Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

Decay Studies of Dmpo-Spin Adducts of Free Radicals Produced by Reactions of Metmyoglobin and Methemoglobin with Hydrogen Peroxide

The 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adduct of myoglobin (Mb) or hemoglobin (Hb) was formed when metmyoglobin (MetMb) or methemoglobin (MetHb) reacted with H₂O₂ in the presence of DMPO, and both decayed with half-life of a few minutes. The DMPO spin adduct of Mb decayed with biphasic kinetics with k₁ = 0.645 min^-1 and k₂ = 0.012 min^-1, indicating that the spin adduct consisted of two kinetically heterogeneous species, stable and unstable ones. The DPMO spin adduct of Hb, however, was homogeneous. Decay of both spin adducts was accelerated in the presence of tyrosine, tryptophan or cysteine, but not phenylalanine, methionine or histidine. The decay obeyed the first order kinetics at varying concentrations of the spin adducts. The decay was accelerated by denaturation and proteolysis of protein moiety. The decay rate was not affected by the extra addition of MetMb or MetHb to each spin adduct. The decay rate of the spin adduct of Mb was increased by hematin in the presence of H₂O₂ and decreased by catalase. Decay of stable spin adduct of Mb, however, was not significantly changed under any experimental conditions used. These results led us to conclude that instability of the DMPO-spin adducts of Mb and Hb is due to intramolecular redox reactions between the spin adducts and amino acid residues and/or products of the reaction between heme and H₂O₂.     

Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity

Tea (Camellia sinensis) catechins have been studied for disease prevention. These compounds undergo oxidation and produce H₂O₂. We have previously shown that holding tea solution or chewing tea leaves generates high salivary catechin levels. Herein, we examined the generation of H₂O₂ in the oral cavity by green tea solution or leaves. Human volunteers holding green tea solution (0.1-0.6%) developed salivary H₂O₂ with C_max = 2.9-9.6 μM and AUC_0 → ∞ = 8.5-285.3 μM min. Chewing 2 g green tea leaves produced higher levels of H₂O₂ (C_max = 31.2 μM, AUC_0 → ∞ = 1290.9 μM min). Salivary H₂O₂ correlated with catechin levels and with predicted levels of H₂O₂ (C_{max(expected)} = 36 μM vs C_{max(determined)} = 31.2 μM). Salivary H₂O₂ and catechin concentrations were similar to those that are biologically active in vitro. Catechin-generated H₂O₂ may, therefore, have a role in disease prevention by green tea.     

Characterization of H2O2-induced acute apoptosis in cultured neural stem/progenitor cells

In the present study, we characterized hydrogen peroxide (H₂O₂)-induced cell apoptosis and related cell signaling pathways in cultured embryonic neural stem/progenitor cells (NS/PCs). Our data indicated that H₂O₂ induced acute cell apoptosis in NS/PC in concentration- and time-dependent manners and selectively, it transiently increased PI3K-Akt and Mek-Erk1/2 in a dose-dependent manner. Inhibition of PI3K-Akt with wortmannin, a PI3-K inhibitor, was found to significantly increase H₂O₂-induced acute apoptosis and dramatically decrease basal pGSK3β levels. The level of pGSK3β remained unchanged with H₂O₂ exposure. We conclude that the transient activation of PI3K-Akt signaling delays the H₂O₂-induced acute apoptosis in cultured NS/PCs in part through maintaining the basal pGSK3β level and activating other downstream effectors.     

Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4β-8)-epicatechin) – a major polyphenol in cocoa – against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H₂O₂). CPF (1 and 5 μg/ml) and procyanidin B2 (1 and 5 μM) reduced PC12 cell death caused by H₂O₂, as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H₂O₂-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H₂O₂ induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X_L and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H₂O₂ treatment diminished PARP cleavage and increased Bcl-X_L and Bcl-2 expression compared with those only treated with H₂O₂. Activation of caspase-3 by H₂O₂ was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H₂O₂-induced apoptosis involve inhibiting the downregulation of Bcl-X_L and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.     

Haemoglobin and Myoglobin as Inhibitors of Hydroxyl Radical Generation in a Model System of “Iron Redox” Cycle

Methionine was oxidized to ethylene by an “Iron Redox” system containing H₂O₂, Fe-EDTA and ascorbate. generating hydroxyl radicals or another species of similar reactivity. Oxy or met forms of haemoglobin and myoglobin were found to inhibit methionine oxidation. Methionine oxidation was elevated in the “Iron Redox” system by increasing ascorbic acid concentration. However, in the presence of metmyoglobin or methaemoglobin, the increases in ascorbic acid did not lower the haemproteins' inhibitory effects but rather increased them.

The pro-oxidative or anti-oxidative activities of haemproteins in biological oxidative reactions seem to be dependent on compartmentalization and on the presence and concentrations of reducing compounds and H₂O₂.     

