Comparison of the muscarinic receptors in the coronary artery,cerebral artery and atrium of the pig

Summary The affinity of various muscarinic antagonists for the muscarinic receptors mediating contraction (induced by acetyl-\-methylcholine) of the isolated pig coronary and basilar artery was determined in order to compare the muscarinic receptor subtype involved in the contractile response of these arteries. In order to identify the muscarinic receptor subtype(s) involved, the affinity of the antagonists for the M₂ receptor present in the pig atria was also investigated. The following muscarinic antagonists were used: atropine, pirenzepine, AF-DX 116 (11-2{{2-{(diethyl-amino)methyl} -1- piperidinyl}acetyl} - 5, 11- dihydro - 6H - pyrido {2, 3 - b} {, 4}benzodiazepin - 6 - one), 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide), HHSiD (hexahydrosiladifenidol), methoctramine (N, N- bis{6 - {(2 - methoxybenzyl)amino} hexyl} -1, 8 - octane - diamine tetrahydrochloride) and ipratropium.The order of affinity of the antagonists with respect to the muscarinic receptor in the coronary artery was clearly different from that for the muscarinic receptor in the basilar artery. The order of affinity established on the basilar artery closely resembled that for the M2 receptor in the atria.It is concluded that the muscarinic receptors on smooth muscle of the coronary and basilar arteries are not identical. The muscarinic receptor involved in the contraction of the basilar artery adheres to the M2 receptor subtype. A comparison of the selectivity of the antagonists suggests that the muscarinic receptor involved in the contraction of the coronary artery belongs to the M₃ (like in exocrine glands) or M₄ (as found in ileal smooth muscle) receptor subtype.     

Characterization of the muscarinic receptor in human tracheal smooth muscle

Summary Muscarinic receptors in human tracheal smooth muscle were characterized by radioligand binding and functional studies. Specific [³H]-(–)-quinuclidinylbenzilate ([³H]-(–)-QNB) binding to tracheal smooth muscle membranes was reversible, stereoselective and of high affinity (K _d=47±4 pmol/l;R _T=920±120 fmol/g tissue). Inhibition of specific [³H]-(–)-QNB binding by the M-1 selective antagonist pirenzepine was found to occur at relative high concentrations classifying the muscarinic receptor population as belonging to the M-2 subclass.Inhibition of specific [³H]-(–)-QNB binding by muscarinic agonists revealed the presence of high and low affinity sites in nearly equal proportions. 5-Guanylylimidodiphosphate converted high affinity sites into low affinity sites although its effect was minimal. Log dose-contraction curves of methacholine had Hill coefficients of 1.10±0.04 with pD₂-values of 6.75±0.02.Inhibition of specific [³H]-(–)-QNB binding by methacholine, however, was best described by a two binding site model with pK _i-values considerably lower. The difference between these affinity values points to the presence of substantial receptor reserve.     

Characterization of postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle

The present study was designed to characterize the postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle by means of a series of muscarinic agonists and subtype-preferring key muscarinic antagonists. Cumulative addition of muscarinic agonists elicited concentration-dependent contractions with the following rank order of potency (pD₂ values): (+)-muscarine (6.36) ≥ oxotremorine M (6.21) ≥ arecaidine propargyl ester (APE) (6.18) > carbachol (5.68)=(±)-methacholine (5.65) > 4-(4-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride (4-Cl-McN-A-343) (4.28) > 4-(3-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) (3.89). (+)-Muscarine, oxotremorine M, carbachol and (±)-methacholine behaved as full agonists, whereas APE, 4-Cl-McN-A-343 and McN-A-343 displayed partial agonism. The contractile responses of the rat anococcygeus muscle to (±)-methacholine were competitively antagonized by pirenzepine (pA₂=6.92), 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl] 5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one (AQ-RA 741; pA₂=6.75), himbacine (pA₂=7.11), (±)-p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD; pA₂=7.68) and the (R)- and (S)-enantiomers of hexahydro-difenidol [(R)-HHD: pA₂=8.52; (S)-HHD: pA₂=6.06]. A comparison of the pA₂ values derived from studies of contraction in rat anococcygeus muscle with literature binding (pK_i values) and functional affinities (pA₂ values) obtained at native M₁-M₄ receptors strongly suggests that the postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle are of the M₃ subtype. Received: 18 April / Accepted: 18 July 1997     

