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Voltage-dependent Ca2+ - Channel in Cerebral Microvascular Endothelial Cells

We have investigated the presence of voltage-dependent Ca2+-channel, effects of potential vasospastic agents on [Ca2+]I, and endothelin-1 production by cerebral microvascular endothelial cells. Primary cultures of endothelial cells isolated from piglet cerebral microvessels were established and used in these studies. To investigate the role extracellular Ca2+ in vasoactive agents induced ET-1 biosynthesis, confluent endothelial cells were washed with PBS and exposed to either the thromboxane receptor agonist U-46619 (1 uM), 5-HT (0.1 mM), or LPA (1 uM) alone or following pretreatment with the Ca2+-chelating agent EDTA (100 mM), the L-type Ca2+-channel blocker verapamil (10 uM), or the antagonist of receptor operated Ca2+-channel SKF 96365 HCl (10 uM) for 15 min. ET-1 levels were elevated from 1.2±0.2 fmol/ug protein in control to 8.2±2.7 (U-46619), 4.9±1 (5-HT), or 3.9±1.9 (LPA) fmol/ug protein, respectively. Such elevated ET-1 biosynthesis was attenuated following pretreatment of cells with verapamil, EDTA, or SKF 96365 HCL. To investigate the presence of L-type Ca2+-channel in endothelial cells, [Ca2+]i was determined by fluorimeteric measurement using the Ca2+ indicator Fura-2. 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 the voltage-sensitive Ca2+ channel blocker nifedipine (4 uM) diminished increases in [Ca2+]i induced by the vasoactive agents.
The novelty of the present findings are that 1) primary culture of cerebral microvascular endothelial cells expresses receptor-operated and L-type voltage-dependent Ca2+-channels, 2) breakdown products of blood-induced elevation of cytosolic Ca2+ in endothelial cells via both receptor- and voltage-operated Ca2+-channels, and 3) increases in ET-1 production from cerebral microvascular endothelial cells caused by structurally dissimilar vasoactive agents found in blood hemolysates are attenuated by Ca2+-free media, L-type voltage-dependent, and receptor-operated Ca2+-channel blockade. Elevated intracellular Ca2+ plays an important role in the modulation of endothelial functions. Regulations of endothelial cell responses by various extracellular signals are mediated via specific second messenger systems that involve cytosolic Ca2+. The regulation of intracellular Ca2+ signals is the most important functional task of ion channels in endothelial cells. The functional roles of the voltage-dependent Ca2+-channels involves the production and release of many vasoactive endothelial factors that regulate vascular tone as well as control of macromolecular traffics such as endocytosis, exocytosis, biosynthetic-secretory pathway, and transcytosis. Synthesis and release of vascular factors such as ET-1, nitric oxide, and prostacyclin by endothelial cells are regulated by Ca2+ signaling via sustained Ca2+ entry. The presence of voltage-gated Ca2+ channel in endothelial cell is very important for such functions and has wide implications as they could play a significant role in the physiology and pathology of vascular systems. However, the presences of voltage-gated Ca2+-channels in endothelial cells have been a source of controversy in recent time. Endothelial ion channels provide Ca2+-entry pathways or the driving force for the Ca2+-influx through these pathways. Voltage-gated Ca2+-channels are responsible for the long-lasting increase in free [Ca2+]i during different stimuli and provide the signal for maintaining endothelial functions.
In conclusion, elevated intracellular Ca2+ plays a significant role in the increased production of ET-1 caused by specific spasmogenic agents and thus could be involved in the mechanism of hemorrhage-induced alteration of cerebral microvascular reactivities and development of vasospasm. In addition, we have shown the presence of voltage dependent calcium channels in cerebral microvascular endothelial cells. The increases in [Ca2+]i induced by these vasoactive agents are due in part to the activation of these voltage dependent calcium channels. The presence of voltage-gated calcium channel in endothelial cell is very important and has wide implications. Pharmacological manipulation of voltage-gated calcium channel in many cases is readily accomplished, and is also of therapeutic significance. L-type channels are very accessible to pharmacological modification and could be manipulated to influence release of mediators of endothelium-dependent relaxing factors.

Momoh A. Yakubu, PhD
Senior Scientist and Head, Vascular Biology Unit
Center for Cardiovascular Diseases
TSU, Houston

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