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.
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