Muscarinic receptor subtype in porcine coronary artery

The specific binding of (-)-[³H]QNB (quinuclidinyl benzilate) in membrane fractions of porcine coronary artery was saturable, of high affinity and stereoselective. It has been shown that there exist (-)-[³H]QNB binding sites with high (K_i = 12 nM)- and low(K_i = 1010 nM)-affinity for pirenzepine in the coronary artery but predominantly low-affinity sites in cardiac muscle. AF-DX 116 and gallamine showed a lower affinity to ( - )-[³H]QNB binding sites in the coronary artery compared to cardiac muscle. Thus, the present study suggests that porcine coronary artery contains a significant number of muscarinic receptors, probably both M₁ and M₂ subtypes.     

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

Summary The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [³H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [³H]QNB was saturable, reversible and of high affinity (K _D = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M₁), AF-DX 116 (M₂) and 4-DAMP (M₃). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M₃ receptors and a much smaller population (12%) exhibiting characteristics of M₂ receptors. The binding curve for the displacement of [³H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (K _D = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (K _D = 4.4 M). When oxotremorine displacement of [³H]QNB binding was determined in the presence GTPS, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [³²P]NAD caused the [³²P]-labelling of a 40 kD protein that may represent the subunit(s) of G proteins that are known to be NAD-ribosylated by the toxin. We conclude that both M₃ and M₂ receptors may be coupled to G proteins in a pertussis-sensitive manner. Send offprint requests to T. W. Honeyman at the above address     

Effects of muscarinic toxins MT2 and MT7, from green mamba venom, on m1, m3 and m5 muscarinic receptors expressed in Chinese Hamster Ovary cells.

Several small proteins called muscarinic toxins (MTs) have been isolated from venom of green mamba (Dendroaspis angusticeps). They have previously been shown in radioligand binding studies to have high selectivity and affinity for individual muscarinic receptor subtypes, but less is known of their functional effects. This study has examined the actions of two of these MTs, MT2 and MT7, using changes in cytosolic Ca(2+) ([Ca(2+)](i)) measured using the fluorescent indicator fura-2 in Chinese Hamster Ovary (CHO) cells stably transfected with individual muscarinic receptor subtypes, m1, m3 and m5. MT2 activated the m1 receptor: at concentrations above 100 nM it caused significant and concentration-dependent increases in [Ca(2+)](i). From 25 to 800 nM MT2 also produced increases in [Ca(2+)](i) by activating m3 receptors, although these increases in [Ca(2+)](i) were not strictly concentration-dependent with only intermittent responses being recorded (i.e. it was not always possible to obtain a response to the agonist with each application of the compound). MT2 (800-1600 nM) also caused significant increases in [Ca(2+)](i) in CHO cells expressing the m5 muscarinic receptor subtype. MT7 (1 microM) displayed no agonist activity at any of the muscarinic receptors but was a potent non-competitive antagonist (at 20 nM) at the m1 muscarinic receptor subtype. It had no antagonist activity at the m3 or m5 subtypes. These results indicate that MT7 is a highly specific antagonist at the m1 muscarinic receptor subtype as suggested by results from radioligand binding studies. However, MT2 is less selective for the m1 muscarinic receptor than previously described as it also exhibits agonist activity at the m3 and m5 muscarinic receptors, which was not detected in radioligand binding studies.     

Quest for agonist and antagonist selectivity at muscarinic receptors in guinea-pig smooth muscles and cardiac atria

