Physiological regulation and pathological dysfunction of the nitric oxide receptor soluble guanylate cyclase: role in cardiovascular homeostasis and disease (360G-Wellcome-067422_Z_02_A)

Soluble guanylate cyclase (sGC) is a heterodimeric haemoprotein that represents the intracellular receptor for nitric oxide (NO). Activation of the enzyme facilitates formation of the second messenger cyclic GMP and it is this molecule that mediates the majority of biological actions attributed to NO. Due to its ubiquitous nature, inappropriate sGC activity may be fundamental to the aetiology of a wide variety of disease states, exemplified in the cardiovascular system by conditions such as stroke, atherosclerosis and septic shock. Therefore, agents that can modulate sGC function selectively will have considerable therapeutic potential, particularly in the treatment of cardiovascular disease. However, understanding of the expressional, biochemical and pharmacological regulation of sGC under physiological and/or pathological conditions is poor. Therefore, the current proposal is designed to test the hypothesis that regulation of sGC expression and activity is a key determinant of the biological actions of NO and that enzyme dysfunction may contribute to the pathogenesis of vascular disease. The programme of research will comprise a multi-disciplinary approach from molecular studies, in vitro and in vivo pharmacological characterisation to evaluation in humans. Molecular & biochemical studies: Regulation of enzyme expression will be studied by analysis of the promoter regions of the human sGC genes (by luciferase reporter assay) which contain NF-?B, oestrogen, hypoxia and shear stress response elements (each believed to be involved in cardiovascular homeostasis and/or disease). Expression of recombinant human sGC isoforms will yield sufficient quantities of purified protein for studies examining biochemical regulation and enzyme kinetics. In vitro/in vivo pharmacology: Determination of the expression and sensitivity of sGC by pharmacological evaluation using cells in culture, in vitro organ bath/myograph techniques and in vivo studies will provide functional correlates for the molecular and biochemical studies. Relevance to human disease: The presence of polymorphisms within the human sGC genes will be investigated to provide potential links between altered sGC activity and human disease. Studies of platelet aggregation (an index of sGC activity) will be conducted and related to genotype. Polymorphisms identified in the coding region will be expressed as recombinant proteins and enzyme kinetics evaluated; variations in the promoter region will be genetically-engineered and evaluated in the promoter-reporter studies. In summary, the results of this proposal are likely to have important implications for the understanding of the regulation of expression and activity of sGC in health and disease and perhaps provide the rationale for the design of novel therapeutics.