Muscarinic M3 receptors mediate contractions in rabbit endothelium-denuded aorta in vitro

1 Muscarinic receptors mediating contraction of rabbit endothelium-denuded aorta have been characterized functionally, in vitro, using a range of antagonists (atropine, pirenzepine, methoctramine, himbacine, 4-diphenyl-acetoxy-N-methyl piperidine methiodide (4-DAMP) and para-fluoro-hexahydro-siladifenidol (p-F-HHSiD). 2 The non-selective muscarinic agonist, (+)cis-dioxolane, induced concentration-dependent contractions of endothelium-denuded aortic rings. The potency (EC₅₀) of (+)cis-dioxolane was 1.0 ± 0.4 μM and the maximal increase in isometric tension was 944 ± 98 mg (mean ± SEM, n= 25). The concentration–effect curves to (+)cis-dioxolane were shifted to the right in the presence of antagonists, in a concentration-dependent manner. The following affinities (– log K_B) were calculated; atropine, 9.4; pirenzepine, 6.6; methoctramine, 5.9; himbacine, 7.1; 4-DAMP, 9.2; and p-F-HHSiD, 7.7. 3 It is concluded that muscarinic M₃ receptors mediate contractions of endothelium-denuded aorta. The low potency of (+)cis-dioxolane, when compared to its potency in other M₃ receptor assays, suggests that the efficiency of receptor coupling, associated with contraction of this tissue, is poor.     

Muscarinic M3 receptors mediate secretion from sweat glands in the rat

We investigated a series of muscarinic antagonists for their ability to inhibit muscarinic-mediated secretions from salivary and sweat glands in the rat. Their potency in blocking saliva secretion was compatible with the inhibition of M3 receptors. A highly significant correlation was found between their potency in blocking secretion from both salivary and sweat glands. These results indicate that salivary and sweat glands possess the same muscarinic receptors which conform to the M3 subtype.     

Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle

The muscarinic receptor subtype involved in human airway smooth muscle contraction was characterised for the first time, using subtype-selective muscarinic antagonists. It was demonstrated that methacholine-induced contraction of central (trachea) and peripheral (small bronchi) airway smooth muscle preparations was antagonised by pirenzepine, AF-DX 116, 4-DAMP methobromide, hexahydrosiladifenidol, and methoctramine with pA₂-values characteristic of M₃ (smooth muscle/glandular) muscarinic receptors. Since these pA₂-values demonstrate significant correlations with those found in bovine and guinea-pig tracheal smooth muscle contraction, it is concluded that these animal tissues provide a good model for the study of M₃ subtype-selective muscarinic antagonists to be used as bronchodilators.     

Muscarinic M-1 receptors mediate the bronchial hyperresponsiveness during methacholine inhalation

Several data suggest that muscarinic M-1 receptors are located on airway smooth muscle of human beings and dogs. Muscarinic M-1 receptors, whose stimulation is associated with enhancement of acetylcholine release, may also be located in the airway parasympathetic ganglia. To determine if postsynaptic muscarinic M-1 receptors mediate airway constriction, the action was compared of pirenzepine, that antagonizes selectively muscarinic M-1 receptors, and atropine, that blocks both muscarinic M-1 and M-2 receptors, on bronchial responses to methacholine inhalation in seven asthmatic patients. Pirenzepine and atropine gave protection against methacholine; however, the degree of protection by pirenzepine was significantly less than that given by atropine. Some inhibitory effect was obtained in all the patients studied after pirenzepine administration, even though some patients were only slight responders. The significant finding of the study is that pirenzepine partially inhibits the bronchospastic response to methacholine. This result suggests that muscarinic receptors on airway smooth muscle are also of the M-1 subtype.     

M1 muscarinic receptors mediate intracellular calcium release in NB-OK1 human neuroblastoma cells

Summary Muscarine acetylcholine receptors were characterized in NB-OK1 cells using radioligand (³H-NMS) binding experiments and second messenger (calcium and phosphatidylinositol (PI) turnover) studies. In radioligand binding experiments the displacement curves of pirenzepine (K_I = 1.3 × 10^–8 M), AF-Dx 116 (KI = 8.2 × 10^–7 M), methoctramine (KI = 8.4 × 10^–8 M) and parafluorohexahydrosiladifenidol (pF-HHSiD) (K1 = 1.8 × 10^–8 M) were monophasic and indicated the presence of M 1 muscarinic receptors. Schild analysis with the muscarinic antagonists pirenzepine, AF-Dx 116, methoctramine and pF-HHSiD yielded pA₂ values of 8.40 +- 0.13, 6.48 =- 0.09, 7.61 +- 0.12 and 7.22 +- 0.08 in the calcium experiments and pA₂ values of 8.13 +- 0.30 and 6.26 +- 0.26, 7.65 +- 0.16 and 7.46 +- 0.11, respectively, in the PI turnover experiments. These results indicate that both the carbachol-induced increase in intracellular calcium and the increase in PI turnover are mediated by M1 muscarinic receptors. In calcium free buffer, stimulation with carbachol induced similar responses to those seen under control conditions. From functional and radioligand binding experiments we conclude that the muscarinic receptor expressed in NB-OK1 cells is the M1 subtype. In addition, the M1 receptor-induced calcium response is related to PI turnover and is independent on extracellular calcium. Send offprint requests to H. W. G. M. Boddeke at the above address     

