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

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 labelling of other phospholipids. Labelling of phosphatidate in these cells did not continue to increase after about 20 min at 37°C; by this time, radioactivity in phosphatidate was about ten 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 50%. In the presence of 2.5 mM Ca²⁺ a maximum effect was seen with about 0.5 μg of ionophore/ml. The concentration of Ca²⁺ giving half maximal labelling of phosphatidate in the presence of 10 μg of ionophore A23187/ml was about 10 μM. A rapid decrease of ATP content in the cell occurred in ionophore treated cells. Labelling 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 five to seven 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 has not been definitely identified, but its fatty acid composition was similar to that of phosphatidylcholine. However, 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 calcium; in this case it appeared to be produced by phosphatidate breakdown.