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 5-mtroso-N-acetyl-pemcillamine (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 (PO.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-nifrosothiols, consistent with non- enzymatic release of NO. Biotransformation of S-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 acetylcholine- and SPER-NONOate-dependent relaxations in LPS-treated vessels (PO.05). LPS reduced the potency of 8-bromo-cyclic guanosine-3',5'- monophosphate (PO.05), but not forskolin (adenylate cyclase activator) or 8-bromo- cyclic 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 PO.05 versus LPS alone). These results indicate that sGC and pGC signalling are desensitised following exposure to LPS, consistent with a compensatory mechanism offsetting high-output NO production by iNOS. This effect is specific to cGMP-dependent pathways since the cAMP pathway was unaltered. The mechanism of desensitisation is likely to involve a reversible biochemical change, since the responsiveness was restored immediately following removal of excess NO (by 1400W) or cGMP (by ODQ). These observations suggest that inflammatory cardiovascular disorders associated with excessive NO production (i.e. septic shock) are characterised by specific impairment of GC-cGMP-mediated vasorelaxation that is mediated, at least in part by cGMP, and readily reversible. To investigate the mechanism(s) of iNOS-derived NO-mediated desensitisation of sGC and pGC, pharmacological inhibition of phosphodiesterases (type 5 (sildenafil), 1A1 (vinpocetine), and 3 (milrinone)), protein phosphatase 2A (okadaic acid and cantharidic acid), superoxide anion (SOD) and myeloperoxidase (4-aminobenzoic hydrazide) were used to probe the role of these enzymes. Desensitisation of ANP- and NO-mediated dilatation by iNOS-derived NO was unaffected by inhibition of these enzymes (all P > 0.05 versus LPS alone). However, activation of protein kinase C by phorbol 12-myristate 13-acetate and thymeleatoxin caused desensitisation of both guanylate and adenylate cyclases. If PKC activation causes LPS-induced desensitisation of sGC and pGC, then other mechanisms must preserved cAMP-mediated relaxation.

The contribution of different calcium-activated potassium channel subtypes to agonist-evoked whole-cell currents was studied in cultured pig coronary artery endothelial cells. From a resting membrane potential of-5.9 0.5mV (n=102), 1-lOuM ATP, 1-1 OnM substance P and 1-lOOnM bradykinin hyperpolarised cell rafts to -50.7 1.6mV (n=76), -45.7 4.7mV (n=19) and -59.1 3.5mV (n=16), respectively. In small clusters of cells, 1 OuM ATP evoked outward currents which reversed close to EK and were sensitive to both the SKca channel blocker UCL 1848 (IC5o 1.2nM -65% maximal block) and the IKca/BKca channel blocker charybdotoxin (-85% block at 30-100nM). Surprisingly lOuM clotrimazole, a non selective blocker of IKca channels, abolished ATP-evoked currents in a total of three out of five cells. This requires further study. ImM 1-EBIO, which increases the calcium sensitivity of SKca and IKca channels, activated currents which were sensitive to lOOnM UCL 1848 and luM clotrimazole (blocked by 57.0 15.1% (n=3) and 89.0 1.6% (n=4), respectively). When applied in combination, these two blockers essentially abolished 1-EBIO evoked currents. Buffering intracellular calcium to 1.5uM activated outward currents which were sensitive to lOOnM UCL 1848, lOOnM charybdotoxin and lu.M clotrimazole (blocked by 28.3 5.4% (n=27), 101.2 0.5% (n=3), and 82.6 3.7% (n=22), respectively. Plasma membrane delimited expression of the SK3 channel protein was detected using fluorescence immunohistochemistry. In many vessels endothelium-derived hyperpolarising factor (EDHF)- mediated vasodilation is abolished by a combination of SKca and IKca channel blockers, which are frequently ineffective when applied alone. This has led some to suggest the existence of a novel channel with unusual pharmacology. The present study demonstrates, however, that separate SKca and IKca channels contribute to endothelial cell currents underlying the EDHF pathway. Based on protein expression and UCL 1848-sensitivity it is further proposed that the contributing SKca channels are formed of SK3 subunits.

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