Endothelial Na-K-Cl cotransport regulation by tonicity and hormones: phosphorylation of cotransport protein.
Endothelial Na-K-Cl cotransport regulation by tonicity and hormones: phosphorylation of cotransport protein.
复制标题
通过张力和激素调节内皮 Na-K-Cl 共转运：共转运蛋白的磷酸化。
DOI：
10.1152/ajpcell.1995.269.6.c1513
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Sun,D
中科院分区：
文献类型：
--
作者：
O'Donnell,ME;Martinez,A;Sun,D
血管内皮细胞的钠-钾-氯共转运系统在维持和调节细胞内容积方面起着重要作用。协同转运蛋白的活性受激素和细胞外张力的调节。加压素和其他刺激内皮协同转运蛋白的激素通过Ca和钙调蛋白依赖性途径起作用。然而，关于介导细胞收缩诱导的共转运活性刺激的机制知之甚少。在本研究中，我们评估了细胞收缩刺激的Na-K-Cl共转运活性和培养的牛主动脉内皮细胞的细胞体积恢复的钙依赖性，以及蛋白激酶和磷酸酶抑制剂对这些过程的影响。此外，研究的可能性，激素和/或高渗调节内皮细胞钠钾氯共转运通过直接磷酸化的共转运蛋白，我们采用了单克隆抗体的人结肠T84上皮细胞钠钾氯共转运蛋白（T4抗体）的Western印迹分析和免疫沉淀的磷蛋白。我们的研究表明，细胞收缩刺激的净钾吸收和细胞内容积的恢复都是钙依赖的。我们还发现，高渗诱导的共转运活性的刺激被几种钙和钙调蛋白依赖性蛋白激酶的抑制剂所阻断。此外，肌球蛋白轻链激酶的抑制剂阻断细胞收缩刺激的共转运和细胞内容积的恢复，而对加压素刺激的共转运没有影响。牛主动脉和脑微血管内皮细胞膜制剂的Western印迹分析显示，T4抗体识别的170-kDa蛋白。此外，我们发现高渗性诱导内皮共转运蛋白磷酸化显著增加，加压素、缓激肽、冈田酸和calyculin A也是如此。我们的研究结果表明，高渗性和刺激性激素对内皮细胞钠钾氯共转运活性的调节通过涉及钙和钙调蛋白依赖性蛋白激酶和共转运蛋白直接磷酸化的途径发生。
The Na-K-Cl cotransport system of vascular endothelial cells plays a central role in maintenance and regulation of intracellular volume. Activity of the cotransporter is modulated both by hormones and by extracellular tonicity. Vasopressin and other hormones that stimulate the endothelial cotransporter act via a Ca- and calmodulin-dependent pathway. Little is known, however, about the mechanisms that mediate cell shrinkage-induced stimulation of cotransport activity. In the present study, we evaluated the Ca dependence of cell shrinkage-stimulated Na-K-Cl cotransport activity and cell volume recovery of cultured bovine aortic endothelial cells and also the effects of protein kinase and phosphatase inhibitors on these processes. In addition, to investigate the possibility that hormones and/or hypertonicity regulate endothelial Na-K-Cl cotransport via direct phosphorylation of the cotransporter protein, we employed a monoclonal antibody to the human colonic T84 epithelial cell Na-K-Cl cotransport protein (T4 antibody) for Western blot analysis and immunoprecipitation of phosphoprotein. Our studies revealed that both cell shrinkage-stimulated net K uptake and recovery of intracellular volume were Ca dependent. We also found that hypertonicity-induced stimulation of cotransport activity was blocked by several inhibitors of Ca- and calmodulin-dependent protein kinases. Furthermore, inhibitors of myosin light chain kinase blocked cell shrinkage-stimulated cotransport and recovery of intracellular volume, while having no effect on vasopressin-stimulated cotransport. Western blot analysis of bovine aortic and cerebral microvascular endothelial cell membrane preparations revealed a 170-kDa protein recognized by the T4 antibody. In addition, we found that hypertonicity induced a marked increase in phosphorylation of the endothelial cotransport protein, as did vasopressin, bradykinin, okadaic acid, and calyculin A. Our findings indicate that modulation of endothelial cell Na-K-Cl cotransport activity by hypertonicity and by stimulatory hormones occurs via pathways involving Ca- and calmodulin-dependent protein kinases and direct phosphorylation of the cotransport protein.