Functional properties of cultured endothelial cells derived from large microvessels of human brain

This report describes the fractional separation of microvessels from human brain for establishment of segmentally derived endothelial cell (EC) cultures. The investigation comprised evaluation of media constituents and purity of the cell culture and focused on functional biochemical characterization of endothelium derived from large microvessels (EC). Cells contained endothelial marker factor VIII (von Willebrand antigen), secreted endothelin-l (ET-1) and prostaglandins, and took up Rb-86(+) as a measure of K+. Exogenous ET-1 stimulated phosphatidylinositol hydrolysis and K+ uptake; BQ-123 (selective ET(A) receptor antagonist) but not IRL-1038 or BQ-788 (selective ET(B) receptor antagonists) inhibited both. Ouabain (inhibitor of Na+-K+-ATPase) and bumetanide (inhibitor of Na+-K+-Cl- cotransport) reduced (74-80 and 20-40%, respectively) the ET-1-stimulated K+ uptake. Staurosporine [protein kinase C (PKC) inhibitor] selectively reduced Na+-K+-Cl- cotransport, whereas verapamil but not nifedipine (L-type voltage-dependent Ca2+ channel blockers) decreased Na+-K+-ATPase activity induced by ET-1. Phorbol la-myristate 13-acetate (PMA; activator of PKC) stimulated K+ uptake, which was only decreased with bumetanide. N-ethylisopropylamiloride (inhibitor of Na+/H+ exchange) reduced the ET-1-stimulated but not the PMA-induced K+ uptake. Results indicate that phosphatidylinositol hydrolysis and ion transport systems in large microvascular EC are stimulated by ET-1 through activation of ETA receptors. The findings also suggest that the ET-1-stimulated Na+-K+-ATPase activity, in contrast to Na+-K+-Cl- cotransport, is not mediated by PKC. In addition, the data suggest a linkage between Na+-K+-ATPase activity and Na+/H+ exchange.