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1.
The signal transduction pathways that trigger dephosphorylation of cofilin in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) were investigated with a phospho-specific antibody that recognized cofilin only when this protein was phosphorylated on ser-3. Unlