Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux

Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M(1) receptor antagonist, or 4-DAMP, a muscarinic M(3) receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies. In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M(2) receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M(1) and M(3) receptors play a major role in cholinergic-mediated microvascular leakage, whereas M(3) receptors are mainly involved in ACh-induced bronchoconstriction.     

Characteristics of spontaneous and evoked motility in the isolated perfused porcine duodenum.

The aims of the study were to evaluate characteristics of spontaneous motility and of the ascending excitatory peristaltic reflex (AEPR) and intraluminal cross-sectional area in the isolated perfused porcine duodenum. The parameters were measured by an intraluminal catheter by use of the perfused side-hole technique and impedance planimetry. Respiratory parameters such as pH and oxygen consumption and the arterial perfusion pressure were monitored and did not vary significantly throughout the study time. Spontaneous motility was intense at the beginning but declined and disappeared within 45-90 min. It was abolished by atropine, epinephrine, and UK-14,304 (an alpha 2-adrenoceptor agonist). Secondary motility was evoked by intraluminal balloon distensions by raising the balloon pressure to 1.5 kPa for 1-min periods. Reproducible results regarding the AEPR, external balloon diameters to elicit the AEPR, and intraluminal cross-sectional area were obtained. The order of potency (pD2 values) for inhibition of the AEPR was the selective M3-receptor antagonist 4-DAMP greater than atropine greater than the selective M2-receptor antagonist AFDX-116 greater than the selective M1-receptor antagonist pirenzepine greater than hexamethonium. 4-DAMP was 16 and 29 times more potent than AFDX-116 (P less than 0.02) and pirenzepine (P less than 0.02). None of the drugs altered the intraluminal cross-sectional area during the balloon distensions. The model provides the opportunity for physiological and pharmacological studies of duodenal motility and duodenal cross-sectional area devoid of extrinsic neural and endocrine effects. The abolishment of the AEPR by atropine is caused by blockade of the M3-receptor in the porcine duodenum.     

Messenger RNA levels and binding sites of muscarinic acetylcholine receptors in gastrointestinal muscle layers from healthy dairy cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M(1)to M(5) in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [(3)H]-QNB (1-quinuclidinyl-[phenyl-4-(3)H]-benzilate) binding by M antagonists [atropine (M(1 - 5)), pirenzepine (M(1)), methoctramine (M(2)), 4-DAMP (M(3)), and tropicamide (M(4))] was used to identify receptors at the functional level. Maximal binding (B(max)) was determined through saturation binding with atropine as a competitor. The mRNA levels of M(1), M(2), M(3), and M(5) represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M(4) was undetectable. The mRNA levels of M(2) and of M(3) in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [(3)H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [(3)H]-QNB binding. The [(3)H]-QNB binding was dose-dependent and saturable. B(max) in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B(max) and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

Beneficial effect of preconditioning on ischemia-reperfusion injury in the rat bladder in vivo

We investigated the effect of preconditioning on ischemia-reperfusion injury in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder. Twelve-week-old male SD rats were divided into three groups; sham-operated control (Cont), 30 min ischemia-60 min reperfusion (IR) and three times of 5 min ischemia and then 30 min ischemia-60 min reperfusion (PC) groups. The bladder functions were estimated by cystometric and functional studies. Contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists, i.e. atropine (non-selective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective). We also measured tissue levels of malonaldehyde (MDA) and examined possible histological changes in these rats' bladders. Preconditioning partially prevented the reduction of bladder dysfunction induced by ischemia-reperfusion. Estimation of the pA2 values for atropine, pirenzepine, methoctramine, and 4-DAMP indicates that the carbachol-induced contractile response in bladder dome is mediated through the M3 receptor subtype in all groups. The MDA concentration in the IR group was significantly larger than that of the control group, and preconditioning significantly reduced MDA production in the bladder. In histological studies, the ischemia-reperfusion with or without preconditioning caused infiltration of leukocytes and rupture of microcirculation in the regions of submucosa and smooth muscle without a corresponding sloughing of mucosal cells. Our data indicate that preconditioning has a beneficial effect on ischemia-reperfusion injury in the rat bladder.     

