Vasopressin, phorbol diesters and serum elicit choline glycerophospholipid hydrolysis and diacylglycerol formation in nontransformed cells: transformed derivatives do not respond.

REF52, a rat embryo cell line, and several transformed derivatives were used to examine the lipid-related events associated with agonist treatment (phorbol diesters, vasopressin, fetal bovine serum). Exposure of cells, prelabeled with [3H]glycerol, to TPA (12-O-tetradecanoylphorbol 13-acetate) resulted in 3-4-fold increase in the amount of intracellular diacyl[3H]glycerols as early as 10 min after treatment. Continued incubation (up to 60 min) revealed that the diacyl[3H]glycerol formed was under dynamic metabolic regulation as shown by the production of triacyl[3H]glycerols and free [3H]glycerol. Serum and vasopressin likewise induced the generation of intracellular diacyl[3H]glycerol, thereby illustrating that physiological agents provoke a similar reaction. In the three SV-40-transformed variants examined, the diacylglycerol generative-response to TPA, serum and vasopressin, was greatly diminished or totally absent. Experiments employing REF52 cells prelabeled with [3H]choline demonstrated that both TPA and vasopressin induce the hydrolysis of cellular choline-containing glycerophospholipids; this was measured by both a decrease in cell-associated phosphatidylcholine radioactivity and an increase in the production of water-soluble [3H]choline-containing metabolites in the culture medium. 92-97% of the tritium released to the medium was identified as [3H]choline. Vasopressin treatment of REF52 cells prelabeled with [3H]arachidonic acid elicited an increase of more than 11-fold in the amount of cellular diacyl[3H]glycerol and a concomitant release of arachidonic acid to the culture medium that was 12-fold higher than controls. These data demonstrate that tumor-promoting phorbol esters (agonists of protein kinase C), serum and vasopressin, increase the levels of cellular diacylglycerol by stimulating the hydrolysis of choline-containing glycerophospholipids. This agonist-directed mechanism is inoperable in transformed cells. Further, collateral with vasopressin-induced phosphatidylcholine hydrolysis, the cellular release of arachidonic acid occurs. The participation of these lipid-related responses in the signaling of agonist-directed events and their relation to cellular homeostasis is currently being explored.