NA-K+-CL- COTRANSPORTER OF BRAIN CAPILLARY ENDOTHELIAL-CELLS - PROPERTIES AND REGULATION BY ENDOTHELINS, HYPEROSMOLAR SOLUTIONS, CALYCULIN-A, AND INTERLEUKIN-1()

Cultured rat brain capillary endothelial cells expressed a large Rb-86 (+) uptake component that was de pendent on external Na+ and Cl- and t hat was inhibited by loop diuretics with unusual pharmacological prope rties: benzmetanide (IC50 = 1-5 mu M) = bumetanide (IC50 = 1-5 mu M) > piretanide (IC50 = 3-16 mu M) = furosemide (IC50 = 7-11 mu M) It was activated a fold by endothelin-1 (EC(50) = 1 nM) and endothelin-3 (EC( 50) = 9 nM). The actions of endothelins were prevented by BQ-123 (cycl o-(D-Trp-D-AspPro-D-Val-Leu)) in a competitive manner and with a high affinity, thus indicating the involvement of an atypical BQ-123-sensit ive, ET(A)-like receptor that had a high affinity for endothelin-3. Ne ither protein kinase C nor Ca2+-dependent protein kinases mediated the actions of endothelins. Cotransport activity was increased 4-fold by hyperosmotic cell shrinkage. Basal Na+-K+-Cl- cotransport activity was partially inhibited by isoproterenol and was unaffected by agents tha t promoted cGMP formation. Calyculin A, an inhibitor of protein phosph atases, stimulated cotransport activity and potentiated the action of endothelin-1, but not that of cell shrinkage. Basal and stimulated cot ransport activities were inhibited by genistein, a protein kinase inhi bitor with similar potencies, and by staurosporine, which has differen t potencies. Finally, endothelin-1-stimulated ac tivity was partially and specifically inhibited by interleukin-1. It is concluded that rat brain capillary endothelial cells express a Na+-K+-Cl- cotransporter t hat has unique properties and that is regulated by multiple protein ki nase/phosphatase systems. It is a target for low concentrations of end othelins and may play a role in brain-to-blood movements of K+.