A pharmacological study of the responses induced by muscarinic agonists on the isolated superior cervical ganglion of the guinea-pig

We have studied the muscarinic agonist induced responses on the guinea-pig superior cervical ganglion in vitro, as recorded from the internal carotid nerve using a grease-gap. The principal response was a depolarization, but a small hyperpolarizing response could be revealed under certain conditions. We determined the pA2 of a number of muscarinic antagonists against the muscarine induced depolarization. Four selective antagonists and atropine appeared to act competitively. The rank order of their pA2s was 4-DAMP (8.5), atropine (8.4), pirenzepine (8.0), methoctramine (7.2) and AF-DX 116 (6.3). In addition to muscarine, we assessed the potency and relative maximum response of nine other muscarinic compounds to depolarize this preparation: carbachol, 5-methylfurmethide, oxotremorine, oxotremorine-M, pilocarpine, RS 86, AF102B and two novel compounds L-670548 and L-679512. L-670548 was the most potent and AF102B was the least potent agonist tested. Only AF102B evoked a maximum depolarization that was significantly smaller than muscarine. A hyperpolarizing response to carbachol (1 microM) could be recorded when the superfusing medium contained 0.3 microM pirenzepine and only 0.1 mM CaCl2 (cf. usual 2.5 mM). This response was relatively small compared to that evoked on the superior cervical ganglion of the rat. It was blocked by the cardioselective antagonists methoctramine (0.1-0.3 microM) and AF-DX 116 (0.3-1.0 microM). Of the 10 agonists tested, only carbachol, oxotremorine and oxotremorine-M reproducibly evoked a hyperpolarizing response. It was concluded that muscarinic agonists can induce a depolarization of the guinea-pig superior cervical ganglion mediated by M1 receptors. The activation of cardiac-like M2 receptors resulted in a hyperpolarizing response that was relatively small.     

Selective antagonism of muscarinic potentials on the superior cervical ganglion of the rat

Selective antagonists have been used to classify the muscarinic receptors involved in the slow excitatory postsynaptic potential and slow inhibitory postsynaptic potential of the superior cervical ganglia of the rat, as recorded in 1 microM neostigmine, using a grease-gap method. Cardioselective M2 antagonists, e.g. AF-DX 116, depressed the slow inhibitory postsynaptic potential and enhanced the slow excitatory postsynaptic potential. The M1 selective antagonist pirenzepine depressed both potentials equally. The high potency of pirenzepine against the slow excitatory postsynaptic potential, however, indicates that it is mediated by M1 receptors. The slow excitatory and inhibitory postsynaptic potentials were found to be pharmacologically similar to the muscarinic agonist-induced depolarisation and hyperpolarisation of this preparation, respectively. The actions of two muscarinic agonists on the postsynaptic potentials were also studied. It is concluded that the slow excitatory postsynaptic potential is mediated by M1 receptors and the slow inhibitory postsynaptic potential by cardiac-like M2 receptors.     

No evidence for a role of muscarinic M2 receptors in functional antagonism in bovine trachea.

1. The functional antagonism between methacholine- or histamine-induced contraction and beta-adrenoceptor-mediated relaxation was evaluated in bovine tracheal smooth muscle in vitro. In addition, the putative contribution of muscarinic M2 receptors mediating inhibition of beta-adrenoceptor-induced biochemical responses to this functional antagonism was investigated with the selective muscarinic antagonists, pirenzepine (M1 over M2), AF-DX 116 and gallamine (M2 over M3), and hexahydrosiladiphenidol (M3 over M2). 2. By use of isotonic tension measurement, contractions were induced with various concentrations of methacholine or histamine, and isoprenaline concentration-relaxation curves were obtained in the absence or presence of the muscarinic antagonists. Antagonist concentrations were chosen so as to produce selective blockade of M2 receptors (AF-DX 116 0.1 microM, gallamine 30 microM), or half-maximal blockade of M3 receptors (pirenzepine 0.1 microM, AF-DX 116 0.5 microM, hexahydrosiladiphenidol 0.03 microM). Since these latter antagonist concentrations mimicked KB values towards bovine tracheal smooth muscle M3 receptors, antagonist-induced decreases in contractile tone were compensated for by doubling the agonist concentration. 3. It was found that isoprenaline-induced relaxation of bovine tracheal smooth muscle preparations was dependent on the nature and the concentration of the contractile agonist used. Thus, isoprenaline pD2 (-log EC50) values were decreased 3.7 log units as a result of increasing cholinergic tone from 22 to 106%, and 2.4 log units by increasing histamine tone over a similar range. Furthermore, maximal relaxability of cholinergic tone decreased gradually from 100% at low to only 1.3% at supramaximal contraction levels, whereas with histamine almost complete relaxation was maintained at all concentrations applied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of the muscarinic M2 antagonists, AF-DX 116 and gallamine, on single neurons of rabbit sympathetic ganglia

