Identification of muscarinic receptor subtypes involved in catecholamine secretion in adrenal medullary chromaffin cells by genetic deletion.

BACKGROUND AND PURPOSE: Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation. EXPERIMENTAL APPROACH: To identify muscarinic receptors, pharmacological tools and strains of mice where one or several muscarinic receptor subtypes were genetically deleted were used. Cellular responses to muscarinic stimulation in isolated chromaffin cells were studied with the patch clamp technique and amperometry. KEY RESULTS: Muscarinic M1, M4 and M5 receptors were immunologically detected in mouse chromaffin cells, and these receptors disappeared after the appropriate gene deletion. Mouse cells secreted catecholamines in response to muscarinic agonists, angiotensin II and a decrease in external pH. Genetic deletion of M1, but not M3, M4 or M5, receptors in mice abolished secretion in response to muscarine, but not to other stimuli. The muscarine-induced secretion was suppressed by MT7, a snake peptide toxin specific for M1 receptors. Similarly, muscarine failed to induce an inward current in the presence of MT7 in mouse and rat chromaffin cells. The binding affinity of VU0255035 for the inhibition of muscarine-induced currents agreed with that for the M1 receptor. CONCLUSIONS AND IMPLICATIONS: Based upon the effects of genetic deletion of muscarinic receptors and MT7, it is concluded that the M1 receptor alone is responsible for muscarine-induced catecholamine secretion.

Li+ and muscarine cooperatively enhance the cationic tail current in rat cortical pyramidal cells.

Li+ is known to facilitate the onset of status epilepticus induced by cholinergic stimulation, although the underlying mechanisms are not clear. Under whole-cell current clamp conditions with a CsCl-based internal solution, cortical pyramidal cells display a single plateau-spike followed by a slow depolarizing afterpotential (DAP) in response to injection of a short current pulse. However, the same current pulse generated a burst of plateau-spikes after application of Li+ (2 mM) and muscarine (10 microM). As similar bursts of plateau-spikes were generated through an enhancement of the slow DAP when [K+]o was raised (Kang et al. 1998), we have investigated the effects of Li+ and muscarine on the Ca2+-dependent cationic current underlying the slow DAP, measured as the slow tail current evoked after the offset of depolarizing voltage pulses. Muscarine enhanced the amplitudes of both early and late components of the slow tail current. This effect of muscarine was markedly potentiated by Li+, while Li+ by itself affected the slow tail current only slightly. Intracellular application of heparin (0.5-1 mg/mL) suppressed the effect of muscarine in the presence of Li+. These results suggest that inositol-trisphosphate-induced Ca2+ release is involved in the cooperative enhancement of the slow tail current, and this cooperation may be one of the mechanisms underlying facilitation of the onset of epilepsy induced by these agents.

N- and C-terminal substance P fragments modulate striatal dopamine outflow through a cholinergic link mediated by muscarinic receptors.

The present study investigated whether the modulatory effects of substance P and substance P fragments on striatal dopamine release involve a cholinergic link. Rat striatal slices were incubated with substance P, substance P(1-4), substance P(1-7), substance P(5-11) and substance P(8-11) in the absence or presence of various agents which modify cholinergic transmissions, and endogenous dopamine outflow was measured using high-performance liquid chromatography. The incubation of striatal slices with substance P and its N- and C-terminal fragments (1 nM) induced a significant overflow of endogenous dopamine. Neostigmine (150 nM) potentiated the effects of substance P and its fragments, whereas the incubation with hemicholinium-3 (50 microM) abolished the effects of the peptides on dopamine outflow. The acetylcholinesterase inhibitor and the inhibitor of choline uptake did not have intrinsic effects on dopamine outflow. The muscarinic antagonist atropine (1 microM) reversed completely the effects of substance P and its fragments, whereas the nicotinic antagonists dihydro-beta-erythroidine (0.5 microM) and pempidine (10 microM) were devoid of effects. None of the cholinergic antagonists modified dopamine outflow. The results suggest that substance P and several N- and C-terminal substance P fragments activate cholinergic neurons in striatal slices. The released acetylcholine induces an increased dopamine outflow, mediated by muscarinic receptors. These observations represent additional evidence which supports the functional interactions between substance P, acetylcholine and dopamine in the striatum. Furthermore, they show that substance P fragments may exert neuromodulatory effects through mechanisms similar to those underlying the effects of the parent peptide.

