Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.

Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells.

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.

Hemodynamic consequences of chronic parasympathetic blockade with a peripheral muscarinic antagonist.

Whereas the sympathetic nervous system has a well-established role in blood pressure (BP) regulation, it is not clear whether long-term levels of BP are affected by parasympathetic function or dysfunction. We tested the hypothesis that chronic blockade of the parasympathetic nervous system has sustained effects on BP, heart rate (HR), and BP variability (BPV). Sprague-Dawley rats were instrumented for monitoring of BP 22-h per day by telemetry and housed in metabolic cages. After the rats healed from surgery and a baseline control period, scopolamine methyl bromide (SMB), a peripheral muscarinic antagonist, was infused intravenously for 12 days. This was followed by a 10-day recovery period. SMB induced a rapid increase in mean BP from 98 +/- 2 mmHg to a peak value of 108 +/- 2 mmHg on day 2 of the SMB infusion and then stabilized at a plateau value of +3 +/- 1 mmHg above control (P < 0.05). After cessation of the infusion, the mean BP fell by 6 +/- 1 mmHg. There was an immediate elevation in HR that remained significantly above control on the last day of SMB infusion. SMB also induced a decrease in short-term (within 30-min periods) HR variability and an increase in both short-term and long-term (between 30-min periods) BPV. The data suggest that chronic peripheral muscarinic blockade leads to modest, but sustained, increases in BP, HR, and BPV, which are known risk factors for cardiovascular morbidity.

Propiverine and metabolites: differences in binding to muscarinic receptors and in functional models of detrusor contraction.

Propiverine is a commonly used antimuscarinic drug used as therapy for symptoms of an overactive bladder. Propiverine is extensively biotransformed into several metabolites that could contribute to its spasmolytic action. In fact, three propiverine metabolites (M-5, M-6 and M-14) have been shown to affect various detrusor functions, including contractile responses and L-type calcium-currents, in humans, pigs and mice, albeit with different potency. The aim of our study was to provide experimental evidence for the relationship between the binding of propiverine and its metabolites to human muscarinic receptor subtypes (hM(1)-hM(5)) expressed in chinese hamster ovary cells, and to examine the effects of these compounds on muscarinic receptor-mediated detrusor function. Propiverine, M-5, M-6 and M-14 bound to hM(1)-hM(5) receptors with the same order of affinity for all five subtypes: M-6 > propiverine > M-14 > M-5. In HEK-293 cells expressing hM(3), carbachol-induced release of intracellular Ca(2+) ([Ca(2+)](i)) was suppressed by propiverine and its metabolites; the respective concentration-response curves for carbachol-induced Ca(2+)-responses were shifted to the right. At higher concentrations, propiverine and M-14, but not M-5 and M-6, directly elevated [Ca(2+)](i). These results were confirmed for propiverine in human detrusor smooth muscle cells (hDSMC). Propiverine and the three metabolites decreased detrusor contractions evoked by electric field stimulation in a concentration-dependent manner, the order of potency being the same as the order of binding affinity. We conclude that, in comparison with the parent compound, loss of the aliphatic side chain in propiverine metabolites is associated with higher binding affinity to hM(1)-hM(5) receptors and higher functional potency. Change from a tertiary to a secondary amine (M-14) results in lower binding affinity and reduced potency. Oxidation of the nitrogen (M-5) further lowers binding affinity as well as functional potency.

Enhancing effects of nicotine and impairing effects of scopolamine on distinct aspects of performance in computerized attention and working memory tasks in marmoset monkeys.

