Franken C, Tränkle C, Mohr K
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 2000 Feb;361(2):107-12. doi: 10.1007/s002109900176.
Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) are prototype allosteric modulators of the G-protein coupled muscarinic acetylcholine receptor family, especially of the M2-subtype. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H1-receptor antagonist [3H]mepyramine ([3H]MEP) in guinea pig cerebellar homogenates. For the sake of comparison, binding of [3H]N-methylscopolamine ([3H]NMS) at muscarinic M2-receptors was measured in porcine cardiac homogenates under identical conditions. The modulators retarded [3H]NMS dissociation (t1/2 control=1.3 min) concentration-dependently indicating their allosteric action with half-maximum effects for gallamine at EC50,discs=27 microM, for alcuronium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [3H]MEP dissociation from H1-receptors (t1/2,control=2.6 min) remained unchanged up to concentrations of 1 mM of the modulators. Equilibrium binding of [3H]NMS (KD=0.46 nM, Bmax=98 fmol/mg protein) was inhibited by gallamine, elevated by alcuronium and left almost unchanged by W84, indicating negative, positive and nearly neutral cooperativity, respectively, with the radioligand. The ternary complex model of allosteric actions yielded the equilibrium dissociation constants K(A) for the binding of the allosteric modulators to free M2-receptors: K(A,gallamine)=100 nM, K(A,alcuronium)=450 nM, K(A,W84)=69 nM. In H1-receptors, more than 1,000-fold higher concentrations than in M2-receptors were required to elicit an effect on the binding of [3H]MEP (KD=1.2 nM, Bmax=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 microM for W84. In conclusion, the muscarinic modulators have little effect on the histamine H1-receptors.
加拉明、阿库氯铵和W84（己烷-1,6-双[二甲基-3'-邻苯二甲酰亚胺基丙基溴化铵]）是G蛋白偶联毒蕈碱型乙酰胆碱受体家族（尤其是M2亚型）的典型变构调节剂。为了探究毒蕈碱型变构调节的特异性，我们检查了这些药物是否与与毒蕈碱受体具有高度同源性的组胺H1受体相互作用。在豚鼠小脑匀浆中，使用H1受体拮抗剂[3H]美吡拉敏（[3H]MEP）进行结合实验（38 mM Na2HPO4，12 mM KH2PO4，pH 7.5）。为作比较，在相同条件下，在猪心脏匀浆中测量[3H]N-甲基东莨菪碱（[3H]NMS）与毒蕈碱M2受体的结合。这些调节剂浓度依赖性地延缓了[3H]NMS的解离（对照t1/2 = 1.3分钟），表明它们具有变构作用，加拉明的半数最大效应浓度（EC50,diss）为27 microM，阿库氯铵为53 nM，W84为170 nM。相反，直至调节剂浓度达到1 mM时，[3H]MEP从H1受体的解离（t1/2,对照 = 2.6分钟）仍未改变。加拉明抑制了[3H]NMS的平衡结合（KD = 0.46 nM，Bmax = 98 fmol/mg蛋白质），阿库氯铵使其升高，W84几乎未使其改变，分别表明与放射性配体存在负协同性、正协同性和几乎中性的协同性。变构作用的三元复合物模型得出变构调节剂与游离M2受体结合的平衡解离常数K(A)：K(A,加拉明)=100 nM，K(A,阿库氯铵)=450 nM，K(A,W84)=69 nM。在H1受体中，要对[3H]MEP的结合产生影响（KD = 1.2 nM，Bmax = 205 fmol/mg蛋白质），所需浓度比在M2受体中高1000多倍。加拉明在10 mM、阿库氯铵在1 mM、W84在92 microM时观察到半数最大降低。总之，毒蕈碱型调节剂对组胺H1受体几乎没有影响。
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