G-PROTEIN-MEDIATED SIGNALING IN CHOLESTEROL-ENRICHED ARTERIAL SMOOTH-MUSCLE CELLS .2. ROLE OF PROTEIN-KINASE C-DELTA IN THE REGULATION OF EICOSANOID PRODUCTION

PGI(2) generation by the vessel wall is an agonist for cyclic-AMP-depe ndent cholesteryl ester hydrolysis, The process of enhanced PGI(2) syn thesis is stimulated, in part, by G-protein-coupled receptor ligands. Cellular cholesterol enrichment has been hypothesized to alter G-prote in-mediated PGI(2) synthesis. In tile studies reported herein, cells g enerated PGI(2) in response to ALF(4)(-), GTP gamma S, and ATP in a do se-dependent manner, G-protein agonists stimulated eicosanoid producti on principally by activating phospholipase A(2), but not phospholipase C. This is in contrast to PDGF, which stimulated phospholipase A(2) a nd PLC gamma activities. G alpha i subunits mediate G-protein agonist- induced PGI(2) synthesis, since ATP- and PDGF-induced PGI(2) synthesis was inhibited by pertussis toxin. Although cholesterol enrichment red uced arachidonic acid- and PDGF-induced PGI(2) synthesis, cholesterol enrichment enhanced PGI(2) release in response to AIF(4)(-), GTP gamma S, and ATP. The enhancement of PGI(2) release in cholesterol-enriched cells was augmented by mevalonate, which inhibits the ability of chol esterol enrichment to reduce membrane-associated G-protein subunits, S ince cholesterol enrichment inhibited PDGF and AIF(4)(-)-induced MAP k inase activity [Pomerantz, K., Lander, H. M., Summers, B., Robishaw, J . D., Balcueva, E. A., & Hajjar, D. P. (1997) Biochemistry 36: 9523-95 31] (the major mechanism by which phospholipase A(2) is activated), th ese results suggest that cholesterol enrichment induces other alternat ive signaling pathways leading to phospholipase A(2) activation. A PKC -dependent pathway is described herein that is involved in enhanced ei cosanoid production in cholesterol-enriched cells. This conclusion is supported by two observations: (1) G-protein-linked PGI(2) production is inhibited by calphostin, and (2) cholesterol enrichment augments th e specific translocation of the delta- isoform of PKC from the cytosol to the plasma membrane following treatment of cells with phorbol este r. These data support the concept that, in cells possessing normal lev els of cholesterol, MAP-kinase-dependent pathways mediate eicosanoid s ynthesis in response to G-protein activation; however, under condition s of high cellular cholesterol levels, augmented G-protein-linked eico sanoid production results from enhanced PKC delta activity.