand for the nicotinic cholinergic receptor (nAchR) in mouse brain. The tracer was injected intravenously, and radioactivity in brain regions was analyzed. Radioactivity peaked in the brain at 30 minutes. It was highest in the thalamus, intermediate in the superior colliculi, prefrontal cortex and hippocampus, and low in the cerebellum. Pretreatment with unlabeled cytisine inhibited binding in the thalamus, but not in the cerebellum. Binding was displaced by l-nicotine, but not by d-nicotine or dexetimide. The results suggest that cytisine, appropriately labeled with a positron emitting radionuclide, may be useful for study of nicotinic cholinergic receptors in humans by emission computed tomography. PMID:8196506

  7. Nicotine receptors and depression: revisiting and revising the cholinergic hypothesis

    PubMed Central

    Mineur, Yann S.; Picciotto, Marina R.

    2010-01-01

    There is a well-established connection between smoking and depression, with depressed individuals over-represented among smokers and ex-smokers often experiencing increased depressive symptoms immediately after quitting. Nicotine in tobacco binds, activates and desensitizes nicotinic acetylcholine receptors (nAChRs), but it is not known whether activation or desensitization is more important for nicotine’s effects on depressive symptoms. In this article, we review the hypothesis that blockade rather than activation of neuronal nAChRs might be important for the effects of nicotinic agents on depressive symptoms based on clinical and preclinical studies of nicotinic drugs. The endogenous neurotransmitter for nAChRs is acetylcholine, and the effects of nicotine on depression-like behaviors support the idea that dysregulation of the cholinergic system might contribute to the etiology of major depressive disorder. Thus, pharmacological agents that limit acetylcholine signaling through neuronal nAChRs might be promising for the development of novel antidepressant medications. PMID:20965579

  8. Cholinergic modulation of dopamine pathways through nicotinic acetylcholine receptors.

    PubMed

    de Kloet, Sybren F; Mansvelder, Huibert D; De Vries, Taco J

    2015-10-15

    Nicotine addiction is highly prevalent in current society and is often comorbid with other diseases. In the central nervous system, nicotine acts as an agonist for nicotinic acetylcholine receptors (nAChRs) and its effects depend on location and receptor composition. Although nicotinic receptors are found in most brain regions, many studies on addiction have focused on the mesolimbic system and its reported behavioral correlates such as reward processing and reinforcement learning. Profound modulatory cholinergic input from the pedunculopontine and laterodorsal tegmentum to dopaminergic midbrain nuclei as well as local cholinergic interneuron projections to dopamine neuron axons in the striatum may play a major role in the effects of nicotine. Moreover, an indirect mesocorticolimbic feedback loop involving the medial prefrontal cortex may be involved in behavioral characteristics of nicotine addiction. Therefore, this review will highlight current understanding of the effects of nicotine on the function of mesolimbic and mesocortical dopamine projections in the mesocorticolimbic circuit. PMID:26208783

  9. Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors.

    PubMed

    Shytle, R Douglas; Mori, Takashi; Townsend, Kirk; Vendrame, Martina; Sun, Nan; Zeng, Jin; Ehrhart, Jared; Silver, Archie A; Sanberg, Paul R; Tan, Jun

    2004-04-01

    Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia-mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous 'cholinergic anti-inflammatory pathway' regulates systemic inflammatory responses via alpha 7 nicotinic acetylcholinergic receptors (nAChR) found on blood-borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express alpha 7 nAChR subunit as determined by RT-PCR, western blot, immunofluorescent, and immunohistochemistry analyses. Acetylcholine and nicotine pre-treatment inhibit lipopolysaccharide (LPS)-induced TNF-alpha release in murine-derived microglial cells, an effect attenuated by alpha 7 selective nicotinic antagonist, alpha-bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen-activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through alpha 7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine's reported neuroprotective properties.

  10. Effects of beta-amyloid protein on M1 and M2 subtypes of muscarinic acetylcholine receptors in the medial septum-diagonal band complex of the rat: relationship with cholinergic, GABAergic, and calcium-binding protein perikarya.

    PubMed

    González, Iván; Arévalo-Serrano, Juan; Sanz-Anquela, José Miguel; Gonzalo-Ruiz, Alicia

    2007-06-01

    Cortical cholinergic dysfunction has been correlated with the expression and processing of beta-amyloid precursor protein. However, it remains unclear as to how cholinergic dysfunction and beta-amyloid (Abeta) formation and deposition might be related to one another. Since the M1- and M2 subtypes of muscarinic acetylcholine receptors (mAChRs) are considered key molecules that transduce the cholinergic message, the purpose of the present study was to assess the effects of the injected Abeta peptide on the number of M1mAchR- and M2mAChR-immunoreactive cells in the medial septum-diagonal band (MS-nDBB) complex of the rat. Injections of Abeta protein into the retrosplenial cortex resulted in a decrease in M1mAChR and M2mAChR immunoreactivity in the MS-nDBB complex. Quantitative analysis revealed a significant reduction in the number of M1mAChR- and M2mAChR-immunoreactive cells in the medial septum nucleus (MS) and in the horizontal nucleus of the diagonal band of Broca (HDB) as compared to the corresponding hemisphere in control animals and with that seen in the contralateral hemisphere, which corresponds to the PBS-injected side. Co-localization studies showed that the M1mAChR protein is localized in GABA-immunoreactive cells of the MS-nDBB complex, in particular those of the MS nucleus, while M2mAChR protein is localized in both the cholinergic and GABAergic cells. Moreover, GABAergic cells containing M2mAChR are mainly localized in the MS nucleus, while cholinergic cells containing M2mAChR are localized in the MS and the HDB nuclei. Our findings suggest that Abeta induces a reduction in M1mAChR- and M2mAChR-containing cells, which may contribute to impairments of cholinergic and GABAergic transmission in the MS-nDBB complex.

  11. Stress, chemical defense agents, and cholinergic receptors. Midterm report, 1 November 1987-31 July 1989

    SciTech Connect

    Lane, J.D.

    1989-11-30

    This project is assessing the affects of exposure to a chemical defense agent on anxiety and stress, by using rat models of anxiety (conditioned emotional response (CER); conditioned suppression) and unconditioned non-specific stres (exposure to footshock). The specific experiments determined the plasticity of muscarinic cholinergic binding sites in the central nervous system. The neuroanatomical locus and neuropharmacological profile of changes in binding sites were assessed in brain areas enriched in cholinergic markers. Acetylcholine turnover was measured to determine if the receptor response is compensatory or independent. The effects of acute exposure to doses of a chemical defense agent (soman--XGD) on lethality and behaviors were examined. The experiments involved training and conditioning adult rats to CER using standard operant/respondent techniques. The binding of radiolabelled ligand was studied in vitro using brain membranes and tissue sections (autoradiography). The major findings are that CER produces increases in acetylcholine turnover in brain areas involved in anxiety, and that primarily post-synaptic M1 receptors compensatorly decrease in response. These neurochemical phenomena are directly correlated with several behaviors, including onset and extinction of CER and non-specific stress. Followup experiments have been designed to test the interaction of CER, XGD and neurochemistry.

  12. Distribution of cholinergic receptors in the rat and human neocortex.

    PubMed

    Zilles, K; Schröder, H; Schröder, U; Horvath, E; Werner, L; Luiten, P G; Maelicke, A; Strosberg, A D

    1989-01-01

    Autoradiographic labelling of muscarinic (M1, M2, NMS binding sites) and nicotinic receptors shows an inhomogeneous distribution over architectonically identified cortical areas of the rat brain with highest concentrations in the medial prefrontal and frontal areas. Beside this general trend the areal patterns of different receptors are slightly varying. The laminar distribution of these receptors in the rat and human neocortex is characterized by two different patterns, one with highest receptor densities in the supragranular layers (M1 receptors, NMS binding sites), the other with a preferential labelling of layer IV and (with a lower intensity) layer V (M2 and nicotinic receptors). M1 receptors and NMS binding sites are codistributed at the laminar level with each other and with GABAA, D1, 5-HT1 and glutamate receptors; M2 receptors are codistributed only with nicotinic receptors. Immuno-histochemical studies with antibodies against muscarinic and nicotinic receptors demonstrate that these structures occur mainly in pyramidal and spiny stellate cells and to a lesser extent (13%) in a variety of interneurons. The immunoreactivity is visible in the perikaryon, dendrites and postsynaptic membranes. Neurons are found in the human neocortex, which react exclusively with one of the two antibodies, but a fraction of the neurons (about 30%) contains antigenic sites reacting with both antibodies. This is interpreted as colocalization of nicotinic and muscarinic receptors in some cortical neurons.

  13. Cholinergic stimulation of pancreatic amylase release and muscarinic receptors: effect of ionophore A23187

    SciTech Connect

    Larose, L.; Morisset, J.

    1985-07-22

    Dispersed rat pancreatic acini were incubated in 0.5 mM calcium medium with increasing concentrations of carbamylcholine, with or without the ionophore A23187 (10/sup -6/M). Addition of the ionophore reduced maximal amylase release, increased the maximal effective concentration of carbamylcholine and dramatically impaired the agonist's capacity to induce enzyme secretion at low concentration. The ionophore also abolished the inhibition of secretion observed at high carbamylcholine concentrations. These effects of the ionophore on the cholinergic secretory response cannot be explained by interaction at the muscarinic receptor since neither the Bmax, the affinity of the receptor for the (/sup 3/H)QNB nor the binding of carbamylcholine were affected by the ionophore. It is suggested that for the conditions studied, the ionophore can interact with the secretory process at one or several points ulterior to the initial recognition site of carbamylcholine on its receptor. 30 references, 3 figures.

  14. Mixed nicotinic and muscarinic features of cholinergic receptor coupled to secretion in bovine chromaffin cells

    SciTech Connect

    Shirvan, M.H.; Pollard, H.B.; Heldman, E. )

    1991-06-01

    Acetylcholine evokes release from cultured bovine chromaffin cells by a mechanism that is believed to be classically nicotinic. However, the authors found that the full muscarinic agonist oxotremorine-M (Oxo-M) induced a robust catecholamine (CA) secretion. By contrast, muscarine, pilocarpine, bethanechol, and McN-A-343 did not elicit any secretory response. Desensitization of the response to nicotine by Oxo-M and desensitization of the response to Oxo-M by nicotine suggest that both nicotine and Oxo-M were acting at the same receptor. Additional experiments supporting this conclusion show that nicotine-induced secretion and Oxo-M-induced secretion were similarly blocked by various muscarinic and nicotinic antagonists. Moreover, secretion induced by nicotine and Oxo-M were Ca{sup 2+} dependent, and both agonists induced {sup 45}Ca{sup 2+} uptake. Equilibrium binding studies showed that ({sup 3}H)Oxo-M bound to chromaffin cell membranes with a K{sub d} value of 3.08 {times} 10{sup {minus}8}M and a Hill coefficient of 1.00, suggesting one binding site for this ligand. Nicotine inhibited Oxo-M binding in a noncompetitive manner, suggesting that both ligands bind at two different sites on the same receptor. They propose that the receptor on bovine chromaffin cells that is coupled to secretion represents an unusual cholinergic receptor that has both nicotinic and muscarinic features.

  15. Mixed nicotinic and muscarinic features of cholinergic receptor coupled to secretion in bovine chromaffin cells.

    PubMed Central

    Shirvan, M H; Pollard, H B; Heldman, E

    1991-01-01

    Acetylcholine evokes release from cultured bovine chromaffin cells by a mechanism that is believed to be classically nicotinic. However, we found that the full muscarinic agonist oxotremorine-M (Oxo-M) induced a robust catecholamine (CA) secretion. By contrast, muscarine, pilocarpine, bethanechol, and McN-A-343 did not elicit any secretory response. Desensitization of the response to nicotine by Oxo-M and desensitization of the response to Oxo-M by nicotine suggest that both nicotine and Oxo-M were acting at the same receptor. Additional experiments supporting this conclusion show that nicotine-induced secretion and Oxo-M-induced secretion were similarly blocked by various muscarinic and nicotinic antagonists. Moreover, secretion induced by nicotine and Oxo-M were Ca2+ dependent, and both agonists induced 45Ca2+ uptake. Equilibrium binding studies showed that [3H]Oxo-M bound to chromaffin cell membranes with a Kd value of 3.08 x 10(-8) M and a Hill coefficient of 1.00, suggesting one binding site for this ligand. Nicotine inhibited Oxo-M binding in a noncompetitive manner, suggesting that both ligands bind at two different sites on the same receptor. We propose that the receptor on bovine chromaffin cells that is coupled to secretion represents an unusual cholinergic receptor that has both nicotinic and muscarinic features. Images PMID:2052567

  16. Structural characteristics of the recognition site for cholinergic ligands in the nicotinic acetylcholine receptor from squid optical ganglia

    SciTech Connect

    Plyashkevich, Yu.G.; Demushkin, V.P.

    1986-01-20

    The influence of chemical modification on the parameters of the binding of cholinergic ligands by the nicotinic acetylcholine receptor of squid optical ganglia was investigated. The presence of two subpopulations of recognition sites, differing in the composition of the groups contained in them, was detected. It was established with high probability that subpopulation I contains arginine and tyrosine residues and a carboxyl group while subpopulation II contains an amino group, a thyrosine residue, and a carboxyl group. Moreover, in both subpopulations there is an amino group important only for the binding of tubocurarin. On the basis of the results obtained, a model of the recognition sites for cholinergic ligands of the nicotinic acetylcholine receptor of squid optical ganglia is proposed.

  17. Sidedness and chemical and kinetic properties of the vesamicol receptor of cholinergic synaptic vesicles

    SciTech Connect

    Kornreich, W.D.; Parsons, S.M.

    1988-07-12

    Cholinergic synaptic vesicles isolated from Torpedo electric organ contain a receptor for the compound l-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183), which then occupied blocks storage of acetylcholine (AcCh). The inside or outside orientation of the receptor and its chemical and ligand binding kinetics characteristics were studied. Binding of (/sup 3/H)vesamicol to the receptor is inhibited efficiently by the protein modification reagents 4-(chloromercuri)benzenesulfonate and N,N'-dicyclohexylcarbodiimide and by protease treatment of cholate-solubilized receptor. The receptor in native vesicles is resistant to irreversible inactivation by proteases, elevated temperature, or pH extremes. (/sup 3/H)Vesamicol binding depends on deprotonation of a group of pK/sub a/sub 1// = 6.26 +/- 0.03 and protonation of a group of pK/sub a/sub 2// = 10.60 +/- 0.04, which is probably the tertiary amine of the drug molecule itself. The membrane-impermeant zwitterionic vesamicol analgoue dl-trans-4-oxo-4-((5,6,7,8-tetrahydro-6-hydroxy-7-(4-phenyl-1-piperidinyl)-1-naphthalenyl)amino)butanoic acid (TPNB) is an effective inhibitor of AcCh active transport. At 0/sup 0/C, 10 ..mu..M unlabeled vesamicol displaced 36 +/- 2% of a low concentration of bound (/sup 3/H)vesamicol at 0.16 +/- 0.02 min/sup -1/ and 64 +/- 2% at 0.013 +/- 0.001 min/sup -1/. One micromolar unlabeled vesamicol behaved similarly. Several types of receptor heterogeneity are consistent with the data. It is concluded that the vesamicol receptor is a stable protein often exhibiting heterogeneity, which faces the cytoplasmic compartment of the cholinergic nerve terminal. It probably contains a binding site carboxylate in a hydrophobic environment, which ion pairs with the protonated tertiary ammonium group of the drug. It also contains a cytoplasmically oriented sulfhydryl group, which is linked to but not part of the binding site.

  18. Diminished trkA receptor signaling reveals cholinergic-attentional vulnerability of aging

    PubMed Central

    Parikh, Vinay; Howe, William M.; Welchko, Ryan M.; Naughton, Sean X.; D'Amore, Drew E.; Han, Daniel H.; Deo, Monika; Turner, David L.; Sarter, Martin

    2012-01-01

    The cellular mechanisms underlying the exceptional vulnerability of the basal forebrain (BF) cholinergic neurons during pathological aging have remained elusive. Here we employed an adeno-associated viral vector-based RNA interference (AAV-RNAi) strategy to suppress the expression of trkA receptors by cholinergic neurons in the nucleus basalis of Meynert/ substantia innominata (nMB/SI) of adult and aged rats. Suppression of trkA receptor expression impaired attentional performance selectively in aged rats. Performance correlated with trkA levels in the nMB/SI. TrkA knockdown neither affected nMB/SI cholinergic cell counts nor the decrease in cholinergic cell size observed in aged rats. However, trkA suppression augmented an age-related decrease in the density of cortical cholinergic processes and attenuated the capacity of cholinergic neurons to release ACh. The capacity of cortical synapses to release acetylcholine (ACh) in vivo was also lower in aged/trkA-AAV-infused rats than in aged or young controls, and it correlated with their attentional performance. Furthermore, age-related increases in cortical proNGF and p75 receptor levels interacted with the vector-induced loss of trkA receptors to shift NGF signaling toward p75-mediated suppression of the cholinergic phenotype, thereby attenuating cholinergic function and impairing attentional performance. These effects model the abnormal trophic regulation of cholinergic neurons and cognitive impairments in patients with early Alzheimer's disease. This rat model is useful for identifying the mechanisms rendering aging cholinergic neurons vulnerable as well as for studying the neuropathological mechanisms that are triggered by disrupted trophic signaling. PMID:23228124

  19. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    SciTech Connect

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced /sup 155/Eu:/sup 3 +/ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor.

  20. Novel alkoxy-oxazolyl-tetrahydropyridine muscarinic cholinergic receptor antagonists.

    PubMed

    Shannon, H E; Bymaster, F P; Hendrix, J C; Quimby, S J; Mitch, C H

    1995-01-01

    The purpose of the present studies was to compare a novel series of alkoxy-oxazolyl-tetrahydropyridines (A-OXTPs) as muscarinic receptor antagonists. The affinity of these compounds for muscarinic receptors was determined by inhibition of [3H]pirenzepine to M1 receptors in hippocampus, [3H]QNB to M2 receptors in brainstem, and [3H]oxotremorine-M to high affinity muscarinic agonist binding sites in cortex. All of the compounds had higher affinity for [3H]pirenzepine than for [3H]QNB or [3H]oxotremorine-M labeled receptors, consistent with an interpretation that they are relatively selective M1 receptor antagonists, although none were as selective as pirenzepine. In addition, dose-response curves were determined for antagonism of oxotremorine-induced salivation (mediated by M3 receptors) and tremor (mediated by non-M1 receptors) in mice. In general, the A-OXTPs were equipotent and equieffective in antagonizing both salivation and tremor, although there were modest differences for some compounds. Dose-response curves also were determined on behavior maintained under a spatial-alternation schedule of food presentation in rats as a measure of effects on working memory. The A-OXTPs produced dose-related decreases in percent correct responding at doses three- to ten-fold lower than those which decreased rates of responding. However, only one compound, MB-OXTP, produced effects on percent correct responding consistent with a selective effect on memory as opposed to non-memory variables. The present results provide evidence that these alkoxy-oxazolyl-tetrahydropyridines are a novel series of modestly M1-selective muscarinic receptor antagonists, and that one member of the series, MB-OXTP, appears to be more selective in its effects on memory than previously studies muscarinic antagonists. PMID:7753969

  1. Ligands for SPECT and PET imaging of muscarinic-cholinergic receptors of the heart and brain

    SciTech Connect

    Knapp, F.F. Jr.; McPherson, D.W.; Luo, H.

    1995-06-01

    Interest in the potential use of cerebral SPECT and PET imaging for determination of the density and activity of muscarinic-cholinergic receptors (mAChR) has been stimulated by the changes in these receptors which occur in many neurological diseases. In addition, the important involvement of mAChR in modulating negative inotropic cardiac activity suggests that such receptor ligands may have important applications in evaluation of changes which may occur in cardiac disease. In this paper, the properties of several key muscarinic receptor ligands being developed or which have been used for clinical SPECT and PET are discussed. In addition, the ORNL development of the new iodinated IQNP ligand based on QNB and the results of in vivo biodistribution studies in rats, in vitro competitive binding studies and ex vivo autoradiographic experiments are described. The use of radioiodinated IQNP may offer several advantages in comparison to IQNB because of its easy and high yield preparation and high brain uptake and the potential usefulness of the {open_quotes}partial{close_quotes} subtype selective IONP isomers. We also describe the development of new IQNP-type analogues which offer the opportunity for radiolabeling with positron-emitting radioisotopes (carbon-11, fluorine-18 and bromine-76) for potential use with PET.

  2. Nipecotic acid ethyl ester: a cholinergic agonist that may differentiate muscarinic receptor subtypes

    SciTech Connect

    Zorn, S.H.; Duman, R.S.; Enna, S.J.; Krogsgaard-Larsen, P.; Micheletti, R.; Giraldo, E.; Giachetti, A.

    1986-03-05

    Reports indicate that nipecotic acid ethyl ester (NAEE) displays cholinomimetic properties in vivo. In the present study a series of physiological and biochemical tests were conducted to characterize this action. NAEE had a negative inotropic effect on the guinea pig atrium, and stimulated contraction of the guinea pig ileum and isolated mouse stomach strip at concentrations similar to bethanechol (BCH). The atrial and ilial effects were reversed by atropine. Unlike BCH, NAEE had no effect on basal acid secretion in the isolated mouse stomach at concentrations < 100 ..mu..M. NAEE was more potent than carbachol (CCH) in displacing /sup 3/H-ONB binding from rat brain membranes. The potency of NAEE to inhibit antagonist binding in rat heart membranes was enhanced by Mg/sup + +/ (Hill coefficient < 1.0) and reduced by Gpp(NH)p. Like CCH, NAEE inhibited GTP-stimulated adenylate cyclase in rat brain striatal membranes. As compared to CCH, NAEE had little effect (< 5%) as a stimulator of inositol phosphate (IP) production in rat brain slices. The results indicate that NAEE is a direct-acting muscarinic receptor agonist. Moreover, its differential effects on acid secretion, IP accumulation, and adenylate cyclase suggest that it may be useful for defining cholinergic receptor subclasses.

  3. Imaging muscarinic cholinergic receptors using I-123-paraiododexetimide and emission computed tomography

    SciTech Connect

    Frost, J.J.; Wilson, A.A.; Dannals, R.F.; Ravert, H.T.; Smith, A.C.; Wagner, H.N. Jr.

    1985-05-01

    Dexetimide is a high affinity muscarinic cholinergic receptor (MR) antagonist which binds preferentially to MR in vivo. The corresponding optical isomer of dexetimide is levetimide (LEV) which binds to MR with a thousand-fold lower affinity. Paraiododexetimide (IDEX) has an affinity slightly less than dexetimide (KD = 2.7 nM). Following the administration of I-125 IDEX (10 ..mu..g/kg,IV) to mice high radioactivity concentrations in the striatum and cerebral cortex and a low radioactivity concentration in the cerebellum were observed; this distribution corresponds to the known distribution of MR in the brain. Following the injection of I-125-ILEV a low and uniform radioactivity concentration was observed throughout the brain. Preliminary imaging studies using an Anger camera and I-123-IDEX in a baboon showed high radioactivity in the brain and parotid gland. The radioactivity in the parotid gland diminished with time whereas the radioactivity in the brain increased over the 90 min. imaging period, which suggests high affinity binding to MR in the brain. These results demonstrate the feasibility of imaging MR using single photon emission computed tomography and I-123-IDEX.

  4. Chronic aspartame affects T-maze performance, brain cholinergic receptors and Na+,K+-ATPase in rats.

    PubMed

    Christian, Brandon; McConnaughey, Kenneth; Bethea, Elena; Brantley, Scott; Coffey, Amy; Hammond, Leigha; Harrell, Shelly; Metcalf, Kasee; Muehlenbein, Danielle; Spruill, Willie; Brinson, Leslie; McConnaughey, Mona

    2004-05-01

    This study demonstrated that chronic aspartame consumption in rats can lead to altered T-maze performance and increased muscarinic cholinergic receptor densities in certain brain regions. Control and treated rats were trained in a T-maze to a particular side and then periodically tested to see how well they retained the learned response. Rats that had received aspartame (250 mg/kg/day) in the drinking water for 3 or 4 months showed a significant increase in time to reach the reward in the T-maze, suggesting a possible effect on memory due to the artificial sweetener. Using [(3)H]quinuclidinyl benzilate (QNB) (1 nM) to label muscarinic cholinergic receptors and atropine (10(-6) M) to determine nonspecific binding in whole-brain preparations, aspartame-treated rats showed a 31% increase in receptor numbers when compared to controls. In aspartame-treated rats, there was a significant increase in muscarinic receptor densities in the frontal cortex, midcortex, posterior cortex, hippocampus, hypothalamus and cerebellum of 80%, 60%, 61%, 65%, 66% and 60%, respectively. The midbrain was the only area where preparations from aspartame-treated rats showed a significant increase in Na(+),K(+)-ATPase activity. It can be concluded from these data that long-term consumption of aspartame can affect T-maze performance in rats and alter receptor densities or enzymes in brain.

  5. Muscarinic cholinergic and alpha 2-adrenergic receptors in the epithelium and muscularis of the human ileum

    SciTech Connect

    Lepor, H.; Rigaud, G.; Shapiro, E.; Baumann, M.; Kodner, I.J.; Fleshman, J.W. )

    1990-04-01

    The aim of this study was to characterize the binding and functional properties of muscarinic cholinergic (MCh) and alpha 2-adrenergic receptors in the human ileum to provide insight into pharmacologic strategies for managing urinary and fecal incontinence after bladder and rectal replacement with intestinal segments. MCh and alpha 2-adrenergic binding sites were characterized in the epithelium and muscularis of eight human ileal segments with 3H-N-methylscopolamine and 3H-rauwolscine, respectively. The dissociation constant for 3H-N-methylscopolamine in the epithelium and muscularis was 0.32 +/- 0.07 nmol/L and 0.45 +/- 0.10 nmol/L, respectively (p = 0.32). The MCh receptor content was approximately eightfold greater in the muscularis compared with the epithelium (p = 0.008). The dissociation constant for 3H-rauwolscine in the muscularis and epithelium was 2.55 +/- 0.42 nmol/L and 2.03 +/- 0.19 nmol/L, respectively (p = 0.29). The alpha 2-adrenoceptor density was twofold greater in the epithelium compared with the muscularis (p = 0.05). Noncumulative concentration-response experiments were performed with carbachol, an MCh agonist, and UK-14304, a selective alpha 2-adrenergic agonist. The epithelium did not contract in the presence of high concentrations of carbachol and UK-14304. The muscularis preparations were responsive only to carbachol. The muscularis contains primarily MCh receptors mediating smooth muscle contraction. The alpha 2-adrenoceptors are localized primarily to the epithelium and may regulate water secretion in the intestine. The distribution and functional properties of ileal MCh and alpha 2-adrenergic receptors provide a theoretic basis for the treatment of incontinence after bladder and rectal replacement with intestinal segments.

  6. Prenatal exposure of rats to nicotine causes persistent alterations of nicotinic cholinergic receptors

    PubMed Central

    Gold, Allison B.; Keller, Ashleigh B.; Perry, David C.

    2010-01-01

    We examined for immediate and persistent changes in nAChRs in cerebral cortex, thalamus and striatum of male rats caused by prenatal exposure to nicotine from gestational day 3 to postnatal day 10 (PN10), and how such exposure affected the responses of adolescents to subsequent nicotine challenge. Receptor numbers were assessed by [3H]epibatidine binding and receptor function was measured by acetylcholine-stimulated 86Rb efflux (cerebral cortex and thalamus) and nicotine-stimulated dopamine release (striatum). Immediate effects of prenatal nicotine, assessed in PN10 animals, were not detected for any parameter. A subsequent 14 day nicotine exposure in adolescence revealed persistent changes caused by prenatal nicotine exposure. Nicotine exposure in adolescents caused up-regulation of binding in all three regions; however, this up-regulation was lost in thalamus from animals prenatally exposed to nicotine. Nicotine exposure in adolescents caused decreased nicotine-stimulated dopamine release in striatum; this effect was also lost in animals prenatally exposed to nicotine. Comparison of parameters in PN10 and PN42 rats revealed developmental changes in the CNS cholinergic system. In thalamus, binding increased with age, as did the proportion of 86Rb efflux with high sensitivity to acetylcholine. In cortex, binding also increased with age, but there was no change in total 86Rb efflux, and the proportion of high to low sensitivity efflux declined with age. Nicotine-stimulated striatal dopamine release (both total and α-conotoxin MII-resistant release) increased with age in naïve animals, but not in those prenatally exposed to nicotine. These findings demonstrate that prenatal exposure to nicotine causes alterations in the regulation of nAChRs by nicotine that persist into adolescence. These changes may play a role in the increased risk for nicotine addiction observed in adolescent offspring of smoking mothers. PMID:19028470

  7. Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity

    SciTech Connect

    Zarbin, M.A.; Wamsley, J.K.; Kuhar, M.J.

    1982-07-01

    The presence and transport of muscarinic cholinergic binding sites have been detected in the rat vagus nerve. These binding sites accumulate both proximal and distal to ligatures in a time-dependent manner. The results of double ligature and colchicine experiments are compatible with the notion that the anterogradely transported binding sites move by fast transport. Most of the sites accumulating proximal to ligatures bind the agonist carbachol with high affinity, while most of the sites accumulating distally bind carbachol with a low affinity. Also, the receptors transported in the anterograde direction are affected by a guanine nucleotide analogue (GppNHp), while those transported in the retrograde direction are less, or not, affected. The bulk of the sites along the unligated nerve trunk bind carbachol with a low affinity and are less sensitive to GppNHp modulation than the anterogradely transported sites. These results suggest that some receptors in the vagus may undergo axonal transport in association with regulatory proteins and that receptor molecules undergo changes in their binding and regulatory properties during their life cycle. These data also support the notion that the high and low affinity agonist form of the muscarinic receptor represent different modulated forms of a single receptor molecule.

  8. Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats

    SciTech Connect

    Balduini, W.; Murphy, S.D.; Costa, L.G. )

    1990-05-01

    Hydrolysis of phosphoinositides elicited by stimulation of cholinergic muscarinic receptors has been studied in brain from neonatal (7-day-old) rats in order to determine: (1) whether the neonatal rat could provide a good model system to study this signal-transduction pathway; and (2) whether potential differences with adult nerve tissue would explain the differential, age-related effects of cholinergic agonists. Accumulation of (3H) inositol phosphates in (3H)inositol prelabeled slices from neonatal and adult rats was measured as an index of phosphoinositide metabolism. Full (acetylcholine, methacholine, carbachol) and partial (oxotremorine, bethanechol) agonists had qualitatively similar, albeit quantitatively different, effects in neonatal and adult rats. Atropine and pirenzepine effectively blocked the carbachol-induced response with inhibition constants of 1.2 and 20.7 nM, respectively. In all brain areas, response to all agonists was higher in neonatal than adult rats, and in hippocampus and cerebral cortex the response was higher than in cerebellum or brainstem. The relative intrinsic activity of partial agonists was higher in the latter two areas (0.6-0.7) than in the former two (0.3-0.4). Carbachol-stimulated phosphoinositide metabolism in brain areas correlated well with the binding of (3H)QNB (r2 = 0.627) and, particularly, with (3H)pirenzepine (r2 = 0.911). In cerebral cortex the effect of carbachol was additive to that of norepinephrine and glutamate. The presence of calcium (250-500 microM) was necessary for maximal response to carbachol to be elicited; the EC50 value for Ca2+ was 65.4 microM. Addition of EDTA completely abolished the response. Removal of sodium ions from the incubation medium reduced the response to carbachol by 50%.

  9. Affinities of pirenzepine for muscarinic cholinergic receptors in membranes isolated from bovine tracheal mucosa and smooth muscle

    SciTech Connect

    Madison, J.M.; Jones, C.A.; Tom-Moy, M.; Brown, J.K.

    1987-03-01

    Muscarinic cholinergic receptors have been classified into subtypes based on their high (M-1 subtype) or low (M-2 subtype) affinities for the nonclassic antagonist pirenzepine, and this classification has important experimental and therapeutic implications. Because muscarinic receptors are abundant in the airways where they mediate several different cellular responses, the goal of this study was to characterize the affinities of pirenzepine for the muscarinic receptors in bovine tracheal mucosa and smooth muscle. After isolating membrane particulates from mucosa and smooth muscle, as well as from bovine cerebral cortex (a known source of M-1 receptors), we used /sup 3/H-quinuclidinyl benzilate to label muscarinic receptors in the particulates and performed competition radioligand binding assays in the presence of either atropine or pirenzepine. Receptors from all 3 tissues (mucosa, smooth muscle, and cerebral cortex) were of a relatively uniform affinity for atropine (range of KI values: 0.8 +/- 0.4 X 10(-9) to 2.4 +/- 1.7 X 10(-9) M), as would be predicted for this classic muscarinic antagonist. By contrast, affinities for pirenzepine differed depending on the tissue. In cerebral cortex, the majority of receptors were of high affinity for pirenzepine (KI = 1.8 +/- 1.4 X 10(-8) M). In both mucosa and smooth muscle, receptors were of low affinity for pirenzepine (Kl = 4.8 +/- 0.4 to 6.9 +/- 3.8 X 10(-7) M). We conclude that muscarinic cholinergic receptors in bovine tracheal mucosa and smooth muscle are predominantly of the M-2 subtype.

  10. Selective increase of alpha 1-adrenoceptors and muscarinic cholinergic receptors in rat cerebral cortex after chronic haloperidol.

    PubMed

    Pazo, J H; Levi de Stein, M; Jerusalinsky, D; Novas, M L; Raskovsky, S; Tumilasci, O R; Medina, J H; De Robertis, E

    1987-06-30

    The effect of chronic administration of haloperidol on alpha 1-, alpha 2-, and beta-adrenoceptors, cholinergic muscarinic, GABAA and benzodiazepine receptors in the cerebral cortex of the rat was investigated. Doses of 0.3 and 2 mg/kg of haloperidol during 7 days increased markedly the density of alpha 1-adrenoceptors without changes in affinity. The alpha 2- and beta-adrenoceptors were not modified after neuroleptic administration. The number of muscarinic receptors were also increased after haloperidol treatment (2 mg/kg/day). However, the GABAA and benzodiazepine binding sites remained unchanged. In the brainstem an increment in the alpha 1-, but not the beta-adrenoceptors was observed. The well known increase in the dopamine receptors in the striatum was confirmed. These observations demonstrate a multireceptor effect of haloperidol in the cerebral cortex.

  11. Nematode cholinergic pharmacology

    SciTech Connect

    Segerberg, M.A.

    1989-01-01

    Nematode acetylcholine (ACh) receptors were characterized using both biochemical and electrophysiological techniques, including: (1) receptor binding studies in crude homogenates of the free-living nematode Caenorhabditis elegans and the parasitic nematode Ascaris lumbricoides with the high-affinity probe ({sup 3}H)N-methylscopolamine (({sup 3}H)NMS) which binds to muscarinic receptors in many vertebrate and invertebrate tissues (2) measurement of depolarization and contraction induced by a variety of cholinergic agents, including N-methylscopolamine (NMS), in an innervated dorsal muscle strip preparation of Ascaris; (3) examination of the antagonistic actions of d-tubocurarine (dTC) and NMS at dorsal neuromuscular junction; (4) measurement of input resistance changes in Ascaris commissural motorneurons induced by ACh, dTC, NMS, pilocarpine and other cholinergic drugs.

  12. 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.

  13. Immunization Against Specific Fragments of Neurotrophin p75 Receptor Protects Forebrain Cholinergic Neurons in the Olfactory Bulbectomized Mice

    PubMed Central

    Bobkova, Natalia; Vorobyov, Vasily; Medvinskaya, Natalia; Nesterova, Inna; Tatarnikova, Olga; Nekrasov, Pavel; Samokhin, Alexander; Deev, Alexander; Sengpiel, Frank; Koroev, Dmitry; Volpina, Olga

    2016-01-01

    Alzheimer’s disease (AD) is characterized by progressive cognitive impairment associated with marked cholinergic neuron loss and amyloid-β (Aβ) peptide accumulation in the brain. The cytotoxicity in AD is mediated, at least in part, by Aβ binding with the extracellular domain of the p75 neurotrophin receptor (p75NTR), localized predominantly in the membranes of acetylcholine-producing neurons in the basal forebrain. Hypothesizing that an open unstructured loop of p75NTR might be the effective site for Aβ binding, we have immunized both olfactory bulbectomized (OBX) and sham-operated (SO) mice (n = 82 and 49, respectively) with synthetic peptides, structurally similar to different parts of the loops, aiming to block them by specific antibodies. OBX-mice have been shown in previous studies, and confirmed in the present one, to be characterized by typical behavioral, morphological, and biochemical AD hallmarks, including cholinergic deficits in forebrain neurons. Immunization of OBX- or SO-mice with KLH conjugated fragments of p75NTR induced high titers of specific serum antibodies for each of nine chosen fragments. However, maximal protective effects on spatial memory, evaluated in a Morris water maze, and on activity of choline acetyltransferase in forebrain neurons, detected by immunoreactivity to specific antibodies, were revealed only for peptides with amino acid residue sequences of 155–164 and 167–176. We conclude that the approach based on immunological blockade of specific p75NTR sites, linked with the cytotoxicity, is a useful and effective tool for study of AD-associated mechanisms and for development of highly selective therapy of cholinergic malfunctioning in AD patients. PMID:27163825

  14. Cholinergic regulation of the vasopressin neuroendocrine system

    SciTech Connect

    Michels, K.M.

    1987-01-01

    To clarify the physical and functional relationship between the cholinergic system, and the neurodocrine cells of the supraoptic nucleus, a combination of experiments on receptor binding, localization and function were carried out. The putative nicotinic receptor probe (/sup 125/I)alpha bungarotoxin ((/sup 125/I)alpha BTX) bound with high affinity and specificity to the vasopressin and oxytocin magnocellular neurons of the supraoptic nucleus, nucleus circularis, and paraventricular nucleus. Binding of (/sup 125/I)alpha BTX within the neural lobe was very low. In contrast, the muscarinic cholinergic receptor probe (/sup 3/H)quinuclidinylbenzilate ((/sup 3/H)QNB) did not bind to magnocellular vasopressin and oxytocin cell groups. The median eminence, which contains the neurosecretory axons, and the neural lobe of the pituitary contain low levels of (/sup 3/H)QNB binding. The physiological significance of these cholinergic receptors in regulation of vasopressin release was tested using an in vitro preparation of the supraoptic - neural lobe system.

  15. Recovery of some functional properties of the detergent-extracted cholinergic receptor protein from Torpedo marmorata after reintegration into a membrane environment.

    PubMed

    Briley, M S; Changeux, J P

    1978-03-15

    The change of affinity of the acetylcholine receptor for agonists and the influence of local anaesthetics has been studied in detail in receptor-rich membranes. These properties are changed after solubilisation by ionic detergents. A method for reproducibly reintegrating the receptor protein into a lipid environment is described. Reintegration of the receptor results in partial recovery of the binding and fluorescence properties of the membrane-bound receptor protein. In particular, the slow affinity change caused by agonists can be recovered but not the effect of local anaesthetics on this change. The fluorescence response to cholinergic ligands of the reintegrated receptor protein labelled with quinacrine does not appear identical to that found with the native receptor-rich membranes. It is suggested that the failure to recover the sensitivity to local anaesthetics is at the origin of the difficulties to regain functional reconstitution.

  16. Role of muscarinic and nicotinic cholinergic receptors in an experimental model of epilepsy-induced analgesia.

    PubMed

    de Freitas, Renato Leonardo; de Oliveira, Rithiele Cristina; de Carvalho, Andressa Daiane; Felippotti, Tatiana Tocchini; Bassi, Gabriel Shimizu; Elias-Filho, Daoud Hibrahim; Coimbra, Norberto Cysne

    2004-10-01

    The blockade of GABA-mediated Cl(-) influx with pentylenetetrazol (PTZ) was used in the present work to induce seizures in animals. The neurotransmission in the postictal period has been the focus of many studies, and there is evidence suggesting antinociceptive mechanisms following tonic-clonic seizures in both animals and men. The aim of this work was to study the involvement of acetylcholine in the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg/kg). Analgesia was measured by the tail-flick test in eight albino Wistar rats per group. Convulsions were followed by significant increases in tail-flick latencies (TFLs) at least for 120 min of the postictal period. Peripheral administration of atropine (0.25, 1 and 4 mg/kg) caused a significant dose-dependent decrease in the TFL in seizing animals, as compared to controls. These data were corroborated by peripheral administration of mecamylamine, a nicotinic cholinergic receptor blocker, at the same doses (0.25, 1 and 4 mg/kg) used for the muscarinic cholinergic receptor antagonist. The recruitment of the muscarinic receptor was made 10 min postconvulsions and in subsequent periods of postictal analgesia, whereas the involvement of the nicotinic cholinergic receptor was implicated only after 30 min postseizures. The cholinergic antagonists caused a minimal reduction in body temperature, but did not impair baseline TFL, spontaneous exploration or motor coordination in the rotarod test at the maximal dose of 4 mg/kg. These results indicate that acetylcholine may be involved as a neurotransmitter in postictal analgesia.

  17. Cholinergic chemosensory cells of the thymic medulla express the bitter receptor Tas2r131.

    PubMed

    Soultanova, Aichurek; Voigt, Anja; Chubanov, Vladimir; Gudermann, Thomas; Meyerhof, Wolfgang; Boehm, Ulrich; Kummer, Wolfgang

    2015-11-01

    The thymus is the site of T cell maturation which includes positive selection in the cortex and negative selection in the medulla. Acetylcholine is locally produced in the thymus and cholinergic signaling influences the T cell development. We recently described a distinct subset of medullary epithelial cells in the murine thymus which express the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and components of the canonical taste transduction cascade, i.e. transient receptor potential melastatin-like subtype 5 channel (TRPM5), phospholipase Cβ(2), and Gα-gustducin. Such a chemical phenotype is characteristic for chemosensory cells of mucosal surfaces which utilize bitter receptors for detection of potentially hazardous compounds and cholinergic signaling to initiate avoidance reflexes. We here demonstrate mRNA expression of bitter receptors Tas2r105, Tas2r108, and Tas2r131 in the murine thymus. Using a Tas2r131-tauGFP reporter mouse we localized the expression of this receptor to cholinergic cells expressing the downstream elements of the taste transduction pathway. These cells are distinct from the medullary thymic epithelial cells which promiscuously express tissue-restricted self-antigens during the process of negative selection, since double-labeling immunofluorescence showed no colocalization of autoimmune regulator (AIRE), the key mediator of negative selection, and TRPM5. These data demonstrate the presence of bitter taste-sensing signaling in cholinergic epithelial cells in the thymic medulla and opens a discussion as to what is the physiological role of this pathway.

  18. Cholinergic chemosensory cells of the thymic medulla express the bitter receptor Tas2r131.

    PubMed

    Soultanova, Aichurek; Voigt, Anja; Chubanov, Vladimir; Gudermann, Thomas; Meyerhof, Wolfgang; Boehm, Ulrich; Kummer, Wolfgang

    2015-11-01

    The thymus is the site of T cell maturation which includes positive selection in the cortex and negative selection in the medulla. Acetylcholine is locally produced in the thymus and cholinergic signaling influences the T cell development. We recently described a distinct subset of medullary epithelial cells in the murine thymus which express the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and components of the canonical taste transduction cascade, i.e. transient receptor potential melastatin-like subtype 5 channel (TRPM5), phospholipase Cβ(2), and Gα-gustducin. Such a chemical phenotype is characteristic for chemosensory cells of mucosal surfaces which utilize bitter receptors for detection of potentially hazardous compounds and cholinergic signaling to initiate avoidance reflexes. We here demonstrate mRNA expression of bitter receptors Tas2r105, Tas2r108, and Tas2r131 in the murine thymus. Using a Tas2r131-tauGFP reporter mouse we localized the expression of this receptor to cholinergic cells expressing the downstream elements of the taste transduction pathway. These cells are distinct from the medullary thymic epithelial cells which promiscuously express tissue-restricted self-antigens during the process of negative selection, since double-labeling immunofluorescence showed no colocalization of autoimmune regulator (AIRE), the key mediator of negative selection, and TRPM5. These data demonstrate the presence of bitter taste-sensing signaling in cholinergic epithelial cells in the thymic medulla and opens a discussion as to what is the physiological role of this pathway. PMID:26102274

  19. Naltrexone pretreatment blocks microwave-induced changes in central cholinergic receptors

    SciTech Connect

    Lai, H.; Carino, M.A.; Wen, Y.F.; Horita, A.; Guy, A.W. )

    1991-01-01

    Repeated exposure of rats to pulsed, circularly polarized microwaves (2,450-MHz, 2-microseconds pulses at 500 pps, power density 1 mW/cm2, at an averaged, whole-body SAR of 0.6 W/kg) induced biphasic changes in the concentration of muscarinic cholinergic receptors in the central nervous system. An increase in receptor concentration occurred in the hippocampus of rats subjected to ten 45-min sessions of microwave exposure, whereas a decrease in concentration was observed in the frontal cortex and hippocampus of rats exposed to ten 20-min sessions. These findings, which confirm earlier work in the authors' laboratory, were extended to include pretreatment of rats with the narcotic antagonist naltrexone (1 mg/kg, IP) before each session of exposure. The drug treatment blocked the microwave-induced changes in cholinergic receptors in the brain. These data further support the authors' hypothesis that endogenous opioids play a role in the effects of microwaves on central cholinergic systems.

  20. Comparative peptide mapping of adrenergic and cholinergic neutrotransmitter receptors by reverse-HPLC

    SciTech Connect

    Kerlavage, A.R.; Fraser, C.M.; Venter, J.C.; Shreeve, S.M.

    1986-05-01

    The authors have developed a methodology for unambiguously identifying neutrotransmitter receptor proteins and comparing structural features of related receptors as well as those in different classes. These techniques have been applied to the study of the ..cap alpha..- and ..beta..-adrenergic receptors as well as the muscarinic and nicotinic cholinergic receptors. The method involves comparative peptide mapping of total proteolytic digests of receptor proteins by microbore reverse-phase HPLC in conjunction with covalent modification by specific receptor ligands or (/sup 125/I)-labeling. Femtomole amounts of receptor can be analyzed. The maps of all the (/sup 125/I)-labeled receptors contain between 20 and 25 peaks and each receptor has a unique profile although all are similar in the very hydrophobic region of the map. The ..cap alpha../sub 2/-adrenergic receptor from human platelets has a higher total percentage of hydrophilic peaks than either the guinea pig lung ..beta../sub 2/-adrenergic receptor or the porcine atria muscarinic receptor. Two forms of the muscarinic receptor have very similar but clearly distinct profiles. The nicotinic receptor subunits show a higher degree of homology by this method than was revealed by previous mapping studies which utilized SDS-PAGE or thin-layer techniques.

  1. GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: implications for REM sleep control.

    PubMed

    Liang, Chang-Lin; Marks, Gerald A

    2014-01-16

    The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABA(A) receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABA(A) receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABA(A) receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABA(A) receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABA(A) receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABA(A) receptor subunit protein γ2. This finding implicates a specific subtype and location of GABA(A) receptors in PnO of rat in the control of REM sleep. PMID:24141149

  2. Nicotinic cholinergic receptors in rat brain. Annual report No. 2

    SciTech Connect

    Kellar, K.J.

    1985-05-13

    We have conducted experiments to determine if 3H acetylcholine (3Hach) nicotinic recognition sites are located presynaptically on catecholamine and/or serotonin axons. Lesions of these axons by intraventricular injections of neurotoxins resulted in marked decreases in 3Hach binding sites in the striatum and hypothalamus, but not in the cortex or thalamus. These results indicate that 3Hach nicotinic binding sites are located on catecholamine and serotonin axons in specific areas of the brain. In other experiments, we determined that repeated administration of nicotine results in enhanced behavioral responses to a subsequent injection of nicotine, and that there appears to be a correlation between the enhanced response to nicotine and increased 3Hach binding sites in cerebral cortex.

  3. Cholinergic modulation by opioid receptor ligands: potential application to Alzheimer's disease.

    PubMed

    Motel, William C; Coop, Andrew; Cunningham, Christopher W

    2013-03-01

    Morphinans have a storied history in medicinal chemistry as pain management drugs but have received attention as modulators of cholinergic signaling for the treatment of Alzheimer's Disease (AD). Galantamine is a reversible, competitive acetylcholinesterase (AChE) inhibitor and allosteric potentiating ligand of nicotinic acetylcholine receptors (nAChR-APL) that shares many common structural elements with morphinan-based opioids. The structurally diverse opioids codeine and eseroline, like galantamine, are also nAChR-APL that have greatly diminished affinity for AChE, representing potential lead compounds for selective nAChR-APL development. In accordance with the emerging repurposing trend of evaluating known compounds for novel pharmacological activity, ongoing research on augmentation of cholinergic signaling that has been aided by the use of opioids will be reviewed. PMID:22931533

  4. Postlesion estradiol treatment increases cortical cholinergic innervations via estrogen receptor-α dependent nonclassical estrogen signaling in vivo.

    PubMed

    Koszegi, Zsombor; Szego, Éva M; Cheong, Rachel Y; Tolod-Kemp, Emeline; Ábrahám, István M

    2011-09-01

    17β-Estradiol (E2) treatment exerts rapid, nonclassical actions via intracellular signal transduction system in basal forebrain cholinergic (BFC) neurons in vivo. Here we examined the effect of E2 treatment on lesioned BFC neurons in ovariectomized mice and the role of E2-induced nonclassical action in this treatment. Mice given an N-methyl-d-aspartic acid (NMDA) injection into the substantia innominata-nucleus basalis magnocellularis complex (SI-NBM) exhibited cholinergic cell loss in the SI-NBM and ipsilateral cholinergic fiber loss in the cortex. A single injection of E2 after NMDA lesion did not have an effect on cholinergic cell loss in the SI-NBM, but it restored the ipsilateral cholinergic fiber density in the cortex in a time- and dose-dependent manner. The most effective cholinergic fiber restoration was observed with 33 ng/g E2 treatment at 1 h after NMDA lesion. The E2-induced cholinergic fiber restoration was absent in neuron-specific estrogen receptor-α knockout mice in vivo. Selective activation of nonclassical estrogen signaling in vivo by estren induced E2-like restorative actions. Selective blockade of the MAPK or protein kinase A pathway in vivo prevented E2's ability to restore cholinergic fiber loss. Finally, studies in intact female mice revealed an E2-induced restorative effect that was similar to that of E2-treated ovariectomized mice. These observations demonstrate that a single E2 treatment restores the BFC fiber loss in the cortex, regardless of endogenous E2 levels. They also reveal the critical role of nonclassical estrogen signaling via estrogen receptor-α and protein kinase A-MAPK pathways in E2-induced restorative action in the cholinergic system in vivo.

  5. Presynaptic Excitation via GABAB Receptors in Habenula Cholinergic Neurons Regulates Fear Memory Expression.

    PubMed

    Zhang, Juen; Tan, Lubin; Ren, Yuqi; Liang, Jingwen; Lin, Rui; Feng, Qiru; Zhou, Jingfeng; Hu, Fei; Ren, Jing; Wei, Chao; Yu, Tao; Zhuang, Yinghua; Bettler, Bernhard; Wang, Fengchao; Luo, Minmin

    2016-07-28

    Fear behaviors are regulated by adaptive mechanisms that dampen their expression in the absence of danger. By studying circuits and the molecular mechanisms underlying this adaptive response, we show that cholinergic neurons of the medial habenula reduce fear memory expression through GABAB presynaptic excitation. Ablating these neurons or inactivating their GABAB receptors impairs fear extinction in mice, whereas activating the neurons or their axonal GABAB receptors reduces conditioned fear. Although considered exclusively inhibitory, here, GABAB mediates excitation by amplifying presynaptic Ca(2+) entry through Cav2.3 channels and potentiating co-release of glutamate, acetylcholine, and neurokinin B to excite interpeduncular neurons. Activating the receptors for these neurotransmitters or enhancing neurotransmission with a phosphodiesterase inhibitor reduces fear responses of both wild-type and GABAB mutant mice. We identify the role of an extra-amygdalar circuit and presynaptic GABAB receptors in fear control, suggesting that boosting neurotransmission in this pathway might ameliorate some fear disorders. PMID:27426949

  6. Effect of partial volume correction on muscarinic cholinergic receptor imaging with single-photon emission tomography in patients with temporal lobe epilepsy.

    PubMed

    Weckesser, M; Hufnagel, A; Ziemons, K; Griessmeier, M; Sonnenberg, F; Hackländer, T; Langen, K J; Holschbach, M; Elger, C E; Müller-Gärtner, H

    1997-09-01

    Animal experiments and preliminary results in humans have indicated alterations of hippocampal muscarinic acetylcholine receptors (mAChR) in temporal lobe epilepsy. Patients with temporal lobe epilepsy often present with a reduction in hippocampal volume. The aim of this study was to investigate the influence of hippocampal atrophy on the quantification of mAChR with single photon emission tomography (SPET) in patients with temporal lobe epilepsy. Cerebral uptake of the muscarinic cholinergic antagonist [123I]4-iododexetimide (IDex) was investigated by SPET in patients suffering from temporal lobe epilepsy of unilateral (n=6) or predominantly unilateral (n=1) onset. Regions of interest were drawn on co-registered magnetic resonance images. Hippocampal volume was determined in these regions and was used to correct the SPET results for partial volume effects. A ratio of hippocampal IDex binding on the affected side to that on the unaffected side was used to detect changes in muscarinic cholinergic receptor density. Before partial volume correction a decrease in hippocampal IDex binding on the focus side was found in each patient. After partial volume no convincing differences remained. Our results indicate that the reduction in hippocampal IDex binding in patients with epilepsy is due to a decrease in hippocampal volume rather than to a decrease in receptor concentration. PMID:9283110

  7. In vivo biodistribution of two ( sup 18 F)-labelled muscarinic cholinergic receptor ligands: 2-( sup 18 F)- and 4-( sup 18 F)-fluorodexetimide

    SciTech Connect

    Wilson, A.A.; Scheffel, U.A.; Dannals, R.F.; Stathis, M.; Ravert, H.T.; Wagner, H.N. Jr. )

    1991-01-01

    Two ({sup 18}F)-labelled analogues of the potent muscarinic cholinergic receptor (m-AChR) antagonist, dexetimide, were evaluated as potential ligands for imaging m-AChR by positron emission tomography (PET). Intravenous administration of both 2-({sup 18}F)- or 4-({sup 18}F)-fluorodexetimide resulted in high brain uptake of radioactivity in mice. High binding levels were observed in m-AChR rich areas, such as cortex and striatum, with low levels in the receptor-poor cerebellum. Uptake of radioactivity was saturable and could be blocked by pre-administration of dexetimide or atropine. Drugs with different sites of action were ineffective at blocking receptor binding. The results indicate that both radiotracers are promising candidates for use in PET studies.

  8. In vivo biodistribution of two [18F]-labelled muscarinic cholinergic receptor ligands: 2-[18F]- and 4-[18F]-fluorodexetimide.

    PubMed

    Wilson, A A; Scheffel, U A; Dannals, R F; Stathis, M; Ravert, H T; Wagner, H N

    1991-01-01

    Two [18F]-labelled analogues of the potent muscarinic cholinergic receptor (m-AChR) antagonist, dexetimide, were evaluated as potential ligands for imaging m-AChR by positron emission tomography (PET). Intravenous administration of both 2-[18F]- or 4-[18F]-fluorodexetimide resulted in high brain uptake of radioactivity in mice. High binding levels were observed in m-AChR rich areas, such as cortex and striatum, with low levels in the receptor-poor cerebellum. Uptake of radioactivity was saturable and could be blocked by pre-administration of dexetimide or atropine. Drugs with different sites of action were ineffective at blocking receptor binding. The results indicate that both radiotracers are promising candidates for use in PET studies. PMID:2008155

  9. Receptor binding properties of amperozide.

    PubMed

    Svartengren, J; Simonsson, P

    1990-01-01

    The receptor pharmacology of amperozide was investigated with in vitro radioligand binding technique. Amperozide possessed a high affinity to the 5-HT2 receptors (Ki = 16.5 +/- 2.1 nM) and a moderate affinity to alpha 1-adrenergic receptors of rat cerebral cortical membranes (Ki = 172 +/- 14 nM). The affinity of amperozide for striatal and limbic dopamine D2 receptors was low and not significantly different (Ki +/- S.E.M. = 540 +/- 59 nM vs 403 +/- 42 nM; p less than 0.11, n = 4). The affinity for striatal and limbic 5-HT2 receptors was measured as well and found to be very close to the affinity to the cerebral cortical 5-HT2 receptor. The drug affinity for D2 and 5-HT2 receptors seems thus not to be influenced by the location of the receptor moiety. The affinity for several other rat brain receptors such as 5-HT1A, alpha 2-adrenergic, dopamine D1, muscarinic M1 and M2, opiate sigma and beta 2-adrenergic was low. The pseudo-Hill coefficient of the amperozide competition binding curve was consistently higher than one indicating antagonistic and complex interactions with the 5-HT2 receptor or with alpha 1-adrenergic and dopamine D2 receptors. The antagonistic properties of amperozide were investigated by its ability to antagonize the serotonin-induced formation of inositol-1-phosphate in human blood platelets. Amperozide inhibited this 5-HT2 receptor-mediated intracellular response with similar potency as ketanserin. These results suggest that amperozide is a selective 5-HT2 receptor antagonist.