Intracellular recording techniques were used to compare the effects of the M2 muscarinic antagonists, AF-DX 116 and gallamine, on membrane potential (Vm), input resistance (Ri), responses induced by methacholine, muscarinic slow postsynaptic potentials and action potentials in the superior cervical ganglion of the rabbit. Gallamine or AF-DX 116 antagonized methacholine-induced or synaptically-evoked muscarinic hyperpolarization, without having significant effect on depolarization induced by methacholine or synaptically. The drug AF-DX 116 reduced evoked muscarinic hyperpolarizing potentials, without significant change in Vm or Ri, recorded in the absence of muscarinic stimulation. In contrast to AF-DX 116, gallamine elicited a concentration-dependent depolarization of the membrane, with a corresponding increase in Ri, when tested in the absence of muscarinic stimulation. These effects of gallamine were accompanied by an increase in duration and decrease in the slope of the descending phase of the action potential. Blockade by gallamine of evoked hyperpolarization was independent of membrane depolarization and readily occurred when gallamine-induced depolarization was prevented by clamping Vm at its pre-gallamine level. The effects of gallamine were maintained during its presence and reversed upon washing with gallamine-free physiological solution. These results indicate that AF-DX 116 and gallamine have a specificity for antagonism of muscarinic responses, mediated by receptors of the M2 type in the superior cervical ganglion. However, gallamine, while an effective antagonist of M2 responses, also has the ability to modify the electrical characteristics of ganglion cells and thus may modify ganglionic transmission by mechanisms other than antagonism of receptors.     

Pertussis toxin sensitivity of drug-induced potentials on the rat superior cervical ganglion

We have investigated the action of pertussis toxin on a range of receptor-mediated responses of the rat superior cervical ganglion in vitro. The ganglia were treated with pertussis toxin for 24 h at 37 degrees C using an in vitro method. Appropriate controls were also carried out. Pertussis toxin (1 microgram/ml) reduced ganglionic hyperpolarisations mediated by adenosine, alpha 2, 5-HT1A, M2 and GABAB receptors. The GABAB-mediated hyperpolarisation of this preparation, evoked by baclofen and GABA in a bicuculline-resistant manner, has not previously been reported. Pertussis toxin did not reduce ganglionic depolarisations evoked by potassium chloride and 5-HT3, GABAA and nicotinic receptors. Depolarisations to muscarine and noradrenaline, probably mediated by M1 and beta-receptors, also appeared to be resistant to pertussis toxin. The similar sensitivity of the various ganglionic hyperpolarisations to pertussis toxin indicates that they may all be mediated by similar G-proteins.     

