Activation of soluble guanylate cyclase by carbon monoxide and inhibition by superoxide anion

Abstract

Human platelet soluble guanylate cyclase activity was studied with respect to the function of its hemecontaining regulatory subunit. As an enzyme source, the 10000 x g supernatant was used and, since its specific activity proved to be too low for inhibition studies, also a partially purified preparation was employed. The partially purified enzyme was stimulated about 2.5-fold by carbon monoxide and this effect was abolished by illumination with visible light. Sodium nitroprusside also increased the basal activity about fourfold, which, however, is much less than the > 100-fold stimulation seen with the supernatant. Superoxide anions generated by the xanthine/xanthine-oxidase system were strongly inhibitory in the enriched preparation as well as in the COstimulated platelet supernatant (median effector concentration = 0.1 mUjml). Unlike CO and NO, the effect of superoxide cannot be mediated through the heme-containing regulatory subunit, since heme-free enzyme, which could not be activated by NO or CO, was inhibited to the same extent as the heme-containing enzyme. Superoxide dismutase did not influence the basal activity, but resulted in a synergistic stimulation in the presence of CO. When Mn2+ replaced MgZf as a cofactor, the basal activity was higher but superoxide could not inhibit the enzyme, possibly due to the superoxide-dismutase-like activity of Mn2+. Superoxide turned out to be a potent and reversible inhibitor of soluble guanylate cyclase which, together with endothelium-derived relaxing factor, recently identified as NO, could form a physiologically relevant regulatory effector system. Guanosine 3',5'-(cyc1ic)phosphate (cGMP) is emerging as an important cellular second messenger, although its mode of action and the regulation of soluble guanylate cyclase and particulate guanylate cyclase are only poorly understood (for a review see [l, 21). Soluble guanylate cyclase contains a heme regulatory subunit and represents the dominant enzyme in platelets and smooth muscle. Its activation is associated with inhibition of platelet aggregation [3] and with smooth muscle relaxation [4, 51. Hence its stimulation under conditions of coronary heart diseases is a preferred pharmacological target. Likewise, the potent vasodilatory properties of organic nitrates seem to be mediated by the release of nitric oxide [6, 71. Recently, the endothelium-derived relaxing factor (EDRF) was identified as NO which has therefore gained not only a pharmacological but also a physiological role in the relaxation of vascular tissue [8].When studying platelet aggregation, we obtained evidence that carbon monoxide prevents aggregation of human platelets by stimulation of soluble guanylate cyclase [9]. From the reversibility of this effect by visible light, we concluded GermanyAbbreviations. cGMP, guanosine 3',5'-(cyclic)phosphate; EDRF, cndothelium-derived relaxing factor; EC,,, median effector concentration.Enzj~mes. Creatine kinase (EC 2.7.3.2); xanthine oxidase (EC I .2.3.2); catalase (EC 1 .I 1 .I .6). _ _ _that CO must bind to t...

Heme oxygenase‐1 in growth control and its clinical application to vascular disease

“…CO appears to block vascular SMC growth by increasing the intracellular levels of guanosine 3 0 -5 0 -cyclic monophosphate (cGMP). CO activates both crude and purified preparations of soluble guanylate cyclase and elevates cGMP levels in vascular SMCs (Ramos et al, 1989;Brune et al, 1990;Stone and Marletta, 1994;Christodoulides et al, 1995;. Furthermore, inhibition of soluble guanylate cyclase by ODQ blocks the rise in cGMP and the antiproliferative action of HO-1 (Duckers et al, 2001).…”

Superoxide as a Messenger of Endothelial Function

“…Brune et al (1990;Mülsch et al, 1997) Therefore, it is likely that • O 2 -formation by a proteolytically derived xanthine oxidase occurs as a rather late event in vascular damage in contrast to fast and reversible oxidative conversion.…”

Structural and Functional Characterization of the Dimerization Region of Soluble Guanylyl Cyclase

“…the major endogenous activator of sGC which induces an activity increase of 200 -500-fold (5,9). Carbon monoxide can also stimulate sGC activity though to a lesser extent, up to 5-fold (5,10). The best studied exogenous sGC activators are organic nitrates, which mimic the action of endogenous NO, e.g.…”