Lifting the Fog over Mitochondrial Chloride Channels

The current through mitochondrial chloride channels was first described in 1987. Subsequently, several types of ion channels permeable to chloride and other anions were found in the mitochondria of different origins. The increasing number of electrophysiological studies, however, yielded only more ambiguity rather than order in the field of chloride channels. This uncertainty was slightly reduced by two different studies: experiments that showed a significant role of chloride channels in the process of mitochondrial membrane potential oscillations and experiments that localized chloride intracellular ion channel (CLIC) proteins in cardiac mitochondrial membranes. Our recently published single-channel electrophysiological experiments are well in line with the channel activity of recombinant CLIC proteins. The experimental evidence seems to be inevitably, though slowly converging on a connection between single-channel activity and the identity of the mitochondrial chloride channel protein

Inorganic Reactive Sulfur-Nitrogen Species: Intricate Release Mechanisms or Cacophony in Yellow, Blue and Red?

Since the heydays of Reactive Sulfur Species (RSS) research during the first decade of the Millennium, numerous sulfur species involved in cellular regulation and signalling have been discovered. Yet despite the general predominance of organic species in organisms, recent years have also seen the emergence of inorganic reactive sulfur species, ranging from inorganic polysulfides (HSx−/Sx2−) to thionitrous acid (HSNO) and nitrosopersulfide (SSNO−). These inorganic species engage in a complex interplay of reactions in vitro and possibly also in vivo. Employing a combination of spectrophotometry and sulfide assays, we have investigated the role of polysulfanes from garlic during the release of nitric oxide (•NO) from S-nitrosoglutathione (GSNO) in the absence and presence of thiol reducing agents. Our studies reveal a distinct enhancement of GSNO decomposition by compounds such as diallyltrisulfane, which is most pronounced in the presence of cysteine and glutathione and presumably proceeds via the initial release of an inorganic mono- or polysulfides, i.e., hydrogen sulfide (H2S) or HSx−, from the organic polysulfane. Albeit being of a preliminary nature, our spectrophotometric data also reveals a complicated underlying mechanism which appears to involve transient species such as SSNO−. Eventually, more in depth studies are required to further explore the underlying chemistry and wider biological and nutritional implications of this interplay between edible garlic compounds, reductive activation, inorganic polysulfides and their interplay with •NO storage and release

Inorganic Polysulfides and Related Reactive Sulfur–Selenium Species from the Perspective of Chemistry

Polysulfides (H2Sx) represent a class of reactive sulfur species (RSS) which includes molecules such as H2S2, H2S3, H2S4, and H2S5, and whose presence and impact in biological systems, when compared to other sulfur compounds, has only recently attracted the wider attention of researchers. Studies in this field have revealed a facet-rich chemistry and biological activity associated with such chemically simple, still unusual inorganic molecules. Despite their chemical simplicity, these inorganic species, as reductants and oxidants, metal binders, surfactant-like “cork screws” for membranes, components of perthiol signalling and reservoirs for inorganic hydrogen sulfide (H2S), are at the centre of complicated formation and transformation pathways which affect numerous cellular processes. Starting from their chemistry, the hidden presence and various roles of polysulfides in biology may become more apparent, despite their lack of clear analytical fingerprints and often murky biochemical footprints. Indeed, the biological chemistry of H2Sx follows many unexplored paths and today, the relationship between H2S and its oxidized H2Sx species needs to be clarified as a matter of “unmistaken identity”. Simultaneously, emerging species, such as HSSeSH and SenS8−n, also need to be considered in earnest

EPR Study of KO2 as a Source of Superoxide and •BMPO-OH/OOH Radical That Cleaves Plasmid DNA and Detects Radical Interaction with H2S and Se-Derivatives

: Superoxide radical anion (O2 •−) and its derivatives regulate numerous physiological and pathological processes, which are extensively studied. The aim of our work was to utilize KO2 as a source of O2 •− and the electron paramagnetic resonance (EPR) spin trapping 5-tert-butoxycarbonyl-5- methyl-1-pyrroline N-oxide (BMPO) technique for the preparation of •BMPO-OOH and/or •BMPOOH radicals in water solution without DMSO. The method distinguishes the interactions of various compounds with •BMPO-OOH and/or •BMPO-OH radicals over time. Here, we show that the addition of a buffered BMPO-HCl mixture to powdered KO2 formed relatively stable •BMPO-OOH and •BMPO-OH radicals and H2O2 , where the •BMPO-OOH/OH ratio depended on the pH. At a final pH of ~6.5–8.0, the concentration of •BMPO-OOH radicals was ≥20 times higher than that of •BMPO-OH, whereas at pH 9.0–10.0, the •BMPO-OH radicals prevailed. The •BMPO-OOH/OH radicals effectively cleaved the plasmid DNA. H2S decreased the concentration of •BMPO-OOH/OH radicals, whereas the selenium derivatives 1-methyl-4-(3-(phenylselanyl) propyl) piperazine and 1-methyl-4-(4-(phenylselanyl) butyl) piperazine increased the proportion of •BMPO-OH over the •BMPO-OOH radicals. In conclusion, the presented approach of using KO2 as a source of O2 •−/H2O2 and EPR spin trap BMPO for the preparation of •BMPO-OOH/OH radicals in a physiological solution could be useful to study the biological effects of radicals and their interactions with compounds

The reaction products of sulfide and S-nitrosoglutathione are potent vasorelaxants

The chemical interaction of sodium sulfide (Na2S) with the NO-donor S-nitrosoglutathione (GSNO) has been described to generate new reaction products, including polysulfides and nitrosopersulfide (SSNO-) via intermediacy of thionitrous acid (HSNO). The aim of the present work was to investigate the vascular effects of the longer-lived products of the Sulfide/GSNO interaction. Here we show that the products of this reaction relax precontracted isolated rings of rat thoracic aorta and mesenteric artery (but to a lesser degree rat uterus) with a >2-fold potency compared with the starting material, GSNO (50?nM), whereas Na2S and polysulfides have little effect at 1-5?µM. The onset of vasorelaxation of the reaction products was 7-10 times faster in aorta and mesenteric arteries compared with GSNO. Relaxation to GSNO (100-500?nM) was blocked by an inhibitor of soluble guanylyl cyclase, ODQ (0.1 and 10?µM), and by the NO scavenger cPTIO (100?µM), but less affected by prior acidification (pH 2-4), and unaffected by N-acetylcysteine (1?mM) or methemoglobin (20?µM heme). By contrast, relaxation to the Sulfide/GSNO reaction products (100-500?nM based on the starting material) was inhibited to a lesser extent by ODQ, only slightly decreased by cPTIO, more markedly inhibited by methemoglobin and N-acetylcysteine, and abolished by acidification before addition to the organ bath. The reaction mixture was found to generate NO as detected by EPR spectroscopy using N-(dithiocarboxy)-N-methyl-D-glucamine (MGD2)-Fe2+ as spin trap. In conclusion, the Sufide/GSNO reaction products are faster and more pronounced vasorelaxants than GSNO itself. We conclude that in addition to NO formation from SSNO-, reaction products other than polysulfides may give rise to nitroxyl (HNO) and be involved in the pronounced relaxation induced by the Sulfide/GSNO cross-talk

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Characterization of Rat Cardiovascular System by Anacrotic/Dicrotic Notches in the Condition of Increase/Decrease of NO Bioavailability

We characterized modes of action of NO-donor S-nitrosoglutathione (GSNO) and NO-synthase inhibitor l-NAME derived from dicrotic (DiN) and anacrotic (AnN) notches of rat arterial pulse waveform (APW) in the condition of increased/decreased NO bioavailability. The cross-relationship patterns of DiN and AnN with 34 hemodynamic parameters (HPs) induced by GSNO and l-NAME are presented. After GSNO bolus administration, approximate non-hysteresis relationships were observed in the difference between DiN–AnN (mmHg) blood pressure (BP) and other 19 HPs, suggesting that these HPs, i.e., their signaling pathways, responding to NO concentration, are directly connected. Hysteresis relationships were observed between DiN-AnN (mmHg) and other 14 HPs, suggesting that signaling pathways of these HPs are indirectly connected. The hysteresis relationships were only observed between the time interval DiN-AnN (ms) and other 34 HPs, indicating no direct connection of signaling pathways. The cross-relationship patterns of DiN-AnN (mmHg), but not DiN-AnN (ms), induced by l-NAME were in accordance to the increased NO bioavailability induced by GSNO. In conclusion, we found the non-hysteresis/hysteresis cross-relationship “patterns” of DiN-AnN intervals to other HPs in the presence of GSNO that revealed their direct or indirect signaling pathways connections. This may contribute to our understanding of biological effects of natural substances that modulate NO production and/or NO signaling pathways

Extract of <i>Acanthopanax senticosus</i> and Its Components Interacting with Sulfide, Cysteine and Glutathione Increase Their Antioxidant Potencies and Inhibit Polysulfide-Induced Cleavage of Plasmid DNA

