A comparison of the antimuscarinic effects of pirenzepine and N-methylatropine on ganglionic and vascular muscarinic receptors in the rat

The antimuscarinic properties of pirenzepine and N-methylatropine were evaluated in two intact preparations by measuring A) the inhibition of increase in mean arterial pressure evoked by McN-A-343 in pithed rats through activation of ganglionic muscarinic receptors and B) the inhibition of fall in arterial pressure evoked by methacholine in anaesthetized rats through activation of vascular muscarinic receptors. To characterize the antimuscarinic potencies of pirenzepine and N-methylatropine, for both antagonists doses were calculated that produce a 10-fold shift to the right of the dose-response curves for A) the pressor response to McN-A-343 (i.v. administration) in pithed rats (D10-p.r.) and B) for the depressor effect to methacholine (i.v. administration) in anaesthetized rats (D10-an.r.), respectively. Whereas N-methylatropine was virtually equieffective in blocking both muscarinic responses (D10-an.r./D10-p.r. approximately equal to 1), pirenzepine, however, was considerably more potent at ganglionic than at vascular muscarinic receptors (D10-an.r./D10-p.r. approximately equal to 16). These data confirm the existence of excitatory ganglionic muscarinic receptors with high affinity for pirenzepine (M1) and provide evidence for the presence of M2 receptors - receptors which show a low sensitivity to pirenzepine - on vascular smooth muscle cells. To further characterize the anticholinergic properties of pirenzepine, its effect on the pressor response to DMPP, a nicotinic ganglionic stimulant, was investigated in pithed rats. A high dose of pirenzepine (1.13 mumol/kg), given i.v., did not affect nicotinic ganglionic transmission.     

Muscarinic receptor subtypes: M1 and M2 biochemical and functional characterization

The heterogeneity of muscarinic receptors was examined in sympathetic ganglia and atria by “in vitro” binding techniques and functional studies. As tools we have used the classical antagonist atropine, the selective antagonist pirenzepine and the unique muscarinic agonist McN-A-343. In binding studies atropine showed similar affinities to muscarinic sites in ganglionic and atrial membranes with dissociation constants of 1.1 and 3.2 nM, respectively. In contrast, pirenzepine displayed a distinctly different binding profile. In atria it bound to an homogenous population of low affinity sites (diss. const. 620 nM) while in ganglia it revealed the presence of two sites: a major population of high affinity sites (diss. const. 11 nM) and a minor one of lower affinity (diss. const. 280 nM). The functional correlate of the receptor properties in the two tissues was studied in the pithed rat by measuring A) the increase of arterial pressure evoked by McN-A-343 through selective activation of muscarinic receptors in ganglia and B) the bradycardia elicited by acetylcholine release in the heart through vagal stimulation. Mirroring the “in vitro” binding data atropine inhibited both muscarinic responses in the same narrow range of doses (2–30 μg/kg i.v.) whereas pirenzepine showed similar potency to atropine in inhibiting ganglionic stimulation (ED50 4.1 μg/kg i.v.) but was almost two orders of magnitude weaker in blocking vagal bradycardia (ED50 172 μg/kg i.v.). These data suggest that McN-A-343 and pirenzepine act selectively on a common muscarinic receptor subtype, a finding which agrees with the view that muscarinic receptors are heterogenous and that excitatory ganglionic receptors (Ml) are distinguishable from those (M2) present in effector organs like smooth muscle and heart.     

