PROTEIN KINASE C AND SMOOTH MUSCLE CONTRACTION

这项研究计划的长期目标是确定 存在于牛颈动脉中的蛋白激酶C（PKC）形式 和气管平滑肌，并确定酶的功能 平滑肌收缩的调节。 第一个主要目标是 从这两种细胞中分离和鉴定不同的PKC亚型， 平滑肌类型。 首先，将通过以下方法分离特异性同种型： 标准色谱方法。 然后这些酶就会 通过使用北方和Western印迹分析来表征。 功能 表征将涉及分析的动力学性质， 使用天然膜（人的由内而外的囊泡）的同种型 红细胞膜），以确定Ca2+，甘油二酯和 佛波醇酯和其它脂质对它们磷酸化 内源性膜蛋白（Ca2+泵）和可溶性底物。 的 激动剂如组胺和去甲肾上腺素对细胞的作用 使用细胞分级分离和 还将进行免疫细胞化学。 另夕h PKC活性与四种关键蛋白磷酸化的关系 蛋白质：肌球蛋白轻链、结蛋白、钙桥蛋白和蛋白质#4，低 分子量（20 kDa）胞质蛋白，将进行检查。 的 第二个主要目的是确定PKC在平滑肌细胞中的作用， 功能 这项工作将涉及三种不同的方法。 第一、 在完整组织、α毒素皮肤肌肉和分离的 肌肉细胞将被执行，以进一步确定关系 PKC活性、Ca2+代谢、蛋白磷酸化和 收缩。 第二，药理学研究以及肽和 将进行PKC的蛋白抑制剂。 技术 将采用可逆透化来引入这些蛋白质 和肽抑制剂进入细胞。 第三，影响研究 磷蛋白磷酸酶抑制剂将在一个 的情况数。 通过这些综合方法， 更清楚地定义PKC在平滑肌收缩中的作用。 的 这项工作的结果应该提供新的见解细胞和 平滑肌收缩的分子基础， 更好地理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.