Quinuclidinyl Benzilate Binding Research Articles

Unexpected decrease in in vivo binding of [3H]QNB in the mouse cerebral cortex in the developing brain - A comparison with [11C]NMPB

IntroductionSignificant discrepancies between in vitro and in vivo binding of the muscarinic receptor ligand – 3H-labeled Quinuclidinyl Benzilate (QNB) – have been well documented. Discernable in vivo cerebellar [3H]QNB binding has been observed in mouse brain, despite the maximum number of binding sites (Bmax) being low. In order to understand this unique in vivo binding phenomenon, the binding of two muscarinic receptor ligands – [3H]QNB and N-[11C]methylpiperidyl Benzilate ([11C]NMPB) – were compared in vivo and in vitro in 3- and 8-week-old mice. MethodIn vitro binding parameters of [3H]QNB were determined using brain homogenates. The time course of radioactivity concentration (TACs) in the cerebral cortex and cerebellum was measured following injection of [3H]QNB and [11C]NMPB with or without 3 mg/kg of carrier QNB in 3- and 8 week old mice using a dual tracer administration technique. A graphical method was employed for the quantitative analysis of in vivo binding of these radioligands. ResultsIn vitro, the available number of binding sites for cerebral cortical muscarinic receptors increased by 17% during the developmental period studied. Paradoxically, in vivo, we observed a decrease of [3H]QNB binding in the cerebral cortex, while [11C]NMPB binding was markedly increased. In vivo saturation analysis of [3H]QNB in 3-week-old mice revealed an apparent positive cooperativity of binding in the cerebral cortex. ConclusionsOur results support the hypothesis that microenvironmental factors proximal to muscarinic receptors cause a local decrease in the cortical free-ligand concentration of [3H]QNB and that this ‘ligand barrier’ is modulated during brain development. Advances in knowledgeThe present study demonstrates that the combined use of radiolabeled QNB and NMPB has the potential to reveal the important effects of receptor microenvironmental factors on receptor function in the living brain.

Ketamine prevents seizures and reverses changes in muscarinic receptor induced by bicuculline in rats

The cholinergic system has been implicated in several experimental epilepsy models. In a previous study bicuculline (BIC), known to antagonize GABA-A postsynaptic receptor subtype, was administered to rats at subconvulsant (1mg/kg) and convulsant (7.5mg/kg) doses and quinuclidinyl benzilate ([3H]-QNB) binding to CNS membranes was determined. It was observed that ligand binding to cerebellum increases while it decreases in the case of hippocampus. Saturation binding curves showed that changes were due to the modification of receptor affinity for the ligand without alteration of binding site number. The purpose of this study was to assay muscarinic receptors employing other BIC dose (5mg/kg), which induces seizures and allows the analysis of a postseizure stage as well. To study further muscarinic receptor involvement in BIC induced seizures, KET was also employed since it is a well known anticonvulsant in some experimental models. The administration of BIC at 5mg/kg to rats produced a similar pattern of changes in [3H]-QNB binding to those recorded with 1.0 and 7.5mg/kg doses. Here again, changes were observed in receptor binding affinity without alteration in binding site number for cerebellum or hippocampus membranes. Pretreatment with KET (40mg/kg) prevented BIC seizures and reverted [3H]-QNB binding changes induced by BIC administration. The single administration of KET invariably resulted in [3H]-QNB binding decrease to either cerebellar or hippocampal membranes. KET added in vitro decreased ligand binding likewise. Results of combined treatment with KET plus BIC are hardly attributable to the single reversion of BIC effect since KET alone invariably decreased ligand binding. It is suggested that besides alteration of cholinergic muscarinic receptor other(s) neurotransmitter system(s) may well also be involved.

Muscarinic receptors on rat ileal villus and crypt cells.