With the CAmbridge Neuropsychological Test Automated Battery (CANTAB), computerized neuropsychological tasks can be presented on a touch-sensitive computer screen, and this system has been used to assess cognitive processes in neuropsychiatric patients, healthy volunteers, and species of non-human primate, primarily the rhesus macaque and common marmoset. Recently, we reported that the common marmoset, a small-bodied primate, can be trained to a high and stable level of performance on the CANTAB five-choice serial reaction time (5-CSRT) task of attention, and a novel task of working memory, the concurrent delayed match-to-position (CDMP) task. Here, in order to increase understanding of the specific cognitive demands of these tasks and the importance of acetylcholine to their performance, the effects of systemic delivery of the muscarinic receptor antagonist scopolamine and the nicotinic receptor agonist nicotine were studied. In the 5-CSRT task, nicotine enhanced performance in terms of increased sustained attention, whilst scopolamine led to increased omissions despite a high level of orientation to the correct stimulus location. In the CDMP task, scopolamine impaired performance at two stages of the task that differ moderately in terms of memory retention load but both of which are likely to require working memory, including interference-coping, abilities. Nicotine tended to enhance performance at the long-delay stage specifically but only against a background of relatively low baseline performance. These data are consistent with a dissociation of the roles of muscarinic and nicotinic cholinergic receptors in the regulation of both sustained attention and working memory in primates.

Blood-brain barrier and electromagnetic fields: effects of scopolamine methylbromide on working memory after whole-body exposure to 2.45 GHz microwaves in rats.

We first verified that our 12-arm radial maze test enabled demonstration of memory deficits in rats treated with the muscarinic antagonist scopolamine hydrobromide (0.5mg/kg, i.p.). We then investigated whether a systemically-injected quaternary-ammonium derivate of this antagonist (scopolamine methylbromide; MBR), which poorly crosses the blood-brain barrier (BBB), altered maze performance after a 45-min exposure to 2.45 GHz electromagnetic field (EMF; 2 micros pulse width, 500 pps, whole-body specific energy absorption rate [SAR] of 2.0 W/kg, +/-2dB and brain averaged SAR of 3.0 W/kg, +/-3 dB); if observed, such an alteration would reflect changes in BBB permeability. The drug was injected before or after exposure. Controls were naive rats (no experience of the exposure device) and sham-exposed rats (experience of the exposure device without microwaves). In a final approach, rats were subjected to i.v. injections of Evans blue, a dye binding serum albumin, before or after EMF exposure. Whether scopolamine MBR was injected before or after exposure, the exposed rats did not perform differently from their naive or sham-exposed counterparts. Thus, EMFs most probably failed to disrupt the BBB. This conclusion was further supported by the absence of Evans blue extravasation into the brain parenchyma of our exposed rats.

Carbamoylcholine homologs: novel and potent agonists at neuronal nicotinic acetylcholine receptors.

The classic muscarinic acetylcholine receptor (mAChR) agonist carbamoylcholine (carbachol) does not seem to be the most obvious lead for the development of selective ligands at nicotinic acetylcholine receptors (nAChRs). In the past, however, N-methylations of carbachol have provided N-methylcarbamoylcholine and N,N-dimethylcarbamoylcholine (DMCC), which predominantly display nicotinic activity. In this study, 12 homologous analogs of DMCC and its corresponding tertiary amine, N,N-dimethylcarbamoyl-N,N-dimethylaminoethanol, were synthesized and their binding affinities to native mAChR and nAChR sites estimated. One of the compounds in the series, 3-N,N-dimethylaminobutyl-N,N-dimethylcarbamate (7), displayed low nanomolar binding affinity to nAChRs and a 400-fold selectivity for nAChRs over mAChRs. Hence, a new series of compounds was synthesized in which alkyl and aryl groups and different ring systems were introduced in the carbamate moiety of 7. In a [3H]epibatidine binding assay, the Ki values of 7 and its analogs at rat alpha2beta2, alpha4beta2, alpha2beta4, alpha3beta4, and alpha4beta4 nAChRs, stably expressed in mammalian cell lines, ranged from low nanomolar to midmicromolar concentrations, whereas all of the compounds displayed weak binding to an alpha7/5-HT3 chimera and to native mAChRs. Compound 7 and its analogs were determined to be agonists at the alpha3beta4 nAChR subtype. This series includes the most potent and selective nicotinic agonists structurally derived from ACh to date. Furthermore, the compounds are tertiary amines, implying some advantages in terms of bioavailability pertinent to future in vivo pharmacological studies. Finally, observations made in the study hold promising perspectives for future development of ligands selective for specific nAChR subtypes.

Comparison of the pharmacological antagonism of M2 and M3 muscarinic receptors expressed in isolation and in combination.

We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M(2) and M(3) muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [3H]N-methylscopolamine binding to CHO cells expressing both M(2) and M(3) muscarinic receptors (CHO M(2)+M(3) cells), the competition curves of the subtype-selective muscarinic antagonists were consistent with a two-site model. One site exhibited binding properties identical to those of CHO M(2) cells, whereas the other site exhibited properties like those of CHO M(3) cells. Oxotremorine-M, a muscarinic agonist, elicited a robust, pertussis toxin-insensitive stimulation of phosphoinositide hydrolysis in both CHO M(3) and CHO M(2)+M(3) cells, but not in CHO M(2) cells. The pharmacological antagonism of the phosphoinositide response exhibited similar properties in both CHO M(3) and CHO M(2)+M(3) cells. Oxotremorine-M elicited a pertussis toxin-sensitive, robust inhibition of forskolin-stimulated cyclic AMP (cAMP) accumulation in both CHO M(2) and CHO M(2)+M(3) cells and a less robust inhibition in CHO M(3) cells. At higher concentrations, oxotremorine-M elicited an increase in cAMP accumulation over the maximal inhibition noted at lower concentrations in both CHO M(3) and CHO M(2)+M(3) cells. Following pertussis toxin treatment, only the stimulatory phase of the cAMP response to oxotremorine-M was observed in CHO M(2), CHO M(3), and CHO M(2)+M(3) cells. The pharmacological antagonism of the cAMP response in CHO M(2)+M(3) cells resembled that expected for a response mediated independently by both M(2) and M(3) receptors.

Dual effects of muscarinic M(2) acetylcholine receptors on the synthesis of cyclic AMP in CHO cells: dependence on time, receptor density and receptor agonists.

1. Muscarinic M(2) receptors normally inhibit the production of cyclic AMP via G(i) proteins, but a stimulatory component occurs in their effect at high agonist concentrations, believed to be based on the activation of G(s) proteins. We investigated the conditions which determine the occurrence and extent of the stimulatory component in CHO cells stably expressing muscarinic M(2) receptors. 2. Biphasic concentration-response curves (decline followed by return towards control values) were obtained after 10 min incubation with carbachol, oxotremorine-M, acetylcholine, arecoline and arecaidine propargyl ester, but the upward phase was missing with oxotremorine, methylfurmethide, furmethide and pentylthio-TZTP. Shortening the incubation favoured the occurrence of the stimulatory component. Carbachol (1 mM) and oxotremorine-M (1 mM) brought about net stimulation (above 100% of control) of cyclic AMP synthesis during 2 min incubations. The stimulatory components disappeared after the density of receptors had been lowered with oxyphenonium mustard. 3. All agonists stimulated the synthesis of cyclic AMP in cells pretreated with pertussis toxin. 4. Most differences between agonists regarding the stimulatory component of their effect on cyclic AMP synthesis could be explained by differences in their efficacy and the induced receptor internalization. 5. We propose that the G(s)-mediated stimulatory component of the effect of muscarinic M(2) receptors on cyclic AMP synthesis only occurs if the density of activated receptors is high enough to saturate the G(i) proteins and proportionate to the receptors' low affinity for the G(s) proteins. It tends to be abolished by receptor internalization.

Inhibition of acetylcholine muscarinic M(1) receptor function by the M(1)-selective ligand muscarinic toxin 7 (MT-7).

