Ileal Muscarinic Receptors Research Articles

Binding studies on two functional cardioselective antimuscarinic compounds.

Vecuronium and himbacine are antimuscarinic compounds which in functional studies exhibited a ca 6- and 10-fold higher potency at cardiac muscarinic receptors than at ileal muscarinic receptors. However in binding studies both compounds failed to differentiate between [3H](-)-QNB binding sites in guinea-pig atrial and ileal muscle homogenates. In the latter experiments, the dissociation constants of vecuronium in atria and ileum and that of himbacine in ileum were lower than the values determined functionally. The basis for the lack of cardioselectivity in binding studies is not known. These compounds add to the list of functional cardioselective muscarinic receptor antagonists that failed to display selectivity in binding studies with [3H](-)-QNB.

Characterization of the ileal muscarinic receptor system in 70-week-old type II Goto–Kakizaki diabetic rats; effects of cyclohexenonic long-chain fatty alcohol

As gastrointestinal motility disorders are frequently reported in patients with diabetes, we attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol in type 2 Goto–Kakizaki (GK) diabetic enteropathy. At 40 weeks of age male GK rats divided into three groups (treated with 0, 2 or 8 mg/kg of cyclohexenonic long-chain fatty alcohol; started at the age of 40 weeks). Age-matched male Wistar rats were used in this study. At 70 weeks of age the ileal functions were estimated by organ bath studies using 100 mM KCl and carbachol. The expression levels of muscarinic M 2 and M 3 receptor mRNAs in the ileum were investigated by real-time polymerase chain reaction (PCR). Treatment with cyclohexenonic long-chain fatty alcohol did not alter the diabetic status of the GK rats, i.e., body weight, serum glucose, and serum insulin levels, but significantly ameliorated diabetes-induced hypercontractility of the rat ileum caused by carbachol in a dose-dependent manner. Although there were no significant differences in the expression levels of muscarinic M 3 receptor mRNAs in any of the groups, cyclohexenonic long-chain fatty alcohol reversed the diabetes-induced up-regulation of intestinal muscarinic M 2 receptor mRNAs in treatment groups. These results indicate that cyclohexenonic long-chain fatty alcohol exerts its therapeutic effects on hypercontractility in the ileum of 70-week-old GK type 2 diabetic rats by ameliorating overexpression of muscarinic M 2 receptors.

Participation of nitric oxide synthase and cyclo-oxygenase in the signal transduction pathway of ileal muscarinic acetylcholine receptors

Parasympathetic activation of ileal motility is essential for intestinal physiology. We have previously demonstrated that carbachol activates muscarinic acetylcholine receptors (mAChR) of rat intestine and stimulates ileal motility via phospholipase C. This activation induces phosphoinositide turnover and intracellular calcium mobilization. We show here that carbachol stimulation of rat ileal motility is potentiated by the nitric oxide synthase (NOS) inhibitor NG-monomethyl arginine. Thus, we confirm that carbachol increases, in a dose-dependent manner, the activity of a NOS isoform that depends on calcium–calmodulin binding. Its product, nitric oxide (NO), activates not only guanylyl cyclase, inducing cGMP synthesis, but also cyclo-oxygenase, producing prostaglandin E2. The prostanoid probably cooperates with NO to induce ileal smooth muscle relaxation.

Protein kinase C regulates NO-cGMP pathway in muscarinic receptor activation by HIV+-IgA.

In this work we demostrate that IgA purified from HIV infected patients recognizes a band with a molecular weight corresponding to radiolabelled ileal muscarinic acethylcholine receptors (mAChR) by immunoblotting. HIV+-IgA triggers the signals that are the consequence of mAChR stimulation in the intestine, regulating protein kinase C (PKC) and nitric oxide synthase (NOS) activity as well as 3',5'-cyclic guanosine monophosphate (cGMP) formation. On the one hand PKC activation by HIV+-IgA induces NOS inhibition and as a consequence, low amounts of NO that could improve local immunosuppression in the intestine; on the other hand HIV+-IgA stimulates cGMP production which could potentiate ileal motility and loss of water/electrolytes involved in intestinal damage in AIDS.

