Mechanisms Underlying Activation of Soluble Guanylate Cyclase by the Nitroxyl Donor Angeli's Salt

Abstract

Nitroxyl (HNO) may be formed endogenously by uncoupled nitric-oxide (NO) synthases, enzymatic reduction of NO or as product of vascular nitroglycerin bioactivation. The established HNO donor Angeli's salt (trioxodinitrate, AS) causes cGMPdependent vasodilation through activation of soluble guanylate cyclase (sGC). We investigated the mechanisms underlying this effect using purified sGC and cultured endothelial cells. AS (up to 0.1 mM) had no significant effect on sGC activity in the absence of superoxide dismutase (SOD) or dithiothreitol (DTT). In the presence of SOD, AS caused biphasic sGC activation (apparent EC 50 ϳ10 nM, maximum at 1 M) that was accompanied by the formation of NO. DTT (2 mM) inhibited the effects of Ͻ10 M AS but led to sGC activation and NO release at 0.1 mM AS even without SOD. AS had no effect on ferric sGC, excluding activation of the oxidized enzyme by HNO. The NO scavenger carboxy-PTIO inhibited endothelial cGMP accumulation induced by AS in the presence but not in the absence of SOD (EC 50 ϳ50 nM and ϳ16 M, respectively). Carboxy-PTIO (0.1 mM) inhibited the effect of Յ10 M AS in the presence of SOD but caused NO release from 0.1 mM AS in the absence of SOD. These data indicate that AS activates sGC exclusively via NO, formed either via SOD-catalyzed oxidation of HNO or through a minor AS decomposition pathway that is unmasked in the presence of HNO scavenging thiols.

Pharmacological Characterization of 1-Nitrosocyclohexyl Acetate, a Long-Acting Nitroxyl Donor That Shows Vasorelaxant and Antiaggregatory Effects

“…Furthermore, it has been shown that HNO stimulates sGC to suppress cardiomyocyte hypertrophy (Lin et al, 2012). In contrast, Zeller's group reported an intracellular oxidation of HNO to NO following the activation of sGC (Zeller et al, 2009). Our data do not clarify whether HNO itself directly activated sGC (Fukuto et al, 1992) or went through the intermediary of NO following intracellular oxidation (Zeller et al, 2009).…”

HNO/cGMP-dependent antihypertrophic actions of isopropylamine-NONOate in neonatal rat cardiomyocytes: potential therapeutic advantages of HNO over NO˙

“…We utilized carboxy-PTIO in the present study to reveal potential NO˙-mediated actions (10,42,59) in response to both donors. It should be noted, however, that carboxy-PTIO is a mixed NO˙-peroxynitrite scavenger (41,54,62). We thus cannot exclude the possibility that carboxy-PTIO removed both NO˙and peroxynitrite in the presence of ET 1 and DEA-NO; given that both IPA-NO and DEA-NO markedly suppressed ET 1 -stimulated cardiomyocyte superoxide generation, it is, however, unlikely that there is detectable peroxynitrite in our cardiomyocyte preparations.…”

The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation

“…However, the thiol dithiothreitol (DTT), which blocks nitroxyl (NO À )-mediated cysteine modification (S-nitrosylation) but not NO (NO. )-dependent sGC activation, [34][35][36] did not protect cultured cells from SNAP cytotoxic stimulus (59.5 ± 9.5%, N ¼ 3, one-way analysis of variance (ANOVA)) in E6 purified retinal cultures, which argues against an S-nitrosylation-dependent mechanism for NO to increase death of E6 neurons. To corroborate our pharmacological data further, we used a lentiviralmediated shRNA delivery system to knockdown cGKII specifically in retinal mixed cultures ( Figure 3e) and evaluated SNAP-induced cell death or survival by using ethidium homodimer-1 (EthD1) labeling (Figure 3f) or 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figures 3g and h).…”