Astroglial-mediated phosphorylation of the na-k-cl cotransporter in
brain microvessel endothelial cells.
Sun, Dandan, and Martha E. O'donnell.
Department of Human Physiology, School of Medicine, University of
California, Davis, CA 95616
APStracts 3:0097C, 1996.
Our previous studies have shown that cerebral microvessel endothelial
cells (CMEC) express a Na-K-Cl cotransporter and that exposure of
CMEC to astroglial cells causes a nearly 2-fold increase in activity
of the cotransporter but only 1.5-fold increase in expression of
cotransport protein (Am. J. Physiol. 269:C1506-C1512, 1995). This
finding suggests that the astroglial cell effects may be mediated by
mechanisms involving cotransporter activation in addition to
increased protein expression. In the present study, we evaluated the
role of protein phosphorylation in elevation of CMEC cotransport
activity by astroglial cells and by extracellular hypertonicity. We
also examined the effects of protein phosphatase inhibitors and
protein kinase inhibitors on both cotransporter activity and
phosphorylation in CMEC. The phosphorylation level of Na-K-Cl
cotransport protein was quantitatively evaluated by
immunoprecipitation analysis using monoclonal antibody to the
cotransporter following 32P-labeling of cultured CMEC monolayers.
Activity of the cotransporter was assessed as bumetanide-sensitive K
influx. We found that the phosphatase inhibitors calyculin A and
okadaic acid significantly increased both cotransport activity and
phosphorylation of cotransport protein. Activity and phosphorylation
level of the cotransporter were also markedly increased by exposing
the cells to astroglial cell-conditioned medium or hypertonic medium.
Moreover, the astroglial-induced stimulation of the CMEC
cotransporter was inhibited by the protein kinase inhibitor K-252a.
These findings suggest that phosphorylation of cotransport protein
plays an important role in regulation of Na-K-Cl cotransport activity
and that astroglial-induced elevation of cotransport activity
involves both phosphorylation-associated stimulation of cotransport
activity and increased expression of the cotransporter protein.
Received 3 January 1996; accepted in final form 13 March 1996.
APS Manuscript Number C6-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 27 March 96