Cholinergic modulation of spontaneous hypothalamic-pituitary-adrenal activity and its circadian variation in man.

Controversy still exists regarding the role of cholinergic pathways in the regulation of the hypothalamic-pituitary-adrenal axis in man. We studied the effects of the administration of placebo, pyridostigmine (PD); 120 mg, orally), and the combination of PD and pirenzepine (PZP; 100 mg, orally) on ACTH, cortisol, and GH secretion at 0730 and 2230 h in seven normal males. PD induced a clear decrease in ACTH levels at both times of the day compared to treatment with placebo, producing higher suppression in the nocturnal period (34.4 +/- 5.8% vs. 21.8 +/- 10.7%). The combination PD and PZP prevented the inhibitory action of PD on ACTH secretion in the morning, but not in the evening, when ACTH values showed a decrease similar to that seen after giving PD alone (38.1 +/- 5.6% vs. 34.4 +/- 5.8%, respectively). Cortisol values declined only when the association PD plus PZP was given in the evening. GH levels had a significant increase after PD administration in the morning (4.1 +/- 1.2 ng/mL) and in the evening (10.2 +/- 1.6 ng/mL), confirming that cholinergic stimulation was taking place, whereas the addition of PZP to PD induced a significant attenuation of these responses. It is concluded that cholinergic pathways have a inhibitory role in ACTH secretion in man. M1 muscarinic receptors seem to be involved in the diurnal inhibition of PD, whereas our observations are consistent with the mediation of another type of cholinergic receptors as an explanation for the nocturnal effect of PD on ACTH secretion. PD did not alter the circadian variation in the hypothalamic-pituitary-adrenal axis, whereas the association of PD and PZP increased the differences between diurnal and nocturnal ACTH values, suggesting a modulatory effect of the cholinergic system on the circadian rhythm of ACTH secretion.

Heterogeneity of cholinergic muscarinic receptors coupled to phosphoinositide metabolism in immature rat brain.

The effects of muscarinic agonists and antagonists on phosphoinositide (PtdIns) metabolism were examined in the cerebral cortex and brainstem of 7-day-old rats, in order to evaluate the role of muscarinic receptor subtypes in this process. Additionally, comparative experiments were performed in cortices from adult animals. Accumulation of [3H]inositol phosphates ([3H]InsPs) in [3H]inositol pre-labeled brain slices was taken as an index of PtdIns hydrolysis. In neonatal cortex, maximal stimulation induced by the full agonists acetylcholine, carbachol and methacholine was 8-10 fold over basal [3H]InsPs accumulation. The effect of the partial agonists bethanechol, pilocarpine and oxotremorine varied from 3 to 4 fold over basal. Smaller responses to cholinergic stimulation were found in the brainstem and in the adult cortex. In neonatal cortex, muscarinic antagonists inhibited the stimulatory responses with the following order of potency: 4-DAMP > pirenzepine > AF-DX 116 approximately p-F-HHSiD. Pirenzepine inhibition of full agonist-induced [3H]InsPs accumulation showed biphasic curves, with two thirds of the response being inhibited with high affinity. When partial agonists were used, the resulting pirenzepine curves were better described by interaction at one high affinity site. No differences were found between immature and adult rats in the effect of pirenzepine on [3H]InsPs accumulation induced by carbachol, methacholine, or bethanechol. Inhibition by pirenzepine of PtdIns hydrolysis induced by carbachol or methacholine showed biphasic curves also in the brainstem. In this area, only one third of the response was inhibited with high affinity, and p-F-HHSiD was more potent as an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarine increases a voltage-independent potassium conductance through an M4 receptor in rat dorsolateral septal nucleus neurons.

