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.     

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.     

Inhibition of endothelium-dependent hyperpolarization by endothelial prostanoids in guinea-pig coronary artery

1. In smooth muscle of the circumflex coronary artery of guinea-pig, acetylcholine (ACh, 10(-6) M) produced an endothelium-dependent hyperpolarization consisting of two components. An initial component that occurs in the presence of ACh and a slow component that developed after ACh had been withdrawn. Each component of the hyperpolarization was accompanied by an increase in membrane conductance. 2. Indomethacin (5 x 10(-6) M) or diclofenac (10(-6) M), both inhibitors of cyclooxygenase, abolished only the slow hyperpolarization. The initial hyperpolarization was not inhibited by diclofenac nor by nitroarginine, an inhibitor of nitric oxide synthase. 3. Both components of the ACh-induced hyperpolarization were abolished in the presence of atropine (10(-6) M) or high-K solution ([K+]0 = 29.4 mM). 4. The interval between ACh-stimulation required to generate an initial hyperpolarization of reproducible amplitude was 20 min or greater, but it was reduced to less than 5 min after inhibiting cyclooxygenase activity. Conditioning stimulation of the artery with substance P (10(-7) M) also caused a long duration (about 20 min) inhibition of the ACh-response. 5. The amplitude of the hyperpolarization generated by Y-26763, a K+-channel opener, was reproducible within 10 min after withdrawal of ACh. 6. Exogenously applied prostacyclin (PGI2) hyperpolarized the membrane and reduced membrane resistance in concentrations over 2.8 x 10(-9)M. 7. At concentrations below threshold for hyperpolarization and when no alteration of membrane resistance occurred, PGI2 inhibited the initial component of the ACh-induced hyperpolarization. 8. It is concluded that endothelial prostanoids, possibly PGI2, have an inhibitory action on the release of endothelium-derived hyperpolarizing factor.     

Muscarinic receptor agonist-antagonist interaction in the rat rectum: are there ways of activating the same receptors?

The interaction between the muscarinic receptor agonists, carbachol, acetylcholine (ACh) and methacholine, and antagonists, atropine, gallamine, 4-DAMP and pirenzepine, was studied on the rat isolated rectum preparation. ACh (1.93 X 10(-8)-1.95 X 10(-6) M), methacholine (8.7 X 10(-8)-1.1 X 10(-6) M) and carbachol (1.1 X 10(-7)-3.5 X 10(-6) M) induced contractions that were reversibly antagonized by atropine (1.9 X 10(-9)-4.8 X 10(-8) M), 4-DAMP (1.5 X 10(-8)-2.86 X 10(-7) M) gallamine (1.12 X 10(-6)-1.12 X 10(-4) M) and pirenzepine (2.8 X 10(-7)-7.0 X 10(-6) M). The pA2 values were atropine: 8.99 +/- 0.28, 9.29 +/- 0.14 and 8.86 +/- 0.05; 4-DAMP: 8.39 +/- 0.10, 8.66 +/- 0.15 and 8.26 +/- 0.30, gallamine: 5.85 +/- 0.23, 5.73 +/- 0.25 and 5.96 +/- 0.10 and pirenzepine: 6.85 +/- 0.44, 7.17 +/- 0.13 and 7.21 +/- 0.03 against ACh, methacholine and carbachol, respectively. The experimental dose-ratio (atropine + gallamine) was greater than the expected dose-ratio (as predicted by the Paton & Rang rule) for ACh and methacholine while the experimental dose-ratio closely approximates the expected dose-ratio for carbachol. It is suggested that atropine, 4-DAMP pirenzepine and gallamine act on the same receptors but gallamine allosterically altered the binding of the agonists and antagonists to varying extents.     

Subtypes of muscarinic receptor on cholinergic nerves and atrial cells of chicken and guinea-pig hearts.

1. Electrically driven chicken and guinea-pig atria were used to investigate the negative inotropic effects of the muscarinic agonists methacholine and acetylcholine (ACh). The release of ACh from isolated hearts into the perfusate in response to (preganglionic) vagal or (pre- and postganglionic) field stimulation was bioassayed on the guinea-pig ileum or determined by labelling with [3H]-choline. 2. Concentration-response curves for the negative inotropic effect of methacholine were shifted to the right by pirenzepine in various concentrations (0.03 to 10 mumol l-1). The pA2 values were 7.76 in chicken atria and 6.53 in guinea-pig atria. Pirenzepine and atropine antagonized the negative inotropic response to 0.3 mumol l-1 ACh. The half-maximally effective concentrations (IC50) of pirenzepine (Pz) and atropine were 40 and 5.4 nmol l-1 in chicken atria and 330 and 3.5 nmol l-1, respectively, in guinea-pig atria. Thus, the respective potency ratios (IC50Pz/IC50atropine) were 7.4 and 94.3 in the two species. 3. Pirenzepine in low concentrations increased the release of unlabelled and 3H-labelled ACh from isolated hearts evoked by vagal and field stimulation only in chicken, but not in guinea-pigs. The half-maximally-effective concentration of pirenzepine was about 30 nmol l-1 in the chicken heart, whereas, in the guinea-pig heart, an increased release was observed at 300 nmol l-1. 4. (+)-Tubocurarine [(+)-Tc; 100 mumol l-1] reduced the release of ACh evoked by (preganglionic) vagal stimulation to a (+)-Tc-resistant release of about 30%. The time-course of the neuronal release of [3H]-ACh was markedly altered: the onset was delayed and the termination was extended beyond the period of stimulation (1 min or 5s) by several seconds. The (+)-Tc-resistant release was nearly abolished by 30 nmol l-1 pirenzepine. 5. In conclusion, the pre- and post-synaptic muscarinic receptors of the parasympathetic neuroeffector junction of the heart both belong to the M1-subtype in the chicken and to an M2-subtype in the guinea-pig. Block of the nicotinic ganglionic transmission in the chicken heart by (+)-Tc unmasked a muscarinic transmission, which presumably was mediated through M1-receptors stimulating a low and prolonged postganglionic release of ACh.     

Acetylcholine induces Ca-dependent K currents in rabbit endothelial cells

Effects of acetylcholine (ACh) on the membrane potential and current recorded from endothelial cells dispersed from the rabbit aorta were investigated using the patch-clamp technique. ACh hyperpolarized the endothelial cell membrane. Using the whole-cell voltage-clamp procedure, ACh (10(-6) M) induced an outward current, and this current was blocked by atropine (10(-6) M). Application of either pirenzepine (3 x 10(-7) M) or AF-DX 116 (3 x 10(-6) M) slightly inhibited the ACh-induced outward current, and simultaneous application of these two blockers markedly inhibited the outward current. Application of caffeine (2 x 10(-2) M), ryanodine (10(-5) M) or heparin (10(-5) g/ml) reduced the amplitude of the ACh-induced outward current. A single-channel current recording using the patch-clamp technique revealed that ACh opens a Ca-dependent K-channel with a single-channel current conductance of 9 pS. These results indicate that both M1 and M2 receptor subtypes are present in endothelial cells of the rabbit aorta and that ACh activates the Ca-dependent K channel via release of Ca from intracellular store sites. In addition, methylene blue inhibited the ACh-induced outward current from the outside membrane.     

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.     

Characteristics of acetylcholine-induced phosphorylase a activity in uterine segments as a substitute for contractile response to acetylcholine

Studies were made on whether the ACh-induced phosphorylase a activity in isolated rat uterine muscle segments could be used as a substitute for the contractile response to ACh. This ACh-induced phosphorylase a activity was dependent upon the concentration of ACh and was inhibited by atropine, suggesting that it was linked to muscarinic ACh receptors. Both extracellular calcium and an increase of the intracellular calcium concentration were needed for its activation by ACh. Ca2+-antagonists such as Co2+, diltiazem, nitrendipine and verapamil inhibited the ACh-induced activity, suggesting that the activation by ACh required the influx of calcium ions into the uterine muscle through Ca2+-antagonist sensitive Ca2+ channels. The IC50 values of CoCl2, diltiazem, nitrendipine and verapamil on the ACh-induced phosphorylase a activity were 3.4 x 10(-3) M, 2.5 x 10(-4) M, 2.5 x 10(-5) M and 1.1 x 10(-4) M, respectively. These values were comparable with the IC50 values of these Ca2+-antagonists on the contractile response of isolated rat uterine muscle segments to 3 x 10(-4) M ACh. The inhibitory effects of Co2+, nitrendipine and verapamil, but not diltiazem, on ACh-induced phosphorylase a activity were attenuated by higher concentrations of CaCl2 (0.36 to 2 mM). These findings suggested that the ACh-induced phosphorylase a activity in isolated rat uterine muscle segments could be used as a substitute for the contractile response to ACh.     

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.     

The intracellular pathway of the acetylcholine-induced contraction in cat detrusor muscle cells

1. The present study was aimed to investigate intracellular pathways involved in acetylcholine (ACh)-induced contraction in cat detrusor muscle cells 2. Contraction was expressed as per cent shortening of length of individually isolated smooth muscle cells obtained by enzymatic digestion. Dispersed intact and permeabilized cells were prepared for the treatment of drugs and antibody to enzymes, respectively. Using Western blot, we confirmed the presence of related proteins. 3. The maximal contraction to ACh was generated at 10(-11) M. This response was preferentially antagonized by M3 muscarinic receptor antagonist rho-fluoro-hexahydrosiladifenidol (rhoF-HSD) but not by the M1 antagonist pirenzepine and the M2 muscarinic receptor antagonist methoctramine. We identified G-proteins (Gq/11), (Gs), (G0), (Gi1), (Gi2) and (Gi3) in the bladder detrusor muscle. ACh-induced contraction was selectively inhibited by (Gq/11) antibody but not to other G subunit. 4. The phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor neomycin reduced ACh-induced contraction. However, the inhibitors of the phospholipase D, the phospholipase A2 and protein kinase C did not attenuate the ACh-induced contraction. ACh-induced contraction was inhibited by antibody to PLC-beta1 but not PLC-beta3 and PLC-gamma. Thapsigargin or strontium, which depletes or blocks intracellular calcium release, inhibited ACh-induced contraction. Inositol 1,4,5-triphosphate IP3 receptor inhibitor heparin reduced ACh-induced contraction. 5. These results suggest that in cat detrusor muscle contraction induced by ACh is mediated via M3 muscarinic receptor-dependent activation of Gq/11 and PLC-beta1 and IP3-dependent Ca(2+) release.     

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.     

Evidence for a M(1) muscarinic receptor on the endothelium of human pulmonary veins

1. To characterize the muscarinic receptors on human pulmonary veins associated with the acetylcholine (ACh)-induced relaxation, isolated venous and arterial preparations were pre-contracted with noradrenaline (10 microM) and were subsequently challenged with ACh in the absence or presence of selective muscarinic antagonists. 2. ACh relaxed venous preparations derived from human lung with a pD(2) value of 5.82+/-0.09 (n=16). In venous preparations where the endothelium had been removed, the ACh relaxations were abolished (n=4). ACh relaxed arterial preparations with a pD(2) value of 7. 06+/-0.14 (n=5). 3. Atropine (1 microM), the non selective antagonist for muscarinic receptors, inhibited ACh-induced relaxations in human pulmonary veins. The affinity value (pK(B) value) for atropine was: 8.64+/-0.10 (n=5). The selective muscarinic antagonists (darifenacin (M(3)), himbacine (M(2),M(4)), methoctramine (M(2)) and pFHHSiD (M(1),M(3))) also inhibited ACh-induced relaxations in venous preparations. The pK(B) values obtained for these antagonists were not those predicted for the involvement of M(2 - 5) receptors in the ACh-induced relaxation in human pulmonary veins. 4. The pK(B) value for darifenacin (1 microM) was significantly greater in human pulmonary arterial (8.63+/-0.14) than in venous (7.41+/-0.20) preparations derived from three lung samples. 5. In human pulmonary veins, the pK(B) values for pirenzepine (0.5 and 1 microM), a selective antagonist for M(1) receptors, were: 7.89+/-0.24 (n=7) and 8.18+/-0.22 (n=5), respectively. In the venous preparations, the pK(B) values derived from the functional studies with all the different muscarinic antagonists used were correlated (r=0.89; P=0.04; slope=0.78) with the affinity values (pK(i) values) previously published for human cloned m1 receptors in CHO cells. 6. These results suggest that the relaxations induced by ACh are due to the activation of M(1) receptors on endothelial cells in isolated human pulmonary veins.     

