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1 Departments of Anesthesiology, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA
2 Gen Vec, Inc., Gaithersburg, MD, USA
3 Department of Medicine, National University of Ireland, Galway, Ireland
* To whom correspondence should be addressed. E-mail: katusic.zvonimir{at}mayo.edu.
Tetrathydrobiopterin (BH4) is an essential co-factor for nitric oxide synthase and in particular endothelial
nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. The present study set out to test the hypothesis that in vivo gene transfer of GTPCH I to endothelial cells could increase bioavailability of BH4, enhance biosynthesis of nitric oxide and thereby enhance
endothelium-dependent relaxations mediated by nitric oxide. In vivo gene transfer was carried out by adenovirus(Ad)-mediated delivery into rabbit carotid arteries. Each artery was transduced by 20 min intraluminal incubation of 109 plaque forming units of adenovirus encoding GTPCH I (AdGTPCH) or
-galactosidase as a control. Rabbits were sacrificed 72 hours later and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4 and oxidised biopterin levels were detected using high performance liquid chromatography, and cyclic guanosine monophosphate(cGMP)was measured using a radioimmunoassay. Expression of recombinant proteins was detected predominantly in endothelial cells. Both GTPCH I activity and BH4 levels were increased in arteries transduced with AdGTPCH. However, contraction to phenylephrine (10-5-10-9 M ), endothelium-dependent relaxation to acetylcholine (10-5-10-9 M ) and cGMP levels were not significantly affected by increased expression of GTPCH I. Our results suggest that expression of GTPCH I, in vascular endothelium in vivo, increases intracellular concentration of BH4. However, under physiological conditions it appears that this increase does not affect nitric oxide production in endothelial cells of the carotid artery.
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