In the vasculature it is well established that cGMP is involved in the relaxant response to nitric oxide (NO) and NO donors. However, there is an increasing evidence that alternative/additional pathways that are cGMP-independent may also exist. A key criterion for a response to NO or a NO donor drug to be classified as cGMP-independent is lack of (or incomplete) inhibition by the selective inhibitor of soluble guanylate cyclase, ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). In many blood vessels cGMP-independent mechanisms contribute to the vasorelaxation, and in certain vascular beds cGMP-independent relaxation may be the predominant mechanism of action of NO and NO donors. NO donor drugs that generate NO "spontaneously", like authentic NO (i.e. solutions of NO gas), appear to exhibit a larger component of cGMP-independent vasorelaxation than do those drugs that require bioactivation in the tissue. The long lasting inhibition of responses to vasoconstrictors by S-nitrosothiols, persisting after removal of these NO donors, may be a cGMP-independent process, at least in some vessels. The mechanisms involved in the inhibition of vascular growth by NO and NO donors are predominantly cGMP-independent, as are the mechanisms responsible for the effects of NO donors on apoptosis in vascular smooth muscle and endothelial cells. The ability of NO and NO donors to inhibit platelet aggregation has a significant cGMP-independent component. cGMP-independent pathways are most often, though not exclusively, seen at high concentrations (microM - mM) of NO and NO donors. Hence, in relation to the actions of endogenous NO, these pathways may be particularly important in settings when the inducible isoform of NO-synthase is expressed. Furthermore, cGMP-independent pathways are enhanced in animal models of atherosclerosis and ischaemia. This suggests that it may be possible to target cGMP-independent mechanisms with selected NO donors in disease states.

1 In rat pulmonary artery pre-contracted with phenylephrine, the mechanisms of relaxation to the nitric oxide (NO) donor, spermine NONOate, were investigated. 2 Responses to spermine NONOate were only partially blocked by the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]Oxadiazolo-[4,3,-a]quinoxalin-1-one) at concentrations up to 30 mM. Ten mM ODQ gave maximal inhibition. Endothelium removal had no e ect on the potency of spermine NONOate or its inhibition by ODQ. 3 The protein kinase G inhibitor, Rp-8-Br-cGMPS (100 mM), caused minimal inhibition of spermine NONOate despite causing marked inhibition of glyceryl trinitrate and isosorbide dinitrate. 4 Spermine NONOate (100 mM) caused a 35 fold increase in guanosine 3'5' cyclic monophosphate (cyclic GMP) above basal levels in pulmonary artery rings. ODQ (3 mM) abolished this cyclic GMP production but did not inhibit corresponding relaxant responses. Similar results were seen with another NONOate (MAHMA NONOate; 10 mM). 5 ODQ-resistant relaxation to spermine NONOate (i.e. relaxation seen in the presence of 10 mM ODQ) was inhibited by potassium (80 mM), charybdotoxin (300 nM), iberiotoxin (300 nM), apamin (100 nM), ouabain (1 mM) or thapsigargin (100 nM) but not by 4-aminopyridine (3 mM), glybenclamide (10 mM) or diltiazem (10 mM). 6 Potassium, charybdotoxin, ouabain and thapsigargin also inhibited ODQ-resistant relaxation to FK409 ((+)-E-4-ethyl-2-[E-hydroxyimino]-5-nitro-3-hexenamide). 7 We conclude that, on rat pulmonary artery, spermine NONOate can produce cyclic GMPindependent relaxation that involves, at least in part, activation of Na + /K + -ATPase, sarcoendoplasmic reticulum Ca 2+ -ATPase and calcium-activated potassium channels.

ODQ, (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase) inhibits vasorelaxant responses to nitric oxide (NO)-donor drugs, but the extent of the inhibition varies depending on the NO donor studied. The purpose of this study was to test the hypothesis that these variations in the effects of ODQ reflect differences in the mechanisms whereby each NO donor generates NO. On pulmonary artery preparations pre-contracted submaximally with phenylephrine, ODQ (3 microM) almost abolished the relaxant responses to glyceryl trinitrate, isosorbide dinitrate and nitroprusside; each of these drugs requires activation in the tissue (by enzymes or reducing agents) to generate NO. In contrast, ODQ (3 microM) caused a parallel shift in the concentration-relaxation curves to linsidomine (SIN-1), FK409, MAHMA NONOate and spermine NONOate (1.63 to 2.54 log units) with no depression in maximum response; each of these NO donors generates NO in the physiological bathing solution without requiring tissue activation. For the four drugs in this group, the effects of 10 microM ODQ were not significantly greater than the effects of 3 microM ODQ; thus there was an ODQ-resistant component to the response suggesting that part of the response involved a mechanism that was independent of soluble guanylate cyclase. NO donors that require tissue activation probably generate NO within the smooth-muscle cell, whereas those that do not require tissue activation generate NO outside the cell. Hence it is concluded that the site of NO generation (intra- or extracellular) might determine whether or not there is an ODQ-resistant component in the relaxation response.

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1 Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1- {N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory eect with vasorelaxation, to determine the eects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and to investigate the possible role of activation of sarco-endoplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2 ± 10 mg ml 71) and adenosine diphosphate (ADP; 2 mM) in platelet rich plasma. It was (i) more eective at inhibiting aggregation induced by collagen than by ADP, and (ii) less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3 ODQ (10 mM) caused only a small shift (approximately half a log unit) in the concentrationresponse curve to MAHMA NONOate irrespective of the aggregating agent. 4 The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole; 1 ± 100 mM), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1 ± 1 mM), caused minimal inhibition. 5 On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 mM present) were abolished by thapsigargin (200 nM). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6 Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7 Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQresistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.