Effect of diisopropylfluorophosphate on muscarinic and gamma-aminobutyric acid receptors in visual cortex of cats.

Administration of diisopropylfluorophosphate (DFP), an organophosphorus (OP) compound, irreversibly inhibits acetylcholinesterase (AChE) and results in cholinergic hyperactivity. This study investigated muscarinic and gamma-aminobutyric acid (GABA) receptor changes in visual cortex of cats following an acute exposure to DFP. A single acute administration of DFP (4 mg/kg) decreased the number of muscarinic receptors at 2, 10, and 20 hours after treatment. GABA receptors were elevated at 2 and 10 hours but returned to within control levels at 20 hours. No significant alteration in muscarinic or GABA receptor affinity was noted. In all cases cortical AChE activity was inhibited 60-90%. These findings show a down regulation of muscarinic receptors after DFP associated with low AChE activity. GABA receptors also are altered, and may be part of a compensatory mechanism to counteract excess cholinergic stimulation.     

Effect of acute and chronic cholinesterase inhibition with diisopropylfluorophosphate on muscarinic, dopamine, and GABA receptors of the rat striatum

The effects of acute and chronic administration of diisopropylfluorophosphate (DFP) to rats on acetylcholinesterase (AChE) activity (in striatum, medulla, diencephalon, cortex, and medulla) and muscarinic, dopamine (DA), and gamma-aminobutyric acid (GABA) receptor characteristics (in striatum) were investigated. After a single injection of (acute exposure to) DFP, striatal region was found to have the highest degree of AChE inhibition. After daily DFP injections (chronic treatment), all brain regions had the same degree of AChE inhibition, which remained at a steady level despite the regression of the DFP-induced cholinergic overactivity. Acute administration of DFP increased the number of DA and GABA receptors without affecting the muscarinic receptor characteristics. Whereas chronic administration of DFP for either 4 or 14 days reduced the number of muscarinic sites without affecting their affinity, the DFP treatment caused increase in the number of DA and GABA receptors only after 14 days of treatment; however, the increase was considerably lower than that observed after the acute treatment. The in vitro addition of DFP to striatal membranes did not affect DA, GABA, or muscarinic receptors. The results indicate an involvement of GABAergic and dopaminergic systems in the actions of DFP. It is suggested that the GABAergic and dopaminergic involvement may be a part of a compensatory inhibitory process to counteract the excessive cholinergic activity produced by DFP.     

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

Cholinergic hyperinnervation in the cerebral cortex of microencephalic rats does not result in muscarinic receptor down-regulation or in alteration of receptor-stimulated phosphoinositide metabolism

Administration of methylazoxymethanol (MAM; 25 mg/kg) to pregnant rats at gestational day 15 (GD 15) induces a marked reduction of telencephalic areas of the offspring brain. Previous neurochemical studies demonstrated a marked cholinergic hyperinnervation in the cerebral cortex of microencephalic rats. In this study we have evaluated whether this cholinergic hyperinnervation could result in altered functionality of muscarinic receptors. Acetylcholinesterase activity (AChE) was increased by 69% in the cerebral cortex of MAM treated rats confirming a relative hyperinnervation, whereas in the hippocampus and striatum no significant changes were observed. Despite the marked hyperinnervation, in the cerebral cortex of microencephalic rats neither muscarinic receptor-stimulated phosphoinositide metabolism nor muscarinic, receptor density were altered. No differences in receptor density were also observed in the hippocampus and striatum. Chronic diisopropylfluorophosphate (DFP) administration induced a marked decrease of AChE activity and down-regulation of muscarinic receptors whereas atropine administration resulted in receptor up-regulation in cerebral cortex, striatum and hippocampus of both control and MAM rats. The results confirm a relative cholinergic hyperinnervation in the cerebral cortex of microencephalic rats and demonstrate that the regulation of muscarinic receptor-stimulated phosphoinositide metabolism and muscarinic receptor plasticity is not modified in a condition of increased cholinergic presynaptic terminals.     