Differences in the adenosine receptors modulating inositol phosphates and cyclic AMP accumulation in mammalian cerebral cortex.

1. 2-Chloroadenosine stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation and potentiated (guinea-pig) or inhibited (mouse) the histamine H1-receptor-stimulated hydrolysis of inositol phospholipids in slices of guinea-pig and mouse cerebral cortex. 2. Two xanthine-based adenosine receptor antagonists were identified which were one order of magnitude more potent at the adenosine receptor mediating augmentation of the histamine-stimulated inositol phospholipid hydrolysis than at the receptor linked to cyclic AMP formation in guinea-pig cerebral cortical slices. 3. These compounds, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 7-benzyl-3-(2-methylpropyl)xanthine (BMPX) retained their selectivity under near-identical incubation conditions in both assays. 4. These compounds also showed similar affinities and selectivity for the adenosine receptor mediating inhibition of histamine-stimulated inositol phospholipid hydrolysis in mouse cerebral cortical slices. 5. Inclusion of agents which give rise to, or mimic, high levels of cyclic AMP (forskolin (1 microM), 8-bromo-cyclic AMP (1 mM) and vasoactive intestinal polypeptide (1 microM] in the incubation medium failed to mimic the action of 2-chloroadenosine on inositol phospholipid turnover in cerebral cortical slices from either species. 6. These data suggest that the adenosine receptor modulating the hydrolysis of inositol phospholipid in mouse and guinea-pig cerebral cortical slices is different from the adenosine receptor linked to cyclic AMP formation, and, furthermore, that the modulation of inositol phospholipid metabolism in either species is not mediated via alterations in cyclic AMP levels.     

Muscarinic M3 receptors are coupled to two signal transduction pathways in rat submandibular cells

The receptor subtypes involved in muscarinic-induced phosphoinositide hydrolysis and adenylate cyclase inhibition in rat submandibular acinar cells were characterized by comparing the inhibitory potencies of four muscarinic antagonists on the two signal transduction responses. Carbachol-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was inhibited by all antagonists with a potency rank order of 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP) = atropine much greater than pirenzepine much greater than AF-DX 116 (P less than 0.01). The same rank order was observed in antagonist-reversal of the reduction of cAMP caused by carbachol in the model. These findings suggest that muscarinic effects are mediated by M3 receptors in both the phosphoinositide and adenylate cyclase pathways in the submandibular gland.     

Evidence for spare alpha 1-adrenoceptors for the accumulation of inositol phosphates in smooth muscle

The accumulation of inositol phosphates (IP) in smooth muscle from rat vas deferens and caudal artery was maximally increased 3- to 4-fold in response to exposure of the tissues to 100 microM noradrenaline. Clonidine (up to 3 mM) was a partial agonist. Pretreatment of the tissues with the irreversible alpha-adrenoceptor antagonist phenoxybenzamine (0.3-10 microM) shifted the noradrenaline concentration-response curve to the right before depressing the maximum. The maximum of the clonidine concentration-response curve was depressed without significant change in the EC50 by the same treatment. These data, which are most easily interpreted as demonstrating the presence of a receptor reserve for IP accumulation, are discussed.     

Effects of opioid compounds on basal and muscarinic induced accumulation of inositol phosphates in cultured bovine chromaffin cells

The mammalian adrenal medulla expresses a variety of both opioid peptides and opioid receptors. The function of this adrenal opioid system is, however, largely unknown. We have examined the ability of a number of opioid compounds to influence basal and muscarinic stimulated accumulation of inositol phosphates in cultured bovine chromaffin cells. Muscarine produced a dose-dependent 1.5-fold increase in total inositol phosphates. This response was sensitive to atropine inhibition. The ten opioid compounds examined were chosen because between them they possess selectivity for all of the identified opioid receptor subtypes. However, none of these opioids in the concentration range 10nM-10 microM had any significant effect on either basal or muscarinic induced total inositol phosphate accumulation. We conclude that it is unlikely that opioid peptides released from either the chromaffin cells themselves or the splanchnic nerve can modulate the inositol phosphate second messenger system within the adrenal chromaffin cells.     

M3-subtype muscarinic receptor that controls intracellular calcium release and inositol phosphate accumulation in gastric parietal cells.

The muscarinic receptor subtype which triggers acid secretion was investigated in isolated rabbit gastric parietal cells. Cytosolic free Ca2+ concentration ([Ca2+]i), measured with the fluorescent indicator FURA-2, increased rapidly after full agonist (carbachol) stimulation (6-8 sec), then returned to an intermediate sustained value. Other M2-agonists, oxotremorine and arecoline, produced a partial [Ca2+]i increase, whereas M1-agonists, pilocarpine and [4-m-chlorophenylcarbamoyloxyl]-2-butynyl-trimethylammonium, were without any significant effect. [Ca2+]i rise was inhibited by selective muscarinic antagonists: atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than quinuclidinylbenzilate (QNB) greater than pirenzepine greater than 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one, this sequence being characteristic of the involvement of an M3-subtype. This inhibition was shown to be stereoselective; dexetimide and (-)QNB were more potent than levetimide and (+)QNB. The IC50 values for inhibition of [Ca2+]i increase by muscarinic antagonists were in good agreement with those obtained for inhibition of phospholipase C activation. In conclusion, the muscarinic receptor that controls acid secretion appears to be of the M3-subtype and the biochemical events coupled to the activation of this receptor system are also controlled through the same subtype.     

Muscarinic M1 receptors in the lateral septal area mediate cardiovascular responses to cholinergic agonists and bradykinin: supersensitivity induced by chronic treatment with atropine

The infusion of pilocarpine, acetylcholine, bradykinin and the selective M1 muscarinic agonist McNeil-A-343 into the lateral septal area produced a dose-dependent increase of arterial blood pressure and heart rate. The M1 muscarinic agonist carbamylcholine that causes a rise in arterial blood pressure when injected into the anterior lateral ventricles did not produce any cardiovascular effects when infused into the lateral septal area. Chronic treatment with atropine induced supersensitivity to the muscarinic agonists and a significant increase in the number of muscarinic receptors. In this study bradykinin failed to produce any significant change in cardiovascular activity. Pirenzepine, a M1 muscarinic blocking agent, inhibited completely the effect of both muscarinic agonists and bradykinin on cardiovascular activity. In fact, in vitro studies shows that the displacement of the binding of [3H]QNB by pirenzepine is compatible with the presence of the M1 subtype of muscarinic receptor in the lateral septal area, where it may play a major role on cardiovascular regulation.     

M(3) muscarinic receptors mediate contraction of human urinary bladder

Since muscarinic receptors appear to be the physiologically most important control system for urinary bladder contraction, we have characterized the receptor subtype mediating contraction in response to the muscarinic agonist carbachol in the human bladder. Experiments were based on four antagonists, the non-selective atropine, the M(1)-selective pirenzepine, the M(2)-selective methoctramine and the M(3)-selective darifenacin. All antagonists yielded Schild-plots with a slope close to unity. The order of potency (atropine> or =darifenacin>pirenzepine>methoctramine) as well as the estimated antagonist affinities suggested that contraction of the human bladder occurs predominantly if not exclusively via the M(3) receptor.     

Alpha 1-adrenergic receptor subtypes and formation of inositol phosphates in dispersed hepatocytes and renal cells

The ability of alpha 1a- and alpha 1b-adrenergic receptor subtypes to stimulate [3H]inositol phosphate [( 3H]InsP) formation was examined in collagenase-dispersed hepatocytes and renal cells. alpha 1-Adrenergic receptor binding sites were labeled with 125I-BE 2254, and the proportion of alpha 1a and alpha 1b subtypes was determined with chloroethylclonidine (CEC) and WB 4101. Hepatocytes contained only alpha 1b-adrenergic receptors, whereas renal cells had approximately equal proportions of both subtypes. Pretreatment of renal cells with CEC selectively inactivated the alpha 1b subtype, leaving a homogeneous population of alpha 1a receptors. Norepinephrine stimulated [3H]InsP accumulation to a similar extent in both hepatocytes and renal cells. Pretreatment with CEC inactivated this response completely in hepatocytes but only partially in renal cells. WB 4101 was 1000-fold more potent in inhibiting the [3H]InsP response in renal cells than hepatocytes; however, some of this difference was due to rapid metabolism of WB 4101 by hepatocytes. After correction for metabolism, WB 4101 was still 11-fold more potent in inhibiting norepinephrine-stimulated [3H]InsP formation in hepatocytes (alpha 1b) than in CEC-pretreated renal cells (alpha 1a). These results demonstrate that both alpha 1a- and alpha 1b-adrenergic receptor subtypes activate formation of [3H]InsP, although the molecular mechanisms by which these responses occur remain to be determined.     

