Quantitative effects of some muscarinic agonists on evoked surface-negative field potentials recorded from the guinea-pig olfactory cortex slice

1. The effects of muscarinic receptor agonists on the electrically-evoked surface-negative field potential (N-wave) were measured in the guinea-pig olfactory cortex slice maintained in vitro. 2. Bath-superfusion of (+/-)-muscarine, acetylcholine (ACh), carbachol (CCh), or methacholine (MCh) (10-200 microM) produced reversible, dose-dependent depressions of the N-wave (ACh and MCh effects were observed in the presence of 10 microM neostigmine). The order of potencies (based on agonist dose causing 50% field depression: IC50) was: ACh greater than or equal to muscarine greater than CCh greater than MCh. All four agonists depressed the field potential by 100% at doses greater than 500 microM. 3. Pilocarpine and bethanechol were weak agonists and only produced measurable effects at high doses (1-2 mM). Neither agonist evoked a maximum response at doses up to 10 mM. 4. The muscarinic ganglion stimulant, McN-A-343 yielded inconsistent results, depressing the field potential in some slices, but having no effect in others. Pre-application of a conditioning dose (100 microM) of McN-A-343 reduced subsequent responses to CCh, suggesting possible partial agonist properties. 5. Oxotremorine (up to 100 microM) did not depress the field potential, but it reversibly antagonized the effects of CCh. 6. It is concluded that reproducible, quantifiable responses to muscarinic agonists can be evoked in the olfactory cortex slice. We suggest this preparation may be useful for conducting pharmacological studies of 'intact' central muscarinic receptors.     

Muscarinic inhibition of excitatory neurotransmission in guinea-pig olfactory cortex slices: weak antagonism by M3-muscarinic receptor antagonists.

Dose-dependent depression of the electrically evoked surface-negative field potential (N-wave) produced by bath-superfusion of carbachol was measured in guinea-pig olfactory cortex slices maintained in vitro. The possible involvement of M3 (smooth muscle/glandular) type muscarinic receptors in partly mediating this response was investigated by testing the effectiveness of the muscarinic M3 receptor antagonists hexahydro-sila-difenidol (HHSiD) and p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD). Low doses of HHSiD (10-100 nM) or p-F-HHSiD (up to 1 microM), pre-applied for 30 min, produced no obvious antagonism of carbachol responses. However, a clear competitive-type inhibition of carbachol effects was observed in 250 nM-1 microM HHSiD or 10-50 microM p-F-HHSiD respectively. Schild plot analysis (regression slope constrained to unity) of pooled data yielded pA2 values of 6.6 for HHSiD (n = 6 slices) and 5.5 for p-F-HHSiD (n = 6 slices) respectively, suggesting a weak competitive antagonism by both compounds. In addition, combination experiments using either HHSiD or p-F-HHSiD with atropine, produced dose-ratio shifts close to those predicted for two antagonists competing for a common receptor site. By comparison, another suggested M3-receptor antagonist, 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) was a potent competitive blocker of carbachol responses. Schild analysis for 4-DAMP versus carbachol gave a pA2 of 7.9 (n = 6 slices). It is concluded that the muscarinic receptors involved in the suppression of the olfactory cortical N-wave possess a low affinity for HHSiD and particularly for p-F-HHSiD, but not 4-DAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism of monosynaptic excitations in the mouse olfactory cortex slice by 6,7-dinitroquinoxaline-2,3-dione

The effects of 6,7-dinitroquinoxaline-2,3-dione (DNQX) have been tested in slices of olfactory cortex of the mouse against responses evoked by N-methyl-D-aspartate, kainate and quisqualate and on the surface field potentials evoked on electrical stimulation of the lateral olfactory tract. At a concentration of 5 microM, DNQX competitively antagonized responses evoked by kainate and quisqualate, with only a small reduction in the responses to N-methyl-D-aspartate. In contrast, DL-(+-)-2-amino-5-phosphonopentanoic acid (APP, 50 microM) selectively antagonized depolarizations to N-methyl-D-aspartate. The amplitude of the field potential known as the N-wave was reduced by DNQX in a concentration-dependent reversible manner (IC50 = 2.92 +/- 0.33 microM; mean +/- SE mean, n = 4). DL-(+-)-2-Amino-5-phosphonopentanoic acid (50 microM) did not significantly affect this action of DNQX. It is concluded that DNQX inhibits monosynaptic excitations in the olfactory cortex by selectively blocking kainate and/or quisqualate receptors, although it is unclear whether the receptors are located at pre- and/or postsynaptic sites.     