MT-7 (1 - 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M(1) receptor, inhibited the acetylcholine (ACh)-stimulated [(35)S]-guanosine-5'-O-(3-thio)triphosphate ([(35)S]-GTPgammaS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M(1) receptor subtype. MT-7 failed to affect the ACh-stimulated [(35)S]-GTPgammaS binding in membranes of CHO cells expressing either the M(2), M(3) or M(4) receptor subtype. In N1E-115 neuroblastoma cells endogenously expressing the M(1) and M(4) receptor subtypes, MT-7 (0.3 - 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to affect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. In both CHO/M(1) and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC(50) value. In CHO/M(1) cell membranes, MT-7 (0.05 - 25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [(3)H]-N-methylscopolamine ([(3)H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [(3)H]-NMS by about 5 fold. CHO/M(1) cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [(3)H]-NMS binding for at least 8 h at 30 degrees C. It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic M(1) receptors by binding stably to an allosteric site.

Cholinergic receptor up-regulates COX-2 expression and prostaglandin E(2) production in colon cancer cells.

The M(3) muscarinic cholinergic receptor has important physiological functions on normal colonic cells. It is frequently expressed on human colon cancer cells and is biologically active. Although it is mitogenic in certain cell models, the importance of this receptor on colon carcinogenesis is unknown. In the present study we have determined expression of the M(3) receptor on human colon cancer tissue compared with matched normal tissue and examined the downstream effect of receptor activation in the HT-29 human colon carcinoma cell line. Using reverse transcription-PCR, M(3) receptor RNA expression was detected in all matched colon carcinoma and normal specimens from eight patients. Five of the eight (62%) patients showed an up to 8-fold greater level of M(3) receptor expression in cancer compared with the matched normal tissue. Exposure of HT-29 cells to carbachol, a stable receptor agonist, results in a 10-fold increase in cyclooxygenase-2 (COX-2) protein. This induction of COX-2 protein was dose dependent and was inhibited by the cholinergic receptor antagonist N-methylscopolamine (NMS). Carbachol caused a dose-dependent increase in prostaglandin E(2) (PGE(2)), the main product of cyclooxygenase activity. The maximum stimulatory effect (40-fold increase) was noted with 1mM carbachol. The increase in PGE(2) was completely abolished by NMS and by the COX-2 selective inhibitor NS398. This suggests that the M(3) receptor mediates PGE(2) production by a mechanism involving COX-2. As COX-2 and PGE(2) are known promoters of gastrointestinal cancer, these data suggest that M(3) receptor activation may facilitate progression of colon carcinoma, in part by a COX-2-mediated cellular mechanism.

Probing the selectivity of allosteric modulators of muscarinic receptors at other G-protein-coupled receptors.

1. The aim of the present investigation was to analyse whether three prototype allosteric modulators of ligand binding to muscarinic receptors, i.e. alcuronium, gallamine, and the alkane-bis-ammonium compound W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropylammonium bromide]), may have allosteric effects on radioligand-binding characteristics at other G-protein-coupled receptors, such as cerebral A1 adenosine receptors (Gi-coupled), cardiac left ventricular alpha1-adrenoceptors (Gq), and beta-adrenoceptors (Gs). 2. The modulators were applied at concentrations known to be high with regard to the allosteric delay of the dissociation of the antagonist [3H]-N-methylscopolamine (NMS) from muscarinic M2-receptors: 30 micromol l(-1) W84, 30 micromol l(-1) alcuronium, 1000 micromol l(-1) gallamine. As radioligands, we used the adenosine A1-receptor ligand [3H]-cyclopentyl-dipropylxanthine (CPX), the alpha1-adrenoceptor ligand [3H]-prazosin (PRAZ), and the beta-adrenoceptor ligand (-)-[125I]-iodocyanopindolol (ICYP). Allosteric actions on ligand dissociation and the equilibrium binding were measured in the membrane fractions of rat whole forebrain (CPX) and of rat cardiac left ventricle (PRAZ, ICYP, NMS), respectively. 3. CPX and PRAZ showed a monophasic dissociation with half-lives of 5.88+/-0.15 and 12.27+/-0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influenced by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36+/-2% of the control value with alcuronium and to 42+/-2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately accelerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and after 3 h, more than 50% of the radioligand was still bound, so that a reliable half-life could not be calculated. ICYP dissociation was not affected by W84. In the presence of alcuronium and gallamine, the dissociation curve of ICYP revealed an initial drop from the starting level, followed by the major phase of dissociation being parallel to the control curve. 4. In summary, the allosteric action of the applied agents is not a common feature of G-protein-coupled receptors and appears to be specific for muscarinic receptors.