Ileal villus and crypt cells exhibit morphological and biochemical differences which may be responsible for functional differences in relation to ion transport. Cholinergic agonists act directly on epithelial cell muscarinic receptors, but it is not known if these receptors exist on both villus and crypt cells. Using the potent muscarinic antagonist [3H](-)-quinuclidinyl benzilate (QNB) we have determined the distribution of muscarinic receptors in rat ileal villus and crypt cells. Plasma membrane preparations from ileal villus and crypt cells possessed a specific, saturable, and high affinity QNB binding site with apparent dissociation constants of 0.23 +/- 0.05 and 0.21 +/- 0.04 nM (mean +/- s.e., n = 6) and densities of 92.2 +/- 2.8 and 90.1 +/- 16.2 fmol (mg protein)-1, respectively. Both types of cells showed similar potencies for agonist and antagonist competition of QNB binding. The muscarinic receptors in membrane fractions from villus cells were found primarily on the basolateral membrane rather than on the brush border membrane. Secretion induced by cholinergic stimulation of the small intestine might, therefore, be due to an effect on both villus and crypt cells as both types contain muscarinic receptors. Furthermore, such stimulation also may result in mucin secretion, as goblet cells were present in the preparation we studied, and receptors on these cells may have contributed to the amount of binding of [3H]QNB.

Influence of pinacolyl dimethylphosphinate on soman storage in rats.

Atropinized rats pretreated with pinacolyl dimethylphosphinate (PDP) or saline were intoxicated with 6 X LD50 soman and, after 30 and 60 min, the absolute amounts of soman in diaphragms and lungs were measured gas chromatographically. The absolute amounts of free soman in both types of tissues were similar and were decreased after 60 min intoxication compared with the 30 min intoxication period. Pretreatment of rats with PDP reduced the amount of soman in the tissues by 63-78%. Incubation of isolated diaphragms and lungs with sodium fluoride (2.5 mM) increased the amount of free soman in these tissues by 2.6 X 10(3) and 16 X 10(3)%, respectively. An assay to study the binding of tritiated PDP was developed. Although this assay was suitable to study the binding of tritiated quinuclidinyl benzilate to rat brain, no binding between tritiated PDP and diaphragm or brain tissue could be detected. It was concluded that PDP may counteract soman storage by another mechanism than occupation of binding sites.

Modulation of benzodiazepine receptor, adrenoceptor and muscarinic receptor by diazepam in rat parotid gland

This study investigated the influence of diazepam on the binding characteristics of adrenoceptor, muscarinic and benzodiazepine receptors in rat parotid gland membrane using a radioligand binding assay. At a concentration of > 10 − 6 M, diazepam competed with [ 3H]dihydroalprenolol for β-adrenoceptor, but not [ 3H]prazosin for α-adrenoceptor or [ 3H]quinuclidinyl benzilate for muscarinic receptor. Continuous administration of diazepam at doses of 0.4 mg/kg/day, i.p. for 7 days in rat significantly decreased pilocarpine (4.0 mg/kg, i.p.)-induced parotid salivary flow. Diazepam also produced a significant increase in the dissociation constant (Kd) value for [ 3H]dihydroalprenolol binding, but no change in the maximal binding capacity (Bmax) value, and a decrease in the Kd value for [ 3H]diazepam binding to benzodiazepine receptors, but no change in the Kd or Bmax values for [ 3H]prazosin or [ 3H]quinuclidinyl benzilate binding. These results suggest that continuous administration of diazepam modifies affinity for β-adrenoceptor and benzodiazepine receptor binding sites in parotid gland membrane and that changes in these binding sites may be closely related to diazepam-induced suppression of salivary secretion.

Neurotensin modulates central muscarinic receptors, an effect which does not involve the high-affinity neurotensin receptor (NTS1)

Neurotensin (NT) is a tridecapeptide distributed in central and peripheral nervous systems, which can behave as a neurotransmitter or neuromodulator at central and peripheral levels. Herein we tested the potential effect of this peptide on quinuclidinyl benzilate ([ 3H]-QNB) binding to muscarinic receptor in rat CNS membranes. It was observed that NT decreased up to 50–70% ligand binding at 1 × 10 − 7 M–1 × 10 − 5 M concentration in cerebral cortex, cerebellum and striatum. In the hippocampus, NT exerted a biphasic effect, behaving as a stimulator in the presence of 1 × 10 − 12 M–1 × 10 − 10 M concentration but as an inhibitor at 1 × 10 − 8 M–1 × 10 − 5 M concentration. In order to test the involvement of high-affinity NT receptor (NTS1) in NT inhibitory effect, assays were carried out in the presence of 1 × 10 − 6 M NT and/or SR 48692 (Sanofi-Aventis, U.S., Inc.), a specific antagonist for this receptor, dissolved in dimethylsulfoxide (DMSO) 10% v/v. As controls, membranes incubated with DMSO and/or NT 1 × 10 − 6 M plus DMSO were processed. It was found that NT + DMSO decreased [ 3H]-QNB binding to cerebral cortex, cerebellum and hippocampal membranes by 49%, 32% and 53%, respectively. This inhibition was not observed with the DMSO control group. Membrane preincubation with 1 × 10 − 6 M SR 48692 failed to alter NT effect on binding. SR 48692 at 1 × 10 − 6 M concentration decreased the binding by 50% only in cerebral cortex membranes, suggesting a possible direct effect of the antagonist on muscarinic receptors in this area. It was therefore concluded that the high-affinity NT receptor may not be involved in ligand binding inhibition to muscarinic receptor by NT.

Comparison of Receptor Binding Characteristics of Commonly Used Muscarinic Antagonists in Human Bladder Detrusor and Mucosa

Recent studies have described muscarinic receptors on the mucosa and the detrusor of the human urinary bladder. Muscarinic receptor antagonists are effective in the treatment of overactive bladder (OAB), but their site(s) of action and actual therapeutic target are unclear. Our aim was to compare, in human bladder mucosa and detrusor, the radioligand binding characteristics of newer, clinically effective agents: darifenacin, its hydroxylated metabolite UK-148,993, fesoterodine, solifenacin, tolterodine, and trospium. Specimens were collected from asymptomatic patients (50-72 years old) undergoing open bladder surgery. Radioligand binding studies with the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) were performed separately on detrusor and mucosal membranes. All antagonists displayed high affinity when competing for [3H]QNB binding in both detrusor and mucosa. Inhibition constants were also obtained for all antagonists against individual muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Here, fesoterodine showed anomalous binding results, suggesting that some conversion to its metabolite had occurred. Global nonlinear regression analysis of bladder binding data with five antagonists demonstrated 82% low-affinity sites in mucosa and 78% low-affinity sites in detrusor, probably representing M(2)/M(4) receptors. There was an excellent correlation (r(2) = 0.99) of low-affinity global estimates between detrusor and mucosa, whereas the corresponding high-affinity estimates ( approximately 20% of sites) were dissimilar. In conclusion, commonly used and clinically effective muscarinic receptor antagonists bind to receptors located on the bladder mucosa and the detrusor, providing support for the hypothesis that muscarinic receptors in the mucosa may represent an important site of action for these agents in OAB.

Trypanosoma cruzi infection induces up-regulation of cardiac muscarinic acetylcholine receptors in vivo and in vitro

The pathogenesis of chagasic cardiomyopathy is not completely understood, but it has been correlated with parasympathetic denervation (neurogenic theory) and inflammatory activity (immunogenic theory) that could affect heart muscarinic acetylcholine receptor (mAChR) expression. In order to further understand whether neurogenic and/or immunogenic alterations are related to changes in mAChR expression, we studied two models of Trypanosoma cruzi infection: 1) in 3-week-old male Sprague Dawley rats chronically infected with T. cruzi and 2) isolated primary cardiomyocytes co-cultured with T. cruzi and peripheral blood mononuclear cells (PBMC). Using [3H]-quinuclidinylbenzilate ([3H]-QNB) binding assays, we evaluated mAChR expression in homogenates from selected cardiac regions, PBMC, and cultured cardiomyocytes. We also determined in vitro protein expression and pro-inflammatory cytokine expression in serum and cell culture medium by ELISA. Our results showed that: 1) mAChR were significantly (P < 0.05) up-regulated in right ventricular myocardium (means +/- SEM; control: 58.69 +/- 5.54, N = 29; Chagas: 72.29 +/- 5.79 fmol/mg, N = 34) and PBMC (control: 12.88 +/- 2.45, N = 18; Chagas: 20.22 +/- 1.82 fmol/mg, N = 19), as well as in cardiomyocyte transmembranes cultured with either PBMC/T. cruzi co-cultures (control: 24.33 +/- 3.83; Chagas: 43.62 +/- 5.08 fmol/mg, N = 7 for both) or their conditioned medium (control: 37.84 +/- 3.84, N = 4; Chagas: 54.38 +/- 6.28 fmol/mg, N = 20); 2) [(3)H]-leucine uptake was increased in cardiomyocytes co-cultured with PBMC/T. cruzi-conditioned medium (Chagas: 21,030 +/- 2321; control 10,940 +/- 2385 dpm, N = 7 for both; P < 0.05); 3) plasma IL-6 was increased in chagasic rats, IL-1beta, was increased in both plasma of chagasic rats and in the culture medium, and TNF-alpha level was decreased in the culture medium. In conclusion, our results suggest that cytokines are involved in the up-regulation of mAChR in chronic Chagas disease.

