Effects Of Muscarinic Receptor Antagonists Research Articles

Rapid antidepressant-like effects of muscarinic receptor antagonists require BDNF-dependent signaling in the ventrolateral periaqueductal gray.

Clinical reports reveal that scopolamine, an acetylcholine muscarinic receptor antagonist, exerts rapid antidepressant effects in depressed patients, but the mechanisms underlying the therapeutic effects have not been fully identified. The present study examines the cellular mechanisms by which scopolamine produces antidepressant-like effects through its action in the ventrolateral midbrain periaqueductal gray (vlPAG). We used a well-established mouse model of depression induced by chronic restraint stress (CRS) exposure for 14days. Behaviors were tested using the forced swim test (FST), tail suspension test (TST), female urine sniffing test (FUST), novelty-suppressed feeding test (NSFT), and locomotor activity (LMA). Synaptic transmission in the vlPAG was measured by whole-cell patch-clamp recordings. IntravlPAG microinjection was used to pharmacologically verify the signaling cascades of scopolamine in the vlPAG. The results demonstrated that intraperitoneal injection of scopolamine produced antidepressant-like effects in a dose-dependent manner without affecting locomotor activity. CRS elicited depression-like behaviors, whereas intraperitoneal injection of scopolamine alleviated CRS-induced depression-like behaviors. CRS diminished glutamatergic transmission in the vlPAG, while scopolamine reversed the above effects. Moreover, intravlPAG microinjection of the L-type voltage-dependent calcium channel (VDCC) blocker verapamil, tropomyosin-related kinase B (TrkB) receptor antagonist ANA-12, mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) antagonist CNQX prevented scopolamine-induced antidepressant-like effects. Scopolamine ameliorated CRS-elicited depression-like behavior required activation of VDCC, resulting in activity-dependent release of brain-derived neurotrophic factor (BDNF), engaging the TrkB receptor and downstream mTORC1 signalingin the vlPAG.

Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice

Muscarinic M1/M4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (SD) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M1, M2, or M4 receptors (M1−/−, M2−/−, M4−/−), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M1, M2, or M4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M1/M4 receptors partially substituted for cocaine and increased the SD effect of cocaine, while M2-preferring antagonists did not substitute, and reduced the SD effect of cocaine. The cocaine-like effects of scopolamine were absent in M1−/− mice. The cocaine SD attenuating effects of methoctramine were absent in M2−/− mice and almost absent in M1−/− mice. The findings indicate that the cocaine-like SD effects of muscarinic antagonists are primarily mediated through M1 receptors, with a minor contribution of M4 receptors. The data also support our previous findings that stimulation of M1 receptors and M4 receptors can each attenuate the SD effect of cocaine, and show that this can also be achieved by blocking M2 autoreceptors, likely via increased acetylcholine release.

The effect of hypercholesterolemia on carbachol-induced contractions of the detrusor smooth muscle in rats: increased role of L-type Ca2+ channels

To investigate a possible relation between hypercholesterolemia and detrusor smooth muscle function, we studied the contractile response to potassium challenge, carbachol (CCh), and the components of CCh-induced contractile mechanism in high-cholesterol diet-fed rats. Adult male Sprague-Dawley rats were fed with standard (control group, N = 17) or 4% cholesterol diet (hypercholesterolemia group (HC), N = 16) for 4weeks. Spontaneous contractions of detrusor muscle strips and their responses to potassium chloride (KCl) or cumulative dose-contraction curves to CCh were recorded. The effects of muscarinic receptor antagonists (methoctramin and/or 4-diphenylacetoxy-N-methylpiperidine), L-type Ca(+2) channel blocker (nifedipine), and/or rho-kinase inhibitor Y-27632 were investigated. Blood cholesterol level was increased in the HC group with no sign of atherosclerosis. The KCl-induced detrusor smooth muscle contractions were higher in HC, whereas spontaneous and CCh-induced responses were similar in both groups. Preincubation with receptor antagonist for M(3) but not for M(2) attenuated contraction significantly, shifting the dose-response curve to the right. This response was similar in both groups. Among two effector mechanisms of M(3)-mediated detrusor smooth muscle contraction, rho-kinase pathway was not affected by hypercholesterolemia, whereas blockade of L-type Ca(+2) channels potently reduced contractions. The results of this study point out a relation between hypercholesterolemia and contractile mechanism of detrusor smooth muscle likely to change urinary bladder function, via altering L-type Ca(+2) channels. Taken together with escalating incidence of hypercholesterolemia and lower urinary tract symptoms, it is a field which deserves to be investigated further.

Effects of muscarinic receptor antagonists on acetylcholine‐induced contractions of jejunal smooth muscle in horses

This study investigated the effects of a muscarinic type 1 (M(1)), 2 (M(2)), and 3 (M(3)) antagonists (4-DAMP, pirenzepine, and methoctramine, respectively) on acetylcholine (Ach)-induced contractions of longitudinal jejunal muscle strips of horses. Strips were irrigated with Krebs-Henseleit solution gassed with 95% O(2) and 5% CO(2), and the developed tension in response to Ach was recorded before and after incubation with increasing concentrations of 4-DAMP (10(-8)-10(-6) M), pirenzepine (10(-6)-10(-4) M), and methoctramine (10(-5)-10(-3) M). When competitive antagonism was characterized, the affinity constant (pA(2)) was calculated by Schild plots. A parallel rightward shift in the concentration-response curves was observed after 4-DAMP and pirenzepine. Methoctramine presented a dual effect on the concentration-response curves: lower concentrations induced a parallel rightward shift without altering the maximum intensity of contraction (E(max)), while the highest concentration increased slope of the concentration-response curve and increased E(max). The pA(2) for 4-DAMP and pirenzepine was 9.18 and 7.13, respectively. Acetylcholine-induced contractions of longitudinal jejunal smooth muscle are mediated mainly via M(3) receptors. The complex role of M(2) receptors in jejunal smooth muscle contractions was evident because methoctramine potentiated the contractile response to higher doses of Ach.

Spinal Effects of the Fesoterodine Metabolite 5-Hydroxymethyl Tolterodine and/or Doxazosin in Rats With or Without Partial Urethral Obstruction

The combination of muscarinic receptor and alpha(1)-adrenoceptor antagonists is increasingly used for benign prostatic hyperplasia related lower urinary tract symptoms. In addition to the well established peripheral site of action, little is known about the central effects of muscarinic receptor antagonists and muscarinic receptor/alpha(1)-adrenoceptor antagonist combinations on bladder function, partly due to poor brain penetration after systemic administration. We assessed the effects of intrathecal 5-hydroxymethyl tolterodine, an active metabolite of fesoterodine, in obstructed and nonobstructed rats, and of combined intrathecal 5-hydroxymethyl tolterodine/doxazosin in a rat model of partial urethral obstruction. We used 80 male Sprague-Dawley rats to test various doses of intrathecal 5-hydroxymethyl tolterodine and/or intrathecal doxazosin on urodynamic parameters. Urodynamic evaluation without anesthesia was done 3 days after bladder and intrathecal catheterization. Two weeks before urodynamics 40 rats underwent partial urethral obstruction. Intrathecal 5-hydroxymethyl tolterodine had no urodynamic effects in nonobstructed rats. Compared to controls obstructed rats had increased bladder pressure and weight, and voiding frequency. In obstructed rats 5-hydroxymethyl tolterodine restored urodynamic parameters to those seen in nonobstructed animals. Doxazosin had effects similar to those of intrathecal 5-hydroxymethyl tolterodine. When the 2 drugs were combined, at the doses used only small additional effects were observed. Urodynamic changes in obstructed rats can be normalized by intrathecal 5-hydroxymethyl tolterodine and by intrathecal doxazosin. The central pathways on which the 2 drugs act seem to be up-regulated with partial urethral obstruction and less relevant under normal conditions.