Adenosine selectively depresses muscarinic compared with non-muscarinic receptor mediated depolarisation of the rat superior cervical ganglion

• 1.1. A grease gap d.c. recording technique was used to measure electrophysiological responses of the isolated rat superior cervical ganglion.
• 2.2. Adenosine at 100 μM depressed depolarisations to the muscarinic agonists carbachol, muscarine and methylfurmethide. In contrast adenosine (100μM) did not alter depolarisations to 1,1-dimethyl-4-phenylpiperazinium, 2-methyl-5-hydroxytryptamine and potassium and enhanced depolarisations to 5-hydroxytryptamine and gamma-aminobutyric acid.
• 3.3. Adenosine-induced depressions of the depolarisations to carbachol, muscarine, and methylfurmethide tended to be increased in the presence of 0.3 μM methoctramine (a muscarinic receptor antagonist with slight selectivity for M₂ receptors). The increase was statistically significant (P < 0.01) for carbachol.
• 4.4. Medium containing 0.1 mM Ca²⁺ and 0.3 μM pirenzepine augmented the hyperpolarising phase of the response to carbachol. Adenosine (10–300μM) hyperpolarised ganglia and did not significantly alter the hyperpolarisation to 0.3 or 1 μM carbachol but selectively reduced the depolarisation response to 3 μM carbachol.
• 5.5. Adenosine-induced hyperpolarisations (100 μM) were enhanced when applied during depolarisations to muscarinic agonists (muscarine, pilocarpine, N-methyl-N-(1-methyl-4-pyrrolidine-2-butynyl)acetamide (BM-5)), and other M-current inhibitors, barium and eledoisin-related-peptide. Adenosine induced hyperpolarisations were not affected by d-Ala⁶-luteinizing-hormone-releasing-hormone or uridine 5′-triphosphate which produced small depolarisations.
• 6.6. It is concluded that adenosine acts selectively in opposing mechanisms of depolarisation of the rat SCG that are due to the action of muscarinic agonists (acting via M₁-receptors) and by other M-current inhibitors.

     

Muscarinic receptor heterogeneity in rat central nervous system. I. Binding of four selective antagonists to three muscarinic receptor subclasses: a comparison with M2 cardiac muscarinic receptors of the C type

We previously observed that [3H]NMS recognizes three types of muscarinic receptors in rat brain (one M1 subclass with high affinity for pirenzepine, and two M2 subclasses with low affinities for pirenzepine), based on distinct affinity and kinetic constants of [3H]NMS for these three subclasses. In this work, we investigated the binding of four selective antagonists to these three (the M1 and two M2) subclasses. We were able to demonstrate that cardiac-like M2 receptors with low affinity for pirenzepine and low affinity for N-methylscopolamine were present not only in cerebellum (as previously shown; see introduction) but also in cortex, striatum, and hippocampus, and the two M2 receptor subclasses were discriminated by dicyclomine, 4-DAMP, and gallamine, as well as by AF-DX 116 and [3H]NMS. Our findings also suggested that the biphasic association and dissociation kinetics of [3H]NMS observed in various brain regions reflect sequential binding to the different receptors.     

Allosteric interactions of three muscarine antagonists at bovine tracheal smooth muscle and cardiac M2 receptors

The kinetics of [3H]dexetimide dissociation from muscarine receptors in bovine cardiac left ventricular and tracheal smooth muscle membranes were studied in the absence and presence of three muscarine antagonists. It was found that [3H]dexetimide dissociation from cardiac muscarine receptors was monophasic and very fast (half life less than 1 min) and was slowed by the cardioselective muscarine antagonists, gallamine, methoctramine and AF-DX 116, concentration dependently. [3H]Dexetimide dissociation from tracheal muscarine receptors was biphasic, with a fast phase (half-life less than 1 min) followed after 4-5 min by a slow phase (half-life = 38.5 min). The fast component, but not the slow component, was slowed by the muscarine antagonists with concentration dependencies very similar to those found in the heart. We conclude from these data that the major population of tracheal smooth muscle muscarine receptors resembles the cardiac M2 type not only with respect to equilibrium binding affinities but also with respect to the secondary, allosteric binding site on the muscarine receptor. The results also imply that the cardiac receptor subtype is much more sensitive to allosteric modulation than the glandular/smooth muscle receptor subtype.     