A snake venom phospholipase A 2 with high affinity for muscarinic acetylcholine receptors acts on guinea pig ileum

A group of small proteins, designated as muscarinic toxins (MTs), have been isolated from the venom of African green mamba ( Dendroaspis angusticeps) and documented to bind selectively to individual muscarinic acetylcholine receptor (mAChR) subtypes. These components have less been reported to be isolated from other snake venoms. In this study, we have isolated a snake factor with high affinity for mAChR from the venom of Naja atra (Chinese cobra) by column chromatography on Sephadex G-50, Sephadex G-150, CM-Sepharose Fast Flow and Poros ®CM 4.6/100 Perfusion Chromatography Column. The final preparation was homogeneous as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and HPLC. The isolated active component, which was designated muscarinic protein (MP), was found to displace [ 3H]quinuclidinyl benzilate binding to rat cortex synaptosomes in a dose-dependent manner, and the K i value estimated was 10.1 nM. The isolated MP was determined to have a molecular weight of 13.3 kDa and an N-terminal amino acid sequence of NLYQFKNMIQCTVPSR, which is highly homologous with phospholipase A 2 from the venoms of genus Naja. The N. atra MP could hydrolyze phosphatidylcholine in a dose-dependent manner. In guinea-pig ileum, MP produced an onset and dose-dependent contraction, which could be reversed by atropine indicating the involvement of mAChR. The EC 50 value of MP for guinea-pig ileum contraction was estimated as 30 nM, and the maximum contraction caused by MP was approximately 43% of that obtained from carbachol. These results seem to suggest that the snake venom phospholipase A 2 may not only have high affinity for mAChRs but also have the ability to activate mAChRs. However, it is possible that the toxin caused the contraction in the guinea-pig ileum by inducing acetylcholine release via another mechanism.

Insect Muscarinic Acetylcholine Receptor: Pharmacological and Toxicological Profiles of Antagonists and Agonists

The insect muscarinic acetylcholine receptor (mAChR) is evaluated as a potential target for insecticide action. The mammalian M2/M4-selective antagonist radioligand [3H]AF-DX 384 (a pirenzepine analogue) binds to Drosophila mAChR at a single high-affinity site identical to that for the nonselective antagonist [3H]quinuclidinyl benzilate (QNB) and with a pharmacological profile distinct from that of all mammalian mAChR subtypes. Three nonselective antagonists (QNB, scopolamine, and atropine) show the highest affinity (Ki=0.5-2.4 nM) at the Drosophila target, and AF-DX 384 and M3-selective 4-DAMP (dimethyl-4-(diphenylacetoxy)piperidinium iodide) rank next in potency (Ki=5-18 nM). Eleven muscarinic antagonists generally exhibit higher affinity than eight agonists. On injection into houseflies, the antagonists 4-DAMP and (S)-(+)-dimethindene produce suppressed movement, the agonist (methyloxadiazolyl)quinuclidine causes knockdown and tremors, and all of them inhibit [3H]QNB binding ex vivo, indicating possible mAChR-mediated intoxication. The insect mAChR warrants continuing study in lead generation to discover novel insecticides.