The role of dopamine in behavioral supersensitivity to muscarinic antagonists following cholinesterase inhibition

The role of brain dopamine (DA) in the enhancement of muscarinic antagonist-induced hyperactivity was investigated. The effects of atropine and scopolamine on the concentrations of DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), following DFP administration were determined. In control animals, atropine and scopolamine decreased the concentration of DA and increased the ratios of DOPAC/DA and HVA/DA in the striatum, but not in the N. accumbens - T. olfactorium (mesolimbic) area. Following a single dose of DFP, the two antimuscarinic drugs caused decreases of DA and further increases of the above ratios in both brain regions. However, following repeated DFP treatment for 2 weeks, these antimuscarinic drug-induced changes were observed only in the mesolimbic area, but not in the striatum. It is suggested that an increased DA turnover, indicated by elevated DOPAC/DA and HVA/DA ratios, underlies the muscarinic antagonist-induced hyperactivity. The well-known occurrence of muscarinic receptor down-regulation after DFP administration, could be responsible for the enhancement of the actions of muscarinic antagonists in DFP-treated animals. The observed differential effect on DA turnover in the two broad areas may involve both muscarinic and DA receptors.     

Lack of desensitization of muscarinic receptor-mediated second messenger signals in rat brain upon acute and chronic inhibition of acetylcholinesterase.

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor down-regulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP-induced muscarinic receptor down-regulation and receptor desensitization.     

Decrease in muscarinic acetylcholine receptors in the small intestine of mice subjected to repeated cold stress

The maximal contraction of the small intestine by acetylcholine greatly decreased during repeated cold stress. This change was mainly due to decrease in muscarinic receptors in small intestine, whose amounts were measured by the binding of 3-quinuclidinyl benzilate. Injection of norepinephrine or a tricyclic antidepressant, carpipramine during the exposure to the stress prevented this decrease in muscarinic receptors. The physiological significance of this phenomenon is discussed in relation to vagal hyperactivity under the stress.     

Lack of desensitization of muscarinic receptor–mediated second messenger signals in rat brain upon acute and chronic inhibition of acetylcholinesterase

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor downregulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP-induced muscarinic receptor down-regulation and receptor desensitization.     

Muscarinic receptor plasticity in the brain of senescent rats: down-regulation after repeated administration of diisopropyl fluorophosphate

Potential age-related differences in the response of Fischer 344 rats to subchronic treatment with diisopropylfluorophosphate (DFP) were evaluated in terms of brain cholinesterase (ChE) inhibition and muscarinic receptor sites. Male 3- and 24-month old rats were sc injected with sublethal doses of DFP (first dose 1.6, subsequent doses 1.1 mg/kg on alternate days) for 2 weeks and killed 48 hrs after the last treatment. In the cerebral cortex, hippocampus and striatum of control rats a significant age-related reduction of ChE and of maximum number of 3H-QNB binding sites (Bmax) was observed. The administration of DFP to senescent rats resulted in more pronounced and longer lasting syndrome of cholinergic stimulation, with marked body weight loss and 60% mortality. The percentage inhibition of brain ChE induced by DFP (over 80% in all regions) did not differ between young and senescent rats. As expected, in young rats DFP caused a significant decrease of Bmax (without apparent changes in affinity), which in the cerebral cortex reached about 40%. In the surviving senescent rats, the percentage decrease of Bmax due to DFP with respect to age-matched controls was very similar to that of young animals, especially in the cerebral cortex. Thus, there is great variability in the response of aged rats to DFP treatment, from total failure of adaptive mechanisms resulting in death to considerable muscarinic receptor plasticity. The data support the view that the ability of central neurotransmitter systems to compensate for pathological or xenobiotic induced insult is an essential part of the aging process.     

Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats

Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D₃ in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D₃ treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D₃ supplementation improved the behavioural deficit. In conclusion, vitamin D₃ shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D₃ modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D₃ supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D₃ and muscarinic M3 receptors in regulating insulin secretion from pancreas.     