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.     

A quantitative study of the effects of some muscarinic antagonists on the guinea-pig olfactory cortex slice.

1. Muscarinic depression of the electrically-evoked surface-negative field potential (N-wave) was measured in guinea-pig olfactory cortex slices maintained in vitro. 2. The effects of three muscarinic receptor antagonists, pirenzepine, atropine and gallamine on this muscarinic response were analysed in detail. 3. Pirenzepine was a potent competitive antagonist of carbachol (CCh)-evoked responses. Schild plot analysis yielded a pA2 value of 7.9 (Schild slope constrained to unity). A similar analysis for atropine versus CCh responses gave a pA2 of 8.9. 4. Combination experiments using pirenzepine and atropine produced dose-ratio shifts close to those expected for two antagonists competing for a similar receptor site. 5. Gallamine was only a weak antagonist of responses to CCh. 6. Oxotremorine behaved as a competitive antagonist at this muscarinic receptor (pA2 = 6.1). 7. It is concluded that the presynaptic muscarinic receptor mediating depression of the N-wave in the olfactory cortex slice is of the M1-subtype.     

Endogenous adenosine inhibits excitatory transmission in the rat olfactory cortex slice

Excitatory neurotransmission in the rat olfactory cortex slice has been monitored by measuring the amplitude of the N-wave surface field potential evoked by stimulation of the lateral olfactory tract. Application of exogenous adenosine or aspartate depressed the N-wave amplitude and evoked synthesis of cyclic AMP. These effects were partially antagonized by theophylline and the reduction of amplitude of the N-wave was potentiated by dipyridamole. When the olfactory tract slice was stimulated, dipyridamole alone reduced the amplitude of the N-wave and increased levels of cyclic AMP, both effects being antagonized by theophylline. Exogenous adenosine significantly attenuated the K+-evoked release of [3H]D-aspartate by a mechanism insensitive to either theophylline or dipyridamole. It is concluded that synaptic activation of the olfactory cortex releases adenosine, possibly as the result of the actions of the transmitter candidate of the olfactory tract, aspartate, and that this causes sufficient adenosine to accumulate to depress excitatory transmission and elevate tissue levels of cyclic AMP although there is no positive evidence that these two effects are directly related.     

Pharmacological evidence that NMDA receptors contribute to mono- and di-synaptic potentials in slices of mouse olfactory cortex.

Experiments have been carried out using slices of olfactory cortex of the mouse perfused in solution containing Mg2+ (1 mM) and in which the lateral olfactory tract was stimulated at a frequency of 1 pulse/5 sec to avoid polysynaptic activity. Application of the N-methyl-D-aspartate antagonist D-(-)-2-amino-5-phosphonopentanoic acid (APP, 25 microM) suppressed a low amplitude component of the potential, the latency to onset of which corresponded with that of the monosynaptically evoked N-wave in 14 of the 17 slices tested and duration of which exceeded that of the N-wave. A residual potential, recorded in slices to which the quisqualate-/kainate-selective antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) had been applied at a concentration of 10microM, was identical to the potential suppressed by APP. The residual potential in the presence of DNQX was blocked by APP, 7-chlorokynurenate (25 microM) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801, 0.125-2 microM). It was potentiated in area by exogenous D-serine (1 mM) and in slices preincubated and perfused with Mg(2+)-free solution. It is concluded that, in addition to receptors of the quisqualate/kainate categories, N-methyl-D-aspartate receptors also contribute to both mono- and di-synaptic excitations in the olfactory cortex.     

Sites and mechanisms of action of catechol (1,2-dihydroxybenzene) in the rat olfactory cortex slice.