Summary Potencies of 11 muscarinic agonists in eliciting contraction of smooth muscle in guinea-pig ileum, trachea, urinary bladder and uterus and in inhibiting the rate of contractions of cardiac atria were compared. While acetylcholine (ACh) was the most potent agonist on the ileum, uterus and cardiac atria, cis-l(+)-dioxolane was equally as potent as ACh on the ileum and more potent on the urinary bladder and trachea. Compared to ACh, methylfurmethide, oxotremorine, acetoxybut-2-inyl-trimethylammonium and cis-l(+)-dioxolane acted weakly on the atria. Aceclidine, arecoline and acetyl--methylcholine displayed selectivity for the urinary bladder and pilocarpine for the tracheal and urinary bladder smooth muscles. Oxotremorine had very low activity on the uterus. The stereoselectivity of muscarinic ACh receptors (mAChRs) for cis-l(+)- and cis-d(–)-dioxolane was low in the urinary bladder and uterus and high in the ileum and trachea.Most antagonists showed little selectivity between different organs, but S(–)-phenylcyclohexylglycoloyl choline was 6 times more active on the urinary bladder than on the ileum and AF-DX 116 was 12–30 times more active on the atria than on the smooth muscles. Among the N-alkyl derivatives of benzilylcholine, the octyl derivative was 400 times more active on the ileum than on the atria, while among the N-alkyl derivatives of QNB, the N-decyl derivative was 41 times more active on the ileum.The observed differences in the potency of various agonists and their stereoisomers on different smooth muscles cannot be explained by differences in the accessibility of receptors or in receptor reserve. It is suggested that they reflect the heterogeneity of muscarinic receptors in different smooth muscles and differences between smooth muscles regarding the preferential coupling of their mAChRs to different G proteins. The observed selectivity of S(–)-phenylcyclohexylglycoloyl choline suggests a difference between the mAChRs responsible for the contraction of ileal and urinary bladder smooth muscles.     

Characterization of an endothelial 5-hydroxytryptamine (5-HT) receptor mediating relaxation of the porcine coronary artery

Summary The pharmacological properties of the endothelial 5-hydroxytryptamine (5-HT) receptors involved in relaxation of vascular smooth muscle were determined in rings of pig coronary artery contracted with 10 nmol/1 of the thromboxane A₂ receptor agonist 9,11-dideoxy-11,9-epoxy-methano-prostaglandin F₂ (U 46619).(1) In the presence of 10 mol/l ketanserin, relaxation was obtained with: 5-HT (apparent pD₂ value 7.00), 5-carboxamidotryptamine (5-CONH₂-T; 6.42), 5-aminotryptamine (5-NH₂-T; 5.96), 5-methoxytryptamine (5-OCH₃-T; 5.92), tryptamine, 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo(1,2-a)quinoxaline maleate (CGS 12066 A) and 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole succinate (RU 24969). The maximum relaxation obtainable with the agonists was about 40–60% of the U 46619-induced contraction and the concentration-response curves for 5-HT, 5-NH₂-T and 5-OCH₃-T were bell-shaped. The endothelium-dependence of this effect (i. e. the failure to relax the artery in endothelium-denuded preparations) was demonstrated for 5-HT, 5-CONH₂-T, RU 24969, CGS 12066A and tryptamine.(2) 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 4-hydroxytryptamine, quipazine and yohimbine were ineffective in decreasing the tension of arteries with or without endothelium. Ipsapirone elicited full relaxation of U 46619-induced contraction, but this effect was not endothelium-dependent.(3) Metitepine (0.03-1 mol/l), 6-chloro-2-(1-piperazinyl)pyrazine (MK 212; 10 mol/l), methysergide (1 gmol/l) and cyanopindolol (0.1 mol/l) antagonized the relaxing effect of 5-HT in a non-surmountable manner, whereas metergoline (0.1 mol/l), quipazine (10 mol/l), yohimbine (1 mol/l), propranolol (1 mol/l) and (3-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930; 0.1 mol/l) did not. However, spiroxatrine (0.1 mol/l) and mesulergine (10 mol/l) enhanced the 5-HT-induced relaxation. The endothelium-dependent relaxation induced by 5-CONH₂-T was also inhibited by metitepine 1 gmol/l.(4) The 5-HT-induced relaxation was probably mediated by release of an endothelium-derived relaxing factor (EDRF). Gossypol, an inhibitor of EDRF, virtually abolished the 5-HT-induced relaxation while indometacin, an inhibitor of cyclooxygenase and accordingly of PGI₂ formation, did not.In conclusion, the failure of ketanserin and ICS 205–930 to counteract the relaxant effect of 5-HT receptor agonists excludes the involvement of 5-HT₂ and 5-HT₃ receptors, respectively, in the endothelium-dependent relaxation of the porcine coronary artery. The rather high potency of 5-CONH₂-T and the ability of certain 5-HT receptor antagonists, such as metitepine, methysergide and cyanopindolol, to counteract the effect of 5-HT are compatible with a 5-HT₁ character of the endothelial receptor. However, on the basis of the present data, no final classification, in particular with respect to the known 5-HT₁ receptor subtypes, is possible. Classification is also hampered by the bell-shaped character of the concentration-response curves for 5-HT receptor agonists and by their property to produce only partial relaxation. Send offprint requests to M. Gothert at the above address     

