Properties of the muscarinic cholinergic receptors in rat atrium

Summary 1. Properties of the muscarinic cholinergic receptor sites in the rat atrial homogenate and microsomal fraction were studied by the use of tritium labelled 3-quinuclidinyl benzilate ([³H]-QNB), a potent muscarinic antagonist. 2. The specific [³H]-QNB binding to the receptor sites displayed saturability, stereospecificity as well as reversibility. 3. The competition studies showed that muscarinic antagonists were more potent than muscarinic agonists. 4. Certain neuromuscular blocking agents, antipsychotics, antiarrhythmics and antihistamines also were capable of interacting with the [³H]-QNB binding sites. However, - and -adrenergic agents, calcium antagonist (D-600) and ionophore (A-23187) failed to show any effect. 5. Analyses by the double reciprocal plot, Hill plot and Scatchard plot of the dependence of the specific [³H]-QNB binding on the concentration of QNB suggested that binding was occurring to a single population of receptor sites in the atrial homogenate or microsomal fraction. Further, there was no evidence of any detectable site to site interactions (positive or negative cooperative type). From the Scatchard plot, the equilibrium dissociation constant (K_D) of 1.1 nM was calculated and the Hill coefficient was close to 1.0 6. Interaction of the muscarinic antagonists with the [³H]-QNB sites showed the Hill coefficients close to 1.0 whereas for the agonists, the coefficients were much less than 1.0 indicating that agonist-receptor site interactions have some different characteristics from those following antagonist-receptor site interaction. 7. The rate and the maximal level of QNB binding to the receptor sites was markedly influenced by the temperature; various cations, on the other hand, displayed no effect either on the association or dissociation of QNB binding. The specific QNB binding exhibited a broad pH optimum from pH 6.0–8.5. 8. Treatment of the membrane fraction (or homogenate) with either phospholipase A or C and with p-chloromercuribenzoate caused significant inhibition of [³H]-QNB binding. 9. The QNB binding site was resistant to tryptic digestion. Even when about 40% of the membrane proteins were removed by the tryptic proteolysis, the [³H]-QNB binding ability of the membrane remained unaffected; in fact, the removal of tryptic proteolytic products by centrifugation markedly increased the specific QNB binding to the membrane.     

Interaction of class III antiarrhythmic drugs with muscarinic M2 and M3 receptors: radioligand binding and functional studies

We have recently reported that class III antiarrhythmic drugs inhibit the muscarinic acetylcholine (ACh) receptor-operated K⁺ current (I _K, ACh) in guinea-pig atrial cells by different molecular mechanisms. The data obtained from the patch-clamp study suggest that d,l-sotalol inhibits I _{K, ACh} by blocking the muscarinic receptors, whereas MS-551 inhibits the K⁺ current by blocking the muscarinic receptors and depressing the function of the K⁺ channel itself and/or the guanine nucleotide-binding protein (G protein). This study was undertaken to determine whether the class III antiarrhythmic drugs d,l-sotalol and MS-551 interact with the muscarinic receptors of cardiac and peripheral tissues. Both drugs inhibited concentration dependently the specific [³H]N-methylscopolamine ([³H]-NMS) binding to membrane preparations obtained from guinea-pig atria and submandibular glands. The competition curves of these drugs for [³H]-NMS binding to glandular membranes were monophasic, suggesting competition with [³H]-NMS at a single site. Although the competition curve of d,l-sotalol for [³H]-NMS binding to atrial membranes was monophasic, that of MS-551 was biphasic and showed high- and low-affinity states of binding. d,l-Sotalol showed slightly, but significantly, higher affinity for cardiac-type muscarinic receptors (M₂) than for glandular-type muscarinic receptors (M₃). The inhibition constant (K _i) for MS-551 in glandular membranes was also slightly greater than the high-affinity K _i value for the drug in atrial membranes. In guinea-pig left atria and ilea, d,l-sotalol shifted the concentration-response curves for the negative inotropic effect and the contracting effect of carbachol in a parallel manner. The slopes of Schild plot were not significantly different from unity, suggesting competitive antagonism, and the pA₂ for d,l-sotalol in left atria was slightly greater than that in ilea. MS-551 also shifted the concentration response curve for the negative inotropic effect of carbachol in atrial preparations to a greater extent than that for the contracting effect in ileal preparations, although MS-551 failed to show a pure competitive antagonism. These results suggest that both d,l-sotalol and MS-551 interact with cardiac M₂ and peripheral M₃ receptors, and that at high concentrations they exert anticholinergic activity in cardiac and peripheral tissues.     

Peripheral blood lymphocyte muscarinic cholinergic receptors in airway hyperresponsiveness: a marker of cholinergic dysfunction?

1 Muscarinic cholinergic receptors were assayed in human peripheral blood lymphocytes of healthy control and airway hyperresponsive subjects using a radioligand binding assay technique and the muscarinic cholinergic receptor antagonist [³H]-quinuclidinyl benzilate (QNB) as a radioligand. Subjects investigated were divided in four different groups based on threshold responses to methacholine inhaled as challenge test. 2 [³H]-QNB was bound to human peripheral blood lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. Dissociation constant (K_d) values of [³H]-QNB binding were similar in the different groups examined, whereas maximum density of binding sites (B_max) was increased in airway hyperresponsive subjects in comparison with healthy controls. 3 The above findings indicate that the density of muscarinic cholinergic receptors is increased in peripheral blood lymphocytes of airway hyperresponsive subjects. 4 This suggests that airway hyperresponsiveness is associated with cholinergic hyperreactivity and is probably a systemic cholinergic dysfunction since it is accompanied by changes in the density of muscarinic cholinergic receptors expressed by peripheral blood lymphocytes.     

Discrimination by N-ethylmaleimide between the chronotropic and inotropic response to muscarinic receptor stimulation in rat atrium

Summary N-ethylmaleimide (NEM) rapidly blocked the negative chronotropic effect of carbachol on rat right atrium. In contrast, NEM did not reduce the negative inotropic response to muscarinic (M) receptor stimulation. Carbachol inhibited the specific binding of [³H]-N-methylscopolamine ([³H]-NMS) to membranes of rat atria as reflected by a shallow inhibition curve. Both guanosine triphosphate (GTP) and NEM shifted the [³H]-NMS inhibition curves of carbachol to the right. Pretreatment of the atrial membranes with NEM abolished the GTP-induced rightward shift. However, when instead of the membranes the intact atria were pre-incubated with NEM, no interaction between NEM and GTP in the membranal preparation was observed. The results indicate that NEM sharply discriminated between the inotropic and chronotropic effects to M-receptor stimulation in rat atria. The inhibitory effect of NEM on the M-receptor-mediated negative chronotropic effect in rat atrium cannot be explained by an interaction of the sulfhydryl reagent with GTP-binding proteins, like N_i or N_o.     

Interaction of antihistaminics with muscarinic receptor (III)

The muscarinic antagonist 1-[benzilic 4,4′-³H]quinuclidinyl benzilate ([³H] QNB) bound to a single class of muscarinic receptors with high affinity in rabbit ileal membranes. The K _D and B _max values for [³H]QNB calculated from analysis of saturation isotherms were 52.5 pM and 154 fmol/mg, respectively. Chlorpheniramine (CHP), histamine H₁ blocker, increased K _D value for [³H]QNB without affecting the binding site concentrations and Hill coefficient. The K _i value of CHP for inhibition of [³H]QNB binding in ileal membranes was 1.44μM and the pseudo-Hill coefficient for CHP was close to unit. In the functional assay carbachol, muscarinic agonist, increased the contractile force of ileum with ED₅₀ value of 0.11μM. CHP caused the rightward shift of the dose-response curve to carbachol. The pA₂ value of CHP determined from Schild analysis of carbachol-induced contraction was 5.77 and the slope was unity indicating competitive antagonism with carbachol. The dissociation constant (K _i ) of CHP obtained in competitive experiments with [³H]QNB was similar to the K _A value (1.69μM) of CHP as inhibitor of carbachol-induced contraction in rabbit ileum. This result suggests that the binding of H₁ blocker, CHP, vs [³H]QNB to muscarinic receptors in ileal membranes represents an interaction with a receptor of physiological relevance.     

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.     

Molecular characterization of muscarinic receptor subtypes in bovine cerebral cortex by radiation inactivation and molecular exclusion h.p.l.c.

Muscarinic receptor subtypes in bovine cerebral cortex were investigated by means of radiation inactivation and molecular exclusion high performance liquid chromatography (h.p.l.c.). The functional molecular size of the muscarinic receptor in situ was determined by the radiation inactivation method. The value for the muscarinic receptor labelled with [3H]-quinuclidinylbenzilate ([3H]-QNB) was 91,000 daltons, while that labelled with [3H]-pirenzepine [( 3H]-PZ) was 157,000 daltons. The muscarinic receptor solubilized with digitonin could be labelled with [3H]-PZ as well as with [3H]-QNB. 3-[(3-Cholamidopropyl)-dimethylammonio] - propane sulphonate (CHAPS) solubilized the muscarinic receptor labelled with [3H]-QNB but not that labelled with [3H]-PZ, in agreement with the low affinity of pirenzepine for inhibiting [3H]-QNB binding in CHAPS-solubilized preparations. The size of the muscarinic receptor in solution was estimated by molecular exclusion h.p.l.c. The digitonin-solubilized muscarinic receptor had a molecular weight of 290,000 and the [3H]-QNB and [3H]-PZ binding activities behaved identically. The CHAPS-solubilized muscarinic receptor labelled with [3H]-QNB was apparently of high molecular weight (greater than 1,000,000 Mr), indicating the formation of aggregates and/or micelles. In the presence of digitonin this form was dissociated into a lower molecular weight species (580,000 Mr). These data indicate that the ligand binding component of the muscarinic receptor species labelled by both [3H]-QNB and [3H]-PZ exists on the same receptor protein, but that the [3H]-PZ binding component in situ is probably coupled to other components in the membrane.     

Pharmacokinetics of anticholinergic drugs and brain muscarinic receptor alterations in streptozotocin diabetic rats

We studied the effects of experimental diabetes on the pharmacokinetics of biperiden (BP) and scopolamine (SP) and brain muscarinic receptor alterations in rats after the injection of streptozotocin (STZ) (60mg kg^?1 i.v.). The serum levels of BP and SP differed significantly between the rats 14 weeks after the STZ treatment and age-matched control rats. The values of total body clearance (CL_tot) of BP and SP were significantly increased by STZ treatment. The values of volume of distribution (V_dss) of SP were slightly increased in the STZ-treated rats, although V_dss of BP was decreased. Because of the high lipophilicity of BP, V_dss of BP may be decreased due to the reduced fat tissue volume caused by STZ treatment. The density of the muscarinic receptors in whole brain was measured by a radioligand receptor binding assay using [³H]-quinuclidinyl-benzylate ([³H]-QNB). The density in the diabetic rats two weeks after the STZ treatment was significantly decreased compared to age-matched control rats. However in the diabetic rats 14 weeks after the STZ treatment, there was no difference in the density of muscarinic receptors. The IC₅₀ of muscarinic antagonist for the binding of [³H]-QNB to the receptor did not change on STZ treatment. Modulation of the receptor following repeated anticholinergic drug exposure was studied. In control rats, the number of muscarinic receptors in the brain increased by 6.9% on chronic treatment with BP for two weeks. When diabetic rats were treated with BP and SP, the number of muscarinic receptors in the brain increased by 9.6% and 33.8%, respectively.     

Correlation between cholinergic histamine release and quinuclidinyl-benzilate ([3H]-QNB) binding in mast cell membranes

Isolated purified rat mast cells release histamine when exposed to acetylcholine according to a different pattern of sensitivity. No correlation was found between the release of histamine evoked by acetylcholine and the high affinity binding of [3H]-quinuclidinyl-benzilate (QNB), a specific cholinergic muscarinic ligand, to rat mast cell membranes, since QNB binding was the same in membrane isolated from cells which were sensitive or insensitive to acetylcholine. In murine neoplastic mast cells, a negative correlation was found between histamine release and [3H]-QNB binding, as no evidence of specific [3H]-QNB binding was present in murine neoplastic mast cell membranes which, accordingly, do not release histamine when exposed to acetylcholine. It is concluded that murine neoplastic mast cells are not provided with muscarinic cholinergic receptors. In rat mast cells, muscarinic cholinergic receptors are always present, but not always coupled with the effector mechanisms triggering the exocytosis.     

Stimulus-evoked release of tritiated monoamines from rat periaqueductal gray slices in vitro and its receptor-mediated modulation

Summary The periaqueductal gray is a brain region of considerable interest. It is innervated by monoamine-containing neurons as well as by a variety of peptidergic fiber systems, and it participates in the regulation of various functions. Virtually nothing is known about monoamine release in the periaqueductal gray and its receptor-mediated modulation. We therefore studied the release of radioactivity from periaqueductal gray slices preloaded with tritriated monoamines, using an in vitro superfusion method.The release of radioactivity from superfused periaqueductal gray slices after preloading of the tissue with [³H]noradrenaline increased upon electrical stimulation in a frequency-dependent manner. The stimulus-evoked release of radioactivity was Ca²⁺-dependent. Clonidine reduced and yohimbine enhanced the release. The inhibition curve for the effect of clonidine was shifted to the right in the presence of 10^–6 M yohimbine. While phenylephrine, isoprenaline, SK&F 38393, quinpirole, carbachol, [Arg⁸]vasopressin, -MSH and ACTH-(1-24), at a concentration of 10^–6 M, did not influence the electrically evoked release of radioactivity, [Leu⁵]enkephalin reduced it. The selective -opioid receptor agonists [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin and [d-Arg²,Lys⁴]-dermorphin-(1–4)-amide reduced the release of radioactivity, whereas the selective opioid receptor agonist [d-Pen²,d-Pen⁵]enkephalin and the selective K opioid receptor agonist U-69593 had no effect. In the presence of naloxone, which by itself had no effect on the release of radioactivity, the effect of [d-Arg²,Lys⁴]dermorphin-(1–4)-amide was abolished. These results show that the release of noradrenaline from periaqueductal gray slices is via a Ca²⁺-dependent. exocytotic process, and that it is modulated through ₂-adrenoceptors as well as via -opioid receptors. Though the overflow of radioactivity from slices preloaded with [3H]dopamine in the presence of desipramine was measurable, there are reasons to assume that we are dealing here with the release of tritiated catecholamines from a population of nerve endings consisting of noradrenergic and dopaminergic terminals.The release of radioactivity from periaqueductal gray slices preloaded with [³H]5-hydroxytryptamine upon elevation of the K⁺ concentration in the superfusion medium was much more pronounced than that induced by electrical stimulation. The K⁺-evoked release of radioactivity was almost completely abolished in the absence of Cat²⁺; showing that the release is via a Ca²⁺-dependent process. 5-Hydrotryptamine reduced the K⁺-evoked release of radioactivity in a concentration-dependent manner.Some of these data were presented at the XIth International Congress of Pharmacology, 1–6 July 1990, Amsterdam, The Netherlands (Eur J Pharmacol 183:408) Send offprint requests to D. H. G. Versteeg at the above address     

Berberine possesses muscarinic agonist-like properties in cultured rodent cardiomyocytes

Berberine, a natural product alkaloid, has been shown to display a wide array of pharmacological effects. Generally, the mechanism of action of each of these effects has not been well described. The aim of the present study is to test the hypothesis that some of berberine's cardiovascular effects are mediated through activation of cardiac M2 muscarinic cholinergic receptors. In our studies, we tested the ability of berberine to alter the contraction rate of cultured neonatal rodent cardiomyocytes. In these spontaneously contracting primary cultured cells, berberine reduced the contraction rate in a manner independent of β-adrenergic receptor blockade but sensitive to pertussis toxin, a Gi/o G protein inhibitor. Muscarinic antagonists completely blocked the effect of berberine on contraction rate of cardiomyocytes, whereas the effect of berberine was not opposed by antagonists to opioid, adenosine or α-adrenergic receptors. Further, berberine bound to muscarinic receptors of adult mouse heart membranes with relatively high affinity (K_i = 5.4 × 10^?6 M) comparable to that of the classic muscarinic agonist, carbachol, and to muscarinic M2 receptors exogenously expressed in HEK 293 cells (K_i = 4.9 × 10^?6 M). Therefore, the findings of the present study suggest that berberine is a muscarinic agonist at M2 receptors, potentially explaining some of its reported cardiovascular effects.     