The direct effect of muscarine on neurons of the rat dorsolateral septal nucleus (DLSN) was examined by using conventional microelectrode and voltage-clamp techniques. Muscarine (1-50 microM) caused a hyperpolarization accompanied by an increase of a voltage-independent potassium conductance. Pirenzepine competitively antagonized the muscarine-induced hyperpolarization with an apparent dissociation constant (Kd) value of 54 nM. Furthermore, intracellular loading with GTP gamma S, a non-hydrolyzable GTP analog, blocked irreversibly the muscarine-induced hyperpolarization. In addition, pretreatment of neurons with pertussis toxin (PTX) prevented the hyperpolarization produced by muscarine. These results suggest that muscarine hyperpolarizes DLSN neurons via a voltage-independent potassium conductance by acting at M4 subtype receptors which are coupled to a PTX-sensitive G-protein in DLSN neurons.

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic beta-cells.

We have studied the effects of cholinergic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca2+ ([Ca2+]i) in the beta-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 beta-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increase in DAG. ACh caused a transient rise in [Ca2+]i which was abolished by omission of Ca2+ or by addition of devapamil. Thus, cholinergic stimulation of beta-cell insulin release is associated with changes in both [Ca2+]i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca2+ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca2+]i and DAG had returned to control values suggesting that additional mechanisms may be involved.

Role of the enteric nervous system in the control of migrating spike complexes in the feline small intestine.

The effects of agonists and antagonists of nicotinic, muscarinic (M1 and M2), and adrenergic receptors on migrating spike complexes (MSC) in ileum of fasting cats are reported. Hexamethonium decreased MSC frequency and blocked propagation. Atropine at low concentrations increased MSC frequency and increased velocity of propagation; atropine at high concentration blocked propagation. Pirenzepine (Pz; M1 antagonist) increased MSC frequency and propagation velocity. McNeil A-343 (M1 agonist), by a Pz-sensitive phentolamine-insensitive mechanism, and 4-diethylamine-methylpiperidine (4-DAMP; M2 antagonist) blocked propagation of an ongoing MSC but had no significant effect on frequency or velocity. Bethanechol (M2-receptor agonist) increased phasic spiking by a 4-DAMP-sensitive mechanism and blocked MSC propagation by a Pz-sensitive mechanism. Phenylephrine (alpha 1-adrenoceptor agonist) or oxymetazoline (alpha 2-adrenoceptor agonist) blocked MSC propagation but had no effect on MSC frequency or velocity. Phentolamine (nonselective alpha 1-adrenoceptor antagonist), prazosin (alpha 1-adrenoceptor antagonist), or yohimbine (alpha 2-adrenoceptor antagonist) alone had no effect on MSC activity. The conclusion is that the enteric nervous system controls and regulates the MSC by the following proposed mechanisms. 1) M1-muscarinic receptors, located either on postganglionic inhibitory neurons or presynaptically at a nicotinic synapse and/or neuromuscular junction, are involved in the tonic inhibitory control of MSC initiation and propagation. 2) Nicotinic and M2 muscarinic receptors, located on excitatory postganglionic motoneurons and smooth muscle cells, respectively, are important in the initiation and/or propagation of MSC. 3) alpha 1-Adrenoceptors on the smooth muscle cells and alpha 2-adrenoceptors located presynaptically at the nicotinic ganglionic synapses are not tonically active but inhibit MSC activity (4). Smooth muscle beta-adrenoceptors do not play a significant role in neural control of MSC activity.

Pharmacological characterization of muscarinic receptors in neonatal rat cardiomyocytes.

[N-methyl-3H]scopolamine methylchloride ([3H]NMS) was used to characterize the muscarinic receptors (mAChRs) in the intact cardiomyocytes. The specific binding of [3H]NMS was proportional to cell concentration, saturable with respect to [3H]NMS concentration, and time dependent. Scatchard analysis of binding isotherms showed that [3H]NMS bound to the freshly isolated and cultured cardiomyocytes with dissociation constants of 275 +/- 64 and 207 +/- 20 pM as well as maximum receptor densities of 0.13 +/- 0.09 and 5.36 +/- 0.20 fmol/10(5) cells, respectively. Heterogeneity of mAChRs was demonstrated by competitive binding experiments against [3H]NMS with M2 and M3 antagonists. These receptors (80%) exhibited high affinities for 11-([2-[(diethylamino)methyl]-1-piperidinyl]-acetyl)-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine similar to those of M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors and possessed high affinity for 4-diphenylacetoxyl-N-methylpiperidine (4-DAMP) and hexahydrosiladifenidol. On the basis of biochemical studies, AF-DX-116 blocked adenosine 3',5'-cyclic monophosphate (cAMP) inhibition with high affinity (pKB 7.4), while it antagonized inositol phosphate formation with low affinity (pKB 6.5). 4-DAMP possessed high affinity in blocking inositol phosphate formation (pKB 9.0) and low affinity for antagonism of cAMP inhibition (pKB 7.7). Although no other muscarinic receptor mRNA has been detected in these cells, these data suggest the presence of a second population of mAChRs, which may not be identical to the classical cardiac "M2" receptors.

Fentanyl and pethidine are antagonists on muscarinic receptors in guinea-pig ileum.

In order to evaluate the anticholinergic effect of fentanyl and pethidine, the influence of these drugs on the cumulative dose-response curves of carbacholine on the guinea-pig ileum has been investigated. Fentanyl and pethidine displaced the dose-response curve for carbacholine to the right in a parallel fashion, indicating competitive antagonism. Dissociation constants determined by an agonist EC versus antagonist plot were 0.22 mumol/l for fentanyl and 1.4 mumol/l for pethidine. It is concluded that during high-dose fentanyl anaesthesia, fentanyl may bind to muscarinic receptors and thereby produce a central anticholinergic syndrome. An additional finding was that the maximal response to carbacholine increased significantly when combined with pethidine.

Cholinergic regulation of guinea pig duodenal bicarbonate secretion.

Although it is well known that vagal stimulation induces duodenal HCO3- secretion, there is presently no information about the nature of the cholinoceptor and the intracellular signals involved. In a series of experiments performed in a guinea pig duodenal loop model in situ, intravenous carbachol, atropine, pirenzepine, and hexamethonium were used to determine the extent of cholinergic stimulation and the types of cholinoceptors. Carbachol (2 micrograms.kg-1.5 min-1) stimulated HCO3- secretion threefold, and atropine (0.1 mg.kg-1.5 min-1) and pirenzepine (1 mg.kg-1.5 min-1) both abolished this effect. In addition, hexamethonium (0.3 mg.kg-1.5 min-1) inhibited carbachol-stimulated duodenal HCO3- secretion. Vasoactive intestinal peptide (VIP, 5 micrograms.kg-1.5 min-1) stimulated duodenal HCO3- secretion, and this action was partly inhibited by atropine (0.1 mg.kg-1.5 min-1) but not by pirenzepine (1 mg.kg-1.5 min-1). [4Cl-D-Phe6,Leu17]VIP (3.3 mg/kg), an antagonist to VIP, reduced basal, VIP-stimulated, and carbachol-stimulated HCO3- secretion. To examine the role of Ca2+ in this process, Ca2+ ionophore A23187, verapamil, and nifedipine were employed. A23187 (5, 50, 500 micrograms.kg-1.5 min-1) stimulated duodenal HCO3- secretion, an effect blocked by the VIP antagonist, and modestly augmented the effect of carbachol. Verapamil (0.2 mg.kg-1.5 min-1) and nifedipine (1.7 mg.kg-1.5 min-1) stopped the effect of carbachol on duodenal HCO3- secretion. These results suggest, that in cholinergic regulation of duodenal HCO3- secretion, the M-cholinoceptor pathway, Ca2+, and VIP are involved.