Intracellular Signals Coupled to Different Rat Ileal Muscarinic Receptor Subtypes

Taking into account that the activation of different subtypes of ileal muscarinic acetylcholine receptors (mAChR) regulate gut functions such as tone, motility, and electrolyte secretion, we characterized the expression of mAChR in ileal-purified membranes. We also studied intracellular signals triggered by mAChR activation. Binding parameters obtained from saturation assays with the nonselective tritiated muscarinic antagonist, quinuclidynil benzilate ([ 3H]-QNB), were maximal number of binding sites (Bmax): 30 ± 2 fmol/mg prot and dissociation constant (Kd): 0.2 ± 0.03 nM. The competitive inhibition of [ 3H]-QNB specific binding by various nonlabelled muscarinic antagonists was measured and the rank order of potency was: atropine (ATROP) > 4-DAMP > AF-DX 116 > pirenzepine (PZ). The activation of mAChR by carbachol (CARB) increased ileal motility in a concentration-dependent manner (EC 50 2 × 10 −7 M). The antagonists' order of potency to displace dose-response curve of CARB was: ATROP > 4-DAMP > AF-DX116 > PZ. Optimal concentration of CARB on ileal strips increased phosphoinositide turnover and cGMP levels by activating m 3 receptor subtype and decreased isoproterenol (ISO) stimulated levels of cAMP due to M 2 receptor activation. We can conclude that the activation of different mAchR subtypes triggers different intracellular signals that could regulate intestinal tone and motility.

Antagonist discrimination between ganglionic and ileal muscarinic receptors

Theeffects offourantagonists onthedepolarization of isolated superior cervical ganglia andthecontraction of isolated ileal segments oftheratwerecompared. pA2 values estimated fromSchild plots indicated significantly higher affinities ofstercuronium (x100)and pirenzepine (x23)anda significantly loweraffinity of4-diphenylacetoxy-N-methylpiperidine methiodide (x0.39) fortheganglion thanfortheileum. Theaffinities ofN-methylscopolamine forthetwotissues were notsignificantly different. Itisconcluded that thetwo types ofmuscarinic receptor arenotidentical.

Muscarinic M 1 and M 3 receptor antagonist effects of a new pirenzepine analogue in isolated guinea-pig ileal longitudinal muscle-myenteric plexus

The new pirenzepine analogue DF 545 has been tested for its muscarinic M 1 and M 3 receptor antagonist properties in guinea-pig longitudinal muscle-myenteric plexus preparations. McN-A-343-induced inhibition of twitch contractions was taken as a parameter for muscarinic M 1 receptor activation while electrical and acetylcholine-induced contractions were considered as a model for muscarinic M 3 receptor stimulation. An unexpected contractile effect evoked by McN-A-343 was also investigated. In contrast to pirenzepine, DF 545 only weakly counteracted the M 1-mediated McN-A-343 inhibitory effect but blocked M 3-related twitch- or acetycholine-stimulated responses with a 2-fold higher affinity than pirenzepine. Therefore, in this preparation, our findings suggest that DF 545 does not share the selectivity profile exhibited by pirenzepine at ileal muscarinic receptors. Studies on the McN-A-343 contractile effect provide evidence that this agonist may interact with ileal muscarinic effector sites in a different way from other cholinergic agents.

Urea and 2-imidazolidone derivatives of the muscarinic agents oxotremorine and N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide.

Some urea and 2-imidazolidone analogues of the muscarinic agents oxotremorine (1) and N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide [10; BM-5] have been synthesized and assayed for muscarinic and antimuscarinic activity on the isolated guinea pig ileum. The new compounds (15-24) were found to be muscarinic agonists, partial agonists, or antagonists. The compounds were also tested for in vitro receptor binding to homogenates of the rat cerebral cortex using the muscarinic antagonist [3H]-3-quinuclidinyl benzilate ([3H]QNB) as the ligand. They were found to be less potent than 1 in this assay. On the guinea pig ileum, the N-3-methyl substituted imidazolidone analogue 20 was the most potent agonist of the new compounds studied; 20 was 5-fold more potent in inducing contractions of the ileum and had 4-fold higher affinity for ileal muscarinic receptors than the 3-methyl substituted 2-pyrrolidone 6. However, the N-3-unsubstituted urea and imidazolidone derivatives 15 and 19 were several-fold less potent than the parent acetamide N-methyl-N-(4-pyrrolidino-2-butynyl)acetamide [9; UH-5] and 1, respectively. The urea analogue (16) of the partial muscarinic agonist 10 was devoid of intrinsic activity and displayed 3-fold lower affinity than 10 for ileal muscarinic receptors.

