Hydrogen peroxide-induced cytoskeletal rearrangement in cultured pulmonary endothelial cells

Although the signaling pathways leading to hydrogen peroxide (H₂O₂)-induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H₂O₂ on actin reorganization in BPAEC. H₂O₂ initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a time- and concentration-dependent manner. The H₂O₂-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C. H₂O₂ also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H₂O₂ transiently attenuated PKC activity but slightly increased the phosphorylation of the prominent PKC substrate and actin-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to below basal levels by 30 min. On the other hand, H₂O₂ induced a time- and dose-dependent phosphorylation of myosin light chains which was eliminated by both MLCK and PKC inhibitors. These data suggest that MLCK contributes to H₂O₂-induced myosin light chain phosphorylation and actin rearrangement and that PKC may play a permissive role. Neither of these enzymes appears to be involved in the H₂O₂-induced recruitment of actin to the cytoskeleton. J. Cell. Physiol. 174:370–379, 1998. © 1998 Wiley-Liss, Inc.     

Taurine protection of PC12 cells against endoplasmic reticulum stress induced by oxidative stress

Background

Taurine is a free amino acid present in high concentrations in a variety of organs of mammalians. As an antioxidant, taurine has been found to protect cells against oxidative stress, but the underlying mechanism is still unclear.

Methods

In this report, we present evidence to support the conclusion that taurine exerts a protective function against endoplasmic reticulum (ER) stress induced by H₂O₂ in PC 12 cells. Oxidative stress was introduced by exposure of PC 12 cells to 250 uM H₂O₂ for 4 hours.

Results

It was found that the cell viability of PC 12 cells decreased with an increase of H₂O₂ concentration ranging from approximately 76% cell viability at 100 uM H₂O₂ down to 18% at 500 uM H₂O₂. At 250 uM H₂O₂, cell viability was restored to 80% by taurine at 25 mM. Furthermore, H₂O₂ treatment also caused a marked reduction in the expression of Bcl-2 while no significant change of Bax was observed. Treatment with taurine restored the reduced expression of Bcl-2 close to the control level without any obvious effect on Bax. Furthermore, taurine was also found to suppress up-regulation of GRP78, GADD153/CHOP and Bim induced by H₂O₂, suggesting that taurine may also exert a protective function against oxidative stress by reducing the ER stress.

Conclusion

In summary, taurine was shown to protect PC12 cells against oxidative stress induced by H₂O₂. ER stress was induced by oxidative stress and can be suppressed by taurine.

     

Aerobic hydrogenase activity in Anacystis Nidulans The oxyhydrogen reaction

1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction.2. Besides O₂, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O₂ levels <10–15%; aerobically the artificial acceptors were reduced simultaneously with O₂.3. H₂-supported O₂ uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction.4. The kinetic properties of O₂ uptake by isolated membranes were the same in presence of H₂ and of other respiratory substrates.5. Low rates of H₂ evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction.6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H₂ to O₂ through the respiratory chain.     

Ca<Superscript>2+</Superscript>-mediated Potentiation of the Swelling-induced Taurine Efflux from HeLa Cells: On the Role of Calmodulin and Novel Protein Kinase C Isoforms

The present work sets out to investigate how Ca²⁺ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca²⁺-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation of the release pathway. The accelerated inactivation is not observed in hypotonic Ca²⁺-free or high-K⁺ media. Addition of Ca²⁺-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca²⁺-activated K⁺ channels. The taurine release from control cells and cells exposed to Ca²⁺ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca²⁺-augmented taurine release. Exposure to Ca²⁺-mobilizing agonists prior to a hypotonic challenge also augments a subsequent swelling-induced taurine release even though the intracellular Ca²⁺-concentration has returned to the unstimulated level. The Ca²⁺-induced augmentation of the swelling-induced taurine release is abolished by inhibition of calmodulin, but unaffected by inhibition of calmodulin-dependent kinase II, myosin light chain kinase and calcineurin. The effect of Ca²⁺-mobilizing agonists is mimicked by protein kinase C (PKC) activation and abolished in the presence of the PKC inhibitor Gö6850 and following downregulation of phorbol ester-sensitive PKC isoforms. It is suggested that Ca²⁺ regulates the volume-sensitive taurine-release pathway through activation of calmodulin and PKC isoforms belonging to the novel subclass (nPKC).This revised version was published online in June 2005 with a corrected cover date.     

Effects of culture conditions on taurine uptake by various variants of human endometrial carcinoma cells in culture

Summary. The general properties of the taurine uptake in human endometrial tumoral Ishikawa cells were similar to those usually found in other tissues. Uptake was notably affected by the oxygen pressure, being higher at the physiological pO₂ of the endometrium (40mm Hg, equivalent to 5% O₂) compared to that used under standard experimental culture conditions (160mm Hg or 20% O₂). Uptake of taurine was also density-dependent in Ishikawa cells and was significantly decreased at confluence. Uptake regulation by PKC driven phosphorylation occurs only in growing cells and not in resting cells. The taurine uptake of three Ishikawa cell lines was very different. The taurine uptake of one of the cell lines was affected by estradiol, probably through a non-genomic pathway, whereas tamoxifen had no effect in all cell lines.     

High-fat diet induces metabolic changes and reduces oxidative stress in female mouse hearts

After an acute myocardial infarction, obese patients generally have a better prognosis than their leaner counterparts, known as the “obesity paradox”. In addition, female sex is associated with a lower risk of cardiac ischemic events and smaller infarct size compared to males. The objective of the present work was to study the metabolic phenotype and mitochondrial function associated to female sex and short-term high-fat diet. ¹H NMR spectra of mice heart extracts were analysed by mRMR variable selection and linear discriminant analysis was used to evaluate metabolic changes. In separate experiments, O₂ consumption and H₂O₂ production were measured from isolated mitochondria as well as serum oxidation susceptibility. Fingerprinting showed that male hearts contained more myo-inositol, taurine and glutamate than female hearts. HFD reduced the levels of creatine, taurine citrate and acetate. Profiling showed increased alanine and fumarate in HFD suggesting altered glycolitic and Krebs cycle pathways. Female mice contained less glucose than males. Female sex nor HFD altered mitochondria oxygen consumption but both conditions reduced the amount of H₂O₂ produced in an additive manner. Serum of females had lower oxidation susceptibility than serum from males but there were no differences associated with HFD. In conclusion, female sex and short-term HFD have an effect on the myocardial metabolic pattern and reduce the amount of H₂O₂ produced by mitochondria in an additive manner suggesting different mechanisms of action. This could explain, at least in part, the protection afforded by female sex and the “obesity paradox”.     

UCP3 overexpression neutralizes oxidative stress rather than nitrosative stress in mouse myotubes

The deleterious effects of oxidants on proteins may be modified by overexpression of uncoupling protein 3 (UCP3) in skeletal muscle cells exposed to hyperoxia or H₂O₂. UCP3 overexpression significantly attenuated the increase in protein carbonylation in response to hyperoxia and H₂O₂ exposures. However, antioxidant enzyme content and activity (superoxide dismutases, peroxiredoxins, glutathione peroxidase-I, and catalase) were reduced or not modified in UCP3-overexpressing myotubes exposed to oxidants. Protein nitration increased in UCP3-overexpressing cells exposed to hyperoxia, but not to H₂O₂. We conclude that protein oxidation rather than nitration is neutralized by UPC3 overexpression in mouse myotubes exposed to abundant reactive oxygen species.     

Downregulation of taurine uptake in multidrug resistant Ehrlich ascites tumor cells

Summary.  In daunorubicin resistant Ehrlich ascites tumor cells (DNR), the initial taurine uptake was reduced by 56% as compared to the parental, drug sensitive Ehrlich cells. Kinetic experiments indicated that taurine uptake in Ehrlich cells occurs via both high- and low-affinity transporters. The maximal rate constant for the initial taurine uptake was reduced by 45% (high-affinity system) and 49% (low affinity system) in the resistant subline whereas the affinity of the transporters to taurine was unchanged. By immunoblotting we identified 3 TauT protein bands in the 50–70 kDa region. A visible reduction in the intensity of the band with the lowest molecular weight was observed in resistant cells. Quantitative RT-PCR indicated a significant reduction in the amount of taurine transporter mRNA in the resistant cells. Drug resistance in DNR Ehrlich cells is associated with overexpression of the mdr1 gene product P-glycoprotein (P-gp). Using 5 progressively DNR resistant Ehrlich cell sublines with different P-gp expression pattern no correlation between taurine uptake and P-gp expression was found. Taurine uptake in MDR1 transfected NIH/3T3 mouse fibroblasts was in contrast to the findings in Ehrlich cells increased compared to the parental fibroblasts. It is concluded that the reduced taurine uptake in resistant Ehrlich cells reflects a down regulation of the taurine transporter at the mRNA and protein level and it is most probably not related to P-gp overexpression. Received October 22, 2001 Accepted November 26, 2001     

Lysophosphatidylcholine Induces Taurine Release from HeLa Cells

The putative role of lysophospholipids in activation and regulation of the volume-sensitive taurine efflux was investigated in HeLa cells using tracer technique. Lysophosphatidylcholine (LPC, 10 μm) with oleic acid increased taurine efflux during hypotonic and isotonic conditions. Substituting palmitic or stearic acid for oleic acid enhanced taurine release during isotonic conditions, whereas ethanolamine, serine or inositol containing lysophospholipids were ineffective. High concentrations of LPC (25 μm) induced Ca²⁺ influx, loss of adenosine nucleotides, taurine and the Ca²⁺-sensitive probe Fura-2, and thus reflected a general breakdown of the membrane permeability barrier. Low concentrations of LPC (5–10 μm) solely induced taurine efflux. The LPC-induced taurine release was unaffected by anion channel blockers (DIDS, MK196) and the 5-lipoxygenase inhibitor ETH 615-139, which all blocked the volume sensitive taurine efflux. Furthermore, LPC-induced taurine release was reduced by antioxidants (NDGA, vitamin E) and the protein tyrosine kinase inhibitor genistein. The swelling-induced taurine efflux was in the absence of LPC unaffected by vitamin E, blocked by genistein, and increased by H₂O₂ and the protein tyrosine phosphatase inhibitor vanadate. It is suggested that low concentrations of LPC permeabilizes the plasma membrane in a Ca²⁺-independent process that involves generation of reactive oxygen species and tyrosine phosphorylation, and that LPC is not a second messenger in activation of the volume sensitive taurine efflux in HeLa cells. Received: 17 December 1999/Revised: 13 April 2000     

Effects of arginine,L-alanyl-L-glutamine or taurine on neutrophil (PMN) free amino acid profiles and immune functions <Emphasis Type="Italic">in vitro</Emphasis>

Summary. The objective of this study was to determine the effects of arginine, L-alanyl-L-glutamine (Ala-Gln) or taurine on polymorphonuclear leucocyte (PMN) free amino acid profiles, superoxide anion (O_{2 −}) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase activity (MPO). Arginine led to significant increases in PMN arginine, ornithine, citrulline, aspartate, glutamate and alanine concentrations as well as increased H₂O₂-generation and MPO activity while O_{2 −}-formation was decreased. Ala-Gln caused significant increases in PMN free glutamine, alanine, asparagine, aspartate, glutamate, ornithine, arginine, serine and glycine concentrations and increased PMN immune functions. Taurine significantly increased PMN free taurine profiles, reduced PMN neutral amino acid content and decreased H₂O₂- and O_{2 −}-formation while MPO was increased. Altogether, the pharmacological regimens which enhance the supply of arginine, Ala-Gln or taurine in whole blood significantly affect PMN "susceptible free amino acid pool". This may be one of the determinants in PMN nutrition considerably influencing PMN immune functions. Received October 25, 2000 Accepted March 21, 2001     

Hypochlorous Acid Generated by Neutrophils Inactivates ADAMTS13: AN OXIDATIVE MECHANISM FOR REGULATING ADAMTS13 PROTEOLYTIC ACTIVITY DURING INFLAMMATION*

ADAMTS13 is a plasma metalloproteinase that cleaves large multimeric forms of von Willebrand factor (VWF) to smaller, less adhesive forms. ADAMTS13 activity is reduced in systemic inflammatory syndromes, but the cause is unknown. Here, we examined whether neutrophil-derived oxidants can regulate ADAMTS13 activity. We exposed ADAMTS13 to hypochlorous acid (HOCl), produced by a myeloperoxidase-H₂O₂-Cl⁻ system, and determined its residual proteolytic activity using both a VWF A2 peptide substrate and multimeric plasma VWF. Treatment with 25 nm myeloperoxidase plus 50 μm H₂O₂ reduced ADAMTS13 activity by >85%. Using mass spectrometry, we demonstrated that Met²⁴⁹, Met³³¹, and Met⁴⁹⁶ in important functional domains of ADAMTS13 were oxidized to methionine sulfoxide in an HOCl concentration-dependent manner. The loss of enzyme activity correlated with the extent of oxidation of these residues. These Met residues were also oxidized in ADAMTS13 exposed to activated human neutrophils, accompanied by reduced enzyme activity. ADAMTS13 treated with either neutrophil elastase or plasmin was inhibited to a lesser extent, especially in the presence of plasma. These observations suggest that oxidation could be an important mechanism for ADAMTS13 inactivation during inflammation and contribute to the prothrombotic tendency associated with inflammation.     

