Overexpression of protein kinase Cα enhances lipopolysaccharide-induced nitric oxide formation in vascular smooth muscle cells
✍ Scribed by Shaohua Li; Freesia L. Huang; Qingping Feng; Jie Liu; Sharon X. Fan; Thomas M. McKenna
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 241 KB
- Volume
- 176
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
Our previous studies showed that lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis in cardiovascular tissues is attenuated by protein kinase C (PKC) inhibitors. In the current study, we identify a specific PKC isotype involved in the LPS signal transduction pathway that leads to NO formation in rat vascular smooth muscle cells (VSMC). VSMC were transfected with a mammalian expression vector containing a full length PKCa cDNA insert, and a stable transfectant overexpressing PKCa was obtained as evidenced by increased expression of PKCa mRNA and protein. In response to 100 ng/ml LPS stimulation, the PKCa transfectants showed a 1.8-fold increase in PKC activity at 30 min and a twofold increase in NO production over 24 hr compared with cells transfected with control plasmids. The LPS-stimulated increase in NO synthesis in PKCa transfectants was blocked by the specific PKCa inhibitor Go ¨6976. After 6 hr LPS treatment, PKCa-transfected and control cells showed equivalent increases in mRNA and protein 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 PKCa-transfected and control cells after LPS stimulation. However, mRNA levels for GTP cyclohydrolase I, a key enzyme in (6R)-tetrahydro-L-biopterin synthesis, and cationic amino acid transporter-2, involved in Larginine transport, was enhanced in cells overexpressing PKCa compared with control cells. These results suggest that PKCa plays an important role in LPSinduced 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.