Possible involvement of M5 muscarinic receptor in the enhancing actions of the novel gastroprokinetic agent Z-338 on nifedipine-sensitive voltage-dependent Ca2+ currents in guinea pig stomach

We investigated the effects of the novel gastroprokinetic agent Z-338 (N-(N-N'-diisopropylaminoethyl)-[2-(2-hydroxy-4,5-dimethoxybenzoylamino)-1,3-thiazole-4-yl] carboxyamide monohydrochloride trihydrate) on L-type voltage-dependent Ca2+ currents (ICa) in guinea pig gastric myocytes by using the whole-cell patch clamp technique. Bath-applied acetylcholine (ACh) produced biphasic effects on ICa, i.e., enhancement (1-100 nM) and inhibition (1-100 microM), both of which were abolished by pretreatment with atropine (10 microM) or intracellular perfusion of GDPbetaS (500 microM). Z-338 (> or = 1 nM, ED50: 120 nM) mimicked the enhancing effects of ACh, but did not inhibit ICa. The effects of Z-338 and ACh were non-additive and blocked by atropine and GDPbetaS, but not by pertussis toxin (PTX) pretreatment (500 ng/ml). ACh (> or = 1 microM) induced slow inward currents via activation of the muscarinic receptor/PTX-sensitive G-protein pathway, but Z-338 was devoid of these effects. Neither pirenzepine (1 microM), AF-DX116 (1 microM), nor oxybutynin (100 nM) could prevent Z-338 (1 microM) and ACh (10 nM) from enhancing ICa, whilst 4-DAMP (100 nM) blocked the effects of Z-338 and ACh. Bath-application of protein kinase C (PKC) activator PDBu (phorbol-12,13-dibutyrate) (250 nM) enhanced ICa, and conversely, pipette inclusion of PKC inhibitor peptide (150 microM) abolished the effects of ACh and Z-338 on ICa. These results collectively suggest that although contribution of the M3 receptor is not excluded, the major actions of Z-338 on gastric myocytes are potentiation of ICa through activation of M5-like receptor.     

Modulation of the structure-binding relationships of antagonists for muscarinic acetylcholine receptor subtypes.

1. Membranes from rat cerebral cortex, myocardium and extraorbital lacrimal gland were used as sources of M1, M2 and M3 muscarinic acetylcholine receptors respectively and the affinities of seven antagonists for the three subtypes were examined under different experimental conditions. 2. The affinities for the membrane-bound receptors were measured at different ionic strengths and temperatures and compared with those determined on the receptor solubilised in the neutral detergent digitonin or the zwitterionic detergent, CHAPSO. 3. The range of measured affinity constants of a given antagonist for a specific subtype varied from 2 (atropine at M1 receptors) to 1000 (AF-DX 116 at M2 receptors). 4. As a consequence of these changes in affinity, which were dependent on the drug, the subtype and the experimental conditions, both the structure-binding relationships of a given subtype can be markedly changed as well as the selectivity of a drug for the different subtypes. For example it is possible to change the relative affinities of AF-DX 116 and gallamine at membrane-bound M1 receptors from 50:1 to 1:60. 5. Experimental conditions for the observation of high selectivity of pirenzepine, AF-DX 116, gallamine and hexahydrosiladiphenidol for the three subtypes are given. 6. When the receptors are removed from their membrane environment by solubilisation in detergent, antagonist affinities are changed but the subtypes still retain different structure-binding relationships. 7. In general, AF-DX 116 and the allosteric antagonist, gallamine, behave differently from the other antagonists, suggesting that they bind in different ways to muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic receptors in canine colonic circular smooth muscle. II. Signal transduction pathways.

