Ca 2+-dependent synthesis of prostaglandin I 2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

Abstract

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca 2+ concentration ([Ca 2+] f) on prostaglandin I 2 (PGI 2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 μg/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and β-hexosaminidase. In cells prelabeled with [ 3H]arachidonic acid for 22 h, permeabilization with 20 μg/ml saponin induced PGI 2 synthesis and release of [ 3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca 2+] f in the range 72 nM to 5 μM. Release of [ 3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [ 3H]arachidonic acid and permeabilized with 20 μg/ml saponin. At [Ca 2+] f optimal for PGI 2 synthesis, 16.2% of the total incorporated [ 3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca 2+] f of [ 3H]arachidonic acid release from phospholipids correlated with PGI 2 synthesis. The amount of PGI 2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI 2 synthesis induced by A23187 was associated with less release of [ 3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [ 3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca 2+ dependence observed for PGI 2 synthesis and [ 3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca 2+ which culminate in PGI 2 synthesis.