Ischemia-induced brain edema formation is mediated by increased transport of Na and Cl across an intact blood-brain barrier (BBB). Our previous studies have provided evidence that a luminally located BBB Na-K-Cl cotransporter is stimulated during cerebral ischemia to increase transport of Na and Cl into the brain. The main focus of the present study was to evaluate the effects of arginine vasopressin (AVP), previously shown to be increased in the brain during ischemia and to promote edema formation, on activity of the BBB cotransporter. Cerebral microvascular endothelial cell (CMEC) monolayers were cultured in astroglial cell conditioned medium, and Na-K-Cl cotransporter activity was assessed as bumetanide-sensitive (86)Rb influx. In both human and bovine CMECs, as well as in freshly isolated microvessels, AVP stimulated cotransport activity. This stimulatory effect was mimicked by V(1) but not V(2) vasopressin agonists and was blocked by V(1) but not V(2) vasopressin antagonists. Consistent with a V(1) vasopressin receptor mechanism of action, AVP caused an increase in CMEC intracellular [Ca] that was blocked by a V(1) antagonist. Exposing the cells to [Ca]-free media and/or reducing intracellular [Ca] by BAPTA also blocked AVP stimulation of CMEC cotransporter activity, as did the phospholipase C inhibitor U-73122. Finally, we found that while stimulation of CMEC cotransporter activity by AVP occurred within minutes, it was also sustained for hours in the continued presence of AVP. These findings support the hypothesis that AVP, through a V(1) receptor- and [Ca]-dependent mechanism, stimulates the BBB Na-K-Cl cotransporter to participate in ischemia-induced edema formation.

Steady laminar shear stress has been shown previously to markedly increase Na-K-Cl cotransporter mRNA and protein in human umbilical vein endothelial cells and also to rapidly increase endothelial K(+) and Cl(-) channel conductances. The present study was done to evaluate the effects of shear stress on Na-K-Cl cotransporter activity and protein expression in bovine aortic endothelial cells (BAEC) and to determine whether changes in cotransporter expression may be dependent on early changes in K(+) and Cl(-) channel conductances. Confluent BAEC monolayers were exposed in a parallel-plate flow chamber to either steady shear stress (19 dyn/cm(2)) or purely oscillatory shear stress (0 +/- 19 dyn/cm(2)) for 6-48 h. After shearing, BAEC monolayers were assessed for Na-K-Cl cotransporter activity or were subjected to Western blot analysis of cotransporter protein. Steady shear stress led to a 2- to 4-fold increase in BAEC cotransporter protein levels and a 1.5- to 1.8-fold increase in cotransporter activity, increases that were sustained over the longest time periods studied. Oscillatory flow, in contrast, had no effect on cotransporter protein levels. In the presence of flow-sensitive K(+) and Cl(-) channel pharmacological blockers, the steady shear stress-induced increase in cotransporter protein was virtually abolished. These results suggest that shear stress modulates the expression of the BAEC Na-K-Cl cotransporter by mechanisms that are dependent on flow-activated ion channels.

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