Thermic response of selective muscarinic agonists and antagonists in rat

Effect of some selective agonists and antagonists of cholinergic M receptor subtypes on rectal temperature was investigated in rats at an ambient temperature of 25 degrees +/- 2 degrees C. Centrally administered acetylcholine (ACh) induced transient hypothermia, whereas the muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (McN) (icv), induced sustained and dose-related hypothermia. However, the nonspecific muscarinic receptor agonist, oxotremorine, and physostigmine, induced hypothermia at a lower dose and hyperthermia, accompanied by tremors, at higher doses. The muscarinic M2 receptor agonist, carbachol (icv) also produced a dose-related dual effect, hyperthermia and hypothermia being induced by the lower and higher doses, respectively. The M1 receptor antagonists, scopolamine (ip) and pirenzepine (icv), induced hyperthermia, whereas the M2 receptor antagonists, gallamine (icv) and AF-DX 116 (AFDX) (ip), produced hypothermia. The hypothermic effects of ACh. arecholine, McN, physostigmine, oxotremorine and carbachol were attenuated by scopolamine and pirenzepine. However, although scopolamine also inhibited the hyperthermic and tremorogenic effects of the higher dose of oxotremorine, it had a synergistic effect with the hyperthermia-inducing higher dose of physostigmine. AFDX attenuated the hyperthermic effect of the lower dose of carbachol, indicating that it was M2 receptor-mediated. Hemicholinium, an ACh synthesis inhibitor, had a transient hypothermic effect followed by slight hyperthermia. However, it markedly antagonized the hypothermic effects of gallamine and AFDX, indicating that their effects were dependent upon the availability of neuronal ACh. The results indicate that cholinergic hypothermia is a function of central muscarinic M1 receptors, with the M2 receptors serving as automodulators.(ABSTRACT TRUNCATED AT 250 WORDS)     

An AF-DX 116 sensitive inhibitory mechanism modulates nicotinic and muscarinic transmission in cat superior cervical ganglion in the presence of anticholinesterase.

The effect of the muscarinic receptor antagonist AF-DX 116 on the inhibitory action of muscarinic agonists and on responses mediated by nicotinic or muscarinic ganglionic transmission was studied in the superior cervical ganglion of the anesthetized cat. The postganglionic compound action potential evoked by cervical sympathetic trunk stimulation was depressed by methacholine or acetylcholine (ACh) injected into the ganglionic arterial supply. The depression was blocked by AF-DX 116. The compound action potentials evoked by preganglionic stimulus trains were also depressed when the intratrain frequency was 2 Hz or greater. This intratrain depression was, however, insensitive to AF-DX 116. The anticholinesterase drug physostigmine markedly enhanced the intratrain depression of the compound action potential. This effect was reversed by AF-DX 116. During nicotinic receptor block with hexamethonium, preganglionic stimulus trains with intratrain frequencies of 5 Hz or greater produced nicitating membrane contractions that could be blocked by the M1 muscarinic receptor antagonist pirenzepine. The amplitude of the contractions increased with frequency and reached a maximum at 20-40 Hz. AF-DX 116 had no effect on these responses. After administration of physostigmine, the amplitude of the nictitating membrane responses decreased with increasing intratrain frequency. AF-DX 116 reversed this effect. The data suggest that, in the superior cervical ganglion, AF-DX 116 sensitive muscarinic receptors which depress synaptic transmission are activated by exogenous agonists but not by the ACh released by the preganglionic axon terminals unless cholinesterase activity is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of selective muscarinic receptor agonists and antagonists on active-avoidance learning acquisition in rats

Effect of some selective muscarinic receptor agonists and antagonists was investigated on learning acquisition in an active-avoidance paradigm in rats which records an anticipatory conditioned avoidance apart from the classical conditioned avoidance response. The muscarinic M1 agonists, arecholine, pilocarpine and McN-A-343, facilitated learning acquisition, which was attenuated by the selective M1 antagonist, pirenzepine. On the other hand, M2 receptor agonist, carbachol, and physostigmine, induced a dose-related dual response, with lower doses retarding and higher doses facilitating the learning acquisition. The former effect was attenuated by gallamine, a muscarinic M2 antagonist, while the latter response was inhibited by pirenzepine, indicating that these putative M2 receptor agonist lose their receptor specificity on dose increment. The selective M2 receptor antagonists, gallamine and AF-DX 116, facilitated learning acquisition, which was inhibited by pirenzepine and the acetylcholine synthesis inhibitor hemicholinium. The results support the cholinergic hypothesis of learning and memory and indicate that M1 receptor agonists and M2 receptor antagonists are likely to prove beneficial in memory deficits. The data also indicates that the clinical dose of some drugs, like physostigmine, needs to be carefully established for optimum therapeutic benefit.     