We have, in the accompanying work, demonstrated the coexistence of M2 and M3 muscarinic receptors in the circular smooth muscle of canine colon. In the present study, the effects of muscarinic receptor stimulation on phosphoinositide turnover and adenylate cyclase activity were examined. In myo-[3H]inositol-labeled circular smooth muscle strips, carbachol caused a concentration-dependent (EC50 = 5 microM) increase in [3H]inositol phosphate production. The more M3 receptor-selective muscarinic antagonist pirenzepine (KB = 53 nM) was approximately 60 times more potent than the more M2-selective agent AF-DX 116 (KB = 3 microM) in blocking carbachol-elicited accumulation of [3H]inositol phosphates. The carbachol-stimulated increase in [3H]inositol phosphate accumulation was not affected by pretreatment of the tissue with pertussis toxin (200 ng/ml, 3 hr). Within the first minute, carbachol (100 microM) caused a rapid and transient increase of [3H]inositol 1,4,5-trisphosphate production that oscillated continuously in the presence of agonist (120 min). The accumulation of [3H]inositol 1,3,4-trisphosphate was also extremely rapid, reaching a peak at 15 sec. The accumulation of [3H]inositol monophosphate was delayed and progressively increased over 30 min. [3H]inositol 1,3,4,5-tetrakisphosphate, although not detectable in the first minute, accumulated to significant levels over 30 min in the presence of agonist. Addition of carbachol in the adenylate cyclase assay caused inhibition of forskolin-stimulated [32P]cAMP production and blocked forskolin-stimulated cAMP accumulation in the intact tissue. The inhibitory effects of carbachol on adenylate cyclase were blocked by atropine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methobromide but were unaffected by the more M3-selective agent pirenzepine (1 microM). Pretreatment of tissues with pertussis toxin completely eliminated M2 receptor-mediated inhibition of adenylate cyclase activity, without altering inositol 1,4,5-trisphosphate accumulation. We conclude that muscarinic receptor stimulation of inositol trisphosphate production is mediated by the M3 receptor coupled to a pertussis toxin-insensitive GTP-binding protein and results in the rapid formation of inositol tetrakisphosphate, whereas inhibition of adenylate cyclase activity is mediated by the M2 subtype of muscarinic receptor coupled to the pertussis toxin-sensitive GTP-binding protein Gi.     

Muscarinic M1 and M2 receptor subtypes play opposite roles in LPS-induced septic shock

BackgroundTo compare pharmacologic effects of pirenzepine and AF-DX116, a selective competitive antagonist for M1 and M2 subtype muscarinic cholinergic receptors (mAChRs), respectively, with atropine, a non-selective competitive antagonist for mAChRs, on Lipopolysaccharide (LPS).MethodsMale C57BL/6 mice were used to establish models of LPS-induced experimental endotoxemia. Mice were intraperitoneally injected 10 min prior to LPS injection with control (saline), atropine, pirenzepine and AF-DX116, respectively. Overall survival time was estimated using Kaplan-Meier plots. Inflammatory cytokine tumor necrosis factor-α (TNF-α) was monitored at various intervals after LPS injection and individual reagent administration. Pathological alternations in lungs and liver were analyzed.ResultsPirenzepine and atropine pretreatment improved survival rate of LPS-induced septic shock; in contrast, AF-DX116 accelerated death from sepsis. Moreover, TNF-α plasma level was decreased in response to pirenzepine or atropine, whereas increased in response to AF-DX116. Pirenzepine and atropine relieved whereas AF-DX116 accelerated LPS-induced pulmonary and hepatic injury. Pirenzepine reduced proportion of M1 subtype of macrophages, while AF-DX116 promoted polarization of macrophages to M1 subtype. Pirenzepine pretreatment reduced while AF-DX116 enhanced expression of SOCS3 at mRNA level.ConclusionsThe administration of pirenzepine and atropine may have beneficial effects on septic shock.     

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 suppression of the M-current in the rat sympathetic ganglion is mediated by receptors of the M1-subtype.

