Mechanisms of the interaction of nitroxyl with mitochondria.
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ABSTRACT:
SUBMITTER: Shiva S
PROVIDER: S-EPMC1224084 | biostudies-other | 2004 Apr
REPOSITORIES: biostudies-other
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Project description:The pharmacological properties of nitroxyl (HNO) donors in the gastrointestinal tract are unknown. We investigated the properties of this molecule in the regulation of gastrointestinal contractility focusing on its possible interaction with other gaseous signaling molecules such as NO and H2S. Organ bath, Ca2+ imaging, and microelectrode recordings were performed on rat intestinal samples, using Angeli's salt as HNO donor. Angeli's salt caused a concentration-dependent relaxation of longitudinal or circular muscle strips of the ileum and the proximal colon. This relaxation was strongly inhibited by the Rho-kinase inhibitor Y-27632 (10 μM), by the reducing agent DTT or by the inhibitor of soluble guanylate cyclase (sGC) ODQ (10 μM) alone or in combination with the inhibitors of the endogenous synthesis of H2S β-cyano-L-alanine (5 mM) and amino-oxyacetate (5 mM). Preventing endogenous synthesis of NO by the NO synthase inhibitor L-NAME (200 μM) did not affect the relaxation induced by HNO. HNO induced an increase in cytosolic Ca2+ concentration in colonic myocytes. It also elicited myocyte membrane hyperpolarization that amounted to -10.6 ± 1.1 mV. ODQ (10 μM) and Apamin (1 μM), a selective inhibitor of small conductance Ca2+-activated K+ channels (SKca), strongly antagonized this effect. We conclude that HNO relaxes the gastrointestinal tract musculature by hyperpolarizing myocytes via activation of the sGC/cGMP pathway similarly to NO, not only inhibiting the RhoK and activating MLCP as do both NO and H2S but also increasing cytosolic Ca2+ for activation of SK C a contributing to hyperpolarization.
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Project description:Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO). Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN-N(O)?NO(-)Na(+) (1, IPA/NO) with BrCH(2)OAc produced a stable derivative of structure iPrHN-N(O)?NO-CH(2)OAc (2, AcOM-IPA/NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To account for this surprising result, we propose a mechanism in which base-induced removal of the N-H proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor, CH(3)-C(O)-NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH, in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1 on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead compound for the development of heart failure therapies.
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Project description:Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO(-)), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO(-) is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO(-) synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.
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