Acetylcholine-induced contractions in the perforating branch of the human internal mammary artery: protective role of the vascular endothelium

The effect of acetylcholine (ACh) on the isolated, nonprecontracted perforating branch of the human internal mammary artery (HIMA) was investigated. ACh induced concentration-dependent contractions of nonprecontracted rings with denuded endothelium (pEC(50) = 6.72 +/- 0.02, E(max) = 88.8% of contractions induced by phenylephrine, 10(-5) mol/l) and was without effect on arterial segments with intact endothelium. An inhibitor of nitric oxide synthase, N(G)-monomethyl-L-arginine (L-NMMA), or indometacin, a cyclooxygenase inhibitor, had no effect on acetylcholine-induced contractions of rings of the perforating branch of HIMA with denuded endothelium (pEC(50) = 6.76 +/- 0.03 and 6.62 +/- 0.05, respectively). In the presence of indometacin, ACh did not evoke contractions of arterial segments with intact endothelium. In contrast, in the same type of preparations ACh induced contractions in the presence of L-NMMA (E(max) = 34%). The muscarinic receptor antagonists atropine (no selectivity), pirenzepine (M(1)), methoctramine (M(2)), and p-fluoro-hexahydro-sila-difenidol (M(1)/M(3)) competitively antagonized the response to ACh. The pA(2) values were 9.60 +/- 0.10, 6.99 +/- 0.02, 6.37 +/- 0.17, and 8.02 +/- 0.06, respectively. In conclusion, the results obtained indicate that secretion of nitric oxide from vascular endothelium may protect the perforating branch of HIMA against the contractile effects of ACh. On the basis of differential antagonist affinity, it can be suggested that the muscarinic receptors involved in the ACh-induced contractions of the isolated perforating branch of the HIMA are predominantly of the M(3) subtype.     

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)     

Voltage-independent catecholamine release mediated by the activation of muscarinic receptors in guinea-pig adrenal glands.

1. The differences between the mechanisms of muscarinic and nicotinic receptor-mediated catecholamine secretion with respect to their dependence on voltage changes and extracellular Ca were examined using perfused adrenal glands of the guinea-pig. 2. Acetylcholine (ACh, 10(-6) to 10(-3) M) caused a dose-dependent increase in catecholamine secretion. The ED50 value for ACh was 7 x 10(-5) M. In the presence of atropine (10(-5) M), the dose-response curve for ACh was shifted to the right. Hexamethonium (5 x 10(-4) M) preferentially reduced the responses to higher concentrations of ACh (greater than 10(-5) M). Pilocarpine (5 x 10(-4) M) and nicotine (3 x 10(-5) M) also stimulated catecholamine release. 3. During perfusion with isotonic KCl solution, ACh and pilocarpine, but not nicotine, evoked catecholamine secretion. These responses were abolished by atropine (10(-6) M). Pilocarpine-stimulated catecholamine secretion was enhanced during perfusion with isotonic KCl solution. Under these conditions, hexamethonium (10(-3) M) significantly augmented ACh-evoked catecholamine release. 4. During perfusion with either Ca-free isotonic KCl or Ca-free Locke solution, ACh and pilocarpine caused a partial increase in catecholamine secretion whereas nicotine and high K solution (56 mM) did not. The responses to ACh and pilocarpine were completely inhibited by atropine but not by hexamethonium. 5. When guinea-pig adrenal glands were perfused with isotonic KCl solution containing 2.2 mM Ca which was subsequently removed and replaced with EGTA, ACh-induced catecholamine secretion was similar in magnitude to that observed during perfusion with Locke solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pirenzepine and gallamine on cardiac and pulmonary muscarinic receptors in the rabbit.

