CELL VOLUME REGULATION AND ENDOTHELIAL FUNCTION

内皮素在血管生理学和病理学中起中心作用， 维持血管完整性和控制血管渗透性， 平滑肌张力 内皮功能受两种因素的调节 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 对机械刺激的了解很少。 反应的早期事件 内皮细胞对机械和神经体液刺激的反应 激活离子转运蛋白，其生理意义是 未知 本实验室发现内皮细胞中的离子转运蛋白 细胞体积的变化也会激活细胞， 来恢复细胞体积 我们相信这些传送者 通过改变细胞体积的机械效应激活， 体积敏感的离子迁移提供了一个方便的研究系统 内皮细胞中的机械转导。 我们建议研究 培养牛主动脉内皮细胞的容量调节离子转运蛋白 细胞来确定它们是如何被调节的。 Na-K-2Cl共转运和 阳离子通道将通过86 Rb通量、离子含量和 膜片钳分析 通过改变调节蛋白质磷酸化 将用蛋白激酶、激酶激活剂 以及分离的细胞骨架和完整细胞中的抑制剂。 的 两种蛋白质的磷酸化特异性地响应细胞 收缩将用于探测体积敏感性激酶。 的 将鉴定Na-K-2Cl协同转运蛋白，并将其磷酸化 使用3 H-布美他尼结合和WGA亲和色谱法测定。 将通过操纵以下物质来检查Ca和Cl离子的调节： 使用离子载体和离子取代， 使用荧光染料测量细胞内水平。 结果 将提供关于内皮细胞如何调节 它们的体积以及它们如何感知和响应机械应力。 了解内皮细胞如何处理机械应力将有助于 提供了重要的洞察血管疾病相关的 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.