1. Under voltage-clamp dissociated adult and foetal rat superior cervical ganglion (s.c.g.) cells exhibited a non-inactivating voltage- and time-dependent component of K+ current termed the M-current (IM). IM was detected and measured from the current decay during hyperpolarizing voltage steps applied from potentials where IM was pre-activated. 2. Neither the resting membrane current nor the amplitude of these current decay relaxations were reduced by omitting Ca from the bathing fluid, showing that the M-current was not a 'Ca-activated' K-current dependent on a primary Ca-influx. Concentrations of (+)-tubocurarine sufficient to block the slow Ca-activated K-current IAHP did not inhibit IM or antagonize the effect of muscarinic agonists on IM, showing that IM was not contaminated by IAHP. Tetraethylammonium (1 mM), which blocks the fast Ca-activated K-current IC, produced a small inhibition of IM. This was not due to contamination of IM by IC since muscarinic agonists did not consistently block IC. 3. The muscarinic agonists muscarine, oxotremorine, McN-A-343 and methacholine reversibly suppressed IM, resulting in an inward (depolarizing) current. The rank order of potency was: oxotremorine greater than or equal to muscarine greater than McN-A-343 greater than methacholine. 4. The suppression of IM by muscarine was similar in cultured cells derived from adult and foetal tissue to that seen in the intact ganglia. 5. IM-suppression by muscarine was inhibited by pirenzepine (Pz) and AF-DX 116 with mean pKB values of 7.53 +/- 0.13 (n = 3) and 6.02 +/- 0.13 (n = 4) respectively. 6. The suppression of IM by muscarinic agonists was not affected by gallamine (10-30 microM). 4-Diphenylacetoxy-N-methylpiperidine methiodide inhibited the response at 300 nM. 7. Pirenzepine inhibited the contractions of the guinea-pig isolated ileum produced by muscarine with a mean pKB of 6.37 +/- 0.03 (n = 8). 8. These results suggest that the receptors mediating suppression of the M-current accord with those designated pharmacologically as M1 and that these receptors reach maturity at a very early stage in the development of the rat s.c.g.     

Carbachol-induced potentiation and inhibition of acid secretion by guinea pig gastric gland

The effects of muscarinic ligands on acid secretion were examined by estimating the accumulation of [14C]aminopyrine in gastric glands isolated from guinea pigs. The accumulation of [14C]aminopyrine in the presence of 0.1 mM histamine was potentiated by 1 microM carbachol but suppressed by 1 mM. These two effects of carbachol were abolished by atropine, pirenzepine and AF-DX 116. Assuming that the binding of carbachol to one site (Site 1) increases [14C]aminopyrine accumulation but its binding to the other site (Site 2) reduces [14C]aminopyrine accumulation, we analysed the dose-response curves for the carbachol effects in the absence and presence of different concentrations of atropine, pirenzepine and AF-DX 116. The dissociation constants determined for these ligands at Sites 1 and 2 were as follows: carbachol, 0.28 and 7.1 microM; atropine, 0.28 and 0.54 nM; pirenzepine, 45 and 560 nM; and AF-DX 116, 380 and 4400 nM, respectively. The binding of [3H]N-methylscopolamine to the gastric glands indicated the presence of two populations of binding sites with different affinities for the above ligands, other than atropine. The apparent dissociation constants, which were estimated by analysing the displacement curves for [3H]N-methylscopolamine binding, were as follows: carbachol, 0.18 microM (10%) and 31 microM (90%); atropine, 1.24 nM; pirenzepine, 15 nM (16%) and 220 nM (84%); and AF-DX 116, 370 nM (10%) and 2970 nM (90%). These results suggest that there are two kinds of muscarinic acetylcholine receptors in the guinea pig gastric gland, one potentiating and the other inhibiting the acid secretion induced by histamine.     

Inverse agonist activity of pirenzepine at M2 muscarinic acetylcholine receptors

1. The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. 2. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). 3. The ranking of Ki values for the agonist carbachol was R*R*+R>R (174, 155, 115 nM), suggesting inverse agonism. 4. The Vmax of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt-2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The K(M) value (0.26-0.33 microM) was not modified by mastoparan or GPAnt-2. 5. Carbachol increased the Vmax of GTP hydrolysis (EC50 8.1+/-0.3 microM), whereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine decreased the Vmax of GTP hydrolysis (EC50 77.5+/-10.3 microM). This effect was enhanced when KCI was substituted for NaCl (EC50 11.0+/-0.8 microM) and was antagonized by atropine and AF-DX 116 (IC50 0.91+/-0.71 and 197+/-85 nM). 6. Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M2 receptor.     

