Role of tetrahydrobiopterin in NO biosynthesis

Biomedical research of the last decade in areas as diverse as human memory, penile erection and immune response has revealed an important signal transduction mechanism in which nitric oxide (NO) acts as a cellular messenger. In the vascular system, NO lowers blood pressure and keeps blood vessels patent against overwhelming odds in a number of diseases such as hardening of the vessel wall (atherosclerosis), diabetic angiopathy, and lack of oxygen (ischemic syndromes). The current project aims at enhancing our understanding of NO biosynthesis. NO is synthesized from the amino acid L-arginine by a family of enzymes termed NO synthases (NOS). Three major isoforms of NOS have been described: a Ca2+-dependent enzyme mainly expressed in neuronal cells, an enzyme first identified in vascular endothelial cells which is activated by both Ca2+-dependent and -independent mechanism resulting in dilatation of resistance vessels, and an enzyme induced by cytokines under infectious and inflammatory conditions in macrophages, smooth muscle cells, cardiac myocytes and many other tissues. The NOS reaction is a complex redox process that is only partially understood. Oxidation of L-arginine occurs in two steps via formation of the intermediate NG-hydroxy-L-arginine, which undergoes immediate oxidative cleavage to NO and L-citrulline. Both reaction steps are catalyzed by a cytochrome P450-type prosthetic heme group that requires NADPH-derived electrons for reductive activation of O2 , which serves as a co-substrate. Unlike other P450s, NOS requires the pterin derivative tetrahydrobiopterin (BH4) for activity. Recent studies have revealed a novel redox function of BH4 involving formation of a pterin radical as intermediate of L-arginine oxidation. In the course of the present project we will study the mechanism of the NOS reaction with emphasis given to the processes leading to formation of this pterin radical and its precise function. The experimental work will involve biochemical and biophysical studies with wild-type and mutated recombinant human NOS proteins obtained from appropriate expression systems. The heme complexes and carbon-centered radicals occurring as reaction intermediates will be identified by light absorption, Raman, and EPR spectroscopy. Part of the studies will be done in collaboration with researchers in France, Norway and Japan, who have developed sophisticated methods that allow detection of these species in a subsecond time frame. As NOS is usually not saturated with BH4 when expressed in tissues, we will give special emphasis on the reaction products formed by the enzyme at limited BH4 availability. Under these conditions, NOS may generate reactive oxygen species such as superoxide radical and hydrogen peroxide, which may contribute to tissue injury in cardiovascular disorders. The pathophysiological consequences of limited BH4 availability will be studied in isolated perfused hearts. These experiments will involve the determination of cardiac BH4 levels under normal and pathophysiological conditions, e.g. ischemia/reperfusion injury, and monitoring of cardiac function (myocardial contractility, coronary blood flow) in the presence of agents that either increase or decrease BH4 levels in the heart. In summary, this project is expected to provide important new information on the regulation of NO synthesis by BH4 and to reveal new therapeutic strategies for the treatment of cardiovascular diseases.

Biomedical research of the last decade in areas as diverse as human memory, penile erection and immune response has revealed an important signal transduction mechanism in which nitric oxide (NO) acts as a cellular messenger. In the vascular system, NO lowers blood pressure and keeps blood vessels patent against overwhelming odds in a number of diseases such as hardening of the vessel wall (atherosclerosis), diabetic angiopathy, and lack of oxygen (ischemic syndromes). The current project aims at enhancing our understanding of NO biosynthesis. NO is synthesized from the amino acid L-arginine by a family of enzymes termed NO synthases (NOS). Three major isoforms of NOS have been described: a Ca2+-dependent enzyme mainly expressed in neuronal cells, an enzyme first identified in vascular endothelial cells which is activated by both Ca2+-dependent and -independent mechanism resulting in dilatation of resistance vessels, and an enzyme induced by cytokines under infectious and inflammatory conditions in macrophages, smooth muscle cells, cardiac myocytes and many other tissues. The NOS reaction is a complex redox process that is only partially understood. Oxidation of L-arginine occurs in two steps via formation of the intermediate NG-hydroxy-L-arginine, which undergoes immediate oxidative cleavage to NO and L-citrulline. Both reaction steps are catalyzed by a cytochrome P450-type prosthetic heme group that requires NADPH-derived electrons for reductive activation of O2, which serves as a co-substrate. Unlike other P450s, NOS requires the pterin derivative tetrahydrobiopterin (BH4) for activity. Recent studies have revealed a novel redox function of BH4 involving formation of a pterin radical as intermediate of L-arginine oxidation. In the course of the present project we will study the mechanism of the NOS reaction with emphasis given to the processes leading to formation of this pterin radical and its precise function. The experimental work will involve biochemical and biophysical studies with wild-type and mutated recombinant human NOS proteins obtained from appropriate expression systems. The heme complexes and carbon-centered radicals occurring as reaction intermediates will be identified by light absorption, Raman, and EPR spectroscopy. Part of the studies will be done in collaboration with researchers in France, Norway and Japan, who have developed sophisticated methods that allow detection of these species in a subsecond time frame. As NOS is usually not saturated with BH4 when expressed in tissues, we will give special emphasis on the reaction products formed by the enzyme at limited BH4 availability. Under these conditions, NOS may generate reactive oxygen species such as superoxide radical and hydrogen peroxide, which may contribute to tissue injury in cardiovascular disorders. The pathophysiological consequences of limited BH4 availability will be studied in isolated perfused hearts. These experiments will involve the determination of cardiac BH4 levels under normal and pathophysiological conditions, e.g. ischemia/reperfusion injury, and monitoring of cardiac function (myocardial contractility, coronary blood flow) in the presence of agents that either increase or decrease BH4 levels in the heart. In summary, this project is expected to provide important new information on the regulation of NO synthesis by BH4 and to reveal new therapeutic strategies for the treatment of cardiovascular diseases.