Determinants of Vascular Responsiveness to Endogenous and Exogenous Activators of Guanylate Cyclases

This thesis describes studies to investigate the regulation of guanylate cyclases pathways in blood vessels. Endogenous iS-nitrosothiols have been implicated in the regulation of soluble guanylate cyclase (sGC), by modulating nitric oxide (NO) bioactivity. To determine whether the biotransformation of S-nitrosothiols by isolated rat aorta is enzyme-dependent, stereoisomers of S-nitrosoglutathione (GSNO), S-nitrosocysteine (CYSNO) and S-nitroso-N-acety 1-penicillamine (SNAP) were synthesised and their decomposition and relaxant effects characterised. Decomposition of these S-nitrosothiol stereoisomers by Cu(I), Cu(II), Cu/Zn superoxide dismutase (Cu/Zn SOD) or rat aorta was equivalent (P>0.05). Similarly, the vasorelaxant activity of iS-nitrosothiol stereoisomers was equipotent (P>0.05). The selective Cu(I) chelator, bathocuproine disulfonic acid (BCS), blocked the decomposition of 5'-nitrosothiol stereoisomers by Cu(I), Cu(II), Cu/Zn SOD and rat aorta (P<0.05) and significantly inhibited their relaxant effects (P<0.05). These studies suggest that in rat aorta, there are no stereospecific vasorelaxant effects of S-nitrosothiols, consistent with nonenzymatic release of NO. Biotransformation of iS-nitrosothiols is, in part, dependent on Cu(I) ions. The sensitivity of sGC to NO and particulate guanylate cyclase (pGC) to atrial natriuretic peptide (ANP) regulates vasodilatation in response to these mediators. To determine the role of endogenous NO as a feedback regulator of sGC and pGC, rat aorta was incubated in vitro with bacterial lipopolysaccharide (LPS) to mimic many aspects of sepsis. LPS produced “high output” NO from inducible nitric oxide synthase (iNOS) and reduced the potency of the direct (SPER-NONOate, sodium nitroprusside, GSNO and BAY 58-2667) and indirect (acetylcholine and histamine) sGC activators. iNOS (1400W) but not cyclooxygenase (indomethacin) inhibition, preserved acetylcholineand SPER-NONOate-dependent relaxations in LPS-treated vessels (P<0.05). LPS reduced the potency of 8-bromo-cyclic guanosine-3’,5’monophosphate (P<0.05), but not forskolin (adenylate cyclase activator) or 8-bromocyclic adenosine-3’,5’-monophosphate (P>0.05). LPS also reduced the potency of the pGC activators C-type natriuretic peptide and ANP. 1400W, the sGC inhibitor 1H[l,2,4]Oxadiazolo[4,3-a]quinoxalin-l-one (ODQ) or both, preserved relaxations to ANP in LPS-treated vessels. 1400W and/or ODQ also reversed established desensitisation to ANP within minutes (all P<0.05 versus LPS alone). These results