Characterization of Muscarinic Receptors in Singl Smooth Muscle Cells

对豚鼠内脏肌和酶消化法制备的单个细胞的M受体特性进行了研究。单个平滑肌细胞的制备：用高纯度的胶原酶和木瓜酶联合酶消化，得到对各种激动剂有反应的单个细胞。单个肌肉细胞收缩反应的测定：利用相差显微镜和微型计算机建立了测定单个肌肉细胞收缩反应的方法。单个平滑肌细胞的收缩反应模式：分散的细胞被氨基甲酰胆碱以剂量依赖的方式收缩，表明收缩模式是渐进型的。然而，每个细胞对激动剂的敏感性不同，每个细胞的剂量-反应曲线也不同。这些曲线的叠加变得类似于完整组织的曲线，这表明组织中每个细胞的力量发展会引起组织的收缩。单细胞M受体特性：分离的单细胞在酶消化制备细胞过程中对激动剂的亲和力没有改变。结果表明，内脏肌的M受体由3个亚型组成，每个亚型的pKi分别为7.1、5.8和4.0。磷脂酸在平滑肌收缩中的作用：刺激M受体，激活磷脂酶C和二酰甘油激酶，使细胞内磷脂酸含量增加。酸性刺激细胞内游离钙的释放，从而引起肌肉收缩。

Characteristics of muscarinic receptors in guinea pig visceral muscles and the single cells prepared by enzymatic digestion were investigated.Preparation of single smooth muscle cells : Enzymatic digestion of smooth muscle, with combination of highly purified collagenase and papain, yielded single cells which were responsive to various agonists.Determination of contractile responses of single muscle cells : A procedure for determination of contractile responses of single muscle cells was developed with use of a phase contrast microscope and a microcomputer.Mode of contractile response of single smooth muscle cells : The dispersed cells were contracted by carbachol dose-dependently, indicating that the mode of contraction was graded type. Each cell, however, had different sensitivity to agonists and the dose-response curves were different in each cell. Superimpose of those curves became resembled to that obtained with the intact tissue suggesting that force development of each cell in the tissue evoked contraction of the tissue. Characteristics of the muscarinic receptors in the single cells : Affinity to agonists in the isolated single cells were not altered during preparation of the cells by enzymatic digestions. With use of the single cells, it was revealed that the muscarinic receptors in the visceral muscle were consisted of three subtypes and each pKi was 7.1, 5.8 and 4.0.Role of phosphatidic acids in contraction of smooth muscle : Stimulating the muscarinic receptors, phospholipase C and diacylglycerol Kinase were activated, and content of intracellular phosphatidic acid was increased. The acid stimulated release of intracellular free calcium which induced muscle contraction.