Slower Heart Rates for Healthy Hearts

The management of atrial fibrillation, even in the modern era, remains complex and challenging. Preventing atrial fibrillation occurrence by identifying and favorably improving modifiable risk factors thus assumes great importance. It is well known that hypertension and resultant structural heart disease contributes significantly to the incidence of atrial fibrillation.1,2 The Losartan Intervention For End point reduction in hypertension (LIFE) study thus far has shown us that angiotensin-receptor blockade and reduction in left ventricular hypertrophy, irrespective of blood pressure–lowering, reduces the incidence of new-onset atrial fibrillation.1,3 Article see p 337 In recent years, accumulating evidence has linked high resting sinus heart rates (HR) directly and indirectly to adverse cardiovascular outcomes.4,5 Epidemiological studies show resting HR to be an independent predictor of cardiovascular and all-cause mortality in general population as well as in hypertensive patients.6 The cardiovascular benefits offered by β-blockade in coronary artery disease and heart failure have, in part, been attributed to β-blocker–mediated HR reduction.7,8 Other studies have linked high baseline HR to the development of hypertension, to the progression of coronary artery disease, and to the triggering of myocardial infarction, ventricular dysfunction, and ventricular arrhythmias.6,9–11 In this issue of Circulation: Arrhythmia and Electrophysiology , Okin and colleagues12 examines the relationship of HR changes over time on risk of atrial fibrillation in hypertensive patients as part of the LIFE study. In this post hoc analysis of the prospective LIFE study, 8828 hypertensive patients with left ventricular hypertrophy by ECG but without a history of atrial fibrillation were followed for a mean of 4.7±1.1 years. New onset atrial fibrillation was determined by 12-lead ECG performed on an intermittent, infrequent basis (at baseline, 6 months, and yearly thereafter). Hypertension was treated with losartan or atenolol. Using a variety of analyses, higher in-treatment HR on serial …

Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation

Most colon cancers overexpress M3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M3R antagonist (p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by approximately 40% (P<0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5- and 2-fold, respectively (P<0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes (n=25) whereas half of colon cancer specimens (n=24) exhibited moderate to strong staining (P<0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation.

Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland

The effects of muscarinic receptor antagonists on responses to electrical stimulation of the chorda-lingual nerve were determined in pentobarbitone-anesthetized sheep and correlated to the morphology of tissue specimens. Stimulation at 2 Hz continuously, or in bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar submandibular fluid responses (19 +/- 3 vs. 21 +/- 3 microl x min(-1) x g gland(-1)), whereas vasodilatation was greater during stimulation in bursts (-52 +/- 4 vs. -43 +/- 5%; P < 0.01). Continuous stimulation at 8 Hz induced substantially greater responses (66 +/- 9 microl x min(-1) x g gland(-1) and -77 +/- 3%). While atropine (0.5 mg/kg iv) abolished the secretory response at 2 and 20 Hz (1:10 s), a small response persisted at 8 Hz (<5%). The "M1-selective" antagonist pirenzepine (40 microg/kg iv) reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at 2 Hz (reduced by 69%). Methoctramine ("M2/M4-selective"; 100 microg/kg iv; n = 5) had no effect on fluid or the vascular responses but increased the protein output at 2 (+90%, P < 0.05) and 8 Hz (+45%, P < 0.05). The immunoblotting showed distinct bands for muscarinic M1, M3, M4, and M5 receptors, and immunohistochemistry showed muscarinic M1 and M3 receptors to occur in the parenchyma. Thus muscarinic M1 receptors contribute to the secretory response to parasympathetic stimulation but have little effect on the vasodilatation in the ovine submandibular gland. Increased transmitter release caused by blockade of neuronal inhibitory receptors of the M4 subtype would explain the increase in protein output.

Postjunctional modulation by muscarinic M2 receptors of responses to electrical field stimulation of rat detrusor muscle preparations