苯环喹溴铵的毒蕈碱样乙酰胆碱受体亚型选择性研究

目的探讨苯环喹溴铵对毒蕈碱样乙酰胆碱受体(M受体)亚型的选择性。方法以表达单一M受体亚型的CHO细胞株进行细胞与[3H]-QNB的饱和实验,并进行竞争抑制药和[3H]-QNB与M受体亚型的竞争结合实验,计算Kd值和Ki值。通过离体膀胱肌条实验评价苯环喹溴铵干预乙酰胆碱对离体膀胱肌条的收缩作用,计算pA2值。观察苯环喹溴铵对麻醉犬心率、心电图的影响及其对小鼠气管分泌功能的影响。结果苯环喹溴铵对3种细胞的抑制强度为CHOM3>CHOM1>CHOM2,对CHOM3和CHOM1的抑制能力相似,明显高于对CHOM2的抑制能力(P<0.01)。苯环喹溴铵可拮抗乙酰胆碱对离体膀胱肌条的收缩作用,使量效曲线平行右移,PA2值为7.09±s0.06。与溶媒组相比,苯环喹溴铵经鼻给药后对麻醉犬的心率和心电图无明显影响(P>0.05)。苯环喹溴铵大剂量组给药后1h、2h和中剂量组给药后2h能明显减少小鼠气管分泌物的分泌(P<0.01)。结论苯环喹溴铵是一种可选择性作用于M1和M3受体亚型的拮抗药。     

Influence of eicosanoids on serotonin release in the rat brain: inhibition by prostaglandins E1 and E2

Summary Cardiac alpha- and beta-adrenoceptor sensitivities were examined after chronic pretreatment of rats with reserpine. Increases in sensitivity would indicate that the receptor is under the influence of the sympathetic innervation, removal by catecholamine depletion with reserpine of the tonic effect of neurotransmitter release would permit receptor upregulation. The positive inotropic responses of paced left atria and papillary muscles and the positive chronotropic responses of spontaneously beating right atria were recorded. A concentration-response curve to isoprenaline (beta-adrenoceptor-mediated) was followed, in the presence of beta-blockade, by one to methoxamine (alpha-adrenoceptor-mediated). Methoxamine exerted positive inotropy of left atria and papillary muscles, the maxima being 43.2 ± 2.7 and 26.8 ± 4.4% of the isoprenaline maxima. A small positive chronotropy (16.5 ± 5.6% maximum) of right atria occurred. After pretreatment with reserpine (1.0 mg kg^–1 i.p. daily) for 7 days, the three preparations displayed supersensitivity to isoprenaline, revealed as a significant displacement (P < 0.05) of the concentration-response curves to the left of those for control rats. Reserpine pretreatment, however, had no effect on the sensitivity to methoxamine. The increase in beta-adrenoceptor sensitivity to isoprenaline after reserpine pretreatment was accompanied by a significant 41.3% increase (P < 0.05) in the number of [³H]-dihydroalprenolol ([³H]-DHA) binding sites (B _max) in ventricular membranes, although the dissociation constant (_{K D}) was unaffected. There were more alpha-adrenoceptor [³H]-prazosin binding sites in ventricular than atrial membranes. However, there was no difference in K _D or B _max between reserpine-pretreated and control tissues. It is proposed that the increase in beta-adrenoceptor sensitivity and binding sites arises from the depletion-induced loss of neuronal noradrenaline release onto the beta-adrenoceptors which are therefore directly under the influence of the neurotransmitter. The failure of cardiac alpha-adrenoceptors to exhibit supersensitivity and increased numbers suggests that they are not directly affected by the sympathetic innervation. Send offprint requests to K. J. Broadley at the above address     

The oxime HGG-12 as a muscarinic acetylcholine receptor antagonist without intrinsic activity in cardiac membranes

Direct interactions of the bispyridinium oxime HGG-12 with muscarinic acetylcholine receptors were investigated in porcine cardiac atrial membranes. Competition binding experiments using the radiolabeled muscarinic receptor antagonist (³H)QNB revealed specific binding of HGG-12 to muscarinic acetylcholine receptors of porcine atrial membranes with a dissociation constant of 3.8×10^–7 mol/l. Muscarinic acetylcholine receptor-stimulated binding of the radiolabeled GTP analog (³⁵S)GTP[S] to guanine nucleotide binding proteins (G-proteins) was used to study antagonistic and possible agonistic effects of HGG-12 at muscarinic acetylcholine receptors. HGG-12 completely inhibited carbachol- and oxotremorine-stimulated (³⁵S)GTP[S] binding to pertussis toxin sensitive and insensitive G-proteins in a competitive manner. Inhibition constants (K_I) of HGG-12 for blockade of carbachol- and oxotremorine-stimulated GTP[S]-binding (9.7×10^–7 mol/l and 1.7×10^–6 mol/l, respectively) were higher by about a factor of 100 than those of the muscarinic acetylcholine receptor antagonist atropine. In the absence of muscarinic acetylcholine receptor agonists, HGG-12 by itself had no stimulatory effect on (³⁵S)GTP[S] binding in porcine atrial membranes. The results of this study show that the oxime HGG-12 is a competitive antagonist without intrinsic activity at porcine atrial muscarinic acetylcholine receptors. The stimulatory action of HGG-12 on muscarinic acetylcholine receptors which has been described by several authors is, therefore, suggested to be due to partial inhibition of acetylcholinesterase by the oxime rather than to direct agonism at muscarinic acetylcholine receptors.     

Enhancement of δ- but not μ-opiate agonist binding by calcium

Summary Present evidence for distinction of 2 types of opiate receptor sites in rat brain homogenates originates from different relative affinities of morphine-like alkaloids and enkephalins to -or enkephalin and - or morphine-receptor sites. We now report that Ca²⁺ in a physiological dose range (0.5–3 mM) enhances the binding of ³H-enkephalin in hypotonically treated rat brain membranes, whereas specific binding of ³H-morphine-like alkaloids is not affected. Furthermore, the potency of [d-Ala², d-Leu⁵]-enkephalin to inhibit [³H]-diprenorphine and [³H]-ethylketazocine binding increased in the presence of Ca²⁺, whereas an increase in potency of [d-Ala², d-Leu⁵]-enkephalin to inhibit binding of -receptor ligands was not observed. Kinetic analysis revealed that Ca²⁺ decreased the rate of dissociation of [d-Ala², d-Leu⁵]-enkephalin without affecting the rate of association, thereby increasing the affinity. However, in saturation binding studies, performed in diencephalic membranes, in which [d-Ala², d-Leu⁵]-enkephalin binds predominantly to -receptors, Ca²⁺ also increased the binding affinity of [³H]-[d-Ala², d-Leu⁵]-enkephalin. Double reciprocal analysis suggested a mixed competitive-noncompetitive type of inhibition of [d-Ala², d-Leu⁵]-enkephalin binding by dihydromorphine. Thus, the interactions of - and -opiate ligands with -receptors may involve topographically different, but closely related binding sites, located on a single receptor molecule.Abbreviations DADL [d-Ala², d-Leu⁵]-enkephalin - DHM dihydromorphine - met-enkephalin methionine-enkephalin - leu-enkephalin leucine-enkephaline - FK 33-824 [d-Ala², MePhe⁴, Met(O)-ol]-enkephalin - EGTA ethyleneglycol-bis-(-aminoethylether) N, N'-tetraacetic acid - TRIS Tris (hydroxymethyl)-aminomethan     

Kinetic and molecular evidences that human cardiac muscle express non-M2 muscarinic receptor subtypes that are able to interact themselves.

In heart tissue five isoforms of the muscarinic acetylcholine receptor (mAChR) have been identified, designated m1-m5, of which only M1, M2 and M3 have functional evidences for their role in cardiac physiology. The present study was designed to explore the diversity of mAChR subtypes in human hearts and determine whether these subtypes are able to interact themselves. Expression of mRNAs encoding all five subtypes was readily detected by RT-PCR reaction in both atrial (A) and ventricle (V) samples. Immunoblotting, MABA and ELISA with subtype-specific antibodies confirmed the presence of M1, M2, M3, M4 and M5 proteins in membrane preparations from both A and V. Kinetic characterization using [(3)H]-QNB shown: (1) atrium had greater B(max) than did the ventricle, (2) [(3)H]-QNB behave as an allosteric modulator, inducing cooperativity at high and disclosing heterogeneity at low concentrations, (3) heterogenity was observed in pirenzepine, biperiden and tropicamide competition curves, being the high affinity sites compatible with M1 and M4 muscarinic receptor subtypes and (4) methoctramine competition curves in presence of selective muscarinic receptor subtypes antagonist displayed heterogeneity profile still maintaining cooperativity (n(H)>1), indicating muscarinic receptors subtypes are able to form homo- and hetero-oligomers. In conclusion, our results provide molecular and kinetic evidence for the presence of multiple subtypes of mAChR in human hearts, which are able to undergo discrete transitions from a non-cooperative kinetics of non-interacting monomers to a cooperative kinetics of interacting oligomers.     