1. The effect of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine) were studied on bronchoconstriction and bradycardia elicited by stimulation of the vagal nerves and by i.v. acetylcholine (ACh) in anaesthetized rabbits. 2. Pirenzepine was equipotent as an antagonist of ACh-induced responses at postjunctional muscarinic receptors in the heart, lung and blood vessels, whereas gallamine was at least ten times less potent at pulmonary and vascular muscarinic receptors. Thus, gallamine never caused complete inhibition of bronchoconstrictor or hypotensive responses to i.v. ACh, whereas doses of pirenzepine in excess of 1 mumol kg-1 abolished all muscarinic responses. 3. In the lung, both antagonists inhibited bronchoconstriction caused by vagal stimulation and ACh-induced bronchoconstriction to the same extent (pirenzepine, mean ED50 65 +/- 22 and, 130 +/- 28 nmol kg-1 respectively; gallamine, ED50 greater than 10,000 nmol kg-1 for both responses). Enhancement of vagally-induced bronchoconstriction was never observed. 4. In the heart, however, both pirenzepine and gallamine were ten times less potent as antagonists of vagally-induced bradycardia than of ACh-induced bradycardia. This differential blockade was unaltered by propranolol (1 mg kg-1) pretreatment. 5. It is concluded that there is no evidence for M1 or M2 muscarinic receptors in the pulmonary innervation of the rabbit and the potency of the antagonists in abolishing in abolishing vagally-induced bronchoconstriction was consistent with blockade of M3 muscarinic receptors on airway smooth muscle. 6. The results suggest that M2 muscarinic receptors may exert an inhibitory effect on transmission in the parasympathetic nerves innervating the heart in the rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional muscarinic cholinoceptors in the isolated canine ureter

The purpose of present study was to characterize the functional muscarinic cholinoceptor (mAChR) subtypes in the isolated canine ureter. Carbachol (CCh), a non-selective mAChR agonist, concentration-dependently increased the frequency of the rhythmic contractions in isolated spiral ureteral preparations, the pD(2) value being 5.78+/-0.12. We then evaluated the effects of subtype-selective mAChR antagonists on the CCh-induced rhythmic contractions. The rank order of antagonistic potencies (apparent pA(2)) was 4-diphenylacetoxy- N-methylpiperidinemethiodide (4-DAMP; M3-subtype selective; 9.31+/-0.06) >atropine (non-selective; 9.16+/-0.10) >himbacine (M4-subtype selective; 7.32+/-0.18) >pirenzepine (M1-subtype selective; 6.78+/-0.16) >methoctramine (M2-subtype selective; 5.51+/-0.43). In sharp contrast, CCh concentration-dependently reduced the 80 mM KCl-induced contraction in longitudinal ureteral preparations, the pD(2) value being 4.83+/-0.10. On this CCh-induced ureteral relaxation, the rank order of antagonistic potencies (apparent pA(2)) was atropine (8.56+/-0.09) >4-DAMP (7.63+/-0.21) >himbacine (7.46+/-0.09) >methoctramine (6.54+/-0.18) >pirenzepine (6.33+/-0.22). The nitric-oxide-synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG; 1 x 10(-4) M) had no effect on the CCh-induced ureteral relaxation. These data suggest that the CCh-induced rhythmic contraction in the spiral preparation was mediated via the M3-receptor, while the CCh-induced relaxation in the longitudinal preparation was probably mediated mainly via the M4-receptor.     

