Interaction of neuromuscular blocking drugs with recombinant human m1–m5 muscarinic receptors expressed in Chinese hamster ovary cells
Interaction of neuromuscular blocking drugs with recombinant human m1–m5 muscarinic receptors expressed in Chinese hamster ovary cells
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DOI：
10.1038/sj.bjp.0702166
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发表时间：
1998-11
影响因子：
7.3
通讯作者：
T. Cembala;J. D. Sherwin;M. Tidmarsh;B. Appadu;D. Lambert
中科院分区：
文献类型：
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作者：
T. Cembala;J. D. Sherwin;M. Tidmarsh;B. Appadu;D. Lambert
已知神经肌肉阻断药物（NMBD）会产生心血管副作用，表现为心动过速。在这项研究中，我们研究了一系列甾体NMBD与中国仓鼠卵巢细胞中表达的重组人m1-m5毒蕈碱受体的相互作用。我们的主要假设是NMBD可能与m2（心脏）毒蕈碱受体相互作用。所有的结合研究都是用CHO m1-m5细胞制备的细胞膜进行的，在1 ml体积的20 mMHEPES， 1 mmmgcl2中，pH 7.4, 1小时。用[3H]‐NMS标记毒蕈碱受体，用潘库溴铵、维库溴铵、哌库溴铵、罗库溴铵和胆碱进行置换研究。此外，还包括一系列毒蕈碱受体亚型选择性参比化合物。为了确定任何相互作用的性质，研究了泮库溴铵、罗库溴铵和维库溴铵对福斯克林刺激的CHO m2细胞中甲基胆碱抑制的影响。使用放射受体测定法评估全细胞中环状AMP的形成。所有数据均为平均值±s.e。意思是(它们)。[3H]‐NMS的结合是剂量依赖性的，并且在所有细胞中都是饱和的。m1-m5细胞的bmax和kd值分别为2242±75、165±13、1877±33、458±30、127±2 fmol mg - 1protein和0.11±0.02、0.15±0.01、0.12±0.01、0.12±0.01、0.22±0.01 nm。4 . [3H]‐NMS的结合在CHO m1膜上呈剂量依赖性偏移（pK50）， CHO m2膜上的甲氧曲明（8.55±0.1），CHO m3膜上的4‐二苯基乙酰氧基‐N‐甲基哌啶甲氧胺（4‐DAMP）（9.38±0.03），CHO m4膜上的tropicamide（6.98±0.01）。4‐DAMP、吡renzepine、tropicamide和methoctramine在CHO m5膜上取代[3H]NMS， pk50值分别为9.20±0.14、6.59±0.04、6.89±0.05和7.22±0.01。这些数据证实了CHO m1-m5细胞的同质亚型表达。5[3H]NMS结合被潘库溴铵（m1, 6.43±0.12,m2, 7.68±0.02,m3, 6.53±0.06,m4, 6.56±0.03,m5, 5.79±0.10）、维库溴铵（m1, 6.14±0.04,m2, 6.90±0.05,m3, 6.17±0.04,m4, 7.31±0.02,m5, 6.20±0.07）、哌库溴铵（m1, 6.34±0.11,m2, 6.58±0.03,m3, 5.94±0.01,m4, 6.60±0.06,m5, 4.80±0.03）、罗库溴铵（m1, 5.42±0.01,m2, 5.40±0.02,m3, 4.34±0.02,m4, 5.02±0.04,m5, 5.10±0.03）和胆碱（m1, 6.83±0.05,m2, 7.67±0.04,m3, 6.06±0.06,m4, 6.20±0.03,m1, 6.83±0.05,m2, 7.67±0.04,m3, 6.06±0.06,m4, 6.20±0.03）剂量依赖性取代（pK50）。m5, 5.34±0.03)。6甲基胆碱对CHO m2细胞中环状AMP形成的抑制作用呈剂量依赖性，对照组（pec50）和泮库溴铵（300 nM）分别为6.18±0.34和3.57±0.36。罗库溴铵（1 μM）和维库溴铵（1 μM）不存在和不存在时的甲胆碱剂量-反应曲线无显著差异。泮库溴铵、维库溴铵和罗库溴铵单独不抑制环AMP的形成，表明没有激动剂活性。除罗库溴铵外，所有NMBD均与m2毒蕈碱受体在临床可达到的浓度下显著相互作用，提示与这些药物相关的brady/心动过速可能是与心脏毒蕈碱受体相互作用的结果。此外，在临床上可达到的浓度下，泮库溴铵可拮抗甲基胆碱对CHO m2细胞中环状AMP形成的抑制作用，进一步表明该药物引起的心动过速是由毒蕈碱拮抗引起的。维库溴铵引起心动过缓的机制尚不清楚。英国药理学杂志（1998）125,1088-1094；doi: 10.1038 / sj.bjp.0702166
1Neuromuscular blocking drugs (NMBD's) are known to produce cardiovascular side effects manifesting as brady/tachycardias. In this study we have examined the interaction of a range of steroidal NMBD's with recombinant human m1–m5 muscarinic receptors expressed in Chinese hamster ovary cells. Our main hypothesis is that NMBD's may interact with m2 (cardiac) muscarinic receptors.2All binding studies were performed with cell membranes prepared from CHO m1–m5 cells in 1 ml volumes of 20 mMHEPES, 1 mMMgCl2at pH 7.4 for 1 h. Muscarinic receptors were labelled with [3H]‐NMS and displacement studies