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

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 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 (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 (nϭ8; PϽ0.01). 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 Ϫ10 to 10 Ϫ8 mol/L) were significantly reduced compared with those in control and AdCMVGFP-transduced arteries (nϭ7; PϽ0.05). 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.

In vivo stimulatory effect of erythropoietin on endothelial nitric oxide synthase in cerebral arteries

“…In a series of previous studies, we demonstrated that intracisternal gene delivery resulted in expression of recombinant protein in adventitial fibroblasts of cerebral arteries (3,12,13,25,36,42). In the present study, successful gene transfer was confirmed by the increased levels of Epo in CSF and by the detection of Epo in the AdEpo-transduced basilar arteries.…”

The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo

“…Comparisons with WT mice may be less revealing because the phosphorylation state of the S1179 residue in WT mice cannot be controlled and because the expression levels in the transgenic mice were less than WT eNOS levels. In this regard, reconstitution of eNOS KO mice with low levels of either S1179A or S1179D transgenes may explain why we did not observe compensatory effects of increased transgene expression that have been reported in some (28) but not other (12,27) systems.…”

“…To date, the effects of S1179A and S1179D mutants have been characterized in cultured cells (12,14,26) and using ex vivo gene transfer into isolated vessels (12,27,28). Both S1179A and S1179D eNOS are enzymatically active, so S1179…”

Endothelial nitric oxide synthase is critical for ischemic remodeling, mural cell recruitment, and blood flow reserve

“…Moreover, substitution of aspartate for serine mimics the negative charge imparted by the phosphate and renders eNOS constitutively active by increasing the rate of electron flux through the protein and increases basal NO production severalfold (21). The delivery method and activity of this construct has been well characterized (22)(23)(24). As seen in Fig.…”