Prejunctional muscarine receptors in the rabbit ear artery differ from M1, M2 and M3 muscarine receptors.

The ability of several selective muscarine receptor antagonists to inhibit the effect of carbachol on prejunctional muscarine receptors on sympathetic nerve endings in the rabbit isolated ear artery was investigated to characterise the receptor subtype involved. Carbachol did not reduce responses to exogenous noradrenaline and the inhibitory effect of carbachol on responses to nerve stimulation was unaffected by hexamethonium (10 microM) indicating that the effect of the muscarine agonist was exerted prejunctionally and was not modulated by nicotine receptor stimulation. The dissociation constants or apparent dissociation constants obtained using (+/-)-benzhexol (pKB; 6.63), (R)-benzhexol methiodide (8.11), dicyclomine (5.86), (+/-)-telenzepine (7.34), AF-DX 116 (6.95), himbacine (7.60), (+/-)-hexahydrosiladiphenidol (5.39) and a bisquaternary ammonium compound, heptane-1,7-bis(dimethyl-3'-phthalimidopropyl ammonium bromide) (5.84), indicate that the muscarine receptor subtype involved is not of the M1, M2 or M3 subtype.

Effect of temperature reduction on responsiveness to cholinomimetics in the taenia caeci of the guinea-pig.

1. The effect of temperature reduction on the interaction of carbachol (CCh) and McN-A-343 (McN) with muscarinic receptors in the guinea-pig taenia caeci was investigated. 2. McN, a partial agonist, acted on the smooth muscle to produce contraction. The response was unaffected by tetrodotoxin and the pKB for inhibition by pirenzepine was 6.8, indicating that ganglionic M1 receptors were not involved in the response. 3. Reduction in temperature from 37 degrees C to 18 degrees C for 3 h led to a marked reduction in the contractile response to McN (2-200 microM) but no reduction in the response to CCh (0.1-3 microM). 4. The reduction in temperature was not accompanied by any change in the affinity of McN or CCh for muscarine receptors in binding experiments with [3H]-QNB. 5. The KA value for CCh determined after irreversible receptor inactivation with propylbenzilylcholine mustard followed by ca 60-min wash-out was 7.6 microM, a value similar to that obtained in binding experiments. 6. The EC50 for McN in producing contraction at 37 degrees C (2.1 microM) was similar to the KA value for the partial agonist obtained in experiments with the irreversible antagonist phenoxybenzamine (2.5 microM). It was also similar to the KB value determined at 18 degrees C (3.4 microM) when McN could be used as an antagonist of contractions to CCh. 7. At 18 degrees C, phosphatidylinositol (PI) hydrolysis by CCh was reduced to 23% of that at 37 degrees C. 8. It is concluded that reduction of muscarinic receptor activation of the PI pathway by cholinomimetics with lowering of the temperature could account for the findings with McN on contractility.

Selective inhibition of responses to carbachol and McN-A-343 in the rabbit vas deferens.

1. The effect of several selective muscarine receptor antagonists were evaluated on the responses of carbachol (CCh) and McN-A-343 (McN) during sympathetic nerve stimulation in the rabbit vas deferens. 2. The muscarine M1 receptor antagonist pirenzepine exhibited similar apparent pKB values for antagonism of the prejunctional inhibitory response of either CCh (pKB, 8.2) or McN (pKB, 8.5) on sympathetic nerve stimulation. 3. The muscarine M2 receptor antagonists, pancuronium and the bisalkyl ammonium compound 'C7/3-phth' were selective inhibitors of the postjunctional facilitatory response produced by CCh on sympathetic nerve stimulation. They were also 17- and three-fold, respectively, less potent against the inhibitory responses of McN on sympathetic nerve stimulation. The apparent pKB value of pancuronium on the inhibitory response produced by CCh did not differ significantly (P greater than 0.05) from that using McN. A similar finding was made for C7/3-phth. 4. Selective blockade of the inhibitory response to CCh with pirenzepine (0.03 or 0.5 mumol/L) did not significantly (P greater than 0.05) modify the apparent pKB value for pancuronium on the facilitatory response of CCh. 5. Selective blockade of the facilitatory response to CCh with a low concentration of pancuronium (0.5 mumol/L) did not significantly (P greater than 0.05) modify the apparent pKB value for pancuronium (30 mumol/L) on the inhibitory response of CCh. 6. It is suggested that CCh and McN activate the same prejunctional M1 muscarine receptor and that pancuronium is the most selective of the muscarine M2 receptor antagonists presently tested in this preparation for distinguishing between muscarine M1 and M2 receptors.