Acetylcholine receptor binding and cholinergic interneuron density are unaltered in a genetic animal model of primary paroxysmal dystonia

The underlying pathophysiological mechanisms of hereditary types of paroxysmal dyskinesias are still unknown, but basal ganglia dysfunctions seem to play a critical role. In fact, numerous pharmacological, neurochemical, immunohistochemical and electrophysiological investigations in the dt sz hamsters, a unique rodent model of age-dependent primary paroxysmal dystonia, revealed alterations within the basal ganglia, particularly of the GABAergic and dopaminergic neurotransmitter systems. A deficit in several types of striatal GABAergic interneurons in dt sz mutant hamsters seems to play a crucial pathophysiological role, but deficits in other types of striatal interneurons cannot be excluded by previous studies. In view of ameliorating effects of anti-cholinergic drugs in dystonic patients, we therefore investigated the density of striatal cholinergic interneurons in the present study. These interneurons were marked specifically by the enzyme choline acetyltransferase and counted by using a stereological counting method in a blinded fashion. Additionally, acetylcholine receptor binding was determined in mutant and nondystonic control hamsters by autoradiographic analyses with the nonselective muscarinic ligand [ 3H]-quinuclidinyl benzilate (QNB) in 11 brain (sub)regions. There were no significant differences in the density of striatal cholinergic interneurons between dt sz mutant hamsters (789 ± 39 interneurons/mm 3) and nondystonic controls (807 ± 36 interneurons/mm 3). [ 3H]QNB binding was also comparable between mutant and control hamsters. These results point to an unaltered striatal cholinergic neurotransmitter system in dt sz hamsters under basal conditions.

Mercury but not Organochlorines Inhibits Muscarinic Cholinergic Receptor Binding in the Cerebrum of Ringed Seals (Phoca hispida)

Elevated concentrations of organochlorines and mercury (Hg) have been reported in marine mammals on a global scale. While risk assessments are generally based on quantifying body burdens of toxicants, much less is known about associated adverse health effects and their underlying mechanisms. The purpose of this study was to characterize the inhibitory effects of methylmercury (MeHg+), mercuric chloride (Hg2+), p,p′-DDT, Arochlor 1254, chlordane, dieldrin, lindane, and toxaphene on [3H]quinuclidinyl benzilate ([3H]-QNB) binding to the muscarinic cholinergic (mACh) receptor in cellular membranes isolated from the cerebrum of ringed seals (Phoca hispida). [3H]-QNB binding to the mACh receptor was saturable with a mean receptor density (B max) of 826.9 ± 68.4 fmol/mg and ligand affinity (K d) of 0.31 ± 0.04 nM. MeHg+ and Hg2+ were the only neurotoxicants that inhibited radioligand binding by greater than 50%. Hg2+ was significantly more potent at inhibiting mACh receptor binding than MeHg+ when the IC50 data were compared (IC50 = 1.92 ± 0.06 μM versus 2.75 ± 0.22 μM), but when the data were normalized to derive inhibition constants (K i) there was no statistical difference in inhibition (Hg2+ = 1.38 ± 0.07 μM; MeHg+ = 1.26 ± 0.12 μM). Toxaphene also inhibited mACh receptor binding by 22.4%, but this was only significant at the highest concentration tested (320 μM). Overall, these data suggest that Hg, and not organochlorines, inhibits ligand binding to the mACh receptor. These mechanistic findings may be used to support and develop specific biomarkers of Hg exposure and neurotoxicity in sensitive ecological species. This study was funded by Collaborative Mercury Research Network (COMERN) and National Sciences and Engineering Research Council (NSERC) of Canada Discovery Research Grants to H. M. Chan. Samples were collected under the Northern Contaminants Program, Canada. Assistance by Christopher J. Stamler, Sonja Ostertag, and Donna Leggee is greatly appreciated.

Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression

The ability of tumor cells to stimulate adaptive immunity, particularly by inducing anti-tumor antibodies (Abs), has been extensively reviewed. LM3 is a tumorigenic cell line derived from a murine mammary metastatic adenocarcinoma that spontaneously overexpressed mAchR. Here we investigate the ability of Abs purified from the sera of LM3 tumor-bearing mice, directed against muscarinic acetylcholine receptors (mAchR) to modulate tumor cells' proliferation and angiogenesis. We observed that IgG from early tumor bearers (ETB), 14-day LM3 tumor, and from late tumor bearers (LTB), 28-day LM3 tumor, displaced tritiated quinuclidinyl benzilate binding to LM3 tumor cells, confirming Abs interaction with cholinoceptors, while IgG from normal mice did not modify the antagonist binding to mAchR at any concentration tested. In addition, Abs from ETB and LTB immunoblotted a protein of 70 kDa on murine tumor cells and on heart homogenates that was also recognized by a specific anti-M 2 receptor monoclonal antibody. We also observed that IgG purified from ETB-stimulated LM3 cells' proliferation in a more effective manner than the muscarinic agonist carbachol (CARB) did. IgG from LTB-potentiated LM3 cells induced angiogenesis by increasing the number of blood vessels and VEGF-A production in peritumoral skin “via” mAchR, in an agonist similar manner. All effects were blocked by preincubating cells with the non-selective antagonist atropine. In conclusion, autoAbs purified from LM3 tumor-bearing mice sera exert different pro-tumor actions depending on the stage of tumor development: in ETB, they stimulate tumor cells' proliferation, while in LTB they potentiate tumor neovascularization.

Gβγ Inhibits Gα GTPase-activating Proteins by Inhibition of Gα-GTP Binding during Stimulation by Receptor

Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.

Aceclidine and pilocarpine interact differently with muscarinic receptor in isolated rabbit iris muscle

The relationship between muscarinic receptor affinity states and the contractile response to the muscarinic agonists carbachol, aceclidine, and pilocarpine, has been examined in the isolated rabbit iris muscle. Contraction of the iris muscle by carbachol and aceclidine was more potent and/or more efficacious than the response to pilocarpine. Analysis of [ 3H]-Quinuclidinyl benzilate (QNB) binding showed that while both carbachol and aceclidine bound to high- and low-affinity forms of the muscarinic receptor, pilocarpine bound to one affinity state. The efficacy of carbachol and aceclidine to stimulate contraction of the iris muscle was consistent with receptor occupancy theory only when considering the low-affinity state of the muscarinic receptor, and activation of the low-affinity rather than high-affinity binding state of the receptor is likely to mediate the contraction of iris muscle. Therefore, the typical anti-glaucoma muscarinic agonists aceclidine and pilocarpine may interact differently with their target receptors in isolated rabbit iris muscle.

Effects of Estrogen on Muscarinic Acetylcholine Receptors in the Rat Hippocampus

The aim of the present study was to investigate whether different estrogen manipulations have effects on the expression of muscarinic acetylcholine receptors (mAChRs) in the adult female rat hippocampus. Hippocampus was obtained from rats in proestrus (control), ovariectomized for 2, 10 and 15 days, ovariectomized for 15 days and treated with 17β-estradiol for 7 days, and treated with 17β-estradiol immediately after ovariectomy for 21 days. Rats’ estrogen status was monitored by measuring estradiol plasma levels and uterus relative weight. [³H]quinuclidinyl benzilate ([³H]QNB) binding studies indicated that ovariectomy time-dependently increases the number of mAChRs in hippocampus when compared to those obtained from control rats. Estradiol treatments for 21 days avoid the effect of ovariectomy. However, the estradiol treatments for 7 days after 15 days of ovariectomy slightly change the number of mAChRs. In conclusion, these results showed that ovariectomy time-dependently increases mAChRs number in the rat hippocampus. In addition, these data suggest that treatment with estradiol initiated within a specific period of time after the loss of ovarian function may be effective at preventing specific effects of hormone deprivation on hippocampus.

