cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase.

Abstract

The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound LH- [1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (ICso = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.Nitric oxide (NO), S-nitrosothiols, and other NO donors (1) activate the soluble guanylyl cyclase (SGC) by binding to the heme moiety of the enzyme (2). This results in increased cGMP concentrations that are associated with relaxation in vascular tissue (for review, see ref.3). In addition, these compounds increase cGMP in platelets, and this is thought to be the mechanism by which they inhibit platelet function (4,5).Studies attempting to investigate the role of SGC in the mediation of the physiological actions of NO have been hampered by the lack of a potent and selective inhibitor of this enzyme. Methylene blue has been used as such; however, this compound also inhibits prostacyclin (PGI2) production (6, 7), generates superoxide anions (8-10), and directly inhibits NO synthase (11). Furthermore, LY 83583 (6-anilino-5,8-quinolinedione), which inhibits elevation of cGMP concentrations in a number of tissues (12, 13), probably does so by generating superoxide anions (14).1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) has been shown to inhibit N-methyl-D-aspartate-induced increases in cGMP concentrations in rat brain slices and to inhibit the activity of SGC purified from bovine lung (15). We have now used this compound to characterize the role of SGC in the actions of NO in platelets and vascular tissue.MATERIALS AND METHODS Washed Platelet Suspensions. Human blood was collected, and PGI2-washed platelet (WP) suspensions were prepared as described (16).Platelet cGMP Concentrations. WPs (2-2.5 X 108/ml) were incubated without stirring in a platelet-ionized calcium lumiaggregometer (Chronolog) for 20 min at 37°C in th...

Natriuretic Factors and Nitric Oxide Suppress Plasminogen Activator Inhibitor-1 Expression in Vascular Smooth Muscle Cells

“…42 ODQ prevented SNAPinduced increases in cGMP in RASMCs (data not shown), as described previously. 42 Treatment of RASMCs with ODQ also significantly impaired SNAP's ability to inhibit PAI-1 expression in Ang II-stimulated RASMCs ( Figure 6, bottom left). To examine the potential role of cGMP-dependent protein kinase (cGPK) in the regulation of PAI-1 expression, cells were treated with KT5823, an inhibitor of cGPK previously shown to interfere with a variety of other actions of natriuretic factors and nitric oxide.…”

“…40,41 Nitric oxide activates a soluble guanylyl cyclase, which is the primary mediator of its actions in VSMCs. 42 Because cGMP is a major second messenger for both natriuretic peptides and nitric oxide, we examined the role of cGMP in the regulation of PAI-1 expression. Intracellular cGMP was directly elevated by treatment of cells with the membrane-permeant cGMP analogue 8-Br-cGMP for 15 minutes followed by stimulation with 100 nmol/L Ang II for an additional 3-hour incubation.…”

“…40,42 The role of cGMP in SNAP's inhibitory effect on PAI-1 expression was examined by treating cells with a soluble guanylyl cyclase inhibitor, ODQ, which does not inhibit adenylyl cyclase or membranous guanylyl cyclase activity. 42 ODQ prevented SNAPinduced increases in cGMP in RASMCs (data not shown), as described previously. 42 Treatment of RASMCs with ODQ also significantly impaired SNAP's ability to inhibit PAI-1 expression in Ang II-stimulated RASMCs ( Figure 6, bottom left).…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…However, even in vascular smooth muscle, a significant component of the relaxation response may be cGMP independent. In some vessels and species, the response may even be completely independent of cGMP [5][6][7][8], and it has been shown that animals deficient in a SNO-metabolizing enzyme have low vascular resistance ( [9], JSS unpublished). By analogy, stringent genetic evidence for a role of NO (per se) in blood pressure control has not been provided.…”

A potential role for extracellular nitric oxide generation in cGMP‐independent inhibition of human platelet aggregation: biochemical and pharmacological considerations

“…Plasma, nitric oxide, cGMP and platelet function investigate NO-mediated cGMP-independent antiplatelet effects (Tsikas et al, 1999;Homer & Wanstall, 2002); however, a 100 : 1 excess of the NO donor SNAP can result in a partial reversal of ODQ-mediated inhibition of sGC (Moro et al, 1996). In our experiments, a theoretical maximum of 20 mM NO will be released by 10 mM DEA/NO, which is equivalent to the concentration of ODQ used here, and unlikely to be sufficiently high to overcome ODQ-mediated inhibition.…”