  10. Affinities of brompheniramine, chlorpheniramine, and terfenadine at the five human muscarinic cholinergic receptor subtypes.

    PubMed

    Yasuda, S U; Yasuda, R P

    1999-04-01

    Anticholinergic effects are presumed to be the mechanism for the efficacy of chlorpheniramine in symptomatic relief of the common cold. Terfenadine, a second-generation antihistamine, reportedly lacks anticholinergic side effects. We evaluated affinities of two commonly used over-the-counter antihistamines, brompheniramine and chlorpheniramine, as well as terfenadine in comparison with atropine at the five human muscarinic cholinergic receptor subtypes using CHO cells stably transfected with the individual subtypes. Atropine was more potent than all three drugs at m1-m5 (p<0.01). No significant difference was observed between chlorpheniramine and brompheniramine. Atropine, brompheniramine, and chlorpheniramine could not discriminate between m1-m5. Terfenadine demonstrated subtype selectivity at m3. In vitro comparisons in human muscarinic receptor subtypes could potentially be used to predict clinical anticholinergic effects of antihistamines and to target receptor-specific effects of such agents.

  11. Metabotropic glutamate 2 receptors modulate synaptic inputs and calcium signals in striatal cholinergic interneurons.

    PubMed

    Pisani, Antonio; Bonsi, Paola; Catania, Maria Vincenza; Giuffrida, Raffaella; Morari, Michele; Marti, Matteo; Centonze, Diego; Bernardi, Giorgio; Kingston, Ann E; Calabresi, Paolo

    2002-07-15

    Striatal cholinergic interneurons were recorded from a rat slice preparation. Synaptic potentials evoked by intrastriatal stimulation revealed three distinct components: a glutamatergic EPSP, a GABA(A)-mediated depolarizing potential, and an acetylcholine (ACh)-mediated IPSP. The responses to group II metabotropic glutamate (mGlu) receptor activation were investigated on the isolated components of the synaptic potentials. Each pharmacologically isolated component was reversibly reduced by bath-applied LY379268 and ((2S,1'R,2'R,3'R)-2-(2,3-dicarboxylcyclopropyl)-glycine, group II agonists. In an attempt to define the relevance of group II mGlu receptor activation on cholinergic transmission, we focused on the inhibitory effect on the IPSP, which was mimicked and occluded by omega-agatoxin IVA (omega-Aga-IVA), suggesting a modulation on P-type high-voltage-activated calcium channels. Spontaneous calcium-dependent plateau-potentials (PPs) were recorded with cesium-filled electrodes plus tetraethylammonium and TTX in the perfusing solution, and measurements of intracellular calcium [Ca2+]i changes were obtained simultaneously. PPs and the concomitant [Ca2+]i elevations were significantly reduced in amplitude and duration by LY379268. The mGlu-mediated inhibitory effect on PPs was mimicked by omega-Aga-IVA, suggesting an involvement of P-type channels. Moreover, electrically induced ACh release from striatal slices was reduced by mGlu2 receptor agonists and occluded by omega-Aga-IVA in a dose-dependent manner. Finally, double-labeling experiments combining mGlu2 receptor in situ hybridization and choline acetyltransferase immunocytochemistry revealed a strong mGlu2 receptor labeling on cholinergic interneurons, whereas single-label isotopic in situ hybridization for mGlu3 receptors did not show any labeling in these large striatal interneurons. These results suggest that the mGlu2 receptor-mediated modulatory action on cell excitability would tune striatal ACh release

  12. Positron emission tomographic investigations of central muscarinic cholinergic receptors with three isomers of [76Br]BrQNP.

    PubMed

    Strijckmans, V; Bottlaender, M; Luo, H; Ottaviani, M; McPherson, D W; Loc'h, C; Fuseau, C; Knapp, F F; Mazière, B

    1997-05-01

    We studied the potential of three radiobrominated isomers of BrQNP, (Z(-,-)-[76Br]BrQNP, E(-,-)-[76Br]BrQNP and E(-,+)-[76Br]BrQNP), as suitable radioligands for imaging of central muscarinic cholinergic receptors in the human brain. These radioligands were stereospecifically prepared by electrophilic radiobromodestannylation of the respective tributylstannyl precursors using no-carrier-added [76Br]BrNH4 and peracetic acid. Preliminary pharmacological characterizations were determined by biodistribution, autoradiography, competition, displacement and metabolite studies in rats. The (-,-)-configuration presented important specific uptakes in brain muscarinic cholinergic receptor (mAChR)-rich structures and in heart, low metabolization rates and an apparent M2 selectivity. The (-,+)-configuration revealed more rapid clearance, lower uptake, a higher metabolization rate and an apparent M1 selectivity. Reversibility of the binding was confirmed for the three radiotracers. Positron emission tomography in the living baboon brain revealed high and rapid uptake in the brain and accumulation in the mAChR-rich structures studied. At 30 min p.i., the E(-,-)-radiotracer reached a plateau in cortex, pons and thalamus with concentrations of 29%, 24% and 19% ID/l, respectively. Z(-,-)-[76Br]BrQNP also accumulated in these structures, reaching a maximal uptake (27% ID/l) in the cortex 2 h p.i. At 5 min p.i. a plateau (17% ID/l) was only observed in the cortex for the E(-, +)-[76Br]BrQNP; by contrast, the other structures showed slow washout. After 3 weeks, the (-,-)-radiotracers were studied in the same baboon pretreated with dexetimide (1 mg/kg), a well-known muscarinic antagonist. In all the mAChR structures, the highly reduced uptake observed after this preloading step indicates that these radiotracers specifically bind to muscarinic receptors. Z(-, -)-[76Br]BrQNP, which is displaced in higher amounts from M2 mAChR-enriched structures, reveals an M2 affinity. The two isomers

  13. Bilateral injection of fasciculin into the amygdala of rats: effects on two avoidance tasks, acetylcholinesterase activity, and cholinergic muscarinic receptors.

    PubMed

    Quillfeldt, J; Raskovsky, S; Dalmaz, C; Dias, M; Huang, C; Netto, C A; Schneider, F; Izquierdo, I; Medina, J H; Silveira, R

    1990-11-01

    These experiments examined the effects of the bilateral injection of fasciculin-2 (FAS), a natural acetylcholinesterase (AChE) inhibitory peptide, into the amygdala of rats on acquisition and retention of two avoidance behaviors. Intraamygdala injection of FAS (150 ng/amygdala) produced a pronounced and long-lasting inhibition of AChE activity: 85% and 74% on day 2 and day 5, respectively. After 48 hr, FAS-treated animals showed no changes in training or test session performance in a step-down inhibitory avoidance task (training-test interval was 24 hr). In a 2-way shuttle avoidance task, intraamygdala FAS slightly reduced retention test performance without modifying training session scores. Two and five days after FAS injections into the amygdala, the density of muscarinic receptor decreased about 50% as measured by the specific bindings of 3H-quinuclidinyl benzilate and 3H-oxotremorine. No alterations were observed in the apparent dissociation constants. On the other hand, the central-type benzodiazepine receptor population of the amygdala remained unchanged, suggesting that FAS microinjection did not produce damage to neuronal components of these nuclei. In conclusion, the results presented have indicated that a clear-cut and long-lasting inhibition of AChE activity in the amygdala is not accompanied by a facilitation of learning and memory of two different avoidance tasks. Compensation of the increased cholinergic activity by a down-regulation of muscarinic receptors could account for these findings.

  14. Physical Chemistry to the Rescue: Differentiating Nicotinic and Cholinergic Agonists

    ERIC Educational Resources Information Center

    King, Angela G.

    2005-01-01

    Researches suggest that two agonists can bind to the same binding site of an important transmembrane protein and elicit a biological response through strikingly different binding interactions. Evidence is provided which suggests two possible types of nicotinic acetylcholine receptor agonist binding like acetlycholine (cholinergic) or like nicotine…

  15. Involvement of Cholinergic and Adrenergic Receptors in Pathogenesis and Inflammatory Response Induced by Alpha-Neurotoxin Bot III of Scorpion Venom.

    PubMed

    Nakib, Imene; Martin-Eauclaire, Marie-France; Laraba-Djebari, Fatima

    2016-10-01

    Bot III neurotoxin is the most lethal α neurotoxin purified from Buthus occitanus tunetanus scorpion venom. This toxin binds to the voltage-gated sodium channel of excitable cells and blocks its inactivation, inducing an increased release of neurotransmitters (acetylcholine and catecholamines). This study aims to elucidate the involvement of cholinergic and adrenergic receptors in pathogenesis and inflammatory response triggered by this toxin. Injection of Bot III to animals induces an increase of peroxidase activities, an imbalance of oxidative status, tissue damages in lung parenchyma, and myocardium correlated with metabolic disorders. The pretreatment with nicotine (nicotinic receptor agonist) or atropine (muscarinic receptor antagonist) protected the animals from almost all disorders caused by Bot III toxin, especially the immunological alterations. Bisoprolol administration (selective β1 adrenergic receptor antagonist) was also efficient in the protection of animals, mainly on tissue damage. Propranolol (non-selective adrenergic receptor antagonist) showed less effect. These results suggest that both cholinergic and adrenergic receptors are activated in the cardiopulmonary manifestations induced by Bot III. Indeed, the muscarinic receptor appears to be more involved than the nicotinic one, and the β1 adrenergic receptor seems to dominate the β2 receptor. These results showed also that the activation of nicotinic receptor leads to a significant protection of animals against Bot III toxin effect. These findings supply a supplementary data leading to better understanding of the mechanism triggered by scorpionic neurotoxins and suggest the use of drugs targeting these receptors, especially the nicotinic one in order to counteract the inflammatory response observed in scorpion envenomation. PMID:27395044

  16. Quantitative studies on the localization of the cholinergic receptor protein in the normal and denervated electroplaque from Electrophorus electricus

    PubMed Central

    1978-01-01

    Electroplaques dissected from the electric organ of Electrophorus electricus are labeled by tritiated alpha1-isotoxin from Naja nigricollis, a highly selective reagent of the cholinergic (nicotinic) receptor site. Preincubation of the cell with an excess of unlabeled alpha-toxin and with a covalent affinity reagent or labeling in the presence of 10(-4) M decamethonium reduces the binding of [3H]alpha- toxin by at least 75%. Absolute surface densities of alpha-toxin sites are estimated by high-resolution autoradiography on the basis of silver grain distribution and taking into account the complex geopmetry of the cell surface. Binding of [3H]alpha-toxin on the noninnervated face does not differ from background. Labeled sites are observed on the innervated membrane both between the synapses and under the nerve terminals but the density of sites is approx. 100 times higher at the level of the synapses than in between. Analysis of the distance of silver grains from the innervated membrane shows a symmetrical distribution centered on the postsynaptic plasma membrane under the nerve terminal. In extrasynaptic areas, the barycenter of the distribution lies approximately 0.5 micrometer inside the cell, indicating that alpha-toxin sites are present on the membrane of microinvaginations, or caveolae, abundant in the extrajunctional areas. An absolute density of 49,600 +/- 16,000 sites/micrometer2 of postsynaptic membrane is calculated; it is in the range of that found at the crest of the folds at the neuromuscular junction and expected from a close packing of receptor molecules. Electric organs were denervated for periods up to 142 days. Nerve transmission fails after 2 days, and within a week all the nerve terminals disappear and are subsequently replaced by Schwann cell processes, whereas the morphology of the electroplaque remains unaffected. The denervated electroplaque develops some of the electrophysiological changes found with denervated muscles (increases of membrane

  17. Influence of acetylcholine on binding of 4-[125I]iododexetimide to muscarinic brain receptors.

    PubMed

    Weckesser, M; Fixmann, A; Holschbach, M; Müller-Gärtner, H W

    1998-11-01

    The distribution of nicotinic and muscarinic cholinergic receptors in the human brain in vivo has been successfully characterized using radiolabeled tracers and emission tomography. The effect of acetylcholine release into the synaptic cleft on receptor binding of these tracers has not yet been investigated. The present study examined the influence of acetylcholine on binding of 4-[125I]iododexetimide to muscarinic cholinergic receptors of porcine brain synaptosomes in vitro. 4-Iododexetimide is a subtype-unspecific muscarinic receptor antagonist with high affinity. Acetylcholine competed with 4-[125I]iododexetimide in a dose-dependent manner. A concentration of 500 microM acetylcholine inhibited 50% of total specific 4-[125I]iododexetimide binding to synaptosomes when both substances were given simultaneously. An 800 microM acetylcholine solution reduced total specific 4-[125I]iododexetimide binding by about 35%, when acetylcholine was given 60 min after incubation of synaptosomes with 4-[125I]iododexetimide. Variations in the synaptic acetylcholine concentration might influence muscarinic cholinergic receptor imaging in vivo using 4-[123I]iododexetimide. Conversely, 4-[123I]iododexetimide might be an appropriate molecule to investigate alterations of acetylcholine release into the synaptic cleft in vivo using single photon emission computed tomography. PMID:9863566

  18. Identification of cholinergic and non-cholinergic neurons in the pons expressing phosphorylated cyclic adenosine monophosphate response element-binding protein as a function of rapid eye movement sleep.

    PubMed

    Datta, S; Siwek, D F; Stack, E C

    2009-09-29

    Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of rapid eye movement (REM) sleep. In the present study performed on rats, phosphorylated cyclic AMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high rapid eye movement sleep (HR, approximately 27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With HR, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low rapid eye movement sleep (LR, approximately 2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr=0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr=0.88), PnO (Rsqr=0.87), and SubCD (Rsqr=0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr=0.70) and DRN (Rsqr=0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are

  19. Cholinergic and serotonergic alterations in the rat hippocampus following trimethyltin exposure and fetal neural transplantation.

    PubMed

    Roy, A; Agrawal, A K; Husain, R; Dubey, M P; Seth, P K

    1999-01-15

    Trimethyltin (TMT) apart from causing cholinergic denervation of the hippocampus, damages the serotonergic inputs into the hippocampus as well. In the present study, fetal cholinergic and serotonergic rich neuronal populations from septal and raphe regions, respectively, were transplanted alone or in combination (as co-grafts) in the hippocampus of TMT exposed rats. Neurotransmitter receptor binding and neurotransmitter levels were assayed 6 months post-transplantation. Fetal septal transplants (rich in cholinergic neurons) significantly restored the deficits in cholinergic (muscarinic) receptor binding and acetylcholinesterase activity caused by TMT exposure. Raphe transplants (rich in serotonergic neurons) restored the deficit in serotonergic receptor binding and serotonin levels caused by TMT. Co-grafts of fetal raphe and septal neurons restored both the cholinergic (muscarinic) and serotonergic receptor functions. The results suggest that co-grafting technique could provide a better restoration of functional deficits when more than one type of neuronal population is damaged. PMID:10025586

  20. Protein stability and interaction of the nicotinic acetylcholine receptor with cholinergic ligands studied by Fourier-transform infrared spectroscopy.

    PubMed Central

    Fernandez-Ballester, G; Castresana, J; Arrondo, J L; Ferragut, J A; Gonzalez-Ros, J M

    1992-01-01

    Based on the conformational dependence of the amide-I i.r. band, this paper explores the use of Fourier-transform i.r. spectroscopy methods to probe structural features of proteins present in native membranes from Torpedo highly enriched in acetylcholine receptor (AcChR). The interference of water absorbance on the amide-I spectral region has been eliminated through isotopic exchange by freeze-drying the membranes in the presence of trehalose to avoid protein denaturation induced by drying, followed by resuspension in deuterated water. AcChR-rich membrane samples prepared in such a way maintained an ability to undergo affinity-state transitions and to promote cation translocation in response to cholinergic agonists, which are functional characteristics of native untreated samples. The temperature-dependence of the i.r. spectrum indicates a massive loss of ordered protein structure, occurring at temperatures similar to those reported for thermal denaturation of the AcChR by differential scanning calorimetry and by thermal inactivation of alpha-bungarotoxin-binding sites on the AcChR [Artigues, Villar, Ferragut & Gonzalez-Ros (1987) Arch. Biochem. Biophys. 258, 33-41], thus suggesting that the observed i.r. spectral changes correspond to alterations in the structure of the AcChR protein. Furthermore, the presence of detergents as well as cholinergic agonists and antagonists produces spectral changes that are also consistent with the alterations in AcChR protein structure expected from previous calorimetric studies. In contrast with the information obtained by calorimetry, i.r. spectroscopy allows the contribution of secondary structural changes to be distinguished from the overall change in protein structure. Thus prolonged exposure to cholinergic agonists, which drives the AcChR protein into the desensitized state, produces only negligible alterations in the amide-I band shape, but increases substantially the thermal stability of the protein. This suggests that

  1. Concanavalin A amplifies both beta-adrenergic and muscarinic cholinergic receptor-adenylate cyclase-linked pathways in cardiac myocytes.

    PubMed Central

    Rocha-Singh, K J; Hines, D K; Honbo, N Y; Karliner, J S

    1991-01-01

    Concanavalin A (Con A) is a tetrameric plant lectin that disrupts plasma membrane-cytoskeletal interactions and alters plasma membrane fluidity. We used Con A as a probe to explore beta-adrenergic and muscarinic cholinergic receptor-mediated regulation of cAMP in intact neonatal rat ventricular myocytes. Preincubation with Con A, 0.5 micrograms/ml, attenuated 1 microM (-)-norepinephrine (NE)-induced downregulation of beta-adrenergic receptors and resulted in a 50% augmentation of cAMP accumulation stimulated by 1 microM NE. Con A also augmented forskolin (1-10 microM)-stimulated cAMP accumulation by an average of 37% (P less than 0.05); however, Con A preincubation had no effect on basal or cholera toxin-stimulated cAMP content. The muscarinic cholinergic agonist carbachol (1-100 microM) decreased 1 microM NE-stimulated cAMP generation by an average of 32% (n = 7, P less than 0.05); preincubation with Con A further enhanced the inhibitory effect of carbachol by 18% (n = 7, P less than 0.05). Carbachol (1 microM) for 2 h decreased muscarinic cholinergic receptor density in whole cells by 33%; preincubation with Con A prevented this receptor downregulation. Con A pretreatment did not affect (-)-isoproterenol- or forskolin-stimulated adenylate cyclase activity in cell homogenates, suggesting that an intact cytoarchitecture is necessary for Con A to augment cAMP formation. We conclude that Con A, through its modulation of beta-adrenergic and muscarinic cholinergic receptor signaling, amplifies both stimulatory and inhibitory adenylate cyclase-linked pathways in intact neonatal ventricular myocytes. These data suggest the possibility that plasma membrane-cytoskeletal interaction is an important regulator of transmembrane signaling because interference with this interaction results in alterations in cAMP accumulation mediated by both beta-adrenergic- and muscarinic cholinergic-adenylate cyclase pathways. PMID:1653274

  2. Glucocorticoid receptor transformation and DNA binding

    SciTech Connect

    Tienrungroj, W.

    1986-01-01

    The overall goal is to probe the mechanism whereby glucocorticoid receptors are transformed from a non-DNA-binding form to their active DNA-binding form. The author has examined the effect of an endogenous inhibitor purified from rat liver cytosol on receptor binding to DNA. The inhibitor binds to transformed receptors in whole cytosol and prevent their binding to DNA. He also examined the role of sulfhydryl groups in determining the DNA binding activity of the transformed receptor and in determining the transformation process. Treatment of rat liver cytosol containing temperature-transformed, (/sup 3/H)dexamethasone-bound receptors at 0/sup 0/C with the sulfhydryl modifying reagent methyl methanethiosulfonate inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol. In addition, he has examined the relationship between receptor phosphorylation and DNA binding. Untransformed receptor complexes purified from cytosol prepared from mouse L cells grown in medium containing (/sup 32/P)orthophosphate contain two components, a 100 k-Da and a 90-kDa subunit, both of which are phosphoproteins. On transformation, the receptor dissociates from the 90-kDa protein. Transformation of the complex under cell free conditions does not result in a dephosphorylation of the 100-kDa steroid-binding protein. Transformed receptor that has been bound to DNA and purified by monoclonal antibody is still in a phosphorylated form. These results suggest that dephosphorylation is not required for receptor binding to DNA.

  3. [3H]AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart.

    PubMed

    Wang, J X; Roeske, W R; Gulya, K; Wang, W; Yamamura, H I

    1987-10-01

    The in vitro binding properties of the novel muscarinic antagonist [3H]AF-DX 116 were studied using a rapid filtration technique. Association and dissociation rates of [3H]AF-DX 116 binding were rapid at 25 degrees C (2.74 and 2.70 X 10(7) min-1 M-1 for K+1; 0.87 and 0.93 min-1 for k-1) but 20-40 times slower at 0-4 degrees C (0.13 and 0.096 X 10(7) min-1 M-1 for k+1; 0.031 and 0.022 min-1 for k-1 in cerebral cortical and cardiac membranes, respectively). Kinetic dissociation constants (Kds) were estimated to be 31.8 nM and 30.9 nM at 25 degrees C; 23.1 nM and 0-4 degrees C for the cerebral cortex and heart, respectively. In saturation studies, [3H]AF-DX 116 labeled 29 percent of the total [3H](-)QNB binding sites in the cerebral cortical membranes and 87 percent in the cardiac membranes, with Kd values of 28.9 nM and 17.9 nM, respectively. Muscarinic antagonists inhibited [3H]AF-DX 116 binding in a rank order of potency of atropine greater than dexetimide greater than AF-DX 116 greater than PZ greater than levetimide in both tissues. Except for PZ/[3H]AF-DX 116 and AF-DX 116/[3H]AF-DX 116 in the cerebral cortex, all the antagonist competition curves had Hill coefficients close to one. Carbachol and oxotremorine produced shallow inhibition curves against [3H]AF-DX 116 binding in both tissues. Regional distribution studies with [3H](-)QNB, [3H]PZ and [3H]AF-DX 116 showed that most of the muscarinic receptors in the cerebral cortex, hippocampus, nucleus accumbens and corpus striatum are of the M1 subtype while those in the brainstem, cerebellum and other lower brain regions are of the M2 subtype. These results indicate that [3H]AF-DX 116 is a useful probe for the study of heterogeneity of muscarinic cholinergic receptors. PMID:3657382

  4. Dorsal raphe nucleus acetylcholine-mediated neurotransmission modulates post-ictal antinociception: The role of muscarinic and nicotinic cholinergic receptors.

    PubMed

    de Oliveira, Rithiele Cristina; de Oliveira, Ricardo; Biagioni, Audrey Francisco; Falconi-Sobrinho, Luiz Luciano; Coimbra, Norberto Cysne

    2016-01-15

    The dorsal raphe nucleus (DRN) is a key structure of the endogenous pain inhibitory system. Although the DRN is rich in serotoninergic neurons, cholinergic neurons are also found in that nucleus. Both ictal and inter-ictal states are followed by post-ictal analgesia. The present study investigated the role of cholinergic mechanisms in postictal antinociceptive processes using microinjections of atropine and mecamylamine, muscarinic and nicotinic cholinergic receptor antagonists, respectively, in the DRN of rats. Intraperitoneal injection of pentylenetetrazole (PTZ) (at 64mg/kg) caused tonic and tonic-clonic seizures. The convulsive motor reactions were followed by an increase in pain thresholds, a phenomenon known as post-ictal analgesia. Pre-treatment of the DRN with atropine or mecamylamine at 1µg, 3µg and 5µg/0.2µL decreased the post-ictal antinociceptive phenomenon. The present results showed that the post-ictal analgesia was mediated by muscarinic and nicotinic cholinergic receptors in the DRN, a structure crucially involved in the neural network that organises post-ictal hypoalgesia. PMID:26620541

  5. Muscarinic receptors of the vascular bed: radioligand binding studies on bovine splenic veins.

    PubMed

    Brunner, F; Kukovetz, W R

    1986-01-01

    Despite an obvious lack of parasympathetic innervation to the spleen, pharmacological evidence suggests the presence of cholinergic receptors in isolated bovine splenic veins. We therefore studied muscarinic cholinergic binding sites in a bovine splenic vein preparation by direct radioligand binding techniques using [3H]quinuclidinyl benzilate ([3H]QNB) as radioactive probe. Saturation experiments indicated one homogeneous class of high-affinity binding sites, with a KD of 0.11 nM and a binding site density Bmax of 55 fmol/mg protein. The rate constants at 37 degrees C for formation and dissociation of the [3H]QNB receptor complex were 2.7 X 10(9) M-1 h-1 and 0.38 h-1, respectively, yielding a KD of 0.14 nM. The binding sites showed a high stereospecificity, which was evident from competition experiments with dexetimide (KI = 1.3 nM) and levetimide (KI = 4.6 microM). In competition experiments with muscarinic and nicotinic antagonists and some antidepressants, only one binding site was found, whereas with muscarinic agonists, two binding sites were detected. In the presence of 0.1 mM guanyl-imido-diphosphate, only one binding site could be identified with the muscarinic agonist carbamylcholine. The affinity of [3H]QNB, on the other hand, was slightly decreased, and Bmax values were unchanged. It is concluded that specific, saturable, high-affinity muscarinic binding sites in the bovine splenic vein have been identified and characterized that exhibit properties similar to cholinergic receptors of brain and peripheral tissues and probably mediate acetylcholine-induced relaxation of splenic veins. PMID:2427809

  6. Local heating of human skin causes hyperemia without mediation by muscarinic cholinergic receptors or prostanoids.

    PubMed

    Golay, Sandrine; Haeberli, Christian; Delachaux, Anne; Liaudet, Lucas; Kucera, Paul; Waeber, Bernard; Feihl, François

    2004-11-01

    Local changes in surface temperature have a powerful influence on the perfusion of human skin. Heating increases local skin blood flow, but the mechanisms and mediators of this response (thermal hyperemia response) are incompletely elucidated. In the present study, we examined the possible dependence of the thermal hyperemia response on stimulation of muscarinic cholinergic receptors and on production of vasodilator prostanoids. In 13 male healthy subjects aged 20-30 yr, a temperature-controlled chamber was positioned on the volar face of one forearm and used to raise surface temperature from 34 to 41 degrees C. The time course of the resulting thermal hyperemia response was recorded with a laser-Doppler imager. In one experiment, each of eight subjects received an intravenous bolus of the antimuscarinic agent glycopyrrolate (4 microg/kg) on one visit and saline on the other. The thermal hyperemia response was determined within the hour after the injections. Glycopyrrolate effectively inhibited the skin vasodilation induced by iontophoresis of acetylcholine but did not influence the thermal hyperemia response. In a second experiment, conducted in five other subjects, 1 g of the cyclooxygenase inhibitor aspirin administered orally totally abolished the vasodilation induced in the skin by anodal current but also failed to modify the thermal hyperemia response. The present study excludes the stimulation of muscarinic receptors and the production of vasodilator prostaglandins as essential and nonredundant mechanisms for the vasodilation induced by local heating in human forearm skin. PMID:15247159

  7. Synthesis of radiotracers for studying muscarinic cholinergic receptors in the living human brain using positron emission tomography: [11C]dexetimide and [11C]levetimide.

    PubMed

    Dannals, R F; Långström, B; Ravert, H T; Wilson, A A; Wagner, H N

    1988-01-01

    Dexetimide (Fig. 1a), a potent muscarinic cholinergic receptor antagonist, and levetimide (Fig. 1b), its pharmacologically inactive enantiomer, were labeled with 11C for non-invasive in vivo studies of muscarinic cholinergic receptors in the human brain using positron emission tomography. The syntheses were completed in approximately 32 min using [alpha-11C]benzyl iodide as the precursor. The synthesis, purification, characterization and determination of specific activity are presented and discussed. PMID:2838435

  8. Culture density regulates both the cholinergic phenotype and the expression of the CNTF receptor in P19 neurons.

    PubMed

    Parnas, D; Linial, M

    1997-04-01

    The P19 embryonal carcinoma cells differentiate into neurons, astrocytes, and fibroblast-like cells following induction with retinoic acid. The cells mature into functional neurons, as determined by their ability to release neurotransmitters in a Ca(2+)- and depolarization-dependent manner. P19 neurons in culture represent a mixed population in terms of their neurotransmitter phenotype. The cholinergic phenotype of these neurons is modulated by culture density. Cholinergic markers, such as the vesicular acetylcholine transporter, acetyl cholinesterase, and choline acetyltransferase, are expressed in about 85% of the cells in sparse cultures and are largely suppressed at high cell densities. In contrast, glutamate release is enhanced in dense P19 neuronal cultures. The factor mediating the density effect is concentrated exclusively on the cell membrane of P19 neurons and not on the nonneuronal cells, which also differentiate from P19 embryonal carcinoma cells. This membrane-associated component retains its functionality, even after membrane fixation. The downregulation of the cholinergic properties in dense cultures is paralleled by a downregulation of the alpha subunit of the ciliary neurotrophic factor (CNTF) receptor. Thus, it is suggested that the membrane-associated factor, which mediates the density effect, downregulates the cholinergic phenotype by inhibiting the responsiveness of these neurons to CNTF. We further suggest that the P19 cell line can serve as a model system for the study of neurotransmitter phenotype acquisition and plasticity throughout neuronal differentiation. PMID:9188041

  9. RQ-00201894: A motilin receptor agonist causing long-lasting facilitation of human gastric cholinergically-mediated contractions.

    PubMed

    Broad, John; Takahashi, Nobuyuki; Tajimi, Masaomi; Sudo, Masaki; Góralczyk, Adam; Parampalli, Umesh; Mannur, Kesava; Yamamoto, Toshinori; Sanger, Gareth J

    2016-02-01

    The aim was to characterise RQ-00201894, a novel non-macrolide motilin agonist, using human recombinant receptors and then investigate its ability to facilitate cholinergic activity in human stomach. A reporter gene assay assessed motilin receptor function. Selectivity of action was determined using a panel of different receptors, ion channels, transporters and enzymes. Cholinergically-mediated muscle contractions were evoked by electrical field stimulation (EFS) of human gastric antrum. The results showed that RQ-00201894, motilin and erythromycin acted as full motilin receptor agonists (EC50: 0.20, 0.11, 69 nM, respectively). In this function, RQ-00201894 had >90-fold selectivity of action over its ability to activate the human ghrelin receptor (EC50 19 nM) and greater selectivity over all other receptors/mechanisms tested. In human stomach RQ-00201894 0.1-30 μM concentration-dependently increased EFS-evoked contractions (up to 1209%; pEC50 6.0). At 0.1-10 μM this activity was usually prolonged. At higher concentrations (3-30 μM) RQ-00201894 also caused a short-lasting muscle contraction, temporally disconnected from the increase in EFS-evoked contractions. RQ-00201894 10 μM did not consistently affect submaximal contractions evoked by carbachol. In conclusion, RQ-00201894 potently and selectively activates the motilin receptor and causes long-lasting facilitation of cholinergic activity in human stomach, an activity thought to correlate with an ability to increase gastric emptying. PMID:26685754

  10. Binding of ATP to the progesterone receptor.

    PubMed Central

    Moudgil, V K; Toft, D O

    1975-01-01

    The possible interaction of progesterone--receptor complexes with nucleotides was tested by affinity chromatography. The cytosol progesterone receptor from hen oviduct was partially purified by ammonium sulfate precipitation before use. When progesterone was bound to the receptor, the resulting complex could be selectively adsorbed onto columns of ATP-Sepharose. This interaction was reversible and of an ionic nature since it could be disrupted by high-salt conditions. A competitive binding assay was used to test the specificity of receptor binding to several other nucleotides, including ADP, AMP, and cAMP. A clear specificity for binding ATP was evident from these studies. When ATP was added to receptor preparations, the nucleotide did not affect the sedimentation properties or hormone binding characteristics of the receptor. Although the function of ATP remains unknown, these studies indicate a role of this nucleotide in some aspect of hormone receptor activity. PMID:165493

  11. Parallel maturation of the pancreatic secretory response to cholinergic stimulation and the muscarinic receptor population.

    PubMed Central

    Dumont, Y.; Larose, L.; Morisset, J.; Poirier, G. G.

    1981-01-01

    1 The appearance of pancreatic muscarinic receptors during development has been measured by use of the specific ligand [3H]-quinuclidinyl benzilate ([3H]-QNB). 2 QNB binding sites are present in foetal pancreas; their maximal concentration is attained at the age of 30 days and a significant decrease is observed in one year old animals. 3 Affinity of [3H]-QNB for the muscarinic receptor does not change with age. 4 An evaluation of the pancreatic secretory response to a cholinoceptor agonist as a function of age indicates that the development of this response parallels that of the receptor population. 5 It is suggested that, at all ages from 3 days after birth onwards, the maximal secretory response of the exocrine pancreas to a cholinoceptor agonist mobilizes the same proportion of the total population of QNB binding sites. PMID:6165420

  12. VPAC1 receptors regulate intestinal secretion and muscle contractility by activating cholinergic neurons in guinea pig jejunum.

    PubMed

    Fung, Candice; Unterweger, Petra; Parry, Laura J; Bornstein, Joel C; Foong, Jaime P P

    2014-05-01

    In the gastrointestinal tract, vasoactive intestinal peptide (VIP) is found exclusively within neurons. VIP regulates intestinal motility via neurally mediated and direct actions on smooth muscle and secretion by a direct mucosal action, and via actions on submucosal neurons. VIP acts via VPAC1 and VPAC2 receptors; however, the subtype involved in its neural actions is unclear. The neural roles of VIP and VPAC1 receptors (VPAC1R) were investigated in intestinal motility and secretion in guinea pig jejunum. Expression of VIP receptors across the jejunal layers was examined using RT-PCR. Submucosal and myenteric neurons expressing VIP receptor subtype VPAC1 and/or various neurochemical markers were identified immunohistochemically. Isotonic muscle contraction was measured in longitudinal muscle-myenteric plexus preparations. Electrogenic secretion across mucosa-submucosa preparations was measured in Ussing chambers by monitoring short-circuit current. Calretinin(+) excitatory longitudinal muscle motor neurons expressed VPAC1R. Most cholinergic submucosal neurons, notably NPY(+) secretomotor neurons, expressed VPAC1R. VIP (100 nM) induced longitudinal muscle contraction that was inhibited by TTX (1 μM), PG97-269 (VPAC1 antagonist; 1 μM), and hyoscine (10 μM), but not by hexamethonium (200 μM). VIP (50 nM)-evoked secretion was depressed by hyoscine or PG97-269 and involved a small TTX-sensitive component. PG97-269 and TTX combined did not further depress the VIP response observed in the presence of PG97-269 alone. We conclude that VIP stimulates ACh-mediated longitudinal muscle contraction via VPAC1R on cholinergic motor neurons. VIP induces Cl(-) secretion directly via epithelial VPAC1R and indirectly via VPAC1R on cholinergic secretomotor neurons. No evidence was obtained for involvement of other neural VIP receptors.

  13. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  14. Binding characteristics of swine erythrocyte insulin receptors

    SciTech Connect

    Dieberg, G.; Bryan, G.S.; Sartin, J.L.; Williams, J.C.; Prince, T.J.; Kemppainen, R.J.

    1985-09-01

    Crossbred gilts had 8.8 +/- 1.1% maximum binding of ( SVI)insulin to insulin receptors on erythrocytes. The number of insulin-binding sites per cell was 137 +/- 19, with a binding affinity ranging from 7.4 X 10(7)M-1 to 11.2 X 10(7)M-1 and mean of 8.8 X 10(7)M-1. Pregnant sows had a significant increase in maximum binding due to an increase in number of receptor sites per cell. Lactating sows fed a high-fiber diet and a low-fiber diet did not develop a significant difference in maximum binding of insulin. Sows fed the low-fiber diet had a significantly higher number of binding sites and a significantly lower binding affinity than did sows fed a high-fiber diet. Receptor-binding affinity was lower in the low-fiber diet group than in cycling gilts, whereas data from sows fed the high-fiber diet did not differ from data for cycling gilts. Data from this study indicated that insulin receptors of swine erythrocytes have binding characteristics similar to those in other species. Pregnancy and diet will alter insulin receptor binding in swine.

  15. Nicotinic cholinergic receptors in esophagus: Early alteration during carcinogenesis and prognostic value

    PubMed Central

    Chianello Nicolau, Marina; Pinto, Luis Felipe Ribeiro; Nicolau-Neto, Pedro; de Pinho, Paulo Roberto Alves; Rossini, Ana; de Almeida Simão, Tatiana; Soares Lima, Sheila Coelho

    2016-01-01

    AIM To compare expression of nicotinic cholinergic receptors (CHRNs) in healthy and squamous cell carcinoma-affected esophagus and determine the prognostic value. METHODS We performed RT-qPCR to measure the expression of CHRNs in 44 esophageal samples from healthy individuals and in matched normal surrounding mucosa, and in tumors from 28 patients diagnosed with esophageal squamous cell carcinoma (ESCC). Next, we performed correlation analysis for the detected expression of these receptors with the habits and clinico-pathological characteristics of all study participants. In order to investigate the possible correlations between the expression of the different CHRN subunits in both healthy esophagus and tissues from ESCC patients, correlation matrices were generated. Subsequently, we evaluated whether the detected alterations in expression of the various CHRNs could precede histopathological modifications during the esophageal carcinogenic processes by using receiver operating characteristic curve analysis. Finally, we evaluated the impact of CHRNA5 and CHRNA7 expression on overall survival by using multivariate analysis. RESULTS CHRNA3, CHRNA5, CHRNA7 and CHRNB4, but not CHRNA1, CHRNA4, CHRNA9 or CHRNA10, were found to be expressed in normal (healthy) esophageal mucosa. In ESCC, CHRNA5 and CHRNA7 were overexpressed as compared with patient-matched surrounding non-tumor mucosa (ESCC-adjacent mucosa; P < 0.0001 and P = 0.0091, respectively). Positive correlations were observed between CHRNA3 and CHRNB4 expression in all samples analyzed. Additionally, CHRNB4 was found to be differentially expressed in the healthy esophagus and the normal-appearing ESCC-adjacent mucosa, allowing for distinguishment between these tissues with a sensitivity of 75.86% and a specificity of 78.95% (P = 0.0002). Finally, CHRNA5 expression was identified as an independent prognostic factor in ESCC; patients with high CHRNA5 expression showed an increased overall survival, in comparison with

  16. Nicotinic cholinergic receptors in esophagus: Early alteration during carcinogenesis and prognostic value

    PubMed Central

    Chianello Nicolau, Marina; Pinto, Luis Felipe Ribeiro; Nicolau-Neto, Pedro; de Pinho, Paulo Roberto Alves; Rossini, Ana; de Almeida Simão, Tatiana; Soares Lima, Sheila Coelho

    2016-01-01

    AIM To compare expression of nicotinic cholinergic receptors (CHRNs) in healthy and squamous cell carcinoma-affected esophagus and determine the prognostic value. METHODS We performed RT-qPCR to measure the expression of CHRNs in 44 esophageal samples from healthy individuals and in matched normal surrounding mucosa, and in tumors from 28 patients diagnosed with esophageal squamous cell carcinoma (ESCC). Next, we performed correlation analysis for the detected expression of these receptors with the habits and clinico-pathological characteristics of all study participants. In order to investigate the possible correlations between the expression of the different CHRN subunits in both healthy esophagus and tissues from ESCC patients, correlation matrices were generated. Subsequently, we evaluated whether the detected alterations in expression of the various CHRNs could precede histopathological modifications during the esophageal carcinogenic processes by using receiver operating characteristic curve analysis. Finally, we evaluated the impact of CHRNA5 and CHRNA7 expression on overall survival by using multivariate analysis. RESULTS CHRNA3, CHRNA5, CHRNA7 and CHRNB4, but not CHRNA1, CHRNA4, CHRNA9 or CHRNA10, were found to be expressed in normal (healthy) esophageal mucosa. In ESCC, CHRNA5 and CHRNA7 were overexpressed as compared with patient-matched surrounding non-tumor mucosa (ESCC-adjacent mucosa; P < 0.0001 and P = 0.0091, respectively). Positive correlations were observed between CHRNA3 and CHRNB4 expression in all samples analyzed. Additionally, CHRNB4 was found to be differentially expressed in the healthy esophagus and the normal-appearing ESCC-adjacent mucosa, allowing for distinguishment between these tissues with a sensitivity of 75.86% and a specificity of 78.95% (P = 0.0002). Finally, CHRNA5 expression was identified as an independent prognostic factor in ESCC; patients with high CHRNA5 expression showed an increased overall survival, in comparison with

  17. Differential ontogeny of alpha 1-adrenergic and cholinergic receptor sites in the atria and ventricles of the inbred Dahl hypertension-sensitive (S/JR) and -resistant (R/JR) rat.

    PubMed

    McCaughran, J A; Juno, C J; O'Malley, E

    1987-10-01

    The ontogeny of atrial and ventricular alpha 1-adrenergic and muscarinic cholinergic receptor sites was investigated in inbred Dahl hypertension-sensitive (S/JR) and -resistant (R/JR) rats between 5 and 150 days of age. The density of sites in both cardiac regions was generally greater in the neonate than mature rat. A marked proliferation of sites was observed in neonatal and young adult rats that occurred in the following order: ventricular cholinoceptors----ventricular adrenoceptors----atrial cholinoceptors----atrial adrenoceptors. The density of ventricular adrenoceptors was greater in the S/JR rat than the R/JR rat at 5 days of age. At 150 days of age, the density of sites was less in the S/JR rat than the age-matched R/JR rat or the normotensive 50-day-old S/JR rat. The development of atrial adrenoceptors was similar between the strains, regardless of the blood pressure. The density of ventricular cholinergic receptors was greater in the S/JR strain at 5 and 15 days of age. However, the density of atrial cholinergic sites was consistently greater in the S/JR strain throughout development. The results of this study suggest that: (1) significant prenatal receptor development occurs in the heart; (2) receptor development may precede the functional maturation of postganglionic autonomic efferents; and (3) distinguishing differences in the regional density of alpha 1-adrenergic and muscarinic cholinergic binding sites are present between S/JR and R/JR rats at much earlier points in development than previously shown.

  18. Nicotinic cholinergic receptors in rat brain. Annual report No. 3, 1 May 85-30 Apr 86

    SciTech Connect

    Kellar, K.J.

    1986-05-01

    We have compared the characteristics of the recognition sites for 3(H)acetylcholine and 3H(-)nicotine in rat brain and found that the pharmacology, distribution, disulfide bond requirement, and regulation by chronic administration of nicotine and soman are identical. From these studies we conclude that 3Hacetylcholine and 3H(-)nicotine recognize the same recognition site which has the characteristics expected of a nicotinic cholinergic receptor. We have also determined that 3Hacetylcholine of high specific radioactivity (80 Ci/mmol) is an excellent ligand with which to study muscarinic receptors that have high affinity for agonists. These receptors may represent a subtype of muscarinic receptors found in brain, heart, glands, an some smooth muscle. (JS)

  19. Cholinergic modulation of the medial prefrontal cortex: the role of nicotinic receptors in attention and regulation of neuronal activity

    PubMed Central

    Bloem, Bernard; Poorthuis, Rogier B.; Mansvelder, Huibert D.

    2014-01-01

    Acetylcholine (ACh) release in the medial prefrontal cortex (mPFC) is crucial for normal cognitive performance. Despite the fact that many have studied how ACh affects neuronal processing in the mPFC and thereby influences attention behavior, there is still a lot unknown about how this occurs. Here we will review the evidence that cholinergic modulation of the mPFC plays a role in attention and we will summarize the current knowledge about the role between ACh receptors (AChRs) and behavior and how ACh receptor activation changes processing in the cortical microcircuitry. Recent evidence implicates fast phasic release of ACh in cue detection and attention. This review will focus mainly on the fast ionotropic nicotinic receptors and less on the metabotropic muscarinic receptors. Finally, we will review limitations of the existing studies and address how innovative technologies might push the field forward in order to gain understanding into the relation between ACh, neuronal activity and behavior. PMID:24653678

  20. The anterior cingulate cortex may enhance inhibition of lateral prefrontal cortex via m2 cholinergic receptors at dual synaptic sites.

    PubMed

    Medalla, Maria; Barbas, Helen

    2012-10-31

    The anterior cingulate cortex (ACC) and dorsolateral prefrontal cortices (DLPFC) share robust excitatory connections. However, during rapid eye movement (REM) sleep, when cortical activity is dominated by acetylcholine, the ACC is activated but DLPFC is suppressed. Using pathway tracing and electron microscopy in nonhuman primates (Macaca mulatta), we tested the hypothesis that the opposite states may reflect specific modulation by acetylcholine through strategic synaptic localization of muscarinic m2 receptors, which inhibit neurotransmitter release presynaptically, but are thought to be excitatory postsynaptically. In the ACC pathway to DLPFC (area 32 to area 9), m2 receptors predominated in ACC axon terminals and in more than half of the targeted dendrites of presumed inhibitory neurons, suggesting inhibitory cholinergic influence. In contrast, in a pathway linking the DLPFC area 46 to DLPFC area 9, postsynaptic m2 receptors predominated in targeted spines of presumed excitatory neurons, consistent with their mutual activation in working memory. These novel findings suggest that presynaptic and postsynaptic specificity of m2 cholinergic receptors may help explain the differential engagement of ACC and DLPFC areas in REM sleep for memory consolidation and synergism in awake states for cognitive control.

  1. Stereoselective L-(3H)quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors

    SciTech Connect

    Murray, T.F.; Mpitsos, G.J.; Siebenaller, J.F.; Barker, D.L.

    1985-12-01

    The muscarinic antagonist L-(/sup 3/H)quinuclidinyl benzilate (L-(/sup 3/H)QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-(/sup 3/H)QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-(/sup 3/H)QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-(/sup 3/H)QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-(/sup 3/H)QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-(/sup 3/H)QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-(/sup 3/H)QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-(/sup 3/H)QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.

  2. Stereoselective L-[3H]quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors.

    PubMed

    Murray, T F; Mpitsos, G J; Siebenaller, J F; Barker, D L

    1985-12-01

    The muscarinic antagonist L-[3H]quinuclidinyl benzilate (L-[3H]QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-[3H]QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-[3H]QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-[3H]QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-[3H]QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-[3H]QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-[3H]QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-[3H]QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution. PMID:4078624

  3. AT-1001 Is a Partial Agonist with High Affinity and Selectivity at Human and Rat α3β4 Nicotinic Cholinergic Receptors

    PubMed Central

    Tuan, Edward W.; Horti, Andrew G.; Olson, Thao T.; Gao, Yongiun; Stockmeier, Craig A.; Al-Muhtasib, Nour; Bowman Dalley, Carrie; Lewin, Amanda E.; Wolfe, Barry B.; Sahibzada, Niaz; Xiao, Yingxian

    2015-01-01

    AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at α3β4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat α3β4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human α3β4 and α4β2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for α3β4 receptors over α4β2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat α3β4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for α3β4 nAChRs and its higher affinity for human compared with rat receptors. In patch-clamp electrophysiology studies, AT-1001 was a potent partial agonist with 65–70% efficacy at both human and rat α3β4 nAChRs. It was also a less potent and weaker (18%) partial agonist at α4β2 nAChRs. Both α3β4 and α4β2 nAChRs are upregulated by exposure of cells to AT-1001 for 3 days. Similarly, AT-1001 desensitized both receptor subtypes in a concentration-dependent manner, but it was 10 and 30 times more potent to desensitize human α3β4 receptors than rat α3β4 and human α4β2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human α3β4 nAChR and shortest for the human α4β2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor. PMID:26162864

  4. Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

    PubMed Central

    Gocayne, J; Robinson, D A; FitzGerald, M G; Chung, F Z; Kerlavage, A R; Lentes, K U; Lai, J; Wang, C D; Fraser, C M; Venter, J C

    1987-01-01

    Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene. Images PMID:2825184

  5. Disparate cholinergic currents in rat principal trigeminal sensory nucleus neurons mediated by M1 and M2 receptors: a possible mechanism for selective gating of afferent sensory neurotransmission.

    PubMed

    Kohlmeier, Kristi A; Soja, Peter J; Kristensen, Morten P

    2006-06-01

    Neurons situated in the principal sensory trigeminal nucleus (PSTN) convey orofacial sensory inputs to thalamic relay regions and higher brain centres, and the excitability of these ascending tract cells is modulated across sleep/wakefulness states and during pain conditions. Moreover, acetylcholine release changes profoundly across sleep/wakefulness states and ascending sensory neurotransmission is altered by cholinergic agonists. An intriguing possibility is, therefore, that cholinergic mechanisms mediate such state-dependent modulation of PSTN tract neurons. We tested the hypotheses that cholinergic agonists can modulate PSTN cell excitability and that such effects are mediated by muscarinic receptor subtypes, using patch-clamp methods in rat and mouse. In all examined cells, carbachol elicited an electrophysiological response that was independent of action potential generation as it persisted in the presence of tetrodotoxin. Responses were of three types: depolarization, hyperpolarization or a biphasic response consisting of hyperpolarization followed by depolarization. In voltage-clamp mode, carbachol evoked corresponding inward, outward or biphasic currents. Moreover, immunostaining for the vesicle-associated choline transporter showed cholinergic innervation of the PSTN. Using muscarinic receptor antagonists, we found that carbachol-elicited PSTN neuron hyperpolarization was mediated by M2 receptors and depolarization, in large part, by M1 receptors. These data suggest that acetylcholine acting on M1 and M2 receptors may contribute to selective excitability enhancement or depression in individual, rostrally projecting sensory neurons. Such selective gating effects via cholinergic input may play a functional role in modulation of ascending sensory transmission, including across behavioral states typified by distinct cholinergic tone, e.g. sleep/wakefulness arousal levels or neuropathic pain conditions. PMID:16820015

  6. Orexin Receptor Activation Generates Gamma Band Input to Cholinergic and Serotonergic Arousal System Neurons and Drives an Intrinsic Ca2+-Dependent Resonance in LDT and PPT Cholinergic Neurons

    PubMed Central

    Ishibashi, Masaru; Gumenchuk, Iryna; Kang, Bryan; Steger, Catherine; Lynn, Elizabeth; Molina, Nancy E.; Eisenberg, Leonard M.; Leonard, Christopher S.

    2015-01-01

    A hallmark of the waking state is a shift in EEG power to higher frequencies with epochs of synchronized intracortical gamma activity (30–60 Hz) – a process associated with high-level cognitive functions. The ascending arousal system, including cholinergic laterodorsal (LDT) and pedunculopontine (PPT) tegmental neurons and serotonergic dorsal raphe (DR) neurons, promotes this state. Recently, this system has been proposed as a gamma wave generator, in part, because some neurons produce high-threshold, Ca2+-dependent oscillations at gamma frequencies. However, it is not known whether arousal-related inputs to these neurons generate such oscillations, or whether such oscillations are ever transmitted to neuronal targets. Since key arousal input arises from hypothalamic orexin (hypocretin) neurons, we investigated whether the unusually noisy, depolarizing orexin current could provide significant gamma input to cholinergic and serotonergic neurons, and whether such input could drive Ca2+-dependent oscillations. Whole-cell recordings in brain slices were obtained from mice expressing Cre-induced fluorescence in cholinergic LDT and PPT, and serotonergic DR neurons. After first quantifying reporter expression accuracy in cholinergic and serotonergic neurons, we found that the orexin current produced significant high frequency, including gamma, input to both cholinergic and serotonergic neurons. Then, by using a dynamic clamp, we found that adding a noisy orexin conductance to cholinergic neurons induced a Ca2+-dependent resonance that peaked in the theta and alpha frequency range (4–14 Hz) and extended up to 100 Hz. We propose that this orexin current noise and the Ca2+ dependent resonance work synergistically to boost the encoding of high-frequency synaptic inputs into action potentials and to help ensure cholinergic neurons fire during EEG activation. This activity could reinforce thalamocortical states supporting arousal, REM sleep, and intracortical gamma. PMID

  7. Differential actions of orexin receptors in brainstem cholinergic and monoaminergic neurons revealed by receptor knockouts: implications for orexinergic signaling in arousal and narcolepsy

    PubMed Central

    Kohlmeier, Kristi A.; Tyler, Christopher J.; Kalogiannis, Mike; Ishibashi, Masaru; Kristensen, Morten P.; Gumenchuk, Iryna; Chemelli, Richard M.; Kisanuki, Yaz Y.; Yanagisawa, Masashi; Leonard, Christopher S.

    2013-01-01

    Orexin neuropeptides influence multiple homeostatic functions and play an essential role in the expression of normal sleep-wake behavior. While their two known receptors (OX1 and OX2) are targets for novel pharmacotherapeutics, the actions mediated by each receptor remain largely unexplored. Using brain slices from mice constitutively lacking either receptor, we used whole-cell and Ca2+ imaging methods to delineate the cellular actions of each receptor within cholinergic [laterodorsal tegmental nucleus (LDT)] and monoaminergic [dorsal raphe (DR) and locus coeruleus (LC)] brainstem nuclei—where orexins promote arousal and suppress REM sleep. In slices from OX−/−2 mice, orexin-A (300 nM) elicited wild-type responses in LDT, DR, and LC neurons consisting of a depolarizing current and augmented voltage-dependent Ca2+ transients. In slices from OX−/−1 mice, the depolarizing current was absent in LDT and LC neurons and was attenuated in DR neurons, although Ca2+-transients were still augmented. Since orexin-A produced neither of these actions in slices lacking both receptors, our findings suggest that orexin-mediated depolarization is mediated by both receptors in DR, but is exclusively mediated by OX1 in LDT and LC neurons, even though OX2 is present and OX2 mRNA appears elevated in brainstems from OX−/−1 mice. Considering published behavioral data, these findings support a model in which orexin-mediated excitation of mesopontine cholinergic and monoaminergic neurons contributes little to stabilizing spontaneous waking and sleep bouts, but functions in context-dependent arousal and helps restrict muscle atonia to REM sleep. The augmented Ca2+ transients produced by both receptors appeared mediated by influx via L-type Ca2+ channels, which is often linked to transcriptional signaling. This could provide an adaptive signal to compensate for receptor loss or prolonged antagonism and may contribute to the reduced severity of narcolepsy in single receptor

  8. The role of adrenergic and cholinergic receptors on the antinociception of sildenafil in the spinal cord of rats.

    PubMed

    Park, Chen Hee; Lee, Hyung Gon; Lee, Seong Heon; Chung, Cheol Won; Yoon, Myung Ha

    2011-09-15

    The role played by spinal adrenergic and cholinergic receptors in the antinociceptive effects of intrathecal sildenafil in formalin-induced nociception was examined. Intrathecal catheters were inserted into the subarachnoid space of male Sprague-Dawley rats, and nociception was assessed using the formalin test, consisting of a subcutaneous injection of 50μL of 5% formalin solution into the hind paw. We examined the effects of an alpha 1 adrenergic receptor antagonist (prazosin), an alpha 2 adrenergic receptor antagonist (yohimbine), a muscarinic acetylcholine receptor antagonist (atropine), and a nicotinic acetylcholine receptor antagonist (mecamylamine) on sildenafil-induced antinociception. Intrathecal sildenafil (3, 10, and 30μg) suppressed, in a dose-dependent manner, formalin-induced flinching during phases 1 and 2 of the test. Intrathecal sildenafil (30μg) could not show any effects against intrathecal prazosin (3μg), yohimbine (10μg), atropine (10μg), and mecamylamine (10μg) pretreatment during both phases of the formalin test. These results suggest that intrathecal sildenafil effectively attenuated the pain evoked by formalin injection. Additionally, spinal alpha 1, alpha 2, muscarinic and nicotinic receptors might play a role in sildenafil-induced antinociception.

  9. Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder.

    PubMed

    Silva-Ramos, M; Silva, I; Faria, M; Magalhães-Cardoso, M T; Correia, J; Ferreirinha, F; Correia-de-Sá, P

    2015-12-01

    This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [(3)H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [(3)H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t (1/2) 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A(1) receptor-mediated inhibition of evoked [(3)H]ACh release by adenosine (100 μM), NECA (1 μM), and R-PIA (0.3 μM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A(1) immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL(-1)) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A(1) receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A(1) receptor activation might be useful to control bladder overactivity in BPH patients.

