Selective M1 Muscarinic Receptor Research Articles

Dysfunction of muscarinic M2 receptors after the early allergic reaction: possible contribution to bronchial hyperresponsiveness in allergic guinea-pigs.

1. Using a guinea-pig model of allergic asthma, in which the animals display early (0-5 h) and late phase (8-23 h after antigen challenge) bronchoconstrictor reactions, the function of prejunctional inhibitory M2 and postjunctional M3 receptors in isolated tracheal preparations have been investigated. In addition, cardiac M2 receptor function in vitro and bronchial responsiveness to histamine in vivo were evaluated. 2. Sensitivity to inhaled histamine was increased 3.1 fold and 1.6 fold after the early and late allergic reactions (i.e. at 5 h and 23 h after a single ovalbumin challenge), respectively. At 23 h after the last of four allergen challenges, executed on four consecutive days, bronchial hyperresponsiveness to histamine was diminished to 1.3 fold. 3. After the early response, there was no change in cardiac muscarinic M2 receptor function, since in left atria pD2 (-log EC50) and Emax values of pilocarpine and pKB values of AQ-RA 741, a selective M2 receptor antagonist, were not significantly different from controls (unchallenged sensitized animals), and this also applied to methacholine pD2 values for muscarinic M3 receptors in tracheal smooth muscle. 4. Prejunctional inhibitory muscarinic M2 autoreceptors in airway smooth muscle were markedly dysfunctional after the early allergic response, since potentiation of electrically evoked twitch contractions of tracheal preparations by low concentrations of the M2-selective muscarinic receptor antagonists, gallamine, methoctramine, AQ-RA 741 and AF-DX 116, which is the result of M2 receptor blockade, was clearly and significantly diminished compared to controls. However, after the late response, both in single and repeatedly challenged animals, twitch potentiation was not significantly different from and similar to controls, indicating restoration of M2 receptor function during the late allergic reaction.5. It is concluded that dysfunction of muscarinic M2 autoreceptors in the airways of sensitized and challenged guinea-pigs is already present after the early allergic reaction, and that it has recovered after the late response. Since histamine-induced bronchoconstriction involves vagal pathways, the present results suggest that bronchial hyperresponsiveness to histamine is partly due to M2 auto receptor dysfunction, leading to increased release of acetylcholine.

Novel alkoxy-oxazolyl-tetrahydropyridine muscarinic cholinergic receptor antagonists

The purpose of the present studies was to compare a novel series of alkoxy-oxazolyl-tetrahydropyridines (A-OXTPs) as muscarinic receptor antagonists. The affinity of these compounds for muscarinic receptors was determined by inhibition of [3H]pirenzepine to M1 receptors in hippocampus, [3H]QNB to M2 receptors in brainstem, and [3H]oxotremorine-M to high affinity muscarinic agonist binding sites in cortex. All of the compounds had higher affinity for [3H]pirenzepine than for [3H]QNB or [3H]oxotremorine-M labeled receptors, consistent with an interpretation that they are relatively selective M1 receptor antagonists, although none were as selective as pirenzepine. In addition, dose-response curves were determined for antagonism of oxotremorine-induced salivation (mediated by M3 receptors) and tremor (mediated by non-M1 receptors) in mice. In general, the A-OXTPs were equipotent and equieffective in antagonizing both salivation and tremor, although there were modest differences for some compounds. Dose-response curves also were determined on behavior maintained under a spatial-alternation schedule of food presentation in rats as a measure of effects on working memory. The A-OXTPs produced dose-related decreases in percent correct responding at doses three- to ten-fold lower than those which decreased rates of responding. However, only one compound, MB-OXTP, produced effects on percent correct responding consistent with a selective effect on memory as opposed to non-memory variables. The present results provide evidence that these alkoxy-oxazolyl-tetrahydropyridines are a novel series of modestly M1-selective muscarinic receptor antagonists, and that one member of the series, MB-OXTP, appears to be more selective in its effects on memory than previously studies muscarinic antagonists.