Muscarinic M1 receptors stimulate phosphoinositide hydrolysis in bovine cerebral arteries

The muscarinic agonist oxotremorine-M produced a concentration-dependent increase in phosphoinositide hydrolysis in bovine pial arteries. The maximal effect was 5.9 +/- 0.89 fold over basal levels, and the EC50 for oxotremorine-M was 8.9 x 10(-6) M. The phosphoinositide response in arteries with the luminal endothelium removed was similar to the response in intact arteries. The specific muscarinic antagonists pirenzepine, 4-DAMP and methoctramine produced parallel shifts of the concentration-response curve to oxotremorine-M, with the following order of potency (pKB): 4-DAMP (8.59 +/- 0.10) greater than pirenzepine (8.12 +/- 0.11) greater than methoctramine (6.77 +/- 0.20). These results indicate that muscarinic stimulation activates phosphoinositide hydrolysis in cerebral arteries, and that the muscarinic receptors mediating this increase are similar to the M1 subtype.     

Muscarinic acetylcholine receptors of the chick amnion

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M₁, methoctramine for M₂, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M₃, and tropicamide for M₄ receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC₅₀), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 μM), activator of adenylate cyclase (AC), was unidirectional with ?-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC-or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M₃ acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M₃ acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M₃-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.     

Change in muscarinic modulation of transmitter release in the rat urinary bladder after spinal cord injury

Muscarinic facilitation of 14C-ACh release from post-ganglionic parasympathetic nerve terminals was studied in bladder strips prepared from spinal intact (SI) and spinal cord transected (SCT) rats. The spinal cord was transected at the lower thoracic spinal segments 3 weeks prior to the experiments. Using non-facilitatory stimulation (2 Hz) the release of ACh in spinal intact rats did not change in the presence of a non-specific muscarinic antagonist, atropine (100 nM), an M(1) specific antagonist (pirenzepine, 50 nM) or an M(1)-M(3) specific antagonist (4-DAMP, 5 nM). However, during a facilitatory stimulation paradigm (10 Hz or 40 Hz, 100 shocks) atropine and pirenzepine, but not 4-DAMP inhibited the release of ACh in bladders from spinal intact rats, indicating an M(1) receptor-mediated facilitation. In spinal cord transected rats, 2 Hz stimulation-induced release was significantly inhibited by atropine or 4-DAMP but not by pirenzepine indicating that a pre-junctional facilitatory mechanism mediated via M(3) muscarinic receptors could be induced by a non-facilitatory stimulation paradigm after spinal injury. In bladders of spinal cord transected rats, 10 Hz stimulation-evoked release of ACh was also inhibited by atropine and 4-DAMP (5 nM) but not by pirenzepine (50 nM). These results indicate that pre-junctional muscarinic receptors at cholinergic nerve endings in the bladder change after chronic spinal cord injury. It appears that low affinity M(1) muscarinic receptors are replaced by high affinity M(3) receptors. This change in modulation of ACh release may partly explain the bladder hyperactivity after chronic spinal cord injury.     

Messenger RNA Levels and Binding Sites of Muscarinic Acetylcholine Receptors in Gastrointestinal Muscle Layers from Healthy Dairy Cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M₁to M₅ in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [³H]-QNB (1-quinuclidinyl-[phenyl-4-³H]-benzilate) binding by M antagonists [atropine (M_{1 ? 5}), pirenzepine (M₁), methoctramine (M₂), 4-DAMP (M₃), and tropicamide (M₄)] was used to identify receptors at the functional level. Maximal binding (B_max) was determined through saturation binding with atropine as a competitor. The mRNA levels of M₁, M₂, M₃, and M₅ represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M₄ was undetectable. The mRNA levels of M₂ and of M₃ in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [³H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [³H]-QNB binding. The [³H]-QNB binding was dose-dependent and saturable. B_max in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B_max and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

Pharmacological identification of acetylcholine receptor subtypes in echinoderm smooth muscle (Sclerodactyla briareus)