Effects of testosterone on muscarinic acetylcholine receptors in the rat epididymis

The effect of testosterone on the expression of muscarinic acetylcholine receptor (mAChR) subtypes was studied in the rat epididymis, at mRNA and protein level. The rat androgen status was monitored by measuring plasma testosterone level and caput and cauda epididymis wet weight. Ribonuclease protection assay (RPA) and [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding assay were performed in the caput and cauda epididymis from control (50-day old), castrated, castrated and treated with testosterone and sexually immature (30-day old) rats. The expression of each mAChR transcript subtype differed depending on the epididymal region analyzed and rat testosterone and/or testicular factors status. In control rats, RPA showed the presence of mRNA for M 1, M 2 and M 3 mAChR in the caput and cauda epididymis. The abundance of m2 and m3 transcripts in the cauda was higher than that in the caput epididymis. Low amount of m1 transcript was observed in both regions. Orchidectomy increased m1 mRNA amount in the caput and cauda epididymis when compared to control rats, an effect slightly modified by testosterone replacement. Although orchidectomy down-regulated the level of m2 transcript in both epididymal regions, castration significantly increased m3 mRNA amount in the caput region. These effects on m2 and m3 transcripts were prevented by testosterone replacement to castrated rats. Similar abundance of m3 transcript, however, was detected in the cauda epididymis of all experimental group tested. [ 3H]QNB binding studies revealed that orchidectomy down-regulated the number of mAChR detected in both epididymal regions, an effect also prevented by testosterone replacement. Thus, testosterone and/or testicular factors may play a role in the regulation of mAChR expression in the rat epididymis.

The effects of the neurosteroids: pregnenolone, progesterone and dehydroepiandrosterone on muscarinic receptor-induced responses in Xenopus oocytes expressing M1 and M3 receptors

The neurosteroids pregnenolone, progesterone, and dehydroepiandrosterone (DHEA) occur naturally in the nervous system. They act on neural tissues, participate in neuronal signaling, and are reported to alter neuronal excitability via nongenomic mechanisms. Muscarinic receptors have important roles in neuronal functions in the brain and autonomic nervous system. In this study, we investigated the effects of pregnenolone, progesterone, and DHEA on M(1) and M(3) muscarinic receptors using the Xenopus oocyte expression system. Pregnenolone and progesterone inhibited the acetylcholine (ACh)-mediated responses of M(1) and M(3) receptors expressed in Xenopus oocytes, whereas DHEA did not. The half-maximal inhibitory concentrations (IC(50)) for pregnenolone inhibition of M(1) receptor- and M(3) receptor-mediated currents were 11.4 and 6.0 microM respectively; the IC(50) values for progesterone inhibition of M(1) receptor- and M(3) receptor-mediated currents were 2.5 and 3.0 microM respectively. The selective protein kinase C (PKC) inhibitor GF109203X had little effect on the pregnenolone or progesterone inhibition of the ACh-induced currents in Xenopus oocytes expressing M(1) or M(3) receptors. The inhibitory effects of pregnenolone and progesterone were overcome at higher concentrations of ACh. Pregnenolone and progesterone inhibited the [(3)H]quinuclidinyl benzilate (QNB) binding to M(1) and M(3) receptor expressed in Xenopus oocytes, and Scatchard plot analysis of [(3)H]QNB binding revealed that pregnenolone and progesterone altered the K(d) value and the B(max), indicating noncompetitive inhibition. In conclusion, pregnenolone and progesterone inhibited M(1) and M(3) receptor functions noncompetitively by the mechanism independent of PKC and by interfering with ACh binding to the receptors.

An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl 2) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl 2 and MeHg on [ 3H]-quinuclidinyl benzilate ([ 3H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density ( B max) and ligand affinity ( K d). Subsequently, samples were exposed to HgCl 2 or MeHg to derive IC50 values and inhibition constants ( K i). Results demonstrate that HgCl 2 is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [ 3H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [3H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [3H]QNB was saturable, reversible and of high affinity (KD = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M3 receptors and a much smaller population (12%) exhibiting characteristics of M2 receptors. The binding curve for the displacement of [3H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (KD = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (KD = 4.4 microM). When oxotremorine displacement of [3H]QNB binding was determined in the presence GTP gamma S, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [32P]NAD caused the [32P]-labelling of a 40 dD protein that may represent the alpha subunit(s) of G proteins that are known to by NAD-ribosylated by the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)