Age-related changes in pulmonary muscarinic receptor binding properties.

The goal of this study was to elucidate mechanisms responsible for age-related reductions in responsiveness to cholinergic muscarinic stimulation in guinea pigs, by examining the binding properties of muscarinic receptors and their coupling to guanine nucleotide regulatory proteins as a function of animal age. In addition, the binding constants of three selective muscarinic receptor antagonists pirenzepine, [11-((2-[(diethylamino)methyl]-1-piperidinyl)-acetyl)-5, 11-dihydro-6H-pyrido(2,3)(1,4)benzodiazepine-6-on], and 4-diphenylacetoxy-N-methylpiperidine methobromide were examined. We found that there were no changes in either the receptor density or the affinity of the muscarinic receptor with age. There was a significant reduction in the affinity constant for the high-affinity agonist binding site in the old tissues (7.63 +/- 0.08) compared with the young tissues (8.31 +/- 0.10). Guanine nucleotides lowered agonist affinity for the receptor in young lungs, however, they had no effect on agonist binding in old tissues. Antagonist competition binding curves in young tissues revealed that 73% of the receptors are of the M2 type, with 27% being of the M3 subtype. In contrast, antagonist competition binding curves in the old tissues revealed that 37% of the receptors were of the M2 subtype, 30% were M3, and 33% were of the M1 subtype. Our studies provide evidence that the loss of sensitivity to cholinergic muscarinic stimulation in the senescent lung may be due to changes in both muscarinic receptor subtypes and receptor coupling to G proteins.

M3 muscarinic acetylcholine receptor coupling to PLC in rat exorbital lacrimal acinar cells.

This study was designed to characterize the muscarinic acetylcholine receptor (mAChR) subtype present in rat exorbital lacrimal gland as well as its biochemical coupling. The nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) binds with high affinity to a homogeneous population of binding sites in both membranes [dissociation constant (Kd) = 82.3 +/- 3.2 pM] and acinar cell (Kd = 170.3 +/- 20 pM) preparations. Muscarinic antagonist inhibition of [3H]NMS binding is homogeneous with the following order of potency: atropine > or = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) > pirenzepine > 11-([2-(diethylamino)-ethyl]-1-piperidinyl)-acetyl- 5,11-dihydro-6H-pirido[2,3-b]1,4,benzo diazepine-6-one (AFDX 116). Both the affinity of the selective antagonists 4-DAMP, pirenzepine, and AFDX 116 and Northern blot analysis of lacrimal gland mRNAs show a single mAChR population of the M3 subtype. Muscarinic agonist inhibition of [3H]NMS binding displays both high (approximately 20%)- and low-affinity sites (approximately 80%). Both the receptor occupancy and the stimulation by agonists or the inhibition by antagonists of the accumulation of [3H]inositol phosphate were examined under identical conditions with respect to tissue preparations (acinar cells) and buffer (Krebs-Ringer). Results demonstrate 1) the efficient coupling of the M3 mAChR subtype with the phosphatidylinositol (4,5))bisphosphate-specific phospholipase C activity and 2) that the efficacy of a muscarinic agonist is dependent on its structure. Lastly, comparison of the agonists affinity and potency to trigger the [3H]inositol phosphate accumulation suggests that the occupation of the high-affinity agonist binding state of the M3 mAChR was involved in the cellular response.

Modulation of release of [3H]acetylcholine in the major pelvic ganglion of the rat.