Modulation of the accumulation of inositol phosphates and the mobilization of calcium in aortic myocytes

In vascular smooth muscle cells the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of C-kinase, inhibited the accumulation of inositol phosphates and the mobilization of calcium produced by several agonists. In the same way, TPA inhibited the fluoride-induced activation of phosphoinositide metabolism. These results suggest a C-kinase action at a post-receptor level. Moreover, the fluoride-induced accumulation of inositol phosphates shows the presence of one or more guanine nucleotide-binding proteins (G-proteins) in the regulation of receptor-phospholipase C coupling. This was confirmed by the use of N-ethylmaleimide and pertussis toxin. These results support the view that, in addition to the induction of sustained contractions, C-kinase can activate negative feedback mechanisms in aortic myocytes.     

M3 muscarinic receptors but not M2 mediate contraction of the porcine detrusor muscle in vitro

1. The objective of the study was to determine the role of muscarinic receptor subtypes in mediating contraction of the porcine detrusor smooth muscle in vitro. 2. Strips of pig detrusor muscle were set up in physiological salt solution and the tensions developed by the tissues were recorded. Responses to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (4-DAMP, methoctramine, darifenacin, oxybutynin, tolterodine and pirenzepine). Antagonist affinity values (pKB values) were calculated and compared with those quoted in the literature for these antagonists at each of the muscarinic receptor subtypes. 3. The M3-selective antagonists, 4-DAMP and darifenacin had high affinities (pKB values of 9.4 and 8.6, respectively). Oxybutynin, tolterodine and pirenzepine had affinities of 8.2, 8.1 and 6.8, respectively, whilst the M2-selective agent methoctramine had a relatively low affinity (pKB = 6.1). The rank order of affinities was, therefore, 4-DAMP > darifenacin > oxybutynin > tolterodine > pirenzepine > methoctramine for the pig detrusor. Correlation of the antagonist affinities obtained on the bladder with those published for these antagonists at the five muscarinic receptor subtypes identified the M3(m3)-receptor as the muscarinic subtype mediating detrusor contractile responses in vitro. 4. These data suggest that a small population of M3-muscarinic receptors must mediate direct contractile responses of the pig detrusor muscle to muscarinic receptor stimulation in vitro.     

Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M2 and M3 subtypes.

The parasympathetic neurotransmitter acetylcholine, acting postsynaptically at the smooth muscle muscarinic receptor, is a principle determinant of colonic motility. In order to elucidate the receptor signal-transduction events responsible for muscarinic receptor-induced contraction of colonic circular smooth muscle, we present here and in the accompanying work studies designed to characterize the muscarinic receptors present in colon and to determine their biochemical coupling. Muscarinic receptor subtypes in canine colonic circular smooth muscle were characterized using radioligand binding techniques. The nonselective muscarinic receptor antagonist radioligand [3H]quinuclidinyl benzilate ([3H]QNB) binds rapidly and reversibly to a single class of saturable sites in colon circular smooth muscle membranes, with an affinity (KD) for the antagonist radioligand of 79.8 +/- 12.6 pM and a density of 123.3 +/- 18.7 fmol/mg of protein. Experiments using membranes prepared from isolated cells purified from the circular smooth muscle layer of canine colon (KD = 102.4 +/- 13.5 pM) confirm the smooth muscle origin of the binding and yield a receptor density of 124,340 receptors/cell. The order of potencies of selective muscarinic receptor antagonists in competition with [3H]QNB for binding to colonic receptors is 4-diphenylacetoxy-N-methylpiperidine methobromide greater than methoctramine greater than AF-DX 116 greater than pirenzepine. Unlike other antagonists tested, pirenzepine competition of [3H]QNB binding is biphasic. The high and low affinities deduced from nonlinear fit of the binding data in colon correlate very well with affinities determined for pirenzepine in mixtures of both submandibular gland (M3) and atrium (M2), indicating the presence of two muscarinic receptor subtypes (82% M2, 18% M3) in colon circular smooth muscle. The muscarinic agonist carbachol binds to both high and low affinity sites in colon, and addition of guanine nucleotide (100 microM GTP gamma S) shifts the agonist competition curve to the right, without eliminating high affinity binding sites. Agonist competition studies with a known ratio of M2 and M3 receptors, obtained by mixing pure M2 and M3 populations, predict the result obtained in colon. cDNA probes specific for each of the muscarinic receptors m1 through m4 were hybridized to colon RNA in a Northern blot analysis. Only m2 and m3 probes hybridized to colon RNA, suggesting the presence of both M2 and M3 receptors. Our data demonstrate that the colon circular smooth muscle contains muscarinic receptors of both the M2 and M3 subtypes, which may be coupled to disparate signal transduction pathways important in the physiological actions of acetylcholine in this tissue.