Evidence for diminished inhibitory neuromuscular transmission in distal small intestine

The influence of myenteric nerves on duodenal muscle contraction and relaxation was examined in vitro and compared with muscle responses from the ileum. Proximal and distal rat small intestine was cut into strips measuring 6.0 X 10.0 mm. Strips cut along the oral-caudal axis were called longitudinal strips, whereas those cut 90 degrees to that axis were called circular strips. The strips were stretched to their optimal lengths and subjected to electrical field stimulation (0.1-1.0 msec pulse duration, 30-300 mA, 2-26 Hz) in the presence of Krebs' solution and Krebs' plus atropine, 10(-6) M. Field stimulation produced contraction responses that were inhibited by atropine and relaxation responses that were augmented by atropine. Muscarinic blockade abolished completely contraction in circular muscle, but atropine-resistant contractions persisted in the longitudinal strips. Proximal muscle showed significantly greater relaxation responses compared to distal muscle (P less than .05) at nearly all parameters of pulse duration, current and frequency. Contraction and relaxation amplitudes were significantly greater in longitudinal than respective circular muscle (P less than .05) at either site in the intestine. Thus, not only do the two muscle layers differ in their respective nerve supplies, but inhibitory neuromuscular transmission appears to have a greater influence in proximal than distal intestine.     

Pharmacologic identification, activation and antagonism of two muscarine receptor subtypes in the lower esophageal sphincter

Lower esophageal sphincter pressures were monitored with water-filled catheters in anesthetized opossums. Muscarinic agonists McN-A-343 and bethanechol were administered in the arterial supply of the sphincter. McN-A-343 caused relaxation after a brief contraction of the sphincter. Bethanechol caused a dose-dependent contraction. Tetrodotoxin antagonized the inhibitory effect of McN-A-343 but did not antagonized sphincter contraction caused by McN-A-343 or bethanechol. The mean ED50 values were 6.9 nmol/kg i.a. for McN-A-343-induced relaxation, 10.5 nmol/kg i.a. for McN-A-343-induced contraction and 0.4 nmol/kg i.a. for bethanechol-induced contraction. Atropine caused a dose-dependent rightward shift in the dose-response curves of inhibitory and excitatory effects of the two muscarinic agonists. Pirenzepine caused a dose-dependent rightward shift in the dose-response curves of McN-A-343-induced relaxation. Pirenzepine did not modify sphincter contraction caused by the muscarinic agonists. 4-Diphenylacetoxy-N-methylpiperidine methiodide, on the other hand, did not modify McN-A-343-induced sphincter relaxation but caused dose-dependent rightward shifts in the dose-response curves of sphincter contraction caused by McN-A-343 or bethanechol. These studies suggest that there are two distinct types of muscarine receptors in the opossum lower esophageal sphincter. The M1 muscarine receptors are present on the inhibitory neurons and participate in the synaptic transmission between vagal preganglionic and intramural postganglionic inhibitory neurons. They are activated by McN-A-343 and antagonized by pirenzepine. The M2 muscarine receptors are located directly on the sphincter muscle. They are also activated by McN-A-343, but are selectively activated by bethanechol and are antagonized by 4-diphenylacetoxy-N-methylpiperidine methiodide.     

Vascular muscarinic receptors: pharmacological characterization in the bovine coronary artery

The goal of this study was to make functional comparisons between muscarinic receptors mediating endothelial-dependent relaxation responses in the rabbit ear artery and receptors mediating endothelial-independent contractile responses. Ring segments of the bovine coronary artery with the endothelium removed proved to be an excellent model for studying the properties of muscarinic receptors mediating vascular smooth muscle contraction. Although endothelial-dependent relaxation responses could be seen with the calcium ionophore A-23187, no relaxation responses to cholinergic agonists were seen in the bovine coronary artery, whether or not the endothelium was present or in the presence or absence of smooth muscle tone. In ring segments of the bovine coronary artery or the rabbit ear artery, the cholinergic agonists, acetylcholine, methacholine and carbachol, proved to be approximately equipotent in evoking contraction or relaxation, respectively. In contrast, the putative M1 selective agonist McN-A-343 did not produce any effect in either tissue; nor did McN-A-343 have any effect on a perfused rabbit ear artery segment. Measurement of antagonist affinities indicated that the bovine coronary artery muscarinic receptors show low affinity for both pirenzepine (pKB = 6.9) and AF-DX 116 (11-2-[[2-[diethylaminomethyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one) (pKB = 6.3). Pirenzepine affinity was also low in the perfused rabbit ear artery preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of relaxation induced by K+ and nicotine in dog duodenal longitudinal muscle.

Dog duodenal longitudinal muscle strips precontracted with bradykinin responded to K+ (10 mM) with a transient relaxation, which was abolished by tetrodotoxin and oxyhemoglobin, but not influenced by atropine, ouabain and apamin. The induced relaxation was suppressed by treatment with 10(-5) M NG-nitro-L-arginine (L-NNA) a nitric oxide synthesis inhibitor, but not by the D-enantiomer. The inhibitory effect was antagonized by L- but not D-arginine. High concentrations (20 mM or higher) of K+ produced a relaxation followed by a sustained contraction; nicardipine abolished the contraction, but did not alter the relaxation. Nicotine produced a contraction, which was converted to a relaxation by atropine. The relaxant response was abolished by tetrodotoxin, hexamethonium and oxyhemoglobin, but was unaffected by timolol and phentolamine. L-NNA suppressed the relaxation, and L-arginine reversed the inhibition. The addition of K+ (20 mM) increased the content of cyclic GMP in the strips, the effect being prevented by tetrodotoxin and L-NNA. These findings suggest that K+ selectively stimulates the nonadrenergic inhibitory nerve, whereas nicotine stimulates both the excitatory cholinergic and inhibitory nerves. Nitric oxide released from the inhibitory nerve appears to transmit information to duodenal smooth muscle by increasing the production of cyclic GMP.     

Age-related changes in enteric neuromuscular transmission

Proximal and distal rat small intestine from 4-, 5- and 6-month-old rats were cut into strips measuring 6.0 X 10.0 mm. Longitudinal strips were cut along the oral-caudal axis of the intestine while circular strips were cut 90 degrees to that axis. The strips were stretched to their optimum lengths and subjected to electrical field stimulation (0.1-1.0 msec pulse duration, 30-270 mA, 1-26 Hz) in the presence of Krebs' solution and Krebs' solution plus 10(-6) M atropine. Field stimulation produced atropine-sensitive and atropine-resistant contractions in these tissues. Significant differences among the three groups were found in the amplitudes of atropine-sensitive contractions in strips from proximal longitudinal muscle. The 6-month-old animals showed the highest amplitude contractions and the 4-month-old the lowest, whereas the 5-month-old animals showed contractions that were intermediate in amplitude. No significant differences were noted among the atropine-resistant contractions. Field stimulation delivered at pulse durations of 5.0 and 50.0 msec in the presence of neural blockade with tetrodotoxin (5 X 10(-6) M) produced similar contraction amplitudes among the three groups. These results show that tension development produced by field stimulation of the proximal intestine increases significantly with age. The increases appear to be due to age-related differences in cholinergic neuromuscular transmission.     

Muscarinic M1 receptors stimulate a nonadrenergic noncholinergic inhibitory pathway in the isolated rat duodenum

We examined the effect of the muscarinic agonist McN-A-343 (4-m-chlorophenylcarbamoyloxy-2-butynyl trimethyl ammonium) on in vitro preparations of rat small intestine. McN-A-343 (0.1-10 microM) induced a concentration-dependent relaxation of duodenum, jejunum and ileum. This effect was due to activation of muscarinic receptors of the M1 subtype, inasmuch as it was antagonized by atropine (pA2, 8.93) and by the selective M1 antagonists pirenzepine and dicyclomine with high affinity (pA2, 8.09 and 8.14, respectively). Tetrodotoxin, but not hexamethonium, abolished McN-A-343 relaxation indicating involvement of neural pathways and absence of nicotinic transmission. Moreover, in the presence of apamin (0.1 microM) McN-A-343 induced a contractile response. Unlike McN-A-343, acetylcholine contracted the rat duodenum; its concentration-response curve was significantly potentiated by tetrodotoxin, suggesting stimulation of the McN-A-343 sensitive receptor by acetylcholine (P less than .01). Prior treatment of animals with reserpine reduced the potency of McN-A-343 only by 2.5-fold. The gamma-aminobutyric acid (GABA) antagonist bicuculline inhibited McN-A-343 with an affinity comparable to that found for GABA(A) receptors (pA2, 5.52), indicating involvement of GABAergic fibers. ATP is also likely to play a role, as desensitization experiments showed that alpha-beta-methylene ATP induced a comparable decrease of both McN-A-343 and its own response. In contrast, desensitization induced by GABA occurred to a minor extent. Thus, in the rat duodenum, activation of an M1 muscarinic receptor induces relaxation by releasing GABA, and possibly ATP, from myenteric neurons.     

Pharmacological characterization of the vascular muscarinic receptors mediating relaxation and contraction in rabbit aorta.

Studies were performed in the rabbit aortic rings, precontracted with norepinephrine, to determine the subtype(s) of muscarinic receptors involved in endothelium-dependent relaxation and contraction in the absence of endothelium elicited by cholinergic stimuli. Acetylcholine (ACh) and arecaidine propargyl ester (APE), a M2 and M3 agonist, produced a dose-dependent relaxation and contraction in endothelium-intact and endothelium-denuded rabbit aortic rings, respectively. Both of these responses were blocked by the muscarinic receptor antagonist atropine. M1 selective agonist McN-A-343 [4-[N-(3-chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium+ ++ chloride] did not produce any effect on the tone of precontracted aortic rings. ACh- and APE-induced relaxation in aortic rings with intact endothelium was selectively blocked by M3 receptor antagonists hexahydrosila-difenidol and p-fluoro-hexahydro-sila-difenidol (pA2 of 7.84 and 7.18) but not by M1 antagonist pirenzepine or M2 receptor antagonists AF-DX 116 [11-(2-[(diethylamino)methyl]- 1-piperidinyl]acetyl)-5, 11-dihydro-6H-pyrido-[2,3-b][1,4]-benzo-diazepin-6-one] and methoctramine. ACh- and APE-induced contraction was inhibited by M2 receptor antagonists AF-DX 116 and methoctramine (pA2 of 7.11 and 6.71) but not by pirenzepine, hexahydro-sila-difenidol or p-fluoro-hexahydro-sila-difenidol. ACh- and APE-induced relaxation or contraction were not altered by nicotinic receptor antagonist hexamethonium or cyclooxygenase inhibitor indomethacin. These data suggest that relaxation elicited by cholinergic stimulin in endothelium-intact aortic rings is mediated via release of endothelium-derived relaxing factor consequent to activation of M3 receptors located on endothelial cells, whereas the contraction in aortic rings denuded of their endothelium is mediated via stimulation of M2 receptors located on smooth muscle cells.     

Nature of the vagal inhibitory innervation to the lower esophageal sphincter.

The purpose of the present study was to investigate the nature of the vagal inhibitory innervation to the lower esophageal sphincter in the anesthetized opossum. Sphincter relaxation with electrical stimulation of the vagus was not antagonized by atropine, propranolol, phentolamine, or by catechloamine depletion with reserpine. A combination of atropine and propranolol was also ineffective, suggesting that the vagal inhibitory influences may be mediated by the noncholinergic, nonadrenergic neurons. To determine whether a synaptic link with nicotinic transmission was present, we investigated the effect of hexamethonium on vagal-stimulated lower esophageal sphincter relaxation. Hexamethonium in doses that completely antagonized the sphincter relaxation in response to a ganglionic stimulant, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), did not block the sphincter relaxation in response to vagal stimulation at 10 pulses per second, and optimal frequency of stimulation. A combination of hexamethonium and catecholamine depletion was also ineffective, but hexamethonium plus atropine markedly antagonized sphincter relaxation (P less than 0.001). Moreover, 4-(m-chlorophenyl carbamoyloxy)-2-butyltrimethylammonium chloride (McN-A-343), a muscarinic ganglionic stimulant, also caused relaxation of the lower esophageal sphincter. We suggest from these results that: (a) pthe vagal inhibitory pathway to the sphincter consists of preganglionic fibers which synapse with postganglionic neurons: (b) the synaptic transmission is predominantly cholinergic and utilizes nicotinic as well as muscarinic receptors on the postganglionic neuron, and; (c) postganglionic neurons exert their influence on the sphincter by an unidentified inhibitory transmitter that is neither adrenergic nor cholinergic.     