Resolved pyrrolidine, piperidine, and perhydroazepine analogs of the muscarinic agent N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide

A series of conformationally restricted analogues of the partial muscarinic agonist N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide (BM 5; 1) was synthesized. Three of the racemic derivatives were resolved into the enantiomers. The compounds were investigated for muscarinic and antimuscarinic activity in the isolated guinea pig ileum. They were found to be fairly potent muscarinic antagonists or weak partial agonists. The new compounds were either equally or less potent than 1 in inhibiting (-)-[3H]-N-methylscopolamine binding in homogenates of the rat cerebral cortex. Thus, structural modifications to 1 in which the amide moiety and the methyl group in the butynyl chain have been joined to form a six- or seven-membered ring preserve affinity but abolish efficacy. The R enantiomers were found to have 14-79 times higher affinity to ileal muscarinic receptors than the respective antipodes. The enantiomeric affinity ratios were nearly identical in both preparations studied. As suggested by molecular mechanics calculations, the difference in affinity between the five-membered and the six- and seven-membered ring analogues may be rationalized in conformational terms.

Synthesis and muscarinic activity of a series of tertiary and quaternary N-substituted guvacine esters structurally related to arecoline and arecaidine propargyl ester.

A series of tertiary and quaternary N-substituted guvacine (1,2,5,6-tetrahydro-3-carboxy-pyridine) methyl and propargyl esters have been synthesized and tested for muscarinic/antimuscarinic activity on rat ileum and electrically paced left atria. Arecoline and arecaidine propargyl ester (APE) as well as their corresponding N-demethyl derivatives, guvacoline (norarecoline) and guvacine propargyl ester, acted as full agonists at both atrial and ileal muscarinic receptors (range of pD2-values 6.09-8.07). However, in both preparations arecoline and APE were clearly more potent (up to 15-fold) than their N-demethyl analogues. Replacement of the N-methyl group in arecoline and APE by larger substituents (ethyl, n-propyl, n-butyl, benzyl, phenylethyl) as well as N-methylation resulted in a decrease or even a complete loss of agonistic activity. In both organs, the propargyl esters usually showed higher potency than the corresponding methyl ester analogues. N-Ethylguvacine propargyl ester and APE methiodide displayed pronounced agonistic activity in the atria (pD2 approximately 6.5; intrinsic activity = 0.79 and 0.67, respectively) but behaved as competitive antagonists in the ileum (pA2 = 6.06 and 5.62, respectively). Beside the lower sensitivity to muscarinic agonists of the rat ileum as compared to rat atria, the cardioselective stimulant action of both agents may also be due to their ability to recognize structural differences between atrial M2 alpha and ileal M2 beta muscarinic receptor subtypes.

Molecular requirements of the recognition site of cholinergic receptors. 27. Enantioselectivity of muscarinic antagonists. 2,2-Dicyclohexyl-5-[(dimethylamino)methyl]-1,3-oxathiolane methiodides and related 3-oxides

The enantiomers of three chiral muscarinic antagonists carrying a 1,3-oxathiolane nucleus were prepared and their absolute configuration established. The enantioselectivity and tissue selectivity of such compounds were studied on rat bladder and guinea pig ileum and heart. The results show that introduction of a sulfoxide function brings about a small but definite enantioselectivity in the 1,3-oxathiolane compound (2), which in itself does not show enantioselectivity among the tissues studied. The results obtained point to differences among cardiac and ileal muscarinic receptors. Comparison of the absolute configuration related agonists shows that the most potent isomers of both series share the same absolute stereochemistry.

Effects of of 545 on different ulcer models in rats

The new pirenzepine analogue DF 545 has been tested for its muscarinic M1 and M3 receptor antagonist properties in guinea-pig longitudinal muscle-myenteric plexus preparations. McN-A-343-induced inhibition of twitch contractions was taken as a parameter for muscarinic M1 receptor activation while electrical and acetylcholine-induced contractions were considered as a model for muscarinic M3 receptor stimulation. An unexpected contractile effect evoked by McN-A-343 was also investigated. In contrast to pirenzepine, DF 545 only weakly counteracted the M1-mediated McN-A-343 inhibitory effect but blocked M3-related twitch- or acetycholine-stimulated responses with a 2-fold higher affinity than pirenzepine. Therefore, in this preparation, our findings suggest that DF 545 does not share the selectivity profile exhibited by pirenzepine at ileal muscarinic receptors. Studies on the McN-A-343 contractile effect provide evidence that this agonist may interact with ileal muscarinic effector sites in a different way from other cholinergic agents.

5-Methyl-2-pyrrolidone analogues of oxotremorine as selective muscarinic agonists.

A series of N-(4-amino-2-butynyl)-5-methyl-2-pyrrolidones modified only in the amino group was synthesized. The compounds were agonists, partial agonists, and antagonists on the isolated guinea pig ileum. They had greater affinity and lower intrinsic efficacy at ileal muscarinic receptors than the identically modified N-(4-amino-2-butynyl)-2-pyrrolidones and N-(4-amino-2-butynyl)succinimides. Dissociation constants in the three series were correlated, suggesting that the compounds had similar mode of binding to muscarinic receptors. The 5-methyl-2-pyrrolidones were 10- to 20-fold less potent as muscarinic agonists on the guinea pig urinary bladder than on the ileum and also elicited lower relative maximal responses on the bladder. For example, the trimethylammonium (9) and azetidino (10) analogues were equipotent (EC50 = 0.2 microM) with the selective muscarinic stimulant N-(1-methyl-4-pyrrolidino-2-butynyl)-N-methylacetamide, BM 5 (2), as agonists on the ileum, but on the bladder 9 and 10 were relatively weak partial agonists, whereas 2 was an antagonist. Compound 10, like 2 and the dimethylamino analogue 8, also differentiated between centrally mediated muscarinic effects in vivo as it was potent in producing analgesia and hypothermia but did not elicit tremor. Instead, 10 antagonized oxotremorine-induced tremor. Thus, 10 resembled 2 in its actions except that the greater intrinsic efficacy of 10 shifted the balance between agonist and antagonist properties slightly toward agonism. Manipulation of intrinsic efficacy by minor changes in chemical structure is emphasized as a means of attaining selectivity.

Derivatives of the muscarinic agent N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide

A series of tertiary and quaternary analogues (acyclic imides, sulfonimides, N-acetyl sulfonamides, and trifluoroacetamides) of the selective partial muscarinic agonist N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide (BM 5,35) was synthesized. The compounds were found to be muscarinic agonists, partial agonists, or antagonists in the isolated guinea pig ileum. Replacement of the acetyl group or the N-methyl group of 35 and its analogues by a methanesulfonyl group abolished efficacy and decreased affinity at ileal muscarinic receptors. Trifluoroacetamide analogues of 35 also had lower affinity and efficacy than 35. Substitution of an acetyl group for the N-methyl group in compounds related to 35 decreased efficacy, but had no appreciable effect on affinity. Most of the tertiary amines showed central antimuscarinic activity as they antagonized oxotremorine-induced tremors in mice.

Methoctramine reveals heterogeneity of M 2 muscarinic receptors in longitudinal ileal smooth muscle membranes

Direct binding studies on longitudinal ileal and atrial muscarinic receptors revealed that most of the ileal or atrial selective antagonists identified in functional studies did not differentiate between these muscarinic receptors in direct binding studies. Methoctramine, an atrial selective muscarinic receptor antagonist in functional studies was, however, able to partially discriminate between these two receptors in our binding studies. Furthermore the binding data obtained using this compound indicated that longitudinal ileal muscarinic receptors were heterogeneous. The predominant population of ileal muscarinic receptors displayed a similar pharmacology to the cardiac type M 2 muscarinic receptor. The minor population of muscarinic receptors identified in binding studies displayed a similar pharmacology to the ileal muscarinic receptor identified in functional studies and were pharmacologically similar to the exocrine gland type M 2 muscarinic receptor.

Direct binding studies on ileal and cardiac muscarinic receptors.

1 Functional studies have indicated that muscarinic receptors in cardiac tissue differ from those in the ileum. In the present study ileal and cardiac muscarinic receptors identified by [3H]-N-methyl scopolamine ([3H]-NMS) were characterized and the selectivity of currently available ileal and atrial selective antagonists determined. 2 In terms of the current functional classification of muscarinic receptors both ileal and cardiac muscarinic receptors were of the M2 subtype based upon their low affinity for pirenzepine. 3 Cyclohexylphenyl(2-piperidinoethyl)silanol (CPPS), a highly ileal selective antagonist in functional studies, was unable to distinguish between ileal and atrial muscarinic receptors identified in binding studies. Furthermore, although AF-DX 116 and dicyclomine were able to differentiate atrial and ileal muscarinic receptors, neither compound was more than 2 fold selective. These data indicate that it is not possible to subclassify ileal and atrial muscarinic receptors using direct ligand binding studies with these antagonists. 4 In circular ileal smooth muscle, apparent heterogeneity of the M2 muscarinic receptor population was observed. Thus AF-DX 116 identified two populations of sites with affinities differing by 30 fold. These two populations of M2 muscarinic receptors may represent the typical M2 muscarinic receptors identified in cardiac tissue and the more recently discovered 'gland type' M2 muscarinic receptors. 5 The circular ileal smooth muscle tissue homogenate was able to decrease dramatically the apparent affinity of adiphenine. This activity, which appeared to result from a phenylmethylsulphonylfluoride (PMSF) sensitive protease effect, should be considered when conducting studies using this tissue preparation and compounds of similar structure to adiphenine.

Competitive and non-competitive antagonism exhibited by 'selective' antagonists at atrial and ileal muscarinic receptor subtypes.

The affinity of a number of 'selective' agonists and antagonists has been assessed at atrial or ileal muscarinic receptors by use of in vitro functional analysis. The most selective compound for ileal muscarinic receptors was silabenzhexol (approx. 50 fold), and to a lesser extent benzhexol (approx. 5 fold). Conversely, the most selective compound for the atrial muscarinic receptors was AF-DX 116 (approx. 6 fold). The novel M1-receptor antagonist, telenzepine and other antagonists such as propantheline and isopropamide did not distinguish between atrial and ileal receptors. Dicyclomine, adiphenine, hexahydroadiphenine and oxyphenonium exhibited competitive antagonism at atrial receptors but non-competitive antagonism at ileal receptors. No conclusions could, therefore, be drawn with regard to their selectivity. The agonists, arecaidine propargyl ester (APE), ethoxyethyltriethylammonium (EOE) and carbachol, exhibited some selectivity in potency but little difference in affinity. It is concluded that the study supports the existence of ileal and atrial muscarinic receptor subtypes. However, the use of dicyclomine and related compounds in receptor classification is limited.

Studies on the Affinity and Selectivity of Tiquizium Bromide (HSR-902), a Novel Spasmolytic Agent, for Muscarinic Receptors

The affinity and selectivity of a novel spasmolytic agent, tiquizium bromide (HSR-902), for muscarinic receptors were studied by the radioligand binding technique using 3H-quinuclidinyl benzilate. The parameters (Ki, nH) of HSR-902 obtained from competition experiments in cerebral cortex and heart muscarinic receptors showed that HSR-902 was an atropine-type, nonselective muscarinic antagonist. The affinity of HSR-902 toward the stomach and ileal muscarinic receptors was about 3–4 times more potent than atropine.

Studies on the affinity and selectivity of tiquizium bromide (HSR-902), a novel spasmolytic agent, for muscarinic receptors.

The affinity and selectivity of a novel spasmolytic agent, tiquizium bromide (HSR-902), for muscarinic receptors were studied by the radioligand binding technique using 3H-quinuclidinyl benzilate. The parameters (Ki, nH) of HSR-902 obtained from competition experiments in cerebral cortex and heart muscarinic receptors showed that HSR-902 was an atropine-type, nonselective muscarinic antagonist. The affinity of HSR-902 toward the stomach and ileal muscarinic receptors was about 3-4 times more potent than atropine.

Dissociation constants and relative efficacies of acetylcholine, (+)- and (-)-methacholine at muscarinic receptors in the guinea-pig ileum.

Dissociation constants (KA) and relative efficacies of acetylcholine, (+)-methacholine and (-)-methacholine at muscarinic receptors in the guinea-pig isolated ileum were determined in the absence and presence of the cholinesterase inhibitor diisopropylfluorophosphate. The method used involved analysis of dose-response data before and after fractional inactivation of receptors with propylbenzilylcholine mustard. The KA values, estimated after cholinesterase inhibition, of acetylcholine, (+)- and (-)-methacholine were 1.7, 2.0 and 620 microM, respectively. The large (730 fold) difference in spasmogenic activity between (+)- and (-)-methacholine is due primarily to a difference in affinity for ileal muscarinic receptors although differences in efficacy (2 to 4 fold) also contribute. It is suggested that the methyl group at the chiral centre of (+)-methacholine has no apparent effect on the binding to muscarinic receptors, whereas the corresponding methyl group of (-)-methacholine interferes with binding, presumably by stabilizing a conformation of the drug which does not fit the receptor very well.