Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active α-thrombin: Evidence for regulation at the level of 1,2-diacylglycerol generation
Membrane Transport Proteins
Molecular Dynamics Simulation
α-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of α-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active α-thrombin. When cultures were incubated in the presence of α-thrombin, 1,2-diacylglycerol levels remained elevated for ≥4 h. Inactivation of α-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of α-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the α-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to α-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active α-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to α-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response. © 1991 by The American Society for Cell Biology.
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