Astroglial cell-induced expression of Na-K-Cl cotransporter in brain microvascular endothelial cells

Endothelial cells of the blood-brain barrier (BBB) are characterized by extensive tight junctions and asymmetric distribution of specific enzymes and transport systems. Maintenance of the BBB endothelial phenotype depends on astrocyte-endothelial interactions. We showed previously that cultured cerebral microvascular endothelial cells (CMEC) exhibit robust Na-K-Cl cotransport activity. In the present study, we evaluated the expression of Na-K-Cl cotransport protein in CMEC by quantitative Western blot analysis and found that a protein of approximately 170 kDa was recognized by a monoclonal antibody against the cotransporter. Exposure of CMEC to astroglial cells or their conditioned media increased the expression of the CMEC cotransport protein by approximately 55%. Using a monoclonal antibody against the alpha-subunit of chicken Na-K-ATPase, we found that these treatments also increased expression of Na-K-ATPase protein by a similar amount. By comparing bumetanide-sensitive K influx and [3H]bumetanide binding apical vs. basolateral surfaces of CMEC, we found both cotransporter activity and [3H]bumetanide binding to be approximately 90% apical and 10% basolateral. Coculture of the CMEC with astroglial cells increased cotransport activity and [3H]bumetanide binding at both surfaces, with the asymmetric distribution maintained. These results indicate that the cotransporter is regulated by astroglial cells and that an apically distributed CMEC cotransporter may function in tandem with the basolateral Na-K-ATPase to mediate vectorial transport of Na and Cl across the BBB.