Calcium-Dependent Synthesis of Prostacyclin in ATP-Stimulated Venous Endothelial Cells

Abstract

Abstract-Prostacyclin is a powerful vasodilator that is released from vascular endothelial cells. Previous studies in our laboratory have indicated that arachidonic acid metabolites from venous endothelium play an important role in the dilation of adjacent arterioles during muscle stimulation. Furthermore, recent studies have suggested that ATP released from red blood cells during hypoxia stimulates dilation of arterioles. We tested the hypothesis that an ATP-induced increase in intracellular Ca 2ϩ in venous endothelium promotes prostacyclin synthesis. Small branches of femoral veins were isolated from male golden hamsters, placed in a 1 mL bath, and cannulated for perfusion with 3-(N-morpholino) propanesulfonic acid (MOPS)-buffered physiological salt solution at 37°C. Prostacyclin synthesis was determined by enzyme immunoassay of bath solution. Perfusion of veins with ATP increased prostacyclin synthesis from 50Ϯ5 to 627Ϯ46 pg/mL (nϭ49). ATP-induced prostacyclin synthesis was inhibited by removal of extracellular Ca 2ϩ , chelation of intracellular Ca 2ϩ with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA) (10 mol/L for 10 minutes), and preincubation with cytosolic phospholipase A 2 (PLA 2 ) inhibitors, AACOCF 3 , and bromoenol lactone. Changes in intracellular Ca 2ϩ in cultured human venous endothelial cells were assessed by fura-2 spectrofluorometry. ATP induced a transient Ca 2ϩ peak within seconds, and the subsequent Ca 2ϩ plateau was abolished by removal of extracellular Ca2ϩ . An increase in prostacyclin synthesis was detected in these cells 2 minutes after application of ATP. These findings suggest that the ATP-induced increase in intracellular Ca 2ϩ stimulates prostacyclin synthesis in venous endothelial cells. Vasodilators released from the endothelium include the arachidonic acid metabolite prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor. However, the quantitative role of these vasodilators in different regions of the vasculature has not been characterized. Nitric oxide appears to have a minimal role in regulating arteriolar diameter during an increase in metabolic rate. [3][4][5] Previous studies from our laboratory have shown that arachidonic acid metabolites make a major contribution to the regulation of arteriolar diameter during muscle stimulation. 6,7 Arachidonic acid is released from membrane phospholipids through the direct action of phospholipase A 2 (PLA 2 ). Further metabolism of arachidonic acid generates three main groups of eicosanoids: prostaglandins and thromboxanes by cyclooxygenase; leukotrienes and lipoxins by lipoxygenase; and epoxides by cytochrome P-450 epoxygenase. Prostacyclin (prostaglandin I 2 ) is a very powerful vasodilator that is produced primarily in vascular endothelial cells. 8 There are both secretory and intracellular forms of PLA 2 . 9 The cytosolic form of PLA 2 includes both Ca 2ϩ -dependent and Ca 2ϩ -independent isozymes. However, it is not yet certain which of these enzymes is responsible for the release of arachidonic...

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