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PubMed1997

The effect of ethanol on sphincter of Oddi motility in vitro.

Antioxidant enzymes are present in nerves supplying the sphincter of Oddi and regulate its motor function. Oxygen free radicals (O2.-) produce hydrogen peroxide (H2O2) by the action of superoxide dismutase (SOD). Hydroxyl radical (OH.), an important mediator of H2O2 toxicity, oxidizes ETOH. Thus, the aim of our study was determine the effects of ETOH on sphincter of Oddi motility. The sphincter of Oddi was removed from opossums and force transducers recorded tension in its transverse axis. Alcohol (ETOH) was added to the tissue bath in concentrations from 0.1 to 0.4%. OH. was generated by the addition of 0.01% H2O2. Nitric oxide production was inhibited by the addition of 0.3 mM N omega-nitro-L-arginine (L-NNA). H2O2 increased the frequency of sphincter of Oddi contractions. Concentrations of ETOH > 0.3% decreased contractile frequency; however, 0.2% ETOH alone had no affect on the basal frequency of contraction but inhibited the increase in contractile frequency caused by H2O2. L-NNA also increased the contractile frequency; however, this effect was not inhibited by ETOH. We conclude that ETOH attenuates the effect of H2O2 on sphincter of Oddi motility.

Animals↗
PubMedDate not supplied

Role of cellular defense against hydrogen peroxide-induced inhibition of myocyte respiration.

Hydrogen peroxide (H2O2) serves as a precursor for highly reactive oxygen intermediates. However, the respiratory function of myocytes is relatively resistant to exogenously administered H2O2. In this study, we examined whether or not the reduction of cellular defense increases the toxicity of H2O2. Rat heart myocytes were isolated by collagenase digestion. Respiratory rates of myocytes, suspended in a medium containing sucrose, 3-N-morpholino-propanesulfonic acid, EGTA and bovine serum albumin, were determined polarographically in the presence of pyruvate and malate with or without 2,4-dinitrophenol (DNP). Mitochondrial membrane potentials were measured by using [3H]triphenylmethylphosphonium+. Cellular defense was attenuated by i) inhibiting the catalase activity by 3-amino-1,2,4-triazole (AT), ii) reducing the glutathione concentration by diethyl maleate (DEM) or ethacrinic acid (EA), and iii) permeabilizing the sarcolemmal membrane by saponin. The dose-response relationship between H2O2 (0.1-5 mM) and mitochondrial membrane potential was not greatly affected by these experimental conditions. Myocyte respiration was inhibited by 5 mM H2O2, particularly that measured in the presence of DNP (48% of control). DEM treatment did not significantly affect the respiratory inhibition by H2O2, whereas the degree of inhibition was somewhat greater following EA or AT treatment. By contrast, the sensitivity of cellular respiration to H2O2 was potentiated approximately two orders of magnitude by the permeabilization of sarcolemmal membrane; thus, 100 microM H2O2 inhibited both DNP-stimulated and unstimulated respiration to 17% and 35% of control, respectively. The results indicate that factors existing in the sarcolemma and/or in the cytosol, which become ineffective and/or are diluted, respectively, following permeabilization with saponin, are important cellular defense mechanisms in alleviating the toxic effect of exogenous H2O2 on the respiration of mitochondria in situ in myocytes.

Amitrole↗
PubMed1993

The antioxidant defense system of isolated guinea pig Leydig cells.

Utilization of highly enriched preparations of steroidogenic Leydig cells have proven invaluable for studying the direct effects of various hormones and agents on Leydig cell function in vitro. However, recent work indicates that isolated Leydig cells are often subjected to oxygen (O2) toxicity when cultured at ambient (19%) oxygen concentrations. Because intracellular antioxidants play an important role in protecting cells against oxygen toxicity, we have investigated the intracellular antioxidant defense system of isolated Leydig cells. The cellular levels of several antioxidants including catalase, glucose-6-phosphate dehydrogenase (G-6-PDH), superoxide dismutase (SOD) of the Cu/Zn & Mn variety, glutathione peroxidase, glutathione reductase and total glutathione were quantitated using enriched populations of Leydig cells isolated from adult male guinea pig testes. Compared to whole testicular homogenates, Leydig cells contained significantly (P < 0.01) less G-6-PDH, total SOD, glutathione reductase and total glutathione, but significantly (P < 0.001) more glutathione peroxidase. Compared to hepatic values previously reported in the guinea pig, Leydig cells contain nearly 400 times less catalase, about 14 times less glutathione peroxidase and almost 11 times less glutathione reductase. Since G-6-PDH and glutathione reductase are both necessary to regenerate reduced glutathione (GSH) which couples with glutathione peroxidase to breakdown hydrogen peroxide (H2O2) under normal conditions, it is plausible that the oxygen toxicity observed in isolated Leydig cells is due to the intracellular accumulation of H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Superoxide and hydrogen peroxide-dependent inhibition of iron regulatory protein activity: a protective stratagem against oxidative injury.

Cellular iron homeostasis is regulated by the cytoplasmic iron regulatory protein (IRP), which binds to iron-responsive elements (IRE) of mRNAs, modulating iron uptake and sequestration, respectively. When iron is scarce, IRP binds to IRE and coordinately increases the synthesis of transferrin receptor and decreases that of ferritin, thus providing the cell with readily available free iron. When iron is in excess, IRP does not bind and iron sequestration prevails over iron uptake. We have found that incubation of rat liver lysates with xanthine oxidase (XO), which generates superoxide (O2-.) and hydrogen peroxide (H2O2), caused a remarkable but reversible inhibition of IRP activity, as the formation of IRE-IRP decreased by 70-80% but returned to baseline values upon exposure to a reducing agent like 2-mercaptoethanol. IRP inhibition was prevented by separate or simultaneous addition of superoxide dismutase and catalase, showing that both O2-. and H2O2 were involved. By contrast, iron chelators and hydroxyl radical scavengers did not impede the inhibition of IRP, suggesting that O2-. and H2O2 acted independently of free iron sources. Ferritin enhanced IRP inhibition, but this process involved tightly bound iron centers that shunted reducing equivalents from XO and returned them to oxygen, thus increasing the formation of O2-. In agreement with the exclusive role of O2-. and H2O2, XO also inhibited recombinant human IRP in the absence of iron. These results demonstrate that O2-. and H2O2 can directly but reversibly down-regulate the RNA-binding activity of IRP, causing transient decrease of free iron that otherwise would convert them into more potent oxidants such as hydroxyl radicals or equally aggressive iron-peroxo complexes. This establishes a novel protective stratagem against oxidative injury under pathophysiologic conditions characterized by the excessive generation of O2-. and H2O2.

Animals↗
PubMed1995

Hydrogen peroxide contracts human airways in vitro: role of epithelium.

The effects of hydrogen peroxide (H2O2) on human airway smooth muscle tone were determined in vitro. Treatment with H2O2 led to transient concentration-related contractions in the organ bath, amounting to 118 +/- 14 mg (mean +/- SE; n = 12) at 1 mM H2O2, and to greater and more prolonged contractions under superfusion conditions, amounting to 451 +/- 71 mg (n = 17) at 1 mM H2O2. Epithelial removal augmented the response to H2O2 in both systems. Addition of catalase (500 U/ml) abolished the effects of H2O2. Pretreatment of superfused tissues with indomethacin (3 microM) shifted the concentration-effect curve to H2O2 rightward and almost abolished the response to 1 mM H2O2 in epithelium-intact preparations (n = 16; P < 0.05); the response in epithelium-denuded tissues was also significantly inhibited (n = 16; P < 0.05). Pretreatment of the tissues with the TP prostanoid-receptor antagonist GR-32191B (1 microM) also inhibited the contractile effect of H2O2 in epithelium-intact and -denuded tissues. In separate experiments, H2O2 resulted in concentration-related generation of prostaglandin (PG) D2 from isolated airway preparations. The amount of PGD2 released was not different in tissues with intact epithelium compared with those without (n = 9; NS). We conclude that H2O2 exerts on isolated human airways a contractile effect that is augmented by epithelium removal and is largely mediated by prostanoids. The source of PGD2 does not appear to be the epithelium, which we suggest serves mainly as a barrier against H2O2-mediated bronchoconstriction.

