Abstract
Tetrahydrobiopterin (BH4) is a limiting cofactor for nitric oxide synthase (NOS) catalysed conversion of L-arginine to nitric oxide and citrulline. Content of BH4 in mammalian cells is regulated at many levels, but most important is de novo biosynthesis from GTP. GTP cyclohydrolase (GTPCH) is the rate-limiting enzyme for the de novo synthesis of BH4. While various immunostimulants, hormones and growth factors have been reported to increase GTPCH mRNA levels and intracellular biopterin (BH4 degradation product), it is not known whether these factors act at the level of GTPCH gene transcription. To test this I utilised 1, 3 and 6 kb 5'upstream GTPCH gene sequence in a secreted alkaline phosphatase reporter vector (SEAP). These constructs were stably transfected in PC-12 cells and rat aortic smooth muscle cells, and the cells were treated with various immunostimulants and growth factors in order to determine whether these factors could enhance GTPCH gene transcription. Intracellular biopterin levels were also measured to confirm that the upregulation of the SEAP-reporter correlated with a rise in biopterin. Our investigations conclude that transcriptional regulation of the GTPCH gene is indeed a major site for control of intracellular BH4 levels. In further experiments, we have characterised the binding of [3H]BH4 to endothelial NOS (eNOS) and examined influences of the substrate, arginine, on the BH4 binding. In addition we selected tetrahydropterins (that support NOS catalysis) and dihydropterins (that are catalytically incompetent) to determine the extent to which modifications of BH4 alter pterin binding affinity to eNOS. Dihydropterins are unable to support NOS catalysis. Studies showed for the first time that dihydropterins, but not tetrahydropterins, support superoxide generation by eNOS. We also have determined that eNOS may be able to produce NO in the absence of BH4 cofactor from the reaction intermediate hydroxyarginine. We have characterised this reaction and are able to provide a plausible mechanism for the NOx generation from eNOS in the absence of BH4 cofactor.