Characterization of the interaction of the cervane alkaloid,imperialine, at muscarinic receptors in vitro

Summary The action of the cervane alkaloid, imperialine, has been assessed at M₁, M₂ and M₃ receptors in functional assays and at M₁, M₂, M₃ and putative M₄ sites in binding studies. In functional studies, imperialine acted as a selective surmountable antagonist at M₂ receptors in guinea-pig isolated atria and uterus (–log K_B = 7.7 and 7.4, respectively), in comparison to M₁, receptors in canine isolated saphenous vein (–log K_B = 6.9) or M3 receptors in a range of guinea-pig isolated smooth muscles including ileum, trachea, fundus, seminal vesicle or oesophagus (–log K_B = 6.6–6.8). In rat aorta, the –log K_B value at the M₃ receptor (5.9) was slightly, but significantly, lower.In competition radioligand binding studies, imperialine was also selective toward to M2 sites in rat myocardium (–log K_i = 7.2) with respect to M₁ and M₃ sites (rat cerebral cortex, rat submaxillary gland; –log K_i = 6.1 and 5.7, respectively). However, it did not significantly discriminate between rat cardiac M₂ sites and putative M₄ sites in rabbit lung (–log K_i = 6.9).Imperialine resembles the alkaloid himbacine in terms of its pharmacological profile at muscarinic receptor subtypes in that it acts as an M2 selective antagonist with respect to M₁ or M₃ sites. It may also provide a second, commercially available, antagonist with which to discriminate between M₁ and M₄ receptors. Send offprint requests to R. M. Eglen at the above address     

Determination of muscarinic agonist potencies at M1 and M2 muscarinic receptors in a modified pithed rat preparation

Summary The agonistic potencies of (±)muscarine, (±)cis - 2 - methyl - 5 - [(dimethylamino)methyl] - 1,3 -oxathiolane methiodide (cis-oxathiolane) and its two enantiomers were determined at muscarinic M₁ and M₂ receptors in the pithed rat. In non-pretreated animals, i.v. administration of these agents produced bradycardic effects mediated by cardiac M₂ receptors followed by increases in heart rate mediated by M1 receptors in sympathetic ganglia. As these responses have been shown to partly overlap, “true” M₁ and M₂ potencies were determined after selective blockade of M₁ and M₂ receptors by pirenzepine and methoctramine, respectively. A similar rank order of agonist potencies was obtained at M₁ and M₂ receptors: (+)cis-oxathiolane > (±)cis-oxathiolane > (±)muscarine > (-)cis-oxathiolane. At both receptor subtypes, (+)cis-oxathiolane was considerably more potent (ca. 30-fold) than its corresponding (−) enantiomer indicating that the agonist binding sites of the two receptor subtypes may have similar stereochemical properties. While (±)muscarine showed similar potencies at M₁ and M₂ receptors, racemic cis-oxathiolane and its two enantiomers showed a slight selectivity (3–7 fold) for M₁ receptors indicating the potential usefulness of these compounds in the development of selective M₁ receptor agonists. Send offprint requests to F. Cantalamessa at the above address     

Pharmacological characterization and distribution of muscarinic receptors in human placental syncytiotrophoblast brush-border and basal plasma membranes

