Kinetic and molecular species analyses of mitogen-induced increases in diglycerides: evidence for stimulated hydrolysis of phosphoinositides and phosphatidylcholine.

A wide variety of agonist-induced events appear to be mediated through an increase in cellular diglyceride levels. With regard to the ability of diglycerides to mediate these events, three important parameters must be considered: a) the kinetics of diglyceride generation, b) the absolute mass levels, and c) their molecular species. While this increase is often due to a stimulated hydrolysis of phosphoinositides, there is increasing evidence that the stimulated hydrolysis of phosphatidylcholine also contributes to agonist-induced increases in diglyceride levels. The kinetics of mass increases in diglyceride levels stimulated in cultured fibroblasts are agonist-dependent. High concentrations of alpha-thrombin stimulate a biphasic increase in diglyceride levels with the first phase peaking at 15 s and the second phase peaking at 5 min. In contrast, stimulation with epidermal growth factor, or platelet-derived growth factor, results in a monophasic increase in cellular diglyceride levels. Furthermore, the molecular species and phospholipid source of the stimulated diglycerides are also agonist-dependent. While the hydrolysis of phosphoinositides is major source of diglycerides initially generated in response to some agonists (15 s with alpha-thrombin at 500 ng/ml), phosphatidylcholine is hydrolyzed as well. Following longer incubations, or at all times following stimulation by epidermal growth factor or platelet-derived growth factor, phosphatidylcholine hydrolysis is the principal source of the stimulated diglycerides.