Expression and function of recombinant S1179D endothelial nitric oxide synthase in canine cerebral arteries

Masahiko Akiyama, Daihiko Eguchi, Deborah Weiler, Timothy O'Brien, Imre Kovesdi, Ramona S. Scotland, William C. Sessa, Zvonimir S Katusic

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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 SI179DeNOS 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, NG-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.

Original languageEnglish (US)
Pages (from-to)1071-1076
Number of pages6
JournalStroke
Volume33
Issue number4
DOIs
StatePublished - 2002

Fingerprint

Cerebral Arteries
Nitric Oxide Synthase Type III
Canidae
NG-Nitroarginine Methyl Ester
Endothelin-1
Arteries
Vasoconstrictor Agents
Nitric Oxide
Genes
Cerebrovascular Disorders
Adventitia
Middle Cerebral Artery
Green Fluorescent Proteins
Transgenes
Reporter Genes
Aspartic Acid
Protein Kinases
Serine
Radioimmunoassay
Blood Vessels

Keywords

  • Cerebral arteries
  • Dogs
  • Gene transfer
  • Nitric oxide

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Neuroscience(all)

Cite this

Expression and function of recombinant S1179D endothelial nitric oxide synthase in canine cerebral arteries. / Akiyama, Masahiko; Eguchi, Daihiko; Weiler, Deborah; O'Brien, Timothy; Kovesdi, Imre; Scotland, Ramona S.; Sessa, William C.; Katusic, Zvonimir S.

In: Stroke, Vol. 33, No. 4, 2002, p. 1071-1076.

Research output: Contribution to journalArticle

Akiyama, M, Eguchi, D, Weiler, D, O'Brien, T, Kovesdi, I, Scotland, RS, Sessa, WC & Katusic, ZS 2002, 'Expression and function of recombinant S1179D endothelial nitric oxide synthase in canine cerebral arteries', Stroke, vol. 33, no. 4, pp. 1071-1076. https://doi.org/10.1161/hs0402.105553
Akiyama, Masahiko ; Eguchi, Daihiko ; Weiler, Deborah ; O'Brien, Timothy ; Kovesdi, Imre ; Scotland, Ramona S. ; Sessa, William C. ; Katusic, Zvonimir S. / Expression and function of recombinant S1179D endothelial nitric oxide synthase in canine cerebral arteries. In: Stroke. 2002 ; Vol. 33, No. 4. pp. 1071-1076.
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AU - Akiyama, Masahiko

AU - Eguchi, Daihiko

AU - Weiler, Deborah

AU - O'Brien, Timothy

AU - Kovesdi, Imre

AU - Scotland, Ramona S.

AU - Sessa, William C.

AU - Katusic, Zvonimir S

PY - 2002

Y1 - 2002

N2 - 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 SI179DeNOS 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, NG-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.

AB - 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 SI179DeNOS 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, NG-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.

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KW - Dogs

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