Based on the existence of choline acetyltransferase and acetylcholine in human placenta, we have investigated the presence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta. Radioligand binding assay, using [³H]N-methyl-scopolamine as tracer, showed the existence of acetylcholine muscarinic receptors in brush-border (K_d 0.28±0.04 nM; B_max 9.4±1.6 fmol/mg protein) and basal plasma membranes (K_d 0.24±0.05 nM; B_max 34.3±6.3 fmol/mg protein). In order to perform a pharmacological characterization of these receptors, competition binding experiments were carried out using the muscarinic receptor antagonists pirenzepine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6H-pyrido(14)benzodiazepine (AF-DX 116), himbacine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), dicyclomine and hexahydro-sila-difenidol (HHSD). The results obtained showed that the muscarinic receptors in brush-border and basal plasma membranes belong to different subtypes. In brush-border membranes, the receptor found match in terms of affinity for the antagonists with the muscarinic M₁ receptor subtype (K_i pirenzepine, 13.6±8.2 nM; K_i AF-DX 116, 1680±271 nM; K_i himbacine, 212±6.5 nM; K_i 4-DAMP, 1.5±0.4 nM; K_i dicyclomine, 5.1±0.8 nM; K_i HHSD, 34.3±7.3 nM), whereas the receptor in basal plasma membrane seems to be of the muscarinic M₂ receptor subtype (K_i pirenzepine, 202±48 nM; K_i AF-DX 116, 124±60 nM; K_i himbacine, 20.6±4.8 nM; K_i 4-DAMP, 4.5±1.2 nM; K_i dicyclomine, 54.6±22 nM; K_i HHSD, 89.2±15.8 nM). The results obtained show the existence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta with a different distribution pattern in terms of number of receptors and distribution of different subtypes. The functional significance of these findings is as yet unknown, but these receptors probably mediate different functions as they belong to different subtypes and are coupled to different second messengers.     

Effects of phthalazinol on smooth muscle cells of the porcine coronary artery and on neuromuscular junction on the guinea-pig vas deferens

Summary In smooth muscle cells of the porcine coronary artery, phthalazinol (10^–5 M) did not modify the membrane potential and the membrane resistance. At a concentration of 10^–4 M or higher, it hyperpolarized the membrane, reduced the membrane resistance and enhanced the rectifying property of the membrane. At the concentration of 10^–5 M, phthalazinol raised the threshold for the induction of a contraction and suppressed nonselectively the amplitude of the contraction evoked by application of high [K]₀, acetylcholine or electrical depolarization of the membrane. Phthalazinol (10^–5–10^–4 M) did not modify the membrane properties of smooth muscle cells from the guinea-pig vas deferens. However, it suppressed the amplitude of the excitatory junction potentials and the facilitation phenomenon produced by repetitive stimulation at various rates. The action potential recorded from the adrenergic nerves was not affected in the presence of phthalazinol (10^–5 and 10^–4 M). The mean amplitude of the miniature excitatory junction potentials (m.e.j.p.s.) was not affected by treatment with phthalazinol (10^–5–10^–4 M), but the rate of which of m.e.j.p.s. appeared was reduced by phthalazinol (>5×10^–5 M). These results indicate that the vasodilator property of phthalazinol may result from a suppression of Ca-mobilization in both the smooth muscle cells and the adrenergic nerve terminals. The former affects the mechanical response directly and the latter leads to an inhibition of noradrenaline release.     

Characterization of muscarinic receptors mediating release of epithelial derived relaxant factor (EpDRF) in guinea-pig isolated trachea

Summary Muscarinic receptors mediating the release of epithelial derived relaxant factor (EpDRF) have been studied by using both contractions of the guinea-pig tracheal strip (with epithelium intact or denuded) or a coaxial bioassay assembly (rat anococcygeus-recipient; guinea-pig trachea-donor tissue). Indomethacin (1 M/l) and physostigmine (0.1 M/l) were both present throughout the study.In the tracheal strip studies, the potencies and maximal effects of all agonists studied (acetylcholine, arecoline, bethanechol, carbachol, (+)cis-dioxolane, ethoxyethyltrimethylammonium, L-660,863, (±)methacholine and OXA-22) were not affected or were only slightly (but significantly) reduced by removal of the epithelium. The -log K_B for the muscarinic antagonists, atropine, pirenzepine, methoctramine and 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine) were also not affected and the -log K_B values were consistent with M₃ muscarinic receptor function. However, the -log K_B value of para-fluoro-hexahydro-siladifendol (p-F-HHSiD) was significantly (P < 0.05) increased upon epithelial denudation (epithelium intact, 7.1; epithelium removed, 7.6). The coaxial bioassay assembly provided more convincing evidence for release of EpDRF in that all muscarinic agonists studied caused relaxations of a precontracted anococcygeus tissue. These relaxations were observed only in the presence of a tracheal tube possessing an intact epithelium. The rank order of potencies for agonists at receptors mediating EpDRF dependent relaxation were similar to those estimated at receptors causing contraction. These data suggested that a substantial receptor reserve was associated with the receptors mediating both EpDRF release and contraction. The affinities of the muscarinic antagonists (atropine, pirenzepine, methoctramine, p-F-HHSiD, 4-DAMP and gallamine) indicated that M₃ receptors also mediated EpDRF release.It is concluded that EpDRF release in guinea-pig trachea is a general property of muscarinic agonists and that this process is mediated, like the contractile response, by M₃ receptors. Send offprint requests to R. M. Eglen at the above address     