Evidence that reciprocal hindlimb scratching elicited in mice by intrathecal administration of muscarinic agonists is mediated through M1 type receptors

Intrathecal (IT) administration of pilocarpine to mice produces a vigorous and dose-related reciprocal hindlimb scratching (RHS) response (ED50 = 0.6 microgram) that is potently blocked by simultaneous IT administration of atropine (ID50 = 0.002 microgram). We now report that RHS is (1) also elicited by the more selective M1 agonist McN-A-343-11 (ED50 = 11.6 micrograms), (2) blocked by the selective M1 antagonist pirenzepine (ID50 = 0.001 microgram), and (3) is not blocked by the selective M2 antagonist AF-DX 116 BS at a dose up to 100 times the ID50 dose of pirenzepine. These results extend our earlier findings and suggest that the RHS elicited in mice by IT injection of muscarinic agonists is mediated through pirenzepine-sensitive (presumably M1) receptors and that RHS may be a convenient in vivo centrally mediated M1 endpoint.     

Muscarinic receptors on nerves and muscles in opossum esophagus muscularis mucosa

The muscarinic receptors of muscularis mucosa have some recognition properties that suggest they resemble receptors of the M1 subtype. The nerves of these tissues also contain muscarinic receptors which inhibit tonic contractions caused by release of a substance-P-like material by field stimulation. These receptors also appear to be M1 in type as they are maximally activated by McNeil A343 as well as by carbachol (pD2, 5.5 and 7.5, respectively). They are also inhibited by pirenzepine, as well as by atropine (negative logarithms of the required dose for 50% inhibition or potentiation, 6.6-6.7 compared with 8.2-8.3). Hexahydrosiladifenidol, an antagonist selective or M2 receptors of guinea pig ileum, had a low (approximately 7.1) pA2 value for antagonism of both agonists in smooth muscle in this tissue. However, it was closer to atropine in potency with respect to potentiating tonic responses to field stimulation or to inhibiting phasic responses to field stimulation than it was to antagonizing smooth muscle contractions. Thus, atropine was about 40 times more potent than pirenzepine and 2-5 times more potent than hexahydrosilafenidol. There were some quantitative differences in the effectiveness of these three antagonists in blocking the phasic (acetylcholine-mediated) response to field stimulation. Atropine was 70-100 times more potent than pirenzepine and 8-25 times more potent than hexahydrosiladifenidol. This greater potency difference for inhibition of phasic contractions compared with potentiation of tonic contractions was discussed. This tissue appears to be one of the first smooth muscles in which both nerves and muscles contain muscarinic receptors with some recognition properties resembling those of the M1 subtype.     

Muscarinic regulation of neonatal rat bladder spontaneous contractions

In vitro preparations of whole urinary bladders of neonatal rats exhibit prominent myogenic spontaneous contractions, the amplitude and frequency of which can be increased by muscarinic agonists. The muscarinic receptor subtype responsible for this facilitation was examined in the present experiments. Basal spontaneous contractions in bladders from 1- to 2-wk-old Sprague-Dawley rats were not affected by M2 or M3 receptor antagonists. However, administration of 0.5 microM physostigmine, an anticholinesterase agent that increases the levels of endogenous acetylcholine, or 50-100 nM carbachol, a cholinergic agonist at low concentrations, which did not cause tonic contractions, significantly augmented the frequency and amplitude of spontaneous contractions. Blockade of M2 receptors with 0.1 microM AF-DX 116 or 1 microM methoctramine or blockade of M3 receptors with 50 nM 4-diphenylacetoxy-N-methylpiperidine methiodide or 0.1 microM 4-diphenylacetoxy-N-(2-chloroethyl)piperidine hydrochloride (4-DAMP mustard) reversed the physostigmine and carbachol responses. M2 and M3 receptor blockade did not alter the facilitation of spontaneous contractions induced by 10 nM BAY K 8644, an L-type Ca2+ channel opener, or 0.1 microM iberiotoxin, a large-conductance Ca2+-activated K+ channel blocker. NS-1619 (30 microM), a large-conductance Ca2+-activated K+ channel opener, decreased carbachol-augmented spontaneous contractions. These results suggest that spontaneous contractions in the neonatal rat bladder are enhanced by activation of M2 and M3 receptors by endogenous acetylcholine released in the presence of an anticholinesterase agent or a cholinergic receptor agonist.     

