Integrin-mediated Regulation of Airway Smooth Muscle

项目摘要/摘要 1整合素在气道平滑肌中高表达。我们培育的缺乏这种整合素的小鼠 只有在平滑肌细胞体内有明显的气道高反应性和来自这些细胞的肺切片 小鼠的呼吸道狭窄程度有所增加。我们之前已经证明了9亚单位胞质结构域 直接结合精胺/亚精胺乙酰转移酶(SSAT)，这是分解代谢的限速步骤 这种结合和多胺分解代谢是重要的调节因子 9-1函数。SSAT的药理稳定性也增强了对呼吸道依赖的预防 平滑的肌肉收缩。在当前的应用中，我们将系统地评估9的效果 气道平滑肌对多种收缩激动剂和异丙肾上腺素引起的松弛的反应 使用4个平行实验系统(活体AHR、气管环收缩、肺切片上的气道狭窄 和呼吸道平滑肌细胞的缩短)。我们将利用各种突变和嵌合的结构 9和SSAT，在体内和体外对SSAT基因敲除小鼠、催化失活SSAT突变体和 基因和药物抑制物彻底研究9与SSAT的相互作用有何贡献 对此作出回应。肌动蛋白-肌球蛋白依赖的肌间肌力生成 交叉桥和平行肌动蛋白聚合，我们将系统地评估？9？1对 这些路径中的每一条。因为高阶多胺的两个主要胞浆效应是预防 通过KIR通道的钾外流和脂激酶PIP5K1的激活，我们将特别支付 注意KIR通道和PIP5K1？在这一过程中的作用。拟议的研究将测试整体 假设结扎的SSAT通常通过集中SSAT而起到抑制呼吸道麻醉的作用， 分解多胺，从而抑制钾外流和/或PIP2的产生，导致减少 钙振荡，减少肌动蛋白-肌球蛋白的交叉桥和/或损害肌动蛋白聚合。 这一途径的异常，无论是后天的还是遗传的，都可能导致哮喘等疾病 以增强的呼吸道狭窄为特征。

Project Summary/Abstract The ¿9¿1 integrin is highly expressed in airway smooth muscle. Mice we have generated lacking this integrin only in smooth muscle cells have marked in vivo airway hyperresponsiveness and lung slices from these mice have increased airway narrowing. We have previously shown that the ¿9 subunit cytoplasmic domain directly binds the enzyme spermine/spermidine acetyltransferase (SSAT), the rate limiting step in catabolism of higher order polyamines, and that this association and polyamine catabolism are important modulators of ¿9¿1 function. Pharmacologic stabilization of SSAT also augments ¿9¿1-dependent prevention of airway smooth muscle contraction. In the current application we will systematically evaluate the effects of ¿9¿1 on responses of airway smooth muscle to multiple contractile agonists and to isoproterenol-induced relaxation using 4 parallel experimental systems (in vivo AHR, tracheal ring contraction, airway narrowing in lung slices and shortening of airway smooth muscle cells). We will utilize a variety of mutant and chimeric constructs of ¿9 and SSAT, in vivo and in vitro studies with SSAT knockout mice, catalytically inactive SSAT mutants and genetic and pharmacologic inhibitors to thoroughly examine what contribution interaction of ¿9 with SSAT makes to this response. Force generatioin in smooth muscle depends on calcium-dependent actin-myosin cross-bridging and parallel actin polymerization, and we will systematically evaluate the effects of ¿9¿1 on each of these pathways. Because the two major cytosolic effects of higher order polyamines are prevention of potassium efflux through Kir channels and activation of the lipid kinase, PIP5K1¿, we will pay special attention to the roles of Kir channels and PIP5K1¿ in this process.The proposed studies will test the overall hypothesis that ligated ¿9¿1 normally serves as a brake on airway narowing by concentrating SSAT, catabolizing polyamines and thus inhibiting potassium efflux and/or PIP2 production, resulting in reduced calcium oscillations, decreased actin-myosin cross-bridging and/or impaired actin polymerization. Abnormalities in this pathway, either acquired or genetic, could contribute to diseases such as asthma that are characterized by enhanced airway narrowing.