Selective inactivation of muscarinic receptor subtypes

Muscarinic receptors exist in multiple subtypes, denoted as M₁, M₂ M₃ and M₄, encoded by four distinct but related genes. A fifth gene product, m5, has also been predicted although this sequence awaits a pharmacological equivalent. Many tissues express more than one muscarinic receptor subtype, which may couple to different intracellular effectors and thus have different physiological roles. One way to characterize the role of each receptor is to selectively inactivate one receptor population, thus pharmacologically ‘isolating’ the muscarinic receptor subtype of interest. Selective receptor inactivation can be achieved using either a selective, irreversible antagonist, or protection using a selective, reversible antagonist against a non-selective irreversible antagonist. Therefore, combination of these two approaches may provide optimal selective inactivation. Several muscarinic alkylating agents have been identified, including phenoxybenzamine, EEDQ (N-Ethoxycarbonyl-1-ethoxy-1,2-dihydroquinoline) and propylbenzilylcholine mustard. These irreversible antagonists do not, in general, discriminate between muscarinic receptor subtypes and are frequently used to estimate the affinity and relative efficacy of muscarinic agonists. Consequently, use of these irreversible antagonists provides estimations of the ‘receptor reserve’ associated with a response mediated by muscarinic receptor activation. In contrast, 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)piperidine) selectively inactivates M₃ receptors, but will not discriminate between M₁ M ₂ or M₄ receptors. In the absence of highly selective alkylating agents, receptor protection by reversible antagonists may be used. Thus, reversible antagonists, such as pirenzepine, methoctramine or para-fluorohexahydrosiladifenidol, at appropriate fractional receptor occupancies, may protect M₁ M₂ or M₃ receptors against alkylation by phenoxybenzamine. Selective alkylation of M₃ receptors by 4-DAMP mustard is enhanced with concurrent M₂ protection. This approach has been applied to defining the role of these muscarinic receptor subtypes in the control of ileal smooth muscle tone. These data suggest that, in ileum, M₂ receptors may act to inhibit β-adrenoceptor activation, thereby offsetting relaxation, while M₃ receptors directly mediate contraction.     

Muscarinic receptor reserve of airway smooth muscle and the response to isoproterenol

It has been hypothesized that the muscarinic receptor reserve for contraction of airway smooth muscle is an important determinant of the potency with which isoproterenol relaxes submaximal muscarinic contractions. The goals of this study were to inactivate, with phenoxybenzamine, a fraction of the muscarinic receptors present in canine tracheal smooth muscle, and then to determine whether this decrease in muscarinic receptor reserve altered the potency with which isoproterenol relaxed submaximal muscarinic contractions. Strips of smooth muscle were suspended from force transducers in vitro and preincubated with either vehicle (untreated) or phenoxybenzamine (10(-5) M) for 30 min. For muscarinic contractions induced by carbachol that were approximately 70-80% of maximum, the half-maximally effective concentration of isoproterenol was 2.4 +/- 0.8 x 10(-7) M for untreated strips but 5.8 +/- 1.3 x 10(-9) M for strips treated with phenoxybenzamine (n = 6, P less than 0.05). We concluded that treatment with phenoxybenzamine increased the sensitivity of a submaximal muscarinic contraction to isoproterenol. The results support the hypothesis that the muscarinic receptor reserve for contraction is an important determinant of the potency with which isoproterenol relaxes submaximal muscarinic contractions.     

Functional role of M2 muscarinic receptors in the guinea pig ileum

Muscarinic agonists elicit contraction in the standard guinea pig ileum bioassay through activation of M3 muscarinic receptors that are also linked to phosphoinositide hydrolysis. Surprisingly, the most abundant muscarinic receptor in the ileum is the M2 which causes a specific inhibition of cyclic AMP accumulation elicited by the beta-adrenergic receptor. After most of the M3 receptors are inactivated, the ileum still retains high sensitivity to muscarinic agonists provided that the contractile responses are measured in the presence of histamine and forskolin, which together, have no effect on contraction. Under these conditions, the potencies of antagonists for blocking the contractile response are consistent with those expected for an M2 response. Moreover, the muscarinic contractile response measured in the presence of histamine and forskolin after inactivation of M3 receptors is pertussis toxin sensitive. In contrast, muscarinic contractions in the standard bioassay are pertussis toxin insensitive. These results demonstrate that the M2 muscarinic receptor can cause an indirect contraction of the guinea pig ileum by preventing the relaxing effect of agents that increase cAMP.     

Selective Alkylation of Rat Urinary Bladder Muscarinic Receptors with 4-DAMP Mustard Reveals a Contractile Function for the M2 Muscarinic Receptor

Abstract

Our previous data indicate that M₃ muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M₃ receptors with 4-DAMP mustard and suggest that the M₂ receptor subtype may be involved in inhibition of β-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M₃ receptors and 65% of M₂ receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M₃ receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCI, indicated a contractile function for the M₂ receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M₂ subtype in bladder smooth muscle contraction.     

Long-term regulation of muscarinic acetylcholine receptors on cultured nerve cells.

Incubation of mouse neuroblastoma cells (clone 1LE-115) with the muscarinic agonist, carbachol, resulted in a time-dependent desensitization to muscarinic receptor-mediated cyclic GMP formation and a decrease in the number and affinity of muscarinic receptors as measured by the binding to intact cells of the muscarinic antagonist, [³H]quinuclidinyl benzilate (QNB). The decrease in responsiveness to cyclic GMP formation reached a maximum after a 15-minute exposure to 1 mM carbachol (short-term desensitization) whereas changes in [³H] QNB binding became apparent only after a one hour exposure and reached a maximum after about 12 hrs (long-term desensitization). Recovery of sensitivity after short-term desensitization was rapid, suggesting that this process may involve a conformational change in the muscarinic receptor. In contrast, recovery after long-term desensitization was prolonged and could be slowed by the inhibition of protein synthesis. These results indicate that different cellular mechanisms are involved in the short-term and long-term desensitization of muscarinic receptors.     

