Kinetic analysis of 1,2-diacylglycerol mass levels in cultured fibroblasts. Comparison of stimulation by alpha-thrombin and epidermal growth factor.
We have examined the kinetics of 1,2-diacylglycerol production in quiescent IIC9 fibroblasts. alpha-Thrombin and epidermal growth factor (EGF) both stimulate an increase in the mass of cellular 1,2-diacylglycerol. The generation of 1,2-diacylglycerol is biphasic when stimulated by a high concentration of alpha-thrombin (500 ng/ml), with an early phase peaking at 15 s and a late phase peaking at 5 min. Production of 1,2-diacylglycerol is monophasic when stimulated by: (a) a low concentration of alpha-thrombin (100 pg/ml); (b) a high concentration of alpha-thrombin added to cultures which had been pretreated with chymotrypsin; or (c) EGF. In all cases the stimulation of 1,2-diacylglycerol was sustained for at least 30 min. In a previous report (Raben, D. M., Yasuda, K., and Cunningham, D. D. (1987) Biochemistry 26, 2759-2765), it was demonstrated that alpha-thrombin stimulates lipid metabolism in fibroblasts via two coupling mechanisms designated R1 and R2. We now present evidence that the early phase of alpha-thrombin-stimulated 1,2-diacylglycerol production is related to R1, which is characterized by: 1) increased release of arachidonic acid, 2) hydrolysis of polyphosphoinositides, and 3) inhibition by pretreating cultures with chymotrypsin. The late phase is related to R2 which is characterized by 1,2-diacylglycerol production in the absence of stimulated phosphoinositide hydrolysis and arachidonic acid release. In addition, EGF activates an R2-like mechanism in that it does not stimulate the release of arachidonic acid or hydrolysis of polyphosphoinositides but does stimulate a 2-fold increase in 1,2-diacylglycerol mass.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002460:Cell Line; D004261:DNA Replication; D004075:Diglycerides / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005989:Glycerides / Q000378:metabolism*; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000096:biosynthesis; D007700:Kinetics; D013917:Thrombin / Q000494:pharmacology*
D001095:Arachidonic Acids; D004075:Diglycerides; D005989:Glycerides; D007295:Inositol Phosphates; D016718:Arachidonic Acid; D007294:Inositol; D004815:Epidermal Growth Factor; D013917:Thrombin