Contractile role of M2 and M3 muscarinic receptors in gastrointestinal smooth muscle

Muscarinic agonists elicit contraction through M3 receptors in most isolated preparations of gastrointestinal smooth muscle, and not surprisingly, several investigators have identified M3 receptors in smooth muscle using biochemical, immunological and molecular biological methods. However, these studies have also shown that the M2 receptor outnumbers the M3 by a factor of about four in most instances. In smooth muscle, M3 receptors mediate phosphoinositide hydrolysis and Ca2+ mobilization, whereas M2 receptors mediate an inhibition of cAMP accumulation. The inhibitory effect of the M2 receptor on cAMP levels suggests an indirect role for this receptor; namely, an inhibition of the relaxant action of cAMP-stimulating agents. Such a function has been rigorously demonstrated in an experimental paradigm where gastrointestinal smooth muscle is first incubated with 4-DAMP mustard to inactivate M3 receptors during a Treatment Phase, and subsequently, the contractile activity of muscarinic agonists is characterized during a Test Phase in the presence of histamine and a relaxant agent. When present together, histamine and the relaxant agent (e.g., isoproterenol or forskolin) have no net contractile effect because their actions oppose one another. However, under these conditions, muscarinic agonists elicit a highly potent contractile response through the M2 receptor, presumably by inhibiting the relaxant action of isoproterenol or forskolin on histamine-induced contractions. This contractile response is pertussis toxin-sensitive, unlike the standard contractile response to muscarinic agonists, which is pertussis toxin-insensitive. When measured under standard conditions (i.e., in the absence of histamine and without 4-DAMP mustard-treatment), the contractile response to muscarinic agonists is moderately sensitive to pertussis toxin if isoproterenol or forskolin is present. Also, pertussis toxin-treatment enhances the relaxant action of isoproterenol in the field-stimulated guinea pig ileum. These results demonstrate that endogenous acetylcholine can activate M2 receptors to inhibit the relaxant effects of beta-adrenoceptor activation on M3 receptor-mediated contractions. An operational model for the interaction between M2 and M3 receptors shows that competitive antagonism of the interactive response resembles an M3 profile under most conditions, making it difficult to detect the contribution of the M2 receptor.     

Histamine H3 receptors in the guinea pig ileum: evidence for a postjunctional location.

The effect of the selective histamine H3 receptor agonists (R)alpha-methylhistamine, (R)MHA and immepip (IMM) on intestinal smooth muscle contractility was investigated on isolated cells from the longitudinal muscle of the guinea pig ileum. (R)MHA (10(-13)-10(-8) M) and IMM (10(-13)-10(-8) M) did not significantly modify the basal length of intestinal cells; in contrast both agonists (10(-15)-10(-11) M) prevented the contraction produced by acetylcholine (10(-7) M). The (S)-isomer of alpha-methylhistamine, (S)MHA, was inactive both on basal contractility and on acetylcholine-induced contractions. The relaxant effect of (R)MHA was not modified by famotidine (10(-7) M), but totally prevented by the selective H3 receptor antagonist clobenpropit (10(-8) M), which per se did not modify either basal contractility or the contractile response to acetylcholine. These data indicate that inhibitory histamine H3 receptors are present on smooth muscle cells of the guinea pig ileum and can be activated by very low concentrations of selective agonists. It is not clear, however, whether they can have a functional importance in the regulation of intestinal contractility in an intact system.     

Contractile and relaxant effects of dimaprit on guinea pig isolated intestinal cells

The effect of the histamine H₂ receptor agonist dimaprit on intestinal contractility was characterized on smooth muscle cells isolated from the longitudinal muscle of the guinea pig ileum. Dimaprit exerted two opposite effects on the contractility of isolated muscle cells: relaxation of cholecystokinin octapeptide (CCK-S)-induced contractions in the range of concentrations 10⁻¹⁷-10⁻¹³ M and contraction at concentrations higher than 10⁻¹³ M. The relaxant effect of dimaprit was totally prevented by the H₂ blocker famotidine (10⁻⁷ M), which, at the same time, enhanced the contractile effect of dimaprit, shifting to the left the concentration-response curve to the agonist. This contraction was not modified by the histamine H₁ receptor antagonists pyrilamine and temelastine, tested both at 10⁻⁷ M. By contrast, atropine 10⁻⁸ M abolished the contractile effect of dimaprit, while leaving unchanged the response to CCK-8. Our results clearly indicate that longitudinal muscle cells of the guinea pig ileum possess inhibitory H₂ receptors, which can be activated by very low concentrations of dimaprit; moreover, they revealed that dimaprit can have non-histaminergic effects, probably due to muscarinic receptor activation; however, concentrations about 10000 times higher than those necessary to activate H₂ receptors, are required.     