  10. Direct measurement of agonist binding to genetically engineered peptides of the acetylcholine receptor by selective T sub 1 NMR relaxation

    SciTech Connect

    Fraenkel, Y.; Navon, G. ); Aronheim, A.; Gershoni, J.M. )

    1990-03-13

    Interactions of four ligands of the nicotinic acetylcholine receptor with genetically engineered peptides have been studied by NMR. A recombinant cholinergic binding site was prepared as a fusion protein between a truncated form of the bacterial protein trpE and a peptide corresponding to the sequence {alpha}184-200 from the Torpedo californica receptor. This construct binds {alpha}-bungarotoxin while the trpE protein alone does not, and thus serves as a negative control. In this study agonist binding to {alpha}184-200 is demonstrated by monitoring the T{sub 1} relaxation of the ligand's protons in the presence and absence of the recombinant binding site. This binding is specific as it can be competed with {alpha}-bungarotoxin. Quantitative analyses of such competitions yielded the concentration of binding sites, which corresponded to 3.3% and 16.5% of the total protein, for partially purified and affinity-purified {alpha}184-200 constructs, respectively. The K{sub D} values for the binding of acetylcholine, nicotine, d-tubocurarine, and gallamine to the affinity-purified construct were 1.4, 1.4, 0.20, and 0.21 mM, respectively, while K{sub D}'s with the nontoxin binding protein were all above 10 mM. Thus, this is a direct demonstration that the toxin binding domain {alpha}184-200 may comprise a major component of the cholinergic agonist site.

  11. Neurotrophin receptor p75 mediates the uptake of the amyloid beta (Aβ) peptide, guiding it to lysosomes for degradation in basal forebrain cholinergic neurons.

    PubMed

    Ovsepian, Saak V; Antyborzec, Inga; O'Leary, Valerie B; Zaborszky, Laszlo; Herms, Jochen; Oliver Dolly, J

    2014-09-01

    A fascinating yet perhaps overlooked trait of the p75 neurotrophin receptor (p75(NTR)) is its ability to bind ligands with no obvious neurotrophic function. Using cultured basal forebrain (BF) neurons, this study demonstrates selective internalization of amyloid β (Aβ) 1-42 in conjunction with p75(NTR) (labelled with IgG192-Cy3) by cholinergic cells. Active under resting conditions, this process was enhanced by high K(+) stimulation and was insensitive to inhibitors of regulated synaptic activity-tetrodotoxin or botulinum neurotoxins (BoNT type/A and/B). Blockade of sarco-endoplasmic reticulum (SERCA) Ca(2+) ATPase with thapsigargin and CPA or chelation of Ca(2+) with EGTA-AM strongly suppressed the endocytosis of p75(NTR), implicating the role of ER released Ca(2+). The uptake of IgG192-Cy3 was also reduced by T-type Ca(2+) channel blocker mibefradil but not Cd(2+), an indiscriminate blocker of high voltage-activated Ca(2+) currents. A strong co-localization of IgG192-Cy3 with late endosome (Rab7) or lysosome (Lamp1) qualifier proteins suggest these compartments as the primary destination for internalized IgG192 and Aβ. Selective uptake and labeling of BF cholinergic cells with IgG192-Cy3 injected into the prefrontal cortex was verified also in vivo. The significance of these findings in relation to Aβ clearance in the cerebral cortex and pathophysiology of Alzheimer's disease is discussed. PMID:23716278

  12. Involvement of central cholinergic mechanisms in the effects of oxytocin and an oxytocin receptor antagonist on retention performance in mice.

    PubMed

    Boccia, M M; Baratti, C M

    2000-11-01

    Oxytocin (OT, 0.10 microg/kg, sc) impaired retention of a one-trial step-through inhibitory avoidance task when injected into male Swiss mice 10 min after training, as indicated by retention performance 48 h later. In contrast, the immediate post-training administration of the putative oxytocin receptor antagonist d(CH(2))(5)[Tyr(Me)(2), Thr(4), Thy-NH(9)(2)] OVT (AOT, 0.30 microg/kg, sc) significantly enhanced retention performance. Neither OT nor AOT affected response latencies in mice not given footshock on the training trial, and neither the impairing effects of OT nor the enhancing effects of AOT were seen when the training-treatment interval was 180 min, suggesting that both treatments influenced memory storage. The effects of OT (0.10 microg/kg, sc) on retention were prevented by AOT (0.03 microg/kg, sc) given immediately after training, but 10 min prior to OT treatment. The central acting anticholinesterase physostigmine (35, 70, or 150 microg/kg, i.p.), but not its quaternary analogue neostigmine (150 microg/kg, i.p.), reversed the impairment of retention performance induced by OT, whereas low subeffective doses of the centrally active muscarinic cholinergic antagonist atropine (0.5 mg/kg, i.p.) or the central acting nicotinic cholinergic antagonist mecamylamine (5 mg/kg, i.p.), but not methylatropine (0.5 mg/kg, i.p.) or hexamethonium (5 mg/kg, i.p.) prevented the enhancement of retention performance caused by AOT. We suggest that oxytocin negatively modulates the activity of central cholinergic mechanisms during the posttraining period that follows an aversively motivated learning experience, leading to an impairment of retention performance of the inhibitory avoidance response. PMID:11031128

  13. Two types of functionally different GABAA receptors mediate GABA modulation of cholinergic transmission in cat terminal ileum.

    PubMed

    Radomirov, R; Pencheva, N

    1995-08-01

    1. The effects of GABA (1 microM-2 mM) on longitudinally or circularly oriented organ bath preparations of cat terminal ileum consisted of a relaxation phase with an inhibition of the rhythmic spontaneous phasic contractions, followed by a phase of contractions characterized by an elevation in basal tone and an increase in amplitude of the spontaneous phasic contractions. 2. Muscimol (100 microM), but not baclofen (100 microM), mimicked the relaxation phase of the response to applied GABA (100 microM) in all tissue preparations. In addition, muscimol induced a phase of contractile activity in the circular muscle layer whilst baclofen exerted a 'GABA-like' contractile effect on the longitudinal muscle layer. Bicuculline (30 microM) or picrotoxinin (30 microM) antagonized the GABA- or muscimol-induced relaxations in all preparations and decreased the GABA- but not the baclofen-induced contractions of the longitudinal muscle layer. 3. Tetrodotoxin (0.5 microM) or atropine (0.1 microM) prevented the bicuculline-sensitive phases of the GABA or muscimol effects on both muscle layers but not the contractile effect of baclofen on the longitudinal muscle layer. 4. The bicuculline-sensitive phases of the GABA effect on both muscle layers were almost completely eliminated by 1 nM pirenzepine. At this concentration pirenzepine did not affect the electrically-evoked cholinergic twitch contractions or contractile responses to applied acetylcholine of both muscle layers. 5. During electrically-evoked cholinergic twitch contractions of both muscle layers, GABA (100 microM) had an inhibitory effect. The inhibition occurred in the presence of pirenzepine (1 nM) but not of bicuculline (30 microM). 6. It is suggested that two types of functionally different bicuculline-sensitive GABAA receptors mediate an exitatory presynaptic and an inhibitory prejunctional action of GABA on the cholinergic transmission in cat terminal ileum.

  14. Involvement of central cholinergic mechanisms in the effects of oxytocin and an oxytocin receptor antagonist on retention performance in mice.

    PubMed

    Boccia, M M; Baratti, C M

    2000-11-01

    Oxytocin (OT, 0.10 microg/kg, sc) impaired retention of a one-trial step-through inhibitory avoidance task when injected into male Swiss mice 10 min after training, as indicated by retention performance 48 h later. In contrast, the immediate post-training administration of the putative oxytocin receptor antagonist d(CH(2))(5)[Tyr(Me)(2), Thr(4), Thy-NH(9)(2)] OVT (AOT, 0.30 microg/kg, sc) significantly enhanced retention performance. Neither OT nor AOT affected response latencies in mice not given footshock on the training trial, and neither the impairing effects of OT nor the enhancing effects of AOT were seen when the training-treatment interval was 180 min, suggesting that both treatments influenced memory storage. The effects of OT (0.10 microg/kg, sc) on retention were prevented by AOT (0.03 microg/kg, sc) given immediately after training, but 10 min prior to OT treatment. The central acting anticholinesterase physostigmine (35, 70, or 150 microg/kg, i.p.), but not its quaternary analogue neostigmine (150 microg/kg, i.p.), reversed the impairment of retention performance induced by OT, whereas low subeffective doses of the centrally active muscarinic cholinergic antagonist atropine (0.5 mg/kg, i.p.) or the central acting nicotinic cholinergic antagonist mecamylamine (5 mg/kg, i.p.), but not methylatropine (0.5 mg/kg, i.p.) or hexamethonium (5 mg/kg, i.p.) prevented the enhancement of retention performance caused by AOT. We suggest that oxytocin negatively modulates the activity of central cholinergic mechanisms during the posttraining period that follows an aversively motivated learning experience, leading to an impairment of retention performance of the inhibitory avoidance response.

  15. Pre- and neonatal exposure of the Dahl rat to NaCl: development and regional distribution of myocardial alpha 1-adrenergic and cholinergic receptor sites.

    PubMed

    McCaughran, J A; Juno, C J; Friedman, R

    1986-06-01

    The prenatal and/or postweaning effects of a hypertensinogenic high NaCl-containing diet (8.0% NaCl, w/w) on (1) the regional distribution of alpha 1-adrenoceptors and muscarinic cholinergic receptor sites in the heart and (2) the predisposition/resistance to hypertension (HT) were assessed in the inbred Dahl HT-sensitive (S/JR) and HT-resistant (R/JR) rat. The density of alpha 1-adrenoceptors was reduced in the left ventricle but not consistently affected in the ventricular septum, right ventricle, or atria of S/JR offspring with NaCl-induced HT. Both normotensive and hypertensive S/JR rats also displayed a significantly greater density of cholinergic receptor sites in the atria but few consistent alterations in other regions of the heart, compared to R/JR rats. Maternal diet had no effect on the predisposition/resistance to salt-induced HT and little effect on the regional development of alpha 1-adrenoceptors and cholinergic receptor sites. The results of this study suggest that the reduced density of ventricular alpha 1-adrenoceptors in the S/JR strain is a consequence of HT while the elevated density of cholinergic receptors in the atria may be related to the genetic predisposition/resistance to HT.

  16. Expression of estrogen receptor-like immunoreactivity by different subgroups of basal forebrain cholinergic neurons in gonadectomized male and female rats.

    PubMed

    Gibbs, R B

    1996-05-13

    Recent studies have demonstrated that estrogen administration can produce significant increases in relative levels of choline acetyltransferase (ChAT) mRNA and protein in specific regions of the female, but not the male, rat basal forebrain. In the present study immunocytochemical techniques were used to identify and compare relative numbers of cholinergic neurons containing estrogen receptors within the medial septum, horizontal limb of the diagonal band of Broca, nucleus basalis magnocellularis, and striatum of gonadectomized male and female rats to determine whether there are differences in the percentage of cholinergic neurons expressing estrogen receptors which might contribute to the different regional- and sex-specific effects of estrogen which have been described. Counts of choline acetyltransferase-immunoreactive cells revealed significant regional differences in the average number of cholinergic neurons/section; however, no difference between males and females in the numbers of cholinergic neurons in each of the four regions analyzed was observed. Fifty to eighty percent of the cholinergic neurons detected in both males and females contained estrogen receptor-like immunoreactivity. A small but significant difference between males and females was detected with females having slightly more (10.5%) double-labeled cells than males overall. Individual comparisons revealed that significantly more (18-33%) double-labeled cells were detected in the horizontal limb of the diagonal band, but not in the medial septum, nucleus basalis, or striatum of females vs. males. There was also a small but significant regional difference in the percentage of double-labeled cells with the highest percentage (74.2%) detected in the striatum and the lowest percentage (63.4%) detected in the horizontal limb. None of these differences appear to account for the regional- and sex-specific effects of estrogen on cholinergic neurons which have been observed. We conclude that differences

  17. Receptor-binding sites: bioinformatic approaches.

    PubMed

    Flower, Darren R

    2006-01-01

    It is increasingly clear that both transient and long-lasting interactions between biomacromolecules and their molecular partners are the most fundamental of all biological mechanisms and lie at the conceptual heart of protein function. In particular, the protein-binding site is the most fascinating and important mechanistic arbiter of protein function. In this review, I examine the nature of protein-binding sites found in both ligand-binding receptors and substrate-binding enzymes. I highlight two important concepts underlying the identification and analysis of binding sites. The first is based on knowledge: when one knows the location of a binding site in one protein, one can "inherit" the site from one protein to another. The second approach involves the a priori prediction of a binding site from a sequence or a structure. The full and complete analysis of binding sites will necessarily involve the full range of informatic techniques ranging from sequence-based bioinformatic analysis through structural bioinformatics to computational chemistry and molecular physics. Integration of both diverse experimental and diverse theoretical approaches is thus a mandatory requirement in the evaluation of binding sites and the binding events that occur within them. PMID:16671408

  18. Cholinergic Modulation of Locomotion and Striatal Dopamine Release is Mediated by α6α4* Nicotinic Acetylcholine Receptors

    PubMed Central

    Drenan, Ryan M.; Grady, Sharon R.; Steele, Andrew D.; McKinney, Sheri; Patzlaff, Natalie E.; McIntosh, J. Michael; Marks, Michael J.; Miwa, Julie M.; Lester, Henry A.

    2012-01-01

    Dopamine (DA) release in striatum is governed by firing rates of midbrain DA neurons, striatal cholinergic tone, and nicotinic ACh receptors (nAChRs) on DA presynaptic terminals. DA neurons selectively express α6* nAChRs, which show high ACh and nicotine sensitivity. To help identify nAChR subtypes that control DA transmission, we studied transgenic mice expressing hypersensitive α6L9′S* receptors. α6L9′S mice are hyperactive, travel greater distance, exhibit increased ambulatory behaviors such as walking, turning, and rearing, and show decreased pausing, hanging, drinking, and grooming. These effects were mediated by α6α4* pentamers, as α6L9′S mice lacking α4 subunits displayed essentially normal behavior. In α6L9′S mice, receptor numbers are normal, but loss of α4 subunits leads to fewer and less sensitive α6* receptors. Gain-of-function nicotine-stimulated DA release from striatal synaptosomes requires α4 subunits, implicating α6α4β2* nAChRs in α6L9′S mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by α6L9′S nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in α6L9′S, but not WT or α4KO/α6L9′S, mice. Thus, increased nAChR activity, like decreased activity, leads to enhanced extracellular DA release during phasic firing. Bursts may directly enhance DA release from α6L9′S presynaptic terminals, as there was no difference in striatal DA receptor numbers or DA transporter levels or function in vitro. These results implicate α6α4β2* nAChRs in cholinergic control of DA transmission, and strongly suggest that these receptors are candidate drug targets for disorders involving the DA system. PMID:20660270

  19. Interaction of nicotinic receptor affinity reagents with central nervous system. cap alpha. -bungarotoxin-binding entities

    SciTech Connect

    Lukas, R.J.; Bennett, E.L.

    1980-01-01

    Membrane-bound ..cap alpha..-bungarotoxin-binding entities derived from rat brain are found to interact specifically with the affinity reagents maleimidobenzyltrimethylammonium (MBTA) and bromoacetylcholine (BAC), originally designed to label nicotinic acetylcholine receptors from electroplax and skeletal muscle. Following treatment of membranes with dithiothreitol, all specific toxin binding sites are irreversibly blocked by reaction with MBTA or BAC. Affinity reagent labeling of dithiothreitol-reduced membranes is prevented (toxin binding sites are not blocked) by prior alkylaction with N-ethylmaleimide, by prior oxidation with dithiobis(2-nitrobenzoic acid), or by incubation with neurotoxin. Reversibly associating cholinergic agonists and antagonists retard the rate of affinity reagent interaction with toxin receptors. The apparent rates of affinity reagent alkylation of toxin receptors, and the influences of other sulfhydryl/disulfide reagents on affinity labeling are comparable to those observed for reaction with nicotinic acetylcholine receptors in the periphery. The results provide further evidence that central nervous system ..cap alpha..-bungarotoxin receptors share a remarkable number of biochemical properties with nicotinic receptors from the periphery.

  20. Neuroanatomical and neuropharmacological approaches to postictal antinociception-related prosencephalic neurons: the role of muscarinic and nicotinic cholinergic receptors

    PubMed Central

    de Freitas, Renato Leonardo; Bolognesi, Luana Iacovelo; Twardowschy, André; Corrêa, Fernando Morgan Aguiar; Sibson, Nicola R; Coimbra, Norberto Cysne

    2013-01-01

    Several studies have suggested the involvement of the hippocampus in the elaboration of epilepsy. There is evidence that suggests the hippocampus plays an important role in the affective and motivational components of nociceptive perception. However, the exact nature of this involvement remains unclear. Therefore, the aim of this study was to determine the role of muscarinic and nicotinic cholinergic receptors in the dorsal hippocampus (dH) in the organization of postictal analgesia. In a neuroanatomical study, afferent connections were found from the somatosensory cortex, the medial septal area, the lateral septal area, the diagonal band of Broca, and the dentate gyrus to the dH; all these areas have been suggested to modulate convulsive activity. Outputs to the dH were also identified from the linear raphe nucleus, the median raphe nucleus (MdRN), the dorsal raphe nucleus, and the locus coeruleus. All these structures comprise the endogenous pain modulatory system and may be involved either in postictal pronociception or antinociception that is commonly reported by epileptic patients. dH-pretreatment with cobalt chloride (1.0 mmol/L CoCl2/0.2 μL) to transiently inhibit local synapses decreased postictal analgesia 10 min after the end of seizures. Pretreatment of the dH with either atropine or mecamylamine (1.0 μg/0.2 μL) attenuated the postictal antinociception 30 min after seizures, while the higher dose (5.0 μg/0.2 μL) decreased postictal analgesia immediately after the end of seizures. These findings suggest that the dH exerts a critical role in the organization of postictal analgesia and that muscarinic and nicotinic cholinergic receptor-mediated mechanisms in the dH are involved in the elaboration of antinociceptive processes induced by generalized tonic-clonic seizures. PMID:23785660

  1. Calcium channel blockade with verapamil. Effects on blood pressure, renal, and myocardial adrenergic, cholinergic, and calcium channel receptors in inbred Dahl hypertension-sensitive (S/JR) and hypertension-resistant (R/JR) rats.

    PubMed

    McCaughran, J A; Juno, C J

    1988-07-01

    Verapamil HCl was chronically administered to inbred Dahl S/JR and R/JR rats maintained on a diet containing 8.0% NaCl (w/w) and the effects on blood pressure (BP) and heart rate (HR) were investigated. Treatment over a 4-week period via implanted miniosmotic pumps attenuated but did not prevent the development of salt-induced hypertension (HT) in the S/JR rat. Elevated HR, possibly reflexive in origin, was observed in S/JR rats that received verapamil but not in similarly treated R/JR rats. Although verapamil retarded the development of HT in S/JR rats, BP rose to moderately hypertensive levels, and the ventricle/body weight ratio was elevated by the termination of the study. The effect of verapamil on the density and affinity of alpha 1-, alpha 2-, and beta-adrenergic, muscarinic cholinergic, and calcium channel receptors in renal and ventricular membranes was also assessed. The density of renal and ventricular alpha 1- and beta-adrenoceptors was not affected by chronic drug treatment. The density of renal alpha 2- and beta-adrenoceptors was greater in the S/JR strain than in the R/JR strain, regardless of the treatment. The density of muscarinic cholinergic and calcium channel receptors in the ventricle was not affected by the treatment. The results of this study suggest that the long-term antihypertensive effects of verapamil in the S/JR rat do not involve an alteration in the binding characteristics of adrenergic, cholinergic, or calcium channel receptor sites in ventricular and renal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Reversal in Cognition Impairments, Cholinergic Dysfunction, and Cerebral Oxidative Stress Through the Modulation of Ryanodine Receptors (RyRs) and Cysteinyl Leukotriene-1 (CysLT1) Receptors.

    PubMed

    Singh, Prabhat; Sharma, Bhupesh

    2016-01-01

    Chronic cerebral hypoperfusion (CCH) is a general pathophysiological condition occurring in vascular dementia (VaD) associated with negative impact on cognitive functions. Ryanodine as well as cysteinyl leukotriene-1 receptors (RyRs and CysLT1Rs) are extensively present in the central nervous system, where they participate in regulation of cognition, motivation, inflammation and neurodegeneration. The purpose of this study is to examine the role of ruthenium red; a selective RyR blocker as well as montelukast; a specific CysLT1 antagonist in CCH induced VaD in mice. Two vessel occlusion (2VO) or permanent ligation of bilateral common carotid arteries technique was used to induce CCH in mice. Animals with bilateral carotid arteries occlusion have revealed impaired learning and memory (Morris water maze), cholinergic dysfunction (increased acetylcholinesterase activity) as well as increased brain oxidative stress (reduction in brain superoxide dismutase, glutathione and catalase with an increase in thiobarbituric acid reactive substance level), with increased brain infarct size (2,3,5-triphenylterazolium chloride staining). While, administration of ruthenium red and montelukast considerably attenuated CCH induced cognitive impairments, cholinergic dysfunction, brain oxidative stress as well as brain damage. The results suggest that bilateral carotid arteries occlusion induced CCH has brought out VaD, which was attenuated by treatment with ruthenium red and montelukast. Therefore, modulation of RyRs as well as CysLT1 receptors may provide help in conditions involving CCH such as cognitive impairment and VaD. PMID:26500103

  3. Imaging muscarinic cholinergic receptors in human brain in vivo with Spect, [123I]4-iododexetimide, and [123I]4-iodolevetimide.

    PubMed

    Müller-Gärtner, H W; Wilson, A A; Dannals, R F; Wagner, H N; Frost, J J

    1992-07-01

    A method to image muscarinic acetylcholine receptors (muscarinic receptors) noninvasively in human brain in vivo was developed using [123I]4-iododexetimide ([123I]IDex), [123I]4-iodolevetimide ([123I]ILev), and single photon emission computed tomography (SPECT). [123I]IDex is a high-affinity muscarinic receptor antagonist. [123I]ILev is its pharmacologically inactive enantiomer and measures nonspecific binding of [123I]IDex in vitro. Regional brain activity after tracer injection was measured in four young normal volunteers for 24 h. Regional [123I]IDex and [123I]ILev activities were correlated early after injection, but not after 1.5 h. [123I]IDex activity increased over 7-12 h in neocortex, neostriatum, and thalamus, but decreased immediately after the injection peak in cerebellum. [123I]IDex activity was highest in neostriatum, followed in rank order by neocortex, thalamus, and cerebellum. [123I]IDex activity correlated with muscarinic receptor concentrations in matching brain regions. In contrast, [123I]ILev activity decreased immediately after the injection peak in all brain regions and did not correspond to muscarinic receptor concentrations. [123I]IDex activity in neocortex and neostriatum during equilibrium was six to seven times higher than [123I]ILev activity. The data demonstrate that [123I]IDex binds specifically to muscarinic receptors in vivo, whereas [123I]ILev represents the nonspecific part of [123I]IDex binding. Subtraction of [123I]ILev from [123I]IDex images on a pixel-by-pixel basis therefore reflects specific [123I]IDex binding to muscarinic receptors. Owing to its high specific binding, [123I]IDex has the potential to measure small changes in muscarinic receptor characteristics in vivo with SPECT. The use of stereoisomerism directly to measure nonspecific binding of [123I]IDex in vivo may reduce complexity in modeling approaches to muscarinic acetylcholine receptors in human brain. PMID:1618935

  4. Mechanism for ordered receptor binding by human prolactin.

    PubMed

    Sivaprasad, Umasundari; Canfield, Jeffrey M; Brooks, Charles L

    2004-11-01

    Prolactin, a lactogenic hormone, binds to two prolactin receptors sequentially, the first receptor binding at site 1 of the hormone followed by the second receptor binding at site 2. We have investigated the mechanism by which human prolactin (hPRL) binds the extracellular domain of the human prolactin receptor (hPRLbp) using surface plasmon resonance (SPR) technology. We have covalently coupled hPRL to the SPR chip surface via coupling chemistries that reside in and block either site 1 or site 2. Equilibrium binding experiments using saturating hPRLbp concentrations show that site 2 receptor binding is dependent on site 1 receptor occupancy. In contrast, site 1 binding is independent of site 2 occupancy. Thus, sites 1 and 2 are functionally coupled, site 1 binding inducing the functional organization of site 2. Site 2 of hPRL does not have a measurable binding affinity prior to hPRLbp binding at site 1. After site 1 receptor binding, site 2 affinity is increased to values approaching that of site 1. Corruption of either site 1 or site 2 by mutagenesis is consistent with a functional coupling of sites 1 and 2. Fluorescence resonance energy transfer (FRET) experiments indicate that receptor binding at site 1 induces a conformation change in the hormone. These data support an "induced-fit" model for prolactin receptor binding where binding of the first receptor to hPRL induces a conformation change in the hormone creating the second receptor-binding site.

  5. (/sup 3/H)pirenzepine selectively identifies a high affinity population of muscarinic cholinergic receptors in the rat cerebral cortex

    SciTech Connect

    Watson, M.; Roeske, W.R.; Yamamura, H.I.

    1982-11-01

    The specific binding of (/sup 3/H)pirenzepine was investigated in homogenates of rat cerebral cortex, cerebellar cortex, and heart. Specific binding of (/sup 3/H)pirenzepine in the cerebral cortex as defined by displacement with atropine sulfate (1..mu..M) was of high affinity (K/sub d/ = 4-10 nM, receptor density = 1.06 pmoles/mg protein), stereoselective, and competitive with drugs specific for the muscarinic receptor. In contrast, few (/sup 3/H)pirenzepine binding sites were demonstrated in cerebellar and heart homogenates.

  6. The cholinergic immune regulation mediated by a novel muscarinic acetylcholine receptor through TNF pathway in oyster Crassostrea gigas.

    PubMed

    Liu, Zhaoqun; Zhou, Zhi; Wang, Lingling; Dong, Wenjing; Qiu, Limei; Song, Linsheng

    2016-12-01

    Muscarinic receptors, which selectively take muscarine as their ligand, are critical for the immunological and physiological processes in animals. In the present study, the open region frame (ORF) of a homologue of muscarinic acetylcholine (ACh) receptor (mAChR) was amplified from oyster Crassostrea gigas (named as CgmAChR-1), whose full length was 1983 bp and the protein it encoded contained 660 amino acids with a seven transmembrane region. Phylogeny analysis suggested that CgmAChR-1 shared homology with M5 muscarinic receptor found in invertebrates including Habropoda laboriosa, Acromyrmex echinatior and Echinococcus granulosus. After cell transfection of CgmAChR-1 into HEK293T cells and ACh incubation, the level of intracellular Ca(2+) and cAMP increased significantly (p < 0.05). Such trend could be reverted with the addition of M3 and M5 muscarinic receptor antagonists DAMP and DAR. The CgmAChR-1 transcripts were ubiquitously detectable in seven different tissues with the maximal expression level in adductor muscle. When the oysters received LPS stimulation, CgmAChR-1 mRNA expression in haemocyte was increased to the highest level (6.05-fold, p < 0.05) at 24 h, while blocking CgmAChR-1 using receptor antagonists before LPS stimulation promoted the expression of oyster TNF, resulting in the increase of haemocyte apoptosis index. These results suggested that CgmAChR-1 was the key molecule in cholinergic neuroendocrine-immune system contributing to the regulation of TNF expression and apoptosis process. PMID:27394930

  7. NGF and anti-transferrin receptor antibody conjugate: short and long-term effects on survival of cholinergic neurons in intraocular septal transplants.

    PubMed

    Granholm, A C; Bäckman, C; Bloom, F; Ebendal, T; Gerhardt, G A; Hoffer, B; Mackerlova, L; Olson, L; Söderström, S; Walus, L R

    1994-01-01

    We describe a new molecular carrier system that allows for the transport of nerve growth factor (NGF) across the blood-brain barrier (BBB), as assessed by trophic effects on intraocular forebrain transplants that contain central cholinergic neurons. The carrier system involves monoclonal antibodies (OX-26) directed against the transferrin receptor, to which NGF molecules are covalently linked. Transferrin receptors are highly concentrated on brain blood vessels and participate in the transport of iron across the BBB. Host rats with septal transplants were divided into four groups, which received OX-26-NGF, OX-26, NGF or saline intravenously at 2, 4, 6 and 8 weeks after grafting. Half of the animals were killed directly after the final injection, whereas the other half were allowed to survive for an additional 5 months. Control experiments revealed that blood vessels in mature brain grafts in oculo contained large amounts of transferrin receptors. Covalent binding of NGF to the OX-26 antibodies did not impede OX-26 binding to CNS transferrin receptors, nor did conjugation affect the bioactivity of NGF. A time-dependent increase in host brain NGF levels was found after injection of OX-26-NGF into the tail vein. Host serum contained some NGF antibodies in the short-term OX-26-NGF group that had disappeared in the long-term group; host adrenals showed no differences in wet weight or norepinephrine or epinephrine whole tissue levels in any of the groups. As previously reported, the overall growth of intraocular septal transplants was approximately twice as great in the OX-26-NGF group relative to all other groups. This difference in final size persisted unabated for at least 5 months after the last injection. Furthermore, the significantly higher numbers of choline acetyl transferase immunoreactive neurons in transplants of OX-26-NGF-treated hosts also persisted during the 5-month postinjection interval. Taken together, the data suggest that the OX-26 conjugate may be a

  8. Monitoring cholinergic activity during attentional performance in mice heterozygous for the choline transporter: a model of cholinergic capacity limits.

    PubMed

    Paolone, Giovanna; Mallory, Caitlin S; Koshy Cherian, Ajeesh; Miller, Thomas R; Blakely, Randy D; Sarter, Martin

    2013-12-01

    Reductions in the capacity of the human choline transporter (SLC5A7, CHT) have been hypothesized to diminish cortical cholinergic neurotransmission, leading to risk for cognitive and mood disorders. To determine the acetylcholine (ACh) release capacity of cortical cholinergic projections in a mouse model of cholinergic hypofunction, the CHT+/- mouse, we assessed extracellular ACh levels while mice performed an operant sustained attention task (SAT). We found that whereas SAT-performance-associated increases in extracellular ACh levels of CHT+/- mice were significantly attenuated relative to wildtype littermates, performance on the SAT was normal. Tetrodotoxin-induced blockade of neuronal excitability reduced both dialysate ACh levels and SAT performance similarly in both genotypes. Likewise, lesions of cholinergic neurons abolished SAT performance in both genotypes. However, cholinergic activation remained more vulnerable to the reverse-dialyzed muscarinic antagonist atropine in CHT+/- mice. Additionally, CHT+/- mice displayed greater SAT-disrupting effects of reverse dialysis of the nAChR antagonist mecamylamine. Receptor binding assays revealed a higher density of α4β2* nAChRs in the cortex of CHT+/- mice compared to controls. These findings reveal compensatory mechanisms that, in the context of moderate cognitive challenges, can overcome the performance deficits expected from the significantly reduced ACh capacity of CHT+/- cholinergic terminals. Further analyses of molecular and functional compensations in the CHT+/- model may provide insights into both risk and resiliency factors involved in cognitive and mood disorders.

  9. Monitoring cholinergic activity during attentional performance in mice heterozygous for the choline transporter: a model of cholinergic capacity limits

    PubMed Central

    Cherian, Ajeesh Koshy; Miller, Thomas R.; Blakely, Randy D.; Sarter, Martin

    2013-01-01

    Reductions in the capacity of the human choline transporter (SLC5A7, CHT) have been hypothesized to diminish cortical cholinergic neurotransmission, leading to risk for cognitive and mood disorders. To determine the acetylcholine (ACh) release capacity of cortical cholinergic projections in a mouse model of cholinergic hypofunction, the CHT+/− mouse, we assessed extracellular ACh levels while mice performed an operant sustained attention task (SAT). We found that whereas SAT-performance-associated increases in extracellular ACh levels of CHT+/− mice were significantly attenuated relative to wildtype littermates, performance on the SAT was normal. Tetrodotoxin-induced blockade of neuronal excitability reduced both dialysate ACh levels and SAT performance similarly in both genotypes. Likewise, lesions of cholinergic neurons abolished SAT performance in both genotypes. However, cholinergic activation remained more vulnerable to the reverse-dialyzed muscarinic antagonist atropine in CHT+/− mice. Additionally, CHT+/− mice displayed greater SAT-disrupting effects of reverse dialysis of the nAChR antagonist mecamylamine. Receptor binding assays revealed a higher density of α4β2* nAChRs in the cortex of CHT+/− mice compared to controls. These findings reveal compensatory mechanisms that, in the context of moderate cognitive challenges, can overcome the performance deficits expected from the significantly reduced ACh capacity of CHT+/− cholinergic terminals. Further analyses of molecular and functional compensations in the CHT +/− model may provide insights into both risk and resiliency factors involved in cognitive and mood disorders. PMID:23958450

  10. Radiobrominated triphenylethylenes as estrogen receptor binding radiopharmaceuticals

    SciTech Connect

    Seevers, R.H.; Meese, R.C.; Friedman, A.M.; DeSombre, E.R.

    1985-05-01

    Estrogen receptor binding radiopharmaceuticals have potential for use in the diagnosis and treatment of cancers of the female reproductive system. Tamoxifen is an antiestrogen derived from the triphenylethylene skeleton which is used in the treatment of mammary carcinoma. Hydroxytamoxifen is a metabolite of tamoxifen which binds tightly to the estrogen receptor. Two triphenylethylene derivatives based on the structure of hydroxytamoxifen have been prepared: 1-bromo-1-phenyl-2- (2-dimethylamino)-4-ethoxyphenyl -2-(4-hydroxyphenyl) ethene (1) where the ethyl group of hydroxytamoxifen has been replaced by a bromine, and 1-bromo-1-phenyl-2,2-(4-hydroxyphenyl) ethene (2) with a similar substitution and also lacking the aminoethoxy side chain believed to confer antiestrogenicity. Both 1 and 2 bind strongly to the estrogen receptor. 2 has been labeled with the Auger electron emitting nuclide Br-80m in moderate yields in high specific activity using either N-bromosuccinimide or N-bromophthalimide and shows promise as a potential radiotherapy agent.

  11. Psychopharmacological evidences for the involvement of muscarinic and nicotinic cholinergic receptors on sweet substance-induced analgesia in Rattus norvegicus.

    PubMed

    Irusta, A E; Savoldi, M; Kishi, R; Resende, G C; Freitas, R L; Carvalho, A D; Coimbra, N C

    2001-06-01

    In order to investigate the effects of sweet substance intake on pain modulation, male albino Wistar rats weighing 180-200 g received either tap water or sucrose solutions (250 g/l) for 14 days as their only source of liquid. Each rat consumed an average of 15.6 g sucrose/day. Their tail withdrawal latencies in the tail-flick test (probably a spinal reflex) were measured immediately before and after this treatment. An analgesia index was calculated from the withdrawal latencies before and after treatment. The index (mean +/- SEM, N = 8) for the groups receiving sucrose solution plus saline (NaCl; 0.9%) for 14 days was 0.70 +/- 0.01. Atropine (1 and 2 mg/kg)-treated rats (N = 8) after intake of sucrose exhibited an analgesia index of 0.39 +/- 0.09 and 0.39 +/- 0.08, respectively, while mecamylamine (1 and 2 mg/kg)-treated rats (N = 10) after intake of sucrose had an index of -0.02 +/- 0.07 and 0.03 +/- 0.07, respectively. These results indicate that the effect of sucrose intake on nociceptive thresholds is controlled by neurotransmission of acetylcholine and depends on the nicotinic cholinergic receptors for its major analgesic effect, although muscarinic receptors were also involved in this antinociceptive process.

  12. Modulation of muscarinic and micotinic cholinergic receptor mediated catecholamine secretion in guinea pig chromaffin cells by phorbol esters

    SciTech Connect

    Figueiredo, J.C.; Fisher, S.K.; Horowitz, M.I.

    1986-05-01

    Isolated guinea pig chromaffin cells possess both nicotinic (nAChR) and muscarinic (mAChR) cholinergic receptors that are positively coupled to catecholamine (CA) release. Sixty to 70% of CA release is mediated by nAChRs and 30-40% by mAChRs. In the absence of added calcium, nAChR mediated CA release was reduced by 65% whereas the muscarinic response was unaffected. The addition of 100nM 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), also resulted in an increased CA release. Temporally and quantitatively, this response resembled that of mAChR activation. Addition of optimal concentrations of nicotine (50..mu..M) and TPA (100nM) induced a synergistic increase in CA release. Addition of muscarine (1mM) and TPA resulted in an additive response despite a 40-60% inhibition of mAChR mediated inositol phosphate release by TPA. Thus, in guinea pig chromaffin cells, it appears that PKC activation alone is a sufficient stimulus for CA release and that activation of both nicotinic and muscarinic receptors may further increase this enzyme's activity.

  13. [Allosteric effect of serotonin and mianserin on the kinetics of specific [3H]-ligand binding to adrenergic and muscarinic receptors in the rat cerebral cortex membranes].

    PubMed

    Manukhin, B N; Nesterova, L A

    2015-01-01

    The effects of serotonin receptor activation (by serotonin) and inhibition (by mianserin) on the properties of the α1-, α2-adrenoreceptors, and muscarinic cholinergic receptors in subcellular membrane fractions from the rat cerebral cortex were studied. Experimental data on the kinetics of specific antagonists binding to adrenergic and muscarinic receptors were analyzed by graphical and mathematical methods. The results suggest the presence of allosteric (cross-talk) interaction. In the control, α1- and α2-adrenoreceptors were represented by a single pool, and muscarinic receptors, by two pools. Two pools of adrenoreceptors with different affinity were detected against the background of serotonin. It was found that mianserin induces the formation of two pools of only (α2-receptors and muscarinic receptors are represented by two pools differing in the main parameters, such as dissociation constants and adrenoreceptor concentrations, in the control and experimental groups. It was shown that the allosteric effect of serotonin and mianserin is manifested in the inhibition of muscarinic receptors. It was assumed that the adrenergic and cholinergic receptors exist as dimers. The interaction between the adrenergic, cholinergic, and serotonergic systems is likely to be implemented at the cell membrane level.

  14. Decreased binding capacity (Bmax) of muscarinic acetylcholine receptors in fibroblasts from boys with attention-deficit/hyperactivity disorder (ADHD).

    PubMed

    Johansson, Jessica; Landgren, Magnus; Fernell, Elisabeth; Lewander, Tommy; Venizelos, Nikolaos

    2013-09-01

    Monoaminergic dysregulation is implicated in attention-deficit/hyperactivity disorder (ADHD), and methylphenidate and amphetamines are the most frequently prescribed pharmacological agents for treating ADHD. However, it has recently been proposed that the core symptoms of the disorder might be due to an imbalance between monoaminergic and cholinergic systems. In this study, we used fibroblast cell homogenates from boys with and without ADHD as an extraneural cell model to examine the cholinergic receptor density, that is, muscarinic acetylcholine receptors (mAChRs). We found that the binding capacity (Bmax) of [³H] Quinuclidinyl benzilate (³H-QNB) to mAChRs was decreased by almost 50 % in the children with ADHD (mean = 30.6 fmol/mg protein, SD = 25.6) in comparison with controls [mean = 63.1 fmol/mg protein, SD = 20.5, p ≤ 0.01 (Student's unpaired t test)]. The decreased Bmax indicates a reduced cholinergic receptor density, which might constitute a biomarker for ADHD. However, these preliminary findings need to be replicated in larger ADHD and comparison cohorts.

  15. Melanocortin 4 receptor activation protects against testicular ischemia-reperfusion injury by triggering the cholinergic antiinflammatory pathway.

    PubMed

    Minutoli, Letteria; Bitto, Alessandra; Squadrito, Francesco; Irrera, Natasha; Rinaldi, Mariagrazia; Nicotina, Piero Antonio; Arena, Salvatore; Magno, Carlo; Marini, Herbert; Spaccapelo, Luca; Ottani, Alessandra; Giuliani, Daniela; Romeo, Carmelo; Guarini, Salvatore; Antonuccio, Pietro; Altavilla, Domenica

    2011-10-01

    Melanocortins (MC) trigger a vagus nerve-mediated cholinergic-antiinflammatory pathway projecting to the testis. We tested whether pharmacological activation of brain MC receptors might protect the testis from the damage induced by ischemia-reperfusion. Adult male rats were subjected to 1-h testicular ischemia, followed by 24-h reperfusion [testicular ischemia-reperfusion (TI/R)]. Before TI/R, groups of animals were subjected to bilateral cervical vagotomy, or pretreated with the nicotinic acetylcholine receptor antagonist chlorisondamine or the selective MC(4) receptor antagonist HS024. Immediately after reperfusion, rats were ip treated with saline or the MC analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) (340 μg/kg). We evaluated testicular IL-6 and TNF-α by Western blot analysis and organ damage by light microscopy. Some experimental groups were prepared for neural efferent activity recording along the vagus nerve starting 30 min after treatment with NDP-α-MSH or saline, and for a 30-min period. Additional groups of TI/R rats were treated for 30 d with saline, NDP-α-MSH, chlorisondamine plus NDP-α-MSH, or HS024 plus NDP-α-MSH to evaluate spermatogenesis, organ damage, and the apoptosis machinery. After a 24-h reperfusion, in TI/R saline-treated rats, there was an increase in IL-6 and TNF-α expression and a marked damage in both testes. NDP-α-MSH inhibited IL-6 and TNF-α expression, decreased histological damage, and increased neural efferent activity. Furthermore, NDP-α-MSH administration for 30 d greatly improved spermatogenesis, reduced organ damage, and inhibited apoptosis. All positive NDP-α-MSH effects were abrogated by vagotomy, chlorisondamine, or HS024. Our data suggest that selective MC(4) receptor agonists might be therapeutic candidates for the management of testicular torsion.

  16. Melanocortin 4 Receptor Activation Protects Against Testicular Ischemia-Reperfusion Injury by Triggering the Cholinergic Antiinflammatory Pathway

    PubMed Central

    Minutoli, Letteria; Bitto, Alessandra; Irrera, Natasha; Rinaldi, Mariagrazia; Nicotina, Piero Antonio; Arena, Salvatore; Magno, Carlo; Marini, Herbert; Spaccapelo, Luca; Ottani, Alessandra; Giuliani, Daniela; Romeo, Carmelo; Guarini, Salvatore; Antonuccio, Pietro; Altavilla, Domenica

    2011-01-01

    Melanocortins (MC) trigger a vagus nerve-mediated cholinergic-antiinflammatory pathway projecting to the testis. We tested whether pharmacological activation of brain MC receptors might protect the testis from the damage induced by ischemia-reperfusion. Adult male rats were subjected to 1-h testicular ischemia, followed by 24-h reperfusion [testicular ischemia-reperfusion (TI/R)]. Before TI/R, groups of animals were subjected to bilateral cervical vagotomy, or pretreated with the nicotinic acetylcholine receptor antagonist chlorisondamine or the selective MC4 receptor antagonist HS024. Immediately after reperfusion, rats were ip treated with saline or the MC analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) (340 μg/kg). We evaluated testicular IL-6 and TNF-α by Western blot analysis and organ damage by light microscopy. Some experimental groups were prepared for neural efferent activity recording along the vagus nerve starting 30 min after treatment with NDP-α-MSH or saline, and for a 30-min period. Additional groups of TI/R rats were treated for 30 d with saline, NDP-α-MSH, chlorisondamine plus NDP-α-MSH, or HS024 plus NDP-α-MSH to evaluate spermatogenesis, organ damage, and the apoptosis machinery. After a 24-h reperfusion, in TI/R saline-treated rats, there was an increase in IL-6 and TNF-α expression and a marked damage in both testes. NDP-α-MSH inhibited IL-6 and TNF-α expression, decreased histological damage, and increased neural efferent activity. Furthermore, NDP-α-MSH administration for 30 d greatly improved spermatogenesis, reduced organ damage, and inhibited apoptosis. All positive NDP-α-MSH effects were abrogated by vagotomy, chlorisondamine, or HS024. Our data suggest that selective MC4 receptor agonists might be therapeutic candidates for the management of testicular torsion. PMID:21828180

  17. GABA(A) receptors implicated in REM sleep control express a benzodiazepine binding site.

    PubMed

    Nguyen, Tin Quang; Liang, Chang-Lin; Marks, Gerald A

    2013-08-21

    It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60 nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10(-2)M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

  18. Cholinergic receptor activation induces a relative facilitation of synaptic responses in the entorhinal cortex during theta- and gamma-frequency stimulation of parasubicular inputs.

    PubMed

    Sparks, D W; Chapman, C A

    2013-01-29

    The parasubiculum sends its single major output to layer II of the entorhinal cortex, and it may therefore interact with inputs to the entorhinal cortex from other cortical areas, and help to shape the activity of layer II entorhinal cells that project to the hippocampal formation. Cholinergic inputs are thought to contribute to the generation of theta- and gamma-frequency activities in the parasubiculum and entorhinal cortex, and the present study assessed how cholinergic receptor activation affects synaptic responses of the entorhinal cortex to theta- and gamma-frequency stimulation. Depth profiles of field excitatory postsynaptic potentials (fEPSPs) in acute brain slices showed a short-latency negative fEPSP in layer II, consistent with the activation of excitatory synaptic inputs to layer II. Application of the cholinergic agonist carbachol (CCh) suppressed synaptic responses and enhanced paired-pulse facilitation. CCh also resulted in a marked relative facilitation of synaptic responses evoked during short 5-pulse trains of stimulation at both theta- and gamma-frequencies. Application of the M(1) antagonist pirenzepine, but not the M(2) antagonist methoctramine, blocked the facilitation of responses. Inhibition of the M-current or block of GABA(B) receptors had no effect, but the facilitation effect was partially blocked by the N-methyl-d-aspartate (NMDA) antagonist APV, indicating that NMDA receptors play a role. Application of ZD7288, a selective inhibitor of the hyperpolarization-activated cationic current I(h), almost completely blocked the relative facilitation of responses, and the less potent I(h)-blocker Cs(+) also resulted in a partial block. The relative facilitation of synaptic responses induced by CCh is therefore likely mediated by multiple mechanisms including the cholinergic suppression of transmitter release that enhances transmitter availability during repetitive stimulation, NMDA receptor-mediated effects on pre- or postsynaptic function, and

  19. A comparison of β-adrenoceptors and muscarinic cholinergic receptors in tissues of brown bullhead catfish (Ameiurus nebulosus) from the black river and old woman creek, Ohio

    USGS Publications Warehouse

    Steevens, Jeffery A.; Baumann, Paul C.; Jones, Susan B.

    1996-01-01

    β-Adrenoceptors (βARs) and muscarinic cholinergic receptors were measured in brain, gill, and heart tissues of brown bullhead catfish exposed to polycyclic aromatic hydrocarbons in the Black River, Ohio, USA, and were compared to values from Old Woman Creek, Ohio, a reference site. A decreased number of βARs were found in the gill from Black River fish, possibly indicating a compensatory response subsequent to chemical stress.

  20. A comparison of {beta}-adrenoceptors and muscarinic cholinergic receptors in tissues of brown bullhead catfish (Ameiurus nebulosus) from the Black River and Old Woman Creek, Ohio

    SciTech Connect

    Steevens, J.A.; Baumann, P.C.; Jones, S.B.

    1996-09-01

    {beta}-Adrenoceptors ({beta}ARs) and muscarinic cholinergic receptors were measured in brain, gill, and heart tissues of brown bullhead catfish exposed to polycyclic aromatic hydrocarbons in the Black River, Ohio, USA, and were compared to values from Old Woman Creek, Ohio, a reference site. A decreased number of {beta}ARs were found in the gill from Black River fish, possibly indicating a compensatory response subsequent to chemical stress.

  1. Development of adrenergic and cholinergic receptor cardiovascular regulatory capacity in the Canada goose (Branta canadensis) and domestic goose (Anser anser domesticus).

    PubMed

    Swart, Justin; Tate, Kevin; Crossley, Dane A

    2014-01-01

    Adrenergic control of cardiovascular function is a common component of regulation in embryonic birds studied to date. Our goal was to investigate adrenergic and cholinergic cardiovascular regulation in two goose species, the Canada goose (Branta canadensis) and the related domestic goose (Anser anser domesticus) to determine if these species possess similar regulation during embryonic development. We determined mean arterial pressure (Pm) and heart rate (fH) responses to serial blockade of cholinergic, β-adrenergic and α-adrenergic receptors in B. canadensis and A. domesticus at 70 and 90% of total incubation (28days). Both species possessed cholinergic and β-adrenergic tone on fH at 70% of incubation that increased in intensity with development. In addition, a constant α-adrenergic tone on Pm was present in both species. Our findings indicate that these geese species possess both cholinergic and adrenergic cardiovascular regulation providing information needed for speculation regarding the commonalities in the ontogeny of these cardiovascular regulatory elements in birds.

  2. Libidibia ferrea Mature Seeds Promote Antinociceptive Effect by Peripheral and Central Pathway: Possible Involvement of Opioid and Cholinergic Receptors

    PubMed Central

    Sawada, Luis Armando; Monteiro, Vanessa Sâmia da Conçeição; Rabelo, Guilherme Rodrigues; Dias, Germana Bueno; Da Cunha, Maura; do Nascimento, José Luiz Martins; Bastos, Gilmara de Nazareth Tavares

    2014-01-01

    Libidibia ferrea (LF) is a medicinal plant that holds many pharmacological properties. We evaluated the antinociceptive effect in the LF aqueous seed extract and Lipidic Portion of Libidibia ferrea (LPLF), partially elucidating their mechanisms. Histochemical tests and Gas chromatography of the LPLF were performed to characterize its fatty acids. Acetic acid-induced abdominal constriction, formalin-induced pain, and hot-plate test in mice were employed in the study. In all experiments, aqueous extract or LPLF was administered systemically at the doses of 1, 5, and 10 mg/kg. LF aqueous seed extract and LPLF demonstrated a dose-dependent antinociceptive effect in all tests indicating both peripheral anti-inflammatory and central analgesia properties. Also, the use of atropine (5 mg/kg), naloxone (5 mg/kg) in the abdominal writhing test was able to reverse the antinociceptive effect of the LPLF, indicating that at least one of LF lipids components is responsible for the dose related antinociceptive action in chemical and thermal models of nociception in mice. Together, the present results suggested that Libidibia ferrea induced antinociceptive activity is possibly related to its ability to inhibit opioid, cholinergic receptors, and cyclooxygenase-2 pathway, since its main component, linoleic acid, has been demonstrated to produce such effect in previous studies. PMID:24860820

  3. Alzheimer's Disease: Targeting the Cholinergic System

    PubMed Central

    Ferreira-Vieira, Talita H.; Guimaraes, Isabella M.; Silva, Flavia R.; Ribeiro, Fabiola M.

    2016-01-01

    Acetylcholine (ACh) has a crucial role in the peripheral and central nervous systems. The enzyme choline acetyltransferase (ChAT) is responsible for synthesizing ACh from acetyl-CoA and choline in the cytoplasm and the vesicular acetylcholine transporter (VAChT) uptakes the neurotransmitter into synaptic vesicles. Following depolarization, ACh undergoes exocytosis reaching the synaptic cleft, where it can bind its receptors, including muscarinic and nicotinic receptors. ACh present at the synaptic cleft is promptly hydrolyzed by the enzyme acetylcholinesterase (AChE), forming acetate and choline, which is recycled into the presynaptic nerve terminal by the high-affinity choline transporter (CHT1). Cholinergic neurons located in the basal forebrain, including the neurons that form the nucleus basalis of Meynert, are severely lost in Alzheimer’s disease (AD). AD is the most ordinary cause of dementia affecting 25 million people worldwide. The hallmarks of the disease are the accumulation of neurofibrillary tangles and amyloid plaques. However, there is no real correlation between levels of cortical plaques and AD-related cognitive impairment. Nevertheless, synaptic loss is the principal correlate of disease progression and loss of cholinergic neurons contributes to memory and attention deficits. Thus, drugs that act on the cholinergic system represent a promising option to treat AD patients. PMID:26813123

  4. Cholinergic Neurons in the Basal Forebrain Promote Wakefulness by Actions on Neighboring Non-Cholinergic Neurons: An Opto-Dialysis Study

    PubMed Central

    Zant, Janneke C.; Kim, Tae; Prokai, Laszlo; Szarka, Szabolcs; McNally, James; McKenna, James T.; Shukla, Charu; Yang, Chun; Kalinchuk, Anna V.; McCarley, Robert W.; Brown, Ritchie E.

    2016-01-01

    Understanding the control of sleep–wake states by the basal forebrain (BF) poses a challenge due to the intermingled presence of cholinergic, GABAergic, and glutamatergic neurons. All three BF neuronal subtypes project to the cortex and are implicated in cortical arousal and sleep–wake control. Thus, nonspecific stimulation or inhibition studies do not reveal the roles of these different neuronal types. Recent studies using optogenetics have shown that “selective” stimulation of BF cholinergic neurons increases transitions between NREM sleep and wakefulness, implicating cholinergic projections to cortex in wake promotion. However, the interpretation of these optogenetic experiments is complicated by interactions that may occur within the BF. For instance, a recent in vitro study from our group found that cholinergic neurons strongly excite neighboring GABAergic neurons, including the subset of cortically projecting neurons, which contain the calcium-binding protein, parvalbumin (PV) (Yang et al., 2014). Thus, the wake-promoting effect of “selective” optogenetic stimulation of BF cholinergic neurons could be mediated by local excitation of GABA/PV or other non-cholinergic BF neurons. In this study, using a newly designed opto-dialysis probe to couple selective optical stimulation with simultaneous in vivo microdialysis, we demonstrated that optical stimulation of cholinergic neurons locally increased acetylcholine levels and increased wakefulness in mice. Surprisingly, the enhanced wakefulness caused by cholinergic stimulation was abolished by simultaneous reverse microdialysis of cholinergic receptor antagonists into BF. Thus, our data suggest that the wake-promoting effect of cholinergic stimulation requires local release of acetylcholine in the basal forebrain and activation of cortically projecting, non-cholinergic neurons, including the GABAergic/PV neurons. SIGNIFICANCE STATEMENT Optogenetics is a revolutionary tool to assess the roles of

  5. Cellular and molecular basis of cholinergic function

    SciTech Connect

    Dowdall, M.J.; Hawthorne, J.N.

    1987-01-01

    This book contains 105 selections. Some of the titles are: Functional correlates of brain nicotine receptors; Muscarinic receptor subclasses; Cholinergic innervation and levels of nerve growth factor and its mRNA in the central nervous system; Developmentally regulated neurontrophic activities of Torpedo electric organ tissue; and Association of a regulatory peptide with cholinergic neurons.

  6. Muscarinic cholinergic receptors (MR3) in saliva of patients with oral lichen planus.

    PubMed

    Agha-Hosseini, Farzaneh; Mirzaii-Dizgah, Iraj; Mohammadpour, Neda

    2016-09-01

    Oral lichen planus (OLP) is a relatively common, chronic, and inflammatory mucocutaneous disease. Xerostomia is also a common complaint of most OLP patients. Considering the significant role of M3 muscarinic receptors (M3R) in secretion of saliva, this study sought to compare the level of this receptor in saliva between OLP patients and healthy controls. Forty OLP patients and 40 healthy controls filled out two questionnaires regarding xerostomia to assess its degree of severity. Unstimulated and stimulated salivary samples were obtained of both groups and the stimulated and unstimulated salivary flow rates were calculated. Salivary level of M3 muscarinic receptors was measured using the ELISA kit. Data were analyzed and compared using unpaired student's t test. P < 0.05 was considered significant. Stimulated and unstimulated salivary flow rates and M3 muscarinic receptors levels were significantly lower but degree of xerostomia was significantly higher in OLP patients compared to healthy controls. Salivary M3 muscarinic receptor seems to be low in the patients with OLP and these patients suffer from xerostomia and reduced salivary flow rate. PMID:27371099

  7. The muscarinic receptor of chick embryo cells: correlation between ligand binding and calcium mobilization

    PubMed Central

    1985-01-01

    In this report we characterize muscarinic cholinergic receptor on embryonic cells. We established dose-response curves by fluorometric measurement of Ca2+ mobilization in cell suspensions of whole chick embryos stage 23/24. Ca2+ mobilization was quantitated by standardization of chlorotetracycline (CTC) fluorescence changes after stimulation with muscarinic agonists. We determined ED50 values for the agonists acetylcholine and carbachol as 3.4 X 10(-6) and 2.7 X 10(-5) M, respectively. Pilocarpine and oxotremorine were found to act as reversible competitive antagonists with inhibition constants (Kl) of 5.0 X 10(-6) and 1.4 X 10(-6) M, respectively. Bethanechol, which induced only 23% of the maximal effect obtained by acetylcholine, was a partial agonist with an ED50 of 4.8 X 10(-4) M. Its antagonistic component is expressed by an inhibition constant of 1.9 X 10(-4) M. In parallel, binding studies were performed in a competition assay with [3H]-quinuclidinylbenzilate. For the agonists acetylcholine and carbachol, binding parameters were best fitted by a "two binding-sites model." Comparison with dose-response curves indicated that Ca2+ mobilization was triggered via the high-affinity binding site. The inhibition constants of antagonists derived from the shift of dose- response curves corresponded to the fitted KD values of the binding studies when a "one binding-site model" was applied. Combination of dose-response and binding data showed close proportionality between receptor occupancy and calcium mobilization. No spare receptors were present. PMID:2858487

  8. PPARα regulates cholinergic-driven activity of midbrain dopamine neurons via a novel mechanism involving α7 nicotinic acetylcholine receptors.

