ASTROGLIAL-MEDIATED PHOSPHORYLATION OF THE NA-K-CL COTRANSPORTER IN BRAIN MICROVESSEL ENDOTHELIAL-CELLS

Our previous studies have shown that cerebral microvessel endothelial cells (CMEC) express a Na-K-CI cotransporter and that exposure of CMEC to astroglial cells causes a nearly 2-fold increase in activity of th e cotransporter but only 1.5-fold increase in expression of cotranspor t protein [D. Sun, C. Lytle, and M. E. O'Donnell. Am. J. Physiol. 269 (Cell Physiol. 38):C1506-C1512, 1995]. This finding suggests that the astroglial cell effects maybe mediated by mechanisms involving cotrans porter activation in addition to increased protein expression. In the present study, we evaluated the role of protein phosphorylation in ele vation of CMEC cotransport activity by astroglial cells and extracellu lar hypertonicity. We also examined the effects of protein phosphatase and protein kinase inhibitors on both cotransporter activity and phos phorylation in CMEC. The phosphorylation level of Na-K-Cl cotransport protein was quantitatively evaluated by immunoprecipitation analysis w ith the use of a monoclonal antibody to the cotransporter after P-32 l abeling of cultured CMEC. Activity of the cotransporter was assessed a s bumetanide-sensitive K influx. We found that the phosphatase inhibit ors calyculin A and okadaic acid significantly increased both cotransp ort activity and phosphorylation of cotransport protein. Activity and phosphorylation level of the cotransporter were also markedly increase d by exposing the cells to astroglial cell-conditioned or hypertonic m edium. Moreover, the astroglial-induced stimulation of the CMEC cotran sporter was inhibited by the protein kinase inhibitor K-252a. These fi ndings suggest that phosphorylation of cotransport protein plays an im portant role in regulation of Na-K-Cl cotransport activity and that as troglial-induced elevation of cotransport activity involves both phosp horylation-associated stimulation of cotransport activity and increase d expression of the cotransporter protein.