Muscarinic cholinergic receptors in the human right coronary artery: a receptor binding and autoradiographic study

Summary We used a combination of radioreceptor binding and autoradiographic techniques to study the pharmacological characteristics and anatomical localization of [³H]-quinuclidinyl benzilate (QNB) binding sites in the human right coronary artery. The ligand was bound to sections of the human right coronary artery in a manner consistent with the labelling of muscarinic receptors. The addition of pirenzepine or of carbachol to the incubation medium to generate displacement curves was indicative of the presence of M₁ and M₂ receptors in the right coronary artery. Autoradiography showed the localization of M₁ sites primarily in the medial layer of the right coronary artery. M₂ sites were located primarily in the adventitia. No [³H]-QNB binding sites were observed in the endothelium. A possible role of muscarinic receptors in the pathogenesis of coronary vasospasm is discussed.     

二苯氰胂对毒蕈碱性受体的抑制作用

目的研究了日本在我国遗弃的化学武器———二苯氰胂(DC)对离体豚鼠回肠纵肌和毒蕈碱性受体的作用机制,为评估DC的毒理学作用及其对环境的影响提供依据。方法取豚鼠8只,分离豚鼠回肠纵肌16条,分4组,用累积给药法,从低到高分别给予不同浓度的DC,记录离体豚鼠回肠纵肌收缩的幅度,求出DC作用于离体回肠纵肌的EC50值,并通过DC与乙酰胆碱(AChR)的相互作用,研究DC的作用机制。在放射性配体-受体结合实验中,从10只大鼠大脑中提取受体,通过DC与[3H]QNB的竞争结合作用,研究了DC对毒蕈碱性受体的作用。结果DC可抑制离体豚鼠回肠纵肌的收缩作用,其对离体豚鼠回肠纵肌的EC50值为(7.335±2.377)×10-7mol/L,AChR可收缩离体豚鼠回肠,并可反转DC引起离体豚鼠回肠纵肌的舒张作用。在放射性配体-受体结合实验中,DC与毒蕈碱性受体结合的Ki值为(9.19±1.52)nmol/L,IC50值为(16.00±2.65)nmol/L。结论DC可作用于mAChR受体上,初步断定为AChR的拮抗剂。     

Signal transduction pathway of the muscarinic receptors mediating gallbladder contraction

In gallbladder smooth muscle, carbachol interacts with M₃ receptors to mediate contraction. To examine components of the intracellular second messenger system that is coupled to these receptors we have tested whether carbachol stimulates the formation of inositol phosphates (IP) to cause contraction. Guinea pig gallbladder muscle strips were prelabeled with [³H]inositol and were incubated with 0.1 mmol/l carbachol, a concentration causing maximal contraction. [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates and contraction were measured at various times (0–90 s). To examine whether a pertussis toxin-sensitive guanine nucleotide binding protein is coupled to the muscarinic receptors, guinea pigs were pretreated with pertussis toxin (180 g/kg i.v./24 h). The effectiveness of pertussis toxin treatment was determined by measuring [³²P]ADP-ribosylation of a –40/41 kDa protein from gallbladder homogenates. Carbachol caused a significant time-dependent increase in the formation of [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates. The time course of [³H]inositol trisphosphate turnover caused by carbachol was biphasic, and was detectable at 15 s and maximal at 60 s; at 75 s and 90 s formation of [³H]inositol trisphosphates decreased, whereas the time course of carbachol-induced contraction of the gallbladder smooth muscle strips reached a plateau after 90 s. The effects of carbachol on [³H]inositol trisphosphates and on contraction were abolished by atropine. Pretreatment with pertussis toxin resulted in ADP-ribosylation of a 40/41 kDa protein from gallbladder cell membranes but did not affect the concentration-response or time course of carbachol-induced contraction. These results indicate that carbachol-induced contraction of gallbladder smooth muscle cells is accompanied by the activation of inositol phosphate turnover and does not involve a pertussis toxin-sensitive G-protein.This article is based in part on the doctoral thesis of Burkhard Mackensen at the Faculty of Medicine, University of Hamburg, Germany. Some of the results were presented at the meeting of the American Gastroenterological Association (AGA) in San Francisco 1992 (von Schrenck et al. 1992) Correspondence to: T. von Schrenck at the above address     