    PubMed

    Melis, Miriam; Scheggi, Simona; Carta, Gianfranca; Madeddu, Camilla; Lecca, Salvatore; Luchicchi, Antonio; Cadeddu, Francesca; Frau, Roberto; Fattore, Liana; Fadda, Paola; Ennas, M Grazia; Castelli, M Paola; Fratta, Walter; Schilstrom, Bjorn; Banni, Sebastiano; De Montis, M Graziella; Pistis, Marco

    2013-04-01

    Ventral tegmental area dopamine neurons control reward-driven learning, and their dysregulation can lead to psychiatric disorders. Tonic and phasic activity of these dopaminergic neurons depends on cholinergic tone and activation of nicotinic acetylcholine receptors (nAChRs), particularly those containing the β2 subunit (β2*-nAChRs). Nuclear peroxisome proliferator-activated receptors type-α (PPARα) tonically regulate β2*-nAChRs and thereby control dopamine neuron firing activity. However, it is unknown how and when PPARα endogenous ligands are synthesized by dopamine cells. Using ex vivo and in vivo electrophysiological techniques combined with biochemical and behavioral analysis, we show that activation of α7-nAChRs increases in the rat VTA both the tyrosine phosphorylation of the β2 subunit of nAChRs and the levels of two PPARα endogenous ligands in a Ca(2+)-dependent manner. Accordingly, in vivo production of endogenous PPARα ligands, triggered by α7-nAChR activation, blocks in rats nicotine-induced increased firing activity of dopamine neurons and displays antidepressant-like properties. These data demonstrate that endogenous PPARα ligands are effectors of α7-nAChRs and that their neuromodulatory properties depend on phosphorylation of β2*-nAChRs on VTA dopamine cells. This reveals an autoinhibitory mechanism aimed at reducing dopamine cell overexcitation engaged during hypercholinergic drive. Our results unveil important physiological functions of nAChR/PPARα signaling in dopamine neurons and how behavioral output can change after modifications of this signaling pathway. Overall, the present study suggests PPARα as new therapeutic targets for disorders associated with unbalanced dopamine-acetylcholine systems. PMID:23554501

  9. The chemokine interleukin-8 acutely reduces Ca(2+) currents in identified cholinergic septal neurons expressing CXCR1 and CXCR2 receptor mRNAs.

    PubMed

    Puma, C; Danik, M; Quirion, R; Ramon, F; Williams, S

    2001-09-01

    The chemokine IL-8 is known to be synthesized by glial cells in the brain. It has traditionally been shown to have an important role in neuroinflammation but recent evidence indicates that it may also be involved in rapid signaling in neurons. We investigated how IL-8 participates in rapid neuronal signaling by using a combination of whole-cell recording and single-cell RT-PCR on dissociated rat septal neurons. We show that IL-8 can acutely reduce Ca(2+) currents in septal neurons, an effect that was concentration-dependent, involved the closure of L- and N-type Ca(2+) channels, and the activation of G(ialpha1) and/or G(ialpha2) subtype(s) of G-proteins. Analysis of the mRNAs from the recorded neurons revealed that the latter were all cholinergic in nature. Moreover, we found that all cholinergic neurons that responded to IL-8, expressed mRNAs for either one or both IL-8 receptors CXCR1 and CXCR2. This is the first report of a chemokine that modulates ion channels in neurons via G-proteins, and the first demonstration that mRNAs for CXCR1 are expressed in the brain. Our results suggest that IL-8 release by glial cells in vivo may activate CXCR1 and CXCR2 receptors on cholinergic septal neurons and acutely modulate their excitability by closing calcium channels. PMID:11553670

  10. Morphine dependence and withdrawal induced changes in cholinergic signaling

    PubMed Central

    Neugebauer, Nichole M.; Einstein, Emily B.; Lopez, Maria B.; McClure-Begley, Tristan D.; Mineur, Yann S.; Picciotto, Marina R.

    2013-01-01

    Cholinergic signaling is thought to be involved in morphine dependence and withdrawal, but the specific mechanisms involved remain unclear. The current study aimed to identify alterations in the cholinergic system that may contribute to the development of morphine dependence and withdrawal. Acetylcholinesterase (AChE) activity and [3H]-epibatidine binding were evaluated in order to determine if morphine dependence and withdrawal induces alterations in cholinergic signaling or expression of high affinity nicotinic acetylcholine receptors (nAChRs) in the midbrain (MB), medial habenula (MHb) and interpeduncular nucleus (IPN). The effect of cholinergic signaling through nAChRs on morphine-withdrawal induced jumping behavior was then determined. Lastly, the contribution of β4-containing nAChRs receptors in the MHb to morphine-withdrawal induced jumping behavior and neuronal activity as indicated by c-fos expression was assessed. Chronic morphine administration decreased AChE activity in MB and MHb, an effect that was no longer present following precipitated withdrawal. Morphine dependent mice showed increased nicotinic acetylcholine receptor (nAChR) levels in MB. Further, nicotine (0.4 mg/kg) and lobeline (3 mg/kg) decreased jumping behavior while mecamylamine (1 mg/kg) had no effect. Knock-down of β4 subunit-containing nAChRs in the MHb attenuated c-fos activation, but did not decrease morphine withdrawal-induced jumping. Thus, morphine withdrawal induces cholinergic signaling in the MHb, but this does not appear to be responsible for the effects of cholinergic drugs on somatic signs of opiate withdrawal, as measured by jumping behavior. PMID:23651795

  11. In vitro and in vivo evidence for the existence of presynaptic muscarinic cholinergic receptors in the rat hippocampus.

    PubMed

    Consolo, S; Wang, J X; Fusi, R; Vinci, R; Forloni, G; Ladinsky, H

    1984-08-20

    The intrahippocampal injection of kainic acid cleared 50% of muscarinic receptors and favored the detection of a further 20% loss in hippocampal presynaptic muscarinic receptors produced by electrolytic lesion of the medial septal nucleus as determined by Scatchard analysis of the saturation isotherms of [3H]dexetimide binding. In accordance, a decrease of about 20% in the in vivo accumulation of [3H]dexetimide in the hippocampus was found in animals lesioned in the medial septal nucleus. This effect occurred at both the dose of 5 micrograms/kg and at the saturating dose of 100 micrograms/kg of [3H]dexetimide. The results suggest that the loss was due to decreased receptor number rather than decreased receptor affinity. PMID:6488003

  12. The α7-nicotinic receptor is upregulated in immune cells from HIV-seropositive women: consequences to the cholinergic anti-inflammatory response

    PubMed Central

    Delgado-Vélez, Manuel; Báez-Pagán, Carlos A; Gerena, Yamil; Quesada, Orestes; Santiago-Pérez, Laura I; Capó-Vélez, Coral M; Wojna, Valerie; Meléndez, Loyda; León-Rivera, Rosiris; Silva, Walter; Lasalde-Dominicci, José A

    2015-01-01

    Antiretroviral therapy partially restores the immune system and markedly increases life expectancy of HIV-infected patients. However, antiretroviral therapy does not restore full health. These patients suffer from poorly understood chronic inflammation that causes a number of AIDS and non-AIDS complications. Here we show that chronic inflammation in HIV+ patients may be due to the disruption of the cholinergic anti-inflammatory pathway by HIV envelope protein gp120IIIB. Our results demonstrate that HIV gp120IIIB induces α7 nicotinic acetylcholine receptor (α7) upregulation and a paradoxical proinflammatory phenotype in macrophages, as activation of the upregulated α7 is no longer capable of inhibiting the release of proinflammatory cytokines. Our results demonstrate that disruption of the cholinergic-mediated anti-inflammatory response can result from an HIV protein. Collectively, these findings suggest that HIV tampering with a natural strategy to control inflammation could contribute to a crucial, unresolved problem of HIV infection: chronic inflammation. PMID:26719799

  13. Activation of Alpha 7 Cholinergic Nicotinic Receptors Reduce Blood–Brain Barrier Permeability following Experimental Traumatic Brain Injury

    PubMed Central

    Zhao, Jing; Kobori, Nobuhide; Redell, John B.; Hylin, Michael J.; Hood, Kimberly N.; Moore, Anthony N.

    2016-01-01

    Traumatic brain injury (TBI) is a major human health concern that has the greatest impact on young men and women. The breakdown of the blood–brain barrier (BBB) is an important pathological consequence of TBI that initiates secondary processes, including infiltration of inflammatory cells, which can exacerbate brain inflammation and contribute to poor outcome. While the role of inflammation within the injured brain has been examined in some detail, the contribution of peripheral/systemic inflammation to TBI pathophysiology is largely unknown. Recent studies have implicated vagus nerve regulation of splenic cholinergic nicotinic acetylcholine receptor α7 (nAChRa7) signaling in the regulation of systemic inflammation. However, it is not known whether this mechanism plays a role in TBI-triggered inflammation and BBB breakdown. Following TBI, we observed that plasma TNF-α and IL-1β levels, as well as BBB permeability, were significantly increased in nAChRa7 null mice (Chrna7−/−) relative to wild-type mice. The administration of exogenous IL-1β and TNF-α to brain-injured animals worsened Evans Blue dye extravasation, suggesting that systemic inflammation contributes to TBI-triggered BBB permeability. Systemic administration of the nAChRa7 agonist PNU-282987 or the positive allosteric modulator PNU-120596 significantly attenuated TBI-triggered BBB compromise. Supporting a role for splenic nAChRa7 receptors, we demonstrate that splenic injection of the nicotinic receptor blocker α-bungarotoxin increased BBB permeability in brain-injured rats, while PNU-282987 injection decreased such permeability. These effects were not seen when α-bungarotoxin or PNU-282987 were administered to splenectomized, brain-injured rats. Together, these findings support the short-term use of nAChRa7-activating agents as a strategy to reduce TBI-triggered BBB permeability. SIGNIFICANCE STATEMENT Breakdown of the blood–brain barrier (BBB) in response to traumatic brain injury (TBI

  14. Cholinergic Neurotransmission in the Posterior Insular Cortex Is Altered in Preclinical Models of Neuropathic Pain: Key Role of Muscarinic M2 Receptors in Donepezil-Induced Antinociception

    PubMed Central

    Ferrier, Jérémy; Bayet-Robert, Mathilde; Dalmann, Romain; El Guerrab, Abderrahim; Aissouni, Youssef; Graveron-Demilly, Danielle; Chalus, Maryse; Pinguet, Jérémy; Eschalier, Alain; Richard, Damien; Daulhac, Laurence; Balayssac, David

    2015-01-01

    Neuropathic pain is one of the most debilitating pain conditions, yet no therapeutic strategy has been really effective for its treatment. Hence, a better understanding of its pathophysiological mechanisms is necessary to identify new pharmacological targets. Here, we report important metabolic variations in brain areas involved in pain processing in a rat model of oxaliplatin-induced neuropathy using HRMAS 1H-NMR spectroscopy. An increased concentration of choline has been evidenced in the posterior insular cortex (pIC) of neuropathic animal, which was significantly correlated with animals' pain thresholds. The screening of 34 genes mRNA involved in the pIC cholinergic system showed an increased expression of the high-affinity choline transporter and especially the muscarinic M2 receptors, which was confirmed by Western blot analysis in oxaliplatin-treated rats and the spared nerve injury model (SNI). Furthermore, pharmacological activation of M2 receptors in the pIC using oxotremorine completely reversed oxaliplatin-induced mechanical allodynia. Consistently, systemic treatment with donepezil, a centrally active acetylcholinesterase inhibitor, prevented and reversed oxaliplatin-induced cold and mechanical allodynia as well as social interaction impairment. Intracerebral microdialysis revealed a lower level of acetylcholine in the pIC of oxaliplatin-treated rats, which was significantly increased by donepezil. Finally, the analgesic effect of donepezil was markedly reduced by a microinjection of the M2 antagonist, methoctramine, within the pIC, in both oxaliplatin-treated rats and spared nerve injury rats. These findings highlight the crucial role of cortical cholinergic neurotransmission as a critical mechanism of neuropathic pain, and suggest that targeting insular M2 receptors using central cholinomimetics could be used for neuropathic pain treatment. SIGNIFICANCE STATEMENT Our study describes a decrease in cholinergic neurotransmission in the posterior insular

  15. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    SciTech Connect

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  16. Steroid binding domain of porcine estrogen receptor

    SciTech Connect

    Koike, S.; Nii, A.; Sakai, M.; Muramatsu, M.

    1987-05-05

    For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), the authors have made use of affinity labeling of partially purified ER with (/sup 3/H)tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or ..cap alpha..-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.

  17. Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons.

    PubMed

    Kelly, J F; Furukawa, K; Barger, S W; Rengen, M R; Mark, R J; Blanc, E M; Roth, G S; Mattson, M P

    1996-06-25

    Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C, resulting in the liberation of inositol trisphosphate and Ca2+ release from intracellular stores. We now report that amyloid beta-peptide (Abeta), which forms plaques in the brain in AD, impairs muscarinic receptor activation of G proteins in cultured rat cortical neurons. Exposure of rodent fetal cortical neurons to Abeta25-35 and Abeta1-40 resulted in a concentration and time-dependent attenuation of carbachol-induced GTPase activity without affecting muscarinic receptor ligand binding parameters. Downstream events in the signal transduction cascade were similarly attenuated by Abeta. Carbachol-induced accumulation of inositol phosphates (IP, IP2, IP3, and IP4) was decreased and calcium imaging studies revealed that carbachol-induced release of calcium was severely impaired in neurons pretreated with Abeta. Muscarinic cholinergic signal transduction was disrupted with subtoxic levels of exposure to AP. The effects of Abeta on carbachol-induced GTPase activity and calcium release were attenuated by antioxidants, implicating free radicals in the mechanism whereby Abeta induced uncoupling of muscarinic receptors. These data demonstrate that Abeta disrupts muscarinic receptor coupling to G proteins that mediate induction of phosphoinositide accumulation and calcium release, findings that implicate Abeta in the impairment of cholinergic transmission that occurs in AD. PMID:8692890

  18. Protection against ventricular fibrillation via cholinergic receptor stimulation and the generation of nitric oxide

    PubMed Central

    Kalla, Manish; Chotalia, Minesh; Coughlan, Charles; Hao, Guoliang; Crabtree, Mark J.; Tomek, Jakub; Bub, Gil; Paterson, David J.

    2016-01-01

    Key points Animal studies suggest an anti‐fibrillatory action of the vagus nerve on the ventricle, although the exact mechanism is controversial.Using a Langendorff perfused rat heart, we show that the acetylcholine analogue carbamylcholine raises ventricular fibrillation threshold (VFT) and flattens the electrical restitution curve.The anti‐fibrillatory action of carbamylcholine was prevented by the nicotinic receptor antagonist mecamylamine, inhibitors of neuronal nitric oxide synthase (nNOS) and soluble guanylyl cyclase (sGC), and can be mimicked by the nitric oxide (NO) donor sodium nitroprusside.Carbamylcholine increased NO metabolite content in the coronary effluent and this was prevented by mecamylamine.The anti‐fibrillatory action of both carbamylcholine and sodium nitroprusside was ultimately dependent on muscarinic receptor stimulation as all effects were blocked by atropine.These data demonstrate a protective effect of carbamylcholine on VFT that depends upon both muscarinic and nicotinic receptor stimulation, where the generation of NO is likely to be via a neuronal nNOS–sGC dependent pathway. Abstract Implantable cardiac vagal nerve stimulators are a promising treatment for ventricular arrhythmia in patients with heart failure. Animal studies suggest the anti‐fibrillatory effect may be nitric oxide (NO) dependent, although the exact site of action is controversial. We investigated whether a stable analogue of acetylcholine could raise ventricular fibrillation threshold (VFT), and whether this was dependent on NO generation and/or muscarinic/nicotinic receptor stimulation. VFT was determined in Langendorff perfused rat hearts by burst pacing until sustained VF was induced. Carbamylcholine (CCh, 200 nmol l–1, n = 9) significantly (P < 0.05) reduced heart rate from 292 ± 8 to 224 ± 6 b.p.m. Independent of this heart rate change, CCh caused a significant increase in VFT (control 1.5 ± 0.3 mA, CCh 2.4 ± 0.4 mA, wash 1.1

  19. [Regional distribution of the muscarinic cholinergic receptor in the human brain studied with 11C-benztropine and PET using an anatomical standardization technique].

    PubMed

    Ono, S; Kawashima, R; Ito, H; Koyama, M; Goto, R; Inoue, K; Sato, K; Fujiwara, T; Meguro, K; Yanai, K; Sasaki, H; Ido, T; Ito, M; Fukuda, H

    1996-07-01

    We measured regional distribution of the muscarinic cholinergic receptor in the normal human brain with 11C-benztropine (BZT) and positron emission tomography (PET) using an anatomical standardization technique. Seven normal volunteers who gave informed consents were involved in this study. Immediately after intravenous injection of 11C-BZT into the subjects, we started dynamic PET scanning and serial frequent arterial blood sampling. Analyses of plasma metabolites were also performed at selected time points and plasma time activity curves (TAC) of unmetabolized ligand were generated. From these PET and TAC data, we obtained Patlak plot slope calculation images. Using the HBA (human brain atlas) system, the Patlak plot slope calculation image of each subject was transformed into the shape of the standard brain. Mean and standard deviation (SD) calculation images were generated from those anatomically standardized images. On these mean and SD images, we placed regions of interest which were previously outlined on a MR image of the standard brain. From those data, we found the highest receptor distribution in the striatum and occipital cortex, as well as high distribution in the frontal, parietal, and temporal cortices, which were consistent with previous reports. These results suggested that anatomical standardization of PET receptor images with 11C-BZT will be useful for delineating the physiological or pathological alterations of the muscarinic cholinergic receptor in the human brain.

  20. Nicotinic α4 Receptor-Mediated Cholinergic Influences on Food Intake and Activity Patterns in Hypothalamic Circuits

    PubMed Central

    Schaaf, Laura; Heeley, Nicholas; Heuschmid, Lena; Bai, Yunjing; Barrantes, Francisco J.; Apergis-Schoute, John

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine’s (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4β2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHβE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHβE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHβE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4β2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4β2 nAChR-mediated transmission. PMID:26247203

  1. Cerebellar Nicotinic Cholinergic Receptors are Intrinsic to the Cerebellum: Implications for Diverse Functional Roles

    PubMed Central

    Turner, Jill R.; Ortinski, Pavel I.; Sherrard, Rachel M.

    2016-01-01

    Although recent studies have delineated the specific nicotinic subtypes present in the mammalian cerebellum, very little is known about their location or function within the cerebellum. This is of increased interest since nicotinic receptors (nAChRs) in the cerebellum have recently been implicated in the pathology of autism spectrum disorders. To begin to better understand the roles of these heteromeric nAChRs in the cerebellar circuitry and their therapeutic potential as targets for drug development, we used various chemical and stereotaxic lesion models in conjunction with slice electrophysiology to examine how specific heteromeric nAChR subtypes may influence the surrounding cerebellar circuitry. Using subunit-specific immunoprecipitation of radiolabeled nAChRs in the cerebella following N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride, p-chloroamphetamine, and pendunculotomy lesions, we show that most, if not all, cerebellar nicotinic receptors are present in cells within the cerebellum itself and not in extracerebellar afferents. Furthermore, we demonstrate that the β4-containing, but not the β2-containing, nAChRs intrinsic to the cerebellum can regulate inhibitory synaptic efficacy at two major classes of cerebellar neurons. These tandem findings suggest that nAChRs may present a potential drug target for disorders involving the cerebellum. PMID:21562921

  2. NMDA receptor binding in focal epilepsies

    PubMed Central

    McGinnity, C J; Koepp, M J; Hammers, A; Riaño Barros, D A; Pressler, R M; Luthra, S; Jones, P A; Trigg, W; Micallef, C; Symms, M R; Brooks, D J; Duncan, J S

    2015-01-01

    Objective To demonstrate altered N-methyl-d-aspartate (NMDA) receptor availability in patients with focal epilepsies using positron emission tomography (PET) and [18F]GE-179, a ligand that selectively binds to the open NMDA receptor ion channel, which is thought to be overactive in epilepsy. Methods Eleven patients (median age 33 years, 6 males) with known frequent interictal epileptiform discharges had an [18F]GE-179 PET scan, in a cross-sectional study. MRI showed a focal lesion but discordant EEG changes in two, was non-localising with multifocal EEG abnormalities in two, and was normal in the remaining seven patients who all had multifocal EEG changes. Individual patient [18F]GE-179 volume-of-distribution (VT) images were compared between individual patients and a group of 10 healthy controls (47 years, 7 males) using Statistical Parametric Mapping. Results Individual analyses revealed a single cluster of focal VT increase in four patients; one with a single and one with multifocal MRI lesions, and two with normal MRIs. Post hoc analysis revealed that, relative to controls, patients not taking antidepressants had globally increased [18F]GE-179 VT (+28%; p<0.002), and the three patients taking an antidepressant drug had globally reduced [18F]GE-179 VT (−29%; p<0.002). There were no focal abnormalities common to the epilepsy group. Conclusions In patients with focal epilepsies, we detected primarily global increases of [18F]GE-179 VT consistent with increased NMDA channel activation, but reduced availability in those taking antidepressant drugs, consistent with a possible mode of action of this class of drugs. [18F]GE-179 PET showed focal accentuations of NMDA binding in 4 out of 11 patients, with difficult to localise and treat focal epilepsy. PMID:25991402

  3. Hippocampal α7-nicotinic cholinergic receptors modulate memory reconsolidation: a potential strategy for recovery from amnesia.

    PubMed

    Blake, M G; Boccia, M M; Krawczyk, M C; Baratti, C M

    2013-11-01

    When subjects are exposed to new learning experiences, the novel information could be acquired and eventually stored through memory consolidation process. The exposure of mice to a novel experience (a hole-board) after being trained in an inhibitory avoidance apparatus is followed by impaired performance of the avoidance memory in subsequent tests. The same impairing effect is produced when mice are exposed to the novel environment after the reactivation of the avoidance memory. This interfering effect is due to impaired consolidation or reconsolidation of the avoidance memory. The administration of the α7-nicotinic receptor agonist choline (Ch) in the dorsal hippocampus (0.8 μg/hippocampus) immediately after the inhibitory avoidance memory reactivation, allowed memory recovery. This effect of Ch was time-dependent, and retention performance was not affected in drug-treated mice that were not subjected to memory reactivation, suggesting that the effects on performance are not due to non-specific effects of the drug. The effects of Ch also depended on the age of the reactivated memory. Altogether, our results suggest that Ch exerts its effects by modulating memory reconsolidation, and that the memory impairment induced by new learning is a memory expression failure and not a storage deficit. Therefore, reconsolidation, among other functions, might serve to change whether a memory will be expressed in later tests. Summarizing, our results open new avenues about the behavioral significance and the physiological functions of memory reconsolidation, providing new strategies for recovering memories from some types of amnesia.

  4. Effects of extracellular acetylcholine on muscarinic receptor binding assessed by [125I]dexetimide and a simple probe.

    PubMed

    Sánchez-Roa, P M; Wagner, H N; Villemagne, V L; London, E D; Lever, J R

    1998-10-01

    New pharmacologic approaches to enhance brain cholinergic function focus on increasing intrasynaptic acetylcholine. We examined the usefulness of a simple probe and [125I]dexetimide to evaluate in vivo the effects of extracellular acetylcholine on muscarinic receptor binding in the mouse brain. After radiotracer injection continuous time/activity curves were generated over 330 min. [125I]Dexetimide reached a plateau at 90 min post-injection. To increase extracellular acetylcholine, the anticholinesterase physostigmine was administered at 120 min, producing a reversible decrease in [125I]dexetimide specific binding (23%) for 30 min. These findings demonstrate that dynamic changes in extracellular acetylcholine can be evaluated by displacement of [125I]dexetimide binding in vivo using a simple probe system. PMID:9822886

  5. Cholinergic receptor blockade by scopolamine and mecamylamine exacerbates global cerebral ischemia induced memory dysfunction in C57BL/6J mice.

    PubMed

    Ray, R S; Rai, S; Katyal, A

    2014-12-01

    Global cerebral ischemia/reperfusion (GCI/R) injury encompasses complex pathophysiological sequalae, inducing loss of hippocampal neurons and behavioural deficits. Progressive neuronal death and memory dysfunctions culminate from several different mechanisms like oxidative stress, excitotoxicity, neuroinflammation and cholinergic hypofunction. Experimental evidences point to the beneficial effects of cholinomimetic agents such as rivastigmine and galantamine in improving memory outcomes following GCI/R injury. However, the direct implications of muscarinic and nicotinic receptor blockade during global cerebral ischemia/reperfusion injury have not been investigated. Therefore, we evaluated the relative involvement of muscarinic and nicotinic receptors in spatial/associative memory functions and neuronal damage during global cerebral ischemia reperfusion injury. The outcomes of present study support the idea that preservation of both muscarinic and nicotinic receptor functions is essential to alleviate hippocampal neuronal death in CA1 region following global cerebral ischemia/reperfusion injury. PMID:25168578

  6. Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors.

    PubMed

    Hellmuth, Antje; Calderón Villalobos, Luz Irina A

    2016-01-01

    In receptor-ligand interactions, dissociation constants provide a key parameter for characterizing binding. Here, we describe filter-based radioligand binding assays at equilibrium, either varying ligand concentrations up to receptor saturation or outcompeting ligand from its receptor with increasing concentrations of ligand analogue. Using the auxin coreceptor system, we illustrate how to use a saturation binding assay to determine the apparent dissociation constant (K D (') ) for the formation of a ternary TIR1-auxin-AUX/IAA complex. Also, we show how to determine the inhibitory constant (K i) for auxin binding by the coreceptor complex via a competition binding assay. These assays can be applied broadly to characterize a one-site binding reaction of a hormone to its receptor. PMID:27424743

  7. Differences in (-)citronellal binding to various odorant receptors.

    PubMed

    Stary, Anna; Suwattanasophon, Chonticha; Wolschann, Peter; Buchbauer, Gerhard

    2007-10-01

    To test the hypothesis that olfactory receptors (ORs) recognize different molecular features of odor molecules termed "odotypes", we studied receptor-ligand interactions of two human and two mouse ORs, recognizing (-)citronellal. Structurally similar receptors provide identical binding pockets (OLFR43, OR1A1, and OR1A2), and have comparable EC(50) values. Other ORs with lower sequence identity bind (-)citronellal in a different way, leading to different EC(50) values.

  8. Cholinergic modulation of hippocampal network function

    PubMed Central

    Teles-Grilo Ruivo, Leonor M.; Mellor, Jack R.

    2013-01-01

    Cholinergic septohippocampal projections from the medial septal area to the hippocampus are proposed to have important roles in cognition by modulating properties of the hippocampal network. However, the precise spatial and temporal profile of acetylcholine release in the hippocampus remains unclear making it difficult to define specific roles for cholinergic transmission in hippocampal dependent behaviors. This is partly due to a lack of tools enabling specific intervention in, and recording of, cholinergic transmission. Here, we review the organization of septohippocampal cholinergic projections and hippocampal acetylcholine receptors as well as the role of cholinergic transmission in modulating cellular excitability, synaptic plasticity, and rhythmic network oscillations. We point to a number of open questions that remain unanswered and discuss the potential for recently developed techniques to provide a radical reappraisal of the function of cholinergic inputs to the hippocampus. PMID:23908628

  9. Effects of ginger constituents on the gastrointestinal tract: role of cholinergic M3 and serotonergic 5-HT3 and 5-HT4 receptors.

    PubMed

    Pertz, Heinz H; Lehmann, Jochen; Roth-Ehrang, René; Elz, Sigurd

    2011-07-01

    The herbal drug ginger (Zingiber officinale Roscoe) may be effective for treating nausea, vomiting, and gastric hypomotility. In these conditions, cholinergic M (3) receptors and serotonergic 5-HT (3) and 5-HT (4) receptors are involved. The major chemical constituents of ginger are [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol. We studied the interaction of [6]-gingerol, [8]-gingerol, [10]-gingerol (racemates), and [6]-shogaol with guinea pig M (3) receptors, guinea pig 5-HT (3) receptors, and rat 5-HT (4) receptors. In whole segments of guinea pig ileum (bioassay for contractile M (3) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol slightly but significantly depressed the maximal carbachol response at an antagonist concentration of 10 µM. In the guinea pig myenteric plexus preparation (bioassay for contractile 5-HT (3) receptors), 5-HT maximal responses were depressed by [10]-gingerol from 93 ± 3 % to 65 ± 6 % at an antagonist concentration of 3 µM and to 48 ± 3 % at an antagonist concentration of 5 µM following desensitization of 5-HT (4) receptors and blockade of 5-HT (1) and 5-HT (2) receptors. [6]-Shogaol (3 µM) induced depression to 61 ± 3 %. In rat esophageal tunica muscularis mucosae (bioassay for relaxant 5-HT (4) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol (2-6.3 µM) showed no agonist effects. The maximal 5-HT response remained unaffected in the presence of the compounds. It is concluded that the efficiency of ginger in reducing nausea and vomiting may be based on a weak inhibitory effect of gingerols and shogaols at M (3) and 5-HT (3) receptors. 5-HT (4) receptors, which play a role in gastroduodenal motility, appear not to be involved in the action of these compounds. PMID:21305447

  10. An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors.

    PubMed

    Seol, W; Choi, H S; Moore, D D

    1996-05-31

    SHP is an orphan member of the nuclear hormone receptor superfamily that contains the dimerization and ligand-binding domain found in other family members but lacks the conserved DNA binding domain. In the yeast two-hybrid system, SHP interacted with several conventional and orphan members of the receptor superfamily, including retinoid receptors, the thyroid hormone receptor, and the orphan receptor MB67. SHP also interacted directly with these receptors in vitro. In mammalian cells, SHP specifically inhibited transactivation by the superfamily members with which it interacted. These results suggest that SHP functions as a negative regulator of receptor-dependent signaling pathways. PMID:8650544

  11. Development of gamma-emitting, receptor binding radiotracers for imaging the brain and pancreas

    SciTech Connect

    Reba, R.C.

    1990-01-01

    This progress report covers period from Nov. 1, 1989 to Aug. 31, 1990. The long term objective was to develop receptor-binding radiotracers for SPECT or PET imaging of CNS or peripheral nervous system. The specific chemistry aims, as understood on the basis of past findings, were: to synthesize and develop a more polar analogs of 4IQNB, possessing similar binding characteristics but eliminated more rapidly from the surrounding tissues and the target organ, to design a method of introducing a technetium chelating group onto a molecule or cholinergic agent without drastic lowering of its apparent affinity, to synthesize and develop radiotracers based on m-AChR antagonists selective for one of the subtypes of the receptor. The chemistry service aims were to prepare and characterize (R,R)- and (R,S)-4IQNB and derivatives, to provide the triazene intermediate to other investigators, and to provide ({sup 123}I)4IQNB for in vivo imaging. The biochemistry aims were to characterize the vitro and in vivo properties of novel compounds and to perform the pharmacokinetic studies. 3 refs., 5 tabs.

  12. Modulation of inflammatory pathways by the immune cholinergic system.

    PubMed

    Nizri, Eran; Brenner, Talma

    2013-07-01

    Research done in the past years pointed to a novel function of cholinergic transmission. It has been shown that cholinergic transmission can modulate various aspects of the immune function, whether innate or adaptive. Cholinergic transmission affects immune cell proliferation, cytokine production, T helper differentiation and antigen presentation. Theses effects are mediated by cholinergic muscarinic and nicotinic receptors and other cholinergic components present in immune cells, such as acetylcholinesterase (AChE) and cholineacetyltransferase. The α7 nicotinic acetylcholine receptor was designated anti-inflammatory activity and has shown promise in pre-clinical models of inflammatory disorders. We herein describe the various components of the immune cholinergic system, and specifically the immune suppressive effects of α7 activation. This activation can be accomplished either by direct stimulation or indirectly, by inhibition of AChE. Thus, the presence of the immune cholinergic system can pave the way for novel immunomodulatory agents, or to the broadening of use of known cholinergic agents.

  13. Characterization of pulmonary sigma receptors by radioligand binding.

    PubMed

    Lever, John R; Litton, Tyler P; Fergason-Cantrell, Emily A

    2015-09-01

    This study establishes the expression of appreciable populations of sites on mouse lung membranes that exhibit radioligand binding properties and pharmacology consistent with assignment as sigma1 and sigma2 receptors. Specific binding of the sigma1 receptor radioligand [(3)H](+)-pentazocine reached steady state within 6h at 37°C. Saturation studies revealed high affinity binding to a single class of sites (Kd 1.36±0.04nM; Bmax 967±11fmol/mg protein). Inhibition studies showed appropriate sigma1 receptor pharmacology, including higher affinity for (+)-N-allylnormetazocine with respect to the (-)-enantiomer, and positive allosteric modulation of dextromethorphan binding by phenytoin. Using [(3)H]1,3-di(2-tolyl)guanidine in the presence of (+)-pentazocine to assess sigma2 receptor binding, steady state was achieved within 2min at 25°C. Cold saturation studies revealed one high affinity, low capacity binding site (Kd 31.8±8.3nM; Bmax 921±228fmol/mg protein) that displayed sigma2 receptor pharmacology. A very low affinity, high capacity interaction also was observed that represents saturable, but not sigma receptor specific, binding. A panel of ligands showed rank order inhibition of radioligand binding appropriate for the sigma2 receptor, with ifenprodil displaying the highest apparent affinity. In vivo, dextromethorphan inhibited the specific binding of a radioiodinated sigma1 receptor ligand in lung with an ED50 of 1.2μmol/kg, a value near the recommended dosage for the drug as a cough suppressant. Overall, the present work provides a foundation for studies of drug interactions with pulmonary sigma1 and sigma2 receptors in vitro and in vivo.

  14. Characterization of pulmonary sigma receptors by radioligand binding

    PubMed Central

    Lever, John R.; Litton, Tyler P.; Fergason-Cantrell, Emily A.

    2015-01-01

    This study establishes the expression of appreciable populations of sites on mouse lung membranes that exhibit radioligand binding properties and pharmacology consistent with assignment as sigma1 and sigma2 receptors. Specific binding of the sigma1 receptor radioligand [3H](+)-pentazocine reached steady state within 6 h at 37 °C. Saturation studies revealed high affinity binding to a single class of sites (Kd 1.36 ± 0.04 nM; Bmax 967 ± 11 fmol / mg protein). Inhibition studies showed appropriate sigma1 receptor pharmacology, including higher affinity for (+)-N-allylnormetazocine with respect to the (−)-enantiomer, and positive allosteric modulation of dextromethorphan binding by phenytoin. Using [3H]1,3-di(2-tolyl)guanidine in the presence of (+)-pentazocine to assess sigma2 receptor binding, steady state was achieved within 2 min at 25 °C. Cold saturation studies revealed one high affinity, low capacity binding site (Kd 31.8 ± 8.3 nM; Bmax 921 ± 228 fmol / mg protein) that displayed sigma2 receptor pharmacology. A very low affinity, high capacity interaction also was observed that represents saturable, but not sigma receptor specific, binding. A panel of ligands showed rank order inhibition of radioligand binding appropriate for the sigma2 receptor, with ifenprodil displaying the highest apparent affinity. In vivo, dextromethorphan inhibited the specific binding of a radioiodinated sigma1 receptor ligand in lung with an ED50 of 1.2 µmol / kg, a value near the recommended dosage for the drug as a cough suppressant. Overall, the present work provides a foundation for studies of drug interactions with pulmonary sigma1 and sigma2 receptors in vitro and in vivo. PMID:26004528

  15. Characterization of pulmonary sigma receptors by radioligand binding.

    PubMed

    Lever, John R; Litton, Tyler P; Fergason-Cantrell, Emily A

    2015-09-01

    This study establishes the expression of appreciable populations of sites on mouse lung membranes that exhibit radioligand binding properties and pharmacology consistent with assignment as sigma1 and sigma2 receptors. Specific binding of the sigma1 receptor radioligand [(3)H](+)-pentazocine reached steady state within 6h at 37°C. Saturation studies revealed high affinity binding to a single class of sites (Kd 1.36±0.04nM; Bmax 967±11fmol/mg protein). Inhibition studies showed appropriate sigma1 receptor pharmacology, including higher affinity for (+)-N-allylnormetazocine with respect to the (-)-enantiomer, and positive allosteric modulation of dextromethorphan binding by phenytoin. Using [(3)H]1,3-di(2-tolyl)guanidine in the presence of (+)-pentazocine to assess sigma2 receptor binding, steady state was achieved within 2min at 25°C. Cold saturation studies revealed one high affinity, low capacity binding site (Kd 31.8±8.3nM; Bmax 921±228fmol/mg protein) that displayed sigma2 receptor pharmacology. A very low affinity, high capacity interaction also was observed that represents saturable, but not sigma receptor specific, binding. A panel of ligands showed rank order inhibition of radioligand binding appropriate for the sigma2 receptor, with ifenprodil displaying the highest apparent affinity. In vivo, dextromethorphan inhibited the specific binding of a radioiodinated sigma1 receptor ligand in lung with an ED50 of 1.2μmol/kg, a value near the recommended dosage for the drug as a cough suppressant. Overall, the present work provides a foundation for studies of drug interactions with pulmonary sigma1 and sigma2 receptors in vitro and in vivo. PMID:26004528

  16. [Cholinergic system of the heart].

    PubMed

    Kučera, Matej; Hrabovská, Anna

    2015-12-01

    The cholinergic system of the heart can be either of neuronal or non-neuronal origin. The neuronal cholinergic system in the heart is represented by preganglionic parasympathetic pathways, intracardiac parasympathetic ganglia and postganglionic parasympathetic neurons projecting to the atria, SA node and AV node. The non-neuronal cholinergic system consists of cardiomyocytes that have complete equipment for synthesis and secretion of acetylcholine. Current knowledge suggests that the non-neuronal cholinergic system in the heart affects the regulation of the heart during sympathetic activation. The non-neuronal cholinergic system of the heart plays also a role in the energy metabolism of cardimyocites. Acetylcholine of both neuronal and non-neuronal origin acts in the heart through muscarinic and nicotinic receptors. The effect of acetylcholine in the heart is terminated by cholinesterases acetylcholinesterase and butyrylcholinesterase. Recently, papers suggest that the increased cholinergic tone in the heart by cholinesterase inhibitors has a positive effect on some cardiovascular disorders such as heart failure. For this reason, the cholinesterase inhibitors might be used in the treatment of certain cardiovascular disorders in the future.

  17. Nonequivalence of alpha-bungarotoxin binding sites in the native nicotinic receptor molecule

    SciTech Connect

    Conti-Tronconi, B.M.; Tang, F.; Walgrave, S.; Gallagher, W. )

    1990-01-30

    In the native, membrane-bound form of the nicotinic acetylcholine receptor (M-AcChR) the two sites for the cholinergic antagonist alpha-bungarotoxin (alpha-BGT) have different binding properties. One site has high affinity, and the M-AcChR/alpha-BGT complexes thus formed dissociate very slowly, similar to the complexes formed with detergent-solubilized AcChR (S-AcChR). The second site has much lower affinity (KD approximately 59 +/- 35 nM) and forms quickly reversible complexes. The nondenaturing detergent Triton X-100 is known to solubilize the AcChR in a form unable, upon binding of cholinergic ligands, to open the ion channel and to become desensitized. Solubilization of the AcChR in Triton X-100 affects the binding properties of this second site and converts it to a high-affinity, slowly reversible site. Prolonged incubation of M-AcChR at 4 degrees C converts the low-affinity site to a high-affinity site similar to those observed in the presence of Triton X-100. Although the two sites have similar properties when the AcChR is solubilized in Triton X-100, their nonequivalence can be demonstrated by the effect on alpha-BGT binding of concanavalin A, which strongly reduces the association rate of one site only. The Bmax of alpha-BGT to either Triton-solubilized AcChR or M-AcChR is not affected by the presence of concanavalin A. Occupancy of the high-affinity, slowly reversible site in M-AcChR inhibits the Triton X-100 induced conversion to irreversibility of the second site. At difference with alpha-BGT, the long alpha-neurotoxin from Naja naja siamensis venom (alpha-NTX) binds with high affinity and in a very slowly reversible fashion to two sites in the M-AcChR. We confirm here that Triton-solubilized AcChR or M-AcChR binds in a very slowly reversible fashion the same amount of alpha-NTX.

  18. Cholinergic Targets in Lung Cancer.

    PubMed

    Spindel, Eliot R

    2016-01-01

    Lung cancers express an autocrine cholinergic loop in which secreted acetylcholine can stimulate tumor growth through both nicotinic and muscarinic receptors. Because activation of mAChR and nAChR stimulates growth; tumor growth can be stimulated by both locally synthesized acetylcholine as well as acetylcholine from distal sources and from nicotine in the high percentage of lung cancer patients who are smokers. The stimulation of lung cancer growth by cholinergic agonists offers many potential new targets for lung cancer therapy. Cholinergic signaling can be targeted at the level of choline transport; acetylcholine synthesis, secretion and degradation; and nicotinic and muscarinic receptors. In addition, the newly describe family of ly-6 allosteric modulators of nicotinic signaling such as lynx1 and lynx2 offers yet another new approach to novel lung cancer therapeutics. Each of these targets has their potential advantages and disadvantages for the development of new lung cancer therapies which are discussed in this review. PMID:26818857

  19. The involvement of ventral tegmental area cholinergic muscarinic receptors in classically conditioned fear expression as measured with fear-potentiated startle.

    PubMed

    Greba, Q; Munro, L J; Kokkinidis, L

    2000-07-01

    Accumulating evidence suggests that dopamine (DA) neurons in the ventral tegmental area (VTA) contribute to the complex amygdala-based neurocircuitry that mediates fear-motivated behaviors. Because of acetylcholine's (ACh) role in DA neuronal activation, the involvement of VTA cholinergic muscarinic receptors in Pavlovian conditioned fear responding was evaluated in the present study. Fear-potentiated startle was used to assess the effects of intraVTA infused methylscopolamine on conditioned fear performance in laboratory rats. Application of this nonspecific muscarinic receptor antagonist to VTA neurons was observed to inhibit the ability of a conditioned stimulus (CS) previously paired with footshock to enhance the amplitude of the acoustic startle reflex. Doses of methylscopolamine that blocked conditioned fear expression did not alter baseline sensorimotor responding. These results identify ACh neurotransmission in the VTA as a potential excitatory mechanism underlying the fear-arousing properties of threatening environmental stimuli.

  20. Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

    PubMed

    Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

    2015-02-01

    One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered.

  1. Effects of clobenpropit (VUF-9153), a histamine H3-receptor antagonist, on learning and memory, and on cholinergic and monoaminergic systems in mice.

    PubMed

    Miyazaki, S; Onodera, K; Imaizumi, M; Timmerman, H

    1997-01-01

    The effects of clobenpropit (VUF-9153), a potent histamine H3-receptor antagonist, on a scopolamine-induced learning deficit in the step-through passive avoidance test was studied in mice. Clobenpropit (10 and 20 mg/kg) alone showed a tendency to ameliorate the scopolamine-induced learning deficit, and clobenpropit (10 mg/kg) in combination with zolantidine (20 mg/kg), a histamine H2-receptor antagonist, ameliorated the scopolamine-induced effect. This ameliorating effect was antagonized by (R)-alpha-methylhistamine (20 mg/kg), a histamine H3-receptor agonist and pyrilamine (20 mg/kg), a histamine H1-receptor antagonist, suggesting that clobenpropit in combination with zolantidine showed the ameliorating effect via histamine H3 receptors and/or histamine H1 receptors. We also studied the effects of clobenpropit on cholinergic and monoaminergic systems. Clobenpropit did not show any significant effect on these neuronal systems except the activation of noradrenergic system. The present results suggest that the effect of clobenpropit might be partially involved with the activation of noradrenergic system, and the histaminergic system may play certain important roles in learning and memory.

  2. Mu opioid receptor binding sites in human brain

    SciTech Connect

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand (/sup 3/H)DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of (/sup 3/H)DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas.

  3. Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor

    SciTech Connect

    Dowding, A.J.; Hall, Z.W.

    1987-10-06

    The authors have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of ..cap alpha..-bungarotoxin (..cap alpha..-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block ..cap alpha..-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit ..cap alpha..-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds ..cap alpha..-BuTx with a forward rate constant of 0.98 x 10/sup 5/ M/sup -1/ s/sup -1/, d-tubocurarine (dTC) with a K/sub D/ of (6.8 +/- 0.3) x 10/sup -8/ M, and pancuronium with a K/sub D/ of (1.9 +/- 1.0) x 10/sup -9/ M. The site recognized by B mAbs binds ..cap alpha..-BuTx with a forward rate constant of 9.3 x 10/sup 5/ M/sup -1/ s/sup -1/, dTC with a K/sub D/ of (4.6 +/- 0.3) x 10/sup -6/ M, and pancurionium with a K/sub D/ of (9.3 +/- 0.8) x 10/sup -6/ M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by ..cap alpha..-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.

  4. Modes and Models of Forebrain Cholinergic Neuromodulation of Cognition

    PubMed Central

    Hasselmo, Michael E; Sarter, Martin

    2011-01-01

    As indicated by the profound cognitive impairments caused by cholinergic receptor antagonists, cholinergic neurotransmission has a vital role in cognitive function, specifically attention and memory encoding. Abnormally regulated cholinergic neurotransmission has been hypothesized to contribute to the cognitive symptoms of neuropsychiatric disorders. Loss of cholinergic neurons enhances the severity of the symptoms of dementia. Cholinergic receptor agonists and acetylcholinesterase inhibitors have been investigated for the treatment of cognitive dysfunction. Evidence from experiments using new techniques for measuring rapid changes in cholinergic neurotransmission provides a novel perspective on the cholinergic regulation of cognitive processes. This evidence indicates that changes in cholinergic modulation on a timescale of seconds is triggered by sensory input cues and serves to facilitate cue detection and attentional performance. Furthermore, the evidence indicates cholinergic induction of evoked intrinsic, persistent spiking mechanisms for active maintenance of sensory input, and planned responses. Models have been developed to describe the neuronal mechanisms underlying the transient modulation of cortical target circuits by cholinergic activity. These models postulate specific locations and roles of nicotinic and muscarinic acetylcholine receptors and that cholinergic neurotransmission is controlled in part by (cortical) target circuits. The available evidence and these models point to new principles governing the development of the next generation of cholinergic treatments for cognitive disorders. PMID:20668433

  5. Reductions in (/sup 3/H)nicotinic acetylcholine binding in Alzheimer's disease and Parkinson's disease: an autoradiographic study

    SciTech Connect

    Whitehouse, P.J.; Martino, A.M.; Wagster, M.V.; Price, D.L.; Mayeux, R.; Atack, J.R.; Kellar, K.J.

    1988-05-01

    In Alzheimer's disease (AD) and Parkinson's disease (PD), dysfunction in the basal forebrain cholinergic system is accompanied by a consistent loss of presynaptic cholinergic markers in cortex, but changes in cholinergic receptor binding sites are poorly understood. In the present study, we used receptor autoradiography to map the distribution of nicotinic (/sup 3/H)acetylcholine binding sites in cortices of individuals with AD and PD and matched control subjects. In both diseases, a profound loss of nicotinic receptors occurs in all cortical layers, particularly the deepest layers.

  6. Limited proteolysis for assaying ligand binding affinities of nuclear receptors.

    PubMed

    Benkoussa, M; Nominé, B; Mouchon, A; Lefebvre, B; Bernardon, J M; Formstecher, P; Lefebvre, P

    1997-01-01

    The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

  7. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  8. CONTAMINANT INTERACTIONS WITH STEROID RECEPTORS: EVIDENCE FOR RECEPTOR BINDING.

    EPA Science Inventory

    Steroid receptors are important determinants of endocrine disrupter consequences. As the most frequently proposed mechanism of endocrine-disrupting contaminant (EDC) action, steroid receptors are not only targets of natural steroids but are also commonly sites of nonsteroidal com...

  9. Regional distribution of somatostatin receptor binding and modulation of adenylyl cyclase activity in Alzheimer's disease brain.

    PubMed

    Bergström, L; Garlind, A; Nilsson, L; Alafuzoff, I; Fowler, C J; Winblad, B; Cowburn, R F

    1991-10-01

    We have previously reported a reduction in the inhibitory effect of somatostatin on adenylyl cyclase activity in the superior temporal cortex of a group of Alzheimer's disease cases, compared to a group of matched controls. In the present study, the levels of high affinity 125I-Tyr11-somatostatin-14 binding, its modulation by guanine nucleotides and the effects of somatostatin on adenylyl cyclase activity have been measured in preparations of frontal cortex, hippocampus, caudate nucleus and cerebellum from the same patient and control groups. A significant reduction in 125I-Tyr11-somatostatin-14 binding was observed in the frontal cortex, but not other regions, of the Alzheimer's disease group, compared with control values. The profiles of inhibition of specific 125I-Tyr11-somatostatin-14 binding by Gpp(NH)p were similar in all regions in both groups. No significant differences in basal, forskolin-stimulated, or somatostatin and neuropeptide Y inhibitions of adenylyl cyclase activity were found between the two groups. The pattern of change of somatostatin binding in the Alzheimer's disease cases observed in the present study differs from the reported pattern of loss of somatostatin neurons and may be secondary to the degeneration of somatostatin receptor-bearing cholinergic afferents arising from the nucleus basalis. The results of this study indicate that impaired somatostatin modulation of adenylyl cyclase is not a global phenomenon in Alzheimer's disease brain and also that there are no major disruptions of somatostatin receptor-G-protein coupling or of adenylyl cyclase catalytic activity in this disorder. PMID:1684616

  10. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  11. Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

    NASA Astrophysics Data System (ADS)

    Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

    1983-10-01

    Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

  12. Experiment K-7-18: Effects of Spaceflight in the Muscle Adductor Longus of Rats Flown in the Soviet Biosatellite Cosmos 2044. Part 2; Quantitative Autoradiographic Analysis of Gaba (Benzodiazepine) and Muscarinic (Cholinergic) Receptors in the Forebrain of Rats Flown on Cosmos 2044

    NASA Technical Reports Server (NTRS)

    Wu, L.; Daunton, N. G.; Krasnov, I. B.; DAmelio, F.; Hyde, T. M.; Sigworth, S. K.

    1994-01-01

    Quantitative autoradiographic analysis of receptors for GABA and acetylcholine in the forebrain of rats flown on COSMOS 2044 was undertaken as part of a joint US-Soviet study to determine the effects of microgravity on the central nervous system, and in particular on the sensory and motor portions of the forebrain. Changes in binding of these receptors in tissue from animals exposed to microgravity would provide evidence for possible changes in neural processing as a result of exposure to microgravity. Tritium-labelled diazepam and Quinuclidinyl-benzilate (QNB) were used to visualize GABA (benzodiazepine) and muscarinic (cholinergic) receptors, respectively. The density of tritium-labelled radioligands bound to various regions in the forebrain of both flight and control animals were measured from autoradiograms. Data from rats flown in space and from ground-based control animals that were not exposed to microgravity were compared.

  13. Assaying binding of nerve growth factor to cell surface receptors

    SciTech Connect

    Vale, R.D.; Shooter, E.M.

    1985-01-01

    The paper describes methods both for the radioiodination of nerve growth factor (NGF) and for assaying NFG receptors by reversible binding techniques. Preparation of (/sup 125/I)NGF along with a rapid method for determining the amount of cell-bound ligand have allowed the detection of NGF receptors on a number of cell types.

  14. Muscarinic and nicotinic cholinergic receptor antagonists differentially mediate acquisition of fructose-conditioned flavor preference and quinine-conditioned flavor avoidance in rats.

    PubMed

    Rotella, Francis M; Olsson, Kerstin; Vig, Vishal; Yenko, Ira; Pagirsky, Jeremy; Kohen, Ilanna; Aminov, Alon; Dindyal, Trisha; Bodnar, Richard J

    2015-09-01

    Rats display both conditioned flavor preference (CFP) for fructose, and conditioned flavor avoidance (CFA) following sweet adulteration with quinine. Previous pharmacological analyses revealed that fructose-CFP expression was significantly reduced by dopamine (DA) D1 or D2 antagonists, but not NMDA or opioid antagonists. Fructose-CFP acquisition was significantly reduced by DA D1, DA D2 or NMDA antagonists, but not opioid antagonists. Quinine-CFA acquisition was significantly enhanced and prolonged by DA D1, NMDA or opioid, but not DA D2 antagonists. Cholinergic interneurons and projections interact with DA systems in the nucleus accumbens and ventral tegmental area. Further, both muscarinic and nicotinic cholinergic receptor signaling have been implicated in sweet intake and development of food-related preferences. Therefore, the present study examined whether systemic administration of muscarinic (scopolamine: SCOP) or nicotinic (mecamylamine: MEC) cholinergic receptor antagonists mediated fructose-CFP expression, fructose-CFP acquisition and quinine-CFA acquisition. For fructose-CFP expression, rats were trained over 10 sessions with a CS+ flavor in 8% fructose and 0.2% saccharin and a CS- flavor in 0.2% saccharin. Two-bottle choice tests with CS+ and CS- flavors mixed in 0.2% saccharin occurred following vehicle, SCOP (0.1-10mg/kg) and MEC (1-8mg/kg). For fructose-CFP acquisition, six groups of rats received vehicle, SCOP (1 or 2.5mg/kg), MEC (4 or 6mg/kg) or a limited intake vehicle control 0.5h prior to 10 CS+ and CS- training sessions followed by six 2-bottle CS+ and CS- choice tests in 0.2% saccharin. For quinine-CFA acquisition, five groups of rats received vehicle, SCOP (1 or 2.5mg/kg) or MEC (4 or 6mg/kg) 0.5h prior to 8 one-bottle CS- (8% fructose+0.2% saccharin: FS) and CS+ (fructose+saccharin+quinine (0.030%: FSQ) training sessions followed by six 2-bottle CS- and CS+ choice tests in fructose-saccharin solutions. Fructose-CFP expression was

  15. Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.

    PubMed Central

    Jerva, L. F.; Sullivan, G.; Lolis, E.

    1997-01-01

    Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor. PMID:9260277

  16. Effect of desipramine on dopamine receptor binding in vivo

    SciTech Connect

    Suhara, Tetsuya Jikei Univ., Tokyo ); Inoue, Osamu; Kobayasi, Kaoru )

    1990-01-01

    Effect of desipramine on the in vivo binding of {sup 3}H-SCH23390 and {sup 3}H-N-methylspiperone ({sup 3}H-NMSP) in mouse striatum was studied. The ratio of radioactivity in the striatum to that in the cerebellum at 15 min after i.v. injection of {sup 3}H-SCH23390 or 45 min after injection of {sup 3}H-NMSP were used as indices of dopamine D1 or D2 receptor binding in vivo, respectively. In vivo binding of D1 and D2 receptors was decreased in a dose-dependent manner by acute treatment with desipramine (DMI). A saturation experiment suggested that the DMI-induced reduction in the binding was mainly due to the decrease in the affinity of both receptors. No direct interactions between the dopamine receptors and DMI were observed in vitro by the addition of 1 mM of DMI into striatal homogenate. Other antidepressants such as imipramine, clomipramine, maprotiline and mianserin also decreased the binding of dopamine D1 and D2 receptors. The results indicated an important role of dopamine receptors in the pharmacological effect of antidepressants.

  17. Effects of atropine treatment on in vitro and in vivo binding of 4-[125I]-dexetimide to central and myocardial muscarinic receptors.