Aged↗
PubMed1991

Isoproterenol prevents oxidant-induced injury in isolated rabbit lungs.

Increased vascular permeability in the adult respiratory distress syndrome is due in part to toxic oxygen metabolites. In the present study, we produced lung injury in the isolated rabbit lung with hydrogen peroxide (H2O2) and examined its prevention with isoproterenol. Pulmonary arterial pressure (Ppa) and the fluid filtration coefficient (Kf) were measured as indices of lung injury. Rabbits were divided into two groups, and 7 mmol/l H2O2 was administered in both groups. In one group, isoproterenol (2 micrograms/ml) was administered 10 min before H2O2 injury. Ppa increased transiently after H2O2 administration in the control group but was unchanged in the isoproterenol group. Kf was significantly increased by H2O2 administration in the control group but not in the isoproterenol group. We conclude that H2O2 increases pulmonary vascular permeability and that isoproterenol may protect against H2O2-induced pulmonary injury.

Animals↗
PubMed1998

3'-blocking damage of DNA as a mutagenic lesion caused by hydrogen peroxide in Escherichia coli.

Ionizing radiation and hydrogen peroxide (H2O2) produce many types of oxidative DNA damage such as strand breaks, apurinic/apyrimidinic (AP) sites, base modifications and 3'-blocking damage such as 3'-phosphoglycolated and 3'-phosphorylated termini. AP sites and 3'-blocking damage are repairable by exonuclease III and endonuclease IV in Escherichia coli. XthA-nfo double mutants of E. coli, which are deficient in exonuclease III and endonuclease IV, were highly sensitive to lethal and mutagenic effects of H2O2, compared with the wild-type strains. The pNT180 and pNT186 plasmids containing wild-type nfo and mutant nfo-186 gene, respectively, were introduced into the xthA-nfo mutant. The nfo-186 gene product, Nfo186, retained normal AP endonuclease activity but could not remove 3'-blocking damage from DNA. The pNT180 corrected the sensitivity of the xthA-nfo mutant to lethal and mutagenic effects of H2O2. On the other hand, the pNT186 did not have any complementation effects. From these results it was concluded that 3'-blocking damage rather than an AP site is the primary lesion responsible for both lethal and mutagenic effects of H2O2.

DNA Damage↗
PubMed1998

A sensitive fluorometric assay for determining hydrogen peroxide-mediated sublethal and lethal endothelial cell injury.

A rapid and sensitive quantitative fluorometric assay was developed to measure the response of endothelial cells to hydrogen peroxide (H2O2). The response of an endothelial cell-derived cell line, EA-hy-926, or human umbilical vein endothelial cells to H2O2 was determined using calcein-AM, a dye which becomes fluorescent upon cleavage by intracellular esterase(s). The ability of the cells to take up and convert calcein-AM was measured directly in the wells of 96-well flat-bottomed tissue culture plates with cell monolayers using a computerised microplate fluorimeter, or in cell suspensions using flow cytometry. The results obtained by these techniques were compared with each other and with a standard 51Cr release cytotoxicity assay. We found that calcein-AM is a highly sensitive probe for measuring H2O2-mediated cell injury, as it can not only detect the irreversible cytotoxicity measured by 51Cr release assay, but can also distinguish sublethal and reversible injury seen at low H2O2 concentrations.

Cell Line↗
PubMed1994

Role of hydrogen peroxide in the inhibitory effect of ascorbate on cell growth.

