The interaction of methoctramine and himbacine at atrial, smooth muscle and endothelial muscarinic receptors in vitro.

1. The action of methoctramine and himbacine at muscarinic receptors has been studied using guinea-pig isolated trachea, oesophageal muscularis mucosae, paced left atria, and rat aortic preparations. 2. Methoctramine (1 x 10(-6)-3.2 x 10(-4) M), but not himbacine, elicited positive inotropic responses. These responses were enhanced by pretreating the animals with reserpine. The responses in reserpine-treated animals were not antagonized by phentolamine (1 x 10(-6) M) but were antagonized by propranolol (1 x 10(-6) M). 3. Methoctramine, but not himbacine, exhibited allosteric inhibitory effects at cardiac muscarinic receptors, resulting in a curvilinear Schild plot. Deviations from competitive antagonism were also observed in combination dose-ratio experiments using atropine and methoctramine. At 1 x 10(-6) M, the pKB value for methoctramine was 7.88 +/- 0.15 (mean +/- s.e.mean, n = 5). The pA2 value for himbacine at cardiac muscarinic receptors was 8.52 +/- 0.06 (n = 3). 4. At tracheal and oesophageal muscularis mucosal smooth muscle receptors, the Schild plots for both antagonists were linear. The pA2 values for methoctramine at receptors in these two preparations were similar (6.08 +/- 0.05 and 6.03 +/- 0.09 respectively, n = 4) and were approximately 60 fold less than those values observed at atrial receptors. Himbacine, also exhibited similar values at muscarinic receptors in the trachea and oesophageal muscularis mucosae (7.61 +/- 0.05 and 7.57 +/- 0.04 respectively, n = 4). 5. Muscarinic receptors mediating relaxation of the rat aortic endothelium exhibited pA2 values for methoctramine (5.87 +/- 0.12, n = 6) which were similar to those observed in the smooth muscle, but not the atria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the interaction of the cervane alkaloid,imperialine, at muscarinic receptors in vitro

Summary The action of the cervane alkaloid, imperialine, has been assessed at M₁, M₂ and M₃ receptors in functional assays and at M₁, M₂, M₃ and putative M₄ sites in binding studies. In functional studies, imperialine acted as a selective surmountable antagonist at M₂ receptors in guinea-pig isolated atria and uterus (–log K_B = 7.7 and 7.4, respectively), in comparison to M₁, receptors in canine isolated saphenous vein (–log K_B = 6.9) or M3 receptors in a range of guinea-pig isolated smooth muscles including ileum, trachea, fundus, seminal vesicle or oesophagus (–log K_B = 6.6–6.8). In rat aorta, the –log K_B value at the M₃ receptor (5.9) was slightly, but significantly, lower.In competition radioligand binding studies, imperialine was also selective toward to M2 sites in rat myocardium (–log K_i = 7.2) with respect to M₁ and M₃ sites (rat cerebral cortex, rat submaxillary gland; –log K_i = 6.1 and 5.7, respectively). However, it did not significantly discriminate between rat cardiac M₂ sites and putative M₄ sites in rabbit lung (–log K_i = 6.9).Imperialine resembles the alkaloid himbacine in terms of its pharmacological profile at muscarinic receptor subtypes in that it acts as an M2 selective antagonist with respect to M₁ or M₃ sites. It may also provide a second, commercially available, antagonist with which to discriminate between M₁ and M₄ receptors. Send offprint requests to R. M. Eglen at the above address     

The interaction of amine local anaesthetics with muscarinic receptors.

1 Amine local anaesthetics inhibited the binding of (-)-[3H]-quinuclidinyl benzilate ((-)-[3H]-QNB) to muscarinic receptors in crude synaptosomal preparations from guinea-pig brain. The order of potency was SKF 525A greater than tetracaine greater than procaine congruent to quinidine greater than procainamide greater than bupivacaine greater than lignocaine greater than prilocaine. 2 The concentration of tetracaine or prilocaine causing 50% inhibition of the receptor-specific binding of [3H]-QNB varied linearly with the concentration of [3H]-QNB present for the range of concentrations of prilocaine used and at lower concentrations of tetracaine, thus providing evidence for a competitive interaction. The affinity constant for tetracaine was 2.6 +/- 0.2 x 10(5) M-1 and that for prilocaine 2.6 +/- 0.8 x 10(3) M-1. At higher concentrations of tetracaine the interaction appears to diverge from simple competitive kinetics. 3 The log dose-response curve for the contractile response of longitudinal muscle strips from guinea-pig intestine to carbachol was shifted in a parallel fashion by low concentrations of tetracaine, but flattened by higher doses. A similar effect was observed for both lignocaine and prilocaine. The affinity constants for tetracaine and prilocaine calculated from the parallel shifts, 1 x 10(5) M-1 and 4 x 10(3) M-1, respectively, were in reasonable accord with the binding data. 4 The curve for the inhibition of [3H]-QNB binding by carbachol was not significantly altered, either in position or shape, in the presence of 1 mM prilocaine. Thus there is no evidence that prilocaine, which increases the affinity of nicotinic acetylcholine receptors for agonists, has any similar effect on agonist binding to muscarinic receptors.     

In vitro down-regulation predicts agonist efficacy at central muscarinic cholinergic receptors

Agonist induced short-term down-regulation of central muscarinic cholinergic receptors in mechanically dissociated cells of the mouse brain has been shown to predict the efficacy of agonists at muscarinic receptors. Pretreatment of cells with full agonists such as carbachol or oxotremorine M resulted in a loss of available muscarinic cholinergic receptors of about 30% using [3H]N-methylscopolamine [( 3H]NMS) as radioligand, whereas a second group of agonists e.g. RS 86 were only weakly active in this regard producing a significantly smaller loss of cell surface muscarinic cholinergic receptors. The magnitude of down-regulation of muscarinic receptors induced by pretreatment with several cholinergic drugs correlates fairly well with their ability to stimulate the phosphatidylinositol turnover. It seems that the agonist induced down-regulation of muscarinic cholinergic receptors on mechanically dissociated neurons of the mouse brain is a simple screening method to test for centrally acting cholinergic agonists.     

Competitive interaction of gallamine with multiple muscarinic receptors

Gallamine, a cholinergic antagonist at the (nicotinic) neuromuscular junction, possesses antimuscarinic potency in several systems. We report here that gallamine inhibited the binding of [³H]quinuclidinyl benzilate (QNB) in a competitive manner in the brainstem and forebrain of the rat. The occupancy curves derived from these studies suggest that gallamine has widely varying affinities for different subpopulations of muscarinic receptors, a finding which sets gallamine apart from classical muscarinic antagonists such as atropine and QNB. The greatest difference in affinities for gallamine occurred in the brainstem, where the data could be satisfactorily fitted to a two-site model, with 77% of the receptors having high affinity (K_d = 25 nM) and 23% low affinity (93 μM). Further, these affinities displayed rank order correlation with those of carbachol (an agonist), although gallamine has not, so far, displayed agonist (or partial agonist) activity. The finding that antagonists as well as agonists can display multiple affinities for muscarinic receptors suggests that there are fundamental differences among subpopulations of these receptors.     

The interaction of hexamethonium with muscarinic receptor subtypes in vitro.

1. The action of hexamethonium has been studied at a range of muscarinic receptors in vitro by use of both functional and radioligand binding studies. 2. In functional studies, hexamethonium exhibited little or no significant (P less than 0.05) antagonism of contractile responses to carbachol at muscarinic receptors in the guinea-pig ileum, oesophageal muscularis mucosae, urinary bladder and trachea. However, antagonism was observed at muscarinic receptors in the guinea-pig left atria mediating negative inotropic responses and the calculated pKB value was 3.80. Hexamethonium also antagonized contractile responses to carbachol in the canine saphenous vein. The pKB value at these receptors was 3.75. 3. In the presence of 3.2 mM hexamethonium, the pA2 value for methoctramine at atrial muscarinic receptors was reduced by approximately 10 fold (control pA2 value was 7.81 +/- 0.05; pA2 value in hexamethonium was 6.73 +/- 0.04). In contrast at tracheal muscarinic receptors, the pA2 values for methoctramine were unaffected in the presence of 3.2 mM hexamethonium (control pA2 = 5.58 +/- 0.07; pA2 value in hexamethonium was 5.63 +/- 0.12). All values quoted are mean +/- s.e. mean, n = 8. 4. In competition radioligand binding studies, hexamethonium exhibited a higher affinity for cardiac M2 receptors (pKi = 3.68) than for cerebrocortical M1 receptors (pKi = 3.28) or for submaxillary gland M3 receptors (pKi = 2.61). At M2 receptors hexamethonium at concentrations of 0.1-10 mM, increased the half life of the dissociation rate of [3H]-N-methylscopolamine 1.6-4.3 fold. This was observed at M3 receptors only at 10 mM, when the half life was increased 1.7 fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

