Expression of muscarinic acetylcholine receptors (M1-, M2-, M3- and M4-type) in the neuromuscular junction of the newborn and adult rat

Using intracellular recording and immunohistochemistry, we studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release in the neuromuscular junctions of the newborn (3-6 days postnatal) and adult (30-40 days) rat. In the Levator auris longus muscles of both newborn and adult rats, acetylcholine release was modified by the M1-receptor selective antagonists pirenzepine (10 microM) and MT-7 (100 nM) and by the M2-receptor selective antagonists methoctramine (1 microM) and AF-DX 116 (10 microM). The M4-receptor selective antagonists tropicamide (1 microM) and MT-3 (100 nM) can also modify the neurotransmitter release in certain synapses of the newborn muscles. The neurotransmitter release was not altered by the M3-receptor selective antagonist 4-DAMP (1 microM) in the adult or newborn rats. However, we directly demonstrate by immunocytochemistry the presence of these receptors in the motor endplates and conclude that M1-, M2-, M3- and M4-type muscarinic receptors are present in all the neuromuscular junctions of the rat muscle both in newborn and adult animals. These receptors may be located in the perisynaptic glial cell as well as at the nerve terminals.     

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.     

The Intact Human Neuroblastoma Cell (SH-SY5Y) Exhibits High-Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols

The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.     

A Point Mutation in the Coding Sequence of the β-Hexosaminidase α Gene Results in Defective Processing of the Enzyme Protein in an Unusual GM2-Gangliosidosis Variant

The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine [( 3H]NMS) and [3H]quinuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki = 869-1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 [11-2[[2-[(diethylamino)methyl]-1-piperidinyl]- acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) bound to the mAChRs with a very low affinity (Ki = 6.0 microM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki = 7.2 nM). 4-DAMP was also 1,000-fold more effective than AF-DX 116 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilycholine mustard) suggest that the size of the SK-N-SH mAChR (Mr = 81,000-98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr = 66,000), an M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.     

Distribution of muscarinic receptor subtypes in rat brain as determined in binding studies with AF-DX 116 and pirenzepine

In vitro competition binding experiments with the selective muscarinic antagonists AF-DX 116 and pirenzepine (PZ) vs 3H-N-methylscopolamine as radioligand revealed a characteristic distribution of muscarinic receptor subtypes in different regions of rat brain. Based on non linear least squares analysis, the binding data were compatible with the presence of three different subtypes: the M1 receptor (high affinity for PZ), the cardiac M2 receptor (high affinity for AF-DX 116) and the glandular M2 receptor (low affinity for PZ and AF-DX 116). The highest proportion of M1 receptors was found in the hippocampus, whilst the cerebellum and the hypothalamus were the regions with the largest fraction of the cardiac M2 and glandular M2 receptors, respectively. In certain brain areas, depending on the relative proportions of the subtypes, flat binding curves were seen for AF-DX 116 and PZ. Based on these data, an approximate distribution pattern of the subtypes in the various brain regions is presented.     

Characterization of the subtype of muscarinic receptor coupled to the stimulation of phosphoinositide hydrolysis in 132-1N1 human astrocytoma cells.

