beta-very low density lipoprotein enhances inducible nitric oxide synthase expression in cytokine-stimulated vascular smooth muscle cells.
beta-very low-density lipoprotein (beta-VLDL), a collective term for VLDL and chylomicron remnants, has recently shown to potently promote the development of atherosclerosis. However, the effects of beta-VLDL on the accumulation of nitric oxide (NO) and the expression of inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMC) have not been determined. In this study, we measured the accumulation of nitrite, stable metabolite of NO and examined the expression of iNOS protein and mRNA using Western blotting and RT-PCR, respectively, in VSMC. NF-kappaB activation in VSMC was examined by gel retardation assay. Incubation of cell cultures with interleukin-1beta (IL-1beta) for 24 h caused a significant increase in nitrite accumulation. Although beta-VLDL alone did not increase nitrite accumulation in unstimulated VSMC, beta-VLDL significantly enhanced nitrite accumulation in IL-1beta-stimulated VSMC in a time- and dose-dependent manner. beta-VLDL-induced nitrite accumulation in IL-1beta-stimulated VSMC was accompanied by an increase in iNOS protein and mRNA expression. In addition, IL-1beta induced NF-kappaB activation in VSMC, an effect that was increased by the addition of beta-VLDL. Use of specific tyrosine kinase inhibitor herbimycin A, genistein, or PP2 (Src family kinase inhibitor) indicated that tyrosine kinases are required for IL-1beta-stimulated and beta-VLDL-enhanced nitrite accumulation, while specific inhibition of ERK1/2 or p38-MAP kinase had no effects. Our results suggest that beta-VLDL enhances iNOS expression and nitrite accumulation in IL-1beta-stimulated VSMC through tyrosine kinase(s)-dependent mechanisms.
Takahashi
Masafumi
M
Department of Internal Medicine, Division of Cardiology, Jichi Medical School, Minamikawachi-machi, 329-0498, Tochigi, Japan. masafumi@jichi.ac.jp
Takahashi
Sadao
S
Shimpo
Masahisa
M
Naito
Akitaka
A
Ogata
Yukiyo
Y
Kobayashi
Eiji
E
Ikeda
Uichi
U
Shimada
Kazuyuki
K
D000818:Animals; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology*; D008074:Lipoproteins / Q000494:pharmacology; D008079:Lipoproteins, VLDL / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D016328:NF-kappa B / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; D007375:Interleukin-1; D008074:Lipoproteins; D008079:Lipoproteins, VLDL; D016328:NF-kappa B; D009573:Nitrites; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases