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

Yakubu, Momoh A., and Charles W. Leffler. L-type voltage-dependent Ca2 channels in cerebral microvascular endothelial cells and ET-1 biosynthesis. Am J Physiol Cell Physiol 283: C1687–C1695, 2002. First published August 22, 2002; 10.1152/ajpcell.00071.2002.—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 M), 5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 M) alone or after pretreatment with the Ca2 -chelating agent EDTA (100 mM), the L-type Ca2 channel blocker verapamil (10 M), or the antagonist of receptor-operated Ca2 channel SKF-96365 HCl (10 M) 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/ g 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 M) 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.