The allosteric interaction of otenzepad (AF-DX 116) at muscarinic M2 receptors in guinea pig atria

The effects of the muscarinic receptor antagonist, otenzepad, in combination with the competitive antagonists N-methylscopolamine, dexetimide and atropine, or the allosteric modulators, C(7)/3'-phth, gallamine and alcuronium, were measured in the guinea pig electrically driven left atrium using the agonists, carbachol or acetylcholine. Otenzepad, in combination with C(7)/3'-phth or gallamine, gave concentration-ratios close to additive and in agreement with theoretical model predictions for combination of two allosteric modulators acting at a common site. However, when otenzepad was combined with alcuronium, dexetimide or N-methylscopolamine, supra-additive effects were observed. For either competitive antagonist in combination with otenzepad, the degree of supra-additivity was more evident after 2-h equilibration than after 40 min. When otenzepad was combined with atropine, no supra-additivity was observed with carbachol as the agonist, but was evident with acetylcholine. Otenzepad was also unable to fully inhibit [3H]N-methylscopolamine binding when the radioligand was employed at a concentration of approximately 100 x K(D). It is concluded that the action of otenzepad involves an allosteric site and a number of possibilities are discussed for its location.     

Competitive interaction of pirenzepine with rat brain muscarinic acetylcholine receptors

In the present work, we studied the details of the interaction of the nonclassical muscarinic receptor antagonist pirenzepine with [3H]quinuclidinyl benzilate binding sites in rat brain homogenates. Pirenzepine showed biphasic competition curves with a Hill coefficient lower than unity, and these curves were better described according to a two-site receptor model. The affinities and the relative preponderance of these sites were constant at different ligand concentrations, in accordance with a competitive type of interaction. Similarly, pirenzepine did not influence the rate of dissociation of the [3H]quinuclidinyl benzilate-receptor complex, even at relatively high concentrations. However, although low concentrations of pirenzepine decreased the affinity of [3H]quinuclidinyl benzilate for the receptor without affecting the density of the binding sites, higher concentrations of the antagonist decreased the receptor number in a reversible fashion. Schild plots of these data indicated an apparent deviation from simple competition in this experimental design, an observation which can be attributed to the selectivity of pirenzepine for different receptor subtypes. Furthermore, pirenzepine, at concentrations high enough to saturate both its high- and low-affinity sites protected [3H]quinuclidinyl benzilate binding sites in the brain against irreversible alkylation by propylbenzilylcholine mustard. Therefore, our data support a competitive nature of interaction of pirenzepine with rat brain muscarinic receptors.     

Action of agonists and antagonists at muscarinic receptors present on ileum and atria in vitro.

The action of 'selective' agonists and antagonists at muscarinic receptors mediating ileal contractions, and the rate and force of atrial contractions has been assessed. The effect of nicotinic receptor stimulation, catecholamine release and acetylcholinesterase (AChE) action on muscarinic activity has also been assessed. The nicotinic actions of carbachol did not affect its agonist potency nor the antagonist affinity data obtained when this agonist was used in atrial and ileal preparations. Antagonist data indicated that muscarinic receptors mediating the rate and force of atrial contractions did not differ. Differences in agonist potencies at these two muscarinic receptors were attributable to either differences in intrinsic efficacy or susceptibility to the action of acetylcholinesterase. The small differences in agonist potency observed between atrial and ileal muscarinic receptors were considered not sufficient to indicate receptor heterogeneity. The pirenzepine affinity data indicated that all three receptors are of the M2 type. Affinity data using secoverine and 4-diphenyl-acetoxy-N-methyl piperidine methiodide indicated that ileal and atrial muscarinic receptors differ. Data obtained using gallamine, pancuronium and stercuronium cannot be regarded as indicative of receptor affinity since the antagonism is not competitive; it did nonetheless corroborate the conclusion that ileal and atrial muscarinic receptors are different.     