Synaptic transmission in the isolated olfactory cortex slice from the rat was monitored by recording the surface field potentials evoked on lateral olfactory tract (LOT) stimulation. Catechol (approximately 0.05 to 2 mM) caused a concentration-dependent, partially reversible increase in the amplitudes of all field potentials. In a series of conditioning experiments, catechol (1 mM) potentiated postsynaptic inhibition by a mechanism which was at least partially picrotoxin-insensitive. When the relationship between the stimulus input and evoked output was investigated in picrotoxin-treated slices, for a given tract action potential amplitude, catechol (0.25 and 0.5 mM) increased the amplitude of the field potential known as the N-wave; in contrast, for a given N-wave amplitude, the latency of the population spike was increased. Catechol (1 mM) increased the K+-evoked release of endogenous aspartate by a tetrodotoxin-insensitive mechanism whereas the release of glutamate and gamma-aminobutyric acid (GABA) was unaffected. Catechol (1 mM) had no effect on submaximal depolarizations evoked by L-aspartate, L-glutamate or GABA. It is concluded that catechol potentiates excitatory transmission at the LOT-superficial pyramidal cell synapse, possibly by increasing evoked transmitter release. Other synaptic actions of catechol may be consequent upon this increased excitatory input but the results do not exclude the possibility of separate and distinct actions on polysynaptic transmission.     

Muscarinic receptor agonist-mediated modulation of neuronal activity in rat cerebral cortex.

Multiple cortical neuronal responses were elicited by the iontophoretic application of muscarinic receptor agonists and antagonists in the rat cerebral sensorimotor cortex in vivo. (1) The muscarinic receptor agonist, oxotremorine-M induced a biphasic effect on spontaneous firing. This was evident as an early brief increase in the firing rate over the spontaneous discharge followed by secondary inhibition of spontaneous activity. The excitation could be blocked by the muscarinic receptor non-selective antagonist atropine and by both the M1 receptor antagonist pirenzepine and the M2 receptor antagonists gallamine or methoctramine. Oxotremorine-M inhibition of spontaneous activity was not affected by the M1 receptor antagonist pirenzepine, while evaluation of its sensitivity to gallamine and methoctramine was not possible since these two M2 receptor antagonists also depressed spontaneous activity, unlike pirenzepine. Of the other two muscarinic receptor agonists, oxotremorine had inconsistent and weak excitatory effects whilst McN-A-343 had only weak excitatory or inhibitory effects on spontaneous activity. (2) Oxotremorine-M, oxotremorine and McN-A-343 had a depressant action on neuronal discharges evoked by glutamate or acetylcholine. A depressant effect of oxotremorine-M was also demonstrated on the early excitation evoked by subsequent applications of oxotremorine-M itself. Of the three muscarinic receptor agonists tested, oxotremorine-M was the most potent in evoking a long-term depression of evoked discharges, lasting from several minutes (greater than 5 min) to as long as 40 min. Oxotremorine-M-induced depression of evoked responses was most sensitive to the M2 receptor antagonists, whereas oxotremorine-induced depression was more sensitive to the M1 receptor antagonist pirenzepine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signaling through the muscarinic receptor-adenylate cyclase system of the heart is buffered against GTP over a range of concentrations

The influence of GTP on muscarinic receptor occupancy and inhibition of adenylate cyclase activity was investigated in well washed homogenates of the rat myocardium. In these homogenates, the highly efficacious muscarinic agonist oxotremorine-M was without effect on adenylate cyclase activity in the absence of exogenous GTP but caused a maximal 38% inhibition of the enzyme in the presence of 0.1 microM GTP. Increasing the concentration of GTP to 0.1 mM caused small to moderate increases in the maximal inhibition of adenylate cyclase elicited by oxotremorine-M and in the concentration of this agonist required for half-maximal inhibition of the enzyme. In contrast, the same change in the concentration of GTP (0.1 microM to 0.1 mM) caused a relatively large increase (46-fold) in the concentration of oxotremorine-M necessary for half-maximal receptor occupancy. Similar observations were made for the highly efficacious muscarinic agonist carbachol. Our results show that GTP increases receptor coupling efficiency and decreases agonist affinity and that these two effects oppose one another, so that the level of muscarinic agonist-mediated inhibition of adenylate cyclase activity remains relatively constant over a range of concentrations of GTP. We have also used a model to predict the influence of GTP on receptor binding properties and agonist-mediated inhibition of adenylate cyclase activity and have calculated theoretical results generally consistent with the experimental observations.     