1. The aim of the present study was to examine the modulator influence of muscarinic M(2) receptors on responses of rat urinary bladder detrusor muscle evoked by endogenous stimuli, i.e. by stimulation of the bladder innervation. 2. Responses were evoked by electrical field stimulation (EFS; 2-20 Hz, 0.8 ms, 60 V) of isolated strip preparations mounted in organ baths. The tension of the muscle strips was recorded digitally. EFS was performed by applying stimulation with either a short duration (5 s) or a longer duration (to reach peak response; approximately 20 s). 3. Effects of muscarinic receptor antagonists (muscarinic M(1)/M(3) receptor selective: 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP); muscarinic M(2) receptor selective: methoctramine), a beta-adrenergic antagonist (propranolol) and an adenosine receptor antagonist (8-p-sulfophenyltheophylline) were assessed on contractile activity and on poststimulatory relaxations. 4. Low concentrations of methoctramine (10(-8) m) reduced or tended to reduce the EFS-induced contraction, e.g. at 2 Hz by 12% while methoctramine at 10(-7) m had no significant effect. In addition, in the presence of 4-DAMP (10(-9) m), which tended to inhibit contractions at all frequencies (2-20 Hz; -17 to -25%), methoctramine at 10(-8) and 10(-7) m induced a further reduction of the contractile responses (-5 to -10%; 2-20 Hz). 5. The beta-adrenergic receptor antagonist propranolol (10(-6) m) and the adenosine receptor antagonist 8-p-sulfophenyltheophylline (10(-6) m) both increased contractile responses by 9-21% (2-10 Hz, long duration; P < 0.05-0.001) as a consequence of antagonizing relaxatory stimuli. Neither antagonist affected the contractile responses to EFS with the short duration stimulation. Poststimulatory relaxations were reduced by 30-60% (P < 0.05) by propranolol and by 40-60% (P < 0.001) by 8-p-sulfophenyltheophylline, but for 8-p-sulfophenyltheophylline only after stimulation with the short duration. 6. In the presence of methoctramine (10(-7) m), the 8-p-sulfophenyltheophylline-induced increases of the contractile response to long duration EFS were significantly enhanced at 10 Hz (+12 +/- 4%; P < 0.05), whereas no such enhancement of the propranolol inhibitory effect occurred in the presence of methoctramine. However, poststimulatory beta-adrenoceptor-evoked relaxations after short duration EFS were increased by about 35% in the presence of methoctramine, but not those after long duration. 7. Thus, muscarinic M(2) receptor activation inhibits adenosine receptor- and beta-adrenoceptor-evoked relaxations of the rat detrusor muscle. The inhibition occurs via a transient postjunctional mechanism that mainly affects responses with a short latency.

Association of Muscarinic M1 Receptor Genetic Polymorphisms with Psychiatric Symptoms and Cognitive Function in Schizophrenic Patients

Objective: The cholinergic system is important in the search for the pathophysiology of schizophrenia due to its role in cognitive function, interaction with the dopamine system in brain regions relevant to schizophrenia, side effects of antipsychotic medication and potential antipsychotic effect of muscarinic receptor antagonists. This study investigated the association of type I muscarinic receptor (CHRM1) genetic polymorphisms with the clinical characteristics of chronic schizophrenic inpatients. Methods: We determined the genotype of CHRM1 genetic polymorphisms in 243 schizophrenic patients hospitalized in chronic care wards. Psychotic symptoms were assessed using the Brief Psychiatric Rating Scale (BPRS), and cognitive function was assessed using the Folstein Mini-Mental Status Examination (MMSE) test. Sixty of the 243 subjects also completed the Wisconsin Card Sorting Test (WCST). Results: There was a significant difference in the number of correct responses and the percentage of perseverative errors in the WCST in the CHRM1 C267A genotype group of schizophrenia patients. There was no significant association between age at onset, chlorpromazine equivalents, BPRS scores, MMSE or schizophrenia per se in patients with the CHRM1 C267A genotype. The full exon of the CHRM1 gene was screened out with single-strand conformation polymorphism, and 2 single nucleotide polymorphisms (C267A and C1353T) were identified in our patients and control subjects. These 2 single nucleotide polymorphisms were linked together without exception. Conclusion: This study demonstrated that in schizophrenic patients, the heterozygote group of CHRM1 C267A polymorphism (267C/A) had more correct responses and less perseverative errors on the WCST performance than the 267C/C homozygote group, implicating that this polymorphism may be related to prefrontal cortical function. Our results also suggested that the C267A polymorphism plays no major role in the susceptibility to and clinical manifestations of schizophrenia.

Impairment of male fertility induced by muscarinic receptor antagonists in rats

Male rats were treated with a muscarinic receptor antagonist at 3, 10, and 100 mg/kg/day for 4 weeks prior to mating with untreated females and their reproductive status was determined on gestation days (GD) 15–17. Treatment-related decreases in the pregnancy rate were observed at 100 mg/kg/day without any effects on mating performance. Impairment of male fertility by this compound was also observed after treatment for 1 week, but there were no effects after a 1-week withdrawal period suggesting reversibility of the effect. There were no treatment-related effects on sperm production or motility, or testicular histopathology in any group. In order to determine whether the reduced fertility was a class effect of muscarinic receptor antagonists, atropine was examined. Males received atropine for 1 week at 62.5 and 125 mg/kg/day and were mated with untreated females. A low pregnancy rate associated with a decrease in the number of implantations was observed at 125 mg/kg/day. The effect on implantation was also observed at 62.5 mg/kg/day. These findings suggest that the impairment of fertility in male rats induced by muscarinic receptor antagonists is a class effect, and has a relatively short onset of effect and is quickly reversible.

