Functional properties of cultured endothelial cells derived from
large microvessels of human brain .
Spatz, M., N. Kawai, N. Merkel, J. Bembry, and R. M. McCarron.
Stroke Branch, NINDS, National Institutes of Health, Bethesda, MD
20892-4128
APStracts 3:0235C, 1996.
This report describes the fractional separation of microvessels from
human brain for establishment of segmentally derived endothelial cell
cultures. The investigation comprised evaluation of media
constituents, 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 ET-1 and prostaglandins, and took
up 86Rb+ as a measure of K+. Exogenous ET-1 stimulated
phosphotidylinositol hydrolysis and K+ uptake; BQ123 (selective ETA
receptor antagonist) but not IRL 1038 or BQ788 (selective ETB
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] selevtively reduced
the 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-12-myristate-13-acetate
(PMA, activator of PKC)-stimulated K+ uptake, which was only
decreased with bumetanide. N-Ethyl-isopropyl amiloride [(EIPA);
inhibitor of Na+/H+ exchange] reduced the ET-1-stimulated but not the
PMA-induced K+ uptake. The results indicate that phosphotidylinositol
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 suggests a linkage between Na+,K+-ATPase activity
and Na+/H+ exchange.
Received 20 February 1996; accepted in final form 8 July 1996.
APS Manuscript Number C89-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 25 July 1996