Structure-activity relationships of some Galbulimima alkaloids related to himbacine.

Himbacine, himbeline, N-methylhimandravine and himandravine together with their dihydro-derivatives were evaluated as antagonists of muscarinic receptors in guinea-pig ileal longitudinal muscle and electrically stimulated left atrium. Himbacine was the most potent compound and the 15-fold selectivity exhibited for the M2 muscarinic receptor was greater than that found with any of the other compounds examined. Reduction of the double bond linking the decalin ring system and the piperidine ring almost abolished selectivity in dihydrohimbacine. Removal of the N-methyl group in himbacine to form himbeline was associated with reduced selectivity. However the corresponding change in converting N-methylhimandravine to himandravine was not associated with any change in selectivity suggesting that orientation of the 2-methyl group in the piperidine ring may be important for selectivity.

Pharmacology of hexahydro-difenidol, hexahydro-sila-difenidol and related selective muscarinic antagonists.

A series of hexahydro-difenidol (HHD) and hexahydro-sila-difenidol (HHSiD) analogues modified in the amino group, the phenyl ring and in the alkylene chain were investigated for their binding and functional properties at muscarinic M1, M2 and M3 receptors. Novel muscarinic receptor antagonists were obtained which exhibited different receptor selectivity profiles from the parent compounds HHD and HHSiD (M1 congruent to M3 greater than M2), e.g. HHD and HHSiD methiodides, M1 greater than M2 congruent to M3; p-fluoro-HHSiD, M3 greater than M1 greater than M2; trans-hexbutenol, M1 greater than M3 greater than M2; and (s)-p-fluoro-hexbutinol, M3 greater than M2 congruent to M1. Stereoselectivity ratios [(R)/(S)] for the enantiomers of HHD, hexbutinol and p-fluoro-hexbutinol were highest at M1, intermediate at M3 and lowest at M2 receptors.

Muscarinic receptor differentiation.

1. Several selective antagonists are available to differentiate between muscarinic receptors. 2. Further subdivision of M1 and M2 muscarinic receptors appears possible and is supported by studies with cloned receptors. 3. Reasons for differences between affinity constants determined in functional and binding studies and whether receptor subtypes couple exclusively with a particular cellular mechanism are still to be determined.

Characterization of the antimuscarinic effect of heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl ammonium bromide).

The effect of heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl ammonium bromide) (C7/3'-phthalimidopropyl), an alkane bisquaternary compound with muscarinic receptor blocking activity was studied in guinea-pig atria and ileal longitudinal muscle. C7/3'-phthalimidopropyl was a more potent inhibitor of atrial muscarinic receptors, the cardioselectivity being ca. 32-fold. Previous studies in guinea-pig atria have shown that its antimuscarinic effect was of an allosteric nature. In ileal longitudinal muscle C7/3'-phthalimidopropyl (3 to 100 microM) appeared to behave in a competitive manner towards carbachol but the combination of atropine or homatropine with C7/3'-phthalimidopropyl produced a supra-additive inhibitory effect on the responses to carbachol. In both atria and ileal longitudinal muscle homogenates, C7/3'-phthalimidopropyl also slowed the dissociation rate of [3H]QNB suggesting an allosteric mechanism. In binding studies using either [3H]QNB or [3H]oxo-M, C7/3'-phthalimidopropyl recognized two binding sites in atria and ileum. In both tissues, C7/3'-phthalimidopropyl bound with high affinity (ca. 30-70 nM) to 60-85% of the sites and with low affinity (ca. 1-9 microM) to the remaining sites. Correlation of these affinity constants with the dissociation constants obtained in functional studies in the two tissues is discussed.

