title: Expression and function of recombinant S1179D endothelial nitric oxide synthase in canine cerebral arteries.
abstract: OBJECTIVE
  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 degrees C) to an adenoviral vector (10(10) 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.
authors.affiliation: Department of Anesthesiology, Mayo Clinic and Foundation, Rochester, Minn 55905, USA.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Akiyama
authors.forename: Daihiko
authors.initials: D
authors.lastname: Eguchi
authors.forename: Deborah
authors.initials: D
authors.lastname: Weiler
authors.forename: Timothy
authors.initials: T
authors.lastname: O'Brien
authors.forename: Imre
authors.initials: I
authors.lastname: Kovesdi
authors.forename: Ramona S
authors.initials: RS
authors.lastname: Scotland
authors.forename: William C
authors.initials: WC
authors.lastname: Sessa
authors.forename: Zvonimir S
authors.initials: ZS
authors.lastname: Katusic
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D002536:Cerebral Arteries / Q000187:drug effects / Q000201:enzymology*; D006152:Cyclic GMP / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D017930:Genes, Reporter; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes / Q000502:physiology; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D014666:Vasomotor System / Q000187:drug effects / Q000502:physiology*
chemical_list: D019332:Endothelin-1; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP; D001920:Bradykinin