Organ cultures have become an important method for the study of some of the biochemical reactions in which ascorbate takes part, and there are conflicting views on the stimulatory and inhibitory actions of ascorbate on cell growth. This study aimed to clarify the inhibitory mechanism of ascorbate using 3T6 fibroblasts. Cells were exposed to ascorbate at various concentrations in medium. The results showed that 3T6 fibroblasts were killed in medium containing more than 0.3 mM ascorbate. This lethal effect of ascorbate on cells was inhibited by the addition of catalase, an enzyme that decomposes hydrogen peroxide (H2O2), to the medium. When the medium with ascorbate was incubated in the absence of cells, the amount of H2O2 generation depended on the ascorbate concentration, and decreased in inverse proportion to the serum concentration added to the medium. The addition of albumin, which is the main protein in serum, also inhibited H2O2 generation in the medium with ascorbate. However H2O2 generation was not inhibited completely by serum and albumin. These results indicate that cytotoxicity of ascorbate is induced by H2O2.

3T3 Cells↗
PubMed1996

A positive correlation between catalase activity and ascorbate uptake in the tissues of guinea pigs and cultured cells of mammals.

We recently reported that the concentration of supplemental ascorbate which inhibits cell growth is positively related to intracellular catalase activity. It is assumed that the cells with high catalase activity are resistant to high concentrations of ascorbate since catalase can decompose hydrogen peroxide (H2O2) induced by the auto-oxidation of ascorbate in cultured medium. In this study, we investigated whether intracellular catalase activity affects the uptake of ascorbate into animal tissue and cultured cells. Ascorbate concentrations in the tissues of guinea pigs and various cultured cells, with and without supplementation of ascorbate, were determined to evaluate the efficiency of ascorbate uptake. We found a positive correlation between the efficiency of ascorbate uptake and catalase activity in various tissues of guinea pigs (r = 0.767, p < 0.05). Furthermore, a positive correlation between the two was also found in various species of cultured cells. This study indicates that tissues and cells with higher efficiency of ascorbate uptake are required for higher catalase activity, presumably for the decomposition of H2O2 from ascorbate.

Animals↗
PubMed1990

Similarities in the pharmacological modulation of reactive hyperemia and vasodilation to hydrogen peroxide in rat skeletal muscle arterioles: effects of probes for endothelium-derived mediators.

Our laboratory has demonstrated previously that prostaglandins are partially responsible for the vasodilation of rat cremaster muscle arterioles in vivo to a brief occlusion or hydrogen peroxide (H2O2). In the present study, in pentobarbital-anesthetized rats, we investigated the mechanism of the prostaglandin-independent portion of the dilation to these stimuli by measurement of changes in the diameter of third order cremasteric arterioles (approximately 15 microns) by video microscopy. In the presence of indomethacin suffusion (10 micrograms/ml), arteriolar dilation to the release of a 15-sec occlusion of a single arteriole or to topical application of 0.1 to 1 mM H2O2 (100 microliters) was reduced significantly by suffusion of 20 microM quinacrine or 5 microM methylene blue, whereas vasodilation to adenosine was not affected by these probes. Furthermore, the reactive hyperemia was not altered by suffusion of 50 microM hydroquinone or 0.2 mM NG-monomethyl-L-arginine, inhibitors of the dilation to acetylcholine mediated by the endothelium-derived relaxing factor. Reactive hyperemia was also not affected by 30 microM theophylline (an adenosine antagonist) or suffusion of catalase (160 U/ml) plus superoxide dismutase (240 U/ml). Therefore, the reactive hyperemia does not appear to be mediated through endothelium-derived relaxing factor, adenosine or the extracellular formation of reactive O2 species. However, these observations suggest that the prostaglandin-independent vasodilation to H2O2 and to the release of a brief arteriolar occlusion may be mediated by a common mechanism, possibly involving cyclic GMP. Similarities in pharmacological modulation of the dilation after occlusion and to H2O2 in the cremaster microcirculation suggest consideration of the involvement of H2O2 in the hyperemic response.

Animals↗
PubMed1998

Response of lens epithelial cells to hydrogen peroxide stress and the protective effect of caloric restriction.

Hydrogen peroxide (H2O2) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H2O2 effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H2O2-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H2O2 at 40 or 400 microM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 microM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H2O2. The 400 microM H2O2 severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H2O2 were significantly different. Exposure of LE cells to 20, 40, or 100 microM H2O2 for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H2O2 than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.

Age Factors↗
PubMed1991

Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells.

Hydrogen peroxide (H2O2) is an important mediator of acute oxidative injury to vascular endothelium. Because the plasma membrane is the initial site of interaction between endothelial cells and extracellular H2O2 produced by stimulated neutrophils or macrophages, we evaluated the effect of H2O2 on the physical state, i.e., fluidity, and function of porcine pulmonary artery endothelial cell plasma membranes. Lactate dehydrogenase (LDH) release, 5-hydroxytryptamine (5-HT) uptake, limiting fluorescence anisotropy (r infinity) for trimethylamino-diphenylhexatriene (TMA-DPH), and conjugated dienes were measured 0.5, 6, and 24 hr after cells were exposed for 30 min to 50-microM H2O2 or Hank's Balanced Salt Solution (control). Compared with control cells, H2O2 caused significant increases in LDH release and in 5-HT uptake 6 hr after exposure. The increase in 5-HT uptake was not blocked by imipramine. H2O2 also caused a significant increase in r infinity for TMA-DPH 0.5 hr after exposure and a significant reduction in r infinity for TMA-DPH 6 hr after exposure. Cellular contents of conjugated dienes were increased 0.5 and 6 hr after exposure to H2O2. Twenty-four hours after exposure LDH release, r infinity, 5-HT uptake, and conjugated dienes had returned to control levels. Preincubation with 50-microM alpha-tocopherol (vitamin E) or 1-mM or 10-mM dimethylthiourea (DMTU) for 1 hr or 24 hr prevented endothelial cell injury, whereas addition of vitamin E or DMTU to the medium 1 hr or 3 hr after H2O2 exposure did not protect against injury. These results indicate that H2O2 causes significant damage to the plasma membrane of pulmonary artery endothelial cells in vitro, leading to alterations in fluidity and leakiness of the membrane. This injury is associated with membrane lipid peroxidation, is reversible, and can be prevented by pretreatment, but not by post-treatment, with vitamin E or DMTU.

Animals↗
PubMed1994

[Superoxide formation and lipid peroxidation by the mitochondrial electron-transfer chain].

Isolated mitochondria supplemented with succinate or NAD(+)-linked substrates generate hydrogen peroxide (H2O2) in State 4 and the generation is enhanced by antimycin A, an inhibitor of the respiratory chain. Superoxide is a stoichiometric precursor of mitochondrial H2O2 because the ratio of O2-/H2O2 generation rates is close to 2.0 and is generated by an autoxidizable component in the NADH dehydrogenase and the ubiquinone-cytochrome b site. Lipid peroxidation is a free radical-mediated degradation of polyunsaturated fatty acids. Lipid-peroxidation reactions by bovine submitochondrial particles are supported by NADH or NADPH in the presence of ADP-Fe3+ chelate. Electrons from NADH are supplied to the reactions from a component between the substrate site and the rotenone-sensitive site of the NADH dehydrogenase. The peroxidation is dependent on the rate of electron input into the respiratory chain and on the concentration of reduced ubiquinone. Alteration of inner-membrane components and damage to electron-transfer activities of submitochondrial particles are induced by lipid peroxidation. 1-Melhyl-4-phenylpyridinium (MPP+), a metabolite of a parkinsonism-inducing drug, induces NADH-dependent superoxide formation and enhances NADH-dependent lipid peroxidation in submitochondrial particles, indicating that the oxidative stress induced by MPP+ may potentiate its toxicity in dopamine neurons.

