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)     

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.     

Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.     

Pharmacological characterization of muscarinic receptor subtypes mediating vasoconstriction of human umbilical vein

The present study attempted to pharmacologically characterize the muscarinic receptor subtypes mediating contraction of human umbilical vein (HUV).HUV rings were mounted in organ baths and concentration-response curves were constructed for acetylcholine (ACh) (pEC50: 6.16+/-0.04; maximum response 80.00+/-1.98% of the responses induced by serotonin 10 microM). The absence of endothelium did not modify the contractile responses of ACh in this tissue. The role of cholinesterases was evaluated: neither neostigmine (acetylcholinesterase inhibitor) nor iso-OMPA (butyrylcholinesterase inhibitor) modified ACh responses. When both enzymes were simultaneously inhibited, a significantly but little potentiation was observed (control: pEC50 6.33+/-0.03; double inhibition: pEC50 6.57+/-0.05). Atropine, nonselective muscarinic receptors antagonist, inhibited ACh-induced contraction (pKB 9.67). The muscarinic receptors antagonists pirenzepine (M1), methoctramine (M2) and pFHHSiD (M3) also antagonized responses to ACh. The affinity values estimated for these antagonists against responses evoked by ACh were 7.58, 6.78 and 7.94, respectively. On the other hand, PD 102807 (M4 selective muscarinic receptors antagonist) was ineffective against ACh-induced contraction.In presence of a blocking concentration of pirenzepine, pFHHSiFD produced an additional antagonism activity on ACh-induced responses. The M1 muscarinic receptors agonist McN-A-343 produced similar maximum but less potent responses than ACh in HUV. The calculated pA2 for pirenzepine against McN-A-343 induced responses was 8.54. In conclusion, the data obtained in this study demonstrate the role of M1 muscarinic receptor subtypes and suggest the involvement of M3 muscarinic receptor subtypes in ACh-induced vasoconstriction in HUV rings. In addition, the vasomotor activity evoked by ACh does not seem to be modulated by endothelial factors, and their enzymatic degradation appears to have little functional relevance in this tissue.     

Involvement of M3 muscarinic receptors in ACh-induced increase in membrane-associated RhoA of rat bronchial smooth muscle

It is known that RhoA is translocated from cytoplasm to cell membrane in bronchial smooth muscle when activated by acetylcholine (ACh) stimulation. In the present study, the effects of selective muscarinic receptor antagonist methoctramine, AF-DX116 (for M(2)) and 4-diphenylacetoxy N-methylpiperidine (4-DAMP; for M(3)) on the ACh-induced rat bronchial smooth muscle contraction and increase in membrane-associated RhoA were investigated to elucidate the muscarinic receptor subtype participating in these responses. To evaluate ACh-induced contraction of bronchial smooth muscle, bronchial ring of rat was prepared, suspended in an organ bath and the tension was measured isometrically. To quantify the ACh-induced increase in membrane-associated RhoA protein, western blot analysis was performed by using homogenates of membrane and cytosolic fractions of the rat bronchi. The muscarinic M(2) and M(3) receptors were detected by using RT-PCR in rat bronchial smooth muscle. Both the ACh-induced smooth muscle contraction and increase in membrane-associated RhoA were markedly inhibited by 4-DAMP, but not by methoctramine or AF-DX116. In conclusion, these results indicated contraction for the first time that the activation of RhoA occurs via M(3) receptor in rat bronchial smooth muscle.     

Interaction of selective compounds with muscarinic receptors at dispersed intestinal smooth muscle cells.

1. The characterization of muscarinic receptors on single cells of the guinea-pig ileum longitudinal smooth muscle, devoid of neuronal elements, was functionally studied by estimating the affinities of muscarinic antagonists on acetylcholine-induced contractions. 2. Atropine (5 x 10(-11) to 5 x 10(-6) M), 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP, 5 x 10(-8) to 5 x 10(-6) M), cyclohexyl(4-fluoro-phenyl) (3-piperidinopropyl) silanol (pFHHSiD, 5 x 10(-7) to 5 x 10(-5) M) as well as pirenzepine (5 x 10(-7) to 5 x 10(-5) M) competitively antagonized the acetylcholine-dependent contractions with different affinities (atropine > 4-DAMP > pFHHSiD > pirenzepine). 3. Methoctramine (5 x 10(-7) to 5 x 10(-5) M), and AF-DX 116 (5 x 10(-6) and 5 x 10(-5) M) also showed antagonist properties but these deviated from simple competition. These compounds, which discriminate between M2 and M3 receptors, showed a potency lower than that of pirenzepine, the rank order of potencies being pirenzepine > methoctramine > AF-DX 116. When concentrations of AF-DX 116, methoctramine and pirenzepine were increased an unspecific contractile effect occurred. 4. McN-A-343, a partial agonist on intact guinea-pig longitudinal smooth muscle strips, on this preparation induced a weak contraction (about 7% in comparison to control) that was not reversed by antimuscarinic agents. 5. These data indicate that M3 rather than M2 receptor sites are present on this tissue.     

