L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis.
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 microM), 5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 microM) alone or after pretreatment with the Ca2+-chelating agent EDTA (100 mM), the L-type Ca2+ channel blocker verapamil (10 microM), or the antagonist of receptor-operated Ca2+ channel SKF-96365 HCl (10 microM) 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/microg 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 microM) 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.
Yakubu
Momoh A
MA
Laboratory for Research in Neonatal Physiology, Vascular Biology Program, Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. yakubu_ma@tsu.edu
Leffler
Charles W
CW
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002317:Cardiovascular Agents / Q000494:pharmacology; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation / Q000502:physiology*; D019332:Endothelin-1 / Q000096:biosynthesis*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008833:Microcirculation / Q000502:physiology; D009994:Osmolar Concentration; D013552:Swine
D020746:Calcium Channels, L-Type; D002317:Cardiovascular Agents; D019332:Endothelin-1; D002118:Calcium