Cholinergic modulation of [3H]acetylcholine release evoked by electrical stimulation was studied in the rat major pelvic ganglion, which was prelabeled with [3H]choline. Acetylcholine (ACh) release was independent of the frequency of stimulation; 0.3 Hz produced the same volley output as 10 Hz. Tetrodotoxin (1 microM) or omission of Ca2+ from the medium abolished ACh release. The M1 receptor agonist (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride (McN-A 343, 50 microM) increased release (by 136%), whereas the M2 muscarinic agonist oxotremorine (1 microM) decreased ACh release (by 22%). The muscarinic antagonists, atropine (1 microM) or pirenzepine (M1 selective, 1 microM), did not change ACh release. However, pirenzepine (1 microM) blocked the facilitatory effect of McN-A 343, and atropine (1 microM) blocked the inhibitory effect of oxotremorine. The cholinesterase inhibitor physostigmine (1-5 microM), the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP, 10 microM), and the nicotinic antagonist D-tubocurarine (50 microM) did not change ACh release. 4-Aminopyridine, a K+ channel blocker, significantly increased the release (by 146%). Seven days after decentralization of the major pelvic ganglion, the evoked release of ACh was abolished. It is concluded that release of ACh occurs from the preganglionic nerve terminals rather than from the cholinergic cell bodies and is not modulated by actions of endogenous ACh on either muscarinic or nicotinic autoreceptors. These data confirm and extend previous electrophysiological findings indicating that synapses in the major pelvic ganglion have primarily a relay function.

Pharmacological sensitivity of the articular capsule of the primary spines of Eucidaris tribuloides.

1. This paper describes the effects of several cholinergic agonists and antagonists, and of beta-phenylethylamine (PEA) and some of its derivatives, on the articular capsule, or ligament, of the primary spines of Eucidaris tribuloides. 2. Carbamylcholine (CCh), methacholine (MeACh), nicotine, and muscarine exert a stiffening effect similar to that of acetylcholine (ACh), although the time course of their actions varies widely. 3. Atropine induced stiffening and blocked and responses to muscarine and MeACh. The responses to MeACh were blocked also by 4-diphenylacetoxy-N-methylpiperidine, suggesting the presence in the ligament of type M3 muscarinic receptors, in addition to nicotinic ones. d-Tubocurarine induced stiffness of the ligament and failed to block the responses to ACh and nicotine. 4. While ACh induced only a slight desensitization, CCh caused a long-lasting blockade of the stiffening effects of the cholinergic agonists. This shows that the receptors for ACh have a site or sites that recognize the ester moieties of these molecules. 5. Eserine and neostigmine potentiate the responses to acetylcholine, indicating the presence of acetylcholinesterase in the ligament. 6. beta-Phenylethylamine, epinephrine, norepinephrine, and dopamine induce diphasic responses; usually a brief softening followed by a slow and irreversible stiffening of the ligament. 7. In contrast to the above, tyramine and octopamine elicit a simple softening of ligaments which are stiff as a result of handling or by exposure to cholinergic agonists. However, tyramine and octopamine do not soften ligaments which become stiff as a result of exposure to adrenergic agonists.

The search for prejunctional inhibitory muscarinic receptors in human bronchus.

The purpose of our study was to search for and classify prejunctional inhibitory muscarinic receptors in human bronchus with a wide range of muscarinic antagonists. Human bronchial airway smooth muscle strips, collected at thoracotomy from 54 subjects, 31 patients with chronic airway obstruction and 23 control subjects, were suspended in organ baths and stimulated by electrical field stimulation. The stimulation parameters were: frequency: 32 Hz; Grass reading voltage: 70 V (voltage between the electrodes: 15 V); pulse duration: 0.5 msec and train duration: 10 sec, every minute. Twitch responses of smooth muscle strips of 27 subjects (14 with and 13 without chronic airway obstruction) were sufficiently stable to search for the effects of M1-, M2- and M3-selective antagonists. All antagonists were added to the bath in a cumulative manner and decreased twitch contraction. No convincing evidence was found for the presence of prejunctional inhibitory muscarinic receptors of the M2-subtype, reasons for this obvious absence are discussed. Anaesthetical drugs for example, may mask the prejunctional inhibitory muscarinic receptors. The question remains whether the used electrical field stimulation experiments are suitable to point out prejunctional inhibitory muscarinic receptors. It is concluded that additional experiments with other experimental methods and in patients with different anaesthetical treatment are needed.

