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

Gut Bacteria and Hydrogen Sulfide: The New Old Players in Circulatory System Homeostasis

Accumulating evidence suggests that gut bacteria play a role in homeostasis of the circulatory system in mammals. First, gut bacteria may affect the nervous control of the circulatory system via the sensory fibres of the enteric nervous system. Second, gut bacteria-derived metabolites may cross the gut-blood barrier and target blood vessels, the heart and other organs involved in the regulation of the circulatory system. A number of studies have shown that hydrogen sulfide (H2S) is an important biological mediator in the circulatory system. Thus far, research has focused on the effects of H2S enzymatically produced by cardiovascular tissues. However, some recent evidence indicates that H2S released in the colon may also contribute to the control of arterial blood pressure. Incidentally, sulfate-reducing bacteria are ubiquitous in mammalian colon, and H2S is just one among a number of molecules produced by the gut flora. Other gut bacteria-derived compounds that may affect the circulatory system include methane, nitric oxide, carbon monoxide, trimethylamine or indole. In this paper, we review studies that imply a role of gut microbiota and their metabolites, such as H2S, in circulatory system homeostasis

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

Cardiovascular “Patterns” of H2S and SSNO−-Mix Evaluated from 35 Rat Hemodynamic Parameters

This work is based on the hypothesis that it is possible to characterize the cardiovascular system just from the detailed shape of the arterial pulse waveform (APW). Since H2S, NO donor S-nitrosoglutathione (GSNO) and their H2S/GSNO products (SSNO−-mix) have numerous biological actions, we aimed to compare their effects on APW and to find characteristic “patterns” of their actions. The right jugular vein of anesthetized rats was cannulated for i.v. administration of the compounds. The left carotid artery was cannulated to detect APW. From APW, 35 hemodynamic parameters (HPs) were evaluated. H2S transiently influenced all 35 HPs and from their cross-relationships to systolic blood pressure “patterns” and direct/indirect signaling pathways of the H2S effect were proposed. The observed “patterns” were mostly different from the published ones for GSNO. Effect of SSNO−-mix (≤32 nmol kg−1) on blood pressure in the presence or absence of a nitric oxide synthase inhibitor (L-NAME) was minor in comparison to GSNO, suggesting that the formation of SSNO−-mix in blood diminished the hemodynamic effect of NO. The observed time-dependent changes of 35 HPs, their cross-relationships and non-hysteresis/hysteresis profiles may serve as “patterns” for the conditions of a transient decrease/increase of blood pressure caused by H2S

•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

Parenteral Na2S, a fast-releasing H2S donor, but not GYY4137, a slow-releasing H2S donor, lowers blood pressure in rats

Hydrogen sulfide (H2S) is involved in blood pressure regulation. We evaluated hemodynamic effects of Na2S and morpholin-4-ium (4-methoxyphenyl)(morpholino)phosphinodithioate (GYY4137), H2S donors. GYY4137 is the most widely studied slow-releasing H2S donor, however, its ability to release H2S under physiological conditions is unclear. Hemodynamics were recorded in anaesthetized Wistar-Kyoto rats at baseline and after intravenous (IV) or intraperitoneal (IP) administration of either a vehicle (20% dimethyl sulfoxide), GYY4137 or Na2S. The stability of GYY4137 in buffers and in plasma was evaluated with nuclear magnetic resonance. The vehicle, as well as GYY4137, given IV did not affect mean arterial blood pressure (MABP), whereas Na2S produced a significant decrease in MABP. Similarly, IP given Na2S, but not GYY4137, lowered MABP. In the buffers at pH of 7.4 and 5.5 and in rat plasma no reaction of GYY4137 was found during 18 hours of observation. In contrast, rapid decomposition of GYY4137 occurred in buffers at pH 2.0. In conclusion, parenteral GYY4137 does not exert a hemodynamic effect in Wistar-Kyoto rats. This seems to be due to the high stability of GYY4137 at physiological pH. Therefore, it is likely that widely reported biological effects of GYY4137 are not H2S-dependent but may depend on GYY4137 itself. However, the H2S-dependent biological effects of GYY4137 may be expected in tissues characterized by low pH