Chronic beta 1-adrenoceptor antagonist treatment sensitizes beta 2-adrenoceptors, but desensitizes M2-muscarinic receptors in the human right atrium.

1. In 64 patients undergoing coronary artery bypass grafting the effects of chronic beta 1-adrenoceptor antagonist (metoprolol, atenolol, bisoprolol) treatment on right atrial beta-adrenoceptor and muscarinic M2-receptor number and functional responsiveness were investigated. 2. The beta 1-adrenoceptor antagonists increased right atrial beta 1-adrenoceptor number, did not affect beta 2-adrenoceptor number, and decreased muscarinic M2-receptor number. 3. Concomitantly, activation of right atrial adenylate cyclase by 10 microM GTP, 10 microM isoprenaline and 1 microM forskolin was enhanced and inhibition by 100 microM carbachol was diminished. 4. On isolated, electrically driven right atria the beta 1-adrenoceptor-mediated positive inotropic effect of noradrenaline was - even with beta 1-adrenoceptor number increased - not altered, while the beta 2-adrenoceptor-mediated effect of procaterol was markedly enhanced. However, the carbachol-induced negative inotropic effect was decreased. 5. It is concluded that chronic beta 1-adrenoceptor antagonist treatment increases beta 1-adrenoceptor number and concomitantly sensitizes beta 2-adrenoceptor function, but desensitizes muscarinic M2-receptor function in the human heart.     

Heterogeneity of presynaptic muscarinic receptors mediating inhibition of sympathetic transmitter release: a study with M2- and M4-receptor-deficient mice

1 Presynaptic muscarinic receptors modulate sympathetic transmitter release. The goal of the present study was to identify the muscarinic receptor subtype(s) mediating inhibition of sympathetic transmitter release in mouse atria, urinary bladder and vas deferens. To address this question, electrically evoked noradrenaline release was assessed using tissue preparations from NMRI, M(2)- and M(4)-knockout, and the corresponding M(2)- and M(4)-wildtype mice, after preincubation with (3)H-noradrenaline. 2 The muscarinic agonist carbachol decreased evoked tritium overflow (20 pulses/50 Hz) in each tissue and strain investigated. After deletion of the M(2)-receptor the maximal inhibition by carbachol was significantly reduced (by 41-72%), but not abolished, in all tissues. After deletion of the M(4)-receptor a moderate and significant reduction of the maximal inhibition by carbachol (by 28%) was observed only in the vas deferens. 3 Experiments with the muscarinic antagonists methoctramine and pirenzepine confirmed that the presynaptic muscarinic receptors were predominantly M(2) in atria and bladder and probably a mixture of M(2) and M(4) in the vas deferens. 4 Experiments in the urinary bladder with the cholinesterase inhibitor physostigmine and the muscarinic antagonist ipratropium demonstrated that endogenously released acetylcholine predominantly acted through M(2)-receptors to inhibit noradrenaline release. However, the results do not exclude a minor contribution of M(4)-receptors to this endogenous inhibition. 5 In conclusion, our results clearly indicate that the release-inhibiting muscarinic receptors on postganglionic sympathetic axons in mouse atria, bladder and vas deferens represent mixtures of M(2)- and non-M(2)-receptors. The non-M(2)-receptors remain unknown in atria and the bladder, and may represent primarily M(4)-receptors in the vas deferens. These results reveal an unexpected heterogeneity among the muscarinic receptors mediating inhibition of noradrenaline release.     

In vitro characterisation of the muscarinic receptor partial agonist, sabcomeline, in rat cortical and heart membranes.

