TRPA1 Has a Key Role in the Somatic Pro-Nociceptive Actions of Hydrogen Sulfide

Hydrogen sulfide (H₂S), which is produced endogenously from L-cysteine, is an irritant with pro-nociceptive actions. We have used measurements of intracellular calcium concentration, electrophysiology and behavioral measurements to show that the somatic pronociceptive actions of H₂S require TRPA1. A H₂S donor, NaHS, activated TRPA1 expressed in CHO cells and stimulated DRG neurons isolated from Trpa1^+/+ but not Trpa1^−/− mice. TRPA1 activation by NaHS was pH dependent with increased activity at acidic pH. The midpoint of the relationship between NaHS EC₅₀ values and external pH was pH 7.21, close to the expected dissociation constant for H₂S (pK_a 7.04). NaHS evoked single channel currents in inside-out and cell-attached membrane patches consistent with an intracellular site of action. In behavioral experiments, intraplantar administration of NaHS and L-cysteine evoked mechanical and cold hypersensitivities in Trpa1^+/+ but not in Trpa1^−/− mice. The sensitizing effects of L-cysteine in wild-type mice were inhibited by a cystathionine β-synthase inhibitor, D,L-propargylglycine (PAG), which inhibits H₂S formation. Mechanical hypersensitivity evoked by intraplantar injections of LPS was prevented by PAG and the TRPA1 antagonist AP-18 and was absent in Trpa1^−/− mice, indicating that H₂S mediated stimulation of TRPA1 is necessary for the local pronociceptive effects of LPS. The pro-nociceptive effects of intraplantar NaHS were retained in Trpv1^−/− mice ruling out TRPV1 as a molecular target. In behavioral studies, NaHS mediated sensitization was also inhibited by a T-type calcium channel inhibitor, mibefradil. In contrast to the effects of NaHS on somatic sensitivity, intracolonic NaHS administration evoked similar nociceptive effects in Trpa1^+/+ and Trpa1^−/− mice, suggesting that the visceral pro-nociceptive effects of H₂S are independent of TRPA1. In electrophysiological studies, the depolarizing actions of H₂S on isolated DRG neurons were inhibited by AP-18, but not by mibefradil indicating that the primary excitatory effect of H₂S on DRG neurons is TRPA1 mediated depolarization.     

Urate Crystal Induced Inflammation and Joint Pain Are Reduced in Transient Receptor Potential Ankyrin 1 Deficient Mice – Potential Role for Transient Receptor Potential Ankyrin 1 in Gout

IntroductionIn gout, monosodium urate (MSU) crystals deposit intra-articularly and cause painful arthritis. In the present study we tested the hypothesis that Transient Receptor Poten-tial Ankyrin 1 (TRPA1), an ion channel mediating nociceptive signals and neurogenic in-flammation, is involved in MSU crystal-induced responses in gout by utilizing three experi-mental murine models.MethodsThe effects of selective pharmacological inhibition (by HC-030031) and genetic depletion of TRPA1 were studied in MSU crystal-induced inflammation and pain by using 1) spontaneous weight-bearing test to assess MSU crystal-induced joint pain, 2) subcutaneous air-pouch model resembling joint inflammation to measure MSU crystal-induced cytokine production and inflammatory cell accumulation, and 3) MSU crystal-induced paw edema to assess acute vascular inflammatory responses and swelling.ResultsIntra-articularly injected MSU crystals provoked spontaneous weight shift off from the affected limb in wild type but not in TRPA1 knock-out mice referring alleviated joint pain in TRPA1 deficient animals. MSU crystal-induced inflammatory cell infiltration and accumulation of cytokines MCP-1, IL-6, IL-1beta, MPO, MIP-1alpha and MIP-2 into subcu-taneous air-pouch (resembling joint cavity) was attenuated in TRPA1 deficient mice and in mice treated with the selective TRPA1 inhibitor HC-030031 as compared to control animals. Further, HC-030031 treated and TRPA1 deficient mice developed tempered inflammatory edema when MSU crystals were injected into the paw.ConclusionsTRPA1 mediates MSU crystal-induced inflammation and pain in experimental models supporting the role of TRPA1 as a potential mediator and a drug target in gout flare.     

Methylglyoxal Evokes Pain by Stimulating TRPA1

Diabetic neuropathy is a severe complication of long-standing diabetes and one of the major etiologies of neuropathic pain. Diabetes is associated with an increased formation of reactive oxygen species and the electrophilic dicarbonyl compound methylglyoxal (MG). Here we show that MG stimulates heterologously expressed TRPA1 in CHO cells and natively expressed TRPA1 in MDCK cells and DRG neurons. MG evokes [Ca²⁺]_i-responses in TRPA1 expressing DRG neurons but is without effect in neurons cultured from Trpa1^−/− mice. Consistent with a direct, intracellular action, we show that methylglyoxal is significantly more potent as a TRPA1 agonist when applied to the intracellular face of excised membrane patches than to intact cells. Local intraplantar administration of MG evokes a pain response in Trpa1^+/+ but not in Trpa1^−/− mice. Furthermore, persistently increased MG levels achieved by two weeks pharmacological inhibition of glyoxalase-1 (GLO-1), the rate-limiting enzyme responsible for detoxification of MG, evokes a progressive and marked thermal (cold and heat) and mechanical hypersensitivity in wildtype but not in Trpa1^−/− mice. Our results thus demonstrate that TRPA1 is required both for the acute pain response evoked by topical MG and for the long-lasting pronociceptive effects associated with elevated MG in vivo. In contrast to our observations in DRG neurons, MG evokes indistinguishable [Ca²⁺]_i-responses in pancreatic β-cells cultured from Trpa1^+/+ and Trpa1^−/− mice. In vivo, the TRPA1 antagonist {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681"}}HC030031 impairs glucose clearance in the glucose tolerance test both in Trpa1^+/+ and Trpa1^−/− mice, indicating a non-TRPA1 mediated effect and suggesting that results obtained with this compound should be interpreted with caution. Our results show that TRPA1 is the principal target for MG in sensory neurons but not in pancreatic β-cells and that activation of TRPA1 by MG produces a painful neuropathy with the behavioral hallmarks of diabetic neuropathy.     

Activation of ERK signalling by Src family kinases (SFKs) in DRG neurons contributes to hydrogen peroxide (H2O2)-induced thermal hyperalgesia

Concomitant generation of reactive oxygen species during tissue inflammation has been recognised as a major factor for the development and the maintenance of hyperalgesia, out of which H₂O₂ is the major player. However, molecular mechanism of H₂O₂ induced hyperalgesia is still obscure. The aim of present study is to analyse the mechanism of H₂O₂-induced hyperalgesia in rats. Intraplantar injection of H₂O₂ (5, 10 and 20 µmoles/paw) induced a significant thermal hyperalgesia in the hind paw, confirmed by increased c-Fos activity in dorsal horn of spinal cord. Onset of hyperalgesia was prior to development of oxidative stress and inflammation. Rapid increase in phosphorylation of extracellular signal regulated kinase (ERK) was observed in neurons of dorsal root ganglia after 20?min of H₂O₂ (10 µmoles/paw) administration, which gradually returned towards normal level within 24?h, following the pattern of thermal hyperalgesia. The expression of TNFR1 followed the same pattern and colocalised with pERK. ERK phosphorylation was observed in NF-200-positive and -negative neurons, indicating the involvement of ERK in C-fibres as well as in A-fibres. Intrathecal preadministration of Src family kinases (SFKs) inhibitor (PP1) and MEK inhibitor (PD98059) prevented H₂O₂ induced augmentation of ERK phosphorylation and thermal hyperalgesia. Pretreatment of protein tyrosine phosphatases (PTPs) inhibitor (sodium orthovanadate) also diminished hyperalgesia, although it further increased ERK phosphorylation. Combination of orthovanadate with PP1 or PD98059 did not exhibit synergistic antihyperalgesic effect. The results demonstrate SFKs-mediated ERK activation and increased TNFR1 expression in nociceptive neurons during H₂O₂ induced hyperalgesia. However, the role of PTPs in hyperalgesic behaviour needs further molecular analysis.     

Sensitization by Pulmonary Reactive Oxygen Species of Rat Vagal Lung C-Fibers: The Roles of the TRPV1, TRPA1, and P2X Receptors

Sensitization of vagal lung C-fibers (VLCFs) induced by mediators contributes to the pathogenesis of airway hypersensitivity, which is characterized by exaggerated sensory and reflex responses to stimulants. Reactive oxygen species (ROS) are mediators produced during airway inflammation. However, the role of ROS in VLCF-mediated airway hypersensitivity has remained elusive. Here, we report that inhalation of aerosolized 0.05% H₂O₂ for 90 s potentiated apneic responses to intravenous capsaicin (a TRPV1 receptor agonist), α,β-methylene-ATP (a P2X receptor agonist), and phenylbiguanide (a 5-HT₃ receptor agonist) in anesthetized rats. The apneic responses to these three stimulants were abolished by vagatomy or by perivagal capsaicin treatment, a procedure that blocks the neural conduction of VLCFs. The potentiating effect of H₂O₂ on the apneic responses to these VLCF stimulants was prevented by catalase (an enzyme that degrades H₂O₂) and by dimethylthiourea (a hydroxyl radical scavenger). The potentiating effect of H₂O₂ on the apneic responses to capsaicin was attenuated by HC-030031 (a TRPA1 receptor antagonist) and by iso-pyridoxalphosphate-6-azophenyl-2′,5′-disulphonate (a P2X receptor antagonist). The potentiating effect of H₂O₂ on the apneic responses to α,β-methylene-ATP was reduced by capsazepine (a TRPV1 receptor antagonist), and by HC-030031. The potentiating effect of H₂O₂ on the apneic responses to phenylbiguanide was totally abolished when all three antagonists were combined. Consistently, our electrophysiological studies revealed that airway delivery of aerosolized 0.05% H₂O₂ for 90 s potentiated the VLCF responses to intravenous capsaicin, α,β-methylene-ATP, and phenylbiguanide. The potentiating effect of H₂O₂ on the VLCF responses to phenylbiguanide was totally prevented when all antagonists were combined. Inhalation of 0.05% H₂O₂ indeed increased the level of ROS in the lungs. These results suggest that 1) increased lung ROS sensitizes VLCFs, which leads to exaggerated reflex responses in rats and 2) the TRPV1, TRPA1, and P2X receptors are all involved in the development of this airway hypersensitivity.     

Thoracic Vagal Efferent Nerve Stimulation Evokes Substance P-Induced Early Airway Bronchonstriction and Late Proinflammatory and Oxidative Injury in the Rat Respiratory Tract

Summary Electrical stimulation of efferent thoracic vagus nerve (TVN) evoked neurogenic inflammation in respiratory tract of atropine-treated rats by an undefined mechanism. We explored whether efferent TVN stimulation via substance P facilitates neurogenic inflammation via action of nuclear factor-κB (NF-κB) activation and reactive oxygen species (ROS) production. Our results showed that increased frequency of TVN stimulation concomitantly increased substance P-enhanced hypotension, and bronchoconstriction (increases in smooth muscle electromyographic activity and total pulmonary resistance). The enhanced SP release evoked the appearance of endothelial gap in silver-stained leaky venules, India-ink labeled extravasation, and accumulations of inflammatory cells in the respiratory tract, contributing to trachea plasma extravasation as well as increases in blood O₂⁻ and H₂O₂ ROS amount. L-732138 (NK₁ receptor antagonist), SR-48968 (NK₂ receptor antagonist), dimethylthiourea (H₂O₂ scavenger) or catechins (O₂⁻ and H₂O₂ scavenger) pretreatment reduced efferent TVN stimulation-enhanced hypotension, bronchoconstriction, and plasma extravasation. Increased frequency of TVN stimulation significantly upregulated the expression of nuclear factor-κB (NF-κB) in nuclear protein and intercellular adhesion molecule-1 (ICAM-1) in total protein of the lower respiratory tract tissue. The upregulation of NF-κB and ICAM-1 was attenuated by NK receptor antagonist and antioxidants. In conclusion, TVN efferent stimulation increases substance P release to trigger NF-κB mediated ICAM-1 expression and O₂⁻ and H₂O₂ ROS production in the respiratory tract.     

