GTPCH Expression Research Articles

Glucocorticoids elevate GTP cyclohydrolase I mRNA levels in vivo and in PC12 cells.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the formation of tetrahydrobiopterin, the cofactor for catecholamine, indolamine and nitric oxide biosynthesis. The effect of glucocorticoids on GTPCH gene expression was examined by direct infusion of cortisol to rats and by incubation of PC12 cells with glucocorticoids. Northern blot analysis revealed that infusion of cortisol for 1 or 7 days elevated levels of the 3.6 kb GTPCH mRNA species in rat adrenal medulla, while the 1.2 kb mRNA species were only increased by 1 day cortisol. Cortisol administration to hypophysectomized animals elicited a 4-5-fold elevation in both forms of GTPCH mRNA. These results indicate that glucocorticoids may be directly involved in the regulation of adrenomedullary GTPCH mRNA levels by physiological stress. Incubation of PC12 cells with plasma from immobilized, but not control, animals increased the level of the 3.6 kb mRNA. Treatment of PC12 cells with dexamethasone for 12-48 h elicited a 4-6-fold elevation in both GTPCH mRNAs. Using the nuclear run-on assay, increased transcription of the GTPCH gene was observed in the rat adrenal medulla with immobilization stress, or in PC12 cells treated with dexamethasone. This is the first report that glucocorticoids can alter GTPCH expression.

Vascular inducible nitric oxide synthase gene therapy: Requirement for guanosine triphosphate cyclohydrolase I

Human inducible nitric oxide synthase (iNOS) gene transfer inhibits myointimal hyperplasia in vitro. However, unstimulated vascular smooth muscle cells (SMC) do not synthesize tetrahydrobiopterin (BH4), an essential cofactor for iNOS, which may be an obstacle to successful vascular iNOS gene therapy. We investigated the capacity of gene transfer of guanosine triphosphate (GTP) cyclohydrolase I (GTPCH), the rate-limiting enzyme for BH4 biosynthesis, to supply cofactor for iNOS activity. A human GTPCH expression plasmid (pCIS-GTPCH) was transfected into rat aortic SMC (RAOSMC) and BH4-deficient NIH3T3 cells engineered to stably express human iNOS (3T3-iNOS). GTPCH activity and intracellular biopterins were assessed as a measure of successful transfection, and the capacity of GTPCH to reconstitute iNOS activity was used to determine whether BH4 was made available to the iNOS protein. The pCIS-GTPCH-transfected 3T3 cells had demonstrable GTPCH activity as compared with control cells (169.3 +/- 6.6 pmol/hr/mg versus 0, p < 0.001). Intracellular biopterin levels were also increased in transfected 3T3 and SMC (60.6 +/- 2.6 and 101.7 +/- 28.3 pmol/mg, respectively, versus less than 4 in control cells). GTPCH reconstituted near-maximal iNOS activity in 3T3-iNOS cells despite a gene transfer efficiency of less than 1%. GTPCH and iNOS enzymes did not have to coexist in the same cell for the synthesized BH4 to support iNOS activity. GTPCH gene transfer reconstitutes iNOS activity in BH4-deficient cells despite poor transfer efficiency. GTPCH can deliver a cofactor to targeted cells even if it is synthesized in neighboring cells, and may be a means to concurrently deliver BH4 with iNOS in vivo.