Hypotonicity stimulates phosphatidylcholine hydrolysis and generates diacylglycerol in erythrocytes

Exposure of skate erythrocytes to hypotonic medium stimulates a rapid increase in levels of 1,2-diacylglycerol. Other treatments which produce cell swelling such as replacement of a portion of medium NaCl with the permeant solutes ethylene glycol or ammonium chloride also stimulate increases in diacylglycerol. Whereas the reduction of medium osmolarity to 460 mosm (from 940) stimulated a persistent diacylglycerol increase, the increase after reduction to 660 mosm was transient, peaking at 2.5 min and then slowly declining. This decline could be prevented by preincubation with the diacylglycerol kinase inhibitor R59022. To investigate the source of the increased diacylglycerol, the rate of incorporation of [32P]PO4 into each major phospholipid was measured. Reduction of osmolarity to 660 mosm stimulated the incorporation of phosphate into phosphatidylcholine markedly, with a smaller increase observed into phosphatidylinositol. To demonstrate phosphatidylcholine hydrolysis, erythrocytes were prelabeled with [32P]PO4. Subsequent exposure to hypotonic (660 mosm) medium stimulated a decrease in radioactivity in phosphatidylcholine and a large increase in radioactivity in phosphatidic acid. When stimulated in the presence of ethanol, 32PO4-labeled phosphatidylethanol was formed, suggesting activation of phospholipase D. In addition, the initial formation of 32PO4-labeled phosphatidic acid was not sensitive to inhibition of diacylglycerol kinase, supporting the role of direct activation of phospholipase D. These results indicate that hypotonicity and the accompanying cell swelling induce cell membrane phospholipid turnover, predominantly phosphatidylcholine, and production of the protein kinase C activator, diacylglycerol, which appears to occur via activation of phospholipase D.