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.     

Pharmacological differences between two muscarinic responses of the rat superior cervical ganglion in vitro.

1 Pharmacological differences have been observed between the muscarinic agonist-induced depolarizing and hyperpolarizing responses of the rat isolated superior cervical ganglion. 2 Pirenzepine (0.3 microM) selectively reduced the depolarizing response and unmasked the hyperpolarizing response. No such selectivity was observed with a concentration of N-methylatropine which was equipotent with pirenzepine in antagonizing the depolarizing response. 3 The neuromuscular blocking agents gallamine (10 microM) and pancuronium (3 microM) exhibited the oppositive selectivity to pirenzepine, both dramatically reduced the hyperpolarization but only slightly antagonized the depolarization. 4 The potencies of a range of agonists in evoking the depolarizing and hyperpolarizing responses, the latter in the presence of 0.3 microM pirenzepine, have been determined. Methylfurmethide failed to hyperpolarize the ganglion at concentrations which evoked maximal depolarizations. 5 The muscarinic hyperpolarization did not appear to be mediated by the secondary release of catecholamines. 6 It was concluded that the two muscarinic responses on the rat superior cervical ganglion, the slow depolarization and faster hyperpolarization, are mediated by different muscarinic receptor subtypes.     

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 microM)-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 greater than AF-DX 116 greater than 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 conscious unrestrained rats are of the cardiac M2-subtype.     

Comparison of anti-M2-muscarinic effect of AF-DX 116 on atrioventricular nodal conduction with those of pirenzepine and atropine as antibradyarrhythmic drugs.

Selectivity of antimuscarinic actions of AF-DX 116 (AF-DX) on the atrioventricular (AV) nodal conduction was compared with those of pirenzepine and atropine by using the canine isolated, blood-perfused AV node preparation and the open-chest in situ dog heart. In the isolated AV node preparation, dose-response curves for negative dromotropic effects (prolongation of Atrio-His interval) of carbachol (CCh) injected into the posterior septal artery were shifted to the right in parallel by AF-DX, pirenzepine, and atropine with apparent pA2-values of 13, 27.5, and 0.45 microg, respectively, and slopes of the modified Schild plot of nearly unity. Meanwhile, dose-response curves for coronary vasodilator effects of CCh were shifted to the right by AF-DX, pirenzepine, and atropine with the apparent pA2 values of 68, 12.5, and 0.55 microg, respectively, but the slopes were far from unity. In the in situ open-chest heart, dose-response curves for negative dromotropic effects (prolongation of AV conduction time) of CCh given intravenously were shifted to the right in parallel by AF-DX, pirenzepine, and atropine with apparent pA2 values of 36, 32, and 1.25 microg/kg, respectively, and the slope of nearly unity, whereas dose-response curves for hypotensive effects of CCh were shifted to the right by AF-DX, pirenzepine, and atropine with apparent pA2 values of 105, 15, and 0.65 microg/kg, respectively, but the slopes of AF-DX and pirenzepine were far from unity. In addition, prolongations of AV conduction time by electrical stimulation of the left vagus nerve in the in situ heart were suppressed by AF-DX, pirenzepine, and atropine with the ID50, dose for 50% suppression, of 40, 35, and 1.9 microg/kg, respectively. These results suggest that (a) the potency of antimuscarinic actions of AF-DX on the CCh-induced negative dromotropic effects was almost equal to that of pirenzepine, and approximately 30 times less potent than atropine; (b) the M2-subtype selectivity of AF-DX was considerably higher in comparison with pirenzepine and atropine; and (c) the muscarinic receptor subtype on the canine AV node is entirely of the M2-type, but only sparsely developed in the coronary vascular beds.