    PubMed

    Uno, Y; Matsumura, K; Scheffel, U; Wilson, A A; Dannals, R F; Wagner, H N

    1991-01-01

    Upregulation of muscarinic cholinergic receptors (mAChR) after chronic atropine treatment has been described previously. The present study was designed to evaluate 4-iodine-125 dexetimide as an agent to determine changes in the number of mAChR. Rats were injected subcutaneously with atropine (500 mg/kg) either once or chronically, once daily for 10 days, and sacrificed 24 h later. In vitro binding assays with 4-[125I]-dexetimide showed significant increases in the number of mAChR in cerebra (21%) and ventricles (45%) after chronic atropine treatment but not after acute treatment. The affinity of binding to cerebral and ventricular mAChR declined after acute and chronic atropine treatment. In vivo studies were carried out involving intravenous injection of 4-[125I]-dexetimide 24 h after atropine treatment. Binding was markedly reduced in the brain and heart. Upregulation of mAChR, as seen in in vitro studies, could not be observed because of the remaining atropine. Occupancy of mAChR by atropine persisted as long as 7 days after one dose. The results of these studies indicate that 4-[125I]-dexetimide binding reflects the effects of atropine on central and peripheral muscarinic cholinergic receptors in vitro and in vivo. PMID:1915471

  18. 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.

  19. Extra-helical binding site of a glucagon receptor antagonist.

    PubMed

    Jazayeri, Ali; Doré, Andrew S; Lamb, Daniel; Krishnamurthy, Harini; Southall, Stacey M; Baig, Asma H; Bortolato, Andrea; Koglin, Markus; Robertson, Nathan J; Errey, James C; Andrews, Stephen P; Teobald, Iryna; Brown, Alastair J H; Cooke, Robert M; Weir, Malcolm; Marshall, Fiona H

    2016-05-12

    Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors. PMID:27111510

  20. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  1. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  2. Simultaneous modulation of retrieval by dopaminergic D(1), beta-noradrenergic, serotonergic-1A and cholinergic muscarinic receptors in cortical structures of the rat.

    PubMed

    Barros, D M; Mello e Souza, T; De David, T; Choi, H; Aguzzoli, A; Madche, C; Ardenghi, P; Medina, J H; Izquierdo, I

    2001-09-28

    Retrieval of inhibitory avoidance has been recently shown to require intact glutamate receptors, protein kinases A and C and mitogen-activated protein kinase in the CA1 region of the rat hippocampus and in the entorhinal, posterior parietal and anterior cingulate cortex. These enzymatic activities are known to be modulated by dopamine D(1), beta-noradrenergic, 5HT1A and cholinergic muscarinic receptors. Here we study the effect on retrieval of this task of well-known agonists and antagonists of these receptors infused in the same brain cortical regions and into the basolateral amygdala, in rats. The drugs used were SKF38393 (D(1) agonist), noradrenaline, 8-HO-DPAT (5HT1A agonist), oxotremorine (muscarinic agonist), SCH23390 (D(1) antagonist), timolol (beta antagonist), NAN-190 (5HT1A antagonist) and scopolamine (muscarinic antagonist). All were studied at two different dose levels. The localised infusion of SKF38393, noradrenaline, NAN-190 and oxotremorine into any of the cortical structures mentioned 10 min prior to a 24-h retention test session of one-trial step-down inhibitory avoidance enhanced retention test performance. SCH2330, timolol, 8-HO-DPAT and scopolamine hindered retention test performance. In the basolateral amygdala only an enhancing effect of noradrenaline and an inhibitory effect of timolol were seen. Three hours after the infusions, retention test performance returned to normal in all cases. None of the treatments affected locomotion or rearing in an open field or behaviour in the elevated plus maze. Therefore, their effects on retention testing can be attributed to an influence on retrieval. In conclusion, memory retrieval of this apparently simple task requires the participation of CA1, entorhinal, posterior parietal and anterior cingulate cortex, and is strongly modulated by, dopaminergic D(1), beta-noradrenergic, muscarinic cholinergic and 5HT1A receptors in the four areas. The first three types of receptor enhance, and the latter inhibits

  3. Molecular modulators of benzodiazepine receptor ligand binding

    SciTech Connect

    Villar, H.O.; Loew, G.H. )

    1989-01-01

    Ten derivatives of {beta}-carbolines with known affinities to the GABA{sub A}/BDZ (benzodiazepine) receptor were studied using the Am 1 and MNDO/H Semiempirical techniques to identify and characterize molecular modulators of receptor recognition. Steric, lipophilic, and electrostatic properties of these compounds were calculated and examined for their possible role in recognition. Particular attention was paid to the regions around the two most favorable proton-accepting sites, the ON and the substituent at the C{sub 3} position, already implicated in recognition, as well as to the acidic N9H group that could be a proton donating center. To probe further the role of these three ligand sites in receptor interactions, a model of the receptor using three methanol molecules was made and optimum interactions of these three sites with them characterized. The results indicate some similarity in the shape of these ligands, which could reflect a steric requirement. The receptor affinity appears to be modulated to some extent by the ratio of lipophilic to hydrophilic surface, the negative potential at the {beta}N, provided there is also one at the C{sub 3} substituent confirming the importance of two accepting sites in recognition. The acidic N9H does not appear to be a modulator of affinity or does it form a stable H-bond with methanol as acceptor. The two proton donating molecules do form such a stable complex, and both are needed for high affinity.

  4. Rapid screening of environmental chemicals for estrogen receptor binding capacity.

    PubMed Central

    Bolger, R; Wiese, T E; Ervin, K; Nestich, S; Checovich, W

    1998-01-01

    Over the last few years, an increased awareness of endocrine disrupting chemicals (EDCs) and their potential to affect wildlife and humans has produced a demand for practical screening methods to identify endocrine activity in a wide range of environmental and industrial chemicals. While it is clear that in vivo methods will be required to identify adverse effects produced by these chemicals, in vitro assays can define particular mechanisms of action and have the potential to be employed as rapid and low-cost screens for use in large scale EDC screening programs. Traditional estrogen receptor (ER) binding assays are useful for characterizing a chemical's potential to be an estrogen-acting EDC, but they involve displacement of a radioactive ligand from crude receptor preparations at low temperatures. The usefulness of these assays for realistically determining the ER binding interactions of weakly estrogenic environmental and industrial compounds that have low aqueous solubility is unclear. In this report, we present a novel fluorescence polarization (FP) method that measures the capacity of a competitor chemical to displace a high affinity fluorescent ligand from purified, recombinant human ER-[alpha] at room temperature. The ER-[alpha] binding interactions generated for 15 natural and synthetic compounds were found to be similar to those determined with traditional receptor binding assays. We also discuss the potential to employ this FP technology to binding studies involving ER-ss and other receptors. Thus, the assay introduced in this study is a nonradioactive receptor binding method that shows promise as a high throughput screening method for large-scale testing of environmental and industrial chemicals for ER binding interactions. Images Figure 2 Figure 3 Figure 4 PMID:9721254

  5. Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors.

    PubMed Central

    Peralta, E G; Ashkenazi, A; Winslow, J W; Smith, D H; Ramachandran, J; Capon, D J

    1987-01-01

    To investigate the molecular basis for the diversity in muscarinic cholinergic function, we have isolated the genes encoding the human M1 and M2 muscarinic receptors (mAChR) as well as two previously undiscovered mAChR subtypes, designated HM3 and HM4. The amino acid sequence of each subtype reflects a structure consisting of seven, highly conserved transmembrane segments and a large intracellular region unique to each subtype, which may constitute the ligand-binding and effector-coupling domains respectively. Significant differences in affinity for muscarinic ligands were detected in individual mAChR subtypes produced by transfection of mammalian cells. Each subtype exhibited multiple affinity states for agonists; differences among subtypes in the affinities and proportions of such sites suggest the capacity of mAChR subtypes to interact differentially with the cellular effector-coupling apparatus. Subtype-specific mRNA expression was observed in the heart, pancreas and a neuronal cell line, indicating that the regulation of mAChR gene expression contributes to the differentiation of cholinergic activity. Images Fig. 3. PMID:3443095

  6. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-05-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10/sup -6/ to 10/sup -8/ M. The 100 kDa protein can be affinity-labeled with these /sup 125/I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by /sup 125/I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors.

  7. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

    SciTech Connect

    Kikuchi, M.; Ishii, S. )

    1989-12-01

    By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

  8. Structural Allostery and Binding of the Transferring Receptor Complex

    SciTech Connect

    Xu,G.; Liu, R.; Zak, O.; Aisen, P.; Chance, M.

    2005-01-01

    The structural allostery and binding interface for the human serum transferrin (Tf){center_dot}transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions. The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.

  9. Sexually dimorphic effects of the Lhx7 null mutation on forebrain cholinergic function.

    PubMed

    Fragkouli, A; Stamatakis, A; Zographos, E; Pachnis, V; Stylianopoulou, F

    2006-01-01

    It has been reported recently that mice lacking both alleles of the LIM-homeobox gene Lhx7, display dramatically reduced number of forebrain cholinergic neurons. In the present study, we investigated whether the Lhx7 mutation affects male and female mice differently, given the fact that gender differences are consistently observed in forebrain cholinergic function. Our results show that in adult male as well as female Lhx7 homozygous mutants there is a dramatic loss of choline acetyltransferase immunoreactive forebrain neurons, both projection and interneurons. The reduction of forebrain choline acetyltransferase immunoreactive neurons in Lhx7 homozygous mutants is accompanied by a decrease of acetylcholinesterase histochemical staining in all forebrain cholinergic neuron target areas of both male and female homozygous mutants. Furthermore, there was an increase of M1-, but not M2-, muscarinic acetylcholine receptor binding site density in the somatosensory cortex and basal ganglia of only the female homozygous mutant mice. Such an increase can be regarded as a mechanism acting to compensate for the dramatically reduced cholinergic input, raising the possibility that the forebrain cholinergic system in female mice may be more plastic and responsive to situations of limited neurotransmitter availability. Finally, our study provides additional data for the sexual dimorphism of the forebrain cholinergic system, as female mice appear to have a lower density of M1-muscarinic acetylcholine receptors in the striatal areas of the basal ganglia and a higher density of M2-muscarinic acetylcholine receptors, in a number of cortical areas, as well as the striatal areas of the basal ganglia.

  10. Electron microscopic localization of M2-muscarinic receptors in cholinergic and noncholinergic neurons of the laterodorsal tegmental and pedunculopontine nuclei of the rat mesopontine tegmentum.

    PubMed

    Garzón, Miguel; Pickel, Virginia M

    2016-10-15

    Muscarinic m2 receptors (M2Rs) are implicated in autoregulatory control of cholinergic output neurons located within the pedunculopontine (PPT) and laterodorsal tegmental (LTD) nuclei of the mesopontine tegmentum (MPT). However, these nuclei contain many noncholinergic neurons in which activation of M2R heteroceptors may contribute significantly to the decisive role of the LTD and PPT in sleep-wakefulness. We examined the electron microscopic dual immunolabeling of M2Rs and the vesicular acetylcholine transporter (VAchT) in the MPT of rat brain to identify the potential sites for M2R activation. M2R immunogold labeling was predominately seen in somatodendritic profiles throughout the PPT/LTD complex. In somata, M2R immunogold particles were often associated with Golgi lamellae and cytoplasmic endomembrannes, but were rarely in contact with the plasma membrane, as was commonly seen in dendrites. Approximately 36% of the M2R-labeled somata and 16% of the more numerous M2R-labeled dendrites coexpressed VAchT. M2R and M2R/VAchT-labeled dendritic profiles received synapses from inhibitory- and excitatory-type axon terminals, over 88% of which were unlabeled and others contained exclusively M2R or VAchT immunoreactivity. In axonal profiles M2R immunogold was localized to plasmalemmal and cytoplasmic regions and showed a similar distribution in many VAchT-negative glial profiles. These results provide ultrastructural evidence suggestive of somatic endomembrane trafficking of M2Rs, whose activation serves to regulate the postsynaptic excitatory and inhibitory responses in dendrites of cholinergic and noncholinergic neurons in the MPT. They also suggest the possibility that M2Rs in this brain region mediate the effects of acetylcholine on the release of other neurotransmitters and on glial signaling. J. Comp. Neurol. 524:3084-3103, 2016. © 2016 Wiley Periodicals, Inc.

  11. Modulation of spontaneous intracellular Ca²⁺ fluctuations and spontaneous cholinergic transmission in rat chromaffin cells in situ by endogenous GABA acting on GABAA receptors.

    PubMed

    Tzitzitlini, Alejandre-García; Pedro, Segura-Chama; Martha, Pérez-Armendáriz E; Rodolfo, Delgado-Lezama; Arturo, Hernández-Cruz

    2016-02-01

    Using fluorescence [Ca(2+)]i imaging in rat adrenal slices, we characterized the effects of agonists and antagonists of the GABAA receptor (GABAA-R) on resting intracellular Ca(2+) ([Ca(2+)]i) and spontaneous [Ca(2+)]i fluctuations (SCFs) in hundreds of individual chromaffin cells (CCs) recorded simultaneously in situ. Muscimol, a GABAA-R agonist (20 μM; 25 s), induced an increase of resting [Ca(2+)]i in 43 ± 3 % of CCs, a decrease in 26 ± 2 %, and no response in 30 ± 5 %. In Ca(2+)-free external medium, SCFs ceased completely and muscimol failed to elicit [Ca(2+)]i rises. All muscimol-induced [Ca(2+)]i changes were blocked by the GABAA-R antagonist bicuculline, suggesting that they result from changes in membrane potential depending on the cell's Cl(-) equilibrium potential. Unexpectedly, bicuculline increased the amplitude and frequency of SCFs in 54 % of CCs, revealing a tonic inhibition of SCFs by ambient GABA acting through GABAA-R. Mecamylamine (a specific nicotinic cholinergic blocker) decreased basal SCF activity in 18 % of CCs and inhibited bicuculline-induced SCF intensification, suggesting that spontaneous acetylcholine (ACh) release from nerve endings contributes to SCF generation in CCs in situ and that blockade of presynaptic GABAA-Rs intensifies SCFs in part through the disinhibition of spontaneous cholinergic transmission. Electrophysiological experiments confirmed that spontaneous excitatory postsynaptic currents recorded from CCs in situ were enhanced by bicuculline. To our knowledge, this is the first description of a regulatory effect of endogenous GABA on synaptic currents and SCFs of adrenal CCs. These findings denote a novel GABA-mediated presynaptic and postsynaptic regulatory mechanism of CC activity which may participate in the control of catecholamine secretion.

  12. Electron microscopic localization of M2-muscarinic receptors in cholinergic and noncholinergic neurons of the laterodorsal tegmental and pedunculopontine nuclei of the rat mesopontine tegmentum.

    PubMed

    Garzón, Miguel; Pickel, Virginia M

    2016-10-15

    Muscarinic m2 receptors (M2Rs) are implicated in autoregulatory control of cholinergic output neurons located within the pedunculopontine (PPT) and laterodorsal tegmental (LTD) nuclei of the mesopontine tegmentum (MPT). However, these nuclei contain many noncholinergic neurons in which activation of M2R heteroceptors may contribute significantly to the decisive role of the LTD and PPT in sleep-wakefulness. We examined the electron microscopic dual immunolabeling of M2Rs and the vesicular acetylcholine transporter (VAchT) in the MPT of rat brain to identify the potential sites for M2R activation. M2R immunogold labeling was predominately seen in somatodendritic profiles throughout the PPT/LTD complex. In somata, M2R immunogold particles were often associated with Golgi lamellae and cytoplasmic endomembrannes, but were rarely in contact with the plasma membrane, as was commonly seen in dendrites. Approximately 36% of the M2R-labeled somata and 16% of the more numerous M2R-labeled dendrites coexpressed VAchT. M2R and M2R/VAchT-labeled dendritic profiles received synapses from inhibitory- and excitatory-type axon terminals, over 88% of which were unlabeled and others contained exclusively M2R or VAchT immunoreactivity. In axonal profiles M2R immunogold was localized to plasmalemmal and cytoplasmic regions and showed a similar distribution in many VAchT-negative glial profiles. These results provide ultrastructural evidence suggestive of somatic endomembrane trafficking of M2Rs, whose activation serves to regulate the postsynaptic excitatory and inhibitory responses in dendrites of cholinergic and noncholinergic neurons in the MPT. They also suggest the possibility that M2Rs in this brain region mediate the effects of acetylcholine on the release of other neurotransmitters and on glial signaling. J. Comp. Neurol. 524:3084-3103, 2016. © 2016 Wiley Periodicals, Inc. PMID:27038330

  13. Development of a homogeneous binding assay for histamine receptors.

    PubMed

    Crane, Kathy; Shih, Daw-Tsun

    2004-12-01

    Histamine is critically involved in a wide range of physiological and pathological processes through its actions at different receptors. Thus, histamine receptors have been actively pursued as therapeutic targets in the pharmaceutical industry for the treatment of a variety of diseases. There are currently four histamine receptors that have been cloned, all of which are G protein-coupled receptors. Studies from both academia and pharmaceutical companies have identified compounds that modulate the function of specific histamine receptors. These efforts led to the successful introduction of histamine H(1) and H(2) receptor antagonists for the treatment of allergy and excess gastric acid secretion, respectively. Histamine H(3) receptor ligands are currently under investigation for the treatment of obesity and neurological disorders. The recently identified histamine H(4) receptor is preferentially expressed in the immune tissues, suggesting a potential role in normal immune functions and possibly in the pathogenesis of inflammatory diseases. Even with the long history of histamine research and the important applications of histamine receptor ligands, assays to measure the affinity of compounds binding to histamine receptors are still routinely analyzed using a filtration assay, a very low-throughput assay involving washing and filtration steps. This article describes a simple, robust, and homogeneous binding assay based on the scintillation proximity assay (SPA) technology that provides results equivalent to those obtained using the more complex filtration assay. The SPA format is easily adapted to high-throughput screening because it is amenable to automation. In summary, this technique allows high-throughput screening of compounds against multiple histamine receptors and, thus, facilitates drug discovery efforts.

  14. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands

    PubMed Central

    Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  15. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands.

    PubMed

    Kržan, Mojca; Vianello, Robert; Maršavelski, Aleksandra; Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N-H and O-H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  16. Radioligand binding to muscarinic receptors of bovine aortic endothelial cells.

    PubMed

    Brunner, F; Kukovetz, W R

    1991-02-01

    1. Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (-)-[3H]-N-methyl quinuclidinyl benzilate ([3H]-NMeQNB) was used as radioligand. 2. Binding of [3H]-NMeQNB to crude membranes of freshly isolated endothelial cells was atropine-displaceable and of high affinity (KD = 0.48 nM) to a single class of sites (maximum binding capacity: 14 +/- 3 fmol mg-1 protein). Stereospecificity of the binding sites was demonstrated in experiments in which [3H]-NMeQNB binding was inhibited by dexetimide in the nanomolar range (KI = 0.63 nM) and by levetimide, its stereoisomer in the micromolar range (KI = 3.2 microM) (selectivity factor: approximately 5000). 3. Drug competition curves indicated a single class of binding sites for antagonists and the following apparent affinities (KI, nM): methyl atropine: 1.1: 4-diphenylacetoxy N-methyl piperidine methyl bromide (4-DAMP): 3.4; pirenzepine: 16; 11-[2-diethylamino-methyl)-1-piperidinyl- acetyl]-5,11-dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one (AF-DX 116); 2.500. Competition of acetylcholine with [3H]-NMeQNB was best described by two affinity sites (or states) (KH = 0.82 microM, KL = 1.6 microM). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 microM), acetylcholine affinity (IC50) was slightly, but significantly reduced (factor approximately 4). 4. Binding of [3H]-NMeQNB to freshly harvested intact cells was also atropine-displaceable, stereospecific (selectivity factor: approximately 3500) and of high affinity (KD = 0.35 nM). The maximum binding capacity (9 +/- 2 fmol mg-1 total cell protein) was comparable to that of membranes and corresponded to approximately 900 binding sites per endothelial cell. Binding to enzymatically harvested and cultured endothelial cells, or membranes derived therefrom, showed no atropine-displaceable binding. 5. The results suggest that (1) bovine aortic endothelial cells contain muscarinic binding sites with all necessary

  17. Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover.

    PubMed

    Akiyama, K; Vickroy, T W; Watson, M; Roeske, W R; Reisine, T D; Smith, T L; Yamamura, H I

    1986-03-01

    (-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems. PMID:3005550

  18. Metallosupramolecular receptors for fullerene binding and release.

    PubMed

    García-Simón, Cristina; Costas, Miquel; Ribas, Xavi

    2016-01-01

    Fullerene extracts are easily available from fullerene soot, but finding an efficient strategy to obtain them in pure form remains elusive, especially for higher fullerenes (Cx, x > 70). The properties of the latter remain unclear and their potential application to multiple research fields has not been developed mainly due to their purification difficulties. In this Tutorial Review we cover the use of molecular receptors for the separation of fullerenes by means of host-guest interactions. This strategy allows gaining selectivity, no specialized equipment is required and, ideally, recyclable systems can be designed. We focus on the metallosupramolecular receptors using the metal-ligand coordination approach, which offers a controlled and versatile strategy to design fullerene hosts, and the latest strategies to release the fullerene guest will be described. The field is probably in its beginnings but it is rapidly evolving and we are confident that this tutorial review will help researchers to rapidly gain a general overview of the main works and concepts that are leading this promising strategy and that may lead towards a useful methodology to purify fullerenes.

  19. Evidence for a second receptor binding site on human prolactin.

    PubMed

    Goffin, V; Struman, I; Mainfroid, V; Kinet, S; Martial, J A

    1994-12-23

    The existence of a second receptor binding site on human prolactin (hPRL) was investigated by site-directed mutagenesis. First, 12 residues of helices 1 and 3 were mutated to alanine. Since none of the resulting mutants exhibit reduced bioactivity in the Nb2 cell proliferation bioassay, the mutated residues do not appear to be functionally necessary. Next, small residues surrounding the helix 1-helix 3 interface were replaced with Arg and/or Trp, the aim being to sterically hinder the second binding site. Several of these mutants exhibit only weak agonistic properties, supporting our hypothesis that the channel between helices 1 and 3 is involved in a second receptor binding site. We then analyzed the antagonistic and self-antagonistic properties of native hPRL and of several hPRLs analogs altered at binding site 1 or 2. Even at high concentrations (approximately 10 microM), no self-inhibition was observed with native hPRL; site 2 hPRL mutants self-antagonized while site 1 mutants did not. From these data, we propose a model of hPRL-PRL receptor interaction which slightly differs from that proposed earlier for the homologous human growth hormone (hGH) (Fuh, G., Cunningham, B. C., Fukunaga, R., Nagata, S., and Goeddel, D. V., and Well, J. A. (1992) Science 256, 1677-1680). Like hGH, hPRL would bind sequentially to two receptor molecules, first through site 1, then through site 2, but we would expect the two sites of hPRL to display, unlike the two binding sites of hGH, about the same binding affinity, thus preventing self-antagonism at high concentrations. PMID:7798264

  20. Enhanced alcohol-seeking behavior by nicotine in the posterior ventral tegmental area of female alcohol-preferring (P) rats: modulation by serotonin-3 and nicotinic cholinergic receptors

    PubMed Central

    Deehan, Gerald A.; Toalston, Jamie E.; Bell, Richard L.; McBride, William J.; Rodd, Zachary A.

    2015-01-01

    Rationale Alcohol and nicotine co-use can reciprocally promote self-administration and drug-craving/drug-seeking behaviors. To date, the neurocircuitry in which nicotine influences ethanol (EtOH) seeking has not been elucidated. Clinical and preclinical research has suggested that the activation of the mesolimbic dopamine system is involved in the promotion of drug seeking. Alcohol, nicotine, and serotonin-3 (5-HT3) receptors interact within the posterior ventral tegmental area (pVTA) to regulate drug reward. Recently, our laboratory has reported that systemic administration of nicotine can promote context-induced EtOH seeking. Objectives The goals of the current study were to (1) determine if microinjections of pharmacologically relevant levels of nicotine into the pVTA would enhance EtOH seeking, (2) determine if coadministration of nicotinic cholinergic receptor antagonist (nACh) or 5-HT3 receptor antagonists would block the ability of nicotine microinjected into the pVTA to promote EtOH seeking, and (3) determine if 5-HT3 receptors in the pVTA can modulate EtOH seeking. Results Nicotine (100 and 200 µM) microinjected into the pVTA enhanced EtOH seeking. Coinfusion with 200 µM mecamylamine (nACh antagonist) or 100 and 200 µM zacopride (5-HT3 receptor antagonist) blocked the observed nicotine enhancement of EtOH seeking. The data also indicated that microinjection of 1 µM CPBG (5-HT3 receptor agonist) promotes context-induced EtOH seeking; conversely microinjection of 100 and 200 µM zacopride alone reduced context-induced EtOH seeking. Conclusions Overall, the results show that nicotine-enhanced EtOH-seeking behavior is modulated by 5-HT3 and nACh receptors within the pVTA and that the 5-HT3 receptor system within pVTA may be a potential pharmacological target to inhibit EtOH-seeking behaviors. PMID:24599396

  1. Cannabinoid receptors in developing rats: detection of mRNA and receptor binding.

    PubMed

    McLaughlin, C R; Martin, B R; Compton, D R; Abood, M E

    1994-08-01

    Despite a large body of research directed at assessing the effects of perinatal cannabinoid exposure, little is known about the development of the cannabinoid receptor. Recent advances, including the cloning of the cannabinoid receptor, have afforded us the opportunity to plot the postnatal ontogeny of the cannabinoid receptor and its mRNA in whole brain using the methods of receptor binding and RNA blot hybridization, respectively. Our results indicate that cannabinoid receptor mRNA is present at adult levels as early as postnatal day 3. The Bmax, on the other hand, increases almost fifty percent with increasing postnatal age, while the affinity does not change. The Hill coefficients for all ages studied were approximately 1. These findings suggest the possibility of a developmental progression for cannabinoid receptor development with receptor mRNA appearing first, followed by a period of rapid proliferation of the receptors themselves. PMID:7988356

  2. Ascorbate deficiency impairs the muscarinic-cholinergic and ss-adrenergic receptor signaling systems in the guinea pig submandibular salivary gland.

    PubMed

    Sawiris, P G; Enwonwu, C O

    2000-12-01

    Ascorbic acid is preferentially concentrated in the hypothalamus, pituitary and adrenal glands. Its level in the acini of salivary glands is relatively high. We therefore hypothesized that ascorbate may have a role in salivary gland function. Ascorbate-deficient guinea pigs had lower stimulated whole salivary flow rates than well-fed, age-matched controls (P: < 0.005). Total salivary protein concentration was also markedly (P: < 0.005) reduced in the deficient guinea pigs. SDS-PAGE and densitometric quantification of protein bands confirmed significant reduction in specific salivary proteins (e.g., amylase, proline-rich proteins) in the saliva samples of malnourished guinea pigs. Some protein bands not seen in control saliva were detected in the saliva of malnourished guinea pigs. Ascorbate deficiency also produced a significant (P: < 0.005) reduction in the ss-adrenergic receptor density (subtype 1; 95 +/- 19 fmol/mg protein compared with 179 +/- 27 fmol/mg protein for the controls). No significant difference was observed between the two groups with respect to the ss-adrenergic receptor subtype 2. Additionally, ascorbate-deficient guinea pigs had significantly lower muscarinic-cholinergic receptor densities (50 +/- 5 vs. 74 +/- 8 fmol/mg protein for controls). Our data support the conclusion that diminished membrane receptors might impair the capacity of the transmembrane signaling system, resulting in salivary gland hypofunction in ascorbate-deficient guinea pigs. Without implying extrapolation of our findings in experimental animals to humans, it is perhaps relevant that many conditions often associated with salivary gland hypofunction in humans (e.g., smoking or drug ingestion) deplete cellular ascorbate. PMID:11110840

  3. The EGF receptor is an actin-binding protein

    PubMed Central

    1992-01-01

    In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996. PMID:1383230

  4. Autoradiographic 3H-Gaboxadol Receptor Binding Protocol

    PubMed Central

    Ling, Lynne; Caspary, Donald

    2016-01-01

    Gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, THIP), a GABAA receptor δ-subunit specific agonist, when present at low (μM) concentrations, preferentially binds and activates extrasynaptic (non-γ2, δ-subunit-containing) GABAARs (Storustovu and Ebert, 2006; Richardson et al., 2011, 2013). In this prototype saturation binding experiment, a series of concentrations of [3H]gaboxadol (5, 10, 25, 50, 75, 100, 250 and 400 nM) will be used. GABA at 200 μM will be added into binding mixtures as a cold displacer for [3H]gaboxadol. Slide mailers are used and each requires 7 ml binding mixture. Pre-, post-washing and binding buffer is 50 mM Tris-Citrate (pH 7.1). The detailed procedure is outlined below.

  5. A tricatecholic receptor for carbohydrate recognition: synthesis and binding studies.

    PubMed

    Cacciarini, Martina; Cordiano, Elisa; Nativi, Cristina; Roelens, Stefano

    2007-05-11

    A new tripodal receptor bearing three catechol subunits on a benzene platform has been synthesized in four steps from 1,3,5-triethylbenzene and pyrogallol. The binding ability of the tricatecholic receptor was investigated toward several monosaccharides in CDCl3, where multiple equilibria were detected, and compared to that of a previously reported trisureidic receptor of analogous structure. Association constants were measured by 1H NMR titrations, and the corresponding affinities were assessed through the BC50 parameter, a binding descriptor univocally defining the affinity of a host for a guest in multi-equilibrium systems. Results show that the tripodal catecholic receptor binds the octyl glycosides with affinities ranging from 0.87 to 5.2 mM and with a 6-fold selectivity factor for the alpha-mannoside over the beta-glucoside. Although the affinity for glycosides was not appreciably improved with respect to the ureidic receptor, a significant change in selectivity was obtained by the H-bonding group replacement.

  6. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    SciTech Connect

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  7. Development of prolactin receptor antagonists with reduced pH-dependence of receptor binding.

    PubMed

    Hansen, Mathilde J Kaas; Olsen, Johan G; Bernichtein, Sophie; O'Shea, Charlotte; Sigurskjold, Bent W; Goffin, Vincent; Kragelund, Birthe B

    2011-01-01

    The cytokine hormone prolactin has a vast number of diverse functions. Unfortunately, it also exhibits tumor growth promoting properties, which makes the development of prolactin receptor antagonists a priority. Prolactin binds to its cognate receptor with much lower affinity at low pH than at physiological pH and since the extracellular environment around solid tumors often is acidic, it is desirable to develop antagonists that have improved binding affinity at low pH. The pK(a) value of a histidine side chain is ∼6.8 making histidine residues obvious candidates for examination. From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone-receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity and thermodynamic characterization of receptor binding by isothermal titration calorimetry combined with in vitro bioactivity in living cells. Histidine residue 27 was recognized as a central hot spot for pH sensitivity and conservative substitutions at this site resulted in strong receptor binding at low pH. Pure antagonists were developed earlier and the histidine mutations were introduced within such background. The antagonistic properties were maintained and the high affinity at low pH conserved. The implications of these findings may open new areas of research in the field of prolactin cancer biology.

  8. Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

    SciTech Connect

    Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang

    2010-03-04

    NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.

  9. Pneumocystis carinii glycoprotein A binds macrophage mannose receptors.

    PubMed Central

    O'Riordan, D M; Standing, J E; Limper, A H

    1995-01-01

    Pneumocystis carinii causes life-threatening pneumonia in patients with impaired immunity. Recent studies suggest that alveolar macrophages interact with P. carinii through macrophage mannose receptors. However, the ligand(s) on P. carinii that is recognized by these receptors has not been fully defined. P. carinii contains a major mannose-rich surface antigen complex termed glycoprotein A (gpA). It was therefore hypothesized that gpA binds directly to macrophage mannose receptors and mediates organism attachment to these phagocytes. To assess this, gpA was purified from P. carinii by continuous-elution gel electrophoresis. 125I-labeled gpA bound to alveolar macrophages in a saturable fashion. In addition, gpA binding was substantially inhibited by both alpha-mannan and EDTA, further suggesting that gpA interacts with macrophage mannose receptors. Macrophage membrane proteins capable of binding to gpA were isolated with a gpA-Sepharose column. A 165-kDa membrane-associated protein was specifically eluted from the gpA-Sepharose column with EDTA (20 mM). This protein was identified as the macrophage mannose receptor by immunoprecipitation with a polyclonal anti-mannose receptor antiserum. To further investigate the role of gpA in P. carinii-macrophage interactions, 51Cr-labeled P. carinii cells were incubated with macrophages in the presence of increasing concentrations of soluble gpA, and organism attachment was quantified. Soluble gpA (2.5 mg/dl) competitively inhibited P. carinii attachment to alveolar macrophages by 51.3% +/- 3.7% (P = 0.01). Our findings demonstrate that gpA present on P. carinii interacts directly with mannose receptors, thereby mediating organism attachment to alveolar macrophages. PMID:7868247

  10. Crustacean retinoid-X receptor isoforms: distinctive DNA binding and receptor-receptor interaction with a cognate ecdysteroid receptor.

    PubMed

    Wu, Xiaohui; Hopkins, Penny M; Palli, Subba R; Durica, David S

    2004-04-15

    We have identified cDNA clones that encode homologs of the ecdysteroid receptor (EcR) and retinoid-X receptor (RXR)/USP classes of nuclear receptors from the fiddler crab Uca pugilator (UpEcR and UpRXR). Several UpRXR cDNA splicing variants were found in coding regions that could potentially influence function. A five-amino acid (aa) insertion/deletion is located in the "T" box in the hinge region. Another 33-aa insertion/deletion is found inside the ligand-binding domain (LBD), between helix 1 and helix 3. Ribonuclease protection assays (RPA) showed that four UpRXR transcripts [UpRXR(+5+33), UpRXR(-5+33), UpRXR(+5-33) and UpRXR(-5-33)] were present in regenerating limb buds. UpRXR(-5+33) was the most abundant transcript present in regenerating limb buds in both early blastema and late premolt growth stages. Expression vectors for these UpRXR variants and UpEcR were constructed, and the proteins expressed in E. coli and in vitro expression systems. The expressed crab nuclear receptors were then characterized by electrophoretic mobility shift assay (EMSA) and glutathione S-transferase (GST) pull down experiments. EMSA results showed that UpEcR/UpRXR(-5+33) heterocomplexes bound with a series of hormone response elements (HREs) including eip28/29, IRper-1, DR-4, and IRhsp-1 with appreciable affinity. Competition EMSA also showed that the affinity decreased as sequence composition deviated from a perfect consensus element. Binding to IRper-1 HREs occurred only if the heterodimer partner UpRXR contained the 33-aa LBD insertion. UpRXR lacking both the 5-aa and 33-aa insertion bound to a DR-1G HRE in the absence of UpEcR. The results of GST-pull down experiments showed that UpEcR interacted only with UpRXR variants containing the 33-aa insertion, and not with those lacking the 33-aa insertion. These in vitro receptor protein-DNA and receptor protein-protein interactions occurred in the absence of hormone (20-hydroxyecdysone and 9-cis retinoid acid, 9-cis RA

  11. Inhibition of insulin receptor binding by phorbol esters.

    PubMed

    Thomopoulos, P; Testa, U; Gourdin, M F; Hervy, C; Titeux, M; Vainchenker, W

    1982-12-15

    Phorbol esters inhibit the binding of insulin to its receptors on U-937 monocyte-like and HL-60 promyelocytic leukemia human cell lines. Within 20-30 min, exposure of these cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) at 37 degrees C results in a 50% reduction of the specific binding of 125I-insulin. Half-maximal inhibition occurs at 1 nM TPA. Other tumor-promoting phorbol esters also inhibit 125I-insulin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA does not alter the degradation of the hormone nor does it induce any shedding of its receptors in the medium. The effect of phorbol esters is dependent on temperature and cell type. It is less prominent at 22 degrees C than at 37 degrees C. It is reversible within 2 h at 37 degrees C. TPA reduces the binding of insulin predominantly by increasing its dissociation rate. This effect results in an accelerated turnover of the hormone on its receptors. PMID:6891320

  12. Allelic association of the D2 dopamine receptor gene with receptor-binding characteristics in alcoholism

    SciTech Connect

    Noble, E.P.; Blum, K.; Ritchie, T.; Montgomery, A.; Sheridan, P.J. )

    1991-07-01

    The allelic association of the human D2 dopamine receptor gene with the binding characteristics of the D2 dopamine receptor was determined in 66 brains of alcoholic and non-alcoholic subjects. In a blinded experiment, DNA from the cerebral cortex was treated with the restriction endonuclease Taql and probed with a 1.5-kilobase (kb) digest of a clone (lambda hD2G1) of the human D2 dopamine receptor gene. The binding characteristics (Kd (binding affinity) and Bmax (number of binding sites)) of the D2 dopamine receptor were determined in the caudate nuclei of these brains using tritiated spiperone as the ligand. The adjusted Kd was significantly lower in alcoholic than in nonalcoholic subjects. In subjects with the A1 allele, in whom a high association with alcoholism was found, the Bmax was significantly reduced compared with the Bmax of subjects with the A2 allele. Moreover, a progressively reduced Bmax was found in subjects with A2/A2, A1/A2, and A1/A1 alleles, with subjects with A2/A2 having the highest mean values, and subjects with A1/A1, the lowest. The polymorphic pattern of the D2 dopamine receptor gene and its differential expression of receptors suggests the involvement of the dopaminergic system in conferring susceptibility to at least one subtype of severe alcoholism.

  13. Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor*

    PubMed Central

    An, Bo; Abbonante, Vittorio; Xu, Huifang; Gavriilidou, Despoina; Yoshizumi, Ayumi; Bihan, Dominique; Farndale, Richard W.; Kaplan, David L.; Balduini, Alessandra; Leitinger, Birgit; Brodsky, Barbara

    2016-01-01

    A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities. PMID:26702058

  14. Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor.

    PubMed

    An, Bo; Abbonante, Vittorio; Xu, Huifang; Gavriilidou, Despoina; Yoshizumi, Ayumi; Bihan, Dominique; Farndale, Richard W; Kaplan, David L; Balduini, Alessandra; Leitinger, Birgit; Brodsky, Barbara

    2016-02-26

    A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities.

  15. Defining a minimal estrogen receptor DNA binding domain.

    PubMed Central

    Mader, S; Chambon, P; White, J H

    1993-01-01

    The estrogen receptor (ER) is a transcriptional regulator which binds to cognate palindromic DNA sequences known as estrogen response elements (EREs). A 66 amino acid core region which contains two zinc fingers and is highly conserved among the nuclear receptors is essential for site specific DNA recognition. However, it remains unclear how many flanking amino acids in addition to the zinc finger core are required for DNA binding. Here, we have characterized the minimal DNA binding region of the human ER by analysing the DNA binding properties of a series of deletion mutants expressed in bacteria. We find that the 66 amino acid zinc finger core of the DBD fails to bind DNA, and that the C-terminal end of the minimal ER DBD required for binding to perfectly palindromic EREs corresponds to the limit of 100% amino acid homology between the chicken and human receptors, which represents the boundary between regions C and D in the ER. Moreover, amino acids of region D up to 30 residues C-terminal to the zinc fingers greatly stabilize DNA binding by the DBD to perfectly palindromic EREs and are absolutely required for formation of gel retardation complexes by the DBD on certain physiological imperfectly palindromic EREs. These results indicate that in addition to the zinc finger core, amino acids C-terminal to the core in regions C and D play a key role in DNA binding by the ER, particularly to imperfectly palindromic response elements. The ER DBD expressed in E. coli binds as a dimer to ERE palindromes in a highly cooperative manner and forms only low levels of monomeric protein-DNA complexes on either palindromic or half-palindromic response elements. Conversion of ER amino acids 222 to 226, which lie within region C, to the corresponding residues of the human RAR alpha abolishes formation of dimeric protein-DNA complexes. Conversely, replacement of the same region of RAR alpha with ER residues 222 to 226 creates a derivative that, unlike the RAR alpha DBD, binds

  16. The α2-subunit of the nicotinic cholinergic receptor is specifically expressed in medial subpallium-derived cells of mammalian amygdala.

    PubMed

    Pombero, Ana; Martinez, Salvador

    2015-08-01

    Nicotinic acetylcholine receptor (nAChR) subtypes are expressed in specific neuronal populations, which are involved in numerous neural functions such as sleep, fatigue, anxiety, and cognition, as well as the central processing of pain and food intake. Moreover, mutations in nAChRs subunits have been related to frontal lobe epilepsy, neurodegenerative diseases, and other neurological disorders, including schizophrenia and attention deficit and hyperactivity disorder (ADHD). Previous studies have shown that the α2-subunit of the AChR (Chrna2) is expressed in the basal forebrain, in the septum, and in some amygdalar nuclei in the adult rodent brain. However, although the importance of this amygdalar expression in emotion-related behavior and the physiopathology of neuropsychiatric disorders has been accepted, a detailed study of the Chrna2 expression pattern during development has been lacking. In this study we found that Chrna2 is specifically expressed in medial subpallium-derived amygdalar nuclei from early developmental stages to adult. This finding could help us to better understand the role of Chrna2 in the differentiation and functional maturation of amygdalar neurons involved in cholinergic-regulated emotional behavior. PMID:25641263

  17. Targeting α-7 nicotinic acetylcholine receptor in the enteric nervous system: a cholinergic agonist prevents gut barrier failure after severe burn injury.

    PubMed

    Costantini, Todd W; Krzyzaniak, Michael; Cheadle, Gerald A; Putnam, James G; Hageny, Ann-Marie; Lopez, Nicole; Eliceiri, Brian P; Bansal, Vishal; Coimbra, Raul

    2012-08-01

    We have previously shown that vagal nerve stimulation prevents intestinal barrier loss in a model of severe burn injury in which injury was associated with decreased expression and altered localization of intestinal tight junction proteins. α-7 Nicotinic acetylcholine receptor (α-7 nAchR) has been shown to be necessary for the vagus nerve to modulate the systemic inflammatory response, but the role of α-7 nAchR in mediating gut protection remained unknown. We hypothesized that α-7 nAchR would be present in the gastrointestinal tract and that treatment with a pharmacological agonist of α-7 nAchR would protect against burn-induced gut barrier injury. The effects of a pharmacological cholinergic agonist on gut barrier integrity were studied using an intraperitoneal injection of nicotine 30 minutes after injury. Intestinal barrier integrity was examined by measuring permeability to 4-kDa fluorescein isothiocyanate-dextran and by examining changes in expression and localization of the intestinal tight junction proteins occludin and ZO-1. Nicotine injection after injury prevented burn-induced intestinal permeability and limited histological gut injury. Treatment with nicotine prevented decreased expression and altered localization of occludin and ZO-1, as seen in animals undergoing burn alone. Defining the interactions among the vagus nerve, the enteric nervous system, and the intestinal epithelium may lead to development of targeted therapeutics aimed at reducing gut barrier failure and intestinal inflammation after severe injury.

  18. The α2-subunit of the nicotinic cholinergic receptor is specifically expressed in medial subpallium-derived cells of mammalian amygdala.

    PubMed

    Pombero, Ana; Martinez, Salvador

    2015-08-01

    Nicotinic acetylcholine receptor (nAChR) subtypes are expressed in specific neuronal populations, which are involved in numerous neural functions such as sleep, fatigue, anxiety, and cognition, as well as the central processing of pain and food intake. Moreover, mutations in nAChRs subunits have been related to frontal lobe epilepsy, neurodegenerative diseases, and other neurological disorders, including schizophrenia and attention deficit and hyperactivity disorder (ADHD). Previous studies have shown that the α2-subunit of the AChR (Chrna2) is expressed in the basal forebrain, in the septum, and in some amygdalar nuclei in the adult rodent brain. However, although the importance of this amygdalar expression in emotion-related behavior and the physiopathology of neuropsychiatric disorders has been accepted, a detailed study of the Chrna2 expression pattern during development has been lacking. In this study we found that Chrna2 is specifically expressed in medial subpallium-derived amygdalar nuclei from early developmental stages to adult. This finding could help us to better understand the role of Chrna2 in the differentiation and functional maturation of amygdalar neurons involved in cholinergic-regulated emotional behavior.

  19. Involvement of sigma (sigma) receptors in the acute actions of methamphetamine: receptor binding and behavioral studies.

    PubMed

    Nguyen, Emily C; McCracken, Kari A; Liu, Yun; Pouw, Buddy; Matsumoto, Rae R

    2005-10-01

    Methamphetamine interacts with sigma (sigma) receptors, suggesting that the drug produces some of its physiological and behavioral effects through these sites. Therefore, in the present report, receptor binding and pharmacological studies were performed to characterize the interaction between methamphetamine and sigma receptors. Of the two major sigma receptor subtypes, sigma1 and sigma2, competition binding studies showed that methamphetamine has a 22-fold preferential affinity for the sigma1 subtype. Saturation binding studies using the sigma1 selective radioligand [3H]+-pentazocine showed that in the presence of methamphetamine, there was a significant change in Kd, but not Bmax, suggesting competitive interactions. In behavioral studies, pretreatment of Swiss Webster mice with the sigma1 receptor antagonists, BD1063 or BD1047, significantly attenuated the locomotor stimulatory effects of methamphetamine. Mice that were administered an antisense oligodeoxynucleotide to down-regulate brain sigma1 receptors also exhibited a reduced locomotor stimulatory response to methamphetamine, as compared to control mice receiving mismatch oligonucleotides. Together, the data suggest that sigma1 receptors are involved in the acute actions of methamphetamine and that antagonism of this subtype is sufficient to prevent the locomotor stimulatory effects of methamphetamine. PMID:15939443

  20. MODELING THE BINDING OF THE METABOLITES OF SOME POLYCYCLIC AROMTIC HYDROCARBONS TO THE LIGAND BINDING DOMAIN OF THE ESTROGEN RECEPTOR

    EPA Science Inventory

    Modeling the binding of the metabolites of some Polycyclic Aromatic Hydrocarbons to the ligand binding domain of the estrogen receptor
    James Rabinowitz, Stephen Little, Katrina Brown, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC; Un...

  1. Two types of receptors for 5-hydroxytryptamine on the cholinergic nerves of the guinea-pig myenteric plexus.

    PubMed Central

    Kilbinger, H.; Pfeuffer-Friederich, I.

    1985-01-01

    The effects of 5-hydroxytryptamine (5-HT) on spontaneous and electrically-evoked release of [3H]-acetylcholine (ACh) from guinea-pig myenteric plexus preparations preincubated with [3H]-choline have been investigated in the absence of cholinesterase inhibitors. 5-HT caused a transient increase in spontaneous release and an inhibition of the electrically-evoked release of [3H]-ACh. The 5-HT-induced contractions of the longitudinal muscle were clearly related to the increase in spontaneous release. The inhibitory effect was not due to activation of alpha-adrenoceptors since it was also observed in the presence of tolazoline and on strips from reserpine-pretreated guinea-pigs. After desensitization of the excitatory 5-HT receptors with 5-HT or metoclopramide the effects of 5-HT on spontaneous [3H]-ACh release were largely reduced. A variety of established antagonists at neuronal 5-HT receptors (i.e. metitepine 0.1-1 microM; methysergide 1 microM; ketanserin 0.1-1 microM; MDL 72222 0.1 microM; tropacocaine 1 microM) failed to block the excitation. The inhibition by 5-HT of the electrically evoked [3H]-ACh release was competitively antagonized by metitepine (pA2 7.6) and methysergide (pA2 7.0) but not by ketanserin. Tachyphylaxis to the inhibitory action of 5-HT did not occur. The results suggest that the excitatory 5-HT receptor ('M'-receptor) differs in its pharmacological properties from other neuronal 5-HT receptors. The presynaptically located inhibitory receptor may roughly correspond to the 5-HT1 receptor subtype but probably differs from the 5-HT autoreceptor. PMID:3161573

  2. Cholinergic medial septum neurons do not degenerate in aged 129/Sv control or p75(NGFR)-/-mice.

    PubMed

    Ward, N L; Stanford, L E; Brown, R E; Hagg, T

    2000-01-01

    Cholinergic medial septum neurons express TrkA and p75 nerve growth factor receptor (p75(NGFR)) and interactions between TrkA and p75(NGFR) are necessary for high-affinity binding and signaling of nerve growth factor (NGF) through TrkA. In adult p75(NGFR)-deficient (-/-) mice, retrograde transport of NGF and other neurotrophins by these neurons is greatly reduced, however, these neurons maintain their cholinergic phenotype and size. Reduced transport of NGF has been proposed to play a role in Alzheimer's disease. Here, we investigated whether chronic and long-term absence of p75(NGFR) (and possibly reduced NGF transport and TrkA binding) would affect the cholinergic septohippocampal system during aging in mice. In young (6-8 months), middle aged (12-18 months), and aged (19-23 months) 129/Sv control mice the total number of choline acetyltransferase-positive medial septum neurons and the mean diameter and cross sectional area of the cholinergic cell bodies were similar. The cholinergic hippocampal innervation, as measured by the density of acetylcholinesterase-positive fibers in the outer molecular layer of the dentate gyrus was also similar across all ages. These parameters also did not change during aging in p75(NGFR) -/- mice and the number and size of the choline acetyltransferase-positive neurons and the cholinergic innervation density were largely similar as in control mice at all ages. These results suggest that p75(NGFR) does not play a major role in the maintenance of the number or morphology of the cholinergic basal forebrain neurons during aging of these mice. Alternatively, p75(NGFR) -/- mice may have developed compensatory mechanisms in response to the absence of p75(NGFR).

  3. New Etiology of Cholinergic Urticaria.

    PubMed

    Tokura, Yoshiki

    2016-01-01

    Cholinergic urticaria (CholU) is characterized by pinpoint-sized, highly pruritic wheals occurring upon sweating. Both direct and indirect theories in the interaction of acetylcholine (ACh) with mast cells have been put forward in the sweating-associated histamine release from mast cells. In the mechanism of indirect involvement of ACh, patients are hypersensitive to sweat antigen(s) and develop wheals in response to sweat substances leaking from the syringeal ducts to the dermis, possibly by obstruction of the ducts. Some patients with CholU exhibit a positive reaction to intradermal injection of their own diluted sweat, representing 'sweat allergy (hypersensitivity)'. Regarding the direct interaction theory between ACh and mast cells, we found that CholU with anhidrosis and hypohidrosis lacks cholinergic receptor M3 (CHRM3) expression in eccrine sweat gland epithelial cells. The expression of CHRM3 is completely absent in the anhidrotic areas and lowly expressed in the hypohidrotic areas. In the hypohidrotic area, where CholU occurs, it is hypothesized that ACh released from nerves cannot be completely trapped by cholinergic receptors of eccrine glands and overflows to the adjacent mast cells, leading to wheals. PMID:27584968

  4. Muscarinic M1 receptor partially modulates higher sensitivity to cadmium-induced cell death in primary basal forebrain cholinergic neurons: A cholinesterase variants dependent mechanism.

    PubMed

    Del Pino, Javier; Zeballos, Gabriela; Anadon, María José; Díaz, María Jesús; Moyano, Paula; Díaz, Gloria Gómez; García, Jimena; Lobo, Margarita; Frejo, María Teresa

    2016-06-15

    Cadmium is a toxic compound reported to produce cognitive dysfunctions, though the mechanisms involved are unknown. In a previous work we described how cadmium blocks cholinergic transmission and induces greater cell death in primary cholinergic neurons from the basal forebrain. It also induces cell death in SN56 cholinergic neurons from the basal forebrain through M1R blockage, alterations in the expression of AChE variants and GSK-3β, and an increase in Aβ and total and phosphorylated Tau protein levels. It was observed that the silencing or blockage of M1R altered ChAT activity, GSK-3β, AChE splice variants gene expression, and Aβ and Tau protein formation. Furthermore, AChE-S variants were associated with the same actions modulated by M1R. Accordingly, we hypothesized that cholinergic transmission blockage and higher sensitivity to cadmium-induced cell death of primary basal forebrain cholinergic neurons is mediated by M1R blockage, which triggers this effect through alteration of the expression of AChE variants. To prove this hypothesis, we evaluated, in primary culture from the basal forebrain region, whether M1R silencing induces greater cell death in cholinergic neurons than cadmium does, and whether in SN56 cells M1R mediates the mechanisms described so as to play a part in the cadmium induction of cholinergic transmission blockage and cell death in this cell line through alteration of the expression of AChE variants. Our results prove that M1R silencing by cadmium partially mediates the greater cell death observed on basal forebrain cholinergic neurons. Moreover, all previously described mechanisms for blocking cholinergic transmission and inducing cell death on SN56 cells after cadmium exposure are partially mediated by M1R through the alteration of AChE expression. Thus, our results may explain cognitive dysfunctions observed in cadmium toxicity. PMID:27377441

  5. Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

    SciTech Connect

    Yukawa, T.; Ukena, D.; Kroegel, C.; Chanez, P.; Dent, G.; Chung, K.F.; Barnes, P.J. )

    1990-06-01

    We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand (125I)pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed.

  6. Learning-Related Translocation of δ-Opioid Receptors on Ventral Striatal Cholinergic Interneurons Mediates Choice between Goal-Directed Actions

    PubMed Central

    Bertran-Gonzalez, Jesus; Laurent, Vincent; Chieng, Billy C.; Christie, MacDonald J.

    2013-01-01

    The ability of animals to extract predictive information from the environment to inform their future actions is a critical component of decision-making. This phenomenon is studied in the laboratory using the pavlovian–instrumental transfer protocol in which a stimulus predicting a specific pavlovian outcome biases choice toward those actions earning the predicted outcome. It is well established that this transfer effect is mediated by corticolimbic afferents on the nucleus accumbens shell (NAc-S), and recent evidence suggests that δ-opioid receptors (DORs) play an essential role in this effect. In DOR-eGFP knock-in mice, we show a persistent, learning-related plasticity in the translocation of DORs to the somatic plasma membrane of cholinergic interneurons (CINs) in the NAc-S during the encoding of the specific stimulus–outcome associations essential for pavlovian–instrumental transfer. We found that increased membrane DOR expression reflected both stimulus-based predictions of reward and the degree to which these stimuli biased choice during the pavlovian–instrumental transfer test. Furthermore, this plasticity altered the firing pattern of CINs increasing the variance of action potential activity, an effect that was exaggerated by DOR stimulation. The relationship between the induction of membrane DOR expression in CINs and both pavlovian conditioning and pavlovian–instrumental transfer provides a highly specific function for DOR-related modulation in the NAc-S, and it is consistent with an emerging role for striatal CIN activity in the processing of predictive information. Therefore, our results reveal evidence of a long-term, experience-dependent plasticity in opioid receptor expression on striatal modulatory interneurons critical for the cognitive control of action. PMID:24107940

  7. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

    PubMed Central

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J.

    2016-01-01

    ABSTRACT Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCE We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. PMID:26764003

  8. The effects of caffeine on the cholinergic system.

    PubMed

    Pohanka, Miroslav

    2014-01-01

    Caffeine is a secondary metabolite of tea and coffee plants. It is the active psychostimulant ingredient of widely consumed beverages, chocolate and some drugs as well. The major pathways for caffeine including interaction with adenosine receptors have been identified but caffeine has several minor pathways as well that remain poorly understood including the cholinergic system. Given the role of caffeine in the cholinergic system, some molecular targets have been tracked and a mechanism of its action has been proposed in research studies. However, the biological effect of caffeine on the cholinergic system is not completely understood. The present review focuses on the role of caffeine in the cholinergic system.

  9. Low-level microwave irradiation and central cholinergic systems

    SciTech Connect

    Lai, H.; Carino, M.A.; Horita, A.; Guy, A.W. )

    1989-05-01

    Our previous research showed that 45 min of exposure to low-level, pulsed microwaves (2450-MHz, 2-microseconds pulses, 500 pps, whole-body average specific absorption rate 0.6 W/kg) decreased sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. The effects of microwaves on central cholinergic systems were further investigated in this study. Increases in choline uptake activity in the frontal cortex, hippocampus, and hypothalamus were observed after 20 min of acute microwave exposure, and tolerance to the effect of microwaves developed in the hypothalamus, but not in the frontal cortex and hippocampus, of rats subjected to ten daily 20-min exposure sessions. Furthermore, the effects of acute microwave irradiation on central choline uptake could be blocked by pretreating the animals before exposure with the narcotic antagonist naltrexone. In another series of experiments, rats were exposed to microwaves in ten daily sessions of either 20 or 45 min, and muscarinic cholinergic receptors in different regions of the brain were studied by 3H-QNB binding assay. Decreases in concentration of receptors occurred in the frontal cortex and hippocampus of rats subjected to ten 20-min microwave exposure sessions, whereas increase in receptor concentration occurred in the hippocampus of animals exposed to ten 45-min sessions. This study also investigated the effects of microwave exposure on learning in the radial-arm maze. Rats were trained in the maze to obtain food reinforcements immediately after 20 or 45 min of microwave exposure.

  10. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    NASA Astrophysics Data System (ADS)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  11. A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission

    PubMed Central

    Hernández, Medardo; Barahona, María Victoria; Bustamante, Salvador; García-Sacristán, Albino; Orensanz, Luis M

    1999-01-01

    The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission. In U46619 (10−7  M)-contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10−6  M), adenosine and related analogues induced relaxations with the following potency order: 5′-N-ethylcarboxamidoadenosine (NECA)=5′-(N-cyclopropyl)-carboxamidoadenosine (CPCA)=2-chloroadenosine (2-CA)>adenosine>cyclopentyladenosine (CPA)=N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA)=2-[p-(carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680). Epithelium removal or incubation with indomethacin (3×10−6  M) and L-NG-nitroarginine (L-NOARG, 3×10−5  M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10−8 M) and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 3×10−8  M and 10−7  M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8-phenyltheophylline (8-PT, 10−5  M) and DPCPX (10−6  M), which block A1/A2-receptors, reduced such relaxations. In strips treated with guanethidine (10−5  M), atropine (10−7  M), L-NOARG (3×10−5  M) and indomethacin (3×10−6  M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10−4  M) induced contractions of preparations. 8-PT (10−5  M) increased both contractions. DPCPX (10−8  M), NECA (10−4  M), CPCA, (10−4  M) and 2-CA (10−4  M) did not alter the contractions to EFS. The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids

  12. Computational Reprogramming of T Cell Antigen Receptor Binding Properties.

    PubMed

    Riley, Timothy P; Singh, Nishant K; Pierce, Brian G; Baker, Brian M; Weng, Zhiping

    2016-01-01

    T-cell receptor (TCR) binding to peptide/MHC is key to antigen-specific cellular immunity, and there has been considerable interest in modulating TCR affinity and specificity for the development of therapeutics and imaging reagents. While in vitro engineering efforts using molecular evolution have yielded remarkable improvements in TCR affinity, such approaches do not offer structural control and can adversely affect receptor specificity, particularly if the attraction towards the MHC is enhanced independently of the peptide. Here we describe an approach to computational design that begins with structural information and offers the potential for more controlled manipulation of binding properties. Our design process models point mutations in selected regions of the TCR and ranks the resulting change in binding energy. Consideration is given to designing optimized scoring functions tuned to particular TCR-peptide/MHC interfaces. Validation of highly ranked predictions can be used to refine the modeling methodology and scoring functions, improving the design process. Our approach results in a strong correlation between predicted and measured changes in binding energy, as well as good agreement between modeled and experimental structures. PMID:27094299

  13. Binding Mode Prediction of Evodiamine within Vanilloid Receptor TRPV1

    PubMed Central

    Wang, Zhanli; Sun, Lidan; Yu, Hui; Zhang, Yanhui; Gong, Wuzhuang; Jin, Hongwei; Zhang, Liangren; Liang, Huaping

    2012-01-01

    Accurate assessment of the potential binding mode of drugs is crucial to computer-aided drug design paradigms. It has been reported that evodiamine acts as an agonist of the vanilloid receptor Transient receptor potential vanilloid-1 (TRPV1). However, the precise interaction between evodiamine and TRPV1 was still not fully understood. In this perspective, the homology models of TRPV1 were generated using the crystal structure of the voltage-dependent shaker family K+ channel as a template. We then performed docking and molecular dynamics simulation to gain a better understanding of the probable binding modes of evodiamine within the TRPV1 binding pocket. There are no significant interspecies differences in evodiamine binding in rat, human and rabbit TRPV1 models. Pharmacophore modeling further provided confidence for the validity of the docking studies. This study is the first to shed light on the structural determinants required for the interaction between TRPV1 and evodiamine, and gives new suggestions for the rational design of novel TRPV1 ligands. PMID:22942745

  14. Multivalent Ligand-Receptor Binding on Supported Lipid Bilayers

    PubMed Central

    Jung, Hyunsook; Robison, Aaron D.; Cremer, Paul S.

    2009-01-01

    Fluid supported lipid bilayers provide an excellent platform for studying multivalent protein-ligand interactions because the two-dimensional fluidity of the membrane allows for lateral rearrangement of ligands in order to optimize binding. Our laboratory has combined supported lipid bilayer-coated microfluidic platforms with total internal reflection fluorescence microscopy (TIRFM) to obtain equilibrium dissociation constant (KD) data for these systems. This high throughput, on-chip approach provides highly accurate thermodynamic information about multivalent binding events while requiring only very small sample volumes. Herein, we review some of the most salient findings from these studies. In particular, increasing ligand density on the membrane surface can provide a modest enhancement or attenuation of ligand-receptor binding depending upon whether the surface ligands interact strongly with each other. Such effects, however, lead to little more than one order of magnitude change in the apparent KD values. On the other hand, the lipophilicity and presentation of lipid bilayer-conjugated ligands can have a much greater impact. Indeed, changing the way a particular ligand is conjugated to the membrane can alter the apparent KD value by at least three orders of magnitude. Such a result speaks strongly to the role of ligand availability for multivalent ligand-receptor binding. PMID:19508894

  15. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    SciTech Connect

    Yashima, N.; Wada, A.; Izumi, F.

    1986-04-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of /sup 45/Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of /sup 45/Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of /sup 45/Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the /sup 45/Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the /sup 45/Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of /sup 45/Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of /sup 45/Ca. Based on these findings, the authors suggest that inhibition of the /sup 45/Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels.

  16. Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

    PubMed Central

    Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

    2013-01-01

    The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

  17. (/sup 3/H)AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart

    SciTech Connect

    Wang, J.X.; Roeske, W.R.; Gulya, K.; Wang, W.; Yamamura, H.I.

    1987-10-05

    The in vitro binding properties of the novel muscarinic antagonist (/sup 3/H)AF-DX 116 were studied using a rapid filtration technique. Association and dissociation rates of (/sup 3/H)AF-DX 116 binding were rapid at 25/sup 0/C but 20-40 times slower at 0-4/sup 0/C. Kinetic dissociation constants (K/sub d/s) were estimated to be 31.8 nM and 30.9 nM at 25/sup 0/C; 23.1 nM and 22.9 nM at 0-4/sup 0/C for the cerebral cortex and heart, respectively. In saturation studies, (/sup 3/H)AF-DX 116 labeled 29% of the total (/sup 3/H)(-)QNB binding sites in the cerebral cortical membranes and 87% in the cardiac membranes, with K/sub d/ values of 28.9 nM and 17.9 nM, respectively. Muscarinic antagonists inhibited (/sup 3/H)AF-DX 116 binding in a rank order of potency of atropine > dexetimide > AF-DX 116 > PZ > levetimide in both tissues. Except for PZ/(/sup 3/H)AF-DX 116 and AF-DX 116/(/sup 3/H)AF-DX 116 in the cerebral cortex, all the antagonist competition curves had Hill coefficients close to one. 19 references, 2 figures, 4 tables.

  18. Prebending the estrogen response element destabilizes binding of the estrogen receptor DNA binding domain.

    PubMed Central

    Kim, J; de Haan, G; Nardulli, A M; Shapiro, D J

    1997-01-01

    Binding of many eukaryotic transcription regulatory proteins to their DNA recognition sequences results in conformational changes in DNA. To test the effect of altering DNA topology by prebending a transcription factor binding site, we examined the interaction of the estrogen receptor (ER) DNA binding domain (DBD) with prebent estrogen response elements (EREs). When the ERE in minicircle DNA was prebent toward the major groove, which is in the same direction as the ER-induced DNA bend, there was no significant effect on ER DBD binding relative to the linear counterparts. However, when the ERE was bent toward the minor groove, in a direction that opposes the ER-induced DNA bend, there was a four- to eightfold reduction in ER DBD binding. Since reduced binding was also observed with the ERE in nicked circles, the reduction in binding was not due to torsional force induced by binding of ER DBD to the prebent ERE in covalently closed minicircles. To determine the mechanism responsible for reduced binding to the prebent ERE, we examined the effect of prebending the ERE on the association and dissociation of the ER DBD. Binding of the ER DBD to ERE-containing minicircles was rapid when the EREs were prebent toward either the major or minor groove of the DNA (k(on) of 9.9 x 10(6) to 1.7 x 10(7) M(-1) s(-1)). Prebending the ERE toward the minor groove resulted in an increase in k(off) of four- to fivefold. Increased dissociation of the ER DBD from the ERE is, therefore, the major factor responsible for reduced binding of the ER DBD to an ERE prebent toward the minor groove. These data provide the first direct demonstration that the interaction of a eukaryotic transcription factor with its recognition sequence can be strongly influenced by altering DNA topology through prebending the DNA. PMID:9154816

  19. Genome-Wide Binding Patterns of Thyroid Hormone Receptor Beta

    PubMed Central

    Ayers, Stephen; Switnicki, Michal Piotr; Angajala, Anusha; Lammel, Jan; Arumanayagam, Anithachristy S.; Webb, Paul

    2014-01-01

    Thyroid hormone (TH) receptors (TRs) play central roles in metabolism and are major targets for pharmaceutical intervention. Presently, however, there is limited information about genome wide localizations of TR binding sites. Thus, complexities of TR genomic distribution and links between TRβ binding events and gene regulation are not fully appreciated. Here, we employ a BioChIP approach to capture TR genome-wide binding events in a liver cell line (HepG2). Like other NRs, TRβ appears widely distributed throughout the genome. Nevertheless, there is striking enrichment of TRβ binding sites immediately 5′ and 3′ of transcribed genes and TRβ can be detected near 50% of T3 induced genes. In contrast, no significant enrichment of TRβ is seen at negatively regulated genes or genes that respond to unliganded TRs in this system. Canonical TRE half-sites are present in more than 90% of TRβ peaks and classical TREs are also greatly enriched, but individual TRE organization appears highly variable with diverse half-site orientation and spacing. There is also significant enrichment of binding sites for TR associated transcription factors, including AP-1 and CTCF, near TR peaks. We conclude that T3-dependent gene induction commonly involves proximal TRβ binding events but that far-distant binding events are needed for T3 induction of some genes and that distinct, indirect, mechanisms are often at play in negative regulation and unliganded TR actions. Better understanding of genomic context of TR binding sites will help us determine why TR regulates genes in different ways and determine possibilities for selective modulation of TR action. PMID:24558356

  20. Lack of [3H]quinuclidinyl benzylate binding to biologically relevant binding sites on mononuclear cells.

    PubMed

    Adams, E M; Lubrano, T M; Gordon, J; Fields, J Z

    1992-09-01

    We analyzed the binding characteristics of [3H]quinuclidinyl benzylate ([3H]QNB), a muscarinic cholinergic ligand, to rat and human mononuclear cells (MNC). Under various assay conditions, atropine-sensitive, saturable binding occurred with an apparent Kd of 10 nM. Conditions which disrupted the MNC membrane reduced total binding and eliminated specific binding. Muscarinic agonists were unable to inhibit [3H]QNB binding to MNC at concentrations up to 10(-2) M. Stereoisomers dexetimide and levetimide were equipotent inhibitors of binding (IC50 2 x 10(-5) M). We conclude that, although atropine-sensitive binding of [3H]QNB to MNC occurs, the binding is not consistent with the presence of a biologically relevant muscarinic cholinergic receptor. PMID:1392105

  1. Tumor necrosis factor: receptor binding and expression of receptors in cultured mouse hepatocytes.

    PubMed

    Adamson, G M; Billings, R E

    1994-04-01

    Recombinant murine tumor necrosis factor (TNF-alpha) was labeled with 125I and used to determine the binding characteristics, internalization and intracellular degradation in cultured mouse hepatocytes. [125I]TNF-alpha bound specifically to hepatocytes and Scatchard analysis of the data indicated binding to both a low-affinity (Kd = 20 nM) high capacity (51225 sites/cell) component and high-affinity component (Kd = 4 pM), with low capacity (290 sites/cell). The extent of TNF-alpha binding to hepatocytes correlated closely with its biological activity in hepatocytes, as indexed by depletion of intracellular ATP. At concentrations lower than 0.06 nM there was minimal binding and no effect on cellular ATP, whereas maximal binding at concentrations greater than 45 nM caused 80% depletion (in comparison to controls) of hepatocyte ATP. Incubation at 37 degrees C resulted in rapid uptake, internalization and degradation of [125I]TNF-alpha. This was followed by release of degraded material from hepatocytes. Examination, by reverse transcriptase/polymerase chain reaction technology, of hepatocyte RNA extracted after the 4-hr adherence period revealed that mouse hepatocytes expressed mRNA for both TNF-alpha receptor 1 and TNF-alpha receptor 2, and that the relative abundance of TNF-alpha receptor 1 was approximately 7-fold greater than that for TNF-alpha receptor 2. Because it has been shown that these receptors have different affinities for TNF-alpha, this may explain the high- and low-affinity binding sites present on cultured mouse hepatocytes.

  2. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    SciTech Connect

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  3. Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina

    PubMed Central

    Stanke, Jennifer J; Lehman, Bret; Fischer, Andy J

    2008-01-01

    Background Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT). Results We found that as ChAT-immunoreactive cells differentiate they expressed the homeodomain transcription factors Pax6 and Islet1, and the cell-cycle inhibitor p27kip1. As differentiation proceeds, type-II cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein (CRABP) and neurofilament, while type-I cells in the inner nuclear layer did not. Although there is a 1:1 ratio of type-I to type-II cells in vivo, in dissociated cell cultures the type-I cells (ChAT-positive and CRABP-negative) out-numbered the type-II cells (ChAT and CRABP-positive cells) by 2:1. The relative abundance of type-I to type-II cells was not influenced by Sonic Hedgehog (Shh), but was affected by compounds that act at muscarinic acetylcholine receptors. In addition, the abundance and mosaic patterning of type-II cholinergic amacrine cells is disrupted by interfering with muscarinic signaling. Conclusion We conclude that: (1) during development type-I and type-II cholinergic amacrine cells are not homotypic, (2) the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and (3) appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning. PMID:18254959

  4. 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

    Nery, Arthur A; Magdesian, Margaret H; 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.

  5. Development of Gamma-Emitting Receptor Binding Radiopharmace

    SciTech Connect

    Reba, Richard

    2003-02-20

    The long-term objective is to develop blood-brain barrier (BBB) permeable m2-selective (relative to m1, m3, and m4) receptor-binding radiotracers and utilize these radiotracers for quantifying receptor concentrations obtained from PET or SPECT images of human brain. In initial studies, we concluded that the lipophilicity and high affinity prevented (R,S)-I-QNB from reaching a flow-independent and receptor-dependent state in a reasonable time. Thus, it was clear that (R,S)-I-QNB should be modified. Therefore, during the last portion of this funded research, we proposed that more polar heterocycles should help accomplish that. Since reports of others concluded that radiobromination and radiofluorination of the unactivated phenyl ring is not feasible (Newkome et al,,1982), we, therefore, explored during this grant period a series of analogues of (R)-QNB in which one or both of the six-membered phenyl rings is replaced by a five-membered thienyl (Boulay et al., 1995), or furyl ring. The chemistry specific aims were to synthesize novel compounds designed to be m2-selective mAChR ligands capable of penetrating into the CNS, and develop methods for efficient radiolabeling of promising m2-selective muscarinic ligands. The pharmacology specific aims were to determine the affinity and subtype-selectivity of the novel compounds using competition binding studies with membranes from cells that express each of the five muscarinic receptor subtypes, to determine the ability of the promising non-radioactive compounds and radiolabeled novel compounds to cross the BBB, to determine the biodistribution, in-vivo pharmacokinetics, and in-vitm kinetics of promising m2-selective radioligands and to determine the distribution of receptors for the novel m2-selective radioligands using quantitative autoradiography of rat brain, and compare this distribution to the distribution of known m2-selective compounds.

  6. Analgesic and Antineuropathic Drugs Acting Through Central Cholinergic Mechanisms

    PubMed Central

    Bartolini, Alessandro; Cesare Mannelli, Lorenzo Di; Ghelardini, Carla

    2011-01-01

    The role of muscarinic and nicotinic cholinergic receptors in analgesia and neuropathic pain relief is relatively unknown. This review describes how such drugs induce analgesia or alleviate neuropathic pain by acting on the central cholinergic system. Several pharmacological strategies are discussed which increase synthesis and release of acetylcholine (ACh) from cholinergic neurons. The effects of their acute and chronic administration are described. The pharmacological strategies which facilitate the physiological functions of the cholinergic system without altering the normal modulation of cholinergic signals are highlighted. It is proposed that full agonists of muscarinic or nicotinic receptors should be avoided. Their activation is too intense and un-physiological because neuronal signals are distorted when these receptors are constantly activated. Good results can be achieved by using agents that are able to a) increase ACh synthesis, b) partially inhibit cholinesterase activity c) selectively block the autoreceptor or heteroreceptor feedback mechanisms. Activation of M1 subtype muscarinic receptors induces analgesia. Chronic stimulation of nicotinic (N1) receptors has neuronal protective effects. Recent experimental results indicate a relationship between repeated cholinergic stimulation and neurotrophic activation of the glial derived neurotrophic factor (GDNF) family. At least 9 patents covering novel chemicals for cholinergic system modulation and pain control are discussed. PMID:21585331

  7. Intracerebroventricular injection of mu- and delta-opiate receptor antagonists block 60 Hz magnetic field-induced decreases in cholinergic activity in the frontal cortex and hippocampus of the rat.

    PubMed

    Lai, H; Carino, M

    1998-01-01

    In previous research, we have found that acute exposure to a 60 Hz magnetic field decreased cholinergic activity in the frontal cortex and hippocampus of the rat as measured by sodium-dependent high-affinity choline uptake activity. We concluded that the effect was mediated by endogenous opioids inside the brain because it could be blocked by pretreatment of rats before magnetic field exposure with the opiate antagonist naltrexone, but not by the peripheral antagonist naloxone methiodide. In the present study, the involvement of opiate receptor subtypes was investigated. Rats were pretreated by intracerebroventricular injection of the mu-opiate receptor antagonist, beta-funaltrexamine, or the delta-opiate receptor antagonist, naltrindole, before exposure to a 60 Hz magnetic field (2 mT, 1 hour). It was found that the effects of magnetic field on high-affinity choline uptake in the frontal cortex and hippocampus were blocked by the drug treatments. These data indicate that both mu- and delta-opiate receptors in the brain are involved in the magnetic field-induced decreases in cholinergic activity in the frontal cortex and hippocampus of the rat.

  8. Influence of pharmacological manipulations of NMDA and cholinergic receptors on working versus reference memory in a dual component odor span task.

    PubMed

    MacQueen, David A; Dalrymple, Savannah R; Drobes, David J; Diamond, David M

    2016-06-01

    Developed as a tool to assess working memory capacity in rodents, the odor span task (OST) has significant potential to advance drug discovery in animal models of psychiatric disorders. Prior investigations indicate OST performance is impaired by systemic administration of N-methyl-d-aspartate receptor (NMDA-r) antagonists and is sensitive to cholinergic manipulations. The present study sought to determine whether an impairment in OST performance can be produced by systemic administration of the competitive NMDA-r antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 3, 10, 17 mg/kg i.p.) in a unique dual-component variant of the OST, and whether this impairment is ameliorated by nicotine (0.75 mg/kg i.p.). Male Sprague-Dawley rats were trained to asymptotic level of performance on a 24-trial two-comparison incrementing nonmatching to sample OST. In addition, rats were administered a two-comparison olfactory reference memory (RM) task, which was integrated into the OST. The RM task provided an assessment of the effects of drug administration on global behavioral measures, long-term memory and motivation. Several measures of working memory (span, longest run, and accuracy) were dose dependently impaired by CPP without adversely affecting RM. Analysis of drug effects across trial blocks demonstrated a significant impairment of performance even at low memory loads, suggesting a CPP-induced deficit of olfactory short-term memory that is not load-dependent. Although nicotine did not ameliorate CPP-induced impairments in span or accuracy, it did block the impairment in longest run produced by the 10 mg/kg dose of CPP. Overall, our results indicate that performance in our 24 odor two-comparison OST is capacity dependent and that CPP impaired OST working, but not reference, memory. PMID:27194794

  9. Estrogen receptor-alpha and -beta immunoreactive neurons in the brainstem and spinal cord of male and female mice: relationships to monoaminergic, cholinergic, and spinal projection systems.

    PubMed

    Vanderhorst, Veronique G J M; Gustafsson, Jan-Ake; Ulfhake, Brun

    2005-07-25

    For many populations of estrogen-sensitive neurons it remains unknown how they are associated with central nervous system circuitries that mediate estrogen-induced modulation of behavioral components. With the use of double-labeling immunohistochemistry and tracing techniques, the relationships of estrogen receptor (ER)-alpha- and ER-beta-immunoreactive (IR) neurons in the mouse brainstem and spinal cord to monoaminergic, cholinergic, and spinal projection systems are explored. Similar distributions of ER-IR neurons were present in females and males, with differences in labeling intensity of ER-alpha immunoreactivity among males and estrogen-, and oil-treated females. Barrington's nucleus, the ventrolateral medulla, and the nucleus of the solitary tract contained spinal-projecting ER-alpha-IR neurons, whereas ER-alpha-IR neurons in the periaqueductal gray, parabrachial nucleus, and catecholaminergic A1 cell group received spinal input. Numerous tyrosine hydroxylase (TH)-IR ER-alpha-IR neurons were present in the ventral periaqueductal gray, nucleus of the solitary tract, A1 cell group, and lumbosacral cord. The dorsal raphe nucleus contained ER-alpha-IR and ER-beta-IR neurons that colocalized with serotonin (5HT), and the reticulotegmental nucleus contained 5HT-IR ER-alpha-IR neurons. Fibers IR for vesicular acetylcholine transporter (VAChT), TH, and 5HT were located among ER-alpha-IR neurons in the dorsal horn and spinal autonomic regions. Robust staining for TH and VAChT, but not 5HT, was present among ER-alpha-IR neurons in the lumbosacral lateral collateral pathway. Possible modulatory actions of estrogen on each of these ER-IR populations are discussed in the context of their specific function, including micturition, sexual behavior, ejaculation, cardiovascular and respiratory control, tactile and nociceptive sensory processing, anti-nociception, endocrine regulation, and feeding.

  10. Binding kinetics differentiates functional antagonism of orexin-2 receptor ligands

    PubMed Central

    Mould, R; Brown, J; Marshall, FH; Langmead, CJ

    2014-01-01

    Orexin receptor antagonism represents a novel approach for the treatment of insomnia that directly targets sleep/wake regulation. Several such compounds have entered into clinical development, including the dual orexin receptor antagonists, suvorexant and almorexant. In this study, we have used equilibrium and kinetic binding studies with the orexin-2 (OX2) selective antagonist radioligand, [3H]-EMPA, to profile several orexin receptor antagonists. Furthermore, selected compounds were studied in cell-based assays of inositol phosphate accumulation and ERK-1/2 phosphorylation in CHO cells stably expressing the OX2 receptor that employ different agonist incubation times (30 and 5 min, respectively). EMPA, suvorexant, almorexant and TCS-OX-29 all bind to the OX2 receptor with moderate to high affinity (pKI values ≥ 7.5), whereas the primarily OX1 selective antagonists SB-334867 and SB-408124 displayed low affinity (pKI values ca. 6). Competition kinetic analysis showed that the compounds displayed a range of dissociation rates from very fast (TCS-OX2-29, koff = 0.22 min−1) to very slow (almorexant, koff = 0.005 min−1). Notably, there was a clear correlation between association rate and affinity. In the cell-based assays, fast-offset antagonists EMPA and TCS-OX2-29 displayed surmountable antagonism of orexin-A agonist activity. However, both suvorexant and particularly almorexant cause concentration-dependent depression in the maximal orexin-A response, a profile that is more evident with a shorter agonist incubation time. Analysis according to a hemi-equilibrium model suggests that antagonist dissociation is slower in a cellular system than in membrane binding; under these conditions, almorexant effectively acts as a pseudo-irreversible antagonist. Linked ArticlesThis article is part of a themed section on Orexin Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-2 PMID:23692283

  11. Angiotensin receptor binding and pressor effects in cat subretrofacial nucleus

    SciTech Connect

    Allen, A.M.; Dampney, R.A.L.; Mendelsohn, F.A.O. Univ. of Sydney )

    1988-11-01

    Central administration of angiotensin II (ANG II) increases arterial blood pressure via increased sympathetic activity. The authors have examined the possibility that one site of action of ANG II is the subretrofacial (SRF) nucleus in the rostral ventrolateral medulla, since this nucleus is known to play a critical role in the tonic and phasic control of arterial pressure. In vitro autoradiography, employing {sup 125}I-labeled (Sar{sup 1}, Ile{sup 8})ANG II as radioligand, was used to localize binding sites for ANG-II in the cat ventrolateral medulla. A high density of ANG II-receptor binding sites was found confined to the SRF nucleus. In a second group of experiments in anesthetized cats, microinjections of ANG II, in doses ranging from 10 to 50 pmol, were made into histologically identified sites within and outside the SRF nucleus. Microinjections into the nucleus resulted in a dose-dependent increase in arterial pressure, which was abolished by systemic administration of the ganglion-blocking drug hexamethonium bromide. In contrast, microinjections just outside the SRF nucleus had no effect on arterial pressure. It is concluded that activation of ANG II-receptor binding sites within the SRF nucleus leads to an increase in arterial pressure via increased sympathetic efferent activity.

  12. The glycocalyx promotes cooperative binding and clustering of adhesion receptors.

    PubMed

    Xu, Guang-Kui; Qian, Jin; Hu, Jinglei

    2016-05-18

    Cell adhesion plays a pivotal role in various biological processes, e.g., immune responses, cancer metastasis, and stem cell differentiation. The adhesion behaviors depend subtly on the binding kinetics of receptors and ligands restricted at the cell-substrate interfaces. Although much effort has been directed toward investigating the kinetics of adhesion molecules, the role of the glycocalyx, anchored on cell surfaces as an exterior layer, is still unclear. In this paper, we propose a theoretical approach to study the collective binding kinetics of a few and a large number of binders in the presence of the glycocalyx, representing the cases of initial and mature adhesions of cells, respectively. The analytical results are validated by finding good agreement with our Monte Carlo simulations. In the force loading case, the on-rate and affinity increase as more bonds form, whereas this cooperative effect is not observed in the displacement loading case. The increased thickness and stiffness of the glycocalyx tend to decrease the affinity for a few bonds, while they have less influence on the affinity for a large number of bonds. Moreover, for a flexible membrane with thermally-excited shape fluctuations, the glycocalyx is exhibited to promote the formation of bond clusters, mainly due to the cooperative binding of binders. This study helps to understand the cooperative kinetics of adhesion receptors under physiologically relevant loading conditions and sheds light on the novel role of the glycocalyx in cell adhesion. PMID:27102288

  13. Cholinergic interneurons control local circuit activity and cocaine conditioning.

    PubMed

    Witten, Ilana B; Lin, Shih-Chun; Brodsky, Matthew; Prakash, Rohit; Diester, Ilka; Anikeeva, Polina; Gradinaru, Viviana; Ramakrishnan, Charu; Deisseroth, Karl

    2010-12-17

    Cholinergic neurons are widespread, and pharmacological modulation of acetylcholine receptors affects numerous brain processes, but such modulation entails side effects due to limitations in specificity for receptor type and target cell. As a result, causal roles of cholinergic neurons in circuits have been unclear. We integrated optogenetics, freely moving mammalian behavior, in vivo electrophysiology, and slice physiology to probe the cholinergic interneurons of the nucleus accumbens by direct excitation or inhibition. Despite representing less than 1% of local neurons, these cholinergic cells have dominant control roles, exerting powerful modulation of circuit activity. Furthermore, these neurons could be activated by cocaine, and silencing this drug-induced activity during cocaine exposure (despite the fact that the manipulation of the cholinergic interneurons was not aversive by itself) blocked cocaine conditioning in freely moving mammals.

  14. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.

  15. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site. PMID:27298318

  16. 1918 Influenza receptor binding domain variants bind and replicate in primary human airway cells regardless of receptor specificity.

    PubMed

    Davis, A Sally; Chertow, Daniel S; Kindrachuk, Jason; Qi, Li; Schwartzman, Louis M; Suzich, Jon; Alsaaty, Sara; Logun, Carolea; Shelhamer, James H; Taubenberger, Jeffery K

    2016-06-01

    The 1918 influenza pandemic caused ~50 million deaths. Many questions remain regarding the origin, pathogenicity, and mechanisms of human adaptation of this virus. Avian-adapted influenza A viruses preferentially bind α2,3-linked sialic acids (Sia) while human-adapted viruses preferentially bind α2,6-linked Sia. A change in Sia preference from α2,3 to α2,6 is thought to be a requirement for human adaptation of avian influenza viruses. Autopsy data from 1918 cases, however, suggest that factors other than Sia preference played a role in viral binding and entry to human airway cells. Here, we evaluated binding and entry of five 1918 influenza receptor binding domain variants in a primary human airway cell model along with control avian and human influenza viruses. We observed that all five variants bound and entered cells efficiently and that Sia preference did not predict entry of influenza A virus to primary human airway cells evaluated in this model. PMID:27062579

  17. 1918 Influenza receptor binding domain variants bind and replicate in primary human airway cells regardless of receptor specificity.

    PubMed

    Davis, A Sally; Chertow, Daniel S; Kindrachuk, Jason; Qi, Li; Schwartzman, Louis M; Suzich, Jon; Alsaaty, Sara; Logun, Carolea; Shelhamer, James H; Taubenberger, Jeffery K

    2016-06-01

    The 1918 influenza pandemic caused ~50 million deaths. Many questions remain regarding the origin, pathogenicity, and mechanisms of human adaptation of this virus. Avian-adapted influenza A viruses preferentially bind α2,3-linked sialic acids (Sia) while human-adapted viruses preferentially bind α2,6-linked Sia. A change in Sia preference from α2,3 to α2,6 is thought to be a requirement for human adaptation of avian influenza viruses. Autopsy data from 1918 cases, however, suggest that factors other than Sia preference played a role in viral binding and entry to human airway cells. Here, we evaluated binding and entry of five 1918 influenza receptor binding domain variants in a primary human airway cell model along with control avian and human influenza viruses. We observed that all five variants bound and entered cells efficiently and that Sia preference did not predict entry of influenza A virus to primary human airway cells evaluated in this model.

  18. Effects of neonatal handling on the basal forebrain cholinergic system of adult male and female rats.

    PubMed

    Pondiki, S; Stamatakis, A; Fragkouli, A; Philippidis, H; Stylianopoulou, F

    2006-10-13

    Neonatal handling is an early experience which results in improved function of the hypothalamic-pituitary-adrenal axis, increased adaptability and coping as a response to stress, as well as better cognitive abilities. In the present study, we investigated the effect of neonatal handling on the basal forebrain cholinergic system, since this system is known to play an important role in cognitive processes. We report that neonatal handling results in increased number of choline-acetyl transferase immunopositive cells in the septum/diagonal band, in both sexes, while no such effect was observed in the other cholinergic nuclei, such as the magnocellular preoptic nucleus and the nucleus basalis of Meynert. In addition, neonatal handling resulted in increased M1 and M2 muscarinic receptor binding sites in the cingulate and piriform cortex of both male and female rats. A handling-induced increase in M1 muscarinic receptor binding sites was also observed in the CA3 and CA4 (fields 3 and 4 of Ammon's horn) areas of the hippocampus. Furthermore, a handling-induced increase in acetylcholinesterase staining was found only in the hippocampus of females. Our results thus show that neonatal handling acts in a sexually dimorphic manner on one of the cholinergic parameters, and has a beneficial effect on BFCS function, which could be related to the more efficient and adaptive stress response and the superior cognitive abilities of handled animals.

  19. Binding site structure of one LRP-RAP complex: implications for a common ligand-receptor binding motif.

    PubMed

    Jensen, Gitte A; Andersen, Olav M; Bonvin, Alexandre M J J; Bjerrum-Bohr, Ida; Etzerodt, Michael; Thøgersen, Hans C; O'Shea, Charlotte; Poulsen, Flemming M; Kragelund, Birthe B

    2006-09-29

    The low-density lipoprotein receptor-related protein (LRP) interacts with more than 30 ligands of different sizes and structures that can all be replaced by the receptor-associated protein (RAP). The double module of complement type repeats, CR56, of LRP binds many ligands including all three domains of RAP and alpha2-macroglobulin, which promotes the catabolism of the Abeta-peptide implicated in Alzheimer's disease. To understand the receptor-ligand cross-talk, the NMR structure of CR56 has been solved and ligand binding experiments with RAP domain 1 (RAPd1) have been performed. From chemical shift perturbations of both binding partners upon complex formation, a HADDOCK model of the complex between CR56 and RAPd1 has been obtained. The binding residues are similar to a common binding motif suggested from alpha2-macroglobulin binding studies and provide evidence for an understanding of their mutual cross-competition pattern. The present structural results convey a simultaneous description of both binding partners of an LRP-ligand complex and open a route to a broader understanding of the binding specificity of the LRP receptor, which may involve a general four-residue receptor-ligand recognition motif common to all LRP ligands. The present result may be beneficial in the design of antagonists of ligand binding to the LDL receptor family, and especially of drugs for treatment of Alzheimer's disease.

  20. Menthol Binding and Inhibition of α7-Nicotinic Acetylcholine Receptors

    PubMed Central

    Ashoor, Abrar; Nordman, Jacob C.; Veltri, Daniel; Yang, Keun-Hang Susan; Al Kury, Lina; Shuba, Yaroslav; Mahgoub, Mohamed; Howarth, Frank C.; Sadek, Bassem; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-01-01

    Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner. PMID:23935840

  1. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    PubMed Central

    Hardison, D. Ransom; Holland, William C.; McCall, Jennifer R.; Bourdelais, Andrea J.; Baden, Daniel G.; Darius, H. Taiana; Chinain, Mireille; Tester, Patricia A.; Shea, Damian; Flores Quintana, Harold A.; Morris, James A.; Litaker, R. Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®- PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  2. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    PubMed

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  3. The structural basis of pregnane X receptor binding promiscuity

    PubMed Central

    Ngan, Chi-Ho; Beglov, Dmitri; Rudnitskaya, Aleksandra N.; Kozakov, Dima; Waxman, David J.; Vajda, Sandor

    2009-01-01

    The steroid and xenobiotic-responsive human pregnane X receptor (PXR) binds a broad range of structurally diverse compounds. The structures of the apo and ligand-bound forms of PXR are very similar, in contrast to most promiscuous proteins that generally adapt their shape to different ligands. We investigated the structural origins of PXR's recognition promiscuity using computational solvent mapping, a technique developed for the identification and characterization of hot spots, i.e., regions of the protein surface that are major contributors to the binding free energy. Results reveal that the smooth and nearly spherical binding site of PXR has a well-defined hot spot structure, with four hot spots located on four different sides of the pocket and a fifth close to its center. Three of these hot spots are already present in the ligand-free protein. The most important hot spot is defined by three structurally and sequentially conserved residues, W299, F288, and Y306. This largely hydrophobic site is not very specific, and interacts with all known PXR ligands. Depending on their sizes and shapes, individual PXR ligands extend into 2, 3, or 4 more hot spot regions. The large number of potential arrangements within the binding site explains why PXR is able to accommodate a large variety of compounds. All five hot spots include at least one important residue, which is conserved in all mammalian PXRs, suggesting that the hot spot locations have remained largely invariant during mammalian evolution. The same side chains also show a high level of structural conservation across hPXR structures. However, each of the hPXR hot spots also includes residues with moveable side chains, further increasing the size variation in ligands that PXR can bind. Results also suggest a unique signal transduction mechanism between the PXR homodimerization interface and its co-activator binding site. PMID:19856963

  4. Persistent Binding of Ligands to the Aryl Hydrocarbon Receptor

    PubMed Central

    Bohonowych, Jessica E.; Denison, Michael S.

    2010-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxic effects of halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and other structurally diverse ligands. While HAHs are several orders of magnitude more potent in producing AhR-dependent biochemical effects than PAHs or other AhR agonists, only the HAHs have been observed to produce AhR-dependent toxicity in vivo. Here we have characterized the dissociation of a prototypical HAH ligand ([3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and PAH-like ligand ([3H] β-naphthoflavone [βNF]) from the guinea pig, hamster, mouse, and rat hepatic cytosolic AhR in order to elucidate the relationship between the apparent ligand-binding affinities and the divergent potency of these chemicals. Both compounds dissociated very slowly from the AhR with the amount of specific binding remaining at 96 h ranging from 53% to 70% for [3H]TCDD and 26% to 85% for [3H] βNF, depending upon the species examined. The rate of ligand dissociation was unaffected by protein concentration or incubation temperature. Preincubation of cytosol with 2,3,7,8-tetrachlorodibenzofuran, carbaryl, or primaquine, prior to the addition of [3H]TCDD, shifted the apparent IC50 of these compounds as competitive AhR ligands by ∼10- to 50-fold. Our results support the need for reassessment of previous AhR ligand-binding affinity calculations and competitive binding analysis since these measurements are not carried out at equilibrium binding conditions. Our studies suggest that AhR binding affinity/occupancy has little effect on the observed differences in the persistence of gene expression by HAHs and PAHs. PMID:17431010

  5. Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors.

    PubMed

    Nayak, Tapan K; Bruhova, Iva; Chakraborty, Srirupa; Gupta, Shaweta; Zheng, Wenjun; Auerbach, Anthony

    2014-12-01

    A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔGB1). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔGB1 is ∼-2 kcal/mol more favorable at αγ compared with at αε and αδ. Only three of the aromatics contribute significantly to ΔGB1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔGB1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αε and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αε and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse. PMID:25422413

  6. Radioligand binding to muscarinic receptors of bovine aortic endothelial cells.

    PubMed Central

    Brunner, F.; Kukovetz, W. R.

    1991-01-01

    1. Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (-)-[3H]-N-methyl quinuclidinyl benzilate ([3H]-NMeQNB) was used as radioligand. 2. Binding of [3H]-NMeQNB to crude membranes of freshly isolated endothelial cells was atropine-displaceable and of high affinity (KD = 0.48 nM) to a single class of sites (maximum binding capacity: 14 +/- 3 fmol mg-1 protein). Stereospecificity of the binding sites was demonstrated in experiments in which [3H]-NMeQNB binding was inhibited by dexetimide in the nanomolar range (KI = 0.63 nM) and by levetimide, its stereoisomer in the micromolar range (KI = 3.2 microM) (selectivity factor: approximately 5000). 3. Drug competition curves indicated a single class of binding sites for antagonists and the following apparent affinities (KI, nM): methyl atropine: 1.1: 4-diphenylacetoxy N-methyl piperidine methyl bromide (4-DAMP): 3.4; pirenzepine: 16; 11-[2-diethylamino-methyl)-1-piperidinyl- acetyl]-5,11-dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one (AF-DX 116); 2.500. Competition of acetylcholine with [3H]-NMeQNB was best described by two affinity sites (or states) (KH = 0.82 microM, KL = 1.6 microM). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 microM), acetylcholine affinity (IC50) was slightly, but significantly reduced (factor approximately 4). 4. Binding of [3H]-NMeQNB to freshly harvested intact cells was also atropine-displaceable, stereospecific (selectivity factor: approximately 3500) and of high affinity (KD = 0.35 nM). The maximum binding capacity (9 +/- 2 fmol mg-1 total cell protein) was comparable to that of membranes and corresponded to approximately 900 binding sites per endothelial cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2015420

  7. Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow

    EPA Science Inventory

    In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach ...

  8. Coregulator control of androgen receptor action by a novel nuclear receptor-binding motif.

    PubMed

    Jehle, Katja; Cato, Laura; Neeb, Antje; Muhle-Goll, Claudia; Jung, Nicole; Smith, Emmanuel W; Buzon, Victor; Carbó, Laia R; Estébanez-Perpiñá, Eva; Schmitz, Katja; Fruk, Ljiljana; Luy, Burkhard; Chen, Yu; Cox, Marc B; Bräse, Stefan; Brown, Myles; Cato, Andrew C B

    2014-03-28

    The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

  9. Structural determinants within residues 180-199 of the rodent. alpha. 5 nicotinic acetylcholine receptor subunit involved in. alpha. -bungarotoxin binding

    SciTech Connect

    McLane, K.E.; Xiadong Wu; Conti-Tronconi, B.M. )

    1991-11-05

    Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) {alpha} subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. The authors have previously defined {alpha}-bungarotoxin ({alpha}-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR {alpha} subunit, designated {alpha}5, and between residues 181 and 200 of the chick neuronal {alpha}7 and {alpha}8 subunits. These sequences are relatively divergent compared with the Torpedo and muscle nAChR {alpha}1 {alpha}-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology in general. Given the highly divergent nature of the {alpha}5 sequence, they were interested in determining the critical amino acid residues for {alpha}-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat {alpha}(180-199) sequence were tested, using a competition assay, in which peptides compete for {sup 125}I-{alpha}-BTX binding with native Torpedo nAChR. These results indicate that a disulfide bridge between the vicinal cysteines at positions 191 and 192 of the {alpha}5 sequence is not an absolute requirement for {alpha}-BTX binding activity.

  10. Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

    SciTech Connect

    Johns, Douglas G. . E-mail: Douglas.G.Johns@gsk.com; Ao, Zhaohui; Heidrich, Bradley J.; Hunsberger, Gerald E.; Graham, Taylor; Payne, Lisa; Elshourbagy, Nabil; Lu, Quinn; Aiyar, Nambi; Douglas, Stephen A.

    2007-06-22

    Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [{sup 125}I]-ANP from NPR-C with pM-to-nM K {sub i} values. DNP displaced [{sup 125}I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K {sub i} > 1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.

  11. EFFECTS OF SOG ON DPP-RECEPTOR BINDING*

    PubMed Central

    LOU, YUAN; NIE, QING; WAN, FREDERIC Y. M.

    2007-01-01

    Concentration gradients of morphogens are known to be instrumental in cell signaling and tissue patterning. Of interest here is how the presence of a competitor of BMP ligands affects cell signaling. The effects of Sog on the binding of Dpp with cell receptors are analyzed for dorsal-ventral morphogen gradient formation in vertebrate and Drosophila embryos. This prototype system includes diffusing ligands, degradation of morphogens, and cleavage of Dpp-Sog complexes by Tolloid to free up Dpp. Simple and biologically meaningful necessary and sufficient conditions for the existence of a steady state gradient configuration are established, and existence theorems are proved. For high Sog production rates (relative to the Dpp production rate), it is found that the steady state configuration exhibits a more intense Dpp-receptor concentration near the dorsal midline. Numerical simulations of the evolution of the system show that, beyond some threshold Sog production rate, the transient Dpp-receptor concentration at the dorsal midline would become more intense than that of the steady state, before subsiding and approaching a nonuniform steady state of lower magnitude. The magnitude of the transient concentration has been found to increase by several fold with increasing Sog production rate. The highly intense Dpp activity at and around the dorsal midline is consistent with available experimental observations and other analytical studies. PMID:17377624

  12. Benzodiazepine receptor binding in vivo with (/sup 3/)-Ro 15-1788

    SciTech Connect

    Goeders, N.E.; Kuhar, M.J.

    1985-07-29

    In vivo benzodiazepine receptor binding has generally been studied by ex vivo techniques. In this investigation, the authors identify the conditions where (/sup 3/H)-Ro 15-1788 labels benzodiazepine receptors by true in vivo binding, i.e. where workable specific to nonspecific ratios are obtained in intact tissues without homogenization or washing. (/sup 3/H)-Flunitrazepam and (/sup 3/H)-clonazepam did not exhibit useful in vivo receptor binding. 39 references, 5 figures, 1 table.

  13. Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening

    EPA Science Inventory

    The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across differ...

  14. The opioid receptor selectivity for trimebutine in isolated tissues experiments and receptor binding studies.

    PubMed

    Kaneto, H; Takahashi, M; Watanabe, J

    1990-07-01

    Differences of affinity to and selectivity for trimebutine between peripheral and central opioid receptors have been investigated. Trimebutine inhibited electrically induced contraction of guinea-pig ileum (GPI) and mouse vas deferens (MVD) but not of rabbit vas deferens, and the inhibition was antagonized by naloxone and, to lesser extent, by nor-binaltorphimine (nor-BNI). The pA2 values for morphine and trimebutine with naloxone were higher than the values for these compounds with nor-BNI in both GPI and MVD preparations. GPI preparations incubated with a high concentration of morphine or trimebutine developed tolerance; however, there was no cross-tolerance between them, suggesting difference in the underlying mechanisms. In mouse and guinea-pig brain homogenate trimebutine was about 1/13 as potent as morphine to displace the [3H]naloxone binding, while it has no appreciable affinity for kappa-opioid receptors in [3H]U-69593, a selective kappa-receptor agonist. These results suggest that trimebutine, showing its low affinity to opioid receptors, possesses mu-receptor selective properties rather than those of kappa-opioid receptor in the peripheral tissues and in the central brain homogenate. PMID:1963196

  15. Managing tight-binding receptors for new separations technologies. 1998 annual progress report

    SciTech Connect

    Busch, D.H.; Givens, R.S.

    1998-06-01

    'Whereas such traditional separation methodologies as ion exchange and solvent extraction require rapid interaction between ligands and metal ions, the most strongly binding ligands invariably bind slowly; e.g., cryptates bind and dissociate more slowly than macrocycles, which are slower than open-chain chelating ligands. This project seeks to maximize the binding and dissociation rates for tight-binding receptors in order to make them more useful to separations science. An alternative slow-binding technology is also under exploration.'

  16. Endothelin B receptors on human endothelial and smooth-muscle cells show equivalent binding pharmacology.

    PubMed

    Flynn, M A; Haleen, S J; Welch, K M; Cheng, X M; Reynolds, E E

    1998-07-01

    We have described the pharmacologic profiles of endothelin B receptors in human endothelial cells and vascular and nonvascular smooth-muscle cells. First, by amplifying endothelin B receptor numbers through the use of phosphoramidon and intact cell-binding techniques, we demonstrated the presence of these receptors in human umbilical vein endothelial cells (100% endothelin B receptors), human aortic smooth-muscle cells (22% endothelin B, 78% endothelin A receptors), and human bronchial smooth-muscle cells (55% endothelin B, 45% endothelin A receptors) by using [125I]-endothelin-1 radioligand binding. The typical binding profiles of the endothelin B receptors were established through competition binding curve analysis with endothelin-1, endothelin-3, sarafotoxin 6c, and the endothelin A receptor-selective antagonist BQ-123. In the presence of BQ-123, a diverse group of antagonists, including PD 142893, BQ-788, SB 209670, and Ro 47-0203, were used to probe for binding differences indicative of multiple endothelin B-receptor subtypes. The results indicate a rank order of potency for the antagonists of BQ-788 > SB 209670 > PD 142893 > Ro 47-0203 for each cell line, and that between any of these human cell lines, measurements of [125I]-endothelin-1-binding antagonism for each of the four test compounds differed by less than twofold. Although this study cannot discount the possibility of more than one endothelin B-receptor subtype in humans, it does indicate that these tissues express receptors that show equivalent binding pharmacology. PMID:9676729

  17. Modulation of mood and cognitive performance following acute administration of single doses of Melissa officinalis (Lemon balm) with human CNS nicotinic and muscarinic receptor-binding properties.

    PubMed

    Kennedy, D O; Wake, G; Savelev, S; Tildesley, N T J; Perry, E K; Wesnes, K A; Scholey, A B

    2003-10-01

    Melissa officinalis (Lemon balm) is a herbal medicine that has traditionally been attributed with memory-enhancing properties, but which is currently more widely used as a mild sedative and sleep aid. In a previous study it was demonstrated that a commercial Melissa extract led to dose-specific increases in calmness, and dose-dependent decrements in timed memory task performance. However, the extract utilized in that study did not exhibit in vitro cholinergic receptor-binding properties. The current study involved an initial screening of samples of M. officinalis for human acetylcholinesterase inhibition and cholinergic receptor-binding properties. The cognitive and mood effects of single doses of the most cholinergically active dried leaf were then assessed in a randomized, placebo-controlled, double-blind, balanced crossover study. Following the in vitro analysis, 20 healthy, young participants received single doses of 600, 1000, and 1600 mg of encapsulated dried leaf, or a matching placebo, at 7-day intervals. Cognitive performance and mood were assessed predose and at 1, 3, and 6 h postdose using the Cognitive Drug Research computerized assessment battery and Bond-Lader visual analog scales, respectively. In vitro analysis of the chosen extract established IC(50) concentrations of 0.18 and 3.47 mg ml(-1), respectively, for the displacement of [(3)H]-(N)-nicotine and [(3)H]-(N)-scopolamine from nicotinic and muscarinic receptors in the human cerebral cortex tissue. However, no cholinesterase inhibitory properties were detected. The most notable cognitive and mood effects were improved memory performance and increased 'calmness' at all postdose time points for the highest (1600 mg) dose. However, while the profile of results was overwhelmingly favorable for the highest dose, decrements in the speed of timed memory task performance and on a rapid visual information-processing task increased with decreasing dose. These results suggest that doses of Melissa

  18. Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

    NASA Technical Reports Server (NTRS)

    Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

    1999-01-01

    We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

  19. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    PubMed Central

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  20. Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

    SciTech Connect

    Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Danielsen, Mark . E-mail: dan@bc.georgetown.edu

    2005-08-15

    The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increased ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids.

  1. Chronic ethanol (EtOH) feeding increases muscarinic receptor (mAChR) density in esophagus without parallel change in dose response (D-R) to cholinergic agonists

    SciTech Connect

    Keshavarzian, A.; Gordon, J.H.; Urban, G.; Fields, J.Z. VA Hospital, Hines, IL )

    1991-03-11

    The mAChR/effector pathway for signal transduction is important in the physiology of esophagus and mAChR alterations are involved in EtOH induced changes in several organs. To see if EtOH-induced increases in lower esophageal sphincter pressure (LESP) are due to upregulation of mAChR, the authors evaluated mAChR binding and D-R curves for bethanechol (IV) induced increases in LESP, and compared these values to changes in LESP after acute and chronic EtOH. EtOH was given to cats acutely or chronically. The number of mAChR sites (Bmax) in esophagus was lowered by acute EtOH, withdrawal from chronic EtOH raised Bmax. Acute injection of EtOH to cats in withdrawal reversed this increase in mAChR density. These changes correlated with the earlier data on EtOH-induced changes in LESP. In contrast, the D-R curve for bethanechol shifted to the right. Thus, the withdrawal-associated increase in Bmax is more likely to be a compensatory response to deficits distal to the receptor recognition site than to proximal deficits and doesn't cause LESP hyperactivity. Also, receptor binding changes do not necessarily translate into physiological changes.

  2. Disentangling Viral Membrane Fusion from Receptor Binding Using Synthetic DNA-Lipid Conjugates.

    PubMed

    Rawle, Robert J; Boxer, Steven G; Kasson, Peter M

    2016-07-12

    Enveloped viruses must bind to a receptor on the host membrane to initiate infection. Membrane fusion is subsequently initiated by a conformational change in the viral fusion protein, triggered by receptor binding, an environmental change, or both. Here, we present a strategy to disentangle the two processes of receptor binding and fusion using synthetic DNA-lipid conjugates to bind enveloped viruses to target membranes in the absence of receptor. This permits direct testing of whether receptor engagement affects the fusion mechanism as well as a comparison of fusion behavior across viruses with different receptor binding specificities. We demonstrate this approach by binding X-31 influenza virus to target vesicles and measuring the rates of individual pH-triggered lipid mixing events using fluorescence microscopy. Influenza lipid mixing kinetics are found to be independent of receptor binding, supporting the common yet previously unproven assumption that receptor binding does not produce any clustering or spatial rearrangement of viral hemagglutinin, which affects the rate-limiting step of pH-triggered fusion. This DNA-lipid tethering strategy should also allow the study of viruses where challenging receptor reconstitution has previously prevented single-virus fusion experiments. PMID:27410740

  3. Disentangling Viral Membrane Fusion from Receptor Binding Using Synthetic DNA-Lipid Conjugates.

    PubMed

    Rawle, Robert J; Boxer, Steven G; Kasson, Peter M

    2016-07-12

    Enveloped viruses must bind to a receptor on the host membrane to initiate infection. Membrane fusion is subsequently initiated by a conformational change in the viral fusion protein, triggered by receptor binding, an environmental change, or both. Here, we present a strategy to disentangle the two processes of receptor binding and fusion using synthetic DNA-lipid conjugates to bind enveloped viruses to target membranes in the absence of receptor. This permits direct testing of whether receptor engagement affects the fusion mechanism as well as a comparison of fusion behavior across viruses with different receptor binding specificities. We demonstrate this approach by binding X-31 influenza virus to target vesicles and measuring the rates of individual pH-triggered lipid mixing events using fluorescence microscopy. Influenza lipid mixing kinetics are found to be independent of receptor binding, supporting the common yet previously unproven assumption that receptor binding does not produce any clustering or spatial rearrangement of viral hemagglutinin, which affects the rate-limiting step of pH-triggered fusion. This DNA-lipid tethering strategy should also allow the study of viruses where challenging receptor reconstitution has previously prevented single-virus fusion experiments.

  4. Endosulfan and cholinergic (muscarinic) transmission: effect on electroencephalograms and (/sup 3/H)quinuclidinyl benzilate in pigeon brain

    SciTech Connect

    Anand, M.; Agrawal, A.K.; Gopal, K.; Sur, R.N.; Seth, P.K.

    1986-08-01

    Single exposure of endosulfan (5 mg/kg) to pigeons (Columbia livia) caused neuronal hyperexcitability as evidence by spike discharges of 200-500 ..mu..V in the electroencephalograms (EEG) from the telencephalon and hyperstriatum, but there was not effect on the ectostriatal area. Cholinergic (muscarinic) receptor binding study using (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) as a specific ligand indicated that a single exposure to 5 mg/kg of endosulfan caused a significant increase in (/sup 3/H)QNB binding to the striatal membrane. Behavior study further indicated that a single dose of 200 ..mu..g/kg of oxotremorine produced a significant induction in the tremor in endosulfan-pretreated pigeons. The results of this behavioral and biochemical study indicate the involvement of a cholinergic (muscarinic) transmitter system in endosulfan-induced neurotoxicity.

  5. Chagas’ disease parasite-derived neurotrophic factor activates cholinergic gene expression in neuronal PC12 cells

    PubMed Central

    Akpan, Nsikan; Caradonna, Kacey; Chuenkova, Marina V.; PereiraPerrin, Mercio

    2008-01-01

    A parasite-derived neurotrophic factor (PDNF) produced by the Chagas’ disease parasite Trypanosoma cruzi binds nerve growth factor (NGF) receptor TrkA, increasing receptor autophosphorylation, activating phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK/Erk) pathways, and transcription factor CREB. The end-result is enhanced survival and neuritogenesis of various types of neurons. PDNF also enhances the expression and activity of tyrosine hydroxylase, a rate limiting enzyme in the synthesis of dopamine and other catecholamine neurotransmitters. It remains unknown, however, if PDNF alters expression and metabolism of acetylcholine (ACh), a neurotransmitter thought to play a role in Chagas’ disease progression. Here we demonstrate that PDNF stimulates mRNA and protein expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are critical for synthesis and storage of ACh. Stimulation requires functional TrkA because it did not occur in cell mutants that lack the receptor and in TrkA-expressing wild-type cells treated with K252a, an inhibitor of TrkA kinase activity. It also requires TrkA-dependent PI3K and MAPK/Erk signaling pathways because PDNF stimulation of cholinergic transcripts is abolished by specific pharmacological inhibitors. Furthermore, the cholinergic actions of PDNF were reproduced by PDNF-expressing extracellular T. cruzi trypomastigotes at the start of host cell invasion. In contrast, host cells bearing intracellular T. cruzi showed decreased, rather than increased, cholinergic gene expression. These results suggest that T. cruzi invasion of the nervous system alters cholinergic gene expression and that could play a role in neuropathology, and/or lack thereof, in Chagas’ disease patients. PMID:18502403

  6. Chagas' disease parasite-derived neurotrophic factor activates cholinergic gene expression in neuronal PC12 cells.

    PubMed

    Akpan, Nsikan; Caradonna, Kacey; Chuenkova, Marina V; PereiraPerrin, Mercio

    2008-06-27

    A parasite-derived neurotrophic factor (PDNF) produced by the Chagas' disease parasite Trypanosoma cruzi binds nerve growth factor (NGF) receptor TrkA, increasing receptor autophosphorylation, and activating phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK/Erk) pathways, and transcription factor CREB. The end-result is enhanced survival and neuritogenesis of various types of neurons. PDNF also enhances the expression and activity of tyrosine hydroxylase, a rate limiting enzyme in the synthesis of dopamine and other catecholamine neurotransmitters. It remains unknown, however, if PDNF alters expression and metabolism of acetylcholine (ACh), a neurotransmitter thought to play a role in Chagas' disease progression. Here we demonstrate that PDNF stimulates mRNA and protein expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are critical for synthesis and storage of ACh. Stimulation requires functional TrkA because it did not occur in cell mutants that lack the receptor and in TrkA-expressing wild-type cells treated with K252a, an inhibitor of TrkA kinase activity. It also requires TrkA-dependent PI3K and MAPK/Erk signaling pathways because PDNF stimulation of cholinergic transcripts is abolished by specific pharmacological inhibitors. Furthermore, the cholinergic actions of PDNF were reproduced by PDNF-expressing extracellular T. cruzi trypomastigotes at the start of host cell invasion. In contrast, host cells bearing intracellular T. cruzi showed decreased, rather than increased, cholinergic gene expression. These results suggest that T. cruzi invasion of the nervous system alters cholinergic gene expression and that could play a role in neuropathology, and/or lack thereof, in Chagas' disease patients. PMID:18502403

  7. Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization.