Muscarinic M1 receptor mediated inhibition of GABA release from rat cerebral cortex

Presynaptic modulation of [3H]GABA release was examined using rat cerebral cortical slices. In vitro addition of carbachol, a muscarinic receptor agonist, resulted in a significant suppression of the release of [3H]GABA evoked by high potassium (50 mM) stimulation in a dose dependent manner, while noradrenaline, isoproterenol, dopamine, 5-hydroxytryptamine, histamine and glutamic acid had no significant effect on the evoked release of [3H]GABA. This suppressive effect of carbachol was antagonized invariably by atropine. Furthermore, it was found that the suppressive action of carbachol could be antagonized by pirenzepine, a selective M1 muscarinic receptor antagonist, but not by AF-DX 116 and 4-DAMP, M2 and M3 receptor antagonists, respectively. These results suggest that the release of GABA from cerebral cortical GABA neurons may be modulated by presynaptic M1 muscarinic receptor.

Exocrine pancreatic secretion in man following one week of M1‐muscarinic receptor blockade

A double-blind, randomized, placebo-controlled crossover study was performed to assess the influence of one week of selective M1-muscarinic receptor blockade on pancreatic exocrine secretion in man. Ten healthy subjects received telenzepine (3 mg p.o.) and placebo each for 8 days, with a 6-day drug-free washout interval between treatment sequences. On Day 8 of each sequence, pancreatic secretion was stimulated for 2 h by infusion of submaximal secretin (0.2 U.kg/h) followed by maximal stimulation with secretin (1.0 U.kg/h) and ceruletide (120 ng.kg/h). Telenzepine had no significant effect on secretory parameters during submaximal stimulation with secretin. During maximal stimulation, total protein, secretory volume, and output of amylase, trypsin and bicarbonate were unexpectedly increased by telenzepine. These findings might be partially explained by removal of the inhibitory influence of pancreatic polypeptide, which was depressed by telenzepine. Acute studies have shown that M1-receptor antagonists inhibit exocrine secretion. Our results suggest that adaptation of physiological mechanisms governing the exocrine pancreas may occur after one week of receptor blockade by a therapeutic dosage of telenzepine, to the extent that M1-blockade no longer inhibits secretion.

SDZ ENS 163, a selective muscarinic M 1 receptor agonist, facilitates the induction of long-term potentiation in rat hippocampal slices

The effect of SDZ ENS 163; (+)-(3S,cis)-3-ethyldihydro-4-[(1-methyl-lH-imidazol-5-yl)methyl-2(3H)-thiphenonedihydrogenphosphate], a selective muscarinic M1 agonist, on long-term potentiation (LTP) was studied in a rat hippocampal slice preparation. LTP was induced by theta-burst stimulation (TBS) delivered to the Schaffer/commissural fibers. In untreated slices delivery of 8 or 10 trains at 100 Hz induced a 27 ± 8.3 and 54 ± 7.2% potentiation of the amplitude of the excitatory postsynaptic potential (epsp), respectively (calculated as percentage of the pre-LTP amplitude). In slices pretreated with SDZ ENS 163 (2 × 10 −6 M, -30 min) delivery of 8 or 10 trains at 100 Hz induced a 62 ± 8.4 and 54 t 7.1% potentiation of the epsp amplitude, respectively. In addition, treatment with SDZ ENS 163 (2 x 10 −6 M) increased the N-methyl-D-aspartate receptor-induced component of the epsp response to TBS from 21 ± 3 (control) to 33 ± 2%. Pretreatment with the muscarinic antagonist, scopolamine (6 × 10 −8 M), or with the M1 selective muscarinic receptor antagonist, pirenzepine (6 × 10 −8 M), did not affect LTP in untreated slices but inhibited the enhancement of UP by SDZ ENS 163 (2 x 10 −6 M) completely. AF-DX 116 10 −6 M, −60 min), a selective muscarinic M 2 receptor antagonist did not affect UP in control slices nor in slices treated with SDZ ENS 163 (2 x 10 −6 M). These data suggest that activation of muscarinic M1 receptors by SDZ ENS 163 facilitates the induction of LTP.

Evidence for the absence of a functional role for muscarinic M2 inhibitory receptors in cat trachea in vivo: contrast with in vitro results.

1. The effect of the selective muscarinic M2 receptor antagonist, gallamine and the selective M2 receptor agonist, pilocarpine, on airway constriction induced by vagal stimulation was studied in anaesthetized cats. In addition, the effect of gallamine on contraction of cat isolated tracheal and bronchi preparations induced by electrical field stimulation was also investigated. 2. In in vivo experiments, extrathoracic airway constriction was measured with an electromechanical caliper that was attached to the outer surface of tracheal ring 4. Intrathoracic airway constriction was determined by measuring the changes in total lung resistance and dynamic compliance during vagal stimulation. 3. Intravenous gallamine (0.1, 1, and 10 mg kg-1) augmented the rise in total lung resistance induced by vagal stimulation in a dose- and frequency-dependent manner. At stimulation frequencies of 8 and 12 Hz the fall in dynamic compliance provoked by vagal stimulation was also significantly increased by gallamine (10 mg kg-1). Gallamine was without effect on airway constriction induced by acetylcholine. 4. Vagal stimulation at 4 Hz produced significant tracheal constriction, but the amount of constriction did not change following injection of increasing doses of gallamine. Similarly, there was no difference in tracheal constriction at any frequency of stimulation (0.5-16 Hz) when frequency-response curves before and after gallamine injection (10 mg kg-1) were compared. 5. Pilocarpine (0.01-10 micrograms kg-1, i.v.) diminished changes in total lung resistance and dynamic compliance induced by vagal stimulation, an effect that was reversed by gallamine (10 mg kg-1, i.v.). Vagally-induced tracheal constriction was not significantly affected by any dose of pilocarpine, nor was it modified by gallamine (10mg kg- ') given subsequently.6. Atropine (0.5 mgkg-') completely blocked tracheal constriction induced by vagal stimulation, indicating that the changes in tracheal ring diameter provoked by stimulation were mediated by muscarinic receptors and that intravenous drugs could reach the cervical trachealis muscle.7. In vitro tissue bath studies demonstrated a significant leftward shift of the frequency-response curve to electrical field stimulation in both tracheal strips and bronchial rings following gallamine (10-4M) administration.8. Although the functional presence of muscarinic M2 autoreceptors was demonstrated in feline isolated tracheal and bronchial preparations, a corresponding functional role was not detected in cat trachea in vivo. This was despite repeated demonstration of muscarinic M2 receptor-mediated limitation of airway constriction of intrathoracic airways in vivo.

Effect of a M1-selective muscarinic receptor antagonist (pirenzepine) on basal bronchomotor tone in young women.

To examine the role of muscarinic M1-receptors in vagally mediated basal bronchomotor tone, we studied the effects of inhaled pirenzepine, which is a M1-receptor antagonist, on basal bronchomotor tone in 8 young women. To obtain dose-response curves to inhaled pirenzepine, we measured partial and full flow-volume curves before and 30 min after inhalation of each concentration of pirenzepine (3.25-200 micrograms/ml). Maximum expiratory flow on partial flow-volume curve at 25% forced vital capacity (PEF25) was measured as an index showing basal bronchomotor tone. The dose-response curve of the percent increase in PEF25 by pirenzepine was biphasic in shape. PEF25 was significantly increased by 3.12 (14.5 +/- 5.8%), 6.25 (15.5 +/- 4.2%), 12.5 (20.0 +/- 0.29%) and 200 micrograms/ml (17.3 +/- 5.9%) of pirenzepine, but not by 25 (9.6 +/- 5.6%), 50 (9.4 +/- 6.2%) and 100 micrograms/ml (14.4 +/- 6.2%) of pirenzepine. The percent increase in PEF25 by 40 micrograms of ipratropium bromide was 66.2 +/- 14.2%. These results suggest that the role of M1-receptors in vagally mediated basal bronchomotor tone may be significant but little compared with M3-receptors in normal humans.

Functionalized congener approach to muscarinic antagonists: analogues of pirenzepine.

The M1-selective muscarinic receptor antagonist pirenzepine 6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) was derivatized to explore points of attachment of functionalized side chains for the synthesis of receptor probes and ligands for affinity chromatography. The analogues prepared were evaluated in competitive binding assays versus [3H]-N-methylscopolamine at four muscarinic receptor subtypes (m1AChR-m4AChR) in membranes from rat heart tissue and transfected A9L cells. 9-(Hydroxymethyl)pirenzepine, 8-(methylthio)pirenzepine, and a series of 8-aminosulfonyl derivatives were synthesized. Several 5-substituted analogues of pirenzepine also were prepared. An alternate series of analogues substituted on the 4-position of the piperazine ring was prepared by reaction of 4-desmethylpirenzepine with various electrophiles. An N-chloroethyl analogue of pirenzepine was shown to form a reactive aziridine species in aqueous buffer yet failed to affinity label muscarinic receptors. Within a series of aminoalkyl analogues, the affinity increased as the length of the alkyl chain increased. Shorter chain analogues were generally much less potent than pirenzepine, and longer analogues (7-10 carbons) were roughly as potent as pirenzepine at m1 receptors, but were nonselective. Depending on the methylene chain length, acylation or alkyl substitution of the terminal amine also influenced the affinity at muscarinic receptors.

Role of hypothalamic cholecystokinin octapeptide in the colonic motor response to a meal in rats

The effects of hypothalamic microinfusions of cholecystokinin octapeptide and its antagonist L364,718 on cecocolonic myoelectrical activity were evaluated by electromyography in fasted and fed rats. The rats were chronically fitted with electrodes implanted on the cecum and proximal colon and cannulas placed bilaterally in either the ventromedial or lateral hypothalamus. In fasted rats, microinfusion of cholecystokinin octapeptide (10 ng/kg) into the ventromedial hypothalamus increased the spikeburst frequency of the cecum and the colon by 45.6% and 43.7%, respectively, during the 30-minute period after treatment. The injection of cholecystokinin octapeptide (10 ng/kg) into the lateral hypothalamus had no effect on either cecal or colonic motility. Feeding increased the frequency of cecal and colonic spike bursts by 52.1% and 50.1% for 30 minutes postprandially. When infused bilaterally into the ventromedial hypothalamus 10 minutes before feeding, L364,718 (1 or 5 μg/kg) abolished the increase of the frequency of cecal and colonic contractions induced by the meal. Infused into the lateral hypothalamus at similar dosages, L364,718 had no effect on the postprandial enhancement of cecocolonic motility. Increase of cecocolonic spikeburst frequency induced by feeding or by cholecystokinin octapeptide injected into the ventromedial hypothalamus was abolished by previous intracerebroventricular but not intraperitoneal administration of atropine (1 (μg) and 4-diphenylacetoxy-N-methylpiperidine (1 μg), a selective muscarinic M2-receptor antagonist. In contrast, pirenzepine (1 (μg, intracerebroventricularly) did not significantly reduce the meal- or cholecystokin octapeptideinduced increase in cecal and colonic motility. These results suggest that, in rats, (a) cholecystokinin octapeptide is involved in the generation of the cecocolonic motor response to a meal and these effects are mediated through cholecystokinin octapeptide receptors located in the ventromedial hypothalamic nuclei, and (b) these postprandial colonic motor changes involve central cholinergic activation through muscarinic M2 receptors.

Mad Honey Poisoning in Man and Rat

Grayanotoxins are known to occur in the honey produced from the nectar of Rhododendron ponticum growing on the mountains of the eastern Black Sea region of Turkey and also in Japan, Nepal, Brazil, and some parts of North America and Europe. Two cases of honey intoxication are presented here. Both of the patients experienced severe bradycardia and hypotension after ingestion of honey which was brought from Trabzon, Turkey. Microscopical examination showed Rhododendron ponticum pollen tetrades. Anesthetized albino rats were injected intraperitoneally with toxic honey extract doses equivalent to 1 or 5 g honey/kg. Dose-dependent hypotension, bradycardia and respiratory rate depression were observed. When marked bradycardia (approximately 75% of control value) was reached, rats were given atropine sulfate (2 mg/kg, i.p.) or AF-DX 116 (20 mg/kg, i.p.). Atropine sulfate improved both bradycardia and respiratory rate depression. AF-DX 116, which is a selective M2-muscarinic receptor antagonist, restored only heart rate, but not the respiratory rate depression. These results suggest that M2-muscarinic receptors are involved in cardiotoxicity of grayanotoxin.

Characterization of the Antinociception Induced by Intrathecally Administered Carbachol

The antinociceptive effect of intrathecally administered carbachol at the L1/L2 level in the rat was evaluated using the tail immersion test. A dose dependent increase in the nociceptive reaction times was evident following intrathecal carbachol in the dose range of 2.5-15 micrograms. At doses of 20 micrograms and above, although still effective in the test, motor impairment was pronounced. The antinociception was antagonized with atropine, and with either pirenzepine (PZ) or AFDX 116, which are selective M1 and M2 muscarinic receptor blocking drugs, respectively. Spinal cholinergic pain modulation was also studied in rats pretreated with DSP4 (N-2-chloroethyl-N-ethyl-2-bromobenzylamine), which causes a selective depletion of the noradrenergic nerve fibres in the CNS. The increased latency times after spinal carbachol were attenuated in animals depleted of spinal noradrenaline by DSP4. In conclusion, spinal analgesia by carbachol in the rat may therefore be mediated through both M1 and M2 muscarinic receptor stimulation in the spinal cord. It is also concluded that this spinal cholinergic pain modulation is interacting with spinal noradrenergic nerve terminals, but that the mechanism of the interaction remains to be established.

Cholinergic regulation of arterial pressure by the C1 area of the rostral ventrolateral medulla

In anesthetized, paralyzed rats intravenous administration of the acetylcholinesterase inhibitor physostigmine (PHY) (100 micrograms/kg) evoked a dose-related rise in arterial pressure (AP) and heart rate (HR) and an associated increase in sympathetic nerve activity (SNA). The responses to PHY were blocked by electrolytic lesions of, or microinjection of kainic acid into, a specific site in the rostral ventrolateral medulla containing a cluster of neurons immunoreactive for the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase and corresponding to the C1 area of the nucleus reticularis rostroventrolateralis (RVL). The C1 area and its surround contain a heretofore unrecognized network of varicose neuronal processes and perikarya labeled immunocytochemically with a monoclonal antibody to the ACh-synthesizing enzyme, choline acetyltransferase (CAT). PHY increased, by over 3-fold, the spontaneous activity of reticulospinal cardiovascular neurons in the C1 area which excite preganglionic sympathetic neurons in the intermediolateral spinal column. The effects of PHY on AP, SNA, and the discharge of reticulospinal neurons were blocked by systemic administration of the muscarinic antagonist scopolamine. Microinjections within the C1 area of the RVL of scopolamine, the M2-selective muscarinic receptor antagonist AF-DX 116, or the high-affinity choline uptake inhibitor hemicholinium-3 blocked the pressor effects of PHY. The nicotinic antagonist hexamethonium and the M1-selective muscarinic receptor antagonist pirenzepine were without effect. We conclude that (1) the increases in AP, HR, and SNA elicited by the systemic administration of PHY result from the augmented action of ACh released from cholinergic terminals within the C1 area of the RVL; (2) the locally released ACh acts through muscarinic receptors of the M2 subtype within the C1 area to produce excitation of intrinsic reticulospinal sympathoexcitatory neurons, thereby increasing the activity of sympathetic preganglionic neurons and consequently elevating AP and HR; and (3) while the specific function of the cholinergic innervation of the C1 area in cardiovascular regulation is unknown, it may contribute to the tonic regulation of AP.

The Role of Pirenzepine-sensitive (M1) Muscarinic Receptors in Vagally Mediated Bronchoconstriction in Humans

In a double-blind randomized study, we compared the effects of the M1-selective muscarinic receptor antagonists pirenzepine and the nonselective antagonist ipratropium bromide on bronchoconstriction induced by inhaled sulfur dioxide (SO2) and methacholine in atopic volunteers. Both inhaled pirenzepine (70 micrograms) and ipratropium bromide (7 micrograms) significantly inhibited vagally mediated bronchoconstriction by SO2 to the same extent (p less than 0.02). However, at this dose, pirenzepine had no effect on methacholine-induced bronchoconstriction, whereas ipratropium bromide gave significant protection (p less than 0.02). This indicates that vagally mediated bronchoconstriction in humans can be inhibited by blockade of pirenzepine-sensitive (M1) muscarinic receptors probably present on a different site from muscarinic receptors at the neuromuscular junction and presumably localized to parasympathetic ganglia. Pirenzepine may be useful in investigating ganglionic function and could be beneficial therapeutically in airway disease.

Beneficial Effects of FKS-508 (AF102B), a Selective M1 Agonist, on the Impaired Working Memory in AF64A-Treated Rats

AbstractThe effects of FKS-508 [AF102B; cis-2-methylspiro(1,3-oxathiolane-5,3′)quinuclidine], a selective M1 muscarinic receptor agonist, were examined to predict the possible activity on memory disorders using a T-maze and radial-arm maze task in experimental amnesia models. The amnesia models were produced by bilateral intracerebroventricular injection of ethylcholine aziridinium ion (AF64A). a selective cholinotoxin. in rats. Repeated administrations of FKS-508 (5 mg/kg/day, i.p.) for 5 weeks significantly ameliorated impaired performance of AF64A-treated rats (AF64A-rats) in a delayed alternation task in the T-maze. Repeated administrations of FKS-508 (1 and 5 mg/kg/day. p.o.) for 5 weeks significantly ameliorated acquisition failures of AF64A-rats in a radial-arm maze task. Single administration of FKS-508 (1 and 5 mg/kg. p.o.) significantly reduced the incorrect choices of AF64A-rats in a radial-arm maze task with 6 hr-delay time. No abnormalities in general behaviors, such as loss of appetite and ataxia, were observed in rats treated with FKS-508 repeatedly during 5 weeks. Our present results showed that FKS-508 can ameliorate memory impairments in AF64A-rats with central cholinergic hypofunction without causing any behavioral abnormalities. FKS-508 may be considered as a candidate for the clinical examination of the cholinergic hypothesis of senile dementia of the Alzheimer type

Cholinergic regulation of PP release is mediated through muscarinic M1-receptors.

The effects of the selective muscarinic M1-receptor antagonist pirenzepine and the nonselective muscarinic antagonist atropine on bombesin- and peptone-stimulated release of pancreatic polypeptide (PP) were studied in healthy subjects. To exclude any effect of changes in intraduodenal pH continuous intragastric titration was performed during all tests. Both pirenzepine (i.v. bolus 0.6 mg/kg) and atropine (i.v. bolus 15 micrograms/kg, followed by an infusion of 5 micrograms/kg.h) significantly reduced peptone-induced integrated mean rise of plasma PP from 28 +/- 5 to 8 +/- 4 and -2 +/- 5 pmol/l, respectively (n = 4). Both drugs also reduced bombesin-induced rise of plasma PP in all 4 subjects, from 27 +/- 12 to 6 +/- 4 and 7 +/- 1 pmol/l (0.05 less than p less than 0.1). It is concluded that cholinergic regulation of PP release is mediated mainly through muscarinic M1-receptors.

Muscarinic Receptor Subtypes Mediating Myocardial Blood Flow Redistribution

Selective M1 and M2 muscarinic receptor antagonists pirenzepine and AF-DX 116, respectively, were administered to open-chest, pentobarbital-anesthetized dogs to determine the muscarinic receptor subtype responsible for the preferential vasodilation of subendocardial blood vessels during intracoronary methacholine infusion. There were no changes in systemic hemodynamics responsible for the myocardial blood flow redistribution produced by methacholine. Selective M1 receptor blockade with pirenzepine attenuated increases in transmural perfusion and prevented the redistribution of blood flow favoring the subendocardium during methacholine infusion. M2 receptor blockade with AF-DX 116 also attenuated the increase in transmural perfusion produced by methacholine but did not selectively block increases in subendocardial blood flow. These results support the hypothesis that M1 muscarinic coronary receptors are responsible for the redistribution of blood flow to the subendocardium (increased endo/epi) during cholinergic coronary vasodilation, whereas M2 receptors mediate an increase in total coronary blood flow. In additional experiments in isolated rabbit aorta, AF-DX 116 was 0.38 times less potent than pirenzepine with respect to endothelium-dependent muscarinic vasodilation but 1.47 times more potent with respect to endothelium-independent muscarinic vasoconstriction. This suggests that differences exist in the receptors found on the vascular smooth muscle and endothelium and that the preferential distribution of blood flow to the subendocardium may be due to stimulation of M1 receptors located on the vascular endothelium.

A putative M3 muscarinic cholinergic receptor of high molecular weight couples to phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells.

The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine [( 3H]NMS) and [3H]quinuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki = 869-1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 [11-2[[2-[(diethylamino)methyl]-1-piperidinyl]- acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) bound to the mAChRs with a very low affinity (Ki = 6.0 microM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki = 7.2 nM). 4-DAMP was also 1,000-fold more effective than AF-DX 116 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilycholine mustard) suggest that the size of the SK-N-SH mAChR (Mr = 81,000-98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr = 66,000), an M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.

Effects of Pirenzepine and Atropine on Amylase Response to Various Secretagogues from the Rat Pancreatic Acini

CCK-8, bombesin, secretin and urecholine added in various concentrations to the incubation medium of the dispersed pancreatic acini, obtained from rats, resulted in a dose-dependent stimulation of amylase secretion, the half-maximal response being achieved at 3 X 10(-12), 10(-10), 10(-7) and 10(-5) M, respectively. Pirenzepine (selective muscarinic M1 receptor blocker) or atropine failed to affect the amylase responses to all these secretagogues except that to urecholine, which was reduced by both antimuscarinic agents. The inhibition of urecholine-induced half-maximal amylase secretion by both anticholinergics was concentration-dependent, and the inhibitory potency of pirenzepine was about 1,000 times lower than that of atropine.

The effect of selective and non-selective cholinergic blockade on bombesin- and peptone-stimulated gastrin release.

We have studied the effects of the selective muscarinic M1-receptor antagonist pirenzepine and the non-selective muscarinic antagonist atropine on bombesin- and peptone-stimulated gastrin release in healthy subjects. Pirenzepine (i. v. bolus 0.6 mg/kg) and atropine (i. v. bolus 15 micrograms/kg, followed by an infusion of 5 micrograms.kg-1.h-1) were given in doses equipotent in terms of reduction of gastric acid secretion. Neither affected bombesin- or peptone-stimulated gastrin release. These findings do not support the involvement of M1-receptors in the cholinergic regulation of gastrin release and suggest that the reduction in acid secretion caused by pirenzepine is not mediated by inhibition of gastrin release.