Heterogeneity of muscarinic receptors in lamb isolated coronary resistance arteries.

1. In vitro experiments in a microvascular myograph were designed to characterize postjunctional muscarinic receptors producing contraction both in the presence and absence of the endothelium in coronary resistance arteries (normalized diameter of 150-450 microns), isolated from the left ventricle of hearts from 3-6 month old lambs. Preferential muscarinic receptor antagonists were used to determine the receptor subtype: pirenzepine (M1 receptor), AFDX 116 (M2 receptor), 4-DAMP and pFHHSiD (M3 receptor). 2. The rank order of potency for muscarinic agonist-induced increases in tension in endothelium-intact preparations was oxotremorine-M = methacholine = acetylcholine (ACh) > carbachol. Removal of the endothelium increased the potency of ACh, but this procedure did not change either the sensitivity or maximal response to carbachol. 3. The contractile response to ACh was reproducible. Incubation with 3 x 10(-7)-3 x 10(-6) M pirenzepine induced non-parallel rightward shifts and depressed the maximum of the concentration-response curve to ACh in endothelium-intact arteries. The slope by Schild analysis was 2.9 +/- 0.8 (P < 0.05, n = 7). Atropine, AFDX 116, 4-DAMP and pFHHSiD produced parallel rightward shifts of the curves to ACh and the slopes of the Schild plots were not significantly different from unity. The pKB values for the antagonists from plots constrained to unity in endothelium-intact segments were: atropine (9.4), 4-DAMP (9.0), pFHHSiD (7.9) and AFDX 116 (6.2). 4. In endothelium-denuded arteries, pirenzepine, AFDX 116 and pFHHSiD caused concentration-dependent, parallel rightward displacements of the concentration-response curves to ACh and the slopes of the Schild plots were not significantly different from unity. The plots constrained to a slope of unity gave the following pKB values: pFHHSiD (8.7), pirenzepine (7.5) and AFDX 116 (6.2). 5. In the presence of the endothelium, low concentrations of pirenzepine (10(-9)-10(-7) M) produced leftward shifts of the ACh concentration-response curves. This potentiating effect of pirenzepine was reversed by endothelial cell removal. In preparations precontracted with the thromboxane-mimetic, U46619, the putative M1-selective agonist, McN-A-343, induced a biphasic relaxation with log IC50 of 8.53 +/- 0.14 and 5.02 +/- 0.08 for the first and second phase of the relaxation, respectively, and maximal relaxations of 22.8 +/- 4.3% and 41.1 +/- 5.4% (n = 16). McN-A-343 relaxed the vessels in the presence of 10(-7) M pFHHSiD and 3 x 10(-7) M AFDX 116, but not after incubation with 10(-9) M pirenzepine.(ABSTRACT TRUNCATED AT 400 WORDS)     

M1 and M3 muscarinic receptors in human pulmonary arteries.