    PubMed

    Kuznetsova, Irina M; Sulatskaya, Anna I; Povarova, Olga I; Turoverov, Konstantin K

    2012-01-01

    In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.

  8. Modulation of glutamate transport and receptor binding by glutamate receptor antagonists in EAE rat brain.

    PubMed

    Sulkowski, Grzegorz; Dąbrowska-Bouta, Beata; Salińska, Elżbieta; Strużyńska, Lidia

    2014-01-01

    The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.

  9. Structure of human Aichi virus and implications for receptor binding.

    PubMed

    Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Kotecha, Abhay; Walter, Thomas S; Yuan, Shuai; Yamashita, Teruo; Tuthill, Tobias J; Fry, Elizabeth E; Rao, Zihe; Stuart, David I

    2016-01-01

    Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries(1,2). The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life(3,4). There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the β-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' β-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification. PMID:27595320

  10. THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS

    EPA Science Inventory

    THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS. JG Teeguarden1 and HA Barton2. 1ENVIRON International, Ruston LA; 2US EPA, ORD, NHEERL, ETD, Pharmacokinetics Branch, RTP, NC.

    One measure of th...

  11. Plasma binding proteins for platelet-derived growth factor that inhibit its binding to cell-surface receptors.

    PubMed Central

    Raines, E W; Bowen-Pope, D F; Ross, R

    1984-01-01

    Evidence is presented that the binding of platelet-derived growth factor (PDGF) to plasma constituents inhibits the binding of PDGF to its cell-surface mitogen receptor. Approximately equivalent amounts of PDGF-binding activity were found in plasma from a number of different species known by radioreceptor assay to contain PDGF homologues in their clotted blood. Activation of the coagulation cascade did not significantly alter the PDGF-binding activity of the plasma components. Three molecular weight classes of plasma fractions that inhibit PDGF binding to its cell-surface receptor were defined by gel filtration: approximately equal to 40,000, 150,000, and greater than 500,000. Specific binding of 125I-labeled PDGF to the highest molecular weight plasma fraction could also be demonstrated by gel filtration. The binding of PDGF to these plasma components was reversible under conditions of low pH or with guanidine X HCl, and active PDGF could be recovered from the higher molecular weight fractions. Immunologic and functional evidence is presented that the highest molecular weight plasma fraction may be alpha 2-macroglobulin. A model is proposed in which the activity of PDGF released in vivo may be regulated by association with these plasma binding components and by high-affinity binding to cell-surface PDGF receptors. PMID:6203121

  12. Substance P receptor binding sites are expressed by glia in vivo after neuronal injury

    SciTech Connect

    Mantyh, P.W.; Johnson, D.J.; Boehmer, C.G.; Catton, M.D.; Vinters, H.V.; Maggio, J.E.; Too, Hengphon; Vigna, S.R. )

    1989-07-01

    In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, the authors examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.

  13. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    PubMed

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms. PMID:25330347

  14. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    PubMed

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms.

  15. Competitive inhibition of (TH)dexamethasone binding to mammary glucocorticoid receptor by leupeptin

    SciTech Connect

    Hsieh, L.C.C.; Su, C.; Markland, F.S. Jr.

    1987-03-01

    The inhibitory effect of leupeptin on (TH)dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of (TH)dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of (TH)dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S).

  16. Bioluminescent Ligand-Receptor Binding Assays for Protein or Peptide Hormones.

    PubMed

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    Bioluminescence has been widely used in biomedical research due to its high sensitivity, low background, and broad linear range. In recent studies, we applied bioluminescence to ligand-receptor binding assays for some protein or peptide hormones based on a newly developed small monomeric Nanoluciferase (NanoLuc) reporter that has the so far brightest bioluminescence. The conventional ligand-receptor binding assays rely on radioligands that have drawbacks, such as radioactive hazards and short shelf lives. In contrast, the novel bioluminescent binding assays use the NanoLuc-based protein or peptide tracers that are safe, stable, and ultrasensitive. Thus, the novel bioluminescent ligand-receptor binding assay would be applied to more and more protein or peptide hormones for ligand-receptor interaction studies in future. In the present article, we provided detailed protocols for setting up the novel bioluminescent ligand-receptor binding assays using two representative protein hormones as examples. PMID:27424896

  17. Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function

    SciTech Connect

    Medici, N.; Minucci, S.; Nigro, V.; Abbondanza, C.; Armetta, I.; Molinari, A.M.; Puca, G.A. )

    1989-01-10

    The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Analysis of affinity-labeled ER by ({sup 3}H)tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentration. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors.

  18. Estrogen-Cholinergic Interactions: Implications for Cognitive Aging

    PubMed Central

    Newhouse, Paul; Dumas, Julie

    2015-01-01

    While many studies in humans have investigated the effects of estrogen and hormone therapy on cognition, potential neurobiological correlates of these effects have been less well studied. An important site of action for estrogen in the brain is the cholinergic system. Several decades of research support the critical role of CNS cholinergic systems in cognition in humans, particularly in learning and memory formation and attention. In humans, the cholinergic system has been implicated in many aspects of cognition including the partitioning of attentional resources, working memory, inhibition of irrelevant information, and improved performance on effort-demanding tasks. Studies support the hypothesis that estradiol helps to maintain aspects of attention and verbal and visual memory. Such cognitive domains are exactly those modulated by cholinergic systems and extensive basic and preclinical work over the past several decades has clearly shown that basal forebrain cholinergic systems are dependent on estradiol support for adequate functioning. This paper will review recent human studies from our laboratories and others that have extended preclinical research examining estrogen-cholinergic interactions to humans. Studies examined include estradiol and cholinergic antagonist reversal studies in normal older women, examinations of the neural representations of estrogen-cholinergic interactions using functional brain imaging, and studies of the ability of selective estrogen receptor modulators such as tamoxifen to interact with cholinergic-mediated cognitive performance. We also discuss the implications of these studies for the underlying hypotheses of cholinergic-estrogen interactions and cognitive aging, and indications for prophylactic and therapeutic potential that may exploit these effects. PMID:26187712

  19. Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors

    PubMed Central

    Koshimizu, Taka-aki; Kashiwazaki, Aki; Taniguchi, Junichi

    2016-01-01

    Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

  20. Detection of persistent organic pollutants binding modes with androgen receptor ligand binding domain by docking and molecular dynamics

    PubMed Central

    2013-01-01

    Background Persistent organic pollutants (POPs) are persistent in the environment after release from industrial compounds, combustion productions or pesticides. The exposure of POPs has been related to various reproductive disturbances, such as reduced semen quality, testicular cancer, and imbalanced sex ratio. Among POPs, dichlorodiphenyldichloroethylene (4,4’-DDE) and polychlorinated biphenyls (PCBs) are the most widespread and well-studied compounds. Recent studies have revealed that 4,4’-DDE is an antagonist of androgen receptor (AR). However, the mechanism of the inhibition remains elusive. CB-153 is the most common congener of PCBs, while the action of CB-153 on AR is still under debate. Results Molecular docking and molecular dynamics (MD) approaches have been employed to study binding modes and inhibition mechanism of 4,4’-DDE and CB-153 against AR ligand binding domain (LBD). Several potential binding sites have been detected and analyzed. One possible binding site is the same binding site of AR natural ligand androgen 5α-dihydrotestosterone (DHT). Another one is on the ligand-dependent transcriptional activation function (AF2) region, which is crucial for the co-activators recruitment. Besides, a novel possible binding site was observed for POPs with low binding free energy with the receptor. Detailed interactions between ligands and the receptor have been represented. The disrupting mechanism of POPs against AR has also been discussed. Conclusions POPs disrupt the function of AR through binding to three possible biding sites on AR/LBD. One of them shares the same binding site of natural ligand of AR. Another one is on AF2 region. The third one is in a cleft near N-terminal of the receptor. Significantly, values of binding free energy of POPs with AR/LBD are comparable to that of natural ligand androgen DHT. PMID:24053684

  1. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
    MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
    U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
    Typically, in vitro hazard assessments for ...

  2. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
    Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  3. Treatment of beta amyloid 1–42 (Aβ1–42)-induced basal forebrain cholinergic damage by a non-classical estrogen signaling activator in vivo

    PubMed Central

    Kwakowsky, Andrea; Potapov, Kyoko; Kim, SooHyun; Peppercorn, Katie; Tate, Warren P.; Ábrahám, István M.

    2016-01-01

    In Alzheimer’s disease (AD), there is a loss in cholinergic innervation targets of basal forebrain which has been implicated in substantial cognitive decline. Amyloid beta peptide (Aβ1–42) accumulates in AD that is highly toxic for basal forebrain cholinergic (BFC) neurons. Although the gonadal steroid estradiol is neuroprotective, the administration is associated with risk of off-target effects. Previous findings suggested that non-classical estradiol action on intracellular signaling pathways has ameliorative potential without estrogenic side effects. After Aβ1–42 injection into mouse basal forebrain, a single dose of 4-estren-3α, 17β-diol (estren), the non-classical estradiol pathway activator, restored loss of cholinergic cortical projections and also attenuated the Aβ1–42-induced learning deficits. Estren rapidly and directly phosphorylates c-AMP-response–element-binding-protein and extracellular-signal-regulated-kinase-1/2 in BFC neurons and restores the cholinergic fibers via estrogen receptor-α. These findings indicated that selective activation of non-classical intracellular estrogen signaling has a potential to treat the damage of cholinergic neurons in AD. PMID:26879842

  4. Impact of D2 Receptor Internalization on Binding Affinity of Neuroimaging Radiotracers

    PubMed Central

    Guo, Ningning; Guo, Wen; Kralikova, Michaela; Jiang, Man; Schieren, Ira; Narendran, Raj; Slifstein, Mark; Abi-Dargham, Anissa; Laruelle, Marc; Javitch, Jonathan A; Rayport, Stephen

    2010-01-01

    Synaptic dopamine (DA) levels seem to affect the in vivo binding of many D2 receptor radioligands. Thus, release of endogenous DA induced by the administration of amphetamine decreases ligand binding, whereas DA depletion increases binding. This is generally thought to be due to competition between endogenous DA and the radioligands for D2 receptors. However, the temporal discrepancy between amphetamine-induced increases in DA as measured by microdialysis, which last on the order of 2 h, and the prolonged decrease in ligand binding, which lasts up to a day, has suggested that agonist-induced D2 receptor internalization may contribute to the sustained decrease in D2 receptor-binding potential seen following a DA surge. To test this hypothesis, we developed an in vitro system showing robust agonist-induced D2 receptor internalization following treatment with the agonist quinpirole. Human embryonic kidney 293 (HEK293) cells were stably co-transfected with human D2 receptor, G-protein-coupled receptor kinase 2 and arrestin 3. Agonist-induced D2 receptor internalization was demonstrated by fluorescence microscopy, flow cytometry, and radioligand competition binding. The binding of seven D2 antagonists and four agonists to the surface and internalized receptors was measured in intact cells. All the imaging ligands bound with high affinity to both surface and internalized D2 receptors. Affinity of most of the ligands to internalized receptors was modestly lower, indicating that internalization would reduce the binding potential measured in imaging studies carried out with these ligands. However, between-ligand differences in the magnitude of the internalization-associated affinity shift only partly accounted for the data obtained in neuroimaging experiments, suggesting the involvement of mechanisms beyond competition and internalization. PMID:19956086

  5. Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

    PubMed

    Bittoun, P; Bagheri-Yarmand, R; Chaubet, F; Crépin, M; Jozefonvicz, J; Fermandjian, S

    1999-06-15

    CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

  6. G Protein-Coupled Receptor Heteromerization: A Role in Allosteric Modulation of Ligand BindingS⃞

    PubMed Central

    Gomes, Ivone; IJzerman, Adriaan P.; Ye, Kai; Maillet, Emeline L.

    2011-01-01

    It is becoming increasingly recognized that G protein-coupled receptors physically interact. These interactions may provide a mechanism for allosteric modulation of receptor function. In this study, we examined this possibility by using an established model system of a receptor heteromer consisting of μ and δ opioid receptors. We examined the effect of a number of μ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled δ receptor agonist, [3H]deltorphin II. We also examined the effect of δ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled μ receptor agonist, [3H][d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin ([3H]DAMGO). We show that μ receptor ligands are capable of allosterically enhancing δ receptor radioligand binding and vice versa. Thus, there is strong positive cooperativity between the two receptor units with remarkable consequences for ligand pharmacology. We find that the data can be simulated by adapting an allosteric receptor model previously developed for small molecules, suggesting that the ligand-occupied protomers function as allosteric modulators of the partner receptor's activity. PMID:21415307

  7. Evidence for a vasopressin receptor-GTP binding protein complex

    SciTech Connect

    Fitzgerald, T.J.; Uhing, R.J.; Exton, J.H.

    1986-05-01

    Plasma membranes from the livers of rats were able to hydrolyze the ..gamma..-phosphate from guanosine-5'-triphosphate (GTP). The rate of GTP hydrolysis could be decreased to 10% of its initial rate by the addition of adenosine-5'-triphosphate with a concomitant decrease in the K/sub m/ for GTP from approx. 10/sup -3/ M to 10/sup -6/ M. The low K/sub m/ GTPase activity was inhibited by the addition of nonhydrolyzable analogs of GTP. In addition, the GTPase activity was stimulated from 10 to 30% over basal by the addition of vasopressin. A dose dependency curve showed that the maximum stimulation was obtained with 10/sup -8/ M vasopressin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized in the presence of (Phenylalanyl-3,4,5-/sup 3/H(N))vasopressin were subjected to sucrose gradient centrifugation, the majority of bound (/sup 3/H)vasopressin migrated with an approximate molecular weight of 300,000. Moreover, there was a GTPase activity that migrated with the bound (/sup 3/H)vasopressin. This peak of bound (/sup 3/H)vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10/sup -5/ M guanosine-5'-O-(3-thiotriphosphate). These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

  8. Growth hormone receptor/binding protein: Physiology and function

    SciTech Connect

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P.

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

  9. Effects of mescaline and some of its analogs on cholinergic neuromuscular transmission.

    PubMed

    Ghansah, E; Kopsombut, P; Malleque, M A; Brossi, A

    1993-02-01

    Mescaline (3,4,5-trimethoxyphenylethylamine; MES) and its analogs, anhalinine (ANH) and methylenemescaline trimer (MMT) were investigated, using sciatic-sartorius preparations of the frog and cortical tissue from the rat. The effects of MES and its analogs were examined with respect to muscle twitch, resting membrane potential and nicotinic receptor binding. Mescaline and its analogs (10-100 microM) blocked both directly and neurally evoked twitches but their effects on neurally evoked twitches were greater than those on directly evoked twitches. Mescaline, ANH and MMT decreased amplitude of the miniature endplate and endplate potentials, decreased acetylcholine (ACh) quantal content, hyperpolarized the resting membrane potential and prolonged duration of the action potential. They did not significantly displace the binding of [125I]-alpha-bungarotoxin (alpha-BTX) to nicotinic receptors, at concentrations which blocked neuromuscular transmission. These results suggest that MES and its analogs inhibit cholinergic neuromuscular transmission by blocking release of ACh; they also affect K+ conductance.

  10. Neurotensin receptor binding levels in basal ganglia are not altered in Huntington's chorea or schizophrenia

    SciTech Connect

    Palacios, J.M.; Chinaglia, G.; Rigo, M.; Ulrich, J.; Probst, A. )

    1991-02-01

    Autoradiographic techniques were used to examine the distribution and levels of neurotensin receptor binding sites in the basal ganglia and related regions of the human brain. Monoiodo ({sup 125}I-Tyr3)neurotensin was used as a ligand. High amounts of neurotensin receptor binding sites were found in the substantia nigra pars compacta. Lower but significant quantities of neurotensin receptor binding sites characterized the caudate, putamen, and nucleus accumbens, while very low quantities were seen in both medial and lateral segments of the globus pallidus. In Huntington's chorea, the levels of neurotensin receptor binding sites were found to be comparable to those of control cases. Only slight but not statistically significant decreases in amounts of receptor binding sites were detected in the dorsal part of the head and in the body of caudate nucleus. No alterations in the levels of neurotensin receptor binding sites were observed in the substantia nigra pars compacta and reticulata. These results suggest that a large proportion of neurotensin receptor binding sites in the basal ganglia are located on intrinsic neurons and on extrinsic afferent fibers that do not degenerate in Huntington's disease.

  11. Effects of vitamin B-6 nutrition on benzodiazepine (BDZ) receptor binding in the developing rat brain

    SciTech Connect

    Borek, J.P.; Guilarte, T.R. )

    1990-02-26

    A dietary deficiency of vitamin B-6 promotes seizure activity in neonatal animals and human infants. Previous studied have shown that neonatal vitamin B-6 deprivation results in reduced levels of brain gamma-aminobutyric acid (GABA) and increased binding at the GABA site of the GABA/BDZ receptor complex. Since the GABA and BDZ receptors are allosterically linked, this study was undertaken to determine if vitamin B-6 deprivation had an effect on BDZ receptor binding. Benzodiazepine receptor binding isotherms using {sup 3}H-flunitrazepam as ligand were performed in the presence and absence of 10 {mu}M GABA. The results indicate a significant increase in the binding affinity (Kd) in the presence of GABA in cerebellar membranes from deficient rat pups at 14 days of age with no effect on receptor number (Bmax). By 28 days of age, the increase in Kd was no longer present. No change in Kd or Bmax was observed in cortical tissue from deficient animals at 14 or 28 days of age. Preliminary studies of GABA-enhancement of {sup 3}H-flunitrazepam binding indicate that vitamin B-6 deficiency also induces alterations in the ability of GABA to enhance BZD receptor binding. In summary, these results indicate that the effects of vitamin B-6 deprivation on BDZ receptor binding are region specific and age related.

  12. Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers.

    PubMed

    Zoenen, Maxime; Urizar, Eneko; Swillens, Stéphane; Vassart, Gilbert; Costagliola, Sabine

    2012-01-01

    Glycoprotein hormone receptors show strong negative cooperativity. As a consequence, at physiological hormone concentrations, a single agonist binds to a receptor dimer. Here we present evidence that constitutively active receptors lose cooperative allosteric regulation in direct relation with their basal activity. The most constitutive mutants lost nearly all cooperativity and showed an increase of initial tracer binding, reflecting the ability of each protomer to bind with equal affinity. Allosteric interaction between the protomers takes place at the transmembrane domain. The allosteric message resulting from hormone binding to the ectodomain of one protomer travels 'downward' to its transmembrane domain, before affecting the transmembrane domain of the other protomer. This results in transmission of an 'upward' message lowering the binding affinity of the ectodomain of the second protomer. Our results demonstrate a direct relation between the conformational changes associated with activation of the transmembrane domain and the allosteric behaviour of glycoprotein hormone receptors dimers.

  13. Stacking interaction and its role in kynurenic acid binding to glutamate ionotropic receptors.

    PubMed

    Zhuravlev, Alexander V; Zakharov, Gennady A; Shchegolev, Boris F; Savvateeva-Popova, Elena V

    2012-05-01

    Stacking interaction is known to play an important role in protein folding, enzyme-substrate and ligand-receptor complex formation. It has been shown to make a contribution into the aromatic antagonists binding with glutamate ionotropic receptors (iGluRs), in particular, the complex of NMDA receptor NR1 subunit with the kynurenic acid (KYNA) derivatives. The specificity of KYNA binding to the glutamate receptors subtypes might partially result from the differences in stacking interaction. We have calculated the optimal geometry and binding energy of KYNA dimers with the four types of aromatic amino acid residues in Rattus and Drosophila ionotropic iGluR subunits. All ab initio quantum chemical calculations were performed taking into account electron correlations at MP2 and MP4 perturbation theory levels. We have also investigated the potential energy surfaces (PES) of stacking and hydrogen bonds (HBs) within the receptor binding site and calculated the free energy of the ligand-receptor complex formation. The energy of stacking interaction depends both on the size of aromatic moieties and the electrostatic effects. The distribution of charges was shown to determine the geometry of polar aromatic ring dimers. Presumably, stacking interaction is important at the first stage of ligand binding when HBs are weak. The freedom of ligand movements and rotation within receptor site provides the precise tuning of the HBs pattern, while the incorrect stacking binding prohibits the ligand-receptor complex formation. PMID:21833825

  14. Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

    SciTech Connect

    Lee, J.H.; el-Fakahany, E.E.

    1985-06-01

    The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/sup 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.

  15. The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

    PubMed Central

    Rajendra, S; Vandenberg, R J; Pierce, K D; Cunningham, A M; French, P W; Barry, P H; Schofield, P R

    1995-01-01

    A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily. Images PMID:7621814

  16. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    SciTech Connect

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H. )

    1990-04-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

  17. Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4

    PubMed Central

    Jen, Angela; Parkyn, Celia J.; Mootoosamy, Roy C.; Ford, Melanie J.; Warley, Alice; Liu, Qiang; Bu, Guojun; Baskakov, Ilia V.; Moestrup, Søren; McGuinness, Lindsay; Emptage, Nigel; Morris, Roger J.

    2010-01-01

    For infectious prion protein (designated PrPSc) to act as a template to convert normal cellular protein (PrPC) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrPC is the low-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor clusters 2 and 4, PrPC and PrPSc fibrils bind only to receptor cluster 4. PrPSc fibrils out-compete PrPC for internalization. When endocytosed, PrPSc fibrils are routed to lysosomes, rather than recycled to the cell surface with PrPC. Thus, although LRP1 binds both forms of PrP, it traffics them to separate fates within sensory neurons. The binding of both to ligand cluster 4 should enable genetic modification of PrP binding without disrupting other roles of LRP1 essential to neuronal viability and function, thereby enabling in vivo analysis of the role of this interaction in controlling both prion and LRP1 biology. PMID:20048341

  18. Dye-labeled benzodiazepines: development of small ligands for receptor binding studies using fluorescence correlation spectroscopy.

    PubMed

    Hegener, Oliver; Jordan, Randolf; Häberlein, Hanns

    2004-07-01

    To investigate benzodiazepine receptor binding studies by fluorescence correlation spectroscopy (FCS), the four fluorophores fluorescein, tetramethylrhodamine, Oregon Green 488, and Alexa 532 were coupled to the benzodiazepine Ro 07-1986/602 (Ro). Binding assays to polyclonal antibodies to benzodiazepines and at the native benzodiazepine receptor on the membrane of rat hippocampal neurons were established to examine the dye-labeled ligands for their benzodiazepine character and their binding behavior. Both the fluorescein and the Oregon Green488 moiety led to a loss of the benzodiazepine receptor binding of the corresponding Ro derivatives. Antibody recognition and interactions to the receptor were observed for the tetramethylrhodamine derivative (K(D) = 96.0 +/- 9.5 nM) but with a high amount of nonspecific binding at the cell membrane of about 50%. In saturation experiments a K(D) value of 97.2 +/- 8.5 nM was found for the Alexa Fluor 532 derivative-antibody interaction. Investigation of the binding of this ligand to the benzodiazepine receptor in FCS cell measurements led to confirmation of high specific binding behavior with a K(D) value of 9.9 +/- 1.9 nM. A nonspecific binding of <10% was observed after coincubation with 1 microM of midazolam. The different properties of the labeled benzodiazepine derivatives and the requirements of the fluorophore in small dye-labeled ligands in FCS binding studies, at the membrane of living cells, are discussed.

  19. Cholinergic dysfunction in Parkinson's disease.

    PubMed

    Müller, Martijn L T M; Bohnen, Nicolaas I

    2013-09-01

    There is increasing interest in the clinical effects of cholinergic basal forebrain and tegmental pedunculopontine complex (PPN) projection degeneration in Parkinson's disease (PD). Recent evidence supports an expanded role beyond cognitive impairment, including effects on olfaction, mood, REM sleep behavior disorder, and motor functions. Cholinergic denervation is variable in PD without dementia and may contribute to clinical symptom heterogeneity. Early in vivo imaging evidence that impaired cholinergic integrity of the PPN associates with frequent falling in PD is now confirmed by human post-mortem evidence. Brainstem cholinergic lesioning studies in primates confirm the role of the PPN in mobility impairment. Degeneration of basal forebrain cholinergic projections correlates with decreased walking speed. Cumulatively, these findings provide evidence for a new paradigm to explain dopamine-resistant features of mobility impairments in PD. Recognition of the increased clinical role of cholinergic system degeneration may motivate new research to expand indications for cholinergic therapy in PD. PMID:23943367

  20. Binding of type II nuclear receptors and estrogen receptor to full and half-site estrogen response elements in vitro.

    PubMed Central

    Klinge, C M; Bodenner, D L; Desai, D; Niles, R M; Traish, A M

    1997-01-01

    The mechanism by which retinoids, thyroid hormone (T3) and estrogens modulate the growth of breast cancer cells is unclear. Since nuclear type II nuclear receptors, including retinoic acid receptor (RAR), retinoid X receptor (RXR) and thyroid hormone receptor (TR), bind direct repeats (DR) of the estrogen response elements (ERE) half-site (5'-AGGTCA-3'), we examined the ability of estrogen receptor (ER) versus type II nuclear receptors, i.e. RARalpha, beta and gamma, RXRbeta, TRalpha and TRbeta, to bind various EREs in vitro . ER bound a consensus ERE, containing a perfectly palindromic 17 bp inverted repeat (IR), as a homodimer. In contrast, ER did not bind to a single ERE half-site. Likewise, ER did not bind two tandem (38 bp apart) half-sites, but low ER binding was detected to three tandem copies of the same half-site. RARalpha,beta or gamma bound both ERE and half-site constructs as a homodimer. RXRbeta did not bind full or half-site EREs, nor did RXRbeta enhance RARalpha binding to a full ERE. However, RARalpha and RXRbeta bound a half-site ERE cooperatively forming a dimeric complex. The RARalpha-RXRbeta heterodimer bound the Xenopus vitellogenin B1 estrogen responsive unit, with two non-consensus EREs, with higher affinity than one or two copies of the full or half-site ERE. Both TRalpha and TRbeta bound the full and the half-site ERE as monomers and homodimers and cooperatively as heterodimers with RXRbeta. We suggest that the cellular concentrations of nuclear receptors and their ligands, and the nature of the ERE or half-site sequence and those of its flanking sequences determine the occupation of EREs in estrogen-regulated genes in vivo . PMID:9115356

  1. DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

    SciTech Connect

    Cuthill, S.; Poellinger, L.

    1988-04-19

    The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant (/sup 3/H)dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin.

  2. Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

    PubMed

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

    2012-12-01

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

  3. The effect of maternal smoking and drinking during pregnancy upon (3)H-nicotine receptor brainstem binding in infants dying of the sudden infant death syndrome: initial observations in a high risk population.

    PubMed

    Duncan, Jhodie R; Randall, Leslie L; Belliveau, Richard A; Trachtenberg, Felicia L; Randall, Bradley; Habbe, Donald; Mandell, Federick; Welty, Thomas K; Iyasu, Solomon; Kinney, Hannah C

    2008-01-01

    The high rate of the sudden infant death syndrome (SIDS) in American Indians in the Northern Plains (3.5/1000) may reflect the high incidence of cigarette smoking and alcohol consumption during pregnancy. Nicotine, a neurotoxic component of cigarettes, and alcohol adversely affect nicotinic receptor binding and subsequent cholinergic development in animals. We measured (3)H-nicotine receptor binding in 16 brainstem nuclei in American Indian SIDS (n = 27) and controls (n = 6). In five nuclei related to cardiorespiratory control, (3)H-nicotinic binding decreased with increasing number of drinks (P < 0.03). There were no differences in binding in SIDS compared with controls, except upon stratification of prenatal exposures. In three mesopontine nuclei critical for arousal there were reductions (P < 0.04) in binding in controls exposed to cigarette smoke compared with controls without exposure; there was no difference between SIDS cases with or without exposure. This study suggests that maternal smoking and alcohol affects (3)H-nicotinic binding in the infant brainstem irrespective of the cause of death. It also suggests that SIDS cases are unable to respond to maternal smoking with the "normal" reduction seen in controls. Future studies are needed to establish the role of adverse prenatal exposures in altered brainstem neurochemistry in SIDS. PMID:17924983

  4. The aging human cochlear nucleus: Changes in the glial fibrillary acidic protein, intracellular calcium regulatory proteins, GABA neurotransmitter and cholinergic receptor.

    PubMed

    Sharma, Saroj; Nag, Tapas C; Thakar, Alok; Bhardwaj, Daya N; Roy, Tara Sankar

    2014-03-01

    The human auditory system is highly susceptible to environmental and metabolic insults which further affect the biochemical and physiological milieu of the cells that may contribute to progressive, hearing loss with aging. The cochlear nucleus (CN) is populated by morphologically diverse types of neurons with discrete physiological and neurochemical properties. Between the dorsal and the ventral cochlear nucleus (DCN and VCN), the VCN is further sub-divided into the rostral (rVCN) and caudal (cVCN) sub-divisions. Although, information is available on the age related neurochemical changes in the mammalian CN similar reports on human CN is still sparse. The morphometry and semiquantitative analysis of intensity of expression of glial fibrillary acidic protein (GFAP), calcium binding proteins (calbindin, calretinin and parvalbumin), gamma amino butyric acid (GABA) and nicotinic acetyl choline receptor (nAchR) beta 2 immunostaining were carried out in all three sub-divisions of the human CN from birth to 90 years. There was increased GFAP immunoreactivity in decades 2 and 3 in comparison to decade 1 in the CN. But no change was observed in rVCN from decade 4 onwards, whereas intense staining was also observed in decades 5 and 6 in cVCN and DCN. All three calcium binding proteins were highly expressed in early to middle ages, whereas a significant reduction was found in later decades in the VCN. GABA and nAchR beta 2 expressions were unchanged throughout in all the decades. The middle age may represent a critical period of onset and progression of aging changes in the CN and these alterations may add to the deterioration of hearing responses in the old age.

  5. The aging human cochlear nucleus: Changes in the glial fibrillary acidic protein, intracellular calcium regulatory proteins, GABA neurotransmitter and cholinergic receptor.

    PubMed

    Sharma, Saroj; Nag, Tapas C; Thakar, Alok; Bhardwaj, Daya N; Roy, Tara Sankar

    2014-03-01

    The human auditory system is highly susceptible to environmental and metabolic insults which further affect the biochemical and physiological milieu of the cells that may contribute to progressive, hearing loss with aging. The cochlear nucleus (CN) is populated by morphologically diverse types of neurons with discrete physiological and neurochemical properties. Between the dorsal and the ventral cochlear nucleus (DCN and VCN), the VCN is further sub-divided into the rostral (rVCN) and caudal (cVCN) sub-divisions. Although, information is available on the age related neurochemical changes in the mammalian CN similar reports on human CN is still sparse. The morphometry and semiquantitative analysis of intensity of expression of glial fibrillary acidic protein (GFAP), calcium binding proteins (calbindin, calretinin and parvalbumin), gamma amino butyric acid (GABA) and nicotinic acetyl choline receptor (nAchR) beta 2 immunostaining were carried out in all three sub-divisions of the human CN from birth to 90 years. There was increased GFAP immunoreactivity in decades 2 and 3 in comparison to decade 1 in the CN. But no change was observed in rVCN from decade 4 onwards, whereas intense staining was also observed in decades 5 and 6 in cVCN and DCN. All three calcium binding proteins were highly expressed in early to middle ages, whereas a significant reduction was found in later decades in the VCN. GABA and nAchR beta 2 expressions were unchanged throughout in all the decades. The middle age may represent a critical period of onset and progression of aging changes in the CN and these alterations may add to the deterioration of hearing responses in the old age. PMID:24412669

  6. Adaptation of avian influenza A (H6N1) virus from avian to human receptor-binding preference.

    PubMed

    Wang, Fei; Qi, Jianxun; Bi, Yuhai; Zhang, Wei; Wang, Min; Zhang, Baorong; Wang, Ming; Liu, Jinhua; Yan, Jinghua; Shi, Yi; Gao, George F

    2015-06-12

    The receptor-binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor-binding properties of Taiwan-isolated H6 HAs from 1972 to 2013. We propose that the receptor-binding properties of Taiwan-isolated H6 HAs have undergone three major stages: initially avian receptor-binding preference, secondarily obtaining human receptor-binding capacity, and recently human receptor-binding preference, which has been confirmed by receptor-binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor-binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor-binding site of HA determines the receptor-binding preference change. We conclude that the human-infecting H6N1 evolved into a human receptor preference.

  7. Mu receptor binding of some commonly used opioids and their metabolites

    SciTech Connect

    Chen, Zhaorong; Irvine, R.J. ); Somogyi, A.A.; Bochner, F. Royal Adelaide Hospital )

    1991-01-01

    The binding affinity to the {mu} receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with {sup 3}H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding. Decreasing the length of the alkyl group at position 3 decreased the K{sub i} values (morphine < codeine < ethylmorphine < pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 had relatively weak receptor binding, while their O-demethylated metabolites had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites.

  8. Measuring relative acetylcholine receptor agonist binding by selective proton nuclear magnetic resonance relaxation experiments.

    PubMed Central

    Behling, R W; Yamane, T; Navon, G; Sammon, M J; Jelinski, L W

    1988-01-01

    A method is presented that uses selective proton Nuclear Magnetic Resonance (NMR) relaxation measurements of nicotine in the presence of the acetylcholine receptor to obtain relative binding constants for acetylcholine, carbamylcholine, and muscarine. For receptors from Torpedo californica the results show that (a) the binding constants are in the order acetylcholine greater than nicotine greater than carbamylcholine greater than muscarine; (b) selective NMR measurements provide a rapid and direct method for monitoring both the specific and nonspecific binding of agonists to these receptors and to the lipid; (c) alpha-bungarotoxin can be used to distinguish between specific and nonspecific binding to the receptor; (d) the receptor--substrate interaction causes a large change in the selective relaxation time of the agonists even at concentrations 100x greater than that of the receptor. This last observation means that these measurements provide a rapid method to monitor drug binding when only small amounts of receptor are available. Furthermore, the binding strategies presented here may be useful for the NMR determination of the conformation of the ligand in its bound state. Images FIGURE 1 PMID:3395661

  9. Corelease of acetylcholine and GABA from cholinergic forebrain neurons

    PubMed Central

    Saunders, Arpiar; Granger, Adam J; Sabatini, Bernardo L

    2015-01-01

    Neurotransmitter corelease is emerging as a common theme of central neuromodulatory systems. Though corelease of glutamate or GABA with acetylcholine has been reported within the cholinergic system, the full extent is unknown. To explore synaptic signaling of cholinergic forebrain neurons, we activated choline acetyltransferase expressing neurons using channelrhodopsin while recording post-synaptic currents (PSCs) in layer 1 interneurons. Surprisingly, we observed PSCs mediated by GABAA receptors in addition to nicotinic acetylcholine receptors. Based on PSC latency and pharmacological sensitivity, our results suggest monosynaptic release of both GABA and ACh. Anatomical analysis showed that forebrain cholinergic neurons express the GABA synthetic enzyme Gad2 and the vesicular GABA transporter (Slc32a1). We confirmed the direct release of GABA by knocking out Slc32a1 from cholinergic neurons. Our results identify GABA as an overlooked fast neurotransmitter utilized throughout the forebrain cholinergic system. GABA/ACh corelease may have major implications for modulation of cortical function by cholinergic neurons. DOI: http://dx.doi.org/10.7554/eLife.06412.001 PMID:25723967

  10. An analysis of the binding of the chick oviduct progesterone-receptor to chromatin.

    PubMed

    Jaffe, R C; Socher, S H; O'Malley, B W

    1975-08-13

    The binding of progesterone-receptor complexes to chromatin from target and nontarget tissues was studied in vitro. Chromatin from both target and nontarget tissues responds in a similar manner to saly and cofactors and has the same K(D) (approx. 3.10(-9) M) for the progesterone-receptor complex. The only observed difference in the binding of the progesterone-receptor complex to target and nontarget chromatins is the difference in total number of acceptor sites. oviduct chromatin has approx. 1300 sites/pg DNA, spleen chromatin has approx. 840 sites/pg DNA, and erythrocyte chromatin has about 330 sites/pg DNA. The K(D) and number of acceptor sites for progesterone-receptor complex binding to oviduct chromatin remains the same even after extensive purification of the progesterone-receptor complex. Activation of cytosol labeled with [3H]progesterone by preincubation at 25 degrees C, analogous to that required for maximal nuclear binding, occurs if the binding studies to chromatin are performed in 0.025 M salt. The absence of an observable temperature effect when the studies are performed at 0.15 M salt is due to the activation of the receptor by salt. The dissociation of the progesterone-receptor complex from chromatin exhibits a single dissociation rate and the initial event is the appearance of free progesterone rather than a progesterone-receptor complex. Lastly, the treatment of chromatin with an antibody prepared against either single-stranded DNA or double-stranded DNA does not alter the extent of binding of the progesterone-receptor complex. Similarly, pretreatment of chromatin with a single-stranded nuclease does not inhibit the capacity of chromatin to bind the hormone-receptor complex.

  11. Analyzing machupo virus-receptor binding by molecular dynamics simulations

    PubMed Central

    Sawyer, Sara L.; Ellington, Andrew D.; Wilke, Claus O.

    2014-01-01

    In many biological applications, we would like to be able to computationally predict mutational effects on affinity in protein–protein interactions. However, many commonly used methods to predict these effects perform poorly in important test cases. In particular, the effects of multiple mutations, non alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here an existing method applied in a novel way to a new test case; we interrogate affinity differences resulting from mutations in a host–virus protein–protein interface. We use steered molecular dynamics (SMD) to computationally pull the machupo virus (MACV) spike glycoprotein (GP1) away from the human transferrin receptor (hTfR1). We then approximate affinity using the maximum applied force of separation and the area under the force-versus-distance curve. We find, even without the rigor and planning required for free energy calculations, that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Moreover, we show that this simple SMD scheme correlates well with relative free energy differences computed via free energy perturbation. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our approach provides a framework to compare the effects of multiple mutations, individually and jointly, on protein–protein interactions. PMID:24624315

  12. Analyzing machupo virus-receptor binding by molecular dynamics simulations.

    PubMed

    Meyer, Austin G; Sawyer, Sara L; Ellington, Andrew D; Wilke, Claus O

    2014-01-01

    In many biological applications, we would like to be able to computationally predict mutational effects on affinity in protein-protein interactions. However, many commonly used methods to predict these effects perform poorly in important test cases. In particular, the effects of multiple mutations, non alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here an existing method applied in a novel way to a new test case; we interrogate affinity differences resulting from mutations in a host-virus protein-protein interface. We use steered molecular dynamics (SMD) to computationally pull the machupo virus (MACV) spike glycoprotein (GP1) away from the human transferrin receptor (hTfR1). We then approximate affinity using the maximum applied force of separation and the area under the force-versus-distance curve. We find, even without the rigor and planning required for free energy calculations, that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Moreover, we show that this simple SMD scheme correlates well with relative free energy differences computed via free energy perturbation. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our approach provides a framework to compare the effects of multiple mutations, individually and jointly, on protein-protein interactions.

  13. Striatal cholinergic interneurons Drive GABA release from dopamine terminals.

    PubMed

    Nelson, Alexandra B; Hammack, Nora; Yang, Cindy F; Shah, Nirao M; Seal, Rebecca P; Kreitzer, Anatol C

    2014-04-01

    Striatal cholinergic interneurons are implicated in motor control, associative plasticity, and reward-dependent learning. Synchronous activation of cholinergic interneurons triggers large inhibitory synaptic currents in dorsal striatal projection neurons, providing one potential substrate for control of striatal output, but the mechanism for these GABAergic currents is not fully understood. Using optogenetics and whole-cell recordings in brain slices, we find that a large component of these inhibitory responses derive from action-potential-independent disynaptic neurotransmission mediated by nicotinic receptors. Cholinergically driven IPSCs were not affected by ablation of striatal fast-spiking interneurons but were greatly reduced after acute treatment with vesicular monoamine transport inhibitors or selective destruction of dopamine terminals with 6-hydroxydopamine, indicating that GABA release originated from dopamine terminals. These results delineate a mechanism in which striatal cholinergic interneurons can co-opt dopamine terminals to drive GABA release and rapidly inhibit striatal output neurons.

  14. Nerve growth factor binding domain of the nerve growth factor receptor

    SciTech Connect

    Welcher, A.A.; Bitler, C.M.; Radeke, M.J.; Shooter, E.M. )

    1991-01-01

    A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptors to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a K{sub d} comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cystein-rich sequence or the first and part of the second cystein-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.

  15. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    SciTech Connect

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG{sub 3}k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of ({sup 3}H)naloxone. The antibody which did not inhibit the binding of ({sup 3}H)naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG{sub 3}k antibody that blocked the binding of ({sup 3}H)naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form.

  16. Day length and sociosexual cohabitation alter central oxytocin receptor binding in female meadow voles (Microtus pennsylvanicus).

    PubMed

    Parker, K J; Phillips, K M; Kinney, L F; Lee, T M

    2001-12-01

    In voles (Microtus), central oxytocin (OT) receptor patterns are associated with interspecific social organization. Social, monogamous voles have more OT receptors in the extended amygdala than asocial, nonmonogamous voles. Nonmonogamous meadow voles (Microtus pennsylvanicus), which exhibit seasonal changes in social organization (long day [LD] females are territorial, short day [SD] females live socially), provide a model for examining whether OT receptor patterns are associated with seasonal changes in intraspecific social behaviors. The authors examined whether sexually inexperienced (naive) SD females had more OT receptor binding than naive LD females. Naive SD females had greater OT receptor binding in the lateral septum (LS), lateral amygdala (LatAmyg), and central amygdala (CenAmyg) than less social, naive LD females. Because both SD and LD females acquire partner preferences, the authors assessed whether OT receptor binding was associated with partner preference onset. For LD females, partner preference onset corresponded with greater OT receptor binding in the anterior olfactory nucleus, LS, and bed nucleus of the stria terminalis, compared with naive LD females. In contrast, naive SD females and those exhibiting partner preferences did not differ. However, SD females that failed to acquire partner preferences showed less OT binding in the LatAmyg and CenAmyg. This study is the first to show that central OT receptor patterns are associated with seasonal changes in intraspecific social organization and partner preference onset in a nonmonogamous rodent.

  17. Epidermal growth factor receptors on PC12 cells: alteration of binding properties by lectins

    SciTech Connect

    Vale, R.D.; Shooter, E.M.

    1983-01-01

    The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of /sup 125/I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37 degrees C and 4 degrees C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylation of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with /sup 125/I-NGF binding, WGA but not Con A was found to increase, by severalfold, the proportion of /sup 125/I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.

  18. Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

    SciTech Connect

    McCabe, R.T.; Mahan, D.R.; Smith, R.B.; Wamsley, J.K. )

    1990-10-01

    The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptor sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.

  19. Effect of ethanol administration and withdrawal on GABA receptor binding in rat cerebral cortex

    SciTech Connect

    Volicer, L.; Biagioni, T.M.

    1982-01-01

    Sodium independent GABA receptor binding was measured in synaptosomes prepared from cerebral cortex of rats made ethanol dependent by three daily ethanol administrations. In rats sacrificed 1 hour after the last ethanol dose there was a lower number of low affinity binding sites and lower affinity of the high affinity binding than in controls. The decreased affinity was present only in rats who showed symptoms of ethanol withdrawal during the course of ethanol administration. In rats sacrificed during ethanol withdrawal the affinity of the high affinity binding was lower than in controls and other binding characteristics were unchanged. This decreased binding was normalized by repeated Triton X-100 incubations indicating involvement of an endogenous inhibitor in this ethanol effect. Acute ethanol administration did not change GABA receptor binding.

  20. Identification of a new hormone-binding site on the surface of thyroid hormone receptor.

    PubMed

    Souza, P C T; Puhl, A C; Martínez, L; Aparício, R; Nascimento, A S; Figueira, A C M; Nguyen, P; Webb, P; Skaf, M S; Polikarpov, I

    2014-04-01

    Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily of ligand-activated transcription factors involved in cell differentiation, growth, and homeostasis. Although X-ray structures of many nuclear receptor ligand-binding domains (LBDs) reveal that the ligand binds within the hydrophobic core of the ligand-binding pocket, a few studies suggest the possibility of ligands binding to other sites. Here, we report a new x-ray crystallographic structure of TR-LBD that shows a second binding site for T3 and T4 located between H9, H10, and H11 of the TRα LBD surface. Statistical multiple sequence analysis, site-directed mutagenesis, and cell transactivation assays indicate that residues of the second binding site could be important for the TR function. We also conducted molecular dynamics simulations to investigate ligand mobility and ligand-protein interaction for T3 and T4 bound to this new TR surface-binding site. Extensive molecular dynamics simulations designed to compute ligand-protein dissociation constant indicate that the binding affinities to this surface site are of the order of the plasma and intracellular concentrations of the thyroid hormones, suggesting that ligands may bind to this new binding site under physiological conditions. Therefore, the second binding site could be useful as a new target site for drug design and could modulate selectively TR functions.

  1. Central phencyclidine (PCP) receptor binding is glutamate dependent: evidence for a PCP/excitatory amino acid receptor (EAAR) complex

    SciTech Connect

    Loo, P.; Braunwalder, A.; Lehmann, J.; Williams, M.

    1986-03-01

    PCP and other dissociative anesthetica block the increase in neuronal firing rate evoked by the EAAR agonist, N-methyl-Daspartate. NMDA and other EAAs such as glutamate (glu) have not been previously shown to affect PCP ligand binding. In the present study, using once washed rat forebrain membranes, 10 ..mu..M-glu was found to increase the binding of (/sup 3/H)TCP, a PCP analog, to defined PCP recognition sites by 20%. Removal of glu and aspartate (asp) by extensive washing decreased TCP binding by 75-90%. In these membranes, 10 ..mu..M L-glu increased TCP binding 3-fold. This effect was stereospecific and evoked by other EAAs with the order of activity, L-glu > D-asp > L- asp > NMDA > D-glu > quisqualate. Kainate, GABA, NE, DA, 5-HT, 2-chloroadenosine, oxotremorine and histamine had no effect on TCP binding at concentrations up to 100 ..mu..M. The effects of L-glu were attenuated by the NMDA-type receptor antagonist, 2-amino-7--phosphonoheptanoate (AP7; 10 ..mu..M-1 mM). These findings indicate that EAAS facilitate TCP binding, possibly through NMDA-type receptors. The observed interaction between the PCP receptor and EAARs may reflect the existence of a macromolecular receptor complex similar to that demonstrated for the benzodiazepines and GABA.

  2. Synthesis and binding profile of haloperidol-based bivalent ligands targeting dopamine D(2)-like receptors.

    PubMed

    Salama, Ismail; Löber, Stefan; Hübner, Harald; Gmeiner, Peter

    2014-08-15

    Homodimers of dopamine D2-like receptors are suggested to be of particular importance in the pathophysiology of schizophrenia and, thus, serve as promising targets for the discovery of atypical antipsychotics. This study describes the development of a series of novel bivalent molecules with a pharmacophore derived from the dopamine receptor antagonist haloperidol. These dimers were investigated in comparison to their monomeric analogues for their D2long, D2short, D3, and D4 receptor binding and the ability to bridge two neighboring receptor protomers. Radioligand binding studies provided diagnostic insights when Hill slopes close to two for the bivalent ligand 13 incorporating 22 spacer atoms and a comparative analysis with monovalent control ligands indicated a bivalent binding mode with a simultaneous occupancy of two neighboring binding sites. PMID:25047579

  3. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    NASA Technical Reports Server (NTRS)

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  4. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory

    NASA Astrophysics Data System (ADS)

    Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard; Weikl, Thomas R.

    2015-12-01

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  5. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory.

    PubMed

    Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard; Weikl, Thomas R

    2015-12-28

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  6. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    SciTech Connect

    Daughaday, W.H.; Trivedi, B.

    1987-07-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of /sup 125/I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound /sup 125/I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

  7. Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

    SciTech Connect

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  8. Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

    SciTech Connect

    Zhang, Yanfeng; Buchko, Garry W.; Qin, Lin; Robinson, Howard; Varnum, Susan M.

    2010-10-28

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  9. Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.

    PubMed

    Vishnivetskiy, Sergey A; Hirsch, Joel A; Velez, Maria-Gabriela; Gurevich, Yulia V; Gurevich, Vsevolod V

    2002-11-15

    Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

  10. Stereospecificity in binding studies. A useful criterion though insufficient to prove the presence of receptors.

    PubMed

    Laduron, P M

    1988-01-01

    In binding studies, stereospecificity is not a property restricted to receptor sites; indeed stereospecific binding has also been observed for acceptor sites. Therefore it does not represent a decisive criterion to make a binding site, a receptor site. However, in some well established cases, it can be useful especially when the difference between the active and inactive enantiomer exceeds 1000-fold as is the case for dexetimide and levetimide on muscarinic receptors. Stereospecific effect is also detectable with acceptor sites, e.g. spirodecanone sites, levocabastine displaceable neurotensin and, presumably, many other ones. Since the membrane is chiral (L-aminoacid) one should expect that non-specific displaceable binding would also display stereospecificity. In this regard, as most of the Scatchard plots reported throughout the literature are curvilinear, even if a straight line is drawn, one may assume that this is due to the presence of acceptor sites that are labelled by the ligand in addition to receptor sites. One cannot exclude the repetition of another "levocabastine story" with other neuropeptides. Hence, as the biochemical criteria like high affinity, saturability, reversibility and stereospecificity cannot differentiate a receptor from an acceptor (see Table 1), the most important and decisive criteria remain: (1) the drug displacement with compounds belonging to different pharmacological classes but mostly to different chemical classes, and (2) the functional correlates between the binding affinity and the potency in pharmacological or physiological tests in vitro or in vivo. When these points are fulfilled a binding site may be called a receptor site. PMID:2827683

  11. Evaluation of the In Vivo and Ex Vivo Binding of Novel BC1 Cannabinoid Receptor Radiotracers

    SciTech Connect

    Miller, A.; Gatley, J.; Gifford, A.

    2002-01-01

    The primary active ingredient of marijuana, 9-tetrahydrocannabinol, exerts its psychoactive effects by binding to cannabinoid CB1 receptors. These receptors are found throughout the brain with high concentrations in the hippocampus and cerebellum. The current study was conducted to evaluate the binding of a newly developed putative cannabinoid antagonist, AM630, and a classical cannabinoid 8-tetrahydrocannabinol as potential PET and/or SPECT imaging agents for brain CB1 receptors. For both of these ligands in vivo and ex vivo studies in mice were conducted. AM630 showed good overall brain uptake (as measure by %IA/g) and a moderately rapid clearance from the brain with a half-clearance time of approximately 30 minutes. However, AM630 did not show selective binding to CB1 cannabinoid receptors. Ex vivo autoradiography supported the lack of selective binding seen in the in vivo study. Similar to AM630, 8-tetrahydrocanibol also failed to show selective binding to CB1 receptor rich brain areas. The 8-tetrahydrocanibol showed moderate overall brain uptake and relatively slow brain clearance as compared to AM630. Further studies were done with AM2233, a cannabinoid ligand with a similar structure as AM630. These studies were done to develop an ex vivo binding assay to quantify the displacement of [131I]AM2233 binding by other ligands in Swiss-Webster and CB1 receptor knockout mice. By developing this assay we hoped to determine the identity of an unknown binding site for AM2233 present in the hippocampus of CB1 knockout mice. Using an approach based on incubation of brain slices prepared from mice given intravenous [131I]AM2233 in either the presence or absence of AM2233 (unlabelled) it was possible to demonstrate a significant AM2233-displacable binding in the Swiss-Webster mice. Future studies will determine if this assay is appropriate for identifying the unknown binding site for AM2233 in the CB1 knockout mice.

  12. Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation

    PubMed Central

    Lahti, Jennifer L.; Lui, Bertrand H.; Beck, Stayce E.; Lee, Stephen S.; Ly, Daphne P.; Longaker, Michael T.; Yang, George P.; Cochran, Jennifer R.

    2011-01-01

    Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity. PMID:21439278

  13. Characterization of nicotine binding in mouse brain and comparison with the binding of alpha-bungarotoxin and quinuclidinyl benzilate

    SciTech Connect

    Marks, M.J.; Collins, A.C.

    1982-11-01

    The binding of (/sup 3/H)nicotine to mouse brain has been measured and subsequently compared with the binding of (/sup 125/I)alpha-bungarotoxin (alpha-BTX) and L-(/sup 3/H)quinuclidinyl benzilate (QNB). The binding of nicotine was saturable, reversible, and stereospecific. The average KD and Bmax were 59 nM and 88 fmoles/mg of protein, respectively. Although the rates of association and dissociation of nicotine were temperature-dependent, the incubation temperature had no effect on either KD or Bmax. When measured at 20 degrees or 37 degrees, nicotine appeared to bind to a single class of binding sites, but a second, very low-affinity, binding site was observed at 4 degrees. Nicotine binding was unaffected by the addition of NaCl, KCl, CaCl/sub 2/, or MgSO/sub 4/ to the incubation medium. Nicotinic cholinergic agonists were potent inhibitors of nicotine binding; however, nicotinic antagonists were poor inhibitors. The regional distribution of binding was not uniform: midbrain and striatum contained the highest number of receptors, whereas cerebellum had the fewest. Differences in site densities, regional distribution, inhibitor potencies, and thermal denaturation indicated that nicotine binding was not the same as either QNB or alpha-BTX binding, and therefore that receptors for nicotine may represent a unique population of cholinergic receptors.

  14. In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

    PubMed Central

    Beltzer, J P; Spiess, M

    1991-01-01

    The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

  15. Binding and transactivation of the largemouth bass estrogen receptors by model compounds

    EPA Science Inventory

    Environmental estrogens (EEs) are chemicals in the environment that can elicit adverse effects on estrogen (E2) signaling by binding with the estrogen receptors (ERs). In largemouth bass (LMB), the physiological actions of E2 are primarily mediated via three receptors (ERα, ERßb ...

  16. Development of a Competitive Binding Assay System with Recombinant Estrogen Receptors from Multiple Species

    EPA Science Inventory

    ABSTRACT In the current study, we developed a new system using full-length recombinant baculovirus-expressed estrogen receptors which allows for direct comparison of binding across species. Estrogen receptors representing five vertebrate classes were compared: human (hERα), quai...

  17. Altered (/sup 125/I)epidermal growth factor binding and receptor distribution in psoriasis

    SciTech Connect

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-03-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that (/sup 125/I)EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

  18. [3H]LY341495 binding to group II metabotropic glutamate receptors in rat brain.

