Laminar shear stress up-regulates inducible nitric oxide synthase in the endothelium.
Shear stress caused by blood flow is a potent physiological stimulus for the generation of nitric oxide (NO) in endothelial cells, which is believed to derive from the up-regulation and post-transcriptional activation of endothelial constitutive NO synthase (ecNOS). However, it has yet to be demonstrated that inducible NO synthase (iNOS) plays a significant role in shear stress-induced NO production from endothelial cells. We used parallel plate-type flow chambers that detect fluid shear stress to determine that shear stress, as quantified by a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), increased iNOS gene transcripts in cultured endothelial cells, which resulted in increased NO production. Shear stress-induced iNOS expression was inhibited by pyrrolidine dithiocarbamate (PDTC), an antioxidant and nuclear factor kappaB (NF-kappaB) blocker, and by MG132, an aldehyde peptide proteasome inhibitor that antagonizes I kappaB-kinase. Laminar shear stress increased the transcriptional activity of NF-kappaB, whereas over-expression of an I kappaB-alpha mutant that inhibits the activation of NF-KB in a dominant-negative fashion was found to attenuate the induction of endothelial iNOS by shear stress. The present results demonstrate that shear stress induces iNOS in the endothelium, mainly via the activation of NF-kappaB.
Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan.
Naoko
N
Ozawa
Masayoshi
M
Shichiri
Masatora
M
Iwashina
Nozomi
N
Fukai
Takanobu
T
Yoshimoto
Yukio
Y
Hirata
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D007976:Leupeptins / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013314:Stress, Mechanical; D013859:Thiocarbamates / Q000494:pharmacology; D015854:Up-Regulation / Q000502:physiology
D000975:Antioxidants; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D016328:NF-kappa B; D012333:RNA, Messenger; D013859:Thiocarbamates; C066229:prolinedithiocarbamate; D011392:Proline; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde