CELL VOLUME REGULATION AND ENDOTHELIAL FUNCTION

内皮细胞在血管生理学和病理学中起着核心作用， 维持血管完整性和调节血管通透性 肌肉发达。血管内皮细胞的功能由两者调节 神经、体液和机械刺激。神经体液刺激发生 通过经典的受体介导的途径，但对 人们对机械刺激知之甚少。回应中的早期事件 内皮细胞对机械刺激和神经体液刺激的反应 激活离子转运体，其生理意义是 未知。本实验室发现内皮细胞中的离子转运体 细胞也被细胞体积的变化激活，并负责 用于恢复细胞体积。我们认为这些传送器是 通过改变细胞体积的机械效应激活，从而 体积敏感离子输运为研究提供了一个方便的系统 血管内皮细胞的机械转导。我们建议研究 牛主动脉内皮细胞容量调节性离子转运体的研究 细胞，以确定它们是如何被调控的。Na-K-2Cl共转运和 阳离子通道将通过86Rb通量、离子含量和 膜片钳分析。蛋白质磷酸化的变化调控 在细胞体积中将用蛋白激酶、激酶激活剂进行检测 以及在分离的细胞骨架和完整细胞中的抑制剂。这个 两种蛋白质对细胞的特异性磷酸化 收缩将被用来探测体积敏感的激酶。这个 Na-K-2Cl共转运蛋白的鉴定及其磷酸化 用~3H-丁美尼特结合和WGA亲和层析法测定。 钙和氯离子的调节将通过操纵 使用离子载体和离子替代的细胞内水平 使用荧光染料测量细胞内水平。结果是 将提供有关内皮细胞如何调控的重要信息 它们的体积以及它们如何感知和响应机械应力。 了解内皮细胞如何应对机械应力将 提供与以下相关的血管疾病的重要洞察力 血管内皮损伤，如动脉粥样硬化。

Endothelium plays a central role in vascular physiology and pathology, maintaining vascular integrity and governing vascular permeability and smooth muscle tone. Endothelial function is regulated by both neurohumoral and mechanical stimuli. Neurohumoral stimulation occurs through classic receptor-mediated pathways, but the response to mechanical stimuli is poorly understood. An early event in the response of endothelial cells to mechanical and neurohumoral stimulation is activation of ion transporters, the physiologic significance of which is unknown. This laboratory has found that ion transporters in endothelial cells are also activated by changes in cell volume and are responsible for restoring cell volume. We believe that these transporters are activated via mechanical effects of changing cell volume, so that volume-sensitive ion transport provides a convenient system for studying mechanotransduction in endothelial cells. We propose to study volume-regulatory ion transporters in cultured bovine aortic endothelial cells to determine how they are regulated. Na-K-2Cl cotransport and cation channels will be measured by 86Rb fluxes, ion contents, and patch-clamp analysis. Regulation of protein phosphorylation by changes in cell volume will be examined with protein kinases, kinase activators and inhibitors in isolated cytoskeletons and intact cells. The phosphorylation of two proteins specifically in response to cell shrinkage will be used to probe for volume-sensitive kinases. The Na-K-2Cl cotransporter will be identified and its phosphorylation determined using 3H-bumetanide binding and WGA affinity chromatography. Regulation by Ca and Cl ions will be examined through manipulation of intracellular levels using ionophores and ion substitutions and through measurements of intracellular levels using fluorescent dyes. The results will provide important information about how endothelial cells regulate their volume and how they sense and respond to mechanical stress. Understanding how endothelial cells deal with mechanical stress will provide important insight into vascular diseases associated with endothelial injury such as atherosclerosis.