Streptozotocin Stimulates the Ion Channel TRPA1 Directly: INVOLVEMENT OF PEROXYNITRITE*

Streptozotocin (STZ)-induced diabetes is the most commonly used animal model of diabetes. Here, we have demonstrated that intraplantar injections of low dose STZ evoked acute polymodal hypersensitivities in mice. These hypersensitivities were inhibited by a TRPA1 antagonist and were absent in TRPA1-null mice. In wild type mice, systemic STZ treatment (180 mg/kg) evoked a loss of cold and mechanical sensitivity within an hour of injection, which lasted for at least 10 days. In contrast, Trpa1^−/− mice developed mechanical, cold, and heat hypersensitivity 24 h after STZ. The TRPA1-dependent sensory loss produced by STZ occurs before the onset of diabetes and may thus not be readily distinguished from the similar sensory abnormalities produced by the ensuing diabetic neuropathy. In vitro, STZ activated TRPA1 in isolated sensory neurons, TRPA1 cell lines, and membrane patches. Mass spectrometry studies revealed that STZ oxidizes TRPA1 cysteines to disulfides and sulfenic acids. Furthermore, incubation of tyrosine with STZ resulted in formation of dityrosine, suggesting formation of peroxynitrite. Functional analysis of TRPA1 mutants showed that cysteine residues that were oxidized by STZ were important for TRPA1 responsiveness to STZ. Our results have identified oxidation of TRPA1 cysteine residues, most likely by peroxynitrite, as a novel mechanism of action of STZ. Direct stimulation of TRPA1 complicates the interpretation of results from STZ models of diabetic sensory neuropathy and strongly argues that more refined models of diabetic neuropathy should replace the use of STZ.     

Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages--induction by multiple nonphagocytic stimuli

The ability of a number of stimulants to activate an oxidative burst (OB) in oil-elicited guinea pig peritoneal exudate macrophages (MPs) was examined. The parameters of the OB were the generation and extracellular release of Superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂). We found that: (1) The cocarcinogen and skin irritant phorbol myristate acetate (PMA) was the most potent OB activator—The weak cocarcinogen 4-O-methyl PMA was a proportionally less effective OB activator; (2) The lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean, Lotus, and pokeweed lectins, were also quite effective OB activators—The ability of Con A to stimulate O₂^? production was abolished by succinylation and could be prevented by the presence of α-methyl-D-mannoside; (3) Other stimulators of an OB in MPs were: N-formyl-methionyl peptides, opsonized zymosan, the Ca²⁺ ionophore A23187, phospholipase C, NaF, antimacrophage antibody, microtubule-disrupting drugs, and sodium nitroprusside—O₂^? generation induced by A23187 (but not that stimulated by PMA) was dependent on extracellular Ca²⁺; (4) The amount of O₂^? produced per cell was higher at low cell densities; (5) The addition of Superoxide dismutase (SOD) to the medium totally prevented the detection of O₂^? and augmented twice the amount of H₂O₂ recovered; (6) Pretreatment of MPs with the SOD inhibitor sodium diethyldithiocarbamate had no effect on the release of O₂^? but blocked H₂O₂ release in a dose-dependent manner. These data were interpreted as indicating that the bulk of H₂O₂ was derived by enzymatic dismutation of O₂^?; (7) The common mechanism by which such a variety of stimuli provoke an OB in MPs was not elucidated. No evidence was found to suggest a role for a cyclic nucleotide messenger.     

Leukocytic myeloperoxidase-mediated formation of bromohydrins and lysophospholipids from unsaturated phosphatidylcholines

Using MALDI-TOF mass spectrometry, we have shown that leukocytic myeloperoxidase (MPO) in the presence of its substrates (H₂O₂ and Br^?) does not induce any changes in saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Incubation of liposomes prepared from mono-unsaturated phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) with the (MPO + H₂O₂ + Br^?) system resulted in formation of bromohydrins as the main products. 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysophosphatidylcholine) was the main product of the reaction of polyunsaturated phosphatidylcholine (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) with the (MPO + H₂O₂ + Br^?) system. The formation of lysophospholipids as well as of bromohydrins was not observed when the enzyme or one of its substrates (H₂O₂ or Br^?) was absent from the incubation medium, or if an inhibitor of MPO (sodium azide) or hypobromite scavengers (taurine or methionine) were added. Thus, it can be postulated that the formation of bromohydrins as well as lysophospholipids by the (MPO + H₂O₂ + Br^?) system results from reactions of hypobromite formed during MPO catalysis with double bonds of acyl chains of phosphatidylcholine. Such destructive processes may take place in vivo in membrane-or lipoprotein-associated unsaturated lipids in centers of inflammation.     

A passive apparatus for controlled-flux delivery of biocides: hydrogen peroxide as an example

A new test method has been developed to estimate the required release rate of hydrogen peroxide (H₂O₂) to prevent marine biofouling. The technique exploits a well-defined concentration gradient of biocide across a cellulose acetate membrane. A controlled flux of H₂O₂, an environmentally friendly biocide, was obtained. Larvae of the barnacle, Balanus improvisus, were subjected to known release rates of H₂O₂ from a surface, under laboratory conditions. It was found that the distribution of settled larvae was not significantly different from the controls when H₂O₂ fluxes of 5–8 μg cm^?2 day^?1 were applied. However, release rates of 40 μg cm^?2 day^?1 significantly displaced the distribution of settled larvae towards the area of the chamber farthest away from the membrane. Membrane tests in seawater (Jyllinge Harbour, Denmark) for over 16 weeks showed that release rates of H₂O₂ of approximately 2800 μg cm^?2 day^?1 deterred biofouling efficiently. A H₂O₂ release rate of about 224 μg cm^?2 day^?1 resulted in some slime formation, but it was less than that on the H₂O₂-free control. It appears that to obtain efficient resistance to biofouling in natural seawater requires much higher membrane release rates of H₂O₂ (factor of between 5 and 50) than laboratory membrane exposure assays using barnacle larvae.     

Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen speciesgeneration

The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca²⁺ uptake. Here we sought to elucidate the effects of extramitochondrial Ca²⁺ (e[Ca²⁺]) on ROS production (measured as H₂O₂ release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl₂ and then energized with the complex I substrate Na⁺ pyruvate or the complex II substrate Na⁺ succinate. Mitochondrial H₂O₂ release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca²⁺] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H₂O₂ (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca²⁺], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H₂O₂ release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca²⁺] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H₂O₂ emission from complex III during ischemia.     

Cytochrome c peroxidase is a mitochondrial heme-based H2O2 sensor that modulates antioxidant defense

Hydrogen peroxide (H₂O₂) is a key signaling molecule that also induces apoptosis. Thus, cells must rapidly sense and tightly control H₂O₂ levels. Well-characterized cellular responses to exogenous H₂O₂ involve oxidation of specific cytosolic protein-based thiols but sensing of H₂O₂ generated by mitochondrial respiration is less well described. Here we provide substantial biochemical evidence that the heme enzyme Ccp1 (cytochrome c peroxidase), which is targeted to the intermembrane space, functions primarily as a mitochondrial H₂O₂ sensing and signaling protein in Saccharomyces cerevisiae. Key evidence for a sensing role for Ccp1 is the significantly higher H₂O₂ accumulation in ccp1-null cells(ccp1Δ) vs ccp1^W191F cells producing the catalytically inactive Ccp1^W191F variant. In fact, intracellular H₂O₂ levels (ccp1Δ>wildtype >ccp1^W191F) correlate inversely with the activity of the mitochondrial (and peroxisomal) heme catalase, Cta1 (ccp1Δ<wildtype <ccp1^W191F). Mitochondrial Sod2 activity also varies in the three strains (ccp1Δ>wildtype >ccp1^W191F) and ccp1Δ cells exhibit low superoxide levels. Notably, Ccp1^W191F is a more persistent H₂O₂ signaling protein than wild-type Ccp1, and this enhanced mitochondrial H₂O₂ signaling decreases the mitochondrial fitness of ccp1^W191F cells. However, these cells are fully protected from a bolus (0.4 mM) of exogenous H₂O₂ added after 12 h of growth, whereas the viability of ccp1Δ cells drops below 20%, which additionally associates Ccp1 with Yap1-dependent H₂O₂ signaling. Combined, our results strongly implicate Ccp1, independent of its peroxidase activity, in mitochondrial H₂O₂ sensing and signaling to maintain reactive oxygen species homeostasis.     

Macrophage-mediated cytolysis of erythrocytes in the guinea pig. II. Role of oxidative burst products in macrophage cytotoxicity activated by lectins and phorbol ester derivatives

Guinea pig peritoneal macrophages (GPPM) exhibited enhanced production of O₂^? and H₂O₂, and cytolytic activity toward erythrocytes, in response to reagents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), its methylated derivative 4-O-MeTPA, Con A, wheat germ agglutinin (WGA), and opsonized zymosan. In order to examine the possible role of oxidative burst products such as O₂^? and H₂O₂ in the cytolytic process, we used reagents and enzymes which influence the balance of O₂^? and H₂O₂ outside and inside the GPPM cells. Macrophage-mediated cytolysis (MMC) of erythrocytes in the presence of the activators and modulators was assessed by ⁵¹Cr release assay. MMC activated by TPA and 4-O-MeTPA was inhibited by scavengers of H₂O₂ such as catalase and α-tocopherol, and was augmented by the catalase inhibitor 3-amino-1,2,4-triazole, and by horseradish peroxidase. TPA- and 4-O-MeTPA-activated MMC was only partially inhibited by the O₂^? scavenger cytochrome c and the enzyme superoxide dismutase and unaffected by cytochalasin D (an inhibitor of phagocytosis). MMC activated by the lectins Con A and WGA was unaffected by the scavengers and enzymes used, but markedly inhibited by cytochalasin D. Activation of MMC by TPA, WGA, and phagocytosis of opsonized zymosan, as well as O₂^? and H₂O₂ generation triggered by these reagents, were markedly inhibited by chlorpromazine. The results indicate that GPPM-mediated cytolysis activated by lectins, phorbol ester derivatives, and phagocytosis of opsonized zymosan, is dependent on the generation of oxidative burst products, mainly H₂O₂. TPA- or 4-O-MeTPA-activated MMC is mainly an extracellular event, while lectin-activated MMC may take place within the macrophages.     

Generation of a proton gradient in Desulfovibrio vulgaris

Washed cells of Desulfovibrio vulgaris strain Marburg oxidized H₂, formate, lactate or pyruvate with sulfate, sulfite, trithionate, thiosulfate or oxygen as electron acceptor. CuCl₂ as an inhibitor of periplasmic hydrogenase inhibited H₂ and formate oxidation with sulfur compounds, and lactate oxidation in H₂-grown, but not in lactate-grown cells. H₂ oxidation was sensitive to O₂ concentrations above 2% saturation. Carbon monoxide inhibited the oxidation of all substrates tested. Additions of micromolar H₂ pulses to cells incubated in KCl in the presence of various sulfur compounds (reductant pulse method) resulted in a reversible acidification. This proton release was stimulated by thiocyanate, methyl triphenylphosphonium (MTPP⁺) or valinomycin plus EDTA, and completely inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), CuCl₂ or carbon monoxide. The extrapolated H⁺/H₂ ratios obtained with sulfate, sulfite, trithionate or thiosulfate varied from 1.0 to 1.7. Micromolar additions of O₂ to cells incubated in the presence of excess of electron donor (oxidant pulse method) caused proton translocation with extrapolated H⁺/H₂ ratios of 3.9 with H₂, 1.6 with lactate and 2.4 with pyruvate. Since a periplasmic hydrogenase can release at maximum 2 H⁺/H₂, it is concluded that D. vulgaris is able to generate a proton gradient by vectorial proton translocation across the cytoplasmic membrane and by extracellular proton release by a periplasmic hydrogenase.     

PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus

Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H₂O₂, whereas Dpr (an Fe²⁺-chelating protein)-dependent H₂O₂ protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H₂O₂-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl₂, S. oligofermentans increased survival after H₂O₂ stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn²⁺-mediated H₂O₂ resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn²⁺) unless it was supplemented with ≥2.5 μM MnCl₂ and caused 82 to 95% reduction of the cellular Mn²⁺ level, while mntABC overexpression increased cellular Mn²⁺ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn²⁺ transporter in S. oligofermentans. mntA mutation reduced the survival of H₂O₂-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H₂O₂-challenged survival 12-fold, indicating that MntABC-mediated Mn²⁺ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H₂O₂ pulsing upregulated mntABC, while H₂O₂-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H₂O₂ can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H₂O₂ stress defenses and may be distributed across all oral streptococci lacking catalase.     

Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO₂), reactive oxygen species (ROS) and potassium (K⁺) channels. Here we test the hypothesis that K⁺ channels mediate the effects of pO₂ and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO₂. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO₂-sensitive voltage-gated K⁺ (K_V) channels, or ROS (10–1000μM H₂O₂). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and ⁸⁶Rb (K⁺) efflux assessed to estimate syncytiotrophoblast K⁺ permeability. hCG secretion and ⁸⁶Rb efflux were significantly greater in explants maintained in 21% pO₂ than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO₂, and reduced ⁸⁶Rb efflux at 21% but not 6% pO₂. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO₂ and unaffected by 4-AP/TEA. H₂O₂ stimulated ⁸⁶Rb efflux and hCG secretion at normoxia but decreased ⁸⁶Rb efflux, without affecting hCG secretion, at 21% pO₂. 4-AP/TEA-sensitive K⁺ channels participate in pO₂-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and ⁸⁶Rb efflux are pO₂-dependent but causal links between the two remain to be established.     

Akt1 Intramitochondrial Cycling Is a Crucial Step in the Redox Modulation of Cell Cycle Progression

Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser⁴⁷³ by mTORC2 and Thr³⁰⁸ by PDK1. On these bases, we investigated the mechanistic connection of H₂O₂ yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H₂O₂ entails the entrance of cytosolic P-Akt1 Ser⁴⁷³ to mitochondria, where it is further phosphorylated at Thr³⁰⁸ by constitutive PDK1. Phosphorylation of Thr³⁰⁸ in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H₂O₂, Akt1-PDK1 association is disrupted and P-Akt1 Ser⁴⁷³ accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H₂O₂ effects on Akt1-PDK interaction depend on the selective oxidation of Cys³¹⁰ to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.     

Hydrogen peroxide depolarizes mitochondria and inhibits IP3-evoked Ca2+ release in the endothelium of intact arteries

Hydrogen peroxide (H₂O₂) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H₂O₂ regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H₂O₂ on Ca²⁺ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca²⁺ signals and acetylcholine evoked Ca²⁺ increases were inhibited by H₂O₂. H₂O₂ inhibition of acetylcholine-evoked Ca²⁺ signals was reversed by catalase. H₂O₂ exerts its inhibition on the IP₃ receptor as Ca²⁺ release evoked by photolysis of caged IP₃ was supressed by H₂O₂. H₂O₂ suppression of IP₃-evoked Ca²⁺ signalling may be mediated by mitochondria. H₂O₂ depolarized mitochondria membrane potential. Acetylcholine-evoked Ca²⁺ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP₃-evoked Ca²⁺ release by H₂O₂ arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca²⁺ overload during IP₃-linked Ca²⁺ signals by a H₂O₂ mediated negative feedback depolarization of the organelle and inhibition of IP₃-evoked Ca²⁺ release.     

Role of LytF and AtlS in eDNA Release by Streptococcus gordonii

Extracellular DNA (eDNA) is an important component of the biofilm matrix produced by many bacteria. In general, the release of eDNA is associated with the activity of muralytic enzymes leading to obvious cell lysis. In the Gram-positive oral commensal Streptococcus gordonii, eDNA release is dependent on pyruvate oxidase generated hydrogen peroxide (H₂O₂). Addition of H₂O₂ to cells grown under conditions non-permissive for H₂O₂ production causes eDNA release. Furthermore, eDNA release is maximal under aerobic growth conditions known to induce pyruvate oxidase gene expression and H₂O₂ production. Obvious cell lysis, however, does not occur. Two enzymes have been recently associated with eDNA release in S. gordonii. The autolysin AtlS and the competence regulated murein hydrolase LytF. In the present report, we investigated the role of both proteins in the H₂O₂ dependent eDNA release process. Single and double mutants in the respective genes for LytF and AtlS released less eDNA under normal growth conditions, but the AtlS mutant was still inducible for eDNA release by external H₂O₂. Moreover, we showed that the AtlS mutation interfered with the ability of S. gordonii to produce eDNA release inducing amounts of H₂O₂. Our data support a role of LytF in the H₂O₂ eDNA dependent release of S. gordonii as part of the competence stress pathway responding to oxidative stress.