Protein kinase C and protein tyrosine kinase mediate lipopolysaccharide- and cytokine-induced nitric oxide formation in vascular smooth muscle cells of rats.
Rat aorta media, adventitia and cultured vascular smooth muscle cells (VSMCs) were used in this study to identify the source of nitric oxide (NO) generation from various cell types of vascular tissues and to elucidate the mechanisms involved in NO formation. Treatment of vascular media and VSMCs with lipopolysaccharide (LPS) or cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta)] resulted in a dose-dependent increase of NO release. Inducible nitric oxide synthase (iNOS) in the stimulated VSMCs was significantly upregulated as shown by Western blot analysis. Protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) prevented LPS-, TNF-alpha- and IL-1beta-induced NO production, whereas N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide (HA1004), an H7 analogue with little activity towards PKC, had no inhibition effect. The role of PKC in LPS- and cytokine-induced changes on NO formation was confirmed by using another structurally distinct PKC inhibitor chelerythrine. Treatment of VSMCs with protein tyrosine kinase (PTK) inhibitor genistein or tyrphostin AG18 also reduced the NO production evoked by LPS, TNF-alpha or IL-1beta, which was associated with inhibition of iNOS protein expression. In contrast, PKC inhibitor chelerythrine did not affect iNOS expression. These results suggest that PTK mediates LPS- and cytokine-induced NO formation by upregulation of iNOS expression. PKC may be involved in the post-translational modification of iNOS or the regulation of the availability of iNOS substrates and cofactors.
Han
Ya-Ling
YL
Department of Cardiology, General Hospital of Shenyang, The Institute of Cardiovascular Research, PLA 110016. hanyl@mail.sy.ln.cn
Kang
Jian
J
Li
Shao-Hua
SH
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C