CLINICAL RESEARCH: PRECLINICAL STUDY
Reversal of Endothelial Nitric Oxide Synthase Uncoupling and Up-Regulation of Endothelial Nitric Oxide Synthase Expression Lowers Blood Pressure in Hypertensive Rats
Huige Li, MD, PhD*,*,
Klaus Witte, MD ,
Michael August, MS ,
Isolde Brausch, BS*,
Ute Gödtel- Armbrust, BS*,
Alice Habermeier, BS*,
Ellen I. Closs, PhD*,
Mathias Oelze, PhD,
Thomas Münzel, MD, PhD and
Ulrich Förstermann, MD, PhD*
* Department of Pharmacology
Department of Internal Medicine II, Johannes Gutenberg University, Mainz, Germany
Institute of Pharmacology and Toxicology, Faculty of Clinical Medicine Mannheim, Ruprecht Karls University Heidelberg, Mannheim, Germany
Manuscript received December 15, 2005;
revised manuscript received January 13, 2006,
accepted January 17, 2006.
* Reprint requests and correspondence: Dr. Huige Li, Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany (Email: HuigeLi{at}mail.Uni-Mainz.de).
OBJECTIVES: We sought to examine the hypothesis that a pharmacologic up-regulation of endothelial nitric oxide synthase (eNOS) combined with a reversal of eNOS uncoupling provides a protective effect against cardiovascular disease.
BACKGROUND: Many cardiovascular diseases are associated with oxidant stress involving protein kinase C (PKC) and uncoupling of eNOS.
METHODS: Messenger ribonucleic acid (mRNA) expression was analyzed with RNase protection assay or quantitative real-time polymerase chain reaction, vascular nitric oxide (NO) with spin trapping, and reactive oxygen species (ROS) with dihydroethidium fluorescence.
RESULTS: Aortas of spontaneously hypertensive rats (SHR) showed an elevated production of ROS when compared with aortas of Wistar-Kyoto rats (WKY). The aortic expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox1, Nox2, Nox4, and p22phox) was higher in SHR compared with WKY. In SHR, aortic production of ROS was reduced by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), indicating eNOS "uncoupling" in hypertension. Oral treatment with the PKC inhibitor midostaurin reduced aortic Nox1 expression, diminished ROS production, and reversed eNOS uncoupling in SHR. Aortic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) were significantly reduced in SHR compared with WKY. Midostaurin normalized BH4 levels in SHR. In both WKY and SHR, midostaurin increased aortic expression of eNOS mRNA and protein, stimulated bioactive NO production, and enhanced relaxation of the aorta to acetylcholine. Midostaurin lowered blood pressure in SHR and, to a lesser extent, in WKY; the compound did not change blood pressure in WKY made hypertensive with L-NAME.
CONCLUSIONS: Pharmacologic interventions that combine eNOS up-regulation and reversal of eNOS uncoupling can markedly increase bioactive NO in the vasculature and produce beneficial hemodynamic effects such as a reduction of blood pressure.
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Abbreviations and Acronyms
| | ANOVA = analysis of variance | | BH4 = (6R)-5,6,7,8-tetrahydro-L-biopterin | | eNOS = endothelial nitric oxide synthase | | L-NAME = NG-nitro-L-arginine methyl ester | | NO = nitric oxide | | Nox = NADPH oxidase | | PKC = protein kinase C | | RNA = ribonucleic acid | | ROS = reactive oxygen species | | RT-PCR = real-time polymerase chain reaction | | SHR = spontaneously hypertensive rats | | WKY = Wistar-Kyoto rats |
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