Functional role of muscarinic M(2) receptors in alpha,beta-methylene ATP induced, neurogenic contractions in guinea-pig ileum.

1. The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist alpha,beta-methylene ATP (mATP) were investigated in guinea-pig ileum. 2. mATP (0.1 - 30 microM) elicited a concentration-dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non-selective muscarinic receptor antagonist N-methylscopolamine (NMS). 3. The contractile response to mATP was pertussis toxin-insensitive, irreversibly antagonized by N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), and unaffected by the muscarinic M(2)/M(4) receptor selective antagonist AF-DX 116 (1 microM). 4. When measured in the presence of histamine and isoproterenol after treatment with 4-DAMP mustard, mATP elicited a pertussis toxin-sensitive contractile response potently antagonized by AF-DX 116. 5. Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M(3) receptor and an indirect contractile response through the muscarinic M(2) receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction.

Scopolamine prevents tolerance to the effects of caffeine on rotational behavior in 6-hydroxydopamine-denervated rats.

Continuous administration of caffeine has been shown to induce tolerance to its psychostimulant effects. In this study, using unilateral 6-hydroxydopamine nigrostriatal denervated rats, we tested the hypothesis that the muscarinic receptor antagonist, scopolamine, would prevent the tolerance to caffeine-induced contralateral rotational behavior. For that purpose we administered either caffeine (40 mg/kg) plus saline or scopolamine (5, 10 and 20 mg/kg) plus saline, as well as caffeine in combination with the various doses of scopolamine for 7 consecutive days, and measured ipsilateral and contralateral rotational behavior. The results showed that acute injections of scopolamine plus saline produced similar levels of both ipsilateral and contralateral turning, while caffeine produced more contralateral than ipsilateral turning. Tolerance to caffeine-induced contralateral turning was observed as of the second administration, while scopolamine plus saline injections did not produce significant changes in rotational behavior with repeated treatment. Scopolamine co-administered with caffeine significantly attenuated the increased contralateral turning produced by acute injections of caffeine plus saline, but significantly prevented the tolerance effects with repeated administration. These findings strongly suggest that muscarinic cholinergic processes may be involved in tolerance to caffeine-induced contralateral turning. The results are interpreted in terms of the possible interactions between dopamine, adenosine and acetylcholine neurotransmitter systems within the basal ganglia circuitry involved in motor behavior.

Selectivity profile of muscarinic toxin 3 in functional assays of cloned and native receptors.

By using acetylcholine-induced stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding to membrane G proteins as a functional assay of the cloned human m1-m4 muscarinic receptor subtypes stably expressed in Chinese hamster ovary cells, muscarinic toxin 3 (MT3) was found to block the m4 receptor with a potency (pA2 = 8.33) much higher than those displayed at the m1 (pA2 = 6.78), m3 (pA2 = 6.3), and m2 (pA2 < 6.3) subtypes. In N1E-115 cells, which have been reported to express m4 receptors coupled to inhibition of cAMP, MT3 potently antagonized the carbachol-induced inhibition of adenylyl cyclase with a pA2 of 8. 81 and displayed monophasic inhibitory curves. Unexpectedly, in NG108-15 cells, known to express only m4 receptors, MT3 counteracted the carbachol inhibition of adenylyl cyclase with a lower potency (pA2 = 7.60) and showed a biphasic inhibitory curve, suggesting the participation of both m4 and m2 receptors. This possibility was supported by radioligand binding data showing that MT3 failed to completely displace the binding of [3H]N-methylscopolamine to NG108-15 cell membranes and by reverse transcription-polymerase chain reaction analysis, revealing the presence of mRNAs for both m4 and m2 receptor subtypes. These data demonstrate that MT3 possesses a high functional receptor selectivity for both the cloned and native m4 receptors and that in cell systems containing m4 and m2 receptors coupled to a common response, the toxin constitutes a powerful tool to resolve the relative contribution by each receptor subtype.