1. Acetylcholine (ACh) and the M1 agonists (McN-A-343 or PD142505) relaxed human isolated pulmonary arteries which were pre-contracted with noradrenaline (10 microM). In preparations where the endothelium had been removed ACh induced a contractile response whereas the M1 agonists (McN-A-343 or PD142505) had no effect. 2. ACh- and McN-A-343-induced relaxations were abolished after treatment of endothelium-intact preparations with the drug combination NG-nitro-L-arginine (L-NOARG: 0.1 mM) and indomethacin (1.7 microM). 3. The affinity (pKB value) for pirenzepine was higher in human pulmonary arteries when tissues were relaxed with McN-A-343 as compared with ACh (pKB values, 7.71 +/- 0.30 (n = 4) and 6.68 +/- 0.15 (n = 8), respectively). In addition, the affinity for pFHHSiD against McN-A-343- and ACh-induced relaxations was 6.86 +/- 0.13 (n = 3) and 7.35 +/- 0.11 (n = 9) respectively. 4. The low affinities for methoctramine in human isolated pulmonary arteries with the endothelium either intact or removed, suggested the lack of involvement of M2 and M4 receptors in the Ach responses. 5. Phenoxybenzamine (3 microM: 30 min) abolished both ACh contraction and relaxation in human pulmonary artery. The ACh contraction was present when the phenoxybenzamine treatment was preceded by incubation with pFHHSiD (2 microM) but not with pirenzepine (1 microM). In addition, the ACh relaxation was present when preparations were treated with either pFHHSiD (2 microM) or pirenzepine (1 microM), before exposure to phenoxybenzamine. 6. These results in human isolated pulmonary arteries support the notion that only M3 receptors, on smooth muscle, mediate the ACh-induced contraction whereas M3 and M1 receptors are involved in the endothelium-dependent ACh-induced relaxation.     

Muscarinic receptor subtypes mediating vasorelaxation of the perforating branch of the human internal mammary artery

The effect of acetylcholine (ACh) on the isolated perforating branch of the human internal mammary artery (HIMA) was investigated. ACh induced concentration- and endothelium-dependent relaxation of arterial rings precontracted with phenylephrine (pEC(50) = 6.93 +/- 0.01). The muscarinic receptor antagonist atropine (no selectivity), pirenzepine (M(1)), methoctramine (M(2)), and p-fluoro-hexahydro-siladifenidol (M(1)/M(3)) competitively antagonized the response to ACh. The pA(2) values were 9.81 +/- 0.15, 7.74 +/- 0.08, 6.27 +/- 0.08, and 7.88 +/- 0.04, respectively. In conclusion, this study has shown that ACh induced an endothelium-dependent relaxation of the perforating branch of the HIMA by stimulation of muscarinic receptors on the endothelial cells. On the basis of differential antagonist affinity, we suggest that the muscarinic receptors involved in the ACh-induced relaxation of the isolated perforating branch of HIMA are predominantly of M(1) subtype.     

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.     

Pre- and postjunctional muscarinic receptor subtypes in dog airways.

To examine muscarinic receptor subtypes involved in cholinergically mediated contractions of the airway, we studied the effects of the M1-selective antagonist, pirenzepine, the M2-selective antagonist, AF-DX 116, the M3-selective antagonist, 4-diphenyl-acetoxy-N-methylpiperidine (4-DAMP) methiodide, and the non-selective antagonist, atropine, on acetylcholine (ACh)- and electrically induced contractions in dog bronchi and bronchioles. The relative potencies of the antagonists based on IC50 values of each antagonist for contractions induced by the two concentrations of ACh that produced 50% of the maximum (ED50) and the maximum (EDmax) contractions and the pA2 values were atropine greater than or equal to 4-DAMP methiodide greater than pirenzepine = AF-DX 116 in both the bronchi and bronchioles. The IC50 and pA2 values of each antagonist did not differ significantly between the bronchi and bronchioles. 4-DAMP methiodide significantly inhibited the contractile response to electrical field stimulation (EFS) at 5 Hz at concentrations that did not alter the contractile responses to exogenous ACh in both the bronchi and bronchioles, whereas pirenzepine, AF-DX 116 and atropine inhibited the EFS-induced contraction only at the concentrations that reduced the contraction induced by exogenous ACh. The present results suggest that the cholinergic contraction is mediated via the postsynaptic receptor M3, based on functional potencies of muscarinic antagonists and presynaptic receptor auto-facilitatory M3, based on the suppression of the contractile response to EFS by 4-DAMP methiodide in central and peripheral airways.     

