ENDOTHELIAL NA-K-CL COTRANSPORT REGULATION BY TONICITY AND HORMONES -PHOSPHORYLATION OF COTRANSPORT PROTEIN

The Na-K-CI cotransport system of vascular endothelial cells plays a c entral role in maintenance and regulation of intracellular volume. Act ivity of the cotransporter is modulated both by hormones and by extrac ellular tonicity. Vasopressin and other hormones that stimulate the en dothelial cotransporter act via a Ca- and calmodulin-dependent pathway . Little is known, however, about the mechanisms that mediate cell shr inkage-induced stimulation of cotransport activity. In the present stu dy, we evaluated the Ca dependence of cell shrinkage-stimulated Na-K-C l cotransport activity and cell volume recovery of cultured bovine aor tic endothelial cells and also the effects of protein kinase and phosp hatase inhibitors on these processes. In addition, to investigate the possibility that hormones and/or hypertonicity regulate endothelial Na -K-CI cotransport via direct phosphorylation of the cotransporter prot ein, we employed a monoclonal antibody to the human colonic T84 epithe lial cell Na-K-Cl cotransport protein (T4 antibody) for Western blot a nalysis and immunoprecipitation of phosphoprotein. Our studies reveale d that both cell shrinkage-stimulated net K uptake and recovery of int racellular volume were Ca dependent. We also found that hypertonicity- induced stimulation of cotransport activity was blocked by several inh ibitors of Ca- and calmodulin-dependent protein kinases. Furthermore, inhibitors of myosin light chain kinase blocked cell shrinkage-stimula ted 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 p reparations 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 vasopre ssin, bradykinin, okadaic acid, and calyculin A. Our findings indicate that modulation of endothelial cell Na-K-CI cotransport activity by h ypertonicity and by stimulatory hormones occurs via pathways involving Ca- and calmodulin-dependent protein kinases and direct phosphorylati on of the cotransport protein.