We have investigated the pharmacology of the functionally selective muscarinic M1 receptor partial agonist, sabcomeline [SB-202026 (R-(Z)-(+)-alpha-(methoxyamino)-1-azabicyclo[2.2.2] octane-3-acetonitrile)], in rat cortex and heart using radioligand binding and functional studies. The Quinuclidinyl benzilate/Oxotremorine-M acetate ratio from radioligand binding studies suggested that sabcomeline and xanomeline [3(3-hexyloxy-1,25-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-met hylpyridine] are muscarinic receptor partial agonists in cortical and heart membranes. In [35S]GTPgammaS binding studies in rat cortex, carbachol stimulated binding via muscarinic M2/M4 receptors which could be blocked by sabcomeline with a pA2 of 7.2. In rat heart membranes, carbachol also stimulated [35S]GTPgammaS binding studies through muscarinic M2 receptors. Sabcomeline caused a small stimulation of basal [35S]GTPgammaS binding in both rat and heart tissues. Sabcomeline did not stimulate phosphoinositide hydrolysis in rat cortical slices, but did block the muscarinic M1 receptor-mediated response caused by carbachol with apparent pKb of 6.9. Xanomeline and milameline also had no effect on phosphoinositide hydrolysis up to 100 microM. In adenylyl cyclase studies in rat atria, sabcomeline inhibited forskolin-stimulated adenylyl cyclase activity to a similar extent to that of carbachol, xanomeline and milameline. The present study, using the techniques of radioligand binding, supports previous publications which have claimed that sabcomeline is a muscarinic receptor partial agonist. As expected, this study shows that the functional actions of this compound at muscarinic receptor subtypes and in different tissues will depend on receptor reserve.     

Actions of agonists of metabotropic glutamate receptors on synaptic transmission and transmitter release in the olfactory cortex.

1. The effects of agonists of metabotropic glutamate receptors on the evoked N-wave complex in slices of mouse olfactory cortex have been studied: most experiments were carried out using slices perfused with Mg(2+)-free solution to which 10 microM of either 6,7-dinitroquinoxaline-2,3-dione or 6-cyano-7-nitroquinoxaline-2,3-dione was applied. 2. Following agonist washout, a slowly developing, long lasting potentiation of the complex occurred which was confined to the N-methyl-D-aspartate (NMDA) receptor-mediated component of the potential. The relative agonist potencies were 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 5-250 microM) = quisqualate (5-50 microM) > 1RS,3RS-cis-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 25-1000 microM) > L-glutamate (0.25-2.5 mM); NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and L-aspartate were inactive. 3. Potentiation of the NMDA receptor-mediated component by 1S,3R-ACPD (0.1 mM) was non-competitively antagonised by S-(+)- but not R-(-)-2-amino-3-phosphonopropionate (AP3, 0.125 mM), equally by D-(-) and L-(+)-2-amino-4-phosphonobutyrate (0.25 mM) and also by the protein kinase C inhibitors sphingosine, (25 microM), sangivamycin (25 microM) and 5-(isoquinolinylsulphonyl)-3-methylpiperazine (50 microM). 4. In a series of input-output experiments, 1S,3R-ACPD (0.1 mM) reversibly reduced the latency to peak of the NMDA receptor-mediated component at submaximal stimulus intensities, an effect blocked by S-(+)-AP3 (0.125 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of release-inhibiting muscarinic autoreceptors in heart atria and urinary bladder: a study with M2- and M4-receptor-deficient mice

Release-inhibiting muscarinic autoreceptors were studied in heart atria and the urinary bladder of NMRI mice, M(2)-receptor-deficient mice, M(4)-receptor-deficient mice, and wildtype mice sharing the genetic background of the knockout animals. Segments of the tissues were preincubated with (3)H-choline and then superfused and stimulated electrically.In atrial segments taken from adult mice and stimulated with 120 pulses at 1 Hz, the muscarinic receptor agonist oxotremorine-M reduced the evoked overflow of tritium. Its concentration-response curves in atria from NMRI, M(2)-wildtype, M(4)-wildtype and M(2)-knockout mice were similar, with maximal inhibition by about 75%. In atria from M(4)-knockout mice, the maximal inhibitory effect of oxotremorine-M was reduced to 57%. The concentration-response curves of oxotremorine-M were shifted to the right by ipratropium, methoctramine and pirenzepine. Methoctramine and pirenzepine were approximately equipotent antagonists in all strains except in M(4)-knockout atria in which methoctramine was more potent than pirenzepine. When atria from adult NMRI mice were stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium both in the absence and in the presence of physostigmine (0.1 microM). In atria taken from 1-day-old NMRI mice, oxotremorine-M failed to reduce the evoked overflow of tritium. In bladder segments taken from adult mice, superfused with medium containing oxotremorine-M (1 microM), and stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium. Its concentration-response curves in preparations from NMRI, M(2)-wildtype, M(4)-wildtype and M(2)-knockout mice were similar. There was one exception: ipratropium failed to cause an increase in bladder pieces from M(4)-knockout mice. Methoctramine and pirenzepine also increased the evoked overflow of tritium in all strains except the M(4)-knockout. The two antagonists were approximately equipotent in NMRI, M(4)-wildtype and M(2)-knockout preparations but methoctramine was less potent than pirenzepine in M(2)-wildtype preparations. When bladder pieces from adult NMRI mice were superfused with oxotremorine-M-free medium and stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium in the presence of physostigmine (0.1 microM) but not in its absence. In bladder segments taken from 1-day-old NMRI mice and superfused with medium containing oxotremorine-M (1 microM), ipratropium increased the evoked overflow of tritium in the same way as in adult tissue.It is concluded that NMRI mice and the two wildtype strains are similar in their muscarinic autoreceptors. In atria, the autoreceptors are heterogeneous. Some are M(4). The non-M(4)-autoreceptors probably are M(2). In the bladder, the autoreceptors are exclusively M(4). In both tissues, the autoreceptors are activated by previously released acetylcholine under appropriate conditions.     