    PubMed

    Wright, R A; Arnold, M B; Wheeler, W J; Ornstein, P L; Schoepp, D D

    2001-08-01

    [3H]LY341495 is a highly potent and selective antagonist for group II metabotropic glutamate (mGlu) receptors (mGlu2 and mGlu3), which has been used to label these receptors in cells expressing recombinant receptor subtypes. In this study, we characterized the kinetics, pharmacology, and distribution of [3H]LY341495 binding to mGlu receptors in rat brain tissue. Equilibrium experiments in the rat forebrain demonstrated binding to a single site that was saturable, reversible, and of high affinity (Bmax, 3.9 +/- 0.65 pmol/mg of protein, Kd, 0.84 +/- 0.11 nM). The relative order of potencies for displacement of [3H]LY341495 by mGlu receptor ligands was LY341495 > L-glutamic acid > LY354740 > (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine > 4-(2R,4R)-aminopyrrolidine-2,4-dicarboxylate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (R,S)-alpha-methyl-4-phosphonophenylglycine > (R,S)3,5-dihydroxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. [3H]LY341495 was not displaced by the selective ionotropic glutamate receptor agonists N-methyl-D-aspartic acid, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate at concentrations up to 1 mM. Comparison of [3H]LY341495 binding in rat brain with recombinant mGlu receptor subtypes demonstrated a very high correlation with mGlu3 receptor binding (r2 = 0.957), a significant, but lower, correlation with mGlu2 receptor binding (r2 = 0.869), but no significant correlation to mGlu8 receptor binding (r2 = 0.284). Regional studies using autoradiography showed a similar distribution of [3H]LY341495 binding to that for group II mGlu receptors previously reported by others using immunocytochemical techniques. These studies indicate that [3H]LY341495 selectively labels group II (mGlu2/3) receptors, but under the conditions used, [3H]LY341495 may bind predominately to mGlu3 receptor populations in the rat forebrain. PMID:11454905

  19. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    ERIC Educational Resources Information Center

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  20. Receptor binding sites for atrial natriuretic factor are expressed by brown adipose tissue

    SciTech Connect

    Bacay, A.C.; Mantyh, C.R.; Vigna, S.R.; Mantyh, P.W. )

    1988-09-01

    To explore the possibility that atrial natriuretic factor (ANF) is involved in thermoregulation we used quantitative receptor autoradiography and homogenate receptor binding assays to identify ANF bindings sites in neonatal rat and sheep brown adipose tissue, respectively. Using quantitative receptor autoradiography were were able to localize high levels of specific binding sites for {sup 125}I-rat ANF in neonatal rat brown adipose tissue. Homogenate binding assays on sheep brown fat demonstrated that the radioligand was binding to the membrane fraction and that the specific binding was not due to a lipophilic interaction between {sup 125}I-rat ANF and brown fat. Specific binding of {sup 125}I-rat ANF to the membranes of brown fat cells was inhibited by unlabeled rat ANF with a Ki of 8.0 x 10(-9) M, but not by unrelated peptides. These studies demonstrate that brown fat cells express high levels of ANF receptor binding sites in neonatal rat and sheep and suggest that ANF may play a role in thermoregulation.

  1. Structural Studies of GABAA Receptor Binding Sites: Which Experimental Structure Tells us What?

    PubMed Central

    Puthenkalam, Roshan; Hieckel, Marcel; Simeone, Xenia; Suwattanasophon, Chonticha; Feldbauer, Roman V.; Ecker, Gerhard F.; Ernst, Margot

    2016-01-01

    Atomic resolution structures of cys-loop receptors, including one of a γ-aminobutyric acid type A receptor (GABAA receptor) subtype, allow amazing insights into the structural features and conformational changes that these pentameric ligand-gated ion channels (pLGICs) display. Here we present a comprehensive analysis of more than 30 cys-loop receptor structures of homologous proteins that revealed several allosteric binding sites not previously described in GABAA receptors. These novel binding sites were examined in GABAA receptor homology models and assessed as putative candidate sites for allosteric ligands. Four so far undescribed putative ligand binding sites were proposed for follow up studies based on their presence in the GABAA receptor homology models. A comprehensive analysis of conserved structural features in GABAA and glycine receptors (GlyRs), the glutamate gated ion channel, the bacterial homologs Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus GLIC, and the serotonin type 3 (5-HT3) receptor was performed. The conserved features were integrated into a master alignment that led to improved homology models. The large fragment of the intracellular domain that is present in the structure of the 5-HT3 receptor was utilized to generate GABAA receptor models with a corresponding intracellular domain fragment. Results of mutational and photoaffinity ligand studies in GABAA receptors were analyzed in the light of the model structures. This led to an assignment of candidate ligands to two proposed novel pockets, candidate binding sites for furosemide and neurosteroids in the trans-membrane domain were identified. The homology models can serve as hypotheses generators, and some previously controversial structural interpretations of biochemical data can be resolved in the light of the presented multi-template approach to comparative modeling. Crystal and cryo-EM microscopic structures of the closest homologs that were solved in different conformational

  2. Specific beta-adrenergic receptor binding of carazolol measured with PET

    SciTech Connect

    Berridge, M.S.; Nelson, A.D.; Zheng, L.

    1994-10-01

    Carazolol is a promising high-affinity beta-adrenergic receptor ligand for the noninvasive determination of beta receptor status using PET> Earlier investigations demonstrated specific receptor binding of carazolol in mice. These PET studies with S(-)-[2{double_prime}-{sup 11}C]carazolol in pigs were performed to explore the utility of the tracer for PET receptor studies. Tracer uptake in the heart and lung was measured by PET as a function of time. Receptors were blocked with propranolol and different doses of ICI 118,551 to estimate specific binding. Fluorine-18-1{double_prime}-Fluorocarazolol and the less active R-enantiomer of [{sup 11}C]-carazolol were also studied. Specific receptor binding was 75% of the total uptake in the heart, preventable and displaceable by propranolol. Dose-dependent competition showed that carazolol binds in vivo to {beta}{sub 1} and to {beta}{sub 2} subtypes. Uptake of the labeled R(=) enantiomer of carazolol was not receptor-specific. Carazolol labeled with {sup 11}C or {sup 18}F is a strong candidate for use in receptor estimation with PET. The in vivo observations were consistent with its known high affinity and slow receptor dissociation rate. Its high specific receptor uptake and low metabolism allow existing kinetic models to be applied for receptor measurements. The {sup 11}C label is convenient for repeated administrations, though {sup 13}F allowed the long observation periods necessary for measurement of the receptor dissociation rate. If needed, nonspecific uptake can be estimated without pharmacologic intervention by using the labeled R enantiomer. 32 refs., 11 figs.

  3. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    SciTech Connect

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.

    2010-09-27

    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  4. A Natural Mutation in Helix 5 of the Ligand Binding Domain of Glucocorticoid Receptor Enhances Receptor-Ligand Interaction

    PubMed Central

    Reyer, Henry; Ponsuksili, Siriluck; Kanitz, Ellen; Pöhland, Ralf; Wimmers, Klaus; Murani, Eduard

    2016-01-01

    The glucocorticoid receptor (GR) is a central player in the neuroendocrine stress response; it mediates feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis and physiological actions of glucocorticoids in the periphery. Despite intensive investigations of GR in the context of receptor-ligand interaction, only recently the first naturally occurring gain-of-function substitution, Ala610Val, of the ligand binding domain was identified in mammals. We showed that this mutation underlies a major quantitative trait locus for HPA axis activity in pigs, reducing cortisol production by about 40–50 percent. To unravel the molecular mechanisms behind this gain of function, receptor-ligand interactions were evaluated in silico, in vitro and in vivo. In accordance with previously observed phenotypic effects, the mutant Val610 GR showed significantly increased activation in response to glucocorticoid and non-glucocorticoid steroids, and, as revealed by GR-binding studies in vitro and in pituitary glands, enhanced ligand binding. Concordantly, the protein structure prediction depicted reduced binding distances between the receptor and ligand, and altered interactions in the ligand binding pocket. Consequently, the Ala610Val substitution opens up new structural information for the design of potent GR ligands and to examine effects of the enhanced GR responsiveness to glucocorticoids on the entire organism. PMID:27736993

  5. Platelet-derived growth factor binds specifically to receptors on vascular smooth muscle cells and the binding becomes nondissociable.

    PubMed Central

    Williams, L T; Tremble, P; Antoniades, H N

    1982-01-01

    Radioiodinated platelet-derived growth factor (125I-PDGF) was used in studies of PDGF binding sites on vascular smooth muscle cells. There was an excellent correlation between the ability of 125I-PDGF to stimulate cell proliferation and to bind specifically to cultured vascular smooth muscle cells. The half-maximal concentration for both processes was 0.1 nM. There were 50,000 binding sites per cell. Reduced PDGF, prepared by treatment of PDGF with 20 mM dithiothreitol, had neither the ability to bind to smooth muscle cells nor to stimulate cellular proliferation. Epidermal growth factor, nerve growth factor, fibroblast growth factor, and histone B did not compete for the binding sites at a concentration of 10 nM. 125I-PDGF binding was slowly reversible at 4 degrees C and was rapidly and totally reversible after a 1-min incubation at 37 degrees C. After continued incubation at 37 degrees C, the binding became irreversible. The half-time for formation of the nondissociable state of 125I-PDGF binding was approximately equal to 5 min at 37 degrees C. The nondissociable state of binding was not formed at 4 degrees C even after 1 hr of incubation. These data suggest that the sites we labeled are the PDGF receptors that mediate PDGF's mitogenic action and that a nondissociable state of PDGF binding is formed at 37 degrees C. It is likely that nondissociable PDGF represents internalized ligand or binding to sites that are converted to a high-affinity state after the ligand binds. PMID:6310551

  6. Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

    SciTech Connect

    Gracz, L.M.; Wang, W.; Parsons, S.M.

    1988-07-12

    Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP/sub 1/ synaptic vesicles banding at 1.055 g/mL actively transported (/sup 3/H)acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more (/sup 3/H)AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP/sub 1/ vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP/sub 2/ synaptic vesicle originally described in intact Torpedo marmorata electric organ. The properties of the receptor for vesamicol were studied by measuring binding of (/sup 3/H)vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP/sub 1/ vesicles the VP/sub 2/ vesicles had a ratio of (/sup 3/H)AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. The Hill coefficients ..cap alpha../sub H/ and equilibrium dissociation constants K for vesamicol binding to VP/sub 1/ and VP/sub 2/ vesicles were essentially the same. The positive Hill coefficient suggests that the vesamicol receptor exists as a homotropic oligomeric complex. The results demonstrate that VP/sub 1/ and VP/sub 2/ synaptic vesicles exhibit functional differences in the AcCh transport system, presumably as a result of regulatory phenomena.

  7. Cholinergic functioning in stimulant addiction: implications for medications development.

    PubMed

    Sofuoglu, Mehmet; Mooney, Marc

    2009-11-01

    Acetylcholine, the first neurotransmitter discovered, participates in many CNS functions, including sensory and motor processing, sleep, nociception, mood, stress response, attention, arousal, memory, motivation and reward. These diverse cholinergic effects are mediated by nicotinic- and muscarinic-type cholinergic receptors (nAChR and mAChR, respectively). The goal of this review is to synthesize a growing literature that supports the potential role of acetylcholine as a treatment target for stimulant addiction. Acetylcholine interacts with the dopaminergic reward system in the ventral tegmental area, nucleus accumbens and prefrontal cortex. In the ventral tegmental area, both nAChR and mAChR stimulate the dopaminergic system. In the nucleus accumbens, cholinergic interneurons integrate cortical and subcortical information related to reward. In the prefrontal cortex, the cholinergic system contributes to the cognitive aspects of addiction. Preclinical studies support a facilitative role of nicotinic receptor agonists in the development of stimulant addiction. In contrast, nonselective muscarinic receptor agonists seem to have an inhibitory role. In human studies, acetylcholinesterase inhibitors, which increase synaptic acetylcholine levels, have shown promise for the treatment of stimulant addiction. Further studies testing the efficacy of cholinergic medications for stimulant addiction are warranted. PMID:19845415

  8. DIFFERENCES IN SENSITIVITY BUT NOT SELECTIVITY OF XENOESTROGEN BINDING TO ALLIGATOR VERSUS HUMAN ESTROGEN RECEPTOR ALPHA

    PubMed Central

    Rider, Cynthia V.; Hartig, Phillip C.; Cardon, Mary C.; Lambright, Christy R.; Bobseine, Kathy L.; Guillette, Louis J.; Gray, L. Earl; Wilson, Vickie S.

    2010-01-01

    Reproductive abnormalities in alligators exposed to contaminants in Lake Apopka, Florida, USA represent a clear example of endocrine disruption in wildlife. Several of these contaminants that are not able to bind to mammalian estrogen receptors (such as atrazine and cyanazine) have previously been reported to bind to the alligator estrogen receptor from oviductal tissue. Binding of known Lake Apopka contaminants to full length estrogen receptors alpha from human (hERα) and alligator (aERα) was assessed in a side-by-side comparison within the same assay system. Baculovirus-expressed recombinant hERα and aERα were used in a competitive binding assay. Atrazine and cyanazine were not able to bind to either receptor. p,p′-Dicofol was able to bind to aERα with a concentration inhibiting 50% of binding (IC50) of 4 μM, while only partially displacing 17β-estradiol (E2) from hERα and yielding a projected IC50 of 45 μM. Chemicals that only partially displaced E2 from either receptor, including some dichlorodiphenyltrichloroethane (DDT) metabolites and trans-nonachlor, appeared to have higher affinity for aERα than hERα. p,p′-Dicofol-mediated transcriptional activation through aERα and hERα was assessed to further explore the preferential binding of p,p′-dicofol to aERα over hERα. p,p′-Dicofol was able to stimulate transcriptional activation in a similar manner with both receptors. However, the in vitro results obtained with p,p′-dicofol were not reflected in an in vivo mammalian model, where Kelthane™ (mixed o,p′-and p,p′-dicofol isomers) did not elicit estrogenic effects. In conclusion, although there was no evidence of exclusively species-specific estrogen receptor binders, some xenoestrogens, especially p,p′-dicofol, had a higher affinity for aERα than for hERα. PMID:20821664

  9. Thyroid hormone receptors bind to defined regions of the growth hormone and placental lactogen genes.

    PubMed Central

    Barlow, J W; Voz, M L; Eliard, P H; Mathy-Harter, M; De Nayer, P; Economidis, I V; Belayew, A; Martial, J A; Rousseau, G G

    1986-01-01

    The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[125I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions. PMID:3466175

  10. The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor.

    PubMed Central

    Moore, D D; Marks, A R; Buckley, D I; Kapler, G; Payvar, F; Goodman, H M

    1985-01-01

    Glucocorticoid receptor (GCR) protein stimulates transcription from a variety of cellular genes. We show here that GCR partially purified from rat liver binds specifically to a site within the first intron of the human growth hormone (hGH) gene, approximately 100 base pairs downstream from the start of hGH transcription. GCR binding is selectively inhibited by methylation of two short, symmetrically arranged clusters of guanine residues within this site. A cloned synthetic 24-base-pair deoxyoligonucleotide containing the predicted GCR binding sequence interacts specifically with GCR. The hGH binding site shares sequence homology with a GCR binding site upstream from the human metallothionein II gene and a subset of GCR binding sites from mouse mammary tumor virus. All of these binding sites for this eukaryotic transcriptional regulatory protein show remarkable similarity in overall geometry to the binding sites for several prokaryotic transcriptional regulatory proteins. Images PMID:2983311

  11. Different behavior toward muscarinic receptor binding between quaternary anticholinergics and their tertiary analogues.

    PubMed

    Ensing, K; de Zeeuw, R A

    1986-12-01

    A number of corresponding tertiary and quaternary anticholinergic analogues were examined for their ability to inhibit specific (3)H-dexetimide binding to calf brain muscarinic receptors. In all cases the tertiary antagonists (except pirenzepine) showed steep and monophasic inhibition curves, whereas those of the quaternary derivatives were shallow (thiazinamium, methylbenactyzine) or even biphasic (oxyphenonium, methylatropine, methylscopolamine). These observations show that the addition of a methyl group to the nitrogen atom changes the mode of interaction of the anticholinergics to muscarinic receptor binding sites. Whether there are separate binding sites present or differences in interaction mode for only the quaternary moiety is discussed. PMID:24271831

  12. Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties*

    PubMed Central

    Schuepbach-Mallepell, Sonia; Das, Dolon; Willen, Laure; Vigolo, Michele; Tardivel, Aubry; Lebon, Luc; Kowalczyk-Quintas, Christine; Nys, Josquin; Smulski, Cristian; Zheng, Timothy S.; Maskos, Klaus; Lammens, Alfred; Jiang, Xuliang; Hess, Henry; Tan, Seng-Lai; Schneider, Pascal

    2015-01-01

    The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity. PMID:25953898

  13. Differential receptor binding characteristics of consecutive phenylalanines in micro-opioid specific peptide ligand endomorphin-2.

    PubMed

    Honda, Takeshi; Shirasu, Naoto; Isozaki, Kaname; Kawano, Michiaki; Shigehiro, Daiki; Chuman, Yoshiro; Fujita, Tsugumi; Nose, Takeru; Shimohigashi, Yasuyuki

    2007-06-01

    Endogenous opioid peptides consist of a conserved amino acid residue of Phe(3) and Phe(4), although their binding modes for opioid receptors have not been elucidated in detail. Endomorphin-2, which is highly selective and specific for the mu opioid receptor, possesses two Phe residues at the consecutive positions 3 and 4. In order to clarify the role of Phe(3) and Phe(4) in binding to the mu receptor, we synthesized a series of analogs in which Phe(3) and Phe(4) were replaced by various amino acids. It was found that the aromaticity of the Phe-beta-phenyl groups of Phe(3) and Phe(4) is a principal determinant of how strongly it binds to the receptor, although better molecular hydrophobicity reinforces the activity. The receptor binding subsites of Phe(3) and Phe(4) of endomorphin-2 were found to exhibit different structural requirements. The results suggest that [Trp(3)]endomorphin-2 (native endomorphin-1) and endomorphin-2 bind to different receptor subclasses. PMID:17395470

  14. Position 4 substituted somatostatin analogs: increased binding to somatostatin receptors in pituitary and brain.

    PubMed

    Srikant, C B; Coy, D H; Patel, Y C

    1983-07-11

    Somatostatin (S-14) analogs with Phe4 substitutions bound to pituitary and cerebrocortical S-14 receptors more avidly than did S-14. The 2-4 fold greater affinities of the Phe4 S-14 as well as analogs with structural modification of the Phe4 residue for binding to pituitary S-14 receptors showed good correlation with their reported potencies for in vivo Gh inhibition. In the cerebral cortex, [Phe4] S-14, [Phe4, D-Trp8] S-14 and [F5-Phe4] S14 were 2-3 times more potent while [p-NH2-Phe4] S-14 was 6 times more potent compared to S-14 in binding to S-14 receptors. The increased binding affinities of the Phe4 analogs in these two tissues does not appear to be due to differential stability of the analogs compared to S-14 under the experimental conditions used. [Thr4] S-14 exhibited very low binding in both these tissues. Thus structural modification of the position 4 moiety of the S-14 molecule does not result in dissociated affinities for binding to S-14 receptors in the brain and the pituitary. The increased receptor binding affinities of the Phe4 analogs in the cerebral cortex suggest that they may be more potent than S-14 in the CNS.

  15. Characterization of a second ligand binding site of the insulin receptor

    SciTech Connect

    Hao Caili; Whittaker, Linda; Whittaker, Jonathan . E-mail: jonathan.whittaker@case.edu

    2006-08-18

    Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the {alpha} subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K {sub d} of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site.

  16. The nicotinic cholinergic system function in the human brain.

    PubMed

    Nees, Frauke

    2015-09-01

    Research on the nicotinic cholinergic system function in the brain was previously mainly derived from animal studies, yet, research in humans is growing. Up to date, findings allow significant advances on the understanding of nicotinic cholinergic effects on human cognition, emotion and behavior using a range of functional brain imaging approaches such as pharmacological functional magnetic resonance imaging or positron emission tomography. Studies provided insights across various mechanistic psychological domains using different tasks as well as at rest in both healthy individuals and patient populations, with so far partly mixed results reporting both enhancements and decrements of neural activity related to the nicotinic cholinergic system. Moreover, studies on the relation between brain structure and the nicotinic cholinergic system add important information in this context. The present review summarizes the current status of human brain imaging studies and presents the findings within a theoretical and clinical perspective as they may be useful not only for an advancement of the understanding of basic nicotinic cholinergic-related mechanisms, but also for the development and integration of psychological and pharmacological treatment approaches. Patterns of functional neuroanatomy and neural circuitry across various cognitive and emotional domains may be used as neuropsychological markers of mental disorders such as addiction, Alzheimer's disease, Parkinson disease or schizophrenia, where nicotinic cholinergic system changes are characteristic. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

  17. Cholinergic Plasticity of Oscillating Neuronal Assemblies in Mouse Hippocampal Slices

    PubMed Central

    Zylla, Maura M.; Zhang, Xiaomin; Reichinnek, Susanne; Draguhn, Andreas; Both, Martin

    2013-01-01

    The mammalian hippocampus expresses several types of network oscillations which entrain neurons into transiently stable assemblies. These groups of co-active neurons are believed to support the formation, consolidation and recall of context-dependent memories. Formation of new assemblies occurs during theta- and gamma-oscillations under conditions of high cholinergic activity. Memory consolidation is linked to sharp wave-ripple oscillations (SPW-R) during decreased cholinergic tone. We hypothesized that increased cholinergic tone supports plastic changes of assemblies while low cholinergic tone favors their stability. Coherent spatiotemporal network patterns were measured during SPW-R activity in mouse hippocampal slices. We compared neuronal activity within the oscillating assemblies before and after a transient phase of carbachol-induced gamma oscillations. Single units maintained their coupling to SPW-R throughout the experiment and could be re-identified after the transient phase of gamma oscillations. However, the frequency of SPW-R-related unit firing was enhanced after muscarinic stimulation. At the network level, these changes resulted in altered patterns of extracellularly recorded SPW-R waveforms. In contrast, recording of ongoing SPW-R activity without intermittent cholinergic stimulation revealed remarkably stable repetitive activation of assemblies. These results show that activation of cholinergic receptors induces plasticity at the level of oscillating hippocampal assemblies, in line with the different role of gamma- and SPW-R network activity for memory formation and –consolidation, respectively. PMID:24260462

  18. Acetylcholine receptors in the human retina

    SciTech Connect

    Hutchins, J.B.; Hollyfield, J.G.

    1985-11-01

    Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand TH-propylbenzilylcholine mustard (TH-PrBCM) to label muscarinic receptors. TH- or SVI-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that TH-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer of the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina.

  19. Structure of the bacteriophage T4 long tail fiber receptor-binding tip

    PubMed Central

    Bartual, Sergio G.; Otero, José M.; Garcia-Doval, Carmela; Llamas-Saiz, Antonio L.; Kahn, Richard; Fox, Gavin C.; van Raaij, Mark J.

    2010-01-01

    Bacteriophages are the most numerous organisms in the biosphere. In spite of their biological significance and the spectrum of potential applications, little high-resolution structural detail is available on their receptor-binding fibers. Here we present the crystal structure of the receptor-binding tip of the bacteriophage T4 long tail fiber, which is highly homologous to the tip of the bacteriophage lambda side tail fibers. This structure reveals an unusual elongated six-stranded antiparallel beta-strand needle domain containing seven iron ions coordinated by histidine residues arranged colinearly along the core of the biological unit. At the end of the tip, the three chains intertwine forming a broader head domain, which contains the putative receptor interaction site. The structure reveals a previously unknown beta-structured fibrous fold, provides insights into the remarkable stability of the fiber, and suggests a framework for mutations to expand or modulate receptor-binding specificity. PMID:21041684

  20. Imaging G protein–coupled receptors while quantifying their ligand-binding free-energy landscape

    PubMed Central

    Zhang, Cheng; Spoerri, Patrizia M; Coughlin, Shaun R; Kobilka, Brian K; Müller, Daniel J

    2016-01-01

    Imaging native membrane receptors and testing how they interact with ligands is of fundamental interest in the life sciences but has proven remarkably difficult to accomplish. Here, we introduce an approach that uses force-distance curve–based atomic force microscopy to simultaneously image single native G protein–coupled receptors in membranes and quantify their dynamic binding strength to native and synthetic ligands. We measured kinetic and thermodynamic parameters for individual protease-activated receptor-1 (PAR1) molecules in the absence and presence of antagonists, and these measurements enabled us to describe PAR1’s ligand-binding free-energy landscape with high accuracy. Our nanoscopic method opens an avenue to directly image and characterize ligand binding of native membrane receptors. PMID:26167642

  1. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes. PMID:17168764

  2. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

  3. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    PubMed

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  4. Structure and receptor binding of the hemagglutinin from a human H6N1 influenza virus.

    PubMed

    Tzarum, Netanel; de Vries, Robert P; Zhu, Xueyong; Yu, Wenli; McBride, Ryan; Paulson, James C; Wilson, Ian A

    2015-03-11

    Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and human receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. This binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics. PMID:25766295

  5. Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites.

    PubMed

    Specht, Christian G; Izeddin, Ignacio; Rodriguez, Pamela C; El Beheiry, Mohamed; Rostaing, Philippe; Darzacq, Xavier; Dahan, Maxime; Triller, Antoine

    2013-07-24

    The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on single-molecule detections to characterize the ultrastructure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organization of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5,000-10,000 molecules/μm(2). The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABAA receptor complexes for synaptic binding sites highlights the potential of single-molecule imaging to quantify synaptic plasticity on the nanoscopic scale.

  6. Sedative-Hypnotic and Receptor Binding Studies of Fermented Marine Organisms

    PubMed Central

    Joung, Hye-Young; Kang, Young Mi; Lee, Bae-Jin; Chung, Sun Yong; Kim, Kyung-Soo; Shim, Insop

    2015-01-01

    This study was performed to investigate the sedative-hypnotic activity of γ-aminobutyric acid (GABA)-enriched fermented marine organisms (FMO), including sea tangle (FST) and oyster (FO) by Lactobacillus brevis BJ20 (L. brevis BJ20). FST and FO were tested for their binding activity of the GABAA-benzodiazepine and 5-HT2C receptors, which are well-known molecular targets for sleep aids. We also measured the sleep latency and sleep duration during pentobarbital-induced sleep in mice after oral administration of FST and FO. In GABAA and 5-HT2C receptor binding assays, FST displayed an effective concentration-dependent binding affinity to GABAA receptor, similar to the binding affinity to 5-HT2C receptor. FO exhibited higher affinity to 5-HT2C receptor, compared with the GABAA receptor. The oral administration of FST and FO produced a dose-dependent decrease in sleep latency and increase in sleep duration in pentobarbital-induced hypnosis. The data demonstrate that FST and FO possess sedative-hypnotic activity possibly by modulating GABAA and 5-HT2C receptors. We propose that FST and FO might be effective agents for treatment of insomnia. PMID:26336589

  7. Are receptor concentrations correlated across tissues within individuals? A case study examining glucocorticoid and mineralocorticoid receptor binding.

    PubMed

    Lattin, Christine R; Keniston, Daniel E; Reed, J Michael; Romero, L Michael

    2015-04-01

    Hormone receptors are a necessary (although not sufficient) part of the process through which hormones like corticosterone create physiological responses. However, it is currently unknown to what extent receptor concentrations across different target tissues may be correlated within individual animals. In this study, we examined this question using a large dataset of radioligand binding data for glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) in 13 different tissues in the house sparrow (Passer domesticus) (n=72). Our data revealed that individual house sparrows tended to exhibit higher or lower receptor binding across all tissues, which could be part of what creates the physiological and behavioral syndromes associated with different hormonal profiles. However, although statistically significant, the correlations between tissues were very weak. Thus, when each tissue was independently regressed on receptor concentrations in the other tissues, multivariate analysis revealed significant relationships only for sc fat (for GR) and whole brain, hippocampus, kidney, omental fat, and sc fat (for MR). We also found significant pairwise correlations only between receptor concentrations in brain and hippocampus, and brain and kidney (both for MR). This research reveals that although there are generalized individual consistencies in GR and MR concentrations, possibly due to such factors as hormonal regulation and genetic effects, the ability of 2 different tissues to respond to the same hormonal signal appears to be affected by additional factors that remain to be identified.

  8. Complement Component C3 Binds to Activated Normal Platelets without Preceding Proteolytic Activation and Promotes Binding to Complement Receptor 1

    PubMed Central

    Hamad, Osama A.; Nilsson, Per H.; Wouters, Diana; Lambris, John D.; Ekdahl, Kristina N.; Nilsson, Bo

    2010-01-01

    It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor-activating peptide-activated platelets in lepirudin-anticoagulated platelet-rich plasma (PRP) and whole blood. However, inhibiting complement activation at the C1q or C3 level did not block the binding of C3 to activated platelets. Diluting PRP and chelating divalent cations also had no effect, further indicating that the deposition of complement components was independent of complement activation. Furthermore, washed, activated platelets bound added C1q and C3 to the same extent as platelets in PRP. The use of mAbs against different forms of C3 demonstrated that the bound C3 consisted of C3(H2O). Furthermore, exogenously added soluble complement receptor 1 was shown to bind to this form of platelet-bound C3. These observations indicate that there is no complement activation on the surface of platelets under physiological conditions. This situation is in direct contrast to a number of pathological conditions in which regulators of complement activation are lacking and thrombocytopenia and thrombotic disease are the ultimate result. However, the generation of C3(H2O) represents nonproteolytic activation of C3 and after factor I cleavage may act as a ligand for receptor binding. PMID:20139276

  9. Multiple opioid receptor binding in dissociated intact guinea pig brain cells

    SciTech Connect

    Tam, S.W.; James, D.W.

    1986-03-05

    Dissociated intact guinea pig brain cells were prepared by the method of Rogers and El-Fakahany. Over 95% of these cells are viable as demonstrated by their exclusion of the dye trypan blue. Opioid receptor binding assays were performed in a modified Kreb-Ringers physiological buffer. The following radiolabeled ligands and conditions were used to selectively labeled multiple opioid receptors: mu binding, 1 nM (/sup 3/H)naloxone + 20 nM DADLE + 300 nM U50,488H; kappa binding, 4 nM (-)-(/sup 3/H)-EKC + 100 nM DAGO + 500 nM DADLE; delta binding, 2 nM (/sup 3/H)-DADLE + 100 nM DAGO + 300 nM U50,488H; sigma binding, 4 nM (+)-(/sup 3/H)SKF 10,047. The intact brain cells in physiological buffer demonstrated specific binding for mu, kappa, delta, and sigma receptors. The relative binding potency of naloxone for each of the receptor types is arbitrarily set at 1.

  10. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

    SciTech Connect

    Ayyagari, R.R.; Khan-Dawood, F.S.

    1987-04-01

    Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol /sup 125/I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function.

  11. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

    PubMed Central

    Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

    2016-01-01

    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

  12. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

    SciTech Connect

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai; Tao, Yong-guang; Tolbert, W. David; Simons, Jr., S. Stoney; Xu, H. Eric

    2010-03-08

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

  13. Sequence-specific DNA binding by glucocorticoid receptor "zinc finger peptides".

    PubMed

    Archer, T K; Hager, G L; Omichinski, J G

    1990-10-01

    Steroid hormone receptors can activate or repress transcription from responsive loci by binding to DNA. We have examined the mechanism of DNA binding by individually synthesizing the putative "zinc finger peptides" from the rat glucocorticoid receptor. Atomic absorption studies show that the peptides will bind zinc on an equimolar basis, and circular dichroism experiments demonstrate a significant alteration in secondary structure in the presence of zinc. The results from a series of experiments establish that metal ion is required for binding to DNA and that the amino-terminal zinc finger shows a significantly greater affinity for glucocorticoid response element-containing DNA over control DNA. These observations indicate that a single synthetic "zinc finger peptide" is able to bind to DNA in a sequence-specific manner. PMID:2120703

  14. Characteristics of albumin binding to opossum kidney cells and identification of potential receptors.

    PubMed

    Brunskill, N J; Nahorski, S; Walls, J

    1997-02-01

    Albumin re-absorption in the kidney proximal tubule may be pathophysiological in disease. Opossum kidney (OK) cell monolayers were used to investigate the characteristics of [125I]-labelled albumin binding at 4 degrees C. Two binding sites were identified, one with high affinity (KD 154.8 +/-7 mg/l) and low capacity, the other with low affinity (KD 8300 +/- 1000 mg/l) and high capacity. Binding was sensitive to lectins Glycine max and Ulex europaeus I, but not other lectins, indicating involvement of a glycoprotein(s) in the binding process. Binding was also sensitive to a number of agents known to inhibit binding to scavenger receptors. [125I]-Labelled albumin ligand blotting of OK cell membrane proteins identified several albumin-binding proteins with identical lectin affinities to those proteins mediating albumin binding to OK cell monolayers. These results provide initial evidence of the identity of albumin receptors in kidney tubules, and suggest that they may be members of the family of scavenger receptors. PMID:9000429

  15. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity

    PubMed Central

    Ayres, Cory M.; Scott, Daniel R.; Corcelli, Steven A.; Baker, Brian M.

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

  16. A receptor binding assay applied to monitoring the neurotoxicity of parathion to Peromyscus after oral exposure

    USGS Publications Warehouse

    Jett, D.A.; Eldefrawi, A.T.; Eldefrawi, M.E.

    1993-01-01

    Many naturally occurring toxins, as well as pesticides, metals, and other compounds that occur in our environment from anthropogenic activities, stimulate or antagonize neuro-receptors to produce acute and/or chronic toxicities. Recent advances in laboratory instrumentation and the availability of a variety of radiolabeled ligands and type-specific drugs for numerous receptors make it possible to easily screen large numbers of samples and detect changes in sensitivity and density of receptor types and subtypes. A receptor binding assay for examining the chronic dietary toxicity of parathion will be used as a model to describe the methodology.

  17. Brain Serotonin 1A Receptor Binding as a Predictor of Treatment Outcome in Major Depressive Disorder

    PubMed Central

    Miller, Jeffrey M.; Hesselgrave, Natalie; Ogden, R. Todd; Zanderigo, Francesca; Oquendo, Maria A.; Mann, J. John; Parsey, Ramin V.

    2013-01-01

    Background We previously reported higher serotonin 1A receptor (5-HT1A) binding in subjects with major depressive disorder (MDD) during a major depressive episode using positron emission tomography imaging with [11C]WAY-100635. 5-HT1A receptor binding is also associated with treatment outcome after nonstandardized antidepressant treatment. We examined whether pretreatment 5-HT1A binding is associated with treatment outcome following standardized escitalopram treatment in MDD. We also compared 5-HT1A binding between all MDD subjects in this cohort and a sample of healthy control subjects. Methods Twenty-four MDD subjects in a current major depressive episode and 51 previously studied healthy control subjects underwent positron emission tomography scanning with [11C]WAY-100635, acquiring a metabolite-corrected arterial input function and free-fraction measurement to estimate 5-HT1A binding potential (BPF = Bmax/KD, where Bmax = available receptors and KD = dissociation constant). Major depressive disorder subjects then received 8 weeks of treatment with escitalopram; remission was defined as a posttreatment 24-item Hamilton Depression Rating Scale <10 and ≥50% reduction in Hamilton Depression Rating Scale. Results Remitters to escitalopram had 33% higher baseline 5-HT1A binding in the raphe nuclei than nonremitters (p = .047). Across 12 cortical and subcortical regions, 5-HT1A binding did not differ between remitters and nonremitters (p = .86). Serotonin 1A receptor binding was higher in MDD than control subjects across all regions (p = .0003). Remitters did not differ from nonremitters in several relevant clinical measures. Conclusions Elevated 5-HT1A binding in raphe nuclei is associated with subsequent remission with the selective serotonin reuptake inhibitor escitalopram; this is consistent with data from a separate cohort receiving naturalistic antidepressant treatment. We confirmed our previous findings of higher 5-HT1A binding in current MDD compared with

  18. MANAGING TIGHT BINDING RECEPTORS FOR NEW SPEARATIONS TECHNOLOGIES

    SciTech Connect

    DARYLE H BUSCH RICHARD S GIVENS

    2004-12-10

    Much of the earth's pollution involves compounds of the metallic elements, including actinides, strontium, cesium, technetium, and RCRA metals. Metal ions bind to molecules called ligands, which are the molecular tools that can manipulate the metal ions under most conditions. This DOE-EMSP sponsored program strives (1) to provide the foundations for using the most powerful ligands in transformational separations technologies and (2) to produce seminal examples of their applications to separations appropriate to the DOE EM mission. These ultra tight-binding ligands can capture metal ions in the most competitive of circumstances (from mineralized sites, lesser ligands, and even extremely dilute solutions), but they react so slowly that they are useless in traditional separations methodologies. Two attacks on this problem are underway. The first accommodates to the challenging molecular lethargy by developing a seminal slow separations methodology termed the soil poultice. The second designs ligands that are only tight-binding while wrapped around the targeted metal ion, but can be put in place by switch-binding and removed by switch-release. We envision a kind of molecular switching process to accelerate the union between metal ion and tight-binding ligand. Molecular switching processes are suggested for overcoming the slow natural equilibration rate with which ultra tight-binding ligands combine with metal ions. Ligands that bind relatively weakly combine with metal ions rapidly, so the trick is to convert a ligand from a weak, rapidly binding species to a powerful, slow releasing ligand--during the binding of the ligand to the metal ion. Such switch-binding ligands must react with themselves, and the reaction must take place under the influence of the metal ion. For example, our generation 1 ligands showed that a well-designed linear ligand with ends that readily combine, forms a cyclic molecule when it wraps around a metal ion. Our generation 2 ligands are even

  19. Dopaminergic and cholinergic learning mechanisms in nicotine addiction

    PubMed Central

    Subramaniyan, Manivannan

    2015-01-01

    Nicotine addiction drives tobacco use by one billion people worldwide, causing nearly six million deaths a year. Nicotine binds to nicotinic acetylcholine receptors that are normally activated by the endogenous neurotransmitter acetylcholine. The widespread expression of nicotinic receptors throughout the nervous system accounts for the diverse physiological effects triggered by nicotine. A crucial influence of nicotine is on the synaptic mechanisms underlying learning that contribute to the addiction process. Here, we focus on the acquisition phase of smoking addiction and review animal model studies on how nicotine modifies dopaminergic and cholinergic signaling in key nodes of the reinforcement circuitry: ventral tegmental area, nucleus accumbens (NAc), amygdala, and hippocampus. Capitalizing on mechanisms that subserve natural rewards, nicotine activates midbrain dopamine neurons directly and indirectly, and nicotine causes dopamine release in very broad target areas throughout the brain, including the NAc, amygdala, and hippocampus. In addition, nicotine orchestrates local changes within those target structures, alters the release of virtually all major neurotransmitters, and primes the nervous system to the influence of other addictive drugs. Hence, understanding how nicotine affects the circuitry for synaptic plasticity and learning may aid in developing reasoned therapies to treat nicotine addiction. PMID:26301866

  20. In vivo evidence against the existence of antiprogestins disrupting receptor binding to DNA.

    PubMed Central

    Delabre, K; Guiochon-Mantel, A; Milgrom, E

    1993-01-01

    The binding of a steroid hormone to its receptor elicits a sequence of events: activation of the receptor (probably through dissociation from a complex of heat shock proteins), dimerization, binding to hormone responsive elements, and finally modulation of gene transcription. RU 486, the first antiprogestin studied, has been shown to act at the last step of this sequence: provoking an inefficient binding of the receptor to hormone responsive elements. Recently, based on in vitro studies, it has been proposed that ZK 98299 was the prototype of a second class of antiprogestins that were supposed to act through disruption of the binding to DNA. We have devised methods allowing us to study the various steps of agonist or antagonist action in vivo. We show here that RU 486 and ZK 98299 have the same effects on receptor activation, dimerization, and binding to hormone responsive elements; differences in their action are explained by the 10-fold difference in their affinity for the receptor (ZK 98299 having the lower affinity). Images Fig. 2 Fig. 3 Fig. 4 PMID:8506282

  1. NK cell inhibitory receptor Ly-49C residues involved in MHC class I binding.

    PubMed

    Sundbäck, Jonas; Achour, Adnane; Michaëlsson, Jakob; Lindström, Hannah; Kärre, Klas

    2002-01-15

    Mouse NK cells express Ly-49 receptors specific for classical MHC class I molecules. Several of the Ly-49 receptors have been characterized in terms of function and ligand specificity. However, the only Ly-49 receptor-ligand interaction previously described in detail is that between Ly-49A and H-2D(d), as studied by point mutations in the ligand and the crystal structure of the co-complex of these molecules. It is not known whether other Ly-49 receptors bind MHC class I in a similar manner as Ly-49A. Here we have studied the effect of mutations in Ly-49C on binding to the MHC class I molecules H-2K(b), H-2D(b), and H-2D(d). The MHC class I molecules were used as soluble tetramers to stain transiently transfected 293T cells expressing the mutated Ly-49C receptors. Three of nine mutations in Ly-49C led to loss of MHC class I binding. The three Ly-49C mutations that affected MHC binding correspond to Ly-49A residues that are in contact or close to H-2D(d) in the co-crystal, demonstrating that MHC class I binding by Ly-49C is dependent on residues in the same area as that used by Ly-49A for ligand contacts.

  2. Opioid binding properties of the purified kappa receptor from human placenta

    SciTech Connect

    Ahmed, M.S.; Zhou, D.; Cavinato, A.G.; Maulik, D.

    1989-01-01

    A glycoprotein with a molecular weight of 63,000 has been purified, in an active form, from human placental villus tissue membranes. The binding properties of this glycoprotein to opioid alkaloids and peptides indicates that it is the kappa opiate receptor of human placenta. The receptor binds the tritiated ligands etorphine, bremazocine, ethylketocyclazocine and naloxone specifically and reversibly with Kd values of 3.3, 4.4, 5.1 and 7.0nM, respectively. The binding of /sup 3/H-Bremazocine to the purified receptor is inhibited by the following compounds with the corresponding Ki values EKC, 1.3 x 10/sup -8/M; Dynorphin 1-8, 3.03 x 10/sup -7/; U50,488H, 4.48 x 10/sup -9/; U69-593,2.28 x 10/sup -8/, morphine, 4.05 x 10/sup -6/ DADLE, 6.47 x 10/sup -6/ and naloxone, 2.64 x 10/sup -8/. The purified receptor binds 8 nmole of /sup 3/H-Etorphine and 1.7 nmole /sup 3/H-BZC per mg protein. The theoretical binding capacity of a protein of this molecular weight is 15.8. Although the iodinated purified receptor appears by autoradiography as one band on SDS-PAGE, yet homogeneity of the preparation is not claimed.

  3. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  4. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  5. Angiotensin II receptor binding in the rat hypothalamus and circumventricular organs during dietary sodium deprivation.

    PubMed

    Yamada, H; Mendelsohn, F A

    1989-10-01

    The effect of dietary sodium intake on angiotensin II (Ang II) receptor binding in the rat brain was studied using quantitative in vitro autoradiography. After 2 weeks of sodium deprivation, the peripheral angiotensin system was activated as shown by increased plasma renin activity (4-fold) and plasma aldosterone concentration (approximately 40-fold). At the same time, Ang II receptor binding in the adrenal glomerulosa zone increased by 40%. Frozen brain sections prepared from 12 male Sprague-Dawley rats (6 control, 6 sodium-deprived) were incubated with 125I[Sar1, Ile8] Ang II, exposed to X-ray film, and Ang II receptor binding in individual brain nuclei was quantitated by computerized densitometry. Ang II binding in the area postrema was significantly suppressed in the sodium-deprived rats (60% of control; p less than 0.05). No change was observed in the other circumventricular organs studied, the subfornical organ or organum vasculosum of the lamina terminalis. Ang II binding in the solitary tract nucleus was not affected by the dietary salt treatment. In the hypothalamic paraventricular nucleus, there was a small (9%) but significant (p less than 0.001) increase in Ang II receptor binding in the sodium-deprived group. However, no change was observed in the hypothalamic median preoptic or suprachiasmatic nuclei, areas with similarly high Ang II receptor binding. These results suggest that only a limited subset of brain Ang II receptors respond to sodium deprivation and do so in a region-specific manner. These results support evidence that the central angiotensin system may contribute to the regulation of fluid and electrolyte homeostasis.

  6. Ligand Binding and Subtype Selectivity of the Human A2A Adenosine Receptor

    PubMed Central

    Jaakola, Veli-Pekka; Lane, J. Robert; Lin, Judy Y.; Katritch, Vsevolod; IJzerman, Adriaan P.; Stevens, Raymond C.

    2010-01-01

    The crystal structure of the human A2A adenosine receptor bound to the A2A receptor-specific antagonist, ZM241385, was recently determined at 2.6-Å resolution. Surprisingly, the antagonist binds in an extended conformation, perpendicular to the plane of the membrane, and indicates a number of interactions unidentified before in ZM241385 recognition. To further understand the selectivity of ZM241385 for the human A2A adenosine receptor, we examined the effect of mutating amino acid residues within the binding cavity likely to have key interactions and that have not been previously examined. Mutation of Phe-168 to Ala abolishes both agonist and antagonist binding as well as receptor activity, whereas mutation of this residue to Trp or Tyr had only moderate effects. The Met-177 → Ala mutation impeded antagonist but not agonist binding. Finally, the Leu-249 → Ala mutant showed neither agonist nor antagonist binding affinity. From our results and previously published mutagenesis data, we conclude that conserved residues Phe-168(5.29), Glu-169(5.30), Asn-253(6.55), and Leu-249(6.51) play a central role in coordinating the bicyclic core present in both agonists and antagonists. By combining the analysis of the mutagenesis data with a comparison of the sequences of different adenosine receptor subtypes from different species, we predict that the interactions that determine subtype selectivity reside in the more divergent “upper” region of the binding cavity while the “lower” part of the binding cavity is conserved across adenosine receptor subtypes. PMID:20147292

  7. Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases.

    PubMed

    Floss, Doreen M; Klöcker, Tobias; Schröder, Jutta; Lamertz, Larissa; Mrotzek, Simone; Strobl, Birgit; Hermanns, Heike; Scheller, Jürgen

    2016-07-15

    The interleukin (IL)-12-type cytokines IL-12 and IL-23 are involved in T-helper (Th) 1 and Th17 immunity, respectively. They share the IL-12 receptor β1 (IL-12Rβ1) as one component of their receptor signaling complexes, with IL-12Rβ2 as second receptor for IL-12 and IL-23R for IL-23 signal transduction. Stimulation with IL-12 and IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase tyrosine kinase (Tyk) 2 associates with IL-12Rβ1, whereas Jak2 binds to IL-23R and also to IL-12Rβ2. Receptor association of Jak2 is mediated by Box1 and Box2 motifs located within the intracellular domain of the receptor chains. Here we define the Box1 and Box2 motifs in IL-12Rβ1 and an unusual Jak2-binding site in IL-23R by the use of deletion and site-directed mutagenesis. Our data show that nonfunctional box motifs abolish IL-12- and IL-23-induced STAT3 phosphorylation and cytokine-dependent proliferation of Ba/F3 cells. Coimmunoprecipitation of Tyk2 by IL-12Rβ1 and Jak2 by IL‑23R supported these findings. In addition, our data demonstrate that association of Jak2 with IL-23R is mandatory for IL-12 and/or IL-23 signaling, whereas Tyk2 seems to be dispensable.

  8. Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

    SciTech Connect

    Rivkees, S.A.; Carlson, L.L.; Reppert, S.M. )

    1989-05-01

    Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using {sup 125}I-labeled melatonin ({sup 125}I-Mel), a potent melatonin agonist. {sup 125}I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K{sub d} of 2.3 {plus minus} 1.0 {times} 10{sup {minus}11} M and 2.06 {plus minus} 0.43 {times} 10{sup {minus}10} M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)), significantly reduced the number of high-affinity receptors and increased the dissociation rate of {sup 125}I-Mel from its receptor. Furthermore, GTP({gamma}S) treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of {sup 125}I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M{sub r} > 400,000 and M{sub r} ca. 110,000. This elution profile was markedly altered by pretreatment with GTP({gamma}S) before solubilization; only the M{sub r} 110,000 peak was present in GTP({gamma}S)-pretreated membranes. The results strongly suggest that {sup 125}I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.

  9. Differentiation between ligand trapping into intact cells and binding on muscarinic receptors.

    PubMed

    Gossuin, A; Maloteaux, J M; Trouet, A; Laduron, P

    1984-05-22

    Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells. PMID:6722181

  10. Glycans on influenza hemagglutinin affect receptor binding and immune response

    PubMed Central

    Wang, Cheng-Chi; Chen, Juine-Ruey; Tseng, Yung-Chieh; Hsu, Che-Hsiung; Hung, Yu-Fu; Chen, Shih-Wei; Chen, Chin-Mei; Khoo, Kay-Hooi; Cheng, Ting-Jen; Cheng, Yih-Shyun E.; Jan, Jia-Tsrong; Wu, Chung-Yi; Ma, Che; Wong, Chi-Huey

    2009-01-01

    Recent cases of avian influenza H5N1 and the swine-origin 2009 H1N1 have caused a great concern that a global disaster like the 1918 influenza pandemic may occur again. Viral transmission begins with a critical interaction between hemagglutinin (HA) glycoprotein, which is on the viral coat of influenza, and sialic acid (SA) containing glycans, which are on the host cell surface. To elucidate the role of HA glycosylation in this important interaction, various defined HA glycoforms were prepared, and their binding affinity and specificity were studied by using a synthetic SA microarray. Truncation of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. The contribution of each monosaccharide and sulfate group within SA ligand structures to HA binding energy was quantitatively dissected. It was found that the sulfate group adds nearly 100-fold (2.04 kcal/mol) in binding energy to fully glycosylated HA, and so does the biantennary glycan to the monoglycosylated HA glycoform. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. Thus, removal of structurally nonessential glycans on viral surface glycoproteins may be a very effective and general approach for vaccine design against influenza and other human viruses. PMID:19822741

  11. Increased cytosolic androgen receptor binding in rat striated muscle following denervation and disuse

    NASA Technical Reports Server (NTRS)

    Bernard, P. A.; Fishman, P. S.; Max, S. R.; Rance, N. E.

    1984-01-01

    The effects of denervation and disuse on cytosolic androgen receptor binding by rat striated muscle are investigated. Denervation of the extensor digitorum longus and tibialis anterior muscles caused by a 40-50-percent increase in cytosolic androgen receptor concentration with no change in apparent binding affinity. This effect was evident at 6 h postdenervation, maximal at 24 h, and declined to 120 percent of the control level 72 h after denervation. A 40-percent increase in cytosolic androgen receptor concentration was also noted 24 hr after denervation of the hormone-sensitive levator ani muscle. The effect of denervation on androgen receptors was blocked by in vivo injection of cycloheximide; therefore, de novo receptor synthesis probably is not involved in the observed increase. Disuse, produced by subperineurial injection of tetrodotoxin into the tibial and common peroneal branches of the sciatic nerve, mimicked the effect of denervation on androgen receptor binding, suggesting that neuromuscular activity is important in regulation of receptor concentration. Possible mechanisms subserving this effect are discussed.

  12. Insulin binding and receptor tyrosine kinase activity in skeletal muscle of carnivorous and omnivorous fish.

    PubMed

    Párrizas, M; Planas, J; Plisetskaya, E M; Gutiérrez, J

    1994-06-01

    We characterized the insulin receptors in skeletal muscle from several fish species with different nutritional preferences: brown trout (Salmo trutta fario), gilthead sea bream (Sparus aurata), tilapia (Tilapia mossambica), and carp (Cyprinus carpio), semipurified by affinity chromatography (wheat germ agglutinin-agarose). Total specific binding and number of receptors per unit weight of piscine white skeletal muscle were lower than those values found in mammalian skeletal muscle. The same parameters in carp muscle receptor preparations were severalfold higher than in trout muscle (binding capacity 440 +/- 47 fmol/mg glycoprotein in carp and 82 +/- 23 fmol/mg glycoprotein in trout). Piscine insulin receptors phosphorylated exogenous substrate poly(Glu,Tyr) but less so than mammalian receptors. Tyrosine kinase activity of receptors, calculated as percent of 32P incorporated into substrate in the presence of insulin compared with basal incorporation, was also highest in carp (210 +/- 4%) and lowest in trout (150 +/- 2%). In both trout and carp deprived of food for 15 days, specific binding of insulin decreased. Nevertheless, differences between the two species were retained. Our results demonstrate that particular properties of insulin receptors in fish skeletal muscle may be related to nutritional preferences. This finding coincides with the phenomenon of differential glucose tolerance in fish: carnivorous fish, such as trout, are less tolerant, whereas omnivorous fish, such as carp, readily utilize a carbohydrate-rich diet. PMID:8024051

  13. Chlorpyrifos exposure during neurulation: cholinergic synaptic dysfunction and cellular alterations in brain regions at adolescence and adulthood.

    PubMed

    Qiao, Dan; Seidler, Frederic J; Abreu-Villaça, Yael; Tate, Charlotte A; Cousins, Mandy M; Slotkin, Theodore A

    2004-01-31

    The developmental neurotoxicity of chlorpyrifos (CPF) involves multiple mechanisms, thus rendering the immature brain susceptible to adverse effects over a wide window of vulnerability. Earlier work indicated that CPF exposure at the neural tube stage elicits apoptosis and disrupts mitotic patterns in the brain primordium but that rapid recovery ensues before birth. In the current study, we assessed whether defects in cholinergic synaptic activity emerge later in development. CPF was given to pregnant rats on gestational days 9-12, using regimens devoid of overt maternal or fetal toxicity. We then examined subsequent development of acetylcholine systems and compared the effects to those on general biomarkers of cell development. Choline acetyltransferase (ChAT), a constitutive marker for cholinergic nerve terminals, was increased in the hippocampus and striatum in adolescence and adulthood. In contrast, hemicholinium-3 (HC-3) binding to the presynaptic choline transporter, an index of nerve impulse activity, was markedly subnormal. Furthermore, m2-muscarinic cholinergic receptor binding was significantly reduced, instead of showing the expected compensatory upregulation for reduced neural input. CPF also elicited delayed-onset alterations in biomarkers of cell packing density, cell number, cell size and neuritic projections, involving brain regions both with and without reductions in indices of cholinergic activity. In combination with earlier results, the current findings indicate that the developing brain, and especially the hippocampus, is adversely affected by CPF regardless of whether exposure occurs early or late in brain development, and that defects emerge in adolescence or adulthood even in situations where normative values are initially restored in the immediate post-exposure period.

  14. Distinct ETA Receptor Binding Mode of Macitentan As Determined by Site Directed Mutagenesis

    PubMed Central

    Gatfield, John; Mueller Grandjean, Celia; Bur, Daniel; Bolli, Martin H.; Nayler, Oliver

    2014-01-01

    The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min) occupancy half-lives at the ETA receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ETA receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ETA receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ETA receptor-antagonist interaction modes, we performed functional studies using ETA receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ETA receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that – in contrast to bosentan and ambrisentan - macitentan-ETA receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and insurmountable antagonism. PMID

  15. Distinct ETA receptor binding mode of macitentan as determined by site directed mutagenesis.

    PubMed

    Gatfield, John; Mueller Grandjean, Celia; Bur, Daniel; Bolli, Martin H; Nayler, Oliver

    2014-01-01

    The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min) occupancy half-lives at the ET(A) receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET(A) receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET(A) receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET(A) receptor-antagonist interaction modes, we performed functional studies using ET(A) receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET(A) receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that--in contrast to bosentan and ambrisentan--macitentan-ET(A) receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and insurmountable

  16. A Mollusk Retinoic Acid Receptor (RAR) Ortholog Sheds Light on the Evolution of Ligand Binding

    PubMed Central

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Lima, Daniela; Pierzchalski, Keely; Jones, Jace W.; Kane, Maureen; Nishikawa, Jun-Ichi; Hiromori, Youhei; Nakanishi, Tsuyoshi; Santos, Miguel M.; Castro, L. Filipe C.; Bourguet, William

    2014-01-01

    Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms. PMID:25116705

  17. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

    PubMed Central

    Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  18. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    PubMed

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  19. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    PubMed

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.

  20. Immunochemical analysis of the glucocorticoid receptor: identification of a third domain separate from the steroid-binding and DNA-binding domains.

    PubMed Central

    Carlstedt-Duke, J; Okret, S; Wrange, O; Gustafsson, J A

    1982-01-01

    The glucocorticoid-receptor complex can be subdivided into three separate domains by limited proteolysis with trypsin or alpha-chymotrypsin. The following characteristics can be separated: steroid-binding activity (domain A), DNA-binding activity (domain B), and immunoactivity (domain C). We have previously reported the separation of the steroid-binding domain from the DNA-binding domain by limited proteolysis of the receptor with trypsin. In this paper, we report the detection by immunochemical analysis of a third domain of the glucocorticoid receptor, which does not bind hormone. Immunoactivity was detected by using specific antiglucocorticoid receptor antibodies raised in rabbits against purified rat liver glucocorticoid receptor and the assay used was an enzyme-linked immunosorbent assay. After digestion with alpha-chymotrypsin, the immunoactive region of the receptor (domain C) was separated from the other two domains (A and B). The immunoactive fragment was found to have a Stokes radius of 2.6 nm. Further digestion with alpha-chymotrypsin resulted in separation of the immunoactive fragment to give a fragment having a Stokes radius of 1.4 nm. The immunoactive domain could be separated from the half of the glucocorticoid receptor containing the steroid-binding and the DNA-binding domains (Stokes radius, 3.3 nm), by limited proteolysis of the receptor by alpha-chymotrypsin followed by gel filtration or chromatography on DNA-cellulose. PMID:6181503

  1. 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