Stimulation of muscarinic receptors increases phosphoinositide (PI) hydrolysis in 132-1N1 human astrocytoma cells. To evaluate the subtype of receptors which mediate PI hydrolysis in 132-1N1 cells, the effects of: a) the nonselective M1 agonist, carbachol; b) the selective M1 agonist, 4-hydroxy-2-butynyl-trimethylammonium chloride-m-chlorocarbinilate (McN-343); c) the nonselective antagonists, atropine and scopolamine; d) the relatively selective M1 antagonist, pirenzepine; e) the relatively selective M2 antagonists, AF-DX 116 (11-2-diethylaminomethyl-1-piperidinylacetyl-5, 11-dihydro-6H-pyrido-2,3-b-1,4-benzodiazepine-6-one) and methoctramine and f) the relatively selective M3 antagonist, hexahydrosila-difenidol (HHSiD) on PI hydrolysis in 132-1N1 cells were studied. The cell pools of inositol-phospholipids were prelabelled by incubating 132-1N1 cells in a low inositol containing medium (CMRL-1066) supplemented with [3H]inositol (2 microCi/ml) for 20-24 hours at 37 degrees C. The cells were washed and resuspended in a physiological salt solution, and PI hydrolysis was measured by accumulation of [3H]inositol-1-phosphate (IP) in the presence of 10 mM LiCl. Carbachol produced time and concentration dependent PI hydrolysis (EC50, 37 microM). McN-A343 did not cause significant hydrolysis of PI in 132-1N1 cells indicating that the receptor was not of M1 type. All the above muscarinic antagonists caused a concentration dependent decrease in the level of IP in response to carbachol (100 microM). The rank order of their affinities (pA2 values) was: atropine (8.8) > HHSiD (7.6) > pirenzepine (6.8) > methoctramine (6.0) > AF-DX 116 (5.8). This rank order supports the concept that M3 (other names, M2 beta, glandular M2) receptors are linked to PI hydrolysis in 132-1N1 cells. HHSiD, which is selective for M3 receptors of the smooth muscle has higher affinity for muscarinic receptors in 132-1N1 cells than AF-DX 116 which is selective for M2 receptors in cardiac tissue. If the receptor in 132-1N1 cells had been M2, part of the rank order for affinities would have been methoctramine > AF-DX 116 > HHSiD > pirenzepine. From all of these observations, the muscarinic receptor for PI hydrolysis in 132-1N1 cells is tentatively characterized as of M3 type.     

The muscarinic receptor subtype in mouse pancreatic B-cells

Isolated mouse islets were used to identify the muscarinic receptor subtype present in pancreatic B-cells. We thus compared the inhibitory potencies of atropine (non-specific), of pirenzepine (specific for M1 receptors) and of compound AF-DX 116 (specific for cardiac M2 receptors) on acetylcholine-induced insulin release, 86Rb+ efflux and 45Ca2+ efflux. The three antagonists inhibited all effects of acetylcholine, but EC50 values were markedly different: atropine = 1.5-5 nM, pirenzepine = 0.6-1.7 microM and AF-DX 116 = 1.7-11 microM. The results did not suggest that the various effects of ACh could result from the activation of different subtypes of receptors. It is concluded that muscarinic receptors of pancreatic B-cells belong to an M2 subtype distinct from the cardiac M2 receptors.     

Characterization of muscarinic receptor subtypes in human tissues

The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with [3H]Pirenzepine and [3H]N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M1 neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M1, the cardiac M2 and the glandular M3.     

Subtype of muscarinic receptor coupled to the attenuation of hormone-stimulated cAMP accumulation in NG108-15 neuroblastoma x glioma hybrid cells.

The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Therefore, several selective muscarinic antagonists were used to characterize the subtype of muscarinic receptor coupled to the inhibition of hormone-stimulated cAMP accumulation using NG108-15 neuroblastoma x glioma hybrid cells. These cells were prelabeled with [2-3H]-adenine, washed, and resuspended in a culture medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before they were challenged with agonists. The formation of [3H]-cAMP was activated by PGE1 (1 microM) or forskolin (1 microM). In all cases, [3H]-cAMP formed was separated and measured. Carbachol (100 microM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN-A343 (10 mM), an M1 receptor agonist, was only a partial agonist causing 40% inhibition of cAMP accumulation indicating that this effect was not mediated by an M1 receptor; b) The M1-selective antagonist, pirenzepine, exhibited low affinity (pA2 6.2) further suggesting that an M1 receptor was not coupled to the attenuation of cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methoctramine) and M3 antagonist (HHSiD) were used to further characterize these muscarinic receptors. The order of all antagonists based on their affinities (pA2 values) could be arranged in the following order: atropine (9.0) > methoctramine (7.6) > HHSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the same degree of affinity to M2 receptors of other tissues as it does to those of NG cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of muscarinic receptors coupled to phosphoinositide breakdown in guinea pig brain and peripheral tissues

