OVEREXPRESSION OF PROTEIN-KINASE-C-ALPHA ENHANCES LIPOPOLYSACCHARIDE-INDUCED NITRIC-OXIDE FORMATION IN VASCULAR SMOOTH-MUSCLE CELLS

Our previous studies showed that lipopolysaccharide (LPS)-induced nitr ic oxide (NO) synthesis in cardiovascular tissues is attenuated by pro tein kinase C (PKC) inhibitors. In the current study, we identify a sp ecific PKC isotype involved in the LPS signal transduction pathway tha t leads to NO formation in rat vascular smooth muscle cells (VSMC). VS MC were transfected with a mammalian expression vector containing a fu ll length PKC alpha cDNA insert, and a stable transfectant overexpress ing PKC alpha was obtained as evidenced by increased expression of PKC alpha mRNA and protein. In response to 100 ng/ml LPS stimulation, the PKC alpha transfectants showed a 1.8-fold increase in PKC activity at 30 min and a twofold increase in NO production over 24 hr compared wi th cells transfected with control plasmids. The LPS-stimulated increas e in NO synthesis in PKC alpha transfectants was blocked by the specif ic PKC alpha inhibitor Ga 6976. After 6 hr LPS treatment, PKC alpha-tr ansfected and control cells showed equivalent increases in mRNA and pr otein for the inducible NO synthase. NO synthase activity of the cell extracts assayed in the presence of excess substrate and cofactors was not significantly different between PKC alpha-transfected and control cells after LPS stimulation. However, mRNA levels for GTP cyclohydrol ase I, a key enzyme in (GR)-tetrahydro-L-biopterin synthesis, and cati onic amino acid transporter-2, involved in L-arginine transport, was e nhanced in cells overexpressing PKC alpha compared with control cells. These results suggest that PKC alpha plays an important role in LPS-i nduced NO formation and that a significant portion of this effect may be by means of enhanced substrate availability to the inducible nitric oxide synthase enzyme. (C) 1998 Wiley-Liss, Inc.