Comparison of the muscarinic receptors in the coronary artery,cerebral artery and atrium of the pig

Summary The affinity of various muscarinic antagonists for the muscarinic receptors mediating contraction (induced by acetyl-\-methylcholine) of the isolated pig coronary and basilar artery was determined in order to compare the muscarinic receptor subtype involved in the contractile response of these arteries. In order to identify the muscarinic receptor subtype(s) involved, the affinity of the antagonists for the M₂ receptor present in the pig atria was also investigated. The following muscarinic antagonists were used: atropine, pirenzepine, AF-DX 116 (11-2{{2-{(diethyl-amino)methyl} -1- piperidinyl}acetyl} - 5, 11- dihydro - 6H - pyrido {2, 3 - b} {, 4}benzodiazepin - 6 - one), 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide), HHSiD (hexahydrosiladifenidol), methoctramine (N, N- bis{6 - {(2 - methoxybenzyl)amino} hexyl} -1, 8 - octane - diamine tetrahydrochloride) and ipratropium.The order of affinity of the antagonists with respect to the muscarinic receptor in the coronary artery was clearly different from that for the muscarinic receptor in the basilar artery. The order of affinity established on the basilar artery closely resembled that for the M2 receptor in the atria.It is concluded that the muscarinic receptors on smooth muscle of the coronary and basilar arteries are not identical. The muscarinic receptor involved in the contraction of the basilar artery adheres to the M2 receptor subtype. A comparison of the selectivity of the antagonists suggests that the muscarinic receptor involved in the contraction of the coronary artery belongs to the M₃ (like in exocrine glands) or M₄ (as found in ileal smooth muscle) receptor subtype.     

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.     

Regulation of guinea pig ileal electrolyte transport by M3-muscarinic acetylcholine receptors in vitro

To determine the muscarinic receptor subtype mediating guinea pig ileal mucosal electrolyte secretion, we compared the potencies (Kb) of selective M1 (pirenzepine) (PZ), M2 (AF-DX 116, methoctramine), and M3 [4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladifenidol (HHSiD)] antagonists as inhibitors of carbachol-induced reductions in guinea pig atrial heart rate and ileal longitudinal muscle contractions, responses mediated by M2 and M3 receptors, respectively. Pretreatment with all five muscarinic antagonists shifted the carbachol concentration-response curve to the right, in a manner suggesting competitive antagonism. The following affinity profiles (Kb, nM) were obtained for: 1) ileal mucosa: 4-DAMP (2.7) greater than HHSiD (23.0) greater than PZ (110) greater than or equal to methoctramine (395) greater than AF-DX 116 (784); 2) atrial heart rate: 4-DAMP (9.5) congruent to methoctramine (11) greater than AF-DX 116 (63) greater than HHSiD (222) greater than PZ (256); and 3) ileal longitudinal muscle: 4-DAMP (3.1) greater than HHSiD (21) greater than PZ (143) greater than methoctramine (388) greater than or equal to AF-DX 116 (482). The selectivity profiles of these antagonists suggest that muscarinic receptors in the ileal mucosa more closely resemble those in the ileal muscle (M3) than those in atrial muscle (M2). Moreover, M1-muscarinic receptors appear to be relatively unimportant in mediating the effects of carbachol on short circuit current (ISC). Carbachol-induced increases in ISC were also unaffected by pretreatment with 0.5 microM tetrodotoxin, suggesting that electrolyte transport in the guinea pig ileal mucosa may be mediated, in part, by postsynaptic M3-muscarinic receptors on the enterocytes.     

Mediation by M3-muscarinic receptors of both endothelium-dependent contraction and relaxation to acetylcholine in the aorta of the spontaneously hypertensive rat.

1. Experiments were designed to characterize the subtype(s) of endothelial muscarinic receptor that mediate(s) endothelium-dependent relaxation and contraction in the aorta of spontaneously hypertensive rats (SHR). 2. Rings of SHR aorta with endothelium were suspended in organ baths for the measurement of isometric force. Ecothiopate (an inhibitor of acetylcholinesterase) was present throughout the experiments. Endothelium-dependent contraction to acetylcholine was studied in quiescent aortic rings in the presence of NG-nitro-L-arginine (to prevent the formation of nitric oxide). Endothelium-dependent relaxation to acetylcholine was obtained during contraction to phenylephrine and in the presence of indomethacin (to inhibit cyclo-oxygenase activity). Responses to acetylcholine were assessed against the non-preferential muscarinic receptor antagonist, atropine, and the preferential antagonists pirenzepine (M1), methoctramine (M2) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3). 3. The potency of acetylcholine in inducing endothelium-dependent contraction was 6.54 +/- 0.07 (EC50). Atropine, pirenzepine, methoctramine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent contraction to acetylcholine. The pA2 values for these muscarinic receptor antagonists were estimated from Arunlakshana-Schild plots to be (-log M) 9.48 +/- 0.07, 6.74 +/- 0.22, 6.30 +/- 0.20 and 9.39 +/- 0.22 respectively. The potency of acetylcholine in inducing endothelium-dependent relaxation was 7.82 +/- 0.09 (IC50). Atropine, pirenzepine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent relaxation to acetylcholine but methoctramine had no effect. The pA2 values for atropine and 4-DAMP for the relaxation to acetylcholine were estimated from Arunlakshana-Schild plots to be (-log M) 9.15 +/- 0.23 and 9.63 +/- 0.28, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic receptors that mediate contraction of guinea-pig isolated trachea to choline esters: effect of removing epithelium.