Electron Transport↗
PubMed2000

UVA-induced oxidative damage in retinal pigment epithelial cells after H2O2 or sparfloxacin exposure.

Retinal impairment is one of the leading causes of visual loss in an aging human population. To explore a possible cause for retinal damage in the human population, we have monitored DNA oxidation in human retinal pigment epithelial (RPE) cells after exposure to hydrogen peroxide (H2O2) or the quinolone antibacterial sparfloxacin. When H2O2- or sparfloxacin-exposed cells were further exposed to ultraviolet A (UVA) irradiation, oxidative damage to the DNA of these cells was greatly increased over baseline values. This RPE+pharmaceutical-UVA cell system was developed to mimic in vivo retinal degeneration, seen in mouse studies using quinolone and UVA exposure. DNA damage produced by sparfloxacin and UVA in RPE cells could be remedied by the use of antioxidants, indicating a possible in vivo method for prevention or minimization of retinal damage in humans

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1986

The effects of Con A-induced lymphokines from the T-lymphocyte subpopulations on human monocyte leishmanicidal capacity and H2O2 production.

The effects of concanavilin A (Con A)-induced lymphokines from human peripheral blood mononuclear cells, enriched helper and suppressor lymphocyte populations and recombinant interferon-gamma (IFN-gamma) on the ability of monocytes to promote killing of intracellular Leishmania major (L. major) amastigotes and to induce the production of hydrogen peroxide (H2O2) was examined. All these mitogen-induced supernatants contained active IFN-gamma. There were no differences in the ability of these supernatants or IFN-gamma to promote the monocyte killing of the L. major amastigotes or H2O2 production. An increase in H2O2 production by monocyte monolayers was observed following the addition of either of these supernatants or IFN-gamma alone. In addition, a marked increase in the production of H2O2 was observed following priming with either of these mitogen-induced supernatants or IFN-gamma and the addition of a second stimulus, phorbol myristate acetate (PMA). Monoclonal antibody to IFN-gamma abrogated the increase in production of H2O2 by all these mitogen-induced supernatants; however, this antibody only resulted in partial inhibition of the leishmaniacidal effect of these lymphokines on human monocytes. These results would suggest that IFN-gamma is the component of the lymphokine that is largely or exclusively responsible for H2O2 production, while other factors in addition to IFN-gamma are important in promoting oxygen-independent mechanisms for the killing of intracellular L. major amastigotes.

Cells, Cultured↗
PubMed2000

Epithelial and stromal uterine cells cultured in vitro protect bovine sperm from hydrogen peroxide.

It is known that large amounts of leukocytes colonize the uterus, and that these leukocytes can produce considerable quantities of hydrogen peroxide (H2O2) and other reactive oxygen species that are toxic to sperm. It has been shown recently that oviductal fluid has a catalase that helps to maintain sperm motility. Therefore, the current experiment was performed to determine if a similar mechanism of protection exists against peroxides within uterine cells. Sperm motility and velocity were recorded after a 6h incubation in 1) conditioned media in the presence of endometrial cells, 2) conditioned media without endometrial cells, 3) control media (48h without cells) over endometrial cells, or 4) control media alone. All these treatments were performed in the presence or absence of added catalase. Conditioned media, endometrial cells and catalase had a significant positive effect on the maintenance of sperm motility and velocity. Addition of anti-catalase antibodies did not neutralize the beneficial effect of the conditioned media. However, the concentrations of aromatic amino acids, known substrates for sperm amino acid oxidase, were significantly lower in uterine conditioned media as compared to control medium. This reduction of aromatic amino acids was in correlation with reduced H2O2 production by sperm as estimated by chemiluminescence. These results suggest that epithelial and stromal uterine cells do not maintain sperm motility by secreting catalase in the conditioned media, but rather by reducing the levels of aromatic amino acids and thus of peroxides generated in the presence of spermatozoa.

Animals↗
PubMed1998

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.

Actins↗