MOLECULAR REGULATION OF ENDOTHELIAL NA K 2CL COTRANSPORT

本研究的目的是确定Na-K-2Cl共转运蛋白（NKCCl）在血管内皮细胞中的分子调控。这种转运蛋白在细胞收缩时被激活并迅速恢复细胞体积，因此在减少内皮单层间隙形成从而维持血组织屏障的完整性方面发挥着潜在的重要作用。这种转运体也被生长因子激活，产生细胞体积的迅速增加，以补偿邻近细胞的损失和细胞的生长。内皮细胞或其他细胞中细胞体积和生长因子调节NKCCl的机制尚不清楚。目前的证据表明，NKCCl受磷酸化调节，但负责激酶的身份及其调节模式尚不清楚。本提案的目的是证明存在一种在体外磷酸化NKCCl的体积敏感激酶，鉴定该激酶，证明其在体内调节NKCCl，并证明该激酶受上游的体积敏感激酶调节，该激酶也受生长因子调节。这将通过使用包含NKCCl部分的基因工程融合蛋白进行体外激酶分析，亲和纯化，以及在使用反义寡核苷酸和显性激酶缺陷突变体特异性抑制激酶后进行NKCCl磷酸化和活性的体内分析来完成。该结果将为内皮细胞中这种重要转运蛋白的功能和调控提供重要信息，并确定一种受细胞体积和生长因子双重控制的特定调节激酶途径。

The objective of this proposal is to determine the molecular regulation of the Na-K-2Cl cotransporter (NKCCl) in vascular endothelial cells. This transporter is activated by cell shrinkage and rapidly restores cell volume, thus playing a potentially important role in minimizing gap formation in the endothelial monolayer and thereby maintaining the integrity of the blood-tissue barrier. The transporter is also activated by growth factors to produce rapid increases in cell volume necessary to compensate for loss of adjacent cells and for cell growth. The mechanism by which cell volume and growth factors regulate NKCCl in endothelial cells or other cells is unknown. Current evidence suggests that NKCCl is regulated by phosphorylation but the identity of the responsible kinase and its mode of regulation is unknown. The aims of this proposal are to demonstrate the existence of a volume-sensitive kinase that phosphorylates NKCCl in vitro, identify the kinase, prove that it regulates NKCCl in vivo, and to demonstrate that the kinase is regulated by an upstream, volume-sensitive kinase that is also regulated by growth factors. This will be accomplished with in vitro kinase assays using genetically engineered fusion proteins encompassing portions of NKCCl, affinity purification, and in vivo assays of NKCCl phosphorylation and activity after specific inhibition of kinases with antisense oligonucleotides and dominant kinase-deficient mutants. The results will provide important information on the function and regulation of this important transporter in endothelial cells and define a specific regulatory kinase pathway that is under dual control by cell volume and growth factors.