The subtypes of muscarinic receptors for neurogenic bladder contraction in rats

We evaluated in vivo functional selectivity profiles for muscarinic M(2) and M(3) subtypes of four muscarinic antagonists: Compound A (a novel muscarinic receptor antagonist with M(2)-sparing antagonistic activity), darifenacin, (a muscarinic M(3) receptor antagonist); methoctramine (a muscarinic M(2) receptor antagonist) and tolterodine (a nonselective muscarinic receptor antagonist), and compared the inhibition potency on distention-induced bladder contraction in rats. In an in vivo functional study, Compound A (0.03-10 mg/kg, i.v.) showed antimuscarinic activity with high selectivity for M(3) (salivation) over M(2) (bradycardia) (>100-fold). Darifenacin (0.01-0.3 mg/kg, i.v.) showed only slight selectivity for M(3) over M(2) (3.7-fold). Methoctramine (0.003-1 mg/kg, i.v.) showed the reverse selectivity profile (0.077-fold). Tolterodine (0.003-0.3 mg/kg, i.v.) showed less selectivity (1.2-fold). Compound A at M(3) inhibitory doses (0.1 and 0.3 mg/kg, i.v.) showed inhibition in a distention-induced neurogenic bladder contraction model, and its maximal inhibitory effects were about 60% at an even higher dose (3 mg/kg). Methoctramine at M(2) inhibitory doses (0.03 and 0.1 mg/kg, i.v.) did not significantly affect distention-induced bladder contraction. When tolterodine and darifenacin caused inhibition of distention-induced bladder contraction, its maximal inhibitory effects were similar to that of Compound A. Therefore, these findings suggest that Compound A would be an excellent pharmacological tool to give a better understanding of which subtypes of muscarinic receptors act in bladder function so far, and muscarinic M(3), but not M(2), receptors mainly mediate the cholinergic component of distention-induced bladder contraction.     

Are muscarinic receptors in the central and peripheral nervous system different?

The concentration-dependent binding of atropine-³H to membrane fractions from bovine tracheal muscle, parotid gland and caudate nucleus, measured by equilibrium dialysis, revealed the presence of virtually identical high affinity binding sites in all three tissues. Sch 1000 and Sch 1178, geometrical isomers of N-isopropylatropine bromide with a large potency ratio as antimuscarinics, inhibited atropine binding identically in all three tissues. Differences in properties of muscarinic receptors in these tissues are either non-existent or too small to be detected by the applied techniques.     

Comparative studies of the postjunctional activities of some very potent muscarinic agonists

Summary This study was undertaken to determine the potenties of seven muscarinic agonists (methylfurtrethonium, dioxolane, oxathiolane, carbachol, muscarine, muscarone and oxotremorine) on the postjunctional muscarinic receptors of seven isolated preparations (guinea pig taenia-coli, ileum, jejunum, trachea and atria and rat jejunum and urinary bladder).The results indicate that the rank order of sensitivity of the preparations varies independently of the potency of the agonist used and it is almost the same for all the compounds with the exception of oxotremorine.Muscarone was the most potent compound in all the tissues. Intergroup comparisons in each preparation and the evaluation of the equieffective molar ratios relative to muscarone revealed that carbachol possesses a certain degree of cardioselectivity and oxathiolane, on the other hand, is much less active on the cardiac tissue than on the others.Oxotremorine is a peculiar compound endowed with cardioselectivity.