Abstract
Stimulation of quiescent Swiss 3T3 cells with platelet-derived growth factor (PDGF) increased the initial rate of cytosolic phospholipase A2 activity by 95 +/- 6% over extracts from control cells. Cytosolic phospholipase A2 activity increased rapidly following PDGF treatment (near maximum stimulation by 2.5 min) and was dose-dependent (EC50 = 2 ng/ml). Epidermal growth factor, vasopressin, and phorbol 12,13-dibutyrate also increased cytosolic phospholipase A2 activity but did not produce a sustained mobilization of arachidonic acid in these cells. Detailed kinetic analysis of PDGF-induced arachidonic acid mobilization revealed a biphasic release of 3H radioactivity into the extracellular medium. A first, rapid phase, occurred within 15 min which, like the activation of cytosolic phospholipase A2 activity, was independent of de novo RNA and protein synthesis. After 20 min of stimulation, a second phase became evident which accounts for the majority of arachidonic acid mobilized by PDGF. This second phase was abolished in the presence of either cycloheximide or actinomycin D. Both inhibitors blocked the release of arachidonic acid rather than inhibiting cyclooxygenase activity and consequently prostaglandin E2 production. These findings demonstrate a biphasic mobilization of arachidonic acid in Swiss 3T3 cells by PDGF. Cytosolic phospholipase A2 activity could contribute to the rapid first phase but not the second major phase, which is dependent upon de novo protein synthesis.
Highlights
From the Imperial Cancer Research Fund, Post Office Box 123, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom
The aim of this study was to determine the contribution of cytosolic phospholipase A, activation toward the release of arachidonic acid induced by PDGF in Swiss 3T3 cells, a useful model for studying arachidonic acid-related signaling events (30).We show that PDGFrapidly increases the initial rate of cytosolic phospholipase AP activity
The increase in phospholipase Az activity coincides with the first, cycloheximide-insensitive phase of arachidonic acid release and can be dissociated from the second, cycloheximide-sensitive phase, which accounts for the massive liberation of arachidonic acid and itsmetabolites in PDGF-treated cells
Summary
From the Imperial Cancer Research Fund, Post Office Box 123, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom. After 20 min of stimulation, a second phase became evident which accounts for the majority of arachidonic acid mobilized by PDGF. This secondphase was abolished in the presence of either cycloheximide or actinomycin D. The best known phospholipase AZSareCa2+-dependent, 14-kDa secretory enzymes isolated from snake venom, mammalian pancreas, and othersources (20,21) Because these phospholipase Azsrequire millimolar concentrations of Ca2+for activity anddo not exhibitselectivity toward the fattyacids in the sn-2 position of phosholipids, it is unlikely that their primary function is to initiate arachidonic acid release inside the cell (20, 22). The increase in phospholipase Az activity coincides with the first, cycloheximide-insensitive phase of arachidonic acid release and can be dissociated from the second, cycloheximide-sensitive phase, which accounts for the massive liberation of arachidonic acid and itsmetabolites in PDGF-treated cells
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