Ca ²⁺-mediated ascorbate release from coronary artery endothelial cells

1 The addition of Ca ²⁺ ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca ²⁺-mediated release. An increase in Ca ²⁺-mediated Asc release was observed from PCEC preincubated with Asc, Asc-2-phosphate or dehydroascorbic acid (DHAA). 2 The effects of various ATP analogs and inhibition by suramin were consistent with the ATP-induced release being mediated by P2Y2-like receptors. 3 ATP-stimulated Asc release was Ca ²⁺-mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca ²⁺], (b) Ca ²⁺ ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca ²⁺ and chelating intracellular Ca ²⁺ inhibited the ATP-induced release, and (d) inositol-selective phospholipase C inhibitor U73122 also inhibited this release. 4 Accumulation of Asc by PCEC was examined at Asc concentrations of 10 μM (Na ⁺-Asc symporter not saturated) and 5 mM (Na ⁺-Asc symporter saturated). At 10 μM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. 5 PCEC but not pig coronary artery smooth muscle cells show a Ca ²⁺- mediated Asc release pathway that may be activated by agents such as ATP.