Muscarinic receptors coupled to phosphoinositide hydrolysis (PI) are present in guinea pig bladder and colon. Compared to rat cerebral cortex, an extensively studied muscarinic/PI turnover system, all agonists were more potent and efficacious in both bladder and colon. The "M1-selective antagonists", pirenzepine and dicyclomine, were much more potent (Ki = 1-5 nM) and selective (300 to 500-fold) at both rat and guinea pig brain and guinea pig colon receptors, compared to PI-coupled receptors in guinea pig bladder. In contrast, "M2-selective antagonists", AF-DX 116 and HHSiD, were 2-6 fold more potent in bladder than in brain, while HHSiD was very potent in the colon (50 times more potent than in brain). These results suggest a pharmacological heterogeneity of PI-linked muscarinic receptors. If muscarinic receptors with a low affinity for pirenzepine are defined as M2, these results show that the guinea pig bladder contains PI-linked M2 muscarinic receptors, whereas the guinea pig colon contains PI-linked M1 receptors.     

Potassium- and Carbachol-Evoked Release of [3H]Noradrenaline from Human Neuroblastoma Cells, SH-SY5Y

The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki = 0.33 nM), hexahydrosiladifenidol (Ki = 18 nM), and pirenzepine (Ki = 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists methoctramine (10(-4) M) and AFDX-116 (10(-4) M), by approximately 14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10(-4) M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.     

M1 Muscarinic Acetylcholine Receptor in Cultured Rat Neostriatum Regulates Phosphoinositide Hydrolysis

Abstract: : Muscarinic acetylcholine receptor expression and function in cultured rat neostriatal neurons were examined. All experiments were performed on intact neurons grown in vitro for 12-14 days. The muscarinic antagonist N-[³H]methylscopolamine ([³H]NMS) binds to a single site in cultures with a K_D of 89 pM and a B_max of 187 fmol/mg of protein, or 32,000 sites/neuron. Competition studies using [³H]NMS were performed to determine what receptor sur > types were present. Nonlinear analysis of competition curves was best described with a single binding site for atropine, pirenzepine, and AF-DX 116 {11-[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one}, with K_i values of 0.6, 62, and 758 nM, respectively. These results indicate that the muscarinic receptors present in neostriatal cultures are of the M₁subtype, having high affinity for pirenzepine and low affinity for AF-DX 116. In contrast with antagonists, carbachol displaced [³H]NMS from two sites with K_i values of 6.5 and 147 μM, with the higher-affinity form predominant (83% of sites). The M₁ receptor subtype was linked to phosphoinositide turnover. Carbachol stimulated the formation of phosphoinositides with an EC₅₀ of 37 μM and was antagonized by atropine. At equimolar doses, pirenzepine was more potent than AF-DX 116 at antagonizing the response.     

Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists

Heterogeneity of the muscarinic receptor population in the rat central and peripheral lung was found in competition binding experiments against [3H]quinuclidinyl benzilate [( 3H]QNB) using the selective antagonists pirenzepine, AF-DX 116 and hexahydrosiladifenidol (HHSiD). Pirenzepine displaced [3H]QNB with low affinity from preparations of central airways indicating the absence of M1 receptors in the trachea and bronchi. Muscarinic receptors in the central airways are comprised of both M2 and M3 receptors since AF-DX 116, an M2-selective antagonist, bound with high affinity to 70% of the available sites while HHSiD, an M3-selective antagonist bound with high affinity to the remaining binding sites. In the peripheral lung, pirenzepine bound with high affinity to 14% of the receptor population, AF-DX 116 bound with high affinity to 79% of the binding sites while HHSiD bound with high affinity to 18% of the binding sites. The presence of M1 receptors in the peripheral airways but not in the central airways was confirmed using [3H]telenzepine, an M1 receptor ligand. [3H]Telenzepine showed specific saturable binding to 8% of [3H]QNB labeled binding sites in homogenates of rat peripheral lung, while there was no detectable specific binding in homogenates of rat trachea or heart. The results presented here demonstrate that there are three muscarinic receptor subtypes in rat lungs, and that the distribution of the different subtypes varies within the lungs. Throughout the airways, the dominant muscarinic receptor subtype is M2. In the trachea and bronchi the remaining receptors are M3, while in the peripheral lungs, the remaining receptors are both M1 and M3.     