Contractions of an echinoderm (sp. Sclerodactyla briareus) smooth muscle, the longitudinal muscle of the body wall (LMBW), were evoked by acetylcholine (ACh) and agonists: epibatidine, muscarine and nicotine (in order of force generation: ACh>muscarine=epibatidine>nicotine). ACh-induced contractions were blocked by atropine by 50%, and methoctramine, by 30%. ACh responses were also blocked by 25% by methyllycaconitine (MLA) but not by d-tubocurarine (dTC). Muscarine initiated large contractions that were completely blocked by atropine. To elucidate possible muscarinic ACh receptor (mAChR) subtypes, muscarinic agonists (oxotremorine, pilocarpine) and antagonists (methoctramine, pirenzepine) were tested. Oxotremorine, pilocarpine, and pirenzepine each enhanced resting tonus and potentiated ACh-induced contractions (order of potency: pilocarpine>oxotremorine=pirenzepine). Muscarine, oxotremorine or pirenzepine generated phasic, rhythmic contractions. Nicotine-induced contractions were almost completely blocked by dTC but were not altered by atropine. Large contractions evoked by epibatidine were potentiated by dTC whereas atropine had no effect on them. MLA blocked spontaneous rhythmicity. Cholinesterase inhibitors, neostigmine or physostigmine, caused marked potentiation of ACh-induced contractions and initiated rhythmic slow wave contractions in previously quiescent muscles. The present pharmacological evidence points to the co-existence of excitatory nicotinic ACh receptor (nAChRs) and mAChRs where nAChRs possibly modulate tone, and the mAChRs initiate and enhance rhythmicity.     

M3 muscarinic receptor activation of a delayed rectifier potassium current in canine atrial myocytes.

Growing body of evidence indicates that the functional responses of cells to muscarinic acetylcholine receptors (mAChRs) are mediated by multiple receptor subtypes. It is commonly thought that the M2 receptor is the only functional mAChR subtype in the heart and little data regarding the potential roles of other subtypes in cardiac tissues has been reported. In the present study, we provide functional evidence for the presence and physiological function of an M3 receptor in canine atrial myocytes. Using whole-cell patch-clamp techniques, we consistently found that pilocarpine, an mAChR agonist, induced a K+ current similar to but distinct from the classical delayed rectifier K+ current. Same observations were obtained when choline or tetramethylammonium (TMA) was applied to the bath. The currents were abolished by 1 microM atropine. Antagonists selective to M1 (pirenzepine, 100 nM), M2 (methoctramine 100 nM), or M4 (tropicamide 200 nM) receptors failed to alter the currents. Conversely, three different M3-selective inhibitors, p-F-HHSiD (20-200 nM), 4-DAMP methiodide (2-10 nM) and 4-DAMP mustard (4-20 nM), all produced concentration-dependent suppression of the currents. A cDNA fragment representing the M3 receptor was isolated from dog atrial RNA and the mRNA level of this construct was 0.7 +/- 0.1 pg/microg total RNA, as quantified by the competitive RT-PCR methods. Our data strongly suggested that an M3 receptor exists and is coupled to a K+ channel in the heart.     

Cholecystokinin induced gallbladder contraction is influenced by nicotinic and muscarinic receptors

Effects of pirenzepine, known as a muscarinic receptor antagonist, on the contraction of dog gallbladder elicited by cholecystokinin (CCK) were examined in comparison with atropine and hexamethonium ones. Intraluminal gallbladder pressure in an in situ anaesthetized dog model was chosen for studying gallbladder motility. The intravenous administration of pirenzepine (0.75 mg/kg b.wt.), atropine (3 mg/kg b.wt.) or hexamethonium (5 mg/kg b.wt.) elicited a marked decrease in the increase of intraluminal gallbladder pressure induced by intravenous bolus injections of CCK (0.25-2 Ivy dog unit/kg b.wt.) and by continuous infusion of CCK (0.025-0.4 Ivy dog unit/kg b.wt./min). It was concluded that CCK induced gallbladder contractions were influenced by both nicotinic and muscarinic receptors.     

M1 and M3 muscarinic antagonists inhibit human nasal glandular secretion in vitro.