Oxidation of lipids and membranes II: Effects of oxidants on cerebral white matter homogenates

Bovine cerebral white matter homogenates were oxidized by various oxidizing solutions of equal molarity and subsequently extracted with water. Most of the oxidants tested (K-dichromate, FeCl₃, H₂O₂, O₂, and chloroperbenzoic, ascorbic, performic, and periodic acids) rendered the various myelin constituents less extractable than the constituents of unoxidized control homogenates. KMnO₄, and to a lesser degree hemoglobin, rendered myelin constituents more extractable with water than those of the control homogenates. The findings indicate that most of the oxidants produced stabilization of the lamellar pattern, probably through cross-linking and polymerization. KMnO₄ and hemoglobin caused labilization and breakdown of the membranous structure. Proof that stabilization of membranes is caused by some oxidants and that fragmentation occurs by the action of KMnO₄ and hemoglobin was obtained by the light-scattering technique and by electron microscopy of the oxidized homogenates. Evidence obtained indicated that formation of hydrophobic end groups during oxidation favors polymerization, while prevalence of hydrophilic groups is associated with fragmentation.     

Cytochrome c oxidase: the mechanistic significance of structural H+ in energy transduction

Changes in the bulk-phase concentration of O₂ and H⁺ associated with the reduction of O₂ to water are simultaneously determined in reactions catalyzed by fully reduced cytochrome c oxidase both isolated and embedded in liposomes. Consistent with the polyphasic kinetics of electron transfer through the oxidase, the time course of O₂ consumption and H⁺ translocation exhibit the following novel characteristics: (1) The uptake of scalar protons (H_m ⁺), the ejection of vectorial protons (H⁺ _v), and the consumption of O₂, all proceed in a kinetically polyphasic process. (2) During the first phase of the reaction the rates of O₂ uptake and H⁺ transfer are extremely fast and compatible with the rates of electron flow through the oxidase. (3) The K_m of the oxidase for O₂ is close to 75 M, the same for O₂ consumption and scalar H⁺ uptake. The V_max of O₂ reduction to water in reactions catalyzed by the isolated enzyme is, at least, 0.5 × 10⁴ s^–1. (4) The extent of vectorial H⁺ ejection by cytochrome c oxidase embedded in liposomes is an exponential function dependent on both enzyme concentration and extent of O₂ consumption. (5) The H⁺/O stoichiometry of H⁺ ejection is a variable that may reach a maximum value of 4.0 only when the enzyme undergoes net oxidation at extremely high enzyme/O₂ molar ratios. It is postulated that the generation of useful energy at the level of cytochrome c oxidase depends not only on the number of molecules of O₂ reduced to water but also on the extent and state of reduction and/or protonation of the enzyme.     

Glutamate receptor-mediated taurine release from the hippocampus during oxidative stress

Background

Hippocampal slices swell and release taurine during oxidative stress. The influence of cellular signalling pathways on this process is unclear. Glutamate signalling can facilitate volume regulation in other CNS preparations. Therefore, we hypothesize activation of taurine release by oxidative stress results from tissue swelling and is coupled to activation of glutamate receptors.

Methods

Rat hippocampi were incubated at room temperature for 2 hr in artificial cerebrospinal fluid (aCSF) equilibrated with 95% O₂ plus 5% CO₂. For some slices, 1 mM taurine was added to the aCSF to maintain normal tissue taurine content. Slices then were perfused with aCSF at 35° C and baseline data recorded before 2 mM H₂O₂ was added. For some studies, mannitol or inhibitors of glutamate receptors or the volume-regulated anion channel (VRAC) were added before and during H₂O₂ treatment. The intensity of light transmitted through the slice (the intrinsic optical signal, IOS) was determined at 1-min intervals. Samples of perfusate were collected at 2-min intervals and amino acid contents determined by HPLC. Data were analyzed by repeated measures ANOVA and post hoc Dunnett’s test with significance indicated for p<0.05.

Results

IOS of slices prepared without taurine treatment increased significantly by 3.3±1.3% (mean±SEM) during oxidative stress. Little taurine was detected in the perfusate of these slices and the rate of taurine efflux did not change during H₂O₂ exposure. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate antagonist, 25 µM CNQX, but not the N-methyl-D-aspartate (NMDA) receptor antagonist, 10 µM MK-801, inhibited the increase in IOS during H₂O₂ treatment. Taurine-treated slices exposed to H₂O₂ showed no change in IOS; however, taurine efflux increased by 335±178%. When these slices were perfused with hypertonic aCSF (350 mOsm) or exposed to the VRAC inhibitor, 20 µM DCPIB, no increase in the taurine efflux rate was observed during H₂O₂ exposure. Taurine-treated slices perfused with 10 µM MK-801 during H₂O₂ exposure showed a 4.6±1.9% increase in IOS but no increase in the taurine efflux rate.

