PROTEIN KINASE C AND SMOOTH MUSCLE CONTRACTION

这项研究计划的长期目标是确定 牛颈动脉中存在的蛋白激酶C(S) 和气管平滑肌，并确定该酶的功能(S) 在调节平滑肌收缩中起重要作用。第一个主要目标是 分离和鉴定这两种蛋白激酶C的不同亚型 类型为平滑肌型。首先，通过以下方式分离特定的异构体 标准的层析方法。然后这些酶将被 利用Northern和Western印迹分析对其进行表征。功能 表征将涉及对以下动力学性质的分析 使用天然膜(由内向外的人类囊泡)的异构体 红细胞膜)以确定Ca~(2+)、二酰甘油和 佛波酯和其他脂类对其磷酸化能力的影响 固有的膜蛋白(钙泵)和可溶性底物。这个 组胺和去甲肾上腺素等激动剂对细胞的影响 用细胞分级和细胞分级法检测PKC的分布 还将进行免疫细胞化学。此外， 四种关键蛋白的磷酸化与PKC活性的关系 蛋白质：肌球蛋白轻链、结蛋白、钙调蛋白和低蛋白#4 将检测分子量(20 KDa)的胞浆蛋白。这个 第二个主要目的是确定PKC在平滑肌细胞中的作用 功能。这一努力将涉及三种不同的方法。第一, 在完整组织、阿尔法毒素皮肤肌肉和分离的组织中的研究 肌肉细胞将被用来进一步定义这种关系 PKC活性、Ca~(2+)代谢、蛋白质磷酸化与 收缩。第二，药理和多肽的研究 将进行PKC的蛋白质抑制剂的研究。这项技术 可逆渗透作用将被用来引入这些蛋白质。 和多肽抑制剂进入细胞。三、对影响的研究 将在一项 情况的数量。通过这些结合的方法，人们希望 更清楚地定义PKC在平滑肌收缩中的作用。这个 这项工作的结果应该会为细胞和 平滑肌收缩的分子基础可以为我们指明方向 为了更好地了解平滑肌的变化状态 收缩见于哮喘、高血压和脑血管痉挛。

the long term objectives of this research program are to determine the form(s) of protein kinase C (PKC) which exists in bovine carotid artery and tracheal smooth muscle, and to define the functions of the enzyme(s) in the regulation of smooth muscle contraction. The first major aim is to isolate and characterize the different isoforms of PKC from these two types smooth muscle. First, the specific isoforms will be isolated by standard chromatographic methods. these enzymes will then be characterized by use of Northern and Western blot analysis. functional characterization will involve an analysis of the kinetic properties of the isoforms using a natural membrane (inside-out vesicles of human erythrocyte membranes) to define the effects of Ca2+, diacylglycerol and phorbol esters and other lipids on their ability to phosphorylate an intrinsic membrane protein (the Ca2+ pump) and soluble substrates. The effect of agonists such as histamine and norepinephrine on the cellular distribution of PKC using both cell fractionation and immunocytochemistry will also be carried out. Additionally, the relationship of PKC activity to the phosphorylation of four critical proteins: myosin light chain, desmin, caldesmo and protein #4, a low molecular weight (20 kDa) cytosolic protein, will be examined. The second major aim is to define the role of PKC in smooth muscle cell function. This effort will involve three different approaches. First, studies in intact tissue, alpha-toxin skinned muscle, and isolated muscle cells will be performed to further define the relationship between PKC activity, Ca2+ metabolism, protein phosphorylation and contraction. Second, studies of pharmacologic as well as peptide and protein inhibitors of PKC will be conducted. The technique of reversible permeabilization will be employed to introduce these protein and peptide inhibitors into the cells. Third, studies of the influence of phosphoprotein phosphatase inhibitor will be carried out under a number of circumstances. by these combined approaches, it is hoped to define more clearly the role PKC in smooth muscle contraction. The results of this work should provide new insights into the cellular and molecular basis of smooth muscle contraction which could point the way to a better understanding of the altered states of smooth muscle contraction seen in asthma, hypertension and cerebral vascular spasm.