Affinity profile of the novel muscarinic antagonist, guanylpirenzepine

The study reports the functional affinity of an amidino derivative of pirenzepine, guanylpirenzepine, for muscarinic receptors mediating relaxation of rat duodenum, inhibition of rabbit vas deferens twitch contraction (both receptors previously classified as M1), guinea pig negative inotropism (M2) and ileal contraction (M3). Unlike pirenzepine, guanylpirenzepine discriminated between duodenum and vas deferens receptors, with a 30-fold greater affinity for the former subtype. The unique selectivity pattern of guanylpirenzepine (duodenum greater than vas deferens greater than ileum greater than atrium) renders it a promising tool for the classification of muscarinic receptor subtypes.     

Prejunctional inhibitory muscarinic receptors on cholinergic nerves in human and guinea pig airways

We have investigated whether prejunctional inhibitory muscarinic receptors ("autoreceptors") exist on cholinergic nerves in human airways in vitro and whether guinea pig trachea provides a good model for further pharmacological characterization of these receptors. Pilocarpine was used as a selective agonist and gallamine as a selective antagonist of these autoreceptors. Acetylcholine (ACh) release from postganglionic cholinergic nerves was elicited by electrical field stimulation (EFS) (40 V, 0.5 ms, 32 Hz). In human bronchi, pilocarpine inhibited the contractile response to EFS in a dose-related fashion; the dose inhibiting 50% of the control contraction was 2.2 +/- 0.4 x 10(-7) (SE) M (n = 22), and the inhibition was 96% at 3 x 10(-5) M. The inhibitory effects of pilocarpine were antagonized by gallamine in a dose-related fashion. The results were qualitatively the same in the guinea pig. Gallamine significantly enhanced the contractile response to EFS in the guinea pig, whereas pirenzepine failed to do so, which suggests that M2-receptors are involved. We conclude that prejunctional muscarinic receptors that inhibit ACh release are present on cholinergic nerves in human airways and that guinea pig trachea is a good model for further pharmacological characterization of these receptors, which appear to belong to the M2-subtype.     

The effect of calcium antagonists on contractions of the sensitized and normal guinea pig ileum

The purpose of this study was to learn wether a number of Ca²⁺ antagonists were effective in reducing contractile response of the isolated ileum of the sensitized and normal guinea pig. Contractions of the normal ileum in response to LTD₄, acetylcholine, histamine, and potassium chloride were obtained before and after verapamil, diltiazen and papaverine. Ovalbumin-induced contractions of the ovalbumin-sensitized ileum were obtained in the presence of the three Ca²⁺ antagonists. In the normal ileum, all the Ca²⁺ antagonists were highly effective in diminishing the contractile responses to LTD₄, acetylcholine, histamine and potassium chloride. In the sensitized ileum, ovalbumin-evoked contractions, with subsequent release of a potent contractile mediator (presumably SRS-A), were Ca²⁺-dependent since verapamil, diltiazem and papaverine caused a concentration-related reduction of contractions. Thus, the influx of extracellular Ca²⁺ plays a key role in the contractile responses of the normal and sensitized guinea pig ileum when stimulated by various potent agonists acting on specific receptors or on the cell membrane.     

Isolation, partial purification and pharmacodynamics of a slow contractile substance in the venom sac extract of the wasp Vespa cincta Fabr

The venom of V. cincta contains acetylcholine (ACh), histamine and 5-hydroxytryptamine (5-HT). Blockers of these agonists did not block completely the hypotensive and smooth muscle contractile activity of venom. On smooth muscle, there was a residual slow contraction. The active substance which produced this slow contraction was separated by solvent extraction, gel filtration and TLC. The purified material (which has been provisionally designated "Vecikinin") lowered cat, rat and guinea pig blood pressure, increased amplitude of cardiac contraction, and increased capillary permeability. Vecikinin contracted several smooth muscle preparations (rat uterus, rat ascending colon, guinea pig ileum, guinea pig colon and rat ileum), while relaxing rat duodenum. Its contractile activity was not lost on boiling, but acid or alkali-boiling reduced its contractile activity. It was inactivated on incubation with chymotrypsin and carboxypeptidase but not with trypsin, pepsin or leucine aminopeptidase. It is a peptide, appears to be of low molecular weight, and could be distinguished from substance P, angiotensin, bradykinin and hornet or wasp kinin.     

Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.     

Nicotinic action of anatoxin-a on guinea pig ileum antagonized by thymopentin

(+)-Anatoxin-a (ANTX) stimulated guinea pig ileum contraction with a potency similar to that of acetylcholine (ACh); the stimulation was blocked by tubocurarine, hexamethonium, or atropine. Although the contraction stimulated by ANTX was blocked by atropine, no specific inhibition of the binding of [3H]N-methylscopolamine to ileum membranes was observed in the presence of ANTX. Furthermore, ANTX failed to stimulate the secretion of alpha-amylase from pancreatic acinar cells, a process that is activated by cholinergic agonists at the muscarinic receptors. When the ileum itself was stimulated by ACh, the contraction was not blocked by either hexamethonium or tubocurarine. Preincubation of the ileum with hemicholinium caused a 50% reduction in the ability of ANTX to stimulate contraction. Based upon these data, it was inferred that ANTX binds to postganglionic synaptic nicotinic receptors in the ileum, thus releasing endogenous ACh, which in turn causes ileum contraction by interacting with the postsynaptic muscarinic receptors. It was also observed that thymopentin (TP-5), a pentapeptide corresponding to positions 32-36 of thymopoietin, blocked the stimulation of ileum contraction by ANTX.     

Histaminergic effect of crude papaya latex on isolated guinea pig ileal strips.

In the present study, we investigated the effect of the crude latex of Carica papaya L. (CPX) on isolated guinea pig ileal strips. CPX (0.5-512 microg/ml) caused concentration-dependent contraction of ileal strips suspended in Tyrode solution. The concentration of atropine (0.69 microM) that significantly blocked the contractile effect of acetylcholine on the isolated guinea pig ileum showed no significant effect on CPX- and histamine-induced contractions of the ileal strips. Mepyramine (87.6 nM) significantly blocked the contractile effect of histamine and CPX on the ileum. The same concentration of mepyramine, however, had no significant effect on acetylcholine-induced contraction of the isolated ileal strips. Removal of Ca2+ from the bathing medium abolished ileal contractions induced by acetylcholine, histamine and CPX. All the test substances were able to provoke ileal contractions after replacement of the Ca(2+)-free solution with Tyrode solution. Furthermore, 10(-5) M of nifedipine, a Ca(2+)-entry antagonist, reversibly inhibited the contractile effect of all the test substances on the ileal strips. Results of this study together appear to show that CPX-induced contraction of the isolated guinea pig ileum is mediated via H1-receptors and dependent on extracellular Ca2+ influx.     

Anthroylcholine bromide: a fluorescent ligand for the muscarinic receptor.

The action of anthroylcholine bromide, a new fluorescent probe, has been studied at the cellular (contraction of intestinal muscle) and subcellular levels (binding of 3H-quinuclidinyl benzilate to brain cortex membranes, fluorescence and enzyme activity) with the following results: 1. Anthroylcholine bromide competitively antagonized the contractile effect of acetylcholine in isolated rat duodenum (pA2 = 6.12), but had no effect either on the concentration response curves to histamine or to noradrenaline in isolated guinea pig ileum and rat vas deferens. 2. Anthroylcholine bromide displaced competitively 3H-quinuclidinyl benzilate from brain cortex membranes (Ki = 0.77 mumol/l). 3. Direct binding to the muscarinic site could be measured by exploiting the fluorescence properties of the probe. Binding displaceable by atropine (approximately 20% change in fluorescence) had an apparent affinity constant similar to that found with indirect methods. In contrast, d-tubocurarine did not displace the probe from its site, and atropine- or d-tubocurarine-sensitive binding of anthroylcholine bromide to Torpedo marmorata electric organ membranes, rich in nicotinic receptors, was not observed. These properties suggest the applicability of the probe to study the distribution, structure and/or kinetic properties of the muscarinic receptor.     

Highly selective agonists for substance P receptor subtypes.

The existence of a third tachykinin receptor (SP-N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N-methylation of individual peptide bonds in the C-terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl-[Asp6,Me-Phe8]SP6-11, elicits half-maximal contraction of the guinea pig ileum through the neuronal SP-N receptor at a concentration of 0.5 nM. At least 60,000-fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP-P and SP-E). The action of selective SP-N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP-N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions.     

Effect of L-prolyl-L-leucyl-glycinamide (MIF-I) on some neutrotransmitter-receptor interactions

Some neurotransmitter-receptor interactions have been studied in an attempt to determine how L-prolyl-L-leucyl-glycinamide (MIF-I) exerts its antiparkinson effect. MIF-I affected neither the contractile responses of isolated mouse vas deferens and guinea pig ileum to noradrenaline, acetylcholine, substance P and histamine, nor the inhibitory effects of dopamine and GABA on the rat vas deferens and guinea pig ileum. MIF-I, as well as L-leucine and Pro-Leu, antagonized the contractile response of the ileum to 5-hydroxytryptamine (5-HT). Behavioural tests were used to examine the action of MIF-I on CNS transmitter-receptor interactions. MIF-I did not modify the circling produced by either dopamine agonists in nigro-striatal lesioned rats of 5-HT agonists in rats with a lesion of the medial raphe nucleus. MIF-I affected neither 5-hydroxytryptophan-induced head twitches in mice, which is a measure of 5-HT receptor stimulation, nor striatally-evoked head turning in the rat, which is a model for brain GABA function. It is concluded that MIF-I, at the doses used, does not directly modify the function of any of the CNS transmitter examined. Other possibilities to explain its antiparkinson action are discussed.     

Characterization of ion movements and their relationship to muscarinic receptor binding and excitation-contraction coupling in guinea pig ileal longitudinal smooth muscle

The relationship between ion movements (sodium uptake and potassium release) and agonist-induced contractile responses or muscarinic receptor binding was investigated in the guinea pig ileal longitudinal muscle (GPLM). Sodium uptake and potassium release were agonist-dependent, concentration-dependent, and stereoselective, with the following rank order of maximum ion movement: muscarinic agonists greater than histamine greater than substance P = serotonin. Potassium depolarization did not initiate sodium uptake or potassium release. Sodium uptake was rapid and monophasic, preceding potassium release which was biphasic in nature. Full muscarinic agonists produced equal maximal increases in sodium uptake, while maximal potassium release varied for all muscarinic agonists and in addition differed from sodium uptake in the following ways: time course, stereoselectivity, sensitivity to calcium antagonists, modulation by the guanylyl nucleotide derivative, 5'-guanylylimidodiphosphate (Gpp(NH)p), and inhibition by muscarinic receptor blockade with benzilylcholine mustard. The calcium ionophores A23187 and ionomycin (SQ23377) did not produce any sodium uptake; A23187 but not ionomycin produced potassium release comparable to that evoked by muscarinic agonists. Ion movement in response to combinations of agonists were not additive. Muscarinic agonist binding as measured by competition for [3H]quinuclidinyl benzilate ([3H]QNB) binding, was best described by multiple sites and was regulated by Gpp(NH)p. Excellent correlations were observed between the dissociation constants for binding and sodium uptake, potassium release, and contraction. The best correlations were those between the pharmacologic responses and the high affinity binding site in the absence, and the low affinity site in the presence, of Gpp(NH)p, respectively. Furthermore, the potencies of muscarinic agonists to evoke ion movements and to inhibit [3H]QNB binding were similar, and from one to two orders of magnitude less than those for contraction. It is suggested that contraction and potassium release were mediated by the high affinity, and sodium uptake by the low and average affinity muscarinic agonist binding sites, respectively. These findings suggest an agonist-activated receptor-effector coupling model in GPLM that leads to the activation of sodium uptake, potassium release, and subsequently, contraction.     

Deoxamuscaroneoxime derivatives as useful muscarinic agonists to explore the muscarinic subsite: demox, a modulator of orthosteric and allosteric sites at cardiac muscarinic M2 receptors

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.     

Effect of M2 and M3 muscarinic receptors on airway responsiveness to carbachol in bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

The expression balance of M2 and M3 muscarinic receptor subtypes on the pathogenesis of airway hyperresponsiveness was investigated by using two congenitally related strains of guinea pigs, bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR). CCh-induced airway responses in vivo and in vitro were investigated by comparing the effects of muscarinic receptor subtype antagonists, and the relative amounts of M2 and M3 muscarinic receptor mRNA in tracheal smooth muscle and lung tissue were investigated. After treatment with muscarinic receptor subtype antagonists, the ventilatory mechanics (VT, Raw, and Cdyn) of response to CCh aerosol inhalation were measured by the bodyplethysmograph method. The effects of these antagonists on CCh-induced tracheal smooth muscle contraction were also investigated. The effects of M2 muscarinic receptor blockade were less but the effects of M3 muscarinic receptors blockade on the airway contractile responses were greater in BHS than in BHR. In M3 muscarinic receptor blockades, CCh-induced tracheal contractions in BHS were significantly greater than those in BHR. In tracheal smooth muscle from BHS, the relative amount of M2 muscarinic receptors mRNA was less but that of M3 muscarinic receptor mRNA was more than those in BHR. These results suggest that the high ACh level as a consequence of dysfunction of M2 muscarinic autoreceptors and the excessive effect of M3 muscarinic receptors on the airway smooth muscle may play an important role in the pathogenesis of airway hyperresponsiveness.     

Different mechanisms are responsible for the contractile effects of histaminergic compounds on isolated intestinal smooth muscle cells

The effects of histamine and dimaprit on intestinal smooth muscle contractility were investigated on isolated cells from longitudinal muscle of the guinea pig ileum. Both histamine (10⁻¹⁴–10⁻¹⁰ M) and dimaprit (10⁻¹³–10⁻¹⁰ M) exerted a concentration-dependent contraction of intestinal cells, causing a maximum decrease in cell length of about 20%. This effect was not significantly different from that induced by cholecystokinin-octapeptide (CCK-8) 10⁻⁹ M. The concentration-response curves to histamine and dimaprit were shifted to the left in the presence of the histamine H₂-receptor antagonist famotidine (10⁻⁷ M) indicating the occurrence in the smooth muscle of H₂ receptors mediating relaxation. Whereas the contraction produced by histamine was competitively antagonized by the H₁ receptor antagonist mepyramine (10⁻⁸ M), neither mepyramine (10⁻⁷ M) nor temelastine (10⁻⁷ M) did modify the contractile effect of dimaprit. In contrast, atropine (10⁻⁸ M) significantly depressed the maximum response to dimaprit without affecting that exerted by histamine. These data indicate that histamine and dimaprit can modify intestinal contractility, by acting via different mechanisms; while the contractile action of histamine is related to H₁ receptor activation, that produced by dimaprit involves cholinergic pathways.     

Evidence for the presence of cholinergic muscarinic receptors negatively linked to adenylate cyclase in the iris-ciliary body

Adenylate cyclase activity can be stimulated in the rabbit iris-ciliary body directly by forskolin or through receptor-mediated mechanisms by vasoactive intestinal peptide (VIP) and the β-adrenoreceptor agonists isoproterenol and salbutamol. Increases in the level of c-AMP observed following application of forskolin, isoproterenol and VIP are decreased by carbachol in a dose-dependent manner. The carbachol response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophyline suggesting the involvement of a Gi-protein. Carbachol attenuation of elevated c-AMP levels can be inhibited by the muscarinic antagonist atropine but not by the specific muscarinic receptor antagonist pirenzepine. This is in contrast to carbachol stimulation of inositol phosphate accumulation, where both atropine and pirenzepine inhibit the muscarinic response. Thus there exist two distinct muscarinic receptors in the iris-ciliary body, one linked to adenylate cyclase and the other to the hydrolysis of phosphoinositides.     

Heterogeneity of muscarinic receptors coupled to phosphoinositide breakdown in guinea pig brain and peripheral tissues

Muscarinic receptors coupled to phosphoinositide hydrolysis (PI) are present in guinea pig bladder and colon. Compared to rat cerebral cortex, an extensively studied muscarinic/PI turnover system, all agonists were more potent and efficacious in both bladder and colon. The "M1-selective antagonists", pirenzepine and dicyclomine, were much more potent (Ki = 1-5 nM) and selective (300 to 500-fold) at both rat and guinea pig brain and guinea pig colon receptors, compared to PI-coupled receptors in guinea pig bladder. In contrast, "M2-selective antagonists", AF-DX 116 and HHSiD, were 2-6 fold more potent in bladder than in brain, while HHSiD was very potent in the colon (50 times more potent than in brain). These results suggest a pharmacological heterogeneity of PI-linked muscarinic receptors. If muscarinic receptors with a low affinity for pirenzepine are defined as M2, these results show that the guinea pig bladder contains PI-linked M2 muscarinic receptors, whereas the guinea pig colon contains PI-linked M1 receptors.