Model of rapid gastrointestinal transit in dogs: effects of muscarinic antagonists and a nitric oxide synthase inhibitor.

Our aims were to establish a canine model of rapid gastrointestinal transit, and to test the effects of muscarinic receptor antagonists (atropine, pirenzepine, AF-DX116, and darifenacin), and an NOS inhibitor, L-nitro-N-arginine (L-NNA) in this model. For gastric emptying and small bowel transit, 99mTc-labelled DTPA were added to a meal of skimmed milk (236 mL) that contained 2.4 g of magnesium hydroxide. Regional colonic transit was measured by111In-labelled beads placed in a capsule that released isotope in the proximal colon. Scintiscans were taken at regular intervals and indices of transit were calculated. Drugs were administrated intravenously. Gastric emptying, small bowel and colonic transit were rapid. Atropine and darifenacin (a selective M3 antagonist) delayed gastric emptying and colonic transit, the selective M1 and M2 muscarinic antagonists did not. The muscarinic blockers did not slow small bowel transit. L-NNA delayed small bowel and colonic transit but did not slow gastric emptying. A model suitable for the preclinical study of antidiarrhoeals was established. M3 receptors are important in the control of gastric emptying and colonic transit, and NOS inhibition slowed small bowel and colonic transit.

A new enzymic method for the isolation and culture of human bladder body smooth muscle cells.

Cultured cells of the human urinary bladder smooth muscle are useful for investigating bladder function, but methods for culturing them are not well developed. We have now established a novel enzymic technique. The smooth muscle layer was separated out and incubated with 0.2% trypsin for 30 min at 37 degrees C. The samples were then minced and incubated with 0.1% collagenase for 30 min and centrifuged at 900 g. The pellets were resuspended in RPMI-1640 medium containing 10% fetal calf serum (FCS) and centrifuged at 250 g. The smooth muscle cells from the supernatant were cultured in RPMI-1640 containing 10% FCS. The cells grew to confluence after 7-10 days, forming the "hills and valleys" growth pattern characteristic of smooth muscle cells. Immunostaining with anti-alpha-actin, anti-myosin, and anti-caldesmon antibodies demonstrated that 99% of the cells were smooth muscle cells. To investigate the pharmacological properties of the cultured cells, we determined the inhibitory effect of muscarinic receptor antagonists on the binding of [3H]N-methylscopolamine to membranes from cultured cells. The pKi values obtained for six antagonists agreed with the corresponding values for transfected cells expressing the human muscarinic M2 subtype. Furthermore, carbachol produced an increase in the concentration of cytoplasmic free Ca2+ an action that was blocked by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 selective antagonist. This result suggests that these cells express functional M3 muscarinic receptors, in addition to M2 receptors. The subcultured cells therefore appear to be unaffected by our new isolation method.

Role of spinal muscarinic and nicotinic receptors in clonidine-induced nitric oxide release in a rat model of neuropathic pain

Intrathecal administration of α 2 adrenergic agonists, such as clonidine, is capable of alleviating neuropathic pain. Recent studies suggest that spinal nitric oxide (NO) mediates the analgesic effect of intrathecal clonidine. Furthermore, compared to nicotinic receptors, spinal muscarinic receptors play a greater role in the analgesic effect of intrathecal clonidine. In the present study, we tested a hypothesis that clonidine-evoked NO release is dependent primarily on muscarinic receptors in the spinal cord after nerve injury. A rat model of neuropathic pain was induced by ligation of the left L 5/L 6 spinal nerves. Using an in vitro spinal cord perfusion preparation, the effect of muscarinic and nicotinic receptor antagonists on clonidine-evoked nitrite (a stable product of NO) release was determined. Both muscarinic and nicotinic antagonists dose-dependently attenuated clonidine-elicited nitrite release. In spinal cords from the neuropathic rats, the inhibitory effect of muscarinic receptor antagonists (atropine and scopolamine) on clonidine-elicited nitrite release was more potent than that of nicotinic receptor antagonists (mecamylamine and hexamethonium). However, in spinal cords obtained from sham animals, the inhibitory effect of muscarinic and nicotinic antagonists did not differ significantly. These results indicate that muscarinic, as well as nicotinic, receptors mediate clonidine-induced NO release in the spinal cord. These data also suggest that after nerve injury, the cascade of activation of α 2 adrenergic receptors–muscarinic receptors–NO in the spinal cord likely plays a predominant role in the analgesic effect of intrathecal clonidine on neuropathic pain.

Model of rapid gastrointestinal transit in dogs; effects of muscarinic antagonists and a nitric oxide synthase inhibitor

Abstract Our aims were to establish a canine model of rapid gastrointestinal transit, and to test the effects of muscarinic receptor antagonists (atropine, pirenzepine, AF-DX116, and darifenacin), and an NOS inhibitor, l-nitro-N-arginine (l-NNA) in this model. For gastric emptying and small bowel transit,99mTc-labelled DTPA were added to a meal of skimmed milk (236 mL) that contained 2.4 g of magnesium hydroxide. Regional colonic transit was measured by111In-labelled beads placed in a capsule that released isotope in the proximal colon. Scintiscans were taken at regular intervals and indices of transit were calculated. Drugs were administrated intravenously. Gastric emptying, small bowel and colonic transit were rapid. Atropine and darifenacin (a selective M3antagonist) delayed gastric emptying and colonic transit, the selective M1and M2muscarinic antagonists did not. The muscarinic blockers did not slow small bowel transit. l-NNA delayed small bowel and colonic transit but did not slow gastric emptying. A model suitable for the preclinical study of antidiarrhoeals was established. M3receptors are important in the control of gastric emptying and colonic transit, and NOS inhibition slowed small bowel and colonic transit.

In vivo and in vitro effects of muscarinic receptor antagonists on contractions and release of [ 3H]acetylcholine in the rabbit urinary bladder

The functional effects of muscarinic receptor antagonists were examined in vivo and in vitro on the rabbit urinary bladder. Inhibitory effects on carbachol-evoked contractions of detrusor strips were pronounced for 4-diphenylacetoxy- N-methylpiperidine (4-DAMP; −logIC 50: 8.64), p-fluoro-hexahydro-sila-diphenidol (pFHHSiD; 7.84) and atropine (8.27), while they were less pronounced for pirenzepine (6.62) and methoctramine (5.36). 4-DAMP and methoctramine increased 3H overflow from [ 3H]choline-labelled strips in response to electrical stimulation, contrary to pirenzepine, which decreased the overflow. Concomitant contractions were markedly reduced by 4-DAMP and by pirenzepine, but not by methoctramine. The −logIC 50 estimations for atropine-sensitive electrically evoked contractions revealed methoctramine (4.85) to be less potent on nerve-evoked contractions than on carbachol-evoked contractions, in contrast to pirenzepine (7.15) and 4-DAMP (9.15). The effects of the antagonists in anaesthetized rabbits resembled those in vitro. Thus, muscarinic receptors in the rabbit urinary bladder are heterogeneous; prejunctional facilitatory (M 1) and inhibitory (M 2) for acetylcholine release, and postjunctional muscarinic M 3 receptors mediating contractile responses.

Prejunctional muscarinic autoreceptors on horse airway cholinergic nerves

Muscarinic autoreceptors on horse airway cholinergic nerves were studied by examining the effects of muscarinic receptor antagonists on electrical field stimulation (EFS)-induced acetylcholine (ACh) release in trachealis preparations. All the antagonists including atropine (non-selective), pirenzepine (M 1-selective), AF-DX 116 (M 2-selective), and hexahydrosiladifenidol (M 3-selective) augmented ACh release concentration-dependently. The augmentation was not due to displacement of ACh molecules from tissue receptors into the bath liquid because incubation with atropine after EFS had no influence on the measured amount of ACh. Hexahydrosiladifenidol was more potent in inhibiting ACh-induced muscle contraction, which is known to be mediated by M 3 receptors, than in augmenting ACh release. The maximal ACh release rate in response to the selective antagonists was much less than that following atropine. Furthermore, the concentrations of the selective antagonists required to augment ACh release far exceeded their K ds for m 1, M 2, or M 3 receptors. These observations suggest that the muscarinic autoreceptors on horse airway cholinergic nerves may belong to a novel subtype.

