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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:672-679.)
© 1997 American Heart Association, Inc.

Articles

N--Tosyl-L-Lysine Chloromethylketone Prevents Expression of iNOS in Vascular Smooth Muscle by Blocking Activation of NF-B

Valérie B. Schini-Kerth; Matthias Boese; Rudi Busse; Beate Fisslthaler; ; Alexander Mülsch

From the Center of Physiology, University Clinic of Frankfurt, Frankfurt, Germany.

Correspondence to V.B. Schini-Kerth, PhD, Zentrum der Physiologie, Klinikum der JWG–Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany.

Abstract Certain cytokines and lipopolysaccharide stimulate expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle, an event that is regulated at the transcriptional level and appears to involve several transcription factors, including nuclear factor B (NF-B). Since proteases play an essential role in NF-B activation, experiments were designed to clarify, in both cultured rat aortic smooth muscle cells (SMCs) and isolated rat aortas, whether protease inhibitors affect the interleukin-1ß (IL-1ß)–elicited expression of iNOS. The formation of NO was assessed by nitrite release in cultured SMCs and the attenuation of phenylephrine-induced contraction in aortic rings, the expression of iNOS by Western blot analysis and reverse transcription–polymerase chain reaction, and NF-B activity in nuclear extracts by gel electrophoretic mobility shift assay. Exposure of cultured SMCs to IL-1ß increased NF-B binding activity within 30 minutes and was associated with nitrite accumulation and the appearance of iNOS protein 24 hours later. These responses were abolished in cells that had been exposed to the cytokine in the presence of the protease inhibitor N--tosyl-L-lysine chloromethylketone. Aprotinin and p-toluenesulfonyl-L-arginine methyl ester, two other protease inhibitors, also reduced the cytokine-stimulated release of nitrite and the level of iNOS protein. Exposure of rat aortic segments without endothelium to IL-1ß activated NF-B within 30 minutes and was associated with the appearance of iNOS mRNA and an attenuation of phenylephrine-induced contraction 6 hours later. These responses were blunted when the segments were incubated with the cytokine and N--tosyl-L-lysine chloromethyl ketone. The present observations indicate that protease inhibitors prevent iNOS expression in both cultured and native vascular SMCs by blocking the activation of NF-B.

Key Words: inducible nitric oxide synthase • interleukin-1ß • nuclear factor-B • vascular reactivity • vascular smooth muscle

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