were performed with pancuronium, vecuronium, pipecuronium, rocuronium and gallamine. In addition a range of muscarinic receptor subtype selective reference compounds were included. In order to determine the nature of any interaction the effects of pancuronium, rocuronium and vecuronium on methacholine inhibition of forskolin stimulated cyclic AMP formation in CHO m2 cells was examined. Cyclic AMP formation was assessed in whole cells using a radioreceptor assay. All data are mean±s.e.mean (n5).3The binding of [3H]‐NMS was dose‐dependent and saturable in all cells tested. Bmaxand Kdvalues in m1–m5 cells were 2242±75, 165±13, 1877±33, 458±30, 127±2 fmol mg−1protein and 0.11±0.02, 0.15±0.01, 0.12±0.01, 0.12±0.01, 0.22±0.01 nMrespectively.4The binding of [3H]‐NMS was displaced dose dependently (pK50) by pirenzepine in CHO m1 membranes (7.97±0.04), methoctramine in CHO m2 membranes (8.55±0.1), 4‐diphenylacetoxy‐N‐methyl piperidine methiodide (4‐DAMP) in CHO m3 membranes (9.38±0.03), tropicamide in CHO m4 membranes (6.98±0.01). 4‐DAMP, pirenzepine, tropicamide and methoctramine displaced [3H]NMS in CHO m5 membranes with pK50values of 9.20±0.14, 6.59±0.04, 6.89±0.05 and 7.22±0.01 respectively. These data confirm homogenous subtype expression in CHO m1–m5 cells.5[3H]NMS binding was displaced dose‐dependently (pK50) by pancuronium (m1, 6.43±0.12; m2, 7.68±0.02; m3, 6.53±0.06; m4, 6.56±0.03; m5, 5.79±0.10), vecuronium (m1, 6.14±0.04; m2, 6.90±0.05; m3, 6.17±0.04; m4, 7.31±0.02; m5, 6.20±0.07), pipecuronium (m1, 6.34±0.11; m2, 6.58±0.03; m3, 5.94±0.01; m4, 6.60±0.06; m5, 4.80±0.03), rocuronium (m1, 5.42±0.01; m2, 5.40±0.02; m3, 4.34±0.02; m4, 5.02±0.04; m5, 5.10±0.03) and gallamine (m1, 6.83±0.05; m2, 7.67±0.04; m3, 6.06±0.06; m4, 6.20±0.03; m5, 5.34±0.03).6Cyclic AMP formation was inhibited dose dependently by methacholine in CHO m2 cells pEC50for control and pancuronium (300 nM) treated cells were 6.18±0.34 and 3.57±0.36 respectively. Methacholine dose‐response curves in the absence and presence of rocuronium (1 μM) and vecuronium (1 μM) did not differ significantly. Pancuronium, vecuronium and rocuronium did not inhibit cyclic AMP formation alone indicating no agonist activity.7With the exception of rocuronium there was a significant interaction with m2 muscarinic receptors with all NMBD's at clinically achievable concentrations suggesting that the brady/tachycardias associated with these agents may result from an interaction with cardiac muscarinic receptors. Furthermore pancuronium at clinically achievable concentrations antagonised methacholine inhibition of cyclic AMP formation in CHO m2 cells further suggesting that the tachycardia produced by this agent results from muscarinic antagonism. The mechanism of the bradycardia produced by vecuronium is unclear.British Journal of Pharmacology(1998)125, 1088–1094; doi:10.1038/sj.bjp.0702166