Differences in antagonist affinities at muscarinic receptors in chick and guinea-pig.

1. The antimuscarinic effects of a number of muscarinic antagonists in left atrium and ileum of the chick and guinea-pig were evaluated. 2. In agreement with findings in the guinea-pig, pirenzepine did not differentiate between muscarinic receptors in the left atrium and ileum of the chick. However, its affinity in this species was 5- to 10-fold higher than that observed in similar mammalian tissues. 3. Gallamine, methoctramine and himbacine are cardioselective antagonists in the guinea-pig with a selectivity index of 20, 25 and 10 respectively. However, in the chick, while himbacine maintained its cardioselective effect, gallamine was non-selective and methoctramine was only 4-fold more selective. 4. Secoverine, a non-selective antagonist in guinea-pig exhibited a small degree of cardioselectivity in the chick. 5. The ileoselective effect of 4-DAMP in the chick was much greater than that observed in the guinea-pig. 6. It is concluded that muscarinic receptors in cardiac and smooth muscles of the chick differ from those in the guinea-pig.

Displacement of [3H]oxotremorine-M binding by muscarinic antagonists in guinea-pig atrial and ileal membrane homogenates.

Displacement of [3H]oxotremorine-M [( 3H]oxo-M) binding by muscarinic antagonists that are functionally non-selective (atropine), ileoselective (4-DAMP) or cardioselective (gallamine, pancuronium, vecuronium, himbacine) was investigated in guinea-pig atrial and ileal longitudinal muscle membranes. [3H]Oxo-M bound to a single population of high affinity sites in atrial (KD = 11.40 nM) and ileal (KD = 6.15 nM) membranes. Atropine displaced [3H]oxo-M binding sites in a competitive manner, showing similar affinities in the two tissues. 4-DAMP showed two binding sites in ileum but not in atria. The dissociation constant at the high affinity site in ileum was ca 5-fold lower than the value observed in atria, indicating ileoselectivity. Vecuronium also displaced [3H]oxo-M binding in a competitive manner and exhibited similar affinities in both tissues. Gallamine, pancuronium and himbacine displayed two binding sites in each of the two tissues with the majority of sites (ca. 60-80%) showing high affinity. Overall the cardioselective antagonists do not exhibit any consistent correlation between the affinities found in functional experiments and those determined in binding experiments.

Comparison of the effects of some muscarinic agonists on smooth muscle function and phosphatidylinositol turnover in the guinea-pig taenia caeci.

1. The effects of the muscarinic agonists acetylcholine (ACh), carbachol (CCh), AHR-602, and McN-A-343 on contractility and on inositol phosphate accumulation in the presence of lithium were compared in the taenia of the guinea-pig caecum. 2. Compared to CCh, ACh was a full agonist for contraction but AHR-602 and McN-A-343 were partial agonists producing 80-85% of the maximal response to CCh. Similar to previous findings with CCh, tonic contractions produced by AHR-602 and McN-A-343 were less sensitive to inhibition by nifedipine or verapamil than tonic contractions to ACh. 3. CCh and ACh produced similar increases in inositol phosphate accumulation and the effect of CCh (0.1 mM) was inhibited by atropine (IC50 8.5 nM) and pirenzepine (IC50 450 nM). The accumulation of inositol phosphates in the presence of AHR-602 or McN-A-343 was not significantly different (P greater than 0.05) from basal levels. 4. A concentration of 0.2 mM AHR-602 produced a parallel shift of the concentration-response curve to CCh on inositol phosphate accumulation. The IC50 value for inhibition of CCh (0.1 mM) was greater than 50 fold higher than the EC50 value for contraction produced by the partial agonist. McN-A-343 (20 microM) produced a flattening of the concentration-response curve to CCh for inositol phosphate accumulation. 5. The results suggest that the increase in phosphatidylinositol turnover produced by muscarinic agonists, like the contractile response, involves an M2-muscarinic receptor. AHR-602 and McN-A-343 are partial agonists for the contractile response and while producing no significant increase in phosphatidylinositol turnover inhibit the response to CCh.