Regulation of the substance P-induced contraction via the release of acetylcholine and gamma-aminobutyric acid in the guinea-pig urinary bladder.

1. The action of substance P (SP) on the release of gamma-aminobutyric acid (GABA) and acetylcholine (ACh) and on contraction were studied in strips of the guinea-pig urinary bladder. Substance P induced a dose-dependent contraction of strips of guinea-pig urinary bladder (EC50 = 1.2 x 10(-9) M). This contraction was not altered by tetrodotoxin, but with a dose of 10(-9) M and less, there was a complete inhibition by 10(-6) M) atropine. Contractions initiated by 3 x 10(-9) M) SP or more were partly inhibited by atropine. The EC50 value of substance P in the presence of atropine was 7.0 x 10(-9) M. 2. Substance P induced a Ca2+-dependent and tetrodotoxin-resistant release of [3H]-acetylcholine (ACh) from strips of urinary bladder preloaded with [3H]-choline (EC50 = 4.9 x 10(-10) M), and this release was antagonized by [D-Pro2,D-Trp7,9] substance P. 3. Bicuculline increased the substance P-induced contraction and the release of [3H]-ACh from the strips. 4. Substance P induced a Ca2+-dependent and tetrodotoxin-sensitive release of [3H]-gamma-aminobutyric acid (GABA) from strips preloaded with [3H]-GABA (EC50 = 2.6 x 10(-9) M), and this release was antagonized by [D-Pro2,D-Trp7,9] substance P. 5. Therefore, substance P appears to exert excitatory effects on the contractility of urinary bladder predominantly by stimulating its own receptor located on the cholinergic nerve terminals. GABA released by substance P inhibits stimulation of the cholinergic neurone. However, the direct action of substance P on the cholinergic neurone is more potent that the indirect action via GABA release.     

Characterization of the central muscarinic cholinoceptors involved in the cholinergic pressor response in anesthetized dogs.

Previous reports have shown that an intracisternal (i.c.) injection of acetylcholine in the dog increases both arterial blood pressure and plasma levels of noradrenaline and vasopressin via central muscarinic receptors. The aim of the present study was to characterize the central muscarinic cholinoceptor subtypes involved in such central cholinergic responses in anesthetized male Beagle-Harrier dogs (n = 12). For this purpose, we studied the relative potency of various muscarinic receptor antagonists to block the acetylcholine-induced pressor responses (30 microg kg(-1) i.c.). The acetylcholine-induced pressor response was inhibited in a dose-dependent manner by the i.c. administration of the non-selective muscarinic receptor antagonist atropine (ID50 = 0.5 microg kg(-1)), the muscarinic M receptor antagonist pirenzepine (ID50 = 0.45 microg kg(-1)), the muscarinic M2 receptor antagonist methoctramine (ID50 = 8.5 microg kg(-1)) and the muscarinic M3 receptor antagonist para-fluoro-hexahydro-sila-difenidol (ID50) = 43.7 microg kg(-1)). The order of potency of these four muscarinic receptor antagonists was: atropine = pirenzepine > methoctramine > para-fluoro-hexahydro-sila-difenidol. In order to confirm the selectivity for muscarinic M1 receptors of this dose of pirenzepine, we checked that 40- to 50-fold higher concentrations were necessary to block a typical muscarinic M2 receptor response (bradycardia) and a typical muscarinic M3 receptor response (endothelial vasodilation) compared with methoctramine and para-fluoro-hexahydro-sila-difenidol, respectively. These results suggest that the pressor response elicited by intracisternal injection of acetylcholine in anesthetized Beagle-Harrier dogs is mediated through the activation of the muscarinic M1 cholinoceptor subtype.