Heterologous regulation of muscarinic and beta-adrenergic receptors in rat cardiomyocytes in culture.

Previous work indicated that hyperstimulation of muscarinic receptors brings about profound changes not only in the density of the muscarinic receptors, but also of the beta-adrenoceptors in rat heart atria in vivo. We have now investigated whether a similar receptor cross-regulation occurs in cardiomyocytes in vitro. Cardiomyocytes from 3-4 day old rats were exposed to chemical agents on days 5-6 in culture. Densities of muscarinic and beta-adrenergic receptors were measured according to the binding of N-[3H]methylscopolamine and [ H]CGP 12177, respectively, to cell surface membranes and cell homogenates. Exposure of cells to the muscarinic agonist carbachol (1 mmol/l) brought about a profound decrease in the number of muscarinic receptors. The number of beta-adrenoceptors displayed biphasic changes, being augmented after 24 h (by 20-45% on the cell surface and by 29% in the homogenate) and diminished after 48 h and 72 h (after 48 h, decrease by 44-75% on the cell surface and by 36% in the homogenate). These effects of carbachol were not prevented by dimethylaminopropyl-bis-indolylmaleimide, the inhibitor of protein kinase C. Exposure of cells to the beta-adrenoceptor agonist isoprenaline (0.1 mmol/l) strongly diminished the number of beta-adrenoceptors on the cell surface and in the homogenate. The density of muscarinic receptors on the cell surface was diminished by 24-43% after 24 h exposure to isoprenaline and unchanged after 48 h, whereas the concentration of muscarinic receptors in the homogenate was unchanged after 24 h and increased by 20% after 48 h. The isoprenaline-induced decrease in the density of cell surface muscarinic receptors could not be simulated by forskolin and was not abolished by the protein kinase A inhibitors Rp-cAMPS and HA-1004. Dibutyryl cyclic AMP diminished the density of cell surface muscarinic receptors more than that of the beta-adrenergic receptors. Our data reveal a novel phenomenon of a biphasic change (an increase followed by a loss) in the density of beta-adrenoceptors during exposure of cardiocytes to carbachol. Activation of beta-adrenoceptors brings about less conspicuous changes in the density of muscarinic receptors. The observed phenomena of receptor cross-regulation cannot be explained by simple activations of protein kinases A and C.

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor.

1. Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells. 2. In rat striatum, clozapine (1 nM-10 microM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 microM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA2 value of 7.51. Moreover, clozapine (1 microM) failed to inhibit dopamine D1 receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [3H]-N-methylscopolamine ([3H]-NMS) bound to striatal M4 receptors with a monophasic inhibitory curve and a pKi value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5'-O-(thio)triphosphate (GTPgammaS). 3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC50 of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 microM (EC50=50 nM) and stimulating at higher concentrations (EC50 = 7 microM). Clozapine (0.3-5 microM) antagonized the CCh stimulation of cyclic AMP with a pKi value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes. 4. In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 microM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum. 5. As in rat striatum, in CHO cell membranes the displacement of [3H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPgammaS. 6. Clozapine (10 nM-10 microM) had a small stimulant effect (approximately 20%) on the binding of [35S]-GTPgammaS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM-5 microM) antagonized the CCh-stimulated [35S]-GTPgammaS binding with a pA2 value of 7.48. 7. These results show that at the striatal M4 receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.