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(Stroke. 2002;33:1071.)
© 2002 American Heart Association, Inc.

Original Contributions

Expression and Function of Recombinant S1179D Endothelial Nitric Oxide Synthase in Canine Cerebral Arteries

Masahiko Akiyama, MD; Daihiko Eguchi, MD, PhD; Deborah Weiler, BA, MS; Timothy O’Brien, MD; Imre Kovesdi, PhD; Ramona S. Scotland, PhD; William C. Sessa, PhD Zvonimir S. Katusic, MD, PhD

From the Departments of Anesthesiology and Molecular Pharmacology and Experimental Therapeutics (M.A., D.E., D.W., Z.S.K.) and Division of Endocrinology and Metabolism (T.O’B.), Mayo Clinic and Foundation, Rochester, Minn; GenVec, Inc, Gaithersburg, Md (I.K.); and Department of Pharmacology and Molecular Cardiobiology Program, Yale University School of Medicine, New Haven, Conn (R.S.S., W.C.S.).

Correspondence to Zvonimir S. Katusic, MD, PhD, Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail katusic.zvonimir{at}mayo.edu

Background and Purpose— Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179DeNOS) increases nitric oxide (NO) production constitutively in the absence of agonist stimulus. The present study was designed to determine the effect of mutant S1179DeNOS gene expression on vasomotor function of canine cerebral arteries.

Methods— Isolated basilar and middle cerebral arteries were exposed ex vivo (30 minutes at 37°C) to an adenoviral vector (10¹⁰ plaque-forming units per milliliter) encoding the S1179DeNOS gene (AdCMVS1179DeNOS), the wild-type eNOS gene (AdCMVeNOS), or the green fluorescent protein (GFP) reporter gene (AdCMVGFP). Twenty-four hours after transduction, arteries were suspended in an organ chamber for isometric force recording, and levels of cGMP were measured by radioimmunoassay.

Results— Transgene protein expression was detected mainly in the vascular adventitia. In AdCMVS1179DeNOS-transduced arteries, basal levels of cGMP were significantly elevated compared with those in control (nontransduced), AdCMVGFP-, or AdCMVeNOS-transduced vessels (n=8; P<0.01). The elevation of cGMP was abolished by a NOS inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), or by incubation in the calcium-free medium in the presence of calcium chelators. In AdCMVS1179DeNOS-transduced arteries, contractions to endothelin-1 (10^-10 to 10^-8 mol/L) were significantly reduced compared with those in control and AdCMVGFP-transduced arteries (n=7; P<0.05). The vasoconstrictor effect of endothelin-1 was restored in the presence of the NOS inhibitor L-NAME.

Conclusions— Our results suggest that in cerebral arteries, expression of recombinant S1179DeNOS increases basal production of NO and inhibits the vasoconstrictor effect of endothelin-1. This effect may have therapeutic application in prevention and treatment of cerebrovascular diseases.

Key Words: cerebral arteries • gene transfer • nitric oxide • dogs

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