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Am J Physiol Cell Physiol 283: C1687-C1695, 2002. First published August 22, 2002; doi:10.1152/ajpcell.00071.2002
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Vol. 283, Issue 6, C1687-C1695, December 2002

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

Momoh A. Yakubu and Charles W. Leffler

Laboratory for Research in Neonatal Physiology, Vascular Biology Program, Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

We investigated the role of intracellular calcium concentration ([Ca2+]i) in endothelin-1 (ET-1) production, the effects of potential vasospastic agents on [Ca2+]i, and the presence of L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells. Primary cultures of endothelial cells isolated from piglet cerebral microvessels were used. Confluent cells were exposed to either the thromboxane receptor agonist U-46619 (1 µM), 5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca2+-chelating agent EDTA (100 mM), the L-type Ca2+ channel blocker verapamil (10 µM), or the antagonist of receptor-operated Ca2+ channel SKF-96365 HCl (10 µM) for 15 min. ET-1 production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT), or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1 biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. To investigate the presence of L-type voltage-dependent Ca2+ channels in endothelial cells, the [Ca2+]i signal was determined fluorometrically by using fura 2-AM. Superfusion of confluent endothelial cells with U-46619, 5-HT, or LPA significantly increased [Ca2+]i. Pretreatment of endothelial cells with high K+ (60 mM) or nifedipine (4 µM) diminished increases in [Ca2+]i induced by the vasoactive agents. These results indicate that 1) elevated [Ca2+]i signals are involved in ET-1 biosynthesis induced by specific spasmogenic agents, 2) the increases in [Ca2+]i induced by the vasoactive agents tested involve receptor as well as L-type voltage-dependent Ca2+ channels, and 3) primary cultures of cerebral microvascular endothelial cells express L-type voltage-dependent Ca2+ channels.

voltage-dependent Ca2+ channel; endothelial cell; vasoactive agents; endothelin-1


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