Mucus glycoproteins (MGP) are high-molecular-weight glycoconjugates that are released from submucosal glands and epithelial goblet cells in the respiratory tract. Muscarinic receptors have an important role in the regulation of human nasal glandular secretion and mucus production, but it is not known which of the five muscarinic receptor subtypes are involved. The effect of nonselective and M1-, M2-, and M3-selective muscarinic antagonists on methacholine (MCh)-induced MGP secretion from human nasal mucosal explants was tested in vitro. MGP was assayed by enzyme-linked immunosorbent assay using a specific anti-MGP monoclonal antibody (7F10). MCh (100 microM) induced MGP secretion up to 127% compared with controls. MCh-induced MGP release was significantly inhibited by atropine (100 microM), the M, receptor antagonist pirenzepine (10-100 microM), and the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 1-100 microM). 4-DAMP significantly inhibited MCh-induced MGP release at a lower concentration (1 microM) than pirenzepine (10 microM). The M2 receptor antagonists AF-DX 116 and gallamine (both at 100 microM) had no effect. No antagonist alone had a significant effect on MGP release. These results indicate that the M1 and M3 muscarinic receptor subtypes regulate MGP secretion from human nasal mucosa and suggest that the M3 receptor has the predominant effect.     

Ca(2+) signaling in porcine duodenal glands by muscarinic receptor activation

The duodenal glands have been thought to play an important role in defense against proximal duodenal ulcer; however, the secretory mechanisms of these glands remain to be determined. In isolated duodenal acinar cells of the pig, we investigated the effects of ACh on intracellular Ca(2+) concentration ([Ca(2+)](i)) and on membrane currents with fura 2 fluorometry and the patch clamp technique. ACh caused a transient increase in [Ca(2+)](i), and the increase was markedly inhibited by atropine or 4-diphenylacetoxy-N-methylpiperidine methiodide but not by hexamethonium, pirenzepine, or methoctramine. The expression of mRNA for the M(3) subtype far exceeded that for either M(1) or M(2) as revealed by real-time quantitative PCR and in situ hybridization. The rise in [Ca(2+)](i) evoked by ACh was largely inhibited by thapsigargin but slightly affected by extracellular Ca(2+) deprivation. Caffeine had no effect on [Ca(2+)](i). ACh elicited Ca(2+)-dependent K(+) currents, a finding similar to the response to inositol 1,4,5,-trisphosphate applied intracellularly. These results suggest the presence of M(3) receptors linked to Ca(2+) release in porcine duodenal glands.     

Muscarinic Receptor Stimulated GTPase Activity in Synaptic Membranes from Bovine Retina

GTPase activity has been measured in synaptic membranes from bovine retina, with and without muscarinic receptor stimulation. Maximal stimulation above basal levels was achieved with 5 microM oxotremorine and 100 microM carbachol. (4-Hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride, which is selective for the M1 muscarinic receptor, failed to stimulate GTPase activity. 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) inhibition of oxotremorine stimulation demonstrated the presence of two populations of receptors, a low-affinity site (IC50 +/- SEM, 0.63 +/- 0.18 microM) which accounted for 63% of the inhibition and a high-affinity site (IC50 less than 1 nM) which accounted for the remaining 37%. When carbachol-stimulated GTPase activity was assayed, a single 4-DAMP inhibitory site was apparent (IC50 +/- SEM, 2.0 +/- 0.9 microM). Pirenzepine inhibited GTPase activity at a single site (IC50 values +/- SEM, 46.9 +/- 11 and 25.4 +/- 6.5 microM against oxotremorine and carbachol, respectively). Methoctramine was equipotent against carbachol and oxotremorine stimulation (IC50 values, 4.2 +/- 1.8 and 6.2 +/- 1.5 microM). Inhibition of maximal carbachol and oxotremorine stimulation by muscarinic antagonists at the major site had a rank order of potency of 4-DAMP = methoctramine greater than pirenzepine. Thus, the major site for muscarinic stimulation of GTPase activity in bovine retinal membranes is pharmacologically similar to M2 receptors.     