Biphasic release of endothelium-derived relaxing factor(s) by acetylcholine from perfused canine femoral arteries. Characterization of muscarinic receptors

Experiments were designed to compare the release of endothelium-derived relaxing factor(s) in response to various muscarinic receptor agonists from canine femoral arteries mounted in organ chambers or perfused in a bioassay system. In rings of femoral arteries, suspended for isometric tension recording in organ chambers, acetylcholine induced endothelium-dependent relaxations during contractions evoked by prostaglandin F2 alpha. Atropine and pirenzepine antagonized these relaxations in a competitive manner, atropine with a higher affinity (KB = 1.9 X 10(-9) M) than pirenzepine (KB = 5.4 X 10(-7) M). Carbachol and McN-A-343 also evoked endothelium-dependent relaxations, and pirenzepine inhibited these responses with a similar low potency. The order of relative potency of the agonists in organ chamber studies was acetylcholine = carbachol much greater than McN-A-343. Isolated segments of femoral arteries with endothelium were perfused (2 ml/min) with modified Krebs-Ringer-bicarbonate solution containing indomethacin; the perfusate was bioassayed for endothelium-derived relaxing factor(s) by means of a ring of coronary artery without endothelium. When infused above but not below the femoral artery, low concentrations (10(-8)-10(-7) M) of acetylcholine caused transient relaxations of the bioassay ring contracted with prostaglandin F2 alpha; higher concentrations of acetylcholine caused sustained decreases in tension. Atropine inhibited the two phases of the concentration-relaxation curve with similar potencies. Pirenzepine inhibited both phases in a competitive manner but exhibited significantly higher potency against the first- (ED50, 1.9 X 10(-9) M) than against the second-phase responses (ED50, 2.1 X 10(-7) M). Compound McN-A-343 induced only transient decreases in tension, whereas carbachol caused sustained relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin synthesis elicited by cholinergic stimuli is mediated by activation of M2 muscarinic receptors in rabbit heart

This study was performed to determine the subtype of muscarinic receptors involved in the action of cholinergic stimuli on prostaglandin (PG) synthesis in the isolated rabbit heart perfused at a constant flow rate with Krebs Hanseleit buffer. Acetylcholine (ACh, 1.0-10.0 nmol), an M1 and M2 receptor agonist, and arecaidine propargyl ester (APE, 1.0-5.0 nmol), a selective M2 agonist, produced a dose-related increase in the output of 6-keto-PGF1 alpha and a decrease in heart rate, whereas 4-[m-chlorophenyl carbamoyl]-2-butynyl-trimethylammonium chloride (McN-A-343, 10 nmol-1.0 mumol), a selective M1 receptor agonist, did not alter PG output. The increase in PG output or the decrease in heart rate elicited by ACh or APE was abolished by atropine (0.1 microM), an M1 and M2 receptor antagonist, and by 11-[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6-H-pyrido-[2,3-b] [1,4]-benzodiazepine-6-one (AF-DX-116, 1.0 microM), a selective M2 antagonist, but not by pirenzepine (1.0 microM), a selective M1 antagonist. The developed tension, which was also reduced by ACh and APE, but not by McN-A-343, was minimized by AF-DX-116 and not by lower concentrations of pirenzepine that attenuated the coronary vasodilator effect of McN-A-343. Lower doses of ACh (1.0-5.0 nmol) caused coronary vasodilation, whereas higher doses of ACh (10.0 nmol) and lower as well as higher doses of APE produced a biphasic effect--an initial vasodilation followed by vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