Expression of Cholinergic,Insulin, Vitamin D Receptors and GLUT 3 in the Brainstem of Streptozotocin Induced Diabetic Rats: Effect of Treatment with Vitamin D3

Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B_max of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, α7 nicotinic acetylcholine, insulin, Vitamin D₃ receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and α7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D₃ treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D₃ in managing neurological disorders associated with diabetes.     

Himbacine discriminates between putative muscarinic M1 receptor-mediated responses

This study describes the antagonistic properties of himbacine, in comparison with those of pirenzepine, at muscarinic receptors mediating the depolarization of rat superior cervical ganglion, the inhibition of electrically-induced twitch contractions of rabbit vas deferens and the contraction of dog saphenous vein, currently classified as putative muscarinic M₁ sites. The affinity of himbacine for the vas deferens site (pA₂ 8.08) was nearly ten times higher than those for the M₁ receptors of rat ganglion and dog saphenous vein (pA₂ 7.14 and 7.16, respectively); affinity estimates for pirenzepine were similar throughout the different preparations. The present data are consistent with the allocation of ganglion and saphenous vein receptors into the M₁ subclass; the profile of the vas deferens site, conversely, appears to be different, and possibly more closely related to that reported for the M₄/m4 receptor.     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

Effects of electrical stimulation of the parasympathetic nerve on the levels of cholinergic muscarinic and beta-adrenergic receptors and cyclic nucleotides in rat salivary glands

Density of muscarinic receptors of rat parotid gland, although unchanged after 5 or 10 min of stimulation of the parasympathetic nerve to the gland, showed a decrease of 23% following a 15-min period of stimulation. After 30 min the decrease was 19% but by 60 min density of receptors returned to within 5% of receptor density of the unstimulated gland; there was virtually no change in density of beta adrenoceptors at any time during the 60 min of stimulation. Markedly elevated (30-fold increase) levels of cyclic GMP appeared within 5 min after initiation of nerve stimulation and remained at this level at 10 min, but dropped from 90 to 46 pmol/mg total protein by 15 min, the time at which a decrease in muscarinic receptors first was evident. GMP levels continued to decrease but were still four times basal levels after 60 min of stimulation and did not return to normal concentration until 120 min. Cyclic AMP was generally unchanged. These changes in muscarinic receptors and cyclic GMP are apparently closely related to the kind of neural stimulation, unlike the condition when stimulation of the sympathetic nerve was employed.     

Interaction between the M2- and M3-Receptor Subtypes in Muscarinic Electrical and Mechanical Responses of Intestinal Smooth Muscles

In various gastrointestinal smooth muscles, two different muscarinic receptor subtypes, M₂ and M₃, are expressed; these receptors are the target for the parasympathetic neurotransmitter acetylcholine. Although the number of M₂ receptors is much greater than that of M₃ receptors, the functional role of the former receptor subtype has yet to be fully defined, since pharmacological analyses of the contractile responses to acetylcholine and other muscarinic agonists have revealed that such responses are mediated extensively by the minor M₃ subtype. The M₃ receptor links to Ca²⁺ store release, and the released Ca²⁺ ions may contribute to the contraction. However, many studies indicated the importance of Ca²⁺ influx through voltage-gated Ca²⁺ channels, rather than Ca²⁺ release, in muscarinic contractions, since the contractile responses are markedly inhibited by Ca²⁺ channel blockers. The major M₂ receptors link to the opening of cationic channels leading to the membrane depolarization, which in turn activates voltage-gated Ca²⁺ channels. Thus, there should be somewhere a point of contact between the M₃- and M₂-mediated signal transductions, as if M₃ receptor stimulation is connected with membrane depolarization. Our electrophysiological and pharmacological findings suggest that the M₂-mediated cationic channel opening and a resulting increase in the membrane electrical activity are the primary mechanism for mediating the contractile response to muscarinic agonists. An allosteric interaction between M₂ and M₃ receptors such that M₃ activation intensifies the M₂/cation channel pathway may account at least in part for the failure of many previous analyses to detect M₂ participation in the contractile responses to full agonists.