Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187

Abstract

When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labeling of other phospholipids. Labeling of phosphatidate in these cells did not continue to increase after about 20 min at 37.degree. C; by this time, radioactivity in phosphatidate was about 10.times. higher in ionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by about 50%. In the presence of 2.5 mM-Ca2+, a maximum effect was seen with about 0.5 .mu.g of ionophore/ml. The concentration of Ca2+ giving half-maximal labeling of phosphatidate in the presence of 10.mu.g of ionophore A23187/ml was about 10 .mu.M. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. Labeling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was 5-7.times. that of untreated control cells. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. The source of 1,2-diacylglycerol was not definitely identified, but its fatty acid composition was similar to that of phosphatidylcholine. It had an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. Accumulation of 1,2-diacylglycerol also occurred in energy-starved cells, even in the absence of Ca; in this case it appeared to be produced by phosphatidate breakdown.