Af Roffel

Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle

The muscarinic receptor subtype involved in human airway smooth muscle contraction was characterised for the first time, using subtype-selective muscarinic antagonists. It was demonstrated that methacholine-induced contraction of central (trachea) and peripheral (small bronchi) airway smooth muscle preparations was antagonised by pirenzepine, AF-DX 116, 4-DAMP methobromide, hexahydrosiladifenidol, and methoctramine with pA2-values characteristic of M3 (smooth muscle/glandular) muscarinic receptors. Since these pA2-values demonstrate significant correlations with those found in bovine and guinea-pig tracheal smooth muscle contraction, it is concluded that these animal tissues provide a good model for the study of M3 subtype-selective muscarinic antagonists to be used as bronchodilators.

Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function

Previous work showing that AF-DX 116, a cardioselective muscarinic antagonist in functional experiments, does not discriminate between muscarinic receptors in bovine cardiac and tracheal membranes has been extended. In addition to AF-DX 116 we used the muscarinic antagonists, atropine, pirenzepine, 4-DAMP methobromide, gallamine, hexahydrosiladifenidol and methoctramine, in radioligand binding experiments on bovine cardiac left ventricular and tracheal smooth muscle membranes. The functional antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips was also evaluated. An excellent correlation was found for all compounds between the binding affinities for muscarinic receptors in cardiac and tracheal smooth muscle membranes; moreover, the affinities found in cardiac membranes correspond with the pA2 values reported for atrial preparations of rat and guinea pig. However, significant and occasionally marked discrepancies were found between binding and functional affinities of these muscarinic antagonists on bovine tracheal smooth muscle.

Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle

1 The muscarinic receptor subtype involved in the methacholine‐induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2‐selective antagonist AF‐DX 116 and the M3‐selective antagonist 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP) methobromide, in addition to the M1‐selective antagonist pirenzepine, in a classical Schild analysis. 2 All the antagonists shifted the methacholine dose‐response curve to the right in a parallel and concentration‐dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF‐DX 116 and 4‐DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3 The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.

Muscarinic control of airway function

Muscarinic M1, M2, and M3 receptor subtypes have been shown to be involved in the pre- and postjunctional control of airway diameter of various species, including man. In a guinea pig model of allergic asthma, the prejunctional M2 receptor was shown to become dysfunctional already during the early allergic reaction, thereby contributing to exaggerated vagal reflex activity and airway hyperreactivity. Moreover, a deficiency of endogenous nitric oxide was observed after allergen provocation, which may also contribute to an enhanced postjunctional M3 receptor-mediated cholinergic response. Both in human and in animal airway preparations it was shown that enhanced cholinergic contractions are relatively resistent to beta-adrenoceptor-mediated relaxation. The reduced beta-adrenoceptor function may primarily be due to transductional cross-talk between PI metabolism and adenylyl cyclase, including protein kinase C-induced uncoupling of the beta-adrenoceptor from the effector system. Cross-talk between postjunctional M2 receptor-mediated inhibition and beta-adrenoceptor-induced activation of adenylyl cyclase appears to be of minor functional importance, but could be enhanced in allergic asthma due to increased expression of the inhibitory G protein as induced by cytokines.

The M2 selective antagonist AF-DX 116 shows high affinity for muscarine receptors in bovine tracheal membranes

SummaryWe have characterized the muscarine receptors in bovine tracheal and left ventricular membranes using 3H-dexetimide/pirenzepine and 3H-dexetimide/AF-DX 116 competition studies. Pirenzepinc exhibited low (M2) affinity binding to both preparations; Kd was 590 nM in left ventricle and 463 nM in trachea. AF-DX 116 exhibited high (M2) affinity binding to left ventricle (Kd = 95.6 nM); in tracheal membranes it bound with high (M2) affinity (Kd = 40.7 nM) to 74% of the receptors and with low (M3) affinity (Kd = 2.26 μM) to 26% of the receptors. It is concluded that bovine tracheal muscle membranes contain a heterogeneous population of muscarine binding sites, the majority having M2 (heart) subtype characteristics and being located on the smooth muscle membranes; a minority having M3 (exocrine gland) subtype characteristics and presumed to be located in submucosal glands. This is the first report of high affinity binding of AF-DX 116 to non-cardiac peripheral muscarine receptors.

CHARACTERIZATION OF PRESYNAPTIC VASCULAR MUSCARINIC RECEPTORS INHIBITING ENDOGENOUS NORADRENALINE OVERFLOW IN THE PORTAL-VEIN OF THE FREELY MOVING RAT

1 In the portal vein of permanently cannulated, freely moving, unanaesthetized rats, methacholine (MCh) is able to inhibit the electrically‐evoked endogenous noradrenaline (NA) overflow. This inhibition is mediated by presynaptic inhibitory muscarinic heteroreceptors. 2 By use of pirenzepine, 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP) and AF‐DX 116 as M1‐, M3‐, and M2‐selective antagonists respectively, the MCh (0.1 μm)‐induced inhibition of the electrically‐evoked NA overflow could be reversed to the control stimulation value dose‐dependently. 3 The potency order of the antagonists was: 4‐DAMP > AF‐DX 116 > pirenzepine, pIC50 values being 8.50, 7.96 and 7.01, respectively. 4 From these results it was concluded that the inhibitory presynaptic heteroreceptors in the portal vein of concious unrestrained rats are of the cardiac M2‐subtype.

Conditional involvement of muscarinic M1 receptors in vagally mediated contraction of guinea-pig bronchi

The involvement of ganglionic muscarinic M1 receptors in vagally induced bronchoconstriction in guinea-pig airways is controversial. Therefore, we studied the effects of the M1-selective muscarinic receptor antagonist pirenzepine on vagus nerve (VNS, preganglionic) and electrical field stimulation (EFS, postganglionic)-induced contractions of the guinea-pig main bronchus under various experimental conditions.Using identical stimulation parameters for VNS and EFS (8V, 30 Hz, 0.5 ms, 5s every min), the amplitude of the VNS-induced twitch contractions was 30.4% of the EFS-induced responses, and pirenzepine showed 2.3-fold selectivity (pIC50-values 6.45 and 6.09, respectively) to inhibit vagally induced contractions. With the stimulation frequency for EFS lowered to match contraction levels obtained using VNS, pirenzepine was equipotent to inhibit both types of response at M3 receptor-selective concentrations, suggesting that M1 receptors are not involved. By contrast, when the stimulation episode was prolonged until plateau contraction (10–20 s), in the presence of the nicotinic antagonist hexamethomum (5 μM), the M2 receptor antagonist AQ-RA 741 (0.1 μM) and the β-adrenoceptor antagonist timolol (1 μM), and again using matched VNS- and EFS-induced contraction levels, pirenzepine inhibited nerve stimulation-evoked responses in a biphasic manner, yielding (pIC50-values of 8.12 indicative of M1 receptor blockade) and 6.43 (indicative of M3 receptor blockade) for the first and second phase, respectively, while postganglionic stimulation showed a purely monophasic inhibition (pIC50 = 6.32).These results show that facilitatory muscarinic M1 receptors are involved in vagally mediated contraction of guinea-pig bronchi, under conditions of elevated neurotransmission and partial nicotinic receptor blockade.

Prejunctional histamine H3-receptors inhibit electrically evoked endogenous noradrenaline overflow in the portal vein of freely moving rats

Abstract The effects of intra-arterial injection of different doses of the selective histamine H3-receptor agonist R-α-methylhistamine and the selective histamine H3-receptor antagonist thioperamide on basal and electrically evoked noradrenaline overflow in the portal vein as well as on mean arterial pressure (MAP) and heart rate (HR) were investigated in permanently instrumented freely moving rats. R-α-Methylhistamine (0.01, 0.1 and 1 μmol/kg) inhibited the evoked noradrenaline overflow up to 43%, the ED50 value being 0.013 μmol/kg. Thioperamide (0.1, 0.5 and 1.0 μmol/kg) antagonized the effect of 1.0 μmol/kg R-α-methylhistamine dose-dependently, evoked overflow returning to control values at 1.0 μmol/kg of the antagonist; thioperamide alone had no effect on electrically evoked noradrenaline overflow. Basal noradrenaline levels, blood pressure and heart rate were not at all influenced by R-α-methylhistamine and thioperamide, alone or in combination. The results clearly show the presence of prejunctional histamine H3-receptors inhibiting the electrically evoked noradrenaline overflow from vascular sympathetic nerve terminals in the portal vein of freely moving rats.

Differences in the prejunctional effects of methacholine and pilocarpine on the release of endogenous acetylcholine from guinea-pig trachea

We investigated the effects of the full muscarinic acetylcholine receptor agonist methacholine and the partial and putatively M2-selective agonist pilocarpine on endogenous acetylcholine release from guinea-pig trachea by use of high-performance liquid chromatography with electrochemical detection. Atropine-induced increases in acetylcholine release were used to monitor the system.Electrical field stimulation (8 V, 30 Hz, 0.5 ms for 5 min)-induced acetylcholine release in the presence of neostigmine, with or without preincubation with choline to maximally enhance acetylcholine output, was increased to about 225% by 0.3 μM atropine, indicating functional autoinhibition. However, methacholine (10 μM) did not affect the acetylcholine release, whereas it was enhanced to 166% by 30 μM pilocarpine. When electrical field stimulation was applied at lower intensity (8 V, 16 Hz, 0.1 ms for 5 min) and in the absence of neostigmine, an increase by 0.3 μM atropine (to 177%) but a decrease of the acetylcholine release by 10 μM methacholine (to 65%) and 30 μM pilocarpine (to 63%) were observed. These results clearly demonstrate (i) that inhibition of evoked endogenous acetylcholine release from prejunctional nerve terminals in guinea-pig trachea can only be demonstrated under conditions of low junctional concentrations of acetylcholine, and (ii) that pilocarpine, as a partial muscarinic agonist, behaves as an antagonist under high junctional concentrations of the neurotransmitter.

POSITIVE COOPERATIVE INTERACTION OF QUATERNARY ANTICHOLINERGICS WITH FUNCTIONAL MUSCARINIC RECEPTORS IN BOVINE TRACHEAL SMOOTH-MUSCLE

SummaryThe interaction of quaternary anticholinergics with muscarinic receptors in bovine tracheal smooth muscle strips was investigated because some of these compounds have shown anomalous (biphasic) behaviour in radioligand displacement studies, in contrast to their tertiary analogues. It was found that ipratropium, N-meth-ylscopolamine, oxyphenonium and N-methyldeptropine give Schild plots with slopes significantly greater than unity (up to 2.0) in contrast to 4-DAMP methobromide and thiazinamium, and the tertiary analogues atropine and scopolamine. However, in guinea pig tracheal smooth muscle, ipratropium and N-methyldeptropine behaved as classic antagonists with Schild slopes of unity. The high Schild plot slopes in bovine tracheal smooth muscle could not be solely explained by inadequate equilibration of the antagonists, since increased incubation times (3 or 5 h instead of 30 min) still brought about slopes significantly greater than unity, or by the presence of an atropinesterase in the tissue. However, by using combinations of atropine with ipratropium or oxyphenonium it could be demonstrated that these quaternary antagonists interact with muscarinic M3 receptors in bovine but not in guinea pig tracheal smooth muscle in a positive cooperative fashion.