PROPERTIES OF MUSCARINIC ACETYLCHOLINE RECEPTORS IN HEART CELL-CULTURES
PROPERTIES OF MUSCARINIC ACETYLCHOLINE RECEPTORS IN HEART CELL-CULTURES
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DOI：
10.1073/pnas.75.12.5831
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发表时间：
1978-01-01
影响因子：
11.1
通讯作者：
SMITH, TW
中科院分区：
文献类型：
--
作者：
GALPER, JB;SMITH, TW
乙酰胆碱与完整心脏中的受体结合导致收缩频率（变时效应）和收缩力（离子效应）降低。证明了培养的鸡胚心脏细胞对毒蕈碱激动剂氨甲酰胆碱的变时性反应。该反应在与0.1 mM氨甲酰胆碱孵育3 h后显著降低。为了确定是否激动剂诱导的毒蕈碱受体的改变负责这种减少，孵育的影响与氨甲酰胆碱的结合的3 H-标记的毒蕈碱拮抗剂奎宁环二苯乙酸（QNB）的心脏细胞培养物的匀浆进行了研究。[~ 3 H]QNB结合到鸡胚心脏培养物的匀浆中，其特征在于，并显示出与完整心脏中的毒蕈碱受体相似的性质。结合是特异性的和可饱和的。[3 H]QNB被浓度与其已知效力一致的毒蕈碱激动剂和拮抗剂取代。烟碱拮抗剂D-筒箭毒碱对结合的抑制作用较差。通过毒蕈碱受体的QNB的结合的动力学分析表明，最初的反应通过形成具有1.8 nM的Kd的快速可逆的复合物而进行，该复合物转化为缓慢可逆的形式。心肌细胞培养物中毒蕈碱受体的这些特性与在完整心脏匀浆中观察到的特性惊人地相似。心脏细胞培养物的匀浆结合84 . 6 f[飞摩尔]mol（平均值±。SD）QNB/mg蛋白质。在培养的第4天至第8天，受体的数量保持稳定。用0.1mM氨甲酰胆碱孵育培养物3小时，使QNB结合降低55%，至38 ± 0.5%。5 fmol/mg蛋白质。当首先将细胞培养物均质化，然后与氨甲酰胆碱孵育时，未检测到QNB结合位点的减少。与氨甲酰胆碱孵育导致毒蕈碱结合位点的丧失和对毒蕈碱激动剂的生理反应性降低。
The binding of acetylcholine to receptors in the intact heart causes a decrease in the frequency (chronotropic effect) and force (ionotropic effect) of contraction. A chronotropic response of cultured embryonic chicken heart cells to the muscarinic agonist carbamoylcholine was demonstrated. This response is markedly decreased after a 3 h incubation with 0.1 mM carbamoylcholine. To determine whether agonist-induced alterations in muscarinic receptors were responsible for this decrease, the effects of incubation with carbamoylcholine on the binding of the 3H-labeled muscarinic antagonist quinuclidinyl benzilate (QNB) to homogenates of heart cell cultures were studied. [3H]QNB binding to homogenates of cultures of embryonic hearts of chicks 9 days in ovo was characterized and shown to have properties similar to those of muscarinic receptors in intact hearts. Binding was both specific and saturable. [3H]QNB was displaced by muscarinic agonists and antagonists in concentrations consistent with their known potency. Binding was poorly inhibited by the nicotinic antagonist D-tubocurarine. Kinetic analysis of the binding of QNB by muscarinic receptors showed that initially the reaction proceeds by formation of a rapidly reversible complex with a Kd of 1.8 nM, which is converted to a slowly reversible form. These properties of muscarinic receptors in heart cell cultures are strikingly similar to those observed in homogenates of intact hearts. Homogenates of heart cell cultures bound 84 .+-. 6 f[femtomoles]mol (mean .+-. SD) QNB/mg protein. The number of receptors remained stable from day 4 to day 8 in culture. Incubation of cultures with 0.1 mM carbamoylcholine for 3 h decreased QNB binding by 55%, to 38 .+-. 5 fmol/mg protein. When cell cultures were first homogenized and then incubated with carbamoylcholine, no decrease in QNB binding sites could be detected. Incubation with carbamoylcholine causes loss of muscarinic binding sites and decreased physiologic responsiveness to muscarinic agonists.