Prostacyclin production in rat aortic smooth muscle cells: role of protein kinase C, phospholipase D and cyclooxygenase-2 expression.
OBJECTIVE
The present study was designed to investigate the role of protein kinase C (PKC) and phospholipase D (PLD) in angiotensin II (AngII)- and phorbol ester (PMA)-induced cyclooxygenase-2 (COX-2) expression and prostacyclin (PGI(2)) production in rat aortic smooth muscle cells (VSMC).


METHODS
Prostacyclin production in cultured VSMC was determined by radioimmunoassay. PKC activity was examined by measuring the transfer of 32P from (gamma-32P)ATP to histone III-S. COX-2 expression was determined by Western blotting. To measure PLD activity, thin layer chromatography was used.


RESULTS
AngII (50 nM) and PMA (100 nM) promoted the translocation of PKC activity from the cytosol to the membranes within 30 min, followed by a strong increase in PLD activity as well as COX-2 expression and PGI(2) production. After 48 h exposure to PMA, PKC was downregulated resulting in a complete suppression of its activity. PKC-downregulation and the PKC inhibitor CGP41251 abolished PMA- and AngII-induced PLD activation, suppressed the stimulatory effect of PMA on COX-2 expression and PGI(2) production and strongly inhibited that of AngII. Furthermore, AngII- and PMA-induced PGI(2) production depended on protein synthesis and COX-2 but not COX-1 activity. Inhibition of PLD-mediated phosphatidic acid (PA) formation by 1% 1-butanol abolished AngII-induced COX-2 expression and PGI(2) secretion, while dioctanoyl PA increased COX-2 expression and PGI(2) production in a time- and concentration-dependent manner.


CONCLUSIONS
Our results indicate that in VSMC, AngII promotes PGI(2) production to a large extent through a rise in COX-2 expression which is mediated by PA generated from increased PKC-dependent PLD activity.
Frias
Miguel A
MA
Division of Endocrinology and Diabetology, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland.
Dubouloz
Frédérique
F
Rebsamen
Michela C
MC
Lang
Ursula
U
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D007527:Isoenzymes / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D044062:Prostaglandins I / Q000032:analysis / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D044062:Prostaglandins I; D000804:Angiotensin II; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate