Muscarinic receptor subtypes and calcium signaling in Fischer rat thyroid cells

A specific and saturable binding site for [3H]N-methyl-scopolamine ([3H]NMS) was observed in plasma membrane of Fischer rat thyroid (FRT) cells with an equilibrium dissociation constant (K(d)) of 0.11 +/- 0.02 nM and a concentration of receptor sites (B(max)) of 14.1 +/- 3.9 fmol/mg protein. Pharmacological characterization of this binding site using pirenzepine, himbacine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6H-pyrido(14) benzodiazepine (AF-DX 116), dicyclomine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and hexahydro-sila-difenidol (HHSD) showed clear differences, in terms of affinities, between these muscarinic receptor antagonists. The order of potency for inhibiting [3H]NMS binding was HHSD = dicyclomine > 4-DAMP > pirenzepine = himbacine > AF-DX 116. These findings suggest that the muscarinic receptors found in FRT cells belong to the M3 subtype. Stimulation of FRT cells with carbachol produced a biphasic and dose-dependent increase in the intracellular calcium concentration ([Ca2+]i), which was blocked in pretreated cells with atropine and almost abolished by a low concentration of 4-DAMP and HHSD. Removal of extracellular Ca2+ from the incubation medium reduced the initial transient peak and completely abolished the plateau phase, while the transient phase was markedly reduced by the phospholipase C inhibitor U73122. These data indicate that [Ca2+]i results from both Ca2+ influx across Ca2+ channels and mobilization of Ca2+ from intracellular Ca2+ stores. The present data showed the presence of the M3 muscarinic acetylcholine receptor subtype in plasma membrane of FRT cells, which may influence cellular function via modulation of [Ca2+]i.     

Characterization of the muscarinic receptor subtype that mediates the contractile response of acetylcholine in the swine myometrium

The aim of the present study was to characterize the subtype of muscarinic receptor that mediates acetylcholine-induced contractions in the nonpregnant proestrus swine myometrium by means of mechanical, radioligand ([3H]quinuclidinyl benzilate) binding and biochemical (measurement of cyclic AMP) approaches. Acetylcholine (-logEC50, 6.12), oxotremorine-methiodide (6.47), methacholine (6.35), carbachol (6.18) and muscarine (6.33) caused contractile responses of the uterine circular muscle, with a similar maximum amplitude, but pilocarpine and McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium) were ineffective in causing contraction. The contractile response to acetylcholine was antagonized by the following muscarinic receptor antagonists in a competitive manner (with pA2 values in parentheses): atropine (8.95), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.83), tropicamide (7.07), himbacine (7.01), pirenzepine (6.42) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyri do[2,3 b][1,4]benzodiazepin-6-one (AF-DX116, 5.96). Electrical field stimulation (10 Hz) caused tetrodotoxin- and atropine-sensitive contractions in the circular muscle. All muscarinic receptor antagonists decreased the electrical field stimulation-induced contraction in a concentration-dependent manner. The order of inhibition (-logIC50) was 4-DAMP (8.35) > tropicamide (6.72) > himbacine (6.54) > pirenzepine (6.31)> AF-DX116 (6.13). Acetylcholine did not affect the cytoplasmic cyclic AMP level, regardless of the presence or absence of forskolin, suggesting the absence of functional muscarinic M2 and/or M4 receptors in the swine myometrium. The receptor binding study indicated that circular muscle layers of the swine myometrium contained a single class of [3H]quinuclidinyl benzilate binding site (Kd = 0.92 nM; Bmax = 126.6 fmol/mg protein). Specific binding was displaced by muscarinic receptor antagonists in the following order (with pKi value and Hill coefficient in parentheses): atropine (8.22 and 0.93) > 4-DAMP (8.18 and 0.94) > tropicamide (6.78 and 0.93) > pirenzepine (5.46 and 0.92) > AF-DX116 (5.12 and 0.94). The present results suggest that in circular muscle layers of the swine myometrium, exogenous and endogenous acetylcholine cause contraction through activation of muscarinic M3 receptors present on smooth muscle cells.     

Decreased binding affinity of olanzapine and clozapine for human muscarinic receptors in intact clonal cells in physiological medium

The binding affinity of olanzapine, clozapine and atropine for muscarinic receptor subtypes in clonal Chinese hamster ovary (CHO) cell lines was compared in intact cells in physiological media to disrupted cells in hypotonic buffer. The affinity of olanzapine and clozapine, but not atropine, for muscarinic receptor subtypes (M₁–M₅) was significantly reduced in intact cells in physiological medium compared to disrupted cells in hypotonic buffer. The affinity of olanzapine for muscarinic M₁ receptors was most affected with a reduction of K_i value from 2.5 to 73 nM in intact cells. These data suggest that the affinity of olanzapine and clozapine for muscarinic receptors have been significantly overestimated.     

Presynaptic and postjunctional muscarinic receptors in dog ileum: binding studies

[3H]N-Methylscopolamine identified two distinct populations of muscarinic receptors in membranes derived from the longitudinal smooth muscle/myenteric plexus of dog ileum. In isolated axonal varicosities, the half-maximal saturation of binding sites occurred at 2.38 +/- 0.39 nM [3H]N-methylscopolamine, with maximal binding capacity 140 +/- 35 fmol/mg protein (mean +/- S.D., n = 8). In purified smooth muscle plasma membranes, the Kd value was 16 +/- 3 nM with Bmax 1960 +/- 494 fmol/mg. The displacement potencies of subtype-selective muscarinic antagonists in the fraction of axonal varicosities followed the order 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methiodide much greater than pirenzepine = methoctramine greater than AF-DX 116 with pKi values 7.38, 5.67, 5.70 and 5.13, respectively. Both 4-DAMP methiodide and pirenzepine were approximately 4-fold less potent in displacing the ligand from the receptors in smooth muscle plasma membranes as compared to varicose receptors. The potency ratios of cardioselective antagonists methoctramine and AF-DX 116 on varicose and smooth muscle receptors were 1 and 1.7. It is concluded that presynaptic receptors located on isolated axonal varicosities have pharmacological properties similar to glandular (M3) subtype of muscarinic receptors. The binding properties of receptors present in smooth muscle plasma membranes were found incompatible with those of any of the M1, M2 or M3 subtypes.     

The endogenous muscarinic acetylcholine receptor in Xenopus oocytes is of the M3 subtype

The muscarinic acetylcholine receptor of the Xenopus oocyte was characterized electrophysiologically. Iontophoretic responses to acetylcholine were inhibited by the muscarinic antagonists 4-DAMP, pirenzepine and AF-DX 116 at respective IC50 values of 7 nM, 15 microM and 2 microM. This antagonist sensitivity order indicates that the receptor is of the M3 muscarinic subtype.     

Muscarinic receptor subtype mediating vasodilation feline middle cerebral artery exhibits M3 pharmacology

The nature of the muscarinic receptor subtype mediating the endothelium-dependent relaxation of the cat middle cerebral artery was investigated in vitro by recording the smooth muscle isometric tension of precontracted arterial segments. Relaxation induced by several agonists (acetylcholine (ACh), acetyl-beta-methylcholine, oxotremorine, carbachol and McN-A-343) was recorded. The ability of selective (pirenzepine, dicyclomine, adiphenine, AF-DX 116, methoctramine, gallamine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and hexahydro-sila-difenidol (HHSiD] and non-selective antagonists (atropine, scopolamine and quinuclidinyl benzilate (QNB] to block the relaxation induced by ACh was also estimated. The weak activity of the poorly selective M1 muscarinic receptor as together with the intermediate affinity of pirenzepine and adiphenine tend to exclude the M1 muscarinic receptor as the primary mediator of the cholinergic relaxation. The low affinity of AF-DX 116 and methoctramine further suggested that the cerebrovascular muscarinic receptor does not correspond to the M2 cardiac subtype. In contrast, 4-DAMP and HHSiD potently inhibited the ACh-induced relaxation with affinities similar to those reported at the M3 glandular receptor. We conclude that a similar to the pharmacological M3 muscarinic receptor subtype is responsible for the cholinergic relaxation of the cat middle cerebral artery.     

M3-muscarinic receptor mediates prejunctional inhibition of noradrenaline release and the relaxation in cat femoral artery.

The aim of the present study was to analyse the muscarinic receptors involved in the vasodilation elicited by acetylcholine (ACh) and the carbachol inhibition of electrically-evoked [3H]noradrenaline (NA) release in cat femoral artery. For this purpose, the following receptor antagonists were used, atropine, pirenzepine (M1-antagonist), AF-DX 116 (M2-antagonist) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3-antagonist). The order of potency (pA2 values) of these drugs at postjunctional level was: atropine (9.7) greater than or equal to 4-DAMP (9.6) greater than pirenzepine (7.2) greater than AF-DX 116 (6.0), and at prejunctional level (pIC50 values) was: 4-DAMP (9.3) greater than atropine (8.5) greater than AF-DX 116 (7.1) greater than pirenzepine (5.9). These findings indicate that the muscarinic receptors mediating the vasodilation induced by ACh and the carbachol inhibition of NA release are of the M3-subtype.     

Muscarinic receptor subtypes mediating positive and negative inotropy in the developing chick ventricle

The inotropic response to muscarinic receptor stimulation of isolated chick ventricular myocardium was examined at various developmental stages, and the receptor subtype involved was pharmacologically characterized. In embryonic chick ventricles, carbachol (CCh) produced positive inotropy at micromolar concentrations. In hatched chick ventricles, CCh produced negative inotropy at nanomolar concentrations. Neither positive nor negative inotropy was observed in the 19 - 21-day-old embryos. Both positive and negative inotropy were also observed with acetylcholine and oxotremoline-M. The CCh-induced positive inotropy in 7 - 9-day-old embryonic ventricles and the negative inotropy in 1 - 3-day-old hatched chick ventricles were antagonized by muscarinic receptor antagonists; pA(2) values for the positive and negative responses of pirenzepine were 7.5 and 7.2, those of AF-DX116 (11-[(2-[(diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one) were 6.8 and 6.9, those of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were 9.0 and 8.5, and those of himbacine were 7.0 and 8.0, respectively. CCh had no effect on action potential configuration. In conclusion, the positive inotropy is most likely mediated by muscarinic M(1) receptors and the negative inotropy is mostly likely mediated by muscarinic M(4) receptors.     

Radioligand binding characteristics of the chicken cardiac muscarinic receptor

Summary The muscarinic receptor present in chicken cardiac membranes was characterised using a ligand binding approach and compared to the M1, M2 and M3 receptors that can be identified in ligand binding studies at present. [³H]N-methylscopolamine and [³H]pirenzepine appeared to label the same population of muscarinic receptors in chicken cardiac membranes since the density of sites labeled by the two radioligands was similar. Furthermore, affinity estimates of 8 muscarinic receptor antagonists for chicken cardiac muscarinic receptors were the same irrespective of whether [³H]N-methylscopolamine or [³H]pirenzepine was used as the radioligand. The chicken cardiac muscarinic receptor displayed high affinity for pirenzepine (pK _i = 7.9) and so did not appear to represent an M2 receptor. Despite the high affinity of chicken cardiac muscarinic receptors for pirenzepine, affinity estimates for dicyclomine (pK _i = 8.0), CPPS (pK _i = 8.4) and 4DAMP (pK _i = 8.6) in chicken heart were not consistent with the presence of M1 receptors. The chicken cardiac muscarinic receptor also displayed significant differences to the M3 receptor since it displayed high affinity for AF-DX 116 (pK _i = 7.1) and methoctramine (pK _i = 8.4). Finally, chicken heart muscarinic receptors displayed high affinity for gallamine (pK _i = 7.0) and pirenzepine suggesting that the receptor was different to the M4 muscarinic receptor of the NG108-15 cell line. These findings suggest that chicken heart expresses a novel muscarinic receptor subtype distinct from the M1, M2, M3, and M4 subtypes already described. Send offprint requests to A. Michel.     

Affinity of muscarinic receptor antagonists for three putative muscarinic receptor binding sites

1. A range of muscarinic receptor antagonists were examined for affinity at the M1 muscarinic binding site, present in rat cerebrocortical membranes and the M2 muscarinic binding sites of rat cardiac and submaxillary gland membranes. 2. The results obtained were consistent with the presence of three classes of muscarinic binding site. 3. Both the M1 binding site, labelled by [3H]-pirenzepine ([3H]-Pir) in rat cerebrocortical membranes, and the M2 gland binding site, labelled by [3H]-N-methyl scopolamine ([3H]-NMS) in rat submaxillary gland membranes, displayed higher affinity for pirenzepine, dicyclomine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and cyclohexylphenyl (2-piperidinoethyl) silanol (CPPS) than did the M2 binding sites of cardiac membranes labelled by [3H]-NMS. 4. The M2 cardiac sites displayed higher affinity for methoctramine, himbacine and AF-DX 116 than did either the M1 binding site of cerebrocortical membranes or the M2 gland binding site present in rat submaxillary gland membranes. 5. The M1 and M2 gland binding sites could only be distinguished by considering the absolute affinity of compounds for these two sites. Thus, all compounds, with the exception of 4-DAMP, displayed between a 2 and 8 fold higher affinity for the M1 than for the M2 gland binding site. There were no antagonists with higher M2 gland than M1 affinity.     

Functional and binding studies with muscarinic M2-subtype selective antagonists.

1. The potency of a series of selective muscarinic antagonists has been measured on two functional isolated tissue preparations (rat ileum and atria) and these compared with their potency on a range of binding preparations in order to determine whether the subtypes of M2 receptor measured functionally are the same as those measured in binding studies. 2. On the functional preparations pirenzepine, hexahydrosiladiphenidol (HSD) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were more potent on the ileum than on the atrium (3 fold, 29 fold and 5 fold respectively), whereas himbacine, AF-DX 116 and methoctramine showed the opposite selectivity (5 fold, 3 fold and 56 fold respectively). Atropine had a similar potency on the ileum and atrium. 3. [3H]-N-methyl scopolamine was used to study M2 binding sites on membranes from rat heart and rat submandibular gland. Each preparation appeared to contain a homogeneous binding site population. The potencies of the five M2 selective antagonists (and pirenzepine) in binding studies to heart membranes were very similar to those observed in functional studies of rat atria (correlation coefficient = 0.98). Similarly the binding to submandibular gland membranes was very similar to that observed in functional studies on rat ileum (correlation coefficient = 0.97). 4. [3H]-pirenzepine was used to examine the binding of these antagonists to M1 binding sites on membranes from rat cerebral cortex. The affinities of 4-DAMP, HSD, AF-DX116 and himbacine at M1 sites were similar to their affinities on the gland. Only pirenzepine and methoctramine had higher affinity on M1 sites than on the gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

An M3-like muscarinic autoreceptor regulates the in vivo release of acetylcholine in rat striatum

Selective muscarinic antagonists were used in an attempt to characterize the muscarinic autoreceptor modulating the release of acetylcholine in the striatum of the rat. In vivo microdialysis was applied to infuse atropine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), pirenzepine or AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro[2,3-b][1,4]benzodiazepine-6-one), leading to a dose-dependent increase in the overflow of acetylcholine, the order of potency being: atropine greater than 4-DAMP greater than pirenzepine greater than AF-DX 116. We conclude from these data that the muscarinic receptor modulating release in the striatum is of the M3 type.     

Characterization of muscarinic receptors mediating contractions of circular and longitudinal muscle of human isolated colon.

1. The effects of seven muscarinic receptor antagonists were used to characterize the receptors which mediate carbachol-evoked contractions of intertaenial circular and taenial longitudinal muscle in human isolated colon. The effects of these antagonists were studied upon colon contractions induced by cumulatively added carbachol which had mean EC50 values of 11.7 +/- 2.3 microM (n = 8) and 12.6 +/- 2.3 microM (n = 8) respectively upon circular and longitudinal smooth muscle. 2. All antagonists displaced concentration-response curves to carbachol to the right in a parallel manner. The maximum concentration of each antagonist added (30 nM-10 microM) did not significantly suppress the maximum response. 3. In circular muscle, the M3 muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladiphenidol (HHSiD) and para-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) inhibited responses with pA2 values of 9.41 +/- 0.23, 7.17 +/- 0.07, 6.94 +/- 0.18 respectively. The M2 muscarinic receptor antagonist, AF-DX 116, the M2/M4 muscarinic receptor antagonist, himbacine, and the M1 muscarinic receptor antagonist, pirenzepine, yielded pA2 values of 7.36 +/- 0.43, 7.47 +/- 0.14 and 7.23 +/- 0.48 respectively. The non-selective antagonist, atropine, had a pA2 of 8.72 +/- 0.28. 4. In longitudinal muscle 4-DAMP, HHSiD, p-F-HHSiD, AF-DX 116, himbacine and pirenzepine gave pA2 values of 9.09 +/- 0.16, 7.45 +/- 0.43, 7.44 +/- 0.21, 6.44 +/- 0.1, 7.54 +/- 0.40, 6.87 +/- 0.38 respectively. Atropine yielded a pA2 value of 8.60 +/- 0.08.(ABSTRACT TRUNCATED AT 250 WORDS)     

M3 muscarinic cholinoceptors are linked to phosphoinositide metabolism in rat cerebellar granule cells.

Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 +/- 48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine [( 3H]NMS), with a KD of 0.18 +/- 0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M2 and M3 receptors respectively, revealed low M2 affinity (Ki: 600 nM) and high M3 affinity (Ki: 2.4 nM), indicative of the M3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine (Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca(2+)-homeostatic and neuromodulatory effects may be mediated by this response.     

Binding and functional profiles of the selective M1 muscarinic receptor antagonists trihexyphenidyl and dicyclomine.

The selectivity profiles of the muscarinic receptor antagonists dicyclomine and trihexyphenidyl have been examined in binding and functional studies and compared with those of pirenzepine and atropine. Dicyclomine, trihexyphenidyl and pirenzepine demonstrated the highest affinity for the M1 muscarinic receptor subtype as revealed in competition experiments against [3H]-pirenzepine labelling of cortical membranes. Their affinity values lay in a narrow range (3.7-14 nM) approaching that of atropine (1.6 nM). Competition experiments against [3H]-N-methylscopolamine in cardiac and glandular (salivary) membranes revealed differences between the drugs examined. Dicyclomine, trihexyphenidyl and pirenzepine displayed low affinity for the cardiac and intermediate affinity for the glandular receptors. Thus, the drugs appeared to discriminate between the M1 (cortical) and the peripheral muscarinic subtypes (cardiac and glandular). However, atropine displayed similar affinities for either subtype with IC50s varying only slightly (1.6-4.6 nM). The rank order of selectivity was: pirenzepine greater than dicyclomine greater than trihexyphenidyl greater than atropine. Mirroring the binding data, pirenzepine, dicyclomine and trihexyphenidyl showed a tenfold greater ability at inhibiting M1-receptor mediated ganglionic responses (McN A-343 pressor effect in pithed rats and nictitating membrane contraction in cats) than at inhibiting peripheral muscarinic responses in the heart and cardiovascular smooth muscle (vagal bradycardia in rats and cats and vagally-induced vasodilatation in cats). The muscarinic antagonists so far examined can be categorized into two groups. Trihexyphenidyl, dicyclomine and pirenzepine, included in one group, are characterized by a higher affinity for the neuronal (M1) muscarinic receptor, hence they antagonize functional responses mediated by the M1 subtype. Atropine, a member of the other group, shows essentially no selectivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic receptors mediating endothelium-dependent relaxation of bovine coronary artery.

In order to identify the receptor subtype responsible for acetylcholine (ACh)-induced relaxation of bovine coronary artery, we determined the affinity of six subtype-selective muscarinic antagonists and compared them with affinity estimates obtained for bovine left atria. At low concentrations, ACh potently relaxed circular strips of coronary artery with endothelium (EC50 0.15 microM), but contracted them at higher agonist concentrations with potencies that depended on the presence or absence of endothelium: EC50 1.8 microM (without endothelium); 4.6 microM (with endothelium). The pA2 values obtained for antagonism of relaxant responses to ACh were: pirenzepine (M1-selective) 7.38 +/- 0.12; AF-DX 116 (11-[2-(diethylamino-methyl)-1-piperidinyl-acetyl]-5,11- dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one; M2-selective) 5.79 +/- 0.09; and 4-diphenylacetoxy-N-methyl-piperidine-methobromide (4-DAMP; M3/M1-selective) 9.07 +/- 0.12. The corresponding Schild slopes were 0.98 +/- 0.07 for pirenzepine, 1.17 +/- 0.09 for AF-DX 116 and 1.01 +/- 0.04 for 4-DAMP. For the following three antagonists, pKB values were determined at two different antagonist concentrations: dicyclomine (M1-selective) 7.49 +/- 0.10, cyclohexylphenyl-(2-piperidinoethyl)-silanol (CPPS; M3-selective) 8.0 +/- 0.10, and parafluoro-hexahydrosila-difenidol (pFHHSiD; M3-selective) 7.87 +/- 0.10. For comparison, the antagonism of methacholine-induced negative inotropy in left atria was determined for three antagonists, yielding the following pA2 values: pirenzepine 5.98 +/- 0.14; AF-DX 116 6.81 +/- 0.14 and 4-DAMP 7.99 +/- 0.14. The slopes of the corresponding Schild plots were 1.05 +/- 0.10, 1.14 +/- 0.12 and 1.08 +/- 0.08, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological differences between muscarinic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase inhibition

Pharmacological differences between muscarinic cholinergic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase were studied. Stimulation of muscarinic receptors from SK-N-SH human neuroblastoma cells resulted in phosphoinositide hydrolysis, but not in inhibition of cAMP formation. As has been shown previously, stimulation of muscarinic receptors from NG108-15 neuroblastoma x glioma cells, on the other hand, resulted in inhibition of cAMP formation without any observable phosphoinositide hydrolysis. These two cell lines provide a useful model system in which to study differential coupling of muscarinic cholinergic receptors. Inhibition of [3H]N-methyl scopolamine [( 3H]NMS) binding and inhibition of carbachol-stimulated function by the antagonists pirenzepine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were studied in this system. Pirenzepine inhibited [3H]NMS binding in both cell lines with low affinity (Ki of 130 and 160 nM in NG108-15 and SK-N-SH cells respectively), indicating that both cell lines express M2 receptors. None of the three antagonists studied exhibited any clear selectivity for the receptors in one cell line over those of the other. In contrast, several agonists including acetylcholine, bethanechol and carbachol exhibited pronounced selectivity. These agonists inhibited [3H]NMS binding to membranes from SK-N-SH cells with IC50 values that were 17-, 3- and 38-fold higher, respectively, than those of NG108-15 cells. This selectivity was still observed when whole cells rather than membranes were studied. These findings indicate that pharmacological differences between receptors coupled to phosphoinositide turnover and those coupled to cAMP inhibition can be detected with certain agonists, but not with the antagonists pirenzepine, AF-DX 116 or 4-DAMP.     

Central muscarinic M2 cholinoceptors involved in cholinergic hypertension

Cholinomimetic agents increase blood pressure and heart rate via central muscarinic cholinoceptors in various aspects. It was reported that i.c.v. injection of the muscarinic M₁ and M₃ cholinoceptor selective antagonist, 4-DAMP (4-diphenylacetoxy-N-methyl-piperidine methiodide, inhibited the pressor response to physostigmine, while the M₁ selective antagonist, pirenzepine, was ineffective. In the present study, the involvement of muscarinic M₂ cholinoceptors in central cholinergic hypertension and tachycardia was investigated. Physostigmine (10–80 μg/kg i.v.), a cholinesterase inhibitor, and oxotremorine (20–40 μg/kg i.v.), a direct muscarinic cholinoceptor agonist, caused a dose-dependent increase in blood pressure. Additionally, physostigmine induced dose-dependent tachycardiac responses. I.c.v. administration of the muscarinic M₂ cholinoceptor antagonists, AF-DX 116 and methoctramine, inhibited both physostigmine (60 μg/kg) and oxotremorine (20 μg/kg)-induced pressor responses at their lower doses used in this study (100 nmol/rat and 10 nmol/rat, respectively). These findings indicate the partial involvement of postsynaptic muscarinic M₂ cholinoceptors. The higher doses of the antagonists (AF-DX 116 300 nmol/rat and methoctramine 30 nmol/rat) potentiated the blood pressure increase due to physostigmine but did not affect that due to oxotremorine. The physostigmine-induced tachycardiac responses were influenced similarly by these antagonists. These results suggest the presence and tonic influence of presynaptic inhibitory muscarinic M₂ cholinoceptors.     

Muscarinic receptor subtype in porcine coronary artery

The specific binding of (-)-[³H]QNB (quinuclidinyl benzilate) in membrane fractions of porcine coronary artery was saturable, of high affinity and stereoselective. It has been shown that there exist (-)-[³H]QNB binding sites with high (K_i = 12 nM)- and low(K_i = 1010 nM)-affinity for pirenzepine in the coronary artery but predominantly low-affinity sites in cardiac muscle. AF-DX 116 and gallamine showed a lower affinity to ( - )-[³H]QNB binding sites in the coronary artery compared to cardiac muscle. Thus, the present study suggests that porcine coronary artery contains a significant number of muscarinic receptors, probably both M₁ and M₂ subtypes.