1. The muscarinic receptor subtype that mediates contraction of guinea-pig trachea, in the presence and absence of epithelium, to acetic and carbamic acid choline esters was determined by use of preferential muscarinic receptor antagonists: pirenzepine (M1 receptor), methoctramine (M2 receptor) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3 receptor). 2. Acetylcholine (ACh), methacholine (MeCh), carbachol (CCh), bethanechol (BeCh) and oxotremorine induced concentration-dependent contraction of guinea-pig isolated tracheal strips in the presence and absence of epithelium. Contraction to acetic choline esters (ACh and MeCh) was augmented by removal of the epithelium, whereas contraction to carbamic acid choline esters (CCh and BeCh) and oxotremorine was not influenced by removal of the epithelium. 3. Pirenzepine, methoctramine and 4-DAMP caused parallel rightward displacements of the concentration-contraction curves to the muscarinic agonists. The pA2 values (determined from Arunlakshana-Schild graphs) for pirenzepine and 4-DAMP in guinea-pig trachea in the presence of epithelium were: ACh as the agonist, 7.6 and 9.0, respectively; CCh as the agonist, 7.6 and 9.1, respectively. The apparent pKB values for methoctramine with the same system were: ACh as the agonist, 5.6; CCh as the agonist, 5.6. Similar values were obtained with MeCh, BeCh and oxotremorine as the agonists. These values were agonist- and epithelium-independent. 4. It is concluded from the pA2 and apparent pKB values obtained for the muscarinic receptor antagonists used in this study that contraction of guinea-pig isolated trachea, with and without epithelium, to both acetic and carbamic acid choline esters is mediated via the muscarinic M3 receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of endothelium-derived hyperpolarizing factor (EDHF) by M3 receptor stimulation in guinea-pig coronary artery.

1. The muscarinic receptor subtype(s) involved in the release of endothelium-derived hyperpolarizing factor (EDHF) were studied in the guinea-pig coronary artery by recording the membrane potential in the smooth muscle cells with intracellular microelectrodes. 2. Acetylcholine (ACh, pD2 6.68) was 10 times more potent than the M2 agonist, oxotremorine (pD2 5.65) and 500 fold more potent than the M1 agonist, McN-A-343 (pD2 3.95) in evoking the EDHF hyperpolarization. 3. The M3 muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) was the most potent (pA2 9.5) in inhibiting the release of EDHF evoked by ACh, being more potent than pirenzepine (pA2 6.7), and AFDX-116 (pA2 6.1) which preferentially block M1 and M2 receptors, respectively. 4. These results suggest that EDHF is released from the endothelium of the guinea-pig coronary artery upon the activation of the muscarinic M3 receptor subtype.     

Predominant acetylcholine-induced vasoconstriction in isolated, perfused simian facial veins

Using the cannula insertion method, we investigated the vascular response to acetylcholine (ACh) and other vasoactive substances. ACh consistently induced only vasoconstriction, whereas isoproterenol and norepinephrine usually induced dilatation. Vasoconstriction induced by phenylephrine was less potent than that induced by ACh. Clonidine and xylazine did not induce significant vascular responses. ACh-induced constrictions were readily inhibited by atropine and slightly potentiated by physostigmine. They were slightly but significantly inhibited by pirenzepine (a muscarinic M1-receptor antagonist), but not influenced by AF-DX 116 (a M2-receptor antagonist). 4-DAMP (4-diphenylacetoxy N-methylpiperidine; a M3-receptor antagonist), strongly inhibited the ACh-induced constrictions. They were not modified by bunazosin but slightly suppressed by diltiazem. Removal of the endothelium did not significantly modify the ACh-induced constrictions. From our results, we conclude that the simian facial vein has many constrictory muscarinic receptors, especially of the M3 subtype.     

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.