The influence of beta-adrenoceptor and muscarinic receptor agonists and antagonists on the static urethral closure function in healthy females.

The effects of terbutaline, propranolol, carbachol, and atropine on the static urethral closure function were investigated in 20 healthy women. Terbutaline caused a statistically reduction of the urethral pressure in the high pressure zone, but not at the bladder neck or in the distal urethra. The other drugs caused no significant pressure changes. None of the drugs used produced significant changes in the static viscoelastic properties, elastance, and hysteresis of the resting urethra. It is suggested that terbutaline partly reduces intraurethral pressure by reducing muscular tone in the rhabdosphincter and pelvic floor.

Cholinergic sensitivity of irides from donors with various pathological conditions and lens implants.

In vitro, iris contractions after muscarinic agonists were measured in mg of tension change and the concentration producing 50% of the response was expressed as EC50 mumol/l. Although the average EC50 value of carbachol in the iris sphincter of the donors with diabetes or Parkinson's disease did not change significantly when compared with the control, the maximum contraction of the tissue from the diseased state was increased significantly. Thus, in addition to the well known denervation supersensitivity of the iris-dilator, the iris-sphincter also develops adaptive sensitivity changes. Antimuscarinic drug treatment in some Parkinson's patients interfered with the estimation of supersensitivity in vitro studies. The enhanced response of carbachol at the low temperatures or the relative potency of carbachol and pilocarpine in the tissue obtained from the diseased donors was not significantly different from that of controls. Based on EC50 values, the potency of arecoline on the iris was 1/3 that of carbachol. Significantly lower EC50 values of carbachol were found in irides which were in contact with open loop type anterior chamber lens implants compared with those in contact with the closed loop anterior chamber lens implants. Maximum responses of irides to carbachol were less when the tissue was in contact with open loop lens compared with those in contact with closed loop anterior chamber implants. Irides from many donors having unilateral or bilateral replacement of the artificial lenses responded with EC50 of carbachol which was approximately equal to that of the contralateral eye. The maximum difference between EC50 values of the left and right iris was less than 5 fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptor subtypes in bovine adrenal medulla.

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of different selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60-80%) correspond to M2 muscarinic subtypes, and the rest is constituted by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.

Muscarinic receptors in MDCK cells are coupled to multiple messenger systems.

Our studies on Madin-Darby canine kidney (MDCK) cells have demonstrated that high-affinity specific muscarinic receptors coupled to the phosphoinositide system are present in these cells. To determine whether muscarinic receptors in MDCK cells are linked negatively to the adenylate cyclase system, we measured the effect of muscarinic agonists and antagonists on vasopressin-, isoproterenol-, and forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Vasopressin produced a maximum stimulation of cAMP formation of 13 pmol.10(6) cells-1.2 min-1 at 10(-7) M. Isoproterenol and forskolin stimulated cAMP formation production to 21 pmol.10(6) cells-1.2 min-1 and 64 pmol.10(6) cells-1.10 min-1, respectively, at 10(-4) M. The effects of vasopressin, isoproterenol, and forskolin were blocked by arecoline, a cholinergic agonist, in a concentration-dependent manner. The arecoline response was blocked by treatment of the cells with pertussis toxin. The inhibition by arecoline of forskolin-stimulated cAMP formation was reversed by various muscarinic antagonists in the following order of potency: 4-diphenyl-acetoxy-N-methylpiperidine > p-fluorohexahydrosiladifenidol > pirenzepine > methoctramine. This order of potency of muscarinic antagonists is similar to that observed in our radioligand binding studies and is consistent with the M3 subtype of muscarinic receptors. Our results indicate that muscarinic receptors in MDCK cells are coupled negatively to the adenylate cyclase system via pertussis toxin-sensitive G protein. It is concluded that this intracellular system may at least be partially responsible for the action of cholinergic agonists in these cells and in the kidney.