ASTROGLIAL CELL-INDUCED EXPRESSION OF NA-K-CL COTRANSPORTER IN BRAIN MICROVASCULAR ENDOTHELIAL-CELLS

Endothelial cells of the blood-brain barrier (BBB) are characterized b y extensive tight junctions and asymmetric distribution of specific en zymes and transport systems. Maintenance of the BBB endothelial phenot ype depends on astrocyte-endothelial interactions. We showed previousl y that cultured cerebral microvascular endothelial cells (CMEC) exhibi t robust Na-K-CI cotransport activity. In the present study, we evalua ted the expression of Na-K-CI cotransport protein in CMEC by quantitat ive Western blot analysis and found that a protein of similar to 170 k Da was recognized by a monoclonal antibody against the cotransporter. Exposure of CMEC to astroglial cells or their conditioned media increa sed the expression of the CMEC cotransport protein by similar to 55%. Using a monoclonal antibody against the alpha-subunit of chicken Ma-K- ATPase, we found that these treatments also increased expression of Fi a-K-ATPase protein by a similar amount. Ey comparing bumetanide-sensit ive K influx and [H-3]bumetanide binding of apical vs. basolateral sur faces of CMEC, we found both cotransporter activity and [H-3]bumetanid e binding to be similar to 90% apical and 10% basolateral. Coculture o f the CMEC with astroglial cells increased cotransport activity and [H -3]bumetanide binding at both surfaces, with the asymmetric distributi on maintained. These results indicate that the cotransporter is regula ted by astroglial cells and that an epically distributed CMEC cotransp orter may function in tandem with the basolateral Na-K-ATPase to media te vectorial transport of Na and CI across the BBB.