In vivo expression and function of recombinant GTPCH I in the rabbit carotid artery

1 Departments of Anesthesiology, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905; 2 GenVec, Gaithersburg, Maryland 20878; and 3 Department of Medicine, National University of Ireland, Galway, Ireland Submitted 25 July 2003 ; accepted in final form 5 October 2003 Tetrahydrobiopterin (BH4) is an essential co-factor for endothelial nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. This 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 10 9 plaque-forming units of Ad-encoding GTPCH I (AdGTPCH) or -galactosidase as a control. The rabbits were euthanized 72 h later, and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4, and oxidized biopterin levels were detected with the use of HPLC, and cGMP was measured with the use of 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 to 10 –9 M), endothelium-dependent relaxation to acetylcholine (10 –5 to 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. nitric oxide synthase; tetrahydrobiopterin; adenovirus Address for reprint requests and other correspondence: Z. S. Katusic, Anesthesia Research, 200 First St. SW, Rochester, MN 55905 (E-mail: katusic.zvonimir{at}mayo.edu ).