The effects of substance P and met5-enkephalin in dog ileum

Substance P initiated tonic contraction of dog ileum when administered in doses from 1 pg to 20 micrograms intraarterially (ED50 = 67 ng). Low doses acted to excite cholinergic postganglionic neurones since atropine or tetrodotoxin (TTX) increased the ED50 of substance P about 25-fold, while hexamethonium and local field stimulation had only a small effect to increase the ED50. Also atropine and tetrodotoxin effects were not additive. Higher doses apparently acted to stimulate smooth muscle directly, but no evidence was obtained that local field stimulation could release substance P to act on smooth muscle. Substance P tachyphylaxis prevented substance P actions on cholinergic nerves, but it did not affect responses to intraaterial acetylcholine or block distal inhibition from proximal distention or field stimulation. Met-enkephalin given intraarterially, was also excitatory in doses from 1 ng to 20 micrograms; the amplitude of tonic and phasic contractions produced was significantly decreased by TTX and atropine but was not diminished by hexamethonium or substance P tachyphylaxis. Partial tachyphylaxis to met-enkephalin was produced but was not diminished by hexamethonium or substance P tachyphylaxis. Partial tachyphylaxis to met-enkephalin was produced without affecting the ED50 for substance P. We conclude that substance P acts in small amounts on receptors in myenteric nerves to release acetylcholine by a mechanism, presumably involving postganglionic cholinergic nerves, while met-enkephalin also apparently may act at least in part through a similar TTX- and atropine-sensitive mechanism. These peptides also caused activation of other receptors, probably on smooth muscle by noncholinergic. TTX-insensitive mechanisms. Also the receptors for each peptide which are located on nerves were distinct and independent since tachyphylaxis could be produced to each without affecting the response to the other.     

Characterization of the subtype of muscarinic receptor coupled to the stimulation of phosphoinositide hydrolysis in 132-1N1 human astrocytoma cells.

Stimulation of muscarinic receptors increases phosphoinositide (PI) hydrolysis in 132-1N1 human astrocytoma cells. To evaluate the subtype of receptors which mediate PI hydrolysis in 132-1N1 cells, the effects of: a) the nonselective M1 agonist, carbachol; b) the selective M1 agonist, 4-hydroxy-2-butynyl-trimethylammonium chloride-m-chlorocarbinilate (McN-343); c) the nonselective antagonists, atropine and scopolamine; d) the relatively selective M1 antagonist, pirenzepine; e) the relatively selective M2 antagonists, AF-DX 116 (11-2-diethylaminomethyl-1-piperidinylacetyl-5, 11-dihydro-6H-pyrido-2,3-b-1,4-benzodiazepine-6-one) and methoctramine and f) the relatively selective M3 antagonist, hexahydrosila-difenidol (HHSiD) on PI hydrolysis in 132-1N1 cells were studied. The cell pools of inositol-phospholipids were prelabelled by incubating 132-1N1 cells in a low inositol containing medium (CMRL-1066) supplemented with [3H]inositol (2 microCi/ml) for 20-24 hours at 37 degrees C. The cells were washed and resuspended in a physiological salt solution, and PI hydrolysis was measured by accumulation of [3H]inositol-1-phosphate (IP) in the presence of 10 mM LiCl. Carbachol produced time and concentration dependent PI hydrolysis (EC50, 37 microM). McN-A343 did not cause significant hydrolysis of PI in 132-1N1 cells indicating that the receptor was not of M1 type. All the above muscarinic antagonists caused a concentration dependent decrease in the level of IP in response to carbachol (100 microM). The rank order of their affinities (pA2 values) was: atropine (8.8) > HHSiD (7.6) > pirenzepine (6.8) > methoctramine (6.0) > AF-DX 116 (5.8). This rank order supports the concept that M3 (other names, M2 beta, glandular M2) receptors are linked to PI hydrolysis in 132-1N1 cells. HHSiD, which is selective for M3 receptors of the smooth muscle has higher affinity for muscarinic receptors in 132-1N1 cells than AF-DX 116 which is selective for M2 receptors in cardiac tissue. If the receptor in 132-1N1 cells had been M2, part of the rank order for affinities would have been methoctramine > AF-DX 116 > HHSiD > pirenzepine. From all of these observations, the muscarinic receptor for PI hydrolysis in 132-1N1 cells is tentatively characterized as of M3 type.     

Ca2+ mobilization and activation of extracellular acidification by carbachol in acutely dispersed cells from guinea pig detrusor: Fura 2 fluorometry and microphysiometry using the cytosensor

The study aim was to develop a simple in vitro model for pharmacophysiological investigation of urinary bladder smooth muscles. Smooth muscle cells from guinea pig detrusor were dissociated, and the suspended cells were stimulated with carbachol (CCh), an acetylcholine receptor agonist. Cytosolic Ca2+ levels were determined using Fura 2 fluorescence and extracellular acidification rates were monitored by the Cytosensor microphysiometer. CCh dose-dependently increased cytosolic Ca2+ levels and extracellular acidification rates, with EC50 values of approximately 1 microM. Both the acetylcholine muscarinic receptor antagonist atropine and the M3 muscarinic receptor-preferring antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the effects of CCh, three orders of magnitude more potently than the selective M2 muscarinic receptor antagonist, methoctramine. These data indicate the dominant role of M3 receptors in guinea-pig bladder but fail to show clear evidence of any functional role for M2 receptors. Since this finding agrees with a number of other studies using in vivo and in vitro models (1), cell suspensions such as these may prove to be simple tools for the pharmacological study of urinary bladder smooth muscle tissue.     

Subtype of muscarinic receptor coupled to the attenuation of hormone-stimulated cAMP accumulation in NG108-15 neuroblastoma x glioma hybrid cells.

The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Therefore, several selective muscarinic antagonists were used to characterize the subtype of muscarinic receptor coupled to the inhibition of hormone-stimulated cAMP accumulation using NG108-15 neuroblastoma x glioma hybrid cells. These cells were prelabeled with [2-3H]-adenine, washed, and resuspended in a culture medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before they were challenged with agonists. The formation of [3H]-cAMP was activated by PGE1 (1 microM) or forskolin (1 microM). In all cases, [3H]-cAMP formed was separated and measured. Carbachol (100 microM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN-A343 (10 mM), an M1 receptor agonist, was only a partial agonist causing 40% inhibition of cAMP accumulation indicating that this effect was not mediated by an M1 receptor; b) The M1-selective antagonist, pirenzepine, exhibited low affinity (pA2 6.2) further suggesting that an M1 receptor was not coupled to the attenuation of cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methoctramine) and M3 antagonist (HHSiD) were used to further characterize these muscarinic receptors. The order of all antagonists based on their affinities (pA2 values) could be arranged in the following order: atropine (9.0) > methoctramine (7.6) > HHSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the same degree of affinity to M2 receptors of other tissues as it does to those of NG cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M₃ subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [³H]quinuclidinyl benzilate ([³H]QNB) was most sensitive to atropine and the M₃-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M₂ antagonist AFDX116. This, and the binding characteristics of [³H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M₃ subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 μM) reduced B_max of [³H]4-DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC₅₀ of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β-adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M₃ muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the G_i proteinadenylyl cyclase system, and produces CBC-like action, that is, inhibition of the forskolin-stimulated [³H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.     

Effects of cholinergic agents on the vasopressin-mediated water transport in the isolated toad bladder

The aim of this work was to study the effect of some pharmacological cholinergic agents on the events that follow the interaction of arginine vasopressin with toad bladder membrane receptors related to synthesis of 3′5′_cAMP. The water flow through the membrane was measured gravimetrically in sac preparations of the membrane. In the absence of arginine vasopressin (AVP), carbachol induced a significant increase in the water flow (37%) related to the basal (Ringer's solution). On the other hand, when carbachol and AVP were associated, a significant decrease of AVP hydrosmotic activity occurred (23%). The inhibitory effect of carbachol on the AVP action was almost completely abolished by the cholinergic antagonists atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and the calcium antagonist lanthanum. Similarly, when carbachol and 3′5′ cyclic adenosine monophosphate (3′5′_cAMP) were associated, a decrease of nucleotide hydrosmotic activity was observed (12.80%). This effect was partially restored by the addition of pirenzepine or 4-DAMP in the bath solution. These results suggest a role for muscarinic receptors of sub-type M₁ and M₃, which are involved in the intracellular calcium release. The increase of calcium concentration in the intracellular medium acts as a negative modulator in the hydrosmotic action of antidiuretic hormone.