G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 2. Role of protein kinase C-delta in the regulation of eicosanoid production.
PGI2 generation by the vessel wall is an agonist for cyclic-AMP-dependent cholesteryl ester hydrolysis. The process of enhanced PGI2 synthesis is stimulated, in part, by G-protein-coupled receptor ligands. Cellular cholesterol enrichment has been hypothesized to alter G-protein-mediated PGI2 synthesis. In the studies reported herein, cells generated PGI2 in response to AlF4-, GTPgammaS, and ATP in a dose-dependent manner. G-protein agonists stimulated eicosanoid production principally by activating phospholipase A2, but not phospholipase C. This is in contrast to PDGF, which stimulated phospholipase A2 and PLCgamma activities. Galphai subunits mediate G-protein agonist-induced PGI2 synthesis, since ATP- and PDGF-induced PGI2 synthesis was inhibited by pertussis toxin. Although cholesterol enrichment reduced arachidonic acid- and PDGF-induced PGI2 synthesis, cholesterol enrichment enhanced PGI2 release in response to AlF4-, GTPgammaS, and ATP. The enhancement of PGI2 release in cholesterol-enriched cells was augmented by mevalonate, which inhibits the ability of cholesterol enrichment to reduce membrane-associated G-protein subunits. Since cholesterol enrichment inhibited PDGF and AlF4--induced MAP kinase activity [Pomerantz, K., Lander, H. M., Summers, B., Robishaw, J. D., Balcueva, E. A., & Hajjar, D. P. (1997) Biochemistry 36, 9523-9531] (the major mechanism by which phospholipase A2 is activated), these results suggest that cholesterol enrichment induces other alternative signaling pathways leading to phospholipase A2 activation. A PKC-dependent pathway is described herein that is involved in enhanced eicosanoid production in cholesterol-enriched cells. This conclusion is supported by two observations: (1) G-protein-linked PGI2 production is inhibited by calphostin, and (2) cholesterol enrichment augments the specific translocation of the delta-isoform of PKC from the cytosol to the plasma membrane following treatment of cells with phorbol ester. These data support the concept that, in cells possessing normal levels of cholesterol, MAP-kinase-dependent pathways mediate eicosanoid synthesis in response to G-protein activation; however, under conditions of high cellular cholesterol levels, augmented G-protein-linked eicosanoid production results from enhanced PKCdelta activity.
Pomerantz
K B
KB
Department of Medicine, Cornell University Medical College, 1300 York Avenue, New York, New York 10021, USA.
Lander
H M
HM
Summers
B
B
Hajjar
D P
DP
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D011464:Epoprostenol / Q000096:biosynthesis*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008798:Mevalonic Acid / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D037342:Pertussis Toxin; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011817:Rabbits; D051381:Rats; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007527:Isoenzymes; D010566:Virulence Factors, Bordetella; D002784:Cholesterol; D011464:Epoprostenol; D037342:Pertussis Toxin; C497260:Prkcd protein, rat; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D008798:Mevalonic Acid