REGULATION OF TRACHEAL SMOOTH MUSCLE CONTRACTION

这项拟议的研究的目的是定义 气管平滑肌收缩。流行的模式是一种 激动剂诱导胞浆内游离钙的增加导致激活 肌球蛋白轻链激酶的钙调蛋白依赖酶。这个 由此产生的肌球蛋白轻链(MLC)被认为是 引发和持续收缩的重大事件。最新数据 表明无论是游离钙的增加还是MLC的磷酸化都不是 反对有效。基于对醛固酮激素调节的研究 分泌，以及对血管和气管平滑肌的初步研究， 我们建立了钙信使系统的两相模型。 在持续的细胞反应中发挥作用。一系列的实验是 计划在气管平滑肌上测试该模型，通过分析：1) 卡巴胆碱对聚磷脂酰肌醇周转的影响 肌醇三磷酸和甘油二酯；2)细胞时间进程 和胞浆蛋白的磷酸化对氨甲酸胺的反应；3) 药物的作用：a)增加细胞内游离 CA2(A23187或离子霉素)；b)激活C-激酶(TPA、OAG等 佛波酯)；和/或c)增加质膜钙(Bay K 8644) 收缩反应的时间进程，以及细胞蛋白 4)细胞内游离钙离子浓度的时程变化 对卡巴拉明和这些药物的反应；5)卡巴拉明的作用 和药物组合对C-激酶细胞内分布的影响；以及6) C-激酶的磷酸化蛋白产物对血管活性的影响 “化学剥皮”的平滑肌的收缩反应。一项分析 腺苷环化酶激活剂Forsklin的作用机制， 引起激动剂和药物引起的收缩的松弛 被执行。作为计划研究的一部分，将制定方法以 活性、激动剂反应性分离的血管平滑肌细胞的分离 用于分析细胞的钙代谢，以及细胞的初始速率 CA2内流是对激动剂和药物的反应。实现这些目标 Goals将为研究平滑肌的分子基础提供新的见解 收缩，这将直接关系到临床问题 支气管哮喘和其他状态的气道平滑肌功能障碍。

The aim of the proposed research is to define the biochemical basis of tracheal smooth muscle contraction. The prevailing model is one in which an agonist-induced increase in cytosolic free Ca2+ leads to the activation of the calmodulin-dependent enzyme myosin light chain kinase. The resulting phosphorylation of the myosin light chain (MLC) is thought to be the major event in both initiating and sustaining contraction. Recent data show that neither the increase in free Ca2+ nor in MLC phosphorylation are sustained. Based on studies of the hormonal regulation of aldosterone secretion, and preliminary studies in vascular and tracheal smooth muscle, we have developed a two phase model of the calcium messenger system function during sustained cellular response. A series of experiments are planned to test this model in tracheal smooth muscle by analyzing: 1) the effect of carbachol on the turnover of the polyphosphatidylinositols, inositol triphosphate, and diacylglycerol; 2) the time course of cellular and cytosol protein phosphorylation in response to carbacholamine; 3) the effects of the agents which specifically: a) increase intracellular free Ca2+ (A23187 or ionomycin); b) activate C-kinase (TPA, OAG and other phorbol esters); and/or c) increase plasma membrane Ca2+ (BAY K 8644) on the time course of the contractile response, and cellular protein phosphorylation; 4) the time course change in intracellular free Ca2+ in response to carbacholamine and these drugs; 5) the effect of carbacholamine and drug combinations on the intracellular distribution of C-kinase; and 6) the effects of the phosphoprotein products of the C-kinase enzyme on the contractile responses of 'chemically skinned' smooth muscle. An analysis of the mechanism by which forskolin, an activator of adenylate cyclase, causes a relaxation of agonist-and drug-induced contractions will also be carried out. As part of the planned studies, methods will be developed for the isolation of viable, agonist-responsive isolated smooth muscle cells for use in the analysis of cellular Ca2+ metabolism, and initial rates of Ca2+ influx in response to agonist and drugs. Accomplishment of these goals would provide new insights into the molecular basis of smooth muscle contraction which would be directly relevant to the clinical problems of bronchial asthma and other states of airway smooth muscle dysfunction.