YM905, a novel M3 antagonist, inhibits Ca2+ signaling and c-fos gene expression mediated via muscarinic receptors in human T cells.

Our earlier observations suggest that M3 muscarinic acetylcholine (ACh) receptors (mAChRs) are involved in Ca2+ signaling and regulation of c-fos gene expression in T lymphocytes. Here, we describe the effects of YM905, a novel M3 antagonist, on evoked Ca2+ signaling and c-fos gene expression in CEM human leukemic T cells. YM905 significantly inhibited increases in intracellular free Ca2+ evoked by 10 microM oxotremorine-M, an M1/M3 agonist (IC50=100 nM), and also inhibited 10 microM oxotremorine-M-induced upregulation of c-fos gene expression at 1 microM. These findings demonstrate that YM905 antagonizes the intracellular responses in T cells induced via mAChRs, possibly M3 receptors.     

Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice

The muscarinic heteroreceptors modulating noradrenaline release in atria, urinary bladder and vas deferens were previously studied in mice in which the M(2) or the M(4) muscarinic receptor genes had been disrupted. These experiments showed that these tissues possessed both M(2) and non-M(2) heteroreceptors. The analysis was now extended to mice in which either the M(3), both the M(2) and the M(3), or both the M(2) and the M(4) genes had been disrupted (M(3)-knockout, M(2/3)-knockout and M(2/4)-knockout). Tissues were preincubated with (3)H-noradrenaline and then stimulated electrically (20 pulses per 50 Hz). In wild-type atria, carbachol (0.01-100 microM) decreased the electrically evoked tritium overflow by maximally 60-78%. The maximum inhibition of carbachol was reduced to 57% in M(3)-knockout and to 23% in M(2/4)-knockout atria. Strikingly, the effect of carbachol was abolished in M(2/3)-knockout atria.In wild-type bladder, carbachol (0.01-100 microM) reduced the evoked tritium overflow by maximally 57-71%. This effect remained unchanged in the M(3)-knockout, but was abolished in the M(2/4)-knockout bladder. In wild-type vas deferens, carbachol (0.01-100 microM) reduced the evoked tritium overflow by maximally 34-48%. The maximum inhibition of carbachol was reduced to 40% in the M(3)-knockout and to 18% in the M(2/4)-knockout vas deferens. We conclude that the postganglionic sympathetic axons of mouse atria possess M(2) and M(3), those of the urinary bladder M(2) and M(4), and those of the vas deferens M(2), M(3) and M(4) release-inhibiting muscarinic receptors.     

Cholinergic facilitation of neurotransmission to the smooth muscle of the guinea-pig prostate gland