Calcitonin: antagonism at intestinal muscarinic receptors.

The action of calcitonin was studied on the motility of isolated innervated segments of rabbit and guinea-pig intestines as well as longitudinal muscle with adherent myenteric plexus dissected from the guinea-pig ileum. Calcitonin (0.25 muM) antagonized contractile responses to acetylcholine and the cholinergic response to electrical field stimulation. This hormonal effect was relatively specific since it was not observed at nicotinic receptors or adrenoceptors, nor did calcitonin act as a local anaesthetic or directly on the contractile machinery of smooth muscle. Perivascular adrenergic and intrinsic non-adrenergic inhibitory responses also were unaffected by calcitonin. However, calcitonin did have antihistaminic properties directed against H1-receptors. The concentration of calcitonin required to achieve muscarinic antagonism in our experiments is not reached at the resting level of circulating hormone.     

Gallamine exerts biphasic allosteric effects at muscarinic receptors

Although gallamine and a number of other compounds have been reported to slow the rate of dissociation of labeled ligands, especially [3H]N-methylscopolamine (NMS), from muscarinic receptors of heart and brain, there has been some dispute as to whether the dissociation of [3H]quinuclidinyl benzilate (QNB) is subject to such allosteric regulation. The present studies were intended to determine whether past discrepancies might be due to differences between tissues. We have found that gallamine modulates the dissociation of [3H]QNB from muscarinic receptors of the heart in a biphasic manner. Low concentrations (micromolar) accelerate the rate of dissociation, whereas higher concentrations (millimolar) slow it; at about 0.1 mM, the two effects cancel each other. Similar results were obtained with muscarinic receptors from the brainstem, but gallamine had only marginal effects on the dissociation of [3H]QNB in the forebrain. On the other hand, verapamil exerts only monophasic effects (slowing) on the dissociation of both [3H]NMS and [3H]QNB from heart receptors and gallamine slows the dissociation of [3H]NMS to a similar extent in all three tissues. Thus, it appears that past discrepancies in the literature can be attributed to the tissues and concentrations of gallamine that were used. Furthermore, the biphasic effects of gallamine suggest that there are multiple allosteric regulatory sites associated with muscarinic receptors.     

Characterization of muscarinic cholinergic receptors in intact myocardial cells in vitro.

Muscarinic acetylcholine receptors (mAChR) were studied on heart cells grown in culture by the radioligand binding technique. We used [3H]n-methyl-scopolamine to monitor the level of receptors on intact cardiocytes. The number of mAChR was very low during the first days in culture (23 fmol/dish). It increased gradually until it reached a plateau on the 4th day (180 fmol/dish), where it remained for 1-2 weeks. To determine whether contractile activity affected the level or affinity of mAChR, the cardiocytes were exposed to agents that stimulate or arrest the heart beat. Treatment with triiodothyronine (T3, 10-90 nM) for 48 hr caused a reduction in the level of the receptors by 20-30% without changing significantly the affinity of the receptors. Similarly, electrical stimulation caused a reduction in the level of the receptors by 30-40%, without a significant influence on creatine kinase activity. When the myocardial cells were treated with Ca-channel blocker such as metoxyverapamil (D600) (10-30 micrograms/mL) or diltiazem (10-25 micrograms/mL) the level of the receptors was also reduced by 30-40%. The reduction in the receptor binding sites was accompanied by an increase in Kd from 0.8 to 3.2 nM in D600-treated cells, whereas there was no significant change in the radioligand affinity after application of diltiazem. Treatment with D600 or T3 together with cycloheximide showed that under these experimental conditions the rate of receptor degradation was accelerated. The half-life of the receptors in the control was 27 hr, whereas the half-lives of T3 and D600 were 15 and 18 hr, respectively. It is concluded that regulation of the amount of cholinergic receptors occurs at the level of receptor breakdown, and simple linkage does not exist between the rate of cardiac contractions and the number of mAChR.     

Allosterism at muscarinic receptors: ligands and mechanisms

The evaluation of allosteric ligands at muscarinic receptors is discussed in terms of the ability of the experimental data to be interpreted by the allosteric ternary complex model. The compilation of useful SAR information of allosteric ligands is not simple, especially for muscarinic receptors, where there are multiple allosteric sites and complex interactions.     

Interaction of amoxapine with muscarinic cholinergic receptors: an in vitro assessment

Amoxapine, an antidepressant with a rapid onset of therapeutic efficacy and great utility in psychotic depression, has been reported to produce anticholinergic side effects in man similar to those observed with imipramine and amitriptyline. To establish its cholinergic disposition, amoxapine and its metabolites 7-hydroxyamoxapine and 8-hydroxyamoxapine, have been evaluated by determining their effects on quinuclidinyl benzilate (QNB) binding to membrane fractions of rat and human brain, on the carbamoylcholine-stimulated accumulation of inositol phosphates in rat cerebral cortex and on the acetylcholine-induced contraction of the guinea pig ileum. In all three preparations, amoxapine was found to be a considerably weaker antagonist of muscarinic cholinergic receptors than either imipramine (4-27 fold) or amitriptyline (51-300 fold). These results indicate that for amoxapine, no correlation exists between the magnitude of muscarinic receptor inhibition and the extent of 'anticholinergic' side effects found in the clinic. Neither the metabolites of amoxapine nor species differences could account for this discrepancy.     

Probing the selectivity of allosteric modulators of muscarinic receptors at other G-protein-coupled receptors

1. The aim of the present investigation was to analyse whether three prototype allosteric modulators of ligand binding to muscarinic receptors, i.e. alcuronium, gallamine, and the alkane-bis-ammonium compound W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropylammonium bromide]), may have allosteric effects on radioligand-binding characteristics at other G-protein-coupled receptors, such as cerebral A1 adenosine receptors (Gi-coupled), cardiac left ventricular alpha1-adrenoceptors (Gq), and beta-adrenoceptors (Gs). 2. The modulators were applied at concentrations known to be high with regard to the allosteric delay of the dissociation of the antagonist [3H]-N-methylscopolamine (NMS) from muscarinic M2-receptors: 30 micromol l(-1) W84, 30 micromol l(-1) alcuronium, 1000 micromol l(-1) gallamine. As radioligands, we used the adenosine A1-receptor ligand [3H]-cyclopentyl-dipropylxanthine (CPX), the alpha1-adrenoceptor ligand [3H]-prazosin (PRAZ), and the beta-adrenoceptor ligand (-)-[125I]-iodocyanopindolol (ICYP). Allosteric actions on ligand dissociation and the equilibrium binding were measured in the membrane fractions of rat whole forebrain (CPX) and of rat cardiac left ventricle (PRAZ, ICYP, NMS), respectively. 3. CPX and PRAZ showed a monophasic dissociation with half-lives of 5.88+/-0.15 and 12.27+/-0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influenced by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36+/-2% of the control value with alcuronium and to 42+/-2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately accelerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and after 3 h, more than 50% of the radioligand was still bound, so that a reliable half-life could not be calculated. ICYP dissociation was not affected by W84. In the presence of alcuronium and gallamine, the dissociation curve of ICYP revealed an initial drop from the starting level, followed by the major phase of dissociation being parallel to the control curve. 4. In summary, the allosteric action of the applied agents is not a common feature of G-protein-coupled receptors and appears to be specific for muscarinic receptors.     

The number of muscarinic receptors in chick amnion muscle

1. [(3)H]-Propyl benzilylcholine mustard ([(3)H]-PrBCM) an irreversible specific antagonist of muscarinic acetylcholine receptors, has been used to label the muscarinic receptors in the aneuronal muscle of isolated 11 day amniotic membranes of chick embryos.2. This muscle was found to have about 9 fmol/mg dry weight of receptor material. This is only 1-2% of the amount found in intestinal muscle.3. Pharmacological studies with isolated amniotic membranes using PrBCM show that labelling is to functional receptors and that the receptor reserve is small.4. The significance of the difference in receptor reserve in innervated and aneuronal plain muscle is discussed.