Central muscarinic control of the pattern of small intestinal motility in rats

The effects of central and peripheral administration of muscarinic agonists and antagonists on small intestinal motility were examined in conscious rats chronically fitted with electrodes implanted in the duodeno-jejunal wall and a cannula in a cerebral lateral ventricle. Intracerebroventricular (i.c.v.) administration of either atropine or pirenzepine at doses from 1 to 10 micrograms, 15 min before a 3 and 6 g lab chow meal significantly reduced the duration of the postprandial disruption of the migrating myoelectric complexes (MMC). The reduction was significantly greater for atropine, a mixed M1 and M2 muscarinic receptor antagonist, than for pirenzepine, an antagonist with a high affinity for M1 receptors. At a higher dose (10 micrograms) intra peritoneal (i.p.) administration of atropine or pirenzepine did not modify the postprandial disruption of MMC. Oxotremorine (10 ng) a M2 agonist, but not McNeil A343 (5 micrograms), a selective M1 agonist, given i.c.v. in fasted rats disrupted for 1.5 h the MMC pattern. At the same doses given i.p. oxotremorine and McNeil A343 disrupted the MMC for 15 and 45 min respectively. We conclude that the postprandial changes in the small intestinal motility involve muscarinic receptors, mainly of M2 subtype, at the level of the central nervous system.     

Effects of muscarinic receptor agonists and antagonists on response of non-extensor rats to maximal electroshock

Rats which do not respond consistently to maximal electroshock by exhibiting the classical hindlimb extensor response, are designated as 'flexors', and can serve as a useful experimental model for investigating seizure mechanisms. 20-25% Charles Foster rats exhibit the flexor status and were used in this study. The flexor rats were converted to extensors by acetylcholine (icv), physostigmine (ip) and the selective muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (icv). This conversion of flexors to extensors was significantly attenuated by M1 receptor antagonists scopolamine (ip) and pirenzepine (icv). The M2 receptor agonist, carbachol (icv), had no effect in lower doses but induced conversion of flexor rats to the extensor status only in very high doses which may be due to loss of receptor specificity on dose increment. The M2 receptor antagonists, gallamine (icv) and AF-DX 116 (ip), also induced significant conversion of flexors to extensors, which was dependent upon the availability of neuronal acetylcholine since the effects were attenuated following pretreatment with hemicholinium, an inhibitor of acetylcholine synthesis. The results suggest that the central cholinergic system has a facilitatory pro-convulsant effect, mediated through the muscarinic M1 receptors, an action modulated by the M2 receptors.     

Cardioselective profile of AF-DX 116, a muscarine M2 receptor antagonist

AF-DX 116 (see chemical name below) is a competitive antagonist of muscarine receptors in peripheral organs. In contrast to pirenzepine, its behaviour in functional experiments indicates selectivity for the M2 muscarinic subtype. In pithed rats AF-DX 116 inhibits vagally-induced bradycardia, an M2 response, (ED50 32 micrograms/kg i.v.) in preference to the M1-mediated pressor response to McN-A-343 (ED50 211 micrograms/kg i.v.). AF-DX 116 further discriminates among M2 receptors, showing a high affinity for the cardiac muscarine receptors. In isolated preparations, AF-DX 116 has a tenfold higher affinity for the muscarine receptors of the heart (pA2 7.33) than for those in smooth muscles (pA2 6.39-6.44). The same profile appears from animal studies, where the compound is a more potent antagonist of either endogenously or exogenously activated cardiac muscarine responses as compared to vascular, smooth muscle or secretory responses. In general, the ratios of potencies (ED50) observed in cardiac vs. other muscarine mediated functions ranged between 30 and 50. Atropine showed no discrimination, inhibiting all muscarine responses in the same range of doses. In the conscious dog intravenous AF-DX 116 increased basal heart rate, and completely reversed the reflex bradycardia induced by clonidine. Tachycardia was dose-related (ED50 79 micrograms/kg i.v.), and occurred independently of background sympathetic tone. AF-DX 116 clearly distinguishes between M1- and M2-mediated responses; it also emphasizes the long-recognized heterogeneity among the peripheral M2 subtypes. AF-DX 116, for its pronounced cardioselectivity, may have a therapeutic potential in the treatment of sinus bradycardia.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

McN-A-343, a specific agonist of M1-muscarinic receptors, exerts antinicotinic and antimuscarinic effects in the rat adrenal medulla

Pirenzepine, McN-A-343 and oxotremorine were used to determine the subtypes of muscarinic receptors involved in the secretion of catecholamines from the isolated perfused adrenal gland of the rat. In the presence of 0.1 microM pirenzepine, the concentration-secretion curve for muscarine was shifted in parallel to the right by almost one log unit. With 0.5 microM the shift was over two log units. The apparent dissociation constant for pirenzepine was about 1.12 X 10(-8) M. Perfusion with McN-A-343 (1-30 microM) did not evoke the secretion of catecholamines. A further increase to very high concentrations (100-1000 microM) caused only a modest secretion (about 50 ng/5 min with 300 microM as compared to the same amount of secretion obtained with 1 microM muscarine). Secretion evoked by nicotine was significantly reduced (30%) by 3 microM McN-A-343, and the inhibition increased (90%) with higher concentrations (100 microM). McN-A-343 also produced concentration-dependent inhibition of catecholamine secretion evoked by muscarine. A significant effect was observed at 30 microM and reached a maximum level at 300 microM. Oxotremorine, like McN-A-343 was a partial agonist on the muscarinic receptors; but unlike McN-A-343, did not block the stimulatory effects of nicotine. Although the pirenzepine data suggest that M1 receptors are responsible for the secretion of catecholamines in the rat adrenal medulla, this conclusion is not supported by the results obtained with the M1-receptor agonist, McN-A-343, which proved to be an effective blocker of muscarinic as well as nicotinic receptors.     

Characterization of the muscarinic and serotoninergic receptors of the intestine of the rainbow trout (Salmo gairdneri)

The ability of carbachol and 5-hydroxytryptamine (5-HT) to contract isolated segments of rainbow trout intestine in a concentration-dependent manner indicates the presence of muscarinic and serotoninergic receptors in this tissue. The activity of these agonists appears to be directly on the smooth muscle, since ganglionic blockers and inhibitors of neurotransmission did not inhibit contractions. The carbachol-induced contractions were selectively inhibited by atropine and (+-)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate hydrate, an M-2 muscarinic receptor antagonist. However, the inhibition was not competitive. McN-A-343, an M-1 muscarinic agonist had no effect on intrinsic tone. The 5-HT-induced contractions were selectively inhibited by methysergide and the 5-HT2 receptor blockers, ketanserin and 1-(1-naphthyl)piperazine. Again, the inhibition by these agents was not competitive. 5-HT1 and 5-HT3 receptor antagonists did not inhibit contractions. The results thus suggest that the smooth muscle of the rainbow trout intestine contains M-2 muscarinic and 5-HT2 receptors.     

Central oxotremorine antagonist properties of pirenzepine

Pirenzepine, the prototype M1 muscarinic receptor antagonist, is an important compound for investigating the functional significance of M1 receptors at the integrated level of behavior but may have limitations imposed by its physical chemistry. Like the nonselective antagonist methylatropine, pirenzepine is highly hydrophilic and crosses the blood-brain-barrier with difficulty. We compared methylatropine with pirenzepine, given intraperitonealy, as antagonists of the behavioral effects of peripheral or central muscarinic activation. Lever-press responses of male Sprague-Dawley rats were maintained under a schedule requiring 10 responses for each food delivery. Administration of oxotremorine or the quaternary analog oxotremorine-M decreased rates of responding by at least 90%. Both methylatropine and pirenzepine antagonized the behavioral effects of oxotremorine-M; maximum reversal was 70%. Although methylatropine was about 30 times more potent than pirenzepine as an antagonist of the peripheral muscarinic activity of oxotremorine-M, it was inactive as an antagonist of oxotremorine when given in doses up to 153 mumol/kg. Pirenzepine, however, reversed oxotremorine-induced behavioral effects, with a maximum antagonism of 50%. These results suggest that pirenzepine interacts with central muscarinic receptors when administered systemically without producing marked behavioral effects of its own. Systemically administered pirenzepine may thus be a useful tool in further investigations of the relevance of M1 receptors to behavioral function.     