M1 receptor-mediated nitric oxide-dependent relaxation unmasked in stomach fundus from M3 receptor knockout mice

Muscarinic receptors can mediate both contractile and relaxant responses in smooth muscle. The stomach fundus from wild-type mice possesses a neuronal M(1) receptor that mediates relaxation to carbamylcholine and (4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride (McN-A-343) but is masked by M(3) receptor-mediated contraction to both agonists. When the M(3) receptor was deleted, cholinergic-induced relaxation was unmasked. M(1) receptor antagonism with pirenzepine, nitric oxide (NO) synthase inhibition with N(omega)-nitro-L-arginine methyl ester hydrochloride, and inhibition of neuronal activation with tetrodotoxin abolished relaxation to McN-A-343 in tissues from M(3) receptor knockout mice, supporting the neuronal localization of an M(1) receptor that activated NO release to effect relaxation. However, the cyclooxygenase inhibitor indomethacin did not affect contraction or relaxation to carbamylcholine in stomach fundus from wild-type or M(3) receptor knockout mice, indicating that cyclooxygenase products played no role in these responses. The neuronal M(1) receptor modulated relaxation induced by carbamylcholine and McN-A-343 but not relaxation induced by electric field stimulation of the stomach fundus. These data support the presence of M(1) receptor-mediated relaxation in the stomach and suggest that when the M(3) receptor is eliminated or blocked, M(1) receptor-mediated gastric relaxation may be enhanced, possibly leading to alterations in gastric emptying and subsequent effects on body weight.     

Local actions of trimebutine maleate in canine small intestine

A study of the local actions of trimebutine (TMB) maleate and its N-diesmethyl metabolite (TMB-M) was carried out in the gastrointestinal tract of anesthetized dogs. In the unstimulated small intestine, but not in the stomach or colon, i.a. TMB and TMB-M caused activation of circular muscle. Like the activation by i.a. [Met5]-enkephalin, this was antagonized by naloxone. In field-stimulated segments of stomach and small intestine circular muscle, TMB or TMB-M, like dynorphin-1-13 or [Met5]-enkephalin, inhibited the phasic and tonic contractions which were mediated mostly by cholinergic, postganglionic nerves. However, the inhibitory effects of dynorphin-1-13 or [Met5]-enkephalin on small intestine were antagonized by naloxone whereas those of TMB sometimes or those of TMB-M usually were not. TMB or TMB-M did not affect responses to i.a. acetylcholine, but high doses reduced the contractile responses to subsequent field stimulation and excitatory responses to [Met5]-enkephalin. We concluded that the excitatory local actions of TMB or TMB-M on small intestine involved opioid receptors probably of the mu or delta types. Inhibitory local actions on nerve-mediated responses, however, may not have involved opioid receptors. Comparison of these data to results when TMB or TMB-M were given i.v. suggests that these agents also have peripheral actions to affect gastrointestinal motility at sites outside the gastrointestinal tract.     

M-1 and M-2 muscarinic receptor-mediated inhibition of dopamine-sensitive adenylate cyclase in rat neostriatum: a permissive role for D-2 dopamine receptors

The interactions between dopamine and muscarinic receptor subtypes coupled to adenylate cyclase in superfused rat neostriatal slices were investigated using the efflux of cyclic AMP, in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, as a highly sensitive parameter of cyclic AMP production. Cyclic AMP efflux induced by simultaneous activation of (stimulatory) D-1 and (inhibitory) D-2 dopamine receptors by dopamine was reduced profoundly by the muscarinic receptor agonist oxotremorine and by inhibition of acetylcholinesterase with physostigmine, but not by the M-1 muscarinic receptor agonist McN-A-343. In contrast, upon blockade of D-2 receptors with (-)-sulpiride, dopamine-stimulated cyclic AMP efflux was inhibited by oxotremorine and physostigmine as well as by McN-A-343. Cyclic AMP efflux induced by isoprenaline, adenosine or vasoactive intestinal peptide was not affected by oxotremorine. The M-1 receptor-selective antagonist pirenzepine, unlike the nonselective antagonist atropine, was about 10 times less potent in antagonizing the inhibitory effects of (a near-maximally effective concentration of) oxotremorine upon simultaneous D-1 and D-2 receptor activation that upon selective D-1 receptor activation (i.e., upon blockade of D-2 receptors). In the latter case, pirenzepine was about 5 times more effective as an antagonist when muscarinic receptors were activated by McN-A-343 than upon exposure of the slices to oxotremorine or physostigmine, whereas the potency of atropine was independent of the agonist used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine produces cerebrovascular constriction through activation of muscarinic-1 receptors in the newborn pig

This study was designed to characterize the nature of responses to selective muscarinic (M)-1 and M-2 agonists and to determine the receptor subtype which mediates the vascular response to acetylcholine in the cerebral circulation of the newborn pig. Pial arterioles of anesthetized newborn pigs were observed using the closed cranial window method. Topical McN-A-343 (10(-7), 10(-4) M), an M-1 agonist, produced pial arteriolar constriction (diameters were 140 +/- 8, 127 +/- 9 and 111 +/- 8 microns for control, 10(-7) and 10(-4) M McN-A-343) that was blocked by topical pirenzepine (10(-3) M), an M-1 antagonist. Topical bethanachol (10(-7), 10(-4) M), an M-2 agonist, produced pial arteriolar dilation (diameters were 148 +/- 8, 169 +/- 10 and 181 +/- 10 microns for control, 10(-7) and 10(-4) M bethanachol) that was blocked by 4-DAMP 4-diphenylacetoxy-N'-methylpiperidine methiodide (10(-3) M), an M-2 antagonist. Responses to McN-A-343 were unchanged after 4-DAMP, and responses to bethanachol were unchanged after pirenzepine. Topical McN-A-343 and bethanachol elicited modest increases in cortical periarachnoid cerebrospinal fluid levels of 6-keto-prostaglandin F1 alpha, prostaglandin E2, thromboxane B2 and prostaglandin F2 alpha. Topical acetylcholine (10(-7), 10(-4) M) elicited pial arteriolar constriction and large increases in cerebrospinal fluid prostanoid levels which were blocked by atropine (0.5 mg/kg i.v.) and topical pirenzepine (10(-3) M), but not by topical 4-DAMP. These data demonstrate the existence of M-1 and M-2 receptors in the cerebral circulation of the newborn pig and indicate that acetylcholine elicits cerebrovascular constriction and increased cerebrospinal fluid prostanoid synthesis via activation of M-1 receptors.     

Neural control of the sphincter of Oddi. A physiological role of 5-hydroxytryptamine in the regulation of basal sphincter of Oddi motor activity in the cat.

The effect of 5-hydroxytryptamine (5-HT) on the sphincter of Oddi (SO) was studied in the cat. The SO had two motor responses to 5-HT: the most common was an initial contraction followed by a more prolonged relaxation, and the other was an exclusive relaxation. Tetrodotoxin did not impair the magnitude of the net contraction induced by 5-HT, but it completely blocked the relaxation. Methysergide partially inhibited the SO contraction in response to submaximal doses of 5-HT (5-20 micrograms/kg). Atropine decreased the SO excitatory response to all doses of 5-HT. The combination of atropine and methysergide completely antagonized the 5-HT excitatory effect, which changed the SO biphasic response to an exclusive relaxation. After tetrodotoxin, the effect of 5-HT was almost completely antagonized by methysergide alone. The SO contraction and relaxation caused by 5-HT were almost completely blocked by 5-HT tachyphylaxis. In contrast, a 5-HT depletion with reserpine enhanced the sensitivity of the SO to 5-HT, responding to doses a thousand times smaller than in control animals. Hexamethonium, phentolamine, propranolol, and 5-methoxy-N,N-dimethyltryptamine did not antagonize the 5-HT-induced contraction or relaxation. These findings indicate that 5-HT caused SO contraction by stimulating postganglionic cholinergic neurons and the smooth muscle directly and caused relaxation by stimulating postganglionic, noncholinergic, nonadrenergic inhibitory neurons. 5-HT blockade or depletion resulted in a significant reduction in basal tonic pressures and in the amplitude of phasic contractions, which suggested that serotonergic neurons may play a physiologic role in the regulation of basal SO motor activity.     