Effects of hypoxia on atrial muscarinic cholinergic receptors and cardiac parasympathetic responsiveness

Chronic exposure of rats to hypoxia resulted in a lower resting heart rate and a supranormal increase in heart rate in response to parasympathetic blockade by atropine. The density of muscarinic cholinergic receptors labeled by the antagonist [³H]quinuclidinyl benzilate was elevated significantly in the atria of animals kept hypoxic for 2–4 weeks. Chronic hypoxia did not change the affinity of the receptor for [³H]quinuclidinyl benzilate, the weight of the atria, or the amount of protein per atrial pair. Thus, the decrease in resting heart rate may be explained by the increase in the density of atrial muscarinic cholinergic receptors.     

Pharmacological characterization of muscarinic acetylcholine binding sites in human and bovine cerebral microvessels

The aim of this study was to investigate the presence of muscarinic receptors in human brain microvessels (MVs) and capillaries (CAPS) and, further, to pharmacologically characterize these receptors as well as those in bovine cerebral microvascular beds. For this purpose, the binding of [³H]N-methyl scopolamine ([³H]NMS) was assessed in isolated human and bovine cerebral MVs and CAPS and competition studies were performed against [³H]NMS binding with several well characterized muscarinic antagonists. The antagonist cerebrovascular affinity constants (pK _d ) were determined with the computer-fitting software LIGAND and then compared by correlation analyses to their reported affinities (pK_i) at the five cloned muscarinic receptors.The specific binding of [³H]NMS to human and bovine MVs and CAPs was saturable, of high affinity and competitively inhibited by muscarinic antagonists. Heterogeneous populations of muscarinic binding sites were found in the microvascular tissues from both species. In human cortical MVs, the pharmacological binding profile obtained from various muscarinic receptor antagonists was best correlated to that of the cloned m1 (r = 0.95; p < 0.001) and less so m5 (r = 0.77; p = 0.025) receptor subtypes while in bovine MVs, the presence of the m1 subtype was strongly suggested. Cerebrovascular affinities obtained for selected muscarinic antagonists in single preparations of human and bovine CAPs were suggestive of the presence of M₁/m1 and M3/m3 receptor subtypes, and possibly the m5 subtype in bovine CAPs. The detection of M₁/m1, M₃/m3 and possibly m5 muscarinic receptor subtypes in brain microcirculation is consistent with reports where these receptors have been shown to mediate vasoconstriction, vasodilatation, and activation of nitric oxide synthase, respectively. The physiological role of the putative m5 receptor in the cerebrovascular bed is, as yet, unclear.Altogether, these results indicate the presence of specific muscarinic binding sites in human and bovine brain microcirculation and are supportive of a role for these muscarinic receptors in the neurogenic regulation of cortical cerebral blood flow and possibly bloodbrain-barrier functions by acetylcholine.     

Developmental changes in beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in rat ventricular muscles.

1. In an attempt to explain the previous electrophysiological data on the ontogeny of beta-adrenergic and muscarinic cholinergic interactions on cardiac Ca2+ current, biochemical studies were performed on the ontogeny of beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in cardiac muscle of developing rats: 16-20 days old foetuses, 0-20 days old neonates, and 2-3 months old adults. 2. Developmental changes in cardiac beta-adrenoceptors, muscarinic cholinoceptors, and Ca2+ channels were determined with the use of specific radioligands, [3H]-dihydroalprenolol (DNA), [3H]-quinuclidinyl benzilate (QNB), and [3H]-nitrendipine (NTD), respectively. 3. The Bmax value (fmol mg-1 tissue) for [3H]-DNA binding started to increase on post-gestation day 20, reached almost its maximum level on neonatal day 6, kept almost the same level until neonatal day 20, and then decreased slightly to its adult level. 4. The Bmax value (fmol mg-1 tissue) for [3H]-QNB binding started to increase on post-gestation day 16, reached almost its maximum level on neonatal day 0, remained almost constant until neonatal day 15, and then decreased to its adult level. 5. The Bmax value (fmol mg-1 tissue) for [3H]-NTD binding increased with age between post-gestation day 18 and neonatal day 15, stayed almost constant until neonatal day 20, and then decreased to its adult level. 6. The Kd values for [3H]-DHA, [3H]-QNB, and [3H]-NTD bindings remained almost constant during the developmental period examined.(ABSTRACT TRUNCATED AT 250 WORDS)