Binding studies on two functional cardioselective antimuscarinic compounds.

Vecuronium and himbacine are antimuscarinic compounds which in functional studies exhibited a ca 6- and 10-fold higher potency at cardiac muscarinic receptors than at ileal muscarinic receptors. However in binding studies both compounds failed to differentiate between [3H](-)-QNB binding sites in guinea-pig atrial and ileal muscle homogenates. In the latter experiments, the dissociation constants of vecuronium in atria and ileum and that of himbacine in ileum were lower than the values determined functionally. The basis for the lack of cardioselectivity in binding studies is not known. These compounds add to the list of functional cardioselective muscarinic receptor antagonists that failed to display selectivity in binding studies with [3H](-)-QNB.

Paraoxon-induced desensitization and its reversal by tetrodotoxin.

1. Paraoxon (10 microM for 20 min) induced a desensitization of the taenia caecum of the guinea-pig for contractions produced by a number of cholinomimetics. 2. Tetrodotoxin (0.3 microM) reversed the desensitization suggesting involvement of Na+ channels.

Investigation of the antimuscarinic and other actions of proadifen in-vitro.

The possibility that proadifen (SKF 525A) antagonizes endothelium-dependent relaxations to acetylcholine (ACh) in isolated blood vessel preparations via a muscarinic receptor blocking action has been investigated. In phenylephrine-contracted rat isolated aortic ring preparations (with endothelium), proadifen (10-100 microM) shifts ACh relaxant curves to the right without affecting the maximal response, yet endothelium-dependent relaxations to ATP are unaffected. At lower concentrations, proadifen (1-10 microM) antagonizes negative inotropic responses to ACh and ATP in guinea-pig left atria, antagonizes contractile responses to ACh and elevated [K+] in guinea-pig ileal preparations, displaces (-)-[3H]quinuclidinyl benzilate from muscarinic binding sites in membrane homogenates of guinea-pig ileal longitudinal muscle and reduces contractile responses to elevated [K+] in rat aortic ring preparations. It is concluded that proadifen may possess complex interactions with muscarinic receptors and Ca2+ entry blocking properties in concentrations 10-100 times lower than those reported to inhibit cytochrome P450-catalysed reactions.

The affinity of some selective muscarinic receptor antagonists for the muscarinic receptor mediating endothelial-dependent relaxation of the rabbit and rat thoracic aorta.

The pKB values determined for pirenzepine, 4-DAMP, secoverine and gallamine against acetylcholine-mediated relaxant effects in rabbit aorta indicate that this muscarinic receptor closely resembles that which mediates contraction of non-vascular smooth muscle. The results of the present study argue against the presence of a novel type of muscarinic receptor mediating endothelium-dependent relaxation.

Interaction of some muscarinic agonists and antagonists at the prejunctional muscarinic receptor in the rabbit ear artery preparation.

The inhibitory effect of several muscarinic agonists on responses to sympathetic nerve stimulation of the isolated perfused ear artery of the rabbit was compared to that of acetylcholine in preparations pretreated with dyflos, cocaine and yohimbine. In general the potency of the agonists was similar to that observed at peripheral muscarinic sites except for arecaidine propargyl ester and 4-(m-chlorophenylcarbamoyloxy)-2-butynyl trimethylammonium chloride (McN-A-343). The inhibitory effect observed with N-benzyl-3-pyrrolidyl acetate methobromide (AHR-602) was not exerted via muscarinic receptors. With carbachol (CCh) as an agonist, the antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) was found to have a pKB value of 7.74 and thus was 19 fold less active as an antagonist of responses to the agonist, than previously reported for guinea-pig ileum. When McN-A-343 was used as the agonist, the slope of the Schild plot with the antagonist was significantly less than unity. It is suggested that an allosteric interaction of 4-DAMP may be involved with this agonist. By use of an allosteric model, a pKB of 8.56 for 4-DAMP was obtained. Secoverine produced similar pKB values with either CCh (8.19) or McN-A-343 (8.13) as the agonist.

