Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation.
alpha-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 alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-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 alpha-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 alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-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 alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-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.
Rangan
L A
LA
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Wright
T M
TM
Raben
D M
DM
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002794:Choline / Q000378:metabolism; D006224:Cricetinae; D004075:Diglycerides / Q000378:metabolism*; D006629:Hirudins / Q000494:pharmacology; D007700:Kinetics; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism
C011439:1,2-diacylglycerol; D004075:Diglycerides; D006629:Hirudins; D013917:Thrombin; D002794:Choline; D013936:Thymidine