Biphasic dose-response relationship for acetylcholine in the chick jugular vein

The pharmacological mechanism of biphasic dose-response relationship for acetylcholine (ACh), relaxation at low doses (1 nM to 0.3 μM) and contraction at high doses (1 μM to 30 μM), in the chick jugular vein was investigated. Neither relaxations nor contractions were affected by the treatment with tetrodotoxin, hexamethonium, d-tubocurarine, phentolamine, propranolol, reserpine, or ouabain. Besides, anoxia did not affect the biphasic pattern of dose-response curve. The contraction was attenuated by the treatment with aspirin or indomethacin, but only slightly. The dose-response curves for these responses to acetylcholine were shifted to the right by the treatment with atropine. Methacholine, carbachol, bethanechol, and arecoline caused similar biphasic responses, although contractions caused by highest doses of bethanechol or arecoline were very small in amplitude. On the other hand, pilocarpine and McN-A-343 only relaxed the strips. The dose-response curves for cholinomimetics were all shifted to the right by the treatment with atropine. It was demonstrated that the responses of the chick jugular vein to muscarinic agonists are different from those of mammalian veins. The mechanisms underlying the biphasic response are discussed.     

Positive and negative inotropic effects of muscarinic receptor stimulation in mouse left atria

In isolated mouse left atria, acetylcholine (ACh) produced a biphasic inotropic response; a transient decrease in developed tension was followed by an increase. Both negative and positive responses were concentration dependent and were inhibited by atropine. The negative and positive inotropic responses were also observed with a nonselective muscarinic stimulant, oxotremorine-M, but not with an M1-receptor selective stimulant, McN-A343. Pirenzepine, an M1-receptor antagonist, inhibited both negative and positive inotropic responses at high concentrations. Gallamine, an M2-receptor antagonist, inhibited the negative response. Hexahydro-siladifenidol hydrochloride, p-fluoro analog (p-F-HHSiD), an M3-receptor antagonist, inhibited the positive response with no effect on the negative phase. In pertussis toxin (PTX) treated preparations, negative inotropic response to ACh was not observed. These results suggest that the negative and positive inotropic responses to acetylcholine in mouse atria are mediated by M2 and M3 receptors, respectively. The negative phase, but not the positive phase, was mediated by a PTX-sensitive G protein.     

Evidence for a complex influence of nicotinic acetylcholine receptors on hippocampal serotonin release

The effects of nicotine on 5-hydroxytryptamine (5-HT) release from serotonergic nerve endings in rat dorsal hippocampal slices were studied. Nicotine (50-500 microM:) caused a concentration-dependent increase in 5-HT release. This effect was antagonised by mecamylamine (0.5 microM:), indicating an action at nicotinic receptors. Nicotine-evoked 5-HT release was not affected by tetrodotoxin (3 microM:), cadmium chloride (0.1 mM:), or the absence of Ca(2+) or Na(+) in the superfusion medium. Unexpectedly, higher concentrations of mecamylamine alone (1-50 microM:) increased 5-HT release. This suggested the presence of inhibitory input to 5-HT neurones and that these inhibitory neurones possess tonically active nicotinic receptors. The effect of mecamylamine (50 microM:) on 5-HT release was reduced by the muscarinic M(1) receptor agonist, McN-A-343 (100 microM:), but pirenzepine (0.005-1 microM:), which blocks M(1) receptors, alone increased 5-HT release. Hippocampal serotonergic neurones are known to possess both excitatory nicotinic receptors and inhibitory M(1) receptors. Although there may be several explanations for our results, one possible explanation is that nicotine stimulates 5-HT release by activating nicotinic heteroreceptors on 5-HT terminals. Mecamylamine (0.5 microM:) antagonises this effect, but higher concentrations increase 5-HT release indirectly by blocking the action of endogenous acetylcholine on nicotinic receptors situated on cholinergic neurones that provide muscarinic inhibitory input to 5-HT neurones.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.