Comparison of the affinity constant of some muscarinic receptor antagonists with their displacement of [3H]quinuclidinyl benzilate binding in atrial and ileal longitudinal muscle of the guinea-pig.

The ability of the muscarinic receptor antagonists fenipramide, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) and secoverine to displace [3H]QNB binding was correlated with the inhibition of responses of cholinomimetics at muscarinic receptors in the atria and ileal longitudinal muscle of the guinea-pig. Fenipramide and 4-DAMP exhibited a 2-4 fold higher affinity for muscarinic receptors in ileal longitudinal muscle in both types of experiments. Secoverine exhibited no difference in affinity in the two tissues.

Failure of gallamine and pancuronium to inhibit selectively (-)-[3H]quinuclidinyl benzilate binding in guinea-pig atria.

Binding of (-)-[3H]quinuclidinyl benzilate (QNB) to muscarinic sites in guinea-pig atrial and ileal longitudinal muscle homogenates showed the presence of a single population of binding sites in atria (KD = 41 (32-53) (95% confidence limits) pM; Bmax = 0.225 +/- 0.02 pmol/mg protein (3)) and two binding sites in the ileum (KD = 20.9 (8.8-49) pM and 11.3 nM; Bmax = 0.436 +/- 0.09 and 11.85 +/- 2.63 pmol/mg protein (4), respectively). Atropine, gallamine, and pancuronium displaced (-)-[3H]QNB binding from the high affinity binding sites in the two tissues in a dose-dependent manner with -log Ki values of 8.6, 6.4, and 6.9, respectively, in atria and 8.7, 6.8, and 6.9, respectively, in ileal longitudinal muscle. The lack of selectivity of gallamine and pancuronium in binding experiments differed from results obtained in isolated tissue experiments where these antagonists showed a marked difference in their ability to antagonize cholinomimetics in the two tissues. In addition, the Ki values for gallamine and pancuronium in ileal homogenates were ca. 130- and 16-fold lower, respectively, than their KB values determined from isolated tissue experiments. Attempts to correlate data from binding experiments and isolated tissue experiments using combinations of antagonists led to variable results attributed to differences in the rates of dissociation of the antagonists from muscarinic receptors. It is concluded that the interaction of gallamine or pancuronium with agonists or antagonists at muscarinic receptors is not a simple bimolecular interaction.

The interaction of McN-A-343 with pirenzepine and other selective muscarine receptor antagonists at a prejunctional muscarine receptor.

The effect of several muscarine receptor antagonists on responses to carbachol (CCh) and McN-A-343 (McN) were compared in the perfused rabbit ear artery preparation stimulated via noradrenergic nerves at 3 Hz in the presence of cocaine (10 microM) and yohimbine (1 microM). The slope of the dose-response curve to McN was significantly less (P less than 0.05) than that for CCh although both agonists produced up to 100% inhibition of responses to nervous stimulation. All the antagonists investigated produced parallel shifts of the dose-response curve to the agonists and atropine, fenipramide or stercuronium gave a similar pA2 value with either agonist. Pirenzepine was a competitive antagonist when CCh was used, as judged by a slope of 0.96 +/- 0.10 for the Arunlakshana-Schild (A-S) plot (pKB 6.2). Displacement of 3H-(-)QNB binding by pirenzepine gave a pKI value of 6.0 which was not significantly different to the pKB value. When McN was used as the agonist, the dose-ratios obtained with pirenzepine (0.5 microM) were significantly different (P less than 0.01) to those with CCh as agonist and the slope of the A-S plot over the concentration range of 0.1 to 3 microM was significantly less than 1.0 (P less than 0.01), indicating that the inhibition was not a simple competitive interaction. It is suggested that the interaction of McN and pirenzepine may involve an allosteric mechanism.

Comparison of the affinity of secoverine for some muscarinic receptors.

The selective muscarinic receptor antagonist secoverine was found to have a similar affinity for muscarinic receptors in the guinea-pig left atrium and ileal longitudinal muscle as well as in the rat urinary bladder and ileum.