PHARMACOLOGIC BASIS OF SMOOTH MUSCLE HETEROGENEITY

平滑肌异质性的药理学基础

• 批准号: 2392585
• 负责人: Julius Allen
• 金额: $ 23.96万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392585
• 关键词: HeLa cells; Sf9 cell line; active sites; antisense nucleic acid; biological signal transduction; dogs; enzyme activity; immunoprecipitation; ion transport; isozymes; muscle contraction; ouabain; phosphatase inhibitor; phosphorylation; protein isoforms; protein kinase A; protein kinase C; sodium potassium exchanging ATPase; vascular smooth muscle

The role of the Na pump in regulating vascular smooth muscle (VSM) contractile function status has been studied for many years. Based on a variety of kinetic differences between VSM and other tissues and Na- independent pump activation by numerous agonists in VSM we have identified at least 2 ways the NaK pump in this tissue can be modulated; 1) ion level activation by intracellular Na+ and 2) protein level activation by phosphorylation of the pump complex itself or adjacent membrane proteins which can modulate pump function. Our hypotheses are that the unique Na+ K+ transport properties of VSM listed above are due to 1) the presence of different alpha and or beta isoforms of the Na+ pump. 2) specific effects of PKA and or PKC mediated phosphorylation of the pump complex. We have identified a truncated form of the alpha1 subunit of the Na+ pump (alpha1T) of molecular weight 66KD (compared to 100 KD) and have expressed it in Sf-9 insect cells. The expressed alpha1T complexes with beta subunit and support ouabain inhibited ATPase activity. Specific Aims 1-2 will determine whether alpha 1T can support pump function alone or whether it modulates transport functions of another isoform: 1) express alpha1T alone or with other isoforms in two mammalian expression systems (HeLa cells and COS-1 cells) and measure Na pump function by Na+ K+ ATPase activity. 86Rb uptake ouabain binding and inhibition and Na by SBFI and 2) utilizing the unique sequence of the alpha1T mRNA, and the 3' untranslated region resulting from alternative splicing, determine if alpha1T can function as a pump by antisense inhibition experiments in vascular smooth muscle cells. Specific aims 3-5 will address the specific kinase- phosphatase control mechanisms of the Na pump. 3) Correlate PKA and PKC supported phosphorylation of the alpha1 and alpha1T subunit with the various activities of the Na pump as above by kinase and phosphatase regulation. 4) Identify the phosphorylation sites on the alpha1 or alpha1T subunit by phosphopeptide analysis. 5) Prevent the effects of PKA and PKC supported phosphorylation of alpha1T by antisense inhibition and substrate competition of PKA and PKC sites and the identified alpha1T site. Competitive peptides will be introduced into the cells under study to be used as false substrates for phosphorylation. These data will define control of ion transport present in VSM and will help delineate the Na pump role in contractile regulation and will identify if the alpha 1T functions are specific for vascular smooth muscle.

NA泵在调节血管平滑肌（VSM）中的作用 收缩功能状态已有很多年了。 基于a VSM与其他组织与NA-之间的动力学差异 VSM中众多激动剂的独立泵激活我们已经确定 至少可以调节该组织中的Nak泵； 1）离子 细胞内Na+和2）蛋白质水平激活的水平激活 泵复合物本身或相邻膜蛋白的磷酸化 可以调节泵功能。 我们的假设是独特的NA+ 上面列出的VSM的K+传输属性是由于1）存在 Na+泵的不同α和 /或β同工型。 2）具体效果 PKA和 /或PKC介导的泵复合物的磷酸化。 我们已经确定了Na+泵的Alpha1亚基的截断形式 （α1T）分子量66kD（与100 kD相比），并已表达 它在SF-9昆虫细胞中。 带有β亚基的表达α1T复合物 支持ouabain抑制ATPase活性。 具体目标1-2将 确定alpha 1t是否可以单独支持泵功能或是否支持泵功能 调节另一种同工型的传输功能：1）Express alpha1t 单独或与两个哺乳动物表达系统中的其他同工型（HELA 细胞和cos-1单元）并测量Na+ K+ ATPase的Na泵功能 活动。 86RB摄取Ouabain结合和抑制作用，而Na则通过SBFI和2） 利用alpha1t mRNA的唯一序列和3'未翻译 替代剪接产生的区域，确定alpha1t是否可以 通过反义抑制实验在血管平滑中充当泵 肌肉细胞。 具体目标3-5将解决特定的激酶 - Na泵的磷酸酶控制机制。 3）关联PKA和PKC 支持α1和α1T亚基的磷酸化 Na泵的各种活性，如激酶和磷酸酶上述 规定。 4）确定α1或 通过磷酸肽分析的α1T亚基。 5）防止PKA的影响 PKC通过反义抑制和 PKA和PKC站点的底物竞争以及已确定的alpha1t位点。 竞争性肽将被引入正在研究的细胞中 用作磷酸化的假底物。 这些数据将定义 控制VSM中存在的离子传输，将有助于描绘Na泵 在收缩法规中的作用，将确定alpha 1T是否功能 是针对血管平滑肌的特异性。