Conclusions

Taurine efflux via VRAC is critical for volume regulation of hippocampal slices exposed to oxidative stress. This increased taurine efflux does not result from direct activation of the taurine release pathway by H₂O₂. NMDA receptor activation plays an important role in taurine release during oxidative stress.

     

Methods for detection and measurement of hydrogen peroxide inside and outside of cells

Hydrogen peroxide (H₂O₂) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H₂O₂ requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H₂O₂ in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3’5’-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H₂O₂, methods based on dihydro compounds such as 2’,7’-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H₂O₂. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H₂O₂-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H₂O₂ in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H₂O₂ can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H₂O₂, they cannot be used to monitor transient changes in H₂O₂ concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H₂O₂ and can be targeted to various compartments of the cell, but they are not selective for H₂O₂ because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H₂O₂-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.     

Factors affecting the transport of β-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the β-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na⁺ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K⁺ or Li⁺ do not increase taurine accumulation more than Na⁺-free mannitol, except that the combination of external K⁺ and Na¹ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN⁻ and NO₃⁻) or less permeant (SO₄²⁻), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl⁻ stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO₃ and LiSO₄. Internal LiCl, regardless of the final Na⁺ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO₃ or LiSO₄ with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl⁻ in taurine uptake in addition to Na⁺ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H⁺ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na⁺/H⁺ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na⁺ concentrations. Taurine uptake is blocked only by other β-amino acids and in a competitive fashion. d-glucose and p-aminohippurate at high concentrations (> 10⁻³ M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of d-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal β-amino acid transport system in brush-border vesicles and indicate a role for external Cl in this uptake system.     

Light-dependent reduction of dehydroascorbate and uptake of exogenous ascorbate by spinach chloroplasts

A reconstituted spinach chloroplast system containing thylakoids, stroma and 0.1 mM NADPH supported O₂ evolution in the presence of oxidised glutathione (GSSG). The properties of the reaction were consistent with light-coupled GSSG-reductase activity involving H₂O as eventual electron donor. The reconstituted system also supported dehydroascorbate-dependent O₂ evolution in the presence of 0.6 mM reduced glutathione (GSH) and 0.1 mM NADPH with the concomitant production of ascorbate. The GSSG could replace GSH in which case the production of GSH preceded the accumulation of ascorbate. The data are consistent with the light-dependent reduction of dehydroascorbate using H₂O as eventual electron donor via the sequence H₂O→NADP→GSSG→dehydroascorbate. Approximately 30% of the GSH-dehydrogenase activity of spinach leaf protoplasts is localised in chloroplasts: this could not be attributed to contamination of chloroplasts by activity from the extrachloroplast compartment. Washed intact chloroplasts supported the uptake of ascorbate but the uptake mechanism had a very low affinity for ascorbate (K_m approximately 20 mM). The rate of uptake of ascorbate was less than the rate of light-dependent reduction of dehydroascorbate and too slow to account for the rate of H₂O₂ reduction by washed intact chloroplasts.     

Oxidation of proteins in rat heart and lungs by polymorphonuclear leukocyte oxidants

Summary The ability of the polymorphonuclear leukocyte (PMN) oxidants, hypochlorous acid (HOC1) and hydrogen peroxide (H₂O₂), to oxidize proteins in rat heart and lung tissues was investigated. Cardiac myocytes, heart tissue slices, isolated perfused hearts, and lung tissue slices, were treated with HOCI and H₂O₂ and the extent of methionine and cysteine oxidation was determined in the cellular proteins. Cardiac tissues were found to be highly susceptible to oxidation by physiological concentrations of HOCl. For example, in isolated hearts perfused for 60 min with 100 M HOCI, approximately 18010 of the methionine and 2801o of the cysteine residues were oxidized. Lung tissues, unlike those of the heart, were resistant to physiological concentrations of HOCI, showing no oxidation of proteins. HOCI was much more effective than H₂O₂ in oxidizing proteins, suggesting that HOCI may be the most reactive oxidant produced by activated PMN. These studies show that PMN oxidants, in particular HOC I, can cause significant oxidation of proteins in target tissues, and may therefore constitute a primary cause of tissue injury at sites of inflammation. In addition, these studies show that different tissues may have varying susceptibilities to PMN oxidants.