The hypotensive effect of cisapride in rat

1. Cisapride is a prokinetic agent believed to facilitate acetylcholine release from the myenteric plexus of the gut. The aim of the present study was to investigate the effect of cisapride on blood pressure and the effects of muscarinic receptor antagonists on the cisapride-induced blood pressure changes. 2. Cisapride was given i.v. alone or 10 min after muscarinic receptor antagonists. Cisapride given i.v. produced a significant decrease in blood pressure in a dose-related manner. Atropine, AF-DX 116 and 4-DAMP given 10 min before cisapride injection, partially inhibited the hypotensive response to cisapride. In pithed rat, the effect of cisapride on blood pressure remained unaltered. 3. This study indicates that the action of cisapride is not through central mechanisms and part of cisapride's effect is through the cholinergic system.

Functional characterization of muscarinic receptors in murine airways.

1. The effects of muscarinic receptor antagonists considered to be selective for M1 receptors (pirenzepine; PZ), M2 receptors (AFDX-116), and for M3 receptors (4-diphenyl acetoxy N-methyl-piperidine (4-DAMP)) were used to investigate the existence of muscarinic receptors subtypes in murine airways. Atropine was used as a nonselective antagonist. The effects of these antagonists were studied upon tracheal contractions induced either by EFS (electric field stimulation) or by application of an exogenous cholinoceptor agonist (arecoline). 2. The muscarinic receptor antagonists tested inhibited arecoline-induced tracheal contractions with the following rank order of potency: 4-DAMP = atropine > pirenzepine = AFDX-116. The rank order of potency of the muscarinic antagonists used in inhibiting EFS-induced tracheal contractions was: 4-DAMP = atropine > PZ > AFDX-116. The pA2 values for these antagonists were similar when compared to the pA2 values determined in guinea-pig and bovine airway smooth muscle. 3. In addition to in vitro studies, the effects of inhalation of the different muscarinic antagonists on lung function parameters in vivo were investigated. Inhalation of 4-DAMP induced a decrease in airway resistance and an increase in lung compliance. In contrast, inhalation of AFDX-116 induced an increase in airway resistance and almost no change in lung compliance. Apart from some minor effects of atropine on airway resistance, atropine, PZ, and pilocarpine failed to induce changes in lung mechanics as determined by in vivo lung function measurements. 4. The results provide evidence for the existence of M3 receptors on murine tracheae that are involved in the contraction of tracheal smooth muscle. This is in agreement with other animal species such as the guinea-pig and bovine. In vivo experiments also demonstrated that in the mouse, M3 receptors play an important role in bronchial smooth muscle contraction and thus in bronchoconstriction. Interestingly we have also demonstrated that M2 receptors can play a role in bronchodilatation. Inhalation of an M2 receptor antagonist induced an increase in airway resistance whereas inhalation of an M3 receptor antagonist induced a decrease in airway resistance. It is therefore likely that an M3/M2 receptor balance plays an important role in the regulation of airway function.

Inhibitory and excitatory muscarinic receptors modulating the release of acetylcholine from the postganglionic parasympathetic neuron of the chicken heart.

The effects of muscarinic receptor antagonists on ACh release were studied in the absence or presence of cholinesterase (ChE) inhibition using the isolated perfused chicken heart. Presynaptic inhibitory muscarinic autoreceptor were characterized by determining the potency of various antagonists to enhance [3H]-ACh release evoked by field stimulation (3 Hz, 1 min). The order of potencies was: (+/-)-telenzepine > atropine > 4-DAMP > silahexocyclium > pirenzepine > hexahydro-siladifenid-ol > AF-DX 116. The comparison with known pA2 values for M1-, M2- and M3-receptors revealed that the presynaptic autoreceptor meets the criteria of an M1-receptor. Basal, not electrically evoked overflow of unlabelled ACh into the perfusate was caused by 'leakage' release (non-exocytotic), as it was independent of extracellular Ca2+. Muscarinic receptor antagonists failed to enhance basel overflow. In contrast, when ChE activity was inhibited by 10(-6) M tacrine or pretreatment with 10(-4) M DFP, the ACh overflow was partially Ca(2+)-dependent and was reduced by tetrodotoxine. Moreover, block of the inhibitory muscarinic autoreceptors by (+/-)-telenzepine or pirenzepine caused a several-fold enhancement of the ACh release. The potencies of these antagonists were identical to those found for the electrically evoked [3H]-ACh release. The rate of ACh release enhanced by ChE inhibition plus telenzepine corresponds to about 12% of the total ACh pool per min, which is about the maximum amount of ACh that is available for any kind of stimuli. The release was dependent on the presence of exogenous choline. Hence elevation of ACh release led to a correspondingly enhanced ACh synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)