Oxidation and Cross-Linking of Human Hemoglobins by Activated Oxygen Species

The effect of activated oxygen species on human hemoglobins was studied. All radicals induced polymerization in Hb A both intermolecular and by cross-linking of subunits (intramolecular). However, a system producing mainly superoxide ion gave the most important changes. An oxidation step is necessary to produce polymerization since in the case of cyanmet Hb A (where there is no possible oxidation) no polymerization occurs. The effect of O^-₂ on blocked SH β 93 Hbs or on the abnormal Hbs tested was practically identical to that on Hb A although their autoxidation rates were modified. Consequently the action of radicals is different from autoxidation processes and the modified residues in the abnormal hemoglobins are not involved in the action of superoxide ion on Hb.

The kinetics of oxidation of Hb by H₂O₂ followed two steps: the first is the oxidation of oxy Hb to ferri Hb and the second is hemichrome formation. This last step is independent of the presence of H₂O₂ since it is not inhibited by catalase. The kinetics of oxidation to ferri Hb were of second order and the rate constant was found to be 16 M^-1 sec^-1.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

The Generation of Ferryl or Hydroxyl Radicals During Interaction of Haemproteins with Hydrogen Peroxide

The oxidation of 2-keto-4-thiomethyl butyric acid (KTBA) and methionine to ethylene has been used to evaluate generation of ferryl species or hydroxyl radicals by H₂O₂-_-activated haemproteins or free ferric ions. Hydrogen peroxide was generated by a glucose oxidase-glucose system at a rate of 1 μM/min. Free ferric in the presence of H₂O₂ oxidizes KTBA, and this was highly inhibited by hydroxyl radical scavengers, caeruloplasmin, superoxide dismutase (SOD) and EDTA. However, when metmyoglobin, methaemoglobin (MtHb) or horseradish peroxidase (HRP) were tested in the same model system, hydroxyl radical scavengers suppressed partially KTBA oxidation and caeruloplasmin, SOD and EDTA failed to inhibit the reaction. Cytochrome-c was found to be a weak promoter of KTBA oxidation in the presence of H₂O₂. Methionine was oxidized to ethylene by an active system which generates hydroxyl radicals, but not by H₂O₂-_-activated metmyoglobin. Ferric ions chelated to membranes or ADP in the presence of H₂O₂ generated enzymatically, initiated membranal lipid peroxidation only in the presence of ascorbic acid, and this was inhibited by EDTA. In contrast, metmyoglobin and methaemoglobin activated by H₂O₂ generated by the same system, initiated membranal lipid peroxidation and this was not inhibited by EDTA. It is concluded that ferryl and not HO. is the main oxidant in systems containing myoglobin and haemoglobin activated by low concentrations of H₂O₂.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.     

Inactivation of Heart Dihydrolipoamide Dehydrogenase by Copper Fenton Systems. Effect of Thiol Compounds and Metal Chelators

Copper Fenton systems (Cu(II)/H₂O₂ and Cu(II)/Asc) inactivated the lipoamide reductase and enhanced the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H₂O₂ treatment, the number of titrated thiols in LADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD⁺ or ATP) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H₂O₂. Dihydrolipoamide, dihydrolipoic acid, Captopril, acetylcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanothione prevented LADH inactivation. 100 μM GSH, DL-dithiothreitol, N-(2-mercaptopropionylglicine) and penicillamine protected LADH against Cu(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also protected LADH against Cu(II)/H₂O₂. Allopurinol provided partial protection against Cu(II)/H₂O₂. EthanoI, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H₂O₂ or Cu(II). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H₂O₂. It is concluded that site-specifically generated HO, radicals were responsible for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injury.     

Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds

Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H₂O₂/halide, MPO/NADH/halide and MPO/H₂O₂/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H₂O₂ system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H₂O₂/NaCl system. NaOCl and the MPO/H₂O₂/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/ NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H₂O₂ production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horseradish peroxidase suplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H₂O₂/NaNO₂ system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated “reactive species” oxidize essential thiol groups at LADH catalytic site.     

Why does SOD overexpression sometimes enhance,sometimes decrease,hydrogen peroxide production? A minimalist explanation

Toxic effects of superoxide dismutase (SOD) overexpression are commonly attributed to increased hydrogen peroxide (H₂O₂) production. Still, published experiments yield contradictory evidence on whether SOD overexpression increases or decreases H₂O₂ production. We analyzed this issue using a minimal mathematical model. The most relevant mechanisms of superoxide consumption are treated as pseudo first-order processes, and both superoxide production and the activity of enzymes other than SOD were considered constant. Even within this simple framework, SOD overexpression may increase, hold constant, or decrease H₂O₂ production. At normal SOD levels, the outcome depends on the ratio between the rate of processes that consume superoxide without forming H₂O₂ and the rate of processes that consume superoxide with high (≥ 1) H₂O₂ yield. In cells or cellular compartments where this ratio is exceptionally low (< 1), a modest decrease in H₂O₂ production upon SOD overexpression is expected. Where the ratio is higher than unity, H₂O₂ production should increase, but at most linearly, with SOD activity. The results are consistent with the available experimental observations. According to the minimal model, only where most superoxide is eliminated through H₂O₂-free processes does SOD activity have the moderately large influence on H₂O₂ production observed in some experiments.     

Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H₂O₂, and Cu(II)/H₂O₂/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H₂O₂, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.     

Cu2+-catalyzed oxidative degradation of thyroglobulin

Thyroglobulin (Tg) was subjected to metal-catalyzed oxidation, and the oxidative degradation was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. In contrast to no effect of hydrogen peroxide (H₂O₂) alone on the Tg degradation, the inclusion of Cu²⁺ (30 μM), in combination with 2 mM H₂O₂, caused a remarkable degradation of Tg, time- and concentration-dependent. The action of Cu²⁺ was not mimicked by Fe²⁺, suggesting that Tg may interact selectively with Cu²⁺. A similar degradation of Tg was also observed with Cu²⁺corbate system, and the concentration of Cu²⁺ (5-10 μM), in combination with ascorbate, required for the effective degradation was smaller than that of Cu²⁺ (10-30 μM) in combination with H₂O₂. In support of involvement of H₂O₂ in the Cu²⁺ corbate action, catalase expressed a complete protection. However, hydroxyl radical scavengers such as dimethylsulfoxide or mannitol failed to prevent the oxidation of Tg whereas phenolic compounds, which can interact with Cu²⁺, diminished the oxidative degradation, presumably consistent with the mechanism for Cu²⁺-catalyzed oxidation of protein. Moreover, the amount of carbonyl groups in Tg was increased as the concentration (3-100 μM) of Cu²⁺ was enhanced, while the formation of acid-soluble peptides was not remarkable in the presence of Cu²⁺ up to 200 μM. In further studies, Tg pretreated with heat or trichloroacetic acid seemed to be somewhat resistant to Cu²⁺-catalyzed oxidation, implying a possible involvement of protein conformation in the susceptibility to the oxidation. Based on these observations, it is proposed that Tg could be degraded non-enzymatically by Cu²⁺-catalyzed oxidation.     

Hydrogen sulphide: a novel physiological inhibitor of LDL atherogenic modification by HOCl

Hypochlorite (HOCl), the product of the activated myeloperoxidase/H₂O₂/chloride (MPO/H₂O₂/Cl^- ) system is favored as a trigger of LDL modifications, which may play a pivotal role in early atherogenesis. As HOCl has been shown to react with thiol-containing compounds like glutathione and N-acetylcysteine protecting LDL from HOCl modification, we have tested the ability of hydrogen sulfide (H₂S)—which has recently been identified as an endogenous vasorelaxant—to counteract the action of HOCl on LDL. The results show that H₂S could inhibit the atherogenic modification of LDL induced by HOCl, as measured by apolipoprotein alterations. Beside its HOCl scavenging potential, H₂S was found to inhibit MPO (one may speculate that this occurs via H₂S/heme interaction) and destroy H₂O₂. Thus, H₂S may interfere with the reactants and reaction products of the activated MPO/H₂O₂/Cl^-  system. Our data add to the evidence of an anti-atherosclerotic action of this gasotransmitter taking the role of HOCl in the atherogenic modification of LDL into account.     

Oxidant-sensitive protein phosphorylation in endothelial cells

Reactive oxygen is an important regulator of vascular cell biology; however, the mechanisms involved in transducing signals from oxidants in endothelial cells are poorly defined. Because protein phosphorylation is a major mechanism for signal ransduction, cultured aortic endothelial cells were exposed to nonlethal concentrations of H₂O₂ to examine oxidant-sensitive changes in phosphorylation state. Addition of H₂O₂ increases the phosphorylation of the heat shock protein 27 (HSP27) within 2 min. This response is maximal by 20 min and remains constant for more than 45 min. Levels of intrcellular free Ca²⁺ in endothelial cells did not change following addition of 100 μM H₂O₂, nor did the ability of the cells to respond to bradykinin. H₂O₂-induced phosphorylations were either not affected or were slightly increased in cells pretreated with PKC inhibitors (H-8, staurosporin, or calphostin c). Two-dimensional analysis of phosphoproteins from homogenates of ³²P-labeled cells revealed that phorbol myristate acetate (PMA) did not cause the same degree of HSP27 phosphorylation as H₂O₂. Simultaneous addition of 10 ηM PMA and 50 μM H₂O₂ decreased the oxidant-stimulated phoshorylation of the most acidic HSP27 isoform. These data suggest that signal transduction for H₂O₂-sensitive endothelial cell responses are not only independent of PKC, but may also be suppressed by the action of the kinase.