1. Functional experiments have been conducted to assess the effects of acetylcholine and carbachol, and the receptors on which they act to facilitate neurotransmission to the stromal smooth muscle of the prostate gland of the guinea-pig. 2. Acetylcholine and carbachol (0.1 microM - 0.1 mM) enhanced contractions evoked by trains of electrical field stimulation (20 pulses of 0.5 ms at 10 Hz every 50 s with a dial setting of 60 V) of nerve terminals within the guinea-pig isolated prostate. In these concentrations they had negligible effects on prostatic smooth muscle tone. 3. The facilitatory effects of acetylcholine, but not those of carbachol, were further enhanced in the presence of physostigmine (10 microM). 3. The facilitatory effects of carbachol were unaffected by the neuropeptide Y Y(1) receptor antagonist BIBP 3226 ((R)-N(2)-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-arginina mide) (0.3 microM, n=3) or suramin (100 microM, n=5). Prazosin (0.1 microM, n=5) and guanethidine (10 microM, n=5) alone and in combination (n=4), reduced responses to field stimulation and produced rightward shifts of the log concentration-response curves to carbachol. 4. The rank orders of potency of subtype-preferring muscarinic receptor antagonists in inhibiting the facilitatory actions of acetylcholine and carbachol were: pirenzepine > HHSiD (hexahydrosiladifenidol) > pF-HHSiD (para-fluoro-hexahydrosiladifenidol)>/= 5 himbacine, and pirenzepine > HHSiD > himbacine>/= 5 pF-HHSiD, respectively. These profiles suggest that muscarinic receptors of the M(1)-subtype mediate the facilitatory effects of acetylcholine and carbachol on neurotransmission to the smooth muscle of the guinea-pig prostate.     

Phorbol dibutyrate enhances local anaesthetic action.

1. Synaptically-evoked field responses were elicited by stimulation of the lateral olfactory tract of rat olfactory cortex slices maintained in vitro. 2. Various concentrations of lignocaine (5-500 microM) were applied to the solution bathing the slices. These produced dose-dependent depressions of the synaptically-evoked potential over the concentration range 20-500 microM. The responses completely recovered on washing out the lignocaine. Similar depressions were also noted for procaine (100-1000 microM). 3. In the 47 slices tested, application of beta-phorbol 12,13-dibutyrate (1 microM) increased the amplitude of the synaptic response (from 0.99 +/- 0.05 to 1.36 +/- 0.06 mV). beta-Phorbol 13-monbutyrate (1 microM) had no effect. 4. In the presence of phorbol dibutyrate the depressant effect of lignocaine was increased: the EC50 changed from 91 +/- 10 to 24 +/- 2 microM (a mean potency increase of 3.47 +/- 0.14). A similar increase in potency for procaine was observed with phorbol dibutyrate (from 264 +/- 23 to 49 +/- 9 microM: a 5.49 +/- 0.82 increase in potency). If the tissue was pre-equilibrated in a concentration of lignocaine which produced a 60-80% depression, addition of phorbol ester caused a complete abolition of the evoked potential. 5. beta-Phorbol 13-monobutyrate (1 microM) had no effect on the potency of lignocaine. 6. The Na and K currents generating the action potential in the presynaptic nerve terminals were unaffected by phorbol dibutyrate. The depressant effect of lignocaine on these currents was not modified by phorbol dibutyrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic receptors controlling the carbachol-activated nonselective cationic current in guinea pig gastric smooth muscle cells

Muscarinic receptor subtypes controlling the nonselective cationic current in response to carbachol (ICCh) were studied in circular smooth muscle cells of the guinea pig gastric antrum using putative muscarinic agonists and antagonists. Both oxotremorine-M (an M2-selective agonist) and CCh dose-dependently activated the cationic current with EC50 values of 0.21 +/- 0.01 microm and 0.97 +/- 0.06 microM, respectively. In contrast, pilocarpine and McN-A 343 (an M1-selective and a putative M4 agonist) were weak partial agonists. In response to 10/microM CCh, 4-DAMP, methoctramine and pirenzepine dose-dependently inhibited ICCh and had IC50 values of 1.91 +/- 0.2 nM, 0.46 +/- 0.07 microM and 8.33 +/- 0.4 microM, respectively. 4-DAMP, methoctramine and pirenzepine shifted the concentration-response curves of ICCh to the right without significantly reducing the maximal current. Values of the apparent dissociation constant pA2 obtained from Schild plot analysis were 9.24, 7.72 and 6.62 for 4-DAMP, methoctramine and pirenzepine, respectively. Also, pertussis toxin completely blocked ICCh generation. These results suggest that the M2-subtype plays a crucial role in the activation of the ICCh, and a block of the M3-subtype reduces the sensitivity of the M2-mediated response with no significant reduction of maximum response.