An endogenous factor induces heterogeneity of binding sites of selective muscarinic receptor antagonists in rat heart

According to molecular biological and pharmacological criteria, rat heart membranes normally express only one muscarinic receptor subtype. The selective antagonists pirenzepine and AF-DX 116 bind to this receptor with a single affinity: low and high, respectively. We report here that an endogenous, intracellular factor alters the affinity of selective antagonists for muscarinic receptors in the heart. Thus, when the intracellular fluid is added back to rat heart membranes, both pirenzepine and AF-DX 116 bind to two receptor sites. Approximately 30% of the receptors bind pirenzepine with high affinity and AF-DX 116 with low affinity. Thus, while cardiac muscarinic receptors are coded for by a single mRNA and are therefore genetically homogeneous, the resulting receptor protein might behave like a mixture of receptor subtypes in intact tissues due to the influence of intracellular factors on receptor conformation.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

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

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

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting beta cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal beta cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1--m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 microM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 microM of methoctramine (M2 antagonist) increased ACh (100 microM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.     

Heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex demonstrated by the selective antagonist AF-DX 116

Recent studies have demonstrated that the majority of muscarinic receptors in rabbit peripheral lung homogenates bind pirenzepine with high affinity (putative M1 subtype). In experiments of AF-DX 116 inhibiting [3H](-)quinuclidinyl benzilate or [3H]pirenzepine, we found similar inhibitory constants for AF-DX 116 binding in rat heart and rabbit peripheral lung that were 4-fold smaller (i.e. of higher affinity) than the inhibitory constant for rat cerebral cortex. This result demonstrates heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex.     

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting β cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal β cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1–m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 μM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 μM of methoctramine (M2 antagonist) increased ACh (100 μM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.     

Subacute reserpine treatment reveals preferential coupling between the M3 muscarinic receptor subtype and phosphatidylinositol turnover.

Muscarinic receptors in the rat cerebral cortex, cardiac atria and vas deferens were identified, quantitated, and characterized relative to phosphatidylinositol (PI) turnover as the functional response to stimulation of specific receptor subtypes. Receptor densities as determined by 3H-QNB binding were ranked: cerebral cortex greater than vas deferens greater than heart. Using displacement of 3H-QNB binding by the selective M1 and M2 muscarinic receptor antagonists pirenzepine and 11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido [2,3-b] [1,4] benzodiazepine-6-one (AF-DX 116) respectively, heterogeneous populations were found in the cerebral cortex and vas deferens. The M1 receptor subtype predominated in the former and the M2 predominated in the latter. An homogeneous M2 receptor population was present in the heart. Methacholine-stimulated accumulation of 3H inositol-1-phosphate was greater in the vas deferens than in the cerebral cortex, whereas PI turnover was not enhanced in cardiac atria. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) increased muscarinic receptor density in the vas deferens coincident with a shift in the low affinity pKi for AF-DX 116 to a value comparable to high affinity binding, and abolished the enhanced PI hydrolysis. In the cerebral cortex, reserpine treatment shifted only the early portion of the methacholine dose-response curve to the right. These results are judged to be supportive of preferential coupling between the M3 muscarinic receptor subtype and PI turnover.