Regular ArticleNO+, NO., and NO− Donation by S-Nitrosothiols: Implications for Regulation of Physiological Functions by S-Nitrosylation and Acceleration of Disulfide Formation
Abstract
The biological effects of S-nitrosothiols have been attributed to homolytic cleavage of the S-N bond with release of nitric oxide (NO.). Rates of NO. release from several S-nitrosothiols were determined by monitoring the oxidation of oxymyoglobin to metmyoglobin at pH 7.4; half-lives for oxymyoglobin oxidation ranged from seconds to hours. Transnitrosation reactions between S-nitrosothiols and thiol-containing amino acids, peptides, and proteins, which indicate the ability of nitrosothiols to act as nitrosyl (NO+) donors, occurred more rapidly than spontaneous NO. release. Decomposition of S-nitrosodithiols were examined as models for the reaction of nitrogen oxides with vicinal thiols on proteins. Rapid disulfide formation was accompanied by formation of hydroxylamine and nitrous oxide, indicative of nitroxyl (NO−) release. Taken together, these model studies demonstrate the ability of S-nitrosothiols to act as NO+, NO., and NO− donors under physiological conditions, Transnitrosation and acceleration of disulfide formation suggest mechanisms of regulation of protein function through the intermediacy of nitrosothiols, and support the notion that biological activities of S-nitrosothiols may be associated with heterolytic as well as homolytic mechanisms of decomposition.
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Nitric oxide resistance in type 2 diabetes: Potential implications of HNO donors
2023, Nitric Oxide in Health and Disease: Therapeutic Applications in Cancer and Inflammatory DisordersNitric oxide (NO•) resistance syndrome refers to a state of decreased NO• bioavailability and/or impaired responsiveness to NO•. Excessive production of reactive oxygen species (ROS) scavenging NO• from the environment, oxidation of its intracellular receptor soluble guanylate cyclase (sGC), or impairing the main physiologically relevant NO• signaling cascade (i.e., NO•/cyclic guanosine 5′-monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway) are the leading causes of NO• resistance. The state of NO• resistance in type 2 diabetes mellitus (T2DM), manifested by a decreased responsiveness of the myocardium, vasculature, platelets, skeletal muscle, and vascular smooth muscle to endogenous and exogenous NO•, is associated with future risk of cardiovascular events. Nitroxyl (HNO), a recently highlighted nitrogen oxide species, can effectively circumvent NO• resistance by bypassing the pathways that become relatively nonresponsive to NO• in T2DM.
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The oral microbiome, nitric oxide and exercise performance
2022, Nitric Oxide - Biology and ChemistryThe human microbiome comprises ∼1013–1014 microbial cells which form a symbiotic relationship with the host and play a critical role in the regulation of human metabolism. In the oral cavity, several species of bacteria are capable of reducing nitrate to nitrite; a key precursor of the signaling molecule nitric oxide. Nitric oxide has myriad physiological functions, which include the maintenance of cardiovascular homeostasis and the regulation of acute and chronic responses to exercise. This article provides a brief narrative review of the research that has explored how diversity and plasticity of the oral microbiome influences nitric oxide bioavailability and related physiological outcomes. There is unequivocal evidence that dysbiosis (e.g. through disease) or disruption (e.g. by use of antiseptic mouthwash or antibiotics) of the oral microbiota will suppress nitric oxide production via the nitrate-nitrite-nitric oxide pathway and negatively impact blood pressure. Conversely, there is preliminary evidence to suggest that proliferation of nitrate-reducing bacteria via the diet or targeted probiotics can augment nitric oxide production and improve markers of oral health. Despite this, it is yet to be established whether purposefully altering the oral microbiome can have a meaningful impact on exercise performance. Future research should determine whether alterations to the composition and metabolic activity of bacteria in the mouth influence the acute responses to exercise and the physiological adaptations to exercise training.
Here we show that the fluorescence of fluorescein isothiocyanate (FITC) is not altered by its reaction with primary amines. However, the fluorescence is rapidly quenched upon reaction with small molecular weight thiols including cysteine, glutathione, homocysteine, dithiothreitol, and sulfide. We have taken advantage of the thiol-dependent quenching of FITC to devise a sulfide specific assay by utilizing polydimethylsiloxane (PDMS) membranes that are permeable to hydrogen sulfide but not to larger charged thiols. In addition, we have discovered that the fluorescein dithiocarbamate (FDTC) formed by the reaction with sulfide can specifically react with S-nitrosothiols (RSNO) to regenerate FITC, thus serving as a specific, fluorogenic reagent to detect picomol levels of RSNO. FDTC was tested as an intracellular RSNO-sensor in germinating tomato seedlings (Solanum lycopersicum) via epifluorescence microscopy. Control plant roots exposed to FDTC showed low intracellular fluorescence which increased ∼3-fold upon exposure to extracellular S-nitrosoglutathione and ∼4-fold in the presence of N6022, a S-nitrosoglutathione reductase (GSNOR) inhibitor, demonstrating that FDTC can be used to visualize intracellular RSNO levels.
Buffer concentration dramatically affects the stability of S-nitrosothiols in aqueous solutions
2022, Nitric Oxide - Biology and ChemistryS-nitrosothiols (RSNOs) are an important group of nitric oxide (NO)-donating compounds with low toxicity and wide biomedical applications. In this paper, we, for the first time, demonstrate that the concentration of buffer remarkably affects the stability of RSNOs including naturally occurring S-nitrosoglutathione (GSNO) and synthetic S-nitroso-N-acetylpenicillamine (SNAP). For a solution with a high concentration of GSNO (e.g., 50 mM) and an initial near-neutral pH, the optimal buffer concentration is close to the GSNO concentration under our experimental conditions. A lower buffer concentration does not have adequate buffer capacity to resist the pH drop caused by GSNO decomposition. The decreased solution pH further accelerates GSNO decomposition because GSNO is most stable at near-neutral pH according to our density functional theory (DFT) calculations. A higher-than-optimal buffer concentration also reduces the GSNO stability because buffer ingredients including phosphate, Tris base, and HEPES consume NO/N2O3. In contrast to GSNO, the highest SNAP stability is obtained when the starting solution at a neutral pH does not contain buffer species, and the stability decreases as the buffer concentration increases. This is because SNAP is more stable at mildly acidic pH and the SNAP decomposition-induced pH drop stabilizes the donor. When the RSNO concentration is low (e.g., 1 mM), the buffer concentration also matters because any excess buffer accelerates the donor decomposition. Since the effect of buffer concentration was previously overlooked and suboptimal buffer concentrations were often used, this paper will aid in the formulation of RSNO solutions to obtain the maximum stability for prolonged storage and sustained NO release.
Quantification of intracellular HNO delivery with capillary zone electrophoresis
2022, Nitric Oxide - Biology and ChemistryRedox signaling, wherein reactive and diffusible small molecules are channeled into specific messenger functions, is a critical component of signal transduction. A central principle of redox signaling is that the redox modulators are produced in a highly controlled fashion to specifically modify biotargets. Thiols serve as primary mediators of redox signaling as a function of the rich variety of adducts, which allows initiation of distinct cellular effects. Coupling the inherent reactivity of thiols with highly sensitive and selective chemical analysis protocols can facilitate identification of redox signaling agents, both in solution and in cultured cells. Here, we describe use of capillary zone electrophoresis to both identify and quantify sulfinamides, which are specific markers of the reaction of thiols with nitroxyl (HNO), a putative biologically relevant reactive nitrogen species.