Effects of 5-hydroxytryptamine on the lower esophageal sphincter in vivo: evidence for multiple sites of action.

Intravenous administration of 5-hydroxytryptamine (5-HT) caused a dose-dependent contraction in the lower esophageal sphincter in the opossum. The smallest dose of 5-HT which caused a detectable contraction of the sphincter was 0.5 mug/kg, and a maximal sphincter contraction was produced by a dose of 40 mug/kg. Methysergide converted the contractile effect of 5-HT to a dose-dependent fall in the sphincter pressure; maximal inhibition of 77.2 +/- 7.2% of the resting pressure occurred with a dose of 40 mug/kg. The inhibitory effect of 5-HT was antagonized by tetrodotoxin, 5 MeO-DMT, and 5-HT tachyphylaxis. 5 MeO-DMT enhanced 5-HT-induced contraction of the sphincter. In the presence of 5 MeO-DMT and methysergide, 5-HT still caused a brief contraction of the sphincter; this contraction appeared to be due to stimulation of postganglionic cholinergic neurons as it was antagonized by tetrodotoxin or atropine. Reserpinization caused enhancement of the sphincter contraction by 5-HT. In the reserpinized animals in the presence of methysergide, 5-HT caused a small initial contraction followed by prolonged inhibition; atropine antagonized the initial contraction, while inhibition was antagonized by 5 MeO-DMT. These studies are consistent with the view that 5-HT exerts several different effects on the sphincter. 5-HT causes contraction of the sphincter by its direct action on the muscle and also by stimulation of cholinergic excitatory neurons. In addition, 5-HT inhibits the sphincter by stimulation of nonadrenergic inhibitory neurons.     

Erythromycin inhibits contractions of nerve-muscle preparations of the guinea pig small intestine

The actions of the macrolide antibiotic, erythromycin lactobionate (EM), on nerve-mediated and drug-induced contractions of longitudinal and circular muscle of guinea pig small intestine were studied in vitro. Longitudinal muscle contractions, evoked by single transmural electrical stimuli, were inhibited by EM (10-300 microM) with half-maximal inhibition occurring at 161 microM (EC50). EM-induced inhibition of longitudinal muscle contractions was not affected by the alpha-2 adrenergic antagonist, yohimbine (0.3 microM), by the adrenergic neuronal blocker, guanethidine (10 microM), or the opiate receptor antagonist, naloxone (1.0 microM). Bethanechol-induced longitudinal muscle contractions were also reduced by EM. Noncholinergic longitudinal muscle contractions (1 microM scopolamine present), induced by trains of transmural stimuli, were reduced by EM (EC50, 142 microM); substance P (a mediator of noncholinergic contractions)-induced contractions were also reduced by EM. Circular muscle contractions, evoked by brief trains of transmural stimulation, were inhibited by EM but bethanechol- and substance P-induced contractions of the circular muscle were not altered by EM. Segments (2 cm) of small intestine were used to study reflex circular muscle contractions evoked by distention of the segment aboral to the recording point. Reflex contractions were inhibited by EM (EC50, 84 microM). These data indicate that, in the guinea pig small intestine, EM inhibits nerve-mediated contractions by actions on enteric nerves and on longitudinal but not circular muscle.     

Effect of cholecystokinin and the octapeptide of cholecystokinin on the feline sphincter of Oddi and gallbladder. Mechanisms of action.

We studied the effect of cholecystokinin (CCK) and the octapeptide of cholecystokinin (OP-CCK) on the feline gallbladder and sphinecter of Oddi. Both CCK caused a dose-dependent gallbladder contraction and sphincter of Oddi relaxation. The half-maximal responses of the sphincter of Oddi were 6 ng/kg for OP-CCK and 0.15 Ivy-dog U/kg for CCK, which were lower than those of the gallbladder with 28 ng/kg and 0.32 Ivy-dog U/kg, respectively. The effect of OP-CCK on the gallbladder was partially blocked by tetrodotoxin (P < 0.02), hexamethonium alone (P < 0.05), or a combination of hexamethonium and atropine (P < 0.01). The gallbladder response to CCK was not blocked by either atropine alone (P < 0.60) or adrenergic antagonists (P > 0.40). The sphincter of Oddi response to OP-CCK was blocked by tetrodotoxin (P < 0.001) but it was not blocked by cholinergic (P < 0.20) or adrenergic antagonists (P < 0.60). After complete denervation with tetrodotoxin, OP-CCK caused sphincter of Oddi contraction. These findings indicate that there are two excitatory receptors for CCK in the gallbladder, one at the cholinergic neurons and the other at the level of the gallbladder muscle. There are also two receptors for CCK in the sphincter of Oddi, one that is inhibitory, and present at the noncholinergic, nonadrenergic neurons, and the other, excitatory, at the circular muscle.     

Contraction mediated by Ca++ release in circular and Ca++ influx in longitudinal intestinal muscle cells

The source of Ca++ responsible for contraction was examined in muscle cells isolated separately from the circular and longitudinal muscle layers of guinea pig and human intestine. Contraction was measured by scanning micrometry and cytosolic-free Ca++ ([Ca++]i) with the fluorescent indicator, quin2. In both species, contraction induced in circular muscle cells by cholecystokinin-8 (CCK-8) and acetylcholine was not affected by withdrawal of Ca++ from the medium or addition of the Ca++ channel blocker, methoxyverapamil, whereas contraction induced by both agonists in longitudinal muscle cells and by depolarizing concentrations of K+ in both cell types was abolished. Depletion of intracellular Ca++ stores with caffeine in Ca++-free medium abolished the response in circular muscle cells. Readdition of Ca++ to the medium for 30 sec restored the response in longitudinal but not circular muscle cells. [Ca++]i, measured in guinea pig muscle cells, increased 3- to 4-fold above resting levels (circular, 70.8 +/- 8.1 nM; longitudinal, 77.4 +/- 9.7 nM) in response to all three contractile agents. The increase in [Ca++]i induced by CCK-8 and acetylcholine in circular muscle cells was not affected by withdrawal of Ca++ from the medium or addition of methoxyverapamil, whereas the response to both agonists in longitudinal muscle cells and to 20 mM K+ in both cell types was abolished. It was concluded that cells from adjacent muscle layers of the intestine mobilize Ca++ differently during agonist-induced contraction, i.e., by Ca++ release in circular and Ca++ influx in longitudinal muscle cells.     

Direct determination of acetylcholine release by radioimmunoassay and presence of presynaptic M1 muscarinic receptors in guinea pig ileum

Radioimmunoassay for acetylcholine (ACh) with a sensitivity of 10 pg/tube was applied to the direct determination of ACh output from the nerve endings in longitudinal muscle strips of guinea pig ileum. The strips were preincubated with an irreversible cholinesterase inhibitor and superfused with Krebs' solution under various experimental conditions. Pirenzepine (0.1-10 microM) and atropine (10-100 nM) produced an increase in electrically evoked ACh output through the inhibition of presynaptic muscarinic receptors. Contractile response to endogenous ACh released by electrical stimulation was enhanced by pirenzepine and atropine at lower concentrations, whereas the highest concentrations of pirenzepine (10 microM) and atropine (100 nM) caused a reduction in the enhanced contractile response and a significantly diminished response, respectively. These results demonstrate that the concentrations of pirenzepine and atropine, effective in inhibiting presynaptic muscarinic receptors, differ from those inhibiting postsynaptic muscarinic receptors and suggest the possibility that presynaptic M1 muscarinic receptors regulating ACh output may be present in the guinea pig ileum.