Modulation of Thrombin-Stimulated Lipid Responses in Cultured Fibroblasts. Evidence for Two Coupling Mechanisms

Treatment of cultured fibroblasts with thrombin results in the stimulation of cell division and lipid metabolism. Proteolytically active a-thrombin rapidly stimulates (a) release of arachidonic acid, (b) generation of inositol phosphates, and (c) increase in cellular diacylglycerol levels. Pretreatment of the fibroblasts with chymotrypsin before a-thrombin prevented the first two responses, (a) and (b), and reduced response c. Treatment of fibroblasts with 7-thrombin, a proteolytic derivative of a-thrombin, produced a response indistinguishable from the a-thrombin treatment when preceded by chymotrypsin. These data support a model, similar to one for platelets [McGowan, E. B., & Detwiler, T. C. (1986) J. Biol. Chem. 261, 739–746], that fibroblasts possess two coupling mechanisms for the stimulation of lipid metabolism by thrombin. Similar to platelets, one mechanism, R1, mediates the stimulated release of arachidonic acid and is capable of activating a GTP-binding protein. R1 is inactivated by chymotrypsin and does not respond to 7-thrombin. The other mechanism, R2, responds to 7-thrombin and is not inactivated by chymotrypsin. In contrast to the mechanisms proposed for platelets, we demonstrate that the phospholipase C responsible for the hydrolysis of phosphoinositides is not activated by R2 but is activated via R1. Importantly, stimulation of either mechanism results in the elevation of cellular diacylglycerol. This indicates that the stimulated elevation of diacylglycerol, or those events dependent upon the elevation of diacylglycerol, is not a reliable indicator for establishing the hydrolysis of phosphoinositides. Furthermore, studies with islet activating protein demonstrate that while a protein(s) does (do) not appear to be involved in the stimulated hydrolysis of phosphoinositides, this protein does appear to be involved in at least part of the thrombin-stimulated release of arachidonic acid. A protein(s) may be involved in the metabolism of stimuated diacylglycerol.