Aqueous root extract from Acanthopanax senticosus (ASRE) has a wide range of medicinal effects. The present work was aimed at studying the influence of sulfide, cysteine and glutathione on the antioxidant properties of ASRE and some of its selected phytochemical components. Reduction of the 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide (●cPTIO) stable radical and plasmid DNA (pDNA) cleavage in vitro assays were used to evaluate antioxidant and DNA-damaging properties of ASRE and its individual components. We found that the interaction of ASRE and its two components, caffeic acid and chlorogenic acid (but not protocatechuic acid and eleutheroside B or E), with H2S/HS−, cysteine or glutathione significantly increased the reduction of the ●cPTIO radical. In contrast, the potency of ASRE and its selected components was not affected by Na2S4, oxidized glutathione, cystine or methionine, indicating that the thiol group is a prerequisite for the promotion of the antioxidant effects. ASRE interacting with H2S/HS− or cysteine displayed a bell-shaped effect in the pDNA cleavage assay. However, ASRE and its components inhibited pDNA cleavage induced by polysulfides. In conclusion, we suggest that cysteine, glutathione and H2S/HS− increase antioxidant properties of ASRE and that changes of their concentrations and the thiol/disulfide ratio can influence the resulting biological effects of ASRE

•BMPO-OOH Spin-Adduct as a Model for Study of Decomposition of Organic Hydroperoxides and the Effects of Sulfide/Selenite Derivatives. An EPR Spin-Trapping Approach

Lipid hydroperoxides play an important role in various pathophysiological processes. Therefore, a simple model for organic hydroperoxides could be helpful to monitor the biologic effects of endogenous and exogenous compounds. The electron paramagnetic resonance (EPR) spin-trapping technique is a useful method to study superoxide (O2&bull;&minus;) and hydroxyl radicals. The aim of our work was to use EPR with the spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), which, by trapping O2&bull;&minus; produces relatively stable &bull;BMPO-OOH spin-adduct, a valuable model for organic hydroperoxides. We used this experimental setup to investigate the effects of selected sulfur/selenium compounds on &bull;BMPO-OOH and to evaluate the antioxidant potential of these compounds. Second, using the simulation of time-dependent individual BMPO adducts in the experimental EPR spectra, the ratio of &bull;BMPO-OH/&bull;BMPO-OOH&mdash;which is proportional to the transformation/decomposition of &bull;BMPO-OOH&mdash;was evaluated. The order of potency of the studied compounds to alter &bull;BMPO-OOH concentration estimated from the time-dependent &bull;BMPO-OH/&bull;BMPO-OOH ratio was as follows: Na2S4 &gt; Na2S4/SeO32&minus; &gt; H2S/SeO32&minus; &gt; Na2S2 ~Na2S2/SeO32&minus; ~H2S &gt; SeO32&minus; ~SeO42&minus; ~control. In conclusion, the presented approach of the EPR measurement of the time-dependent ratio of &bull;BMPO-OH/&bull;BMPO-OOH could be useful to study the impact of compounds to influence the transformation of &bull;BMPO-OOH

The Possible Role of the Nitroso-Sulfide Signaling Pathway in the Vasomotoric Effect of Garlic Juice

The beneficial cardiovascular effects of garlic have been reported in numerous studies. The major bioactive properties of garlic are related to organic sulfides. This study aimed to investigate whether garlic juice works exclusively due to its sulfur compounds or rather via the formation of new products of the nitroso-sulfide signaling pathway. Changes in isometric tension were measured on the precontracted aortic rings of adult normotensive Wistar rats. We evaluated NO-donor (S-nitrosoglutathione, GSNO)-induced vasorelaxation and compare it with effects of hydrogen sulfide (H2S)/GSNO and garlic/GSNO. Incubation with garlic juice increased the maximal GSNO-induced relaxation and markedly changed the character of the relaxant response. Although incubation with an H2S donor enhanced the maximal vasorelaxant response of GSNO, neither the absolute nor the relative relaxation changed over time. The mixture of GSNO with an H2S donor evoked a response similar to GSNO-induced relaxation after incubation with garlic juice. This relaxation of the H2S and GSNO mixture was soluble guanylyl cyclase (sGC) dependent, partially reduced by HNO scavenger and it was adenosine triphosphate-sensitive potassium channels (KATP) independent. In this study, we demonstrate for the first time the suggestion that H2S itself is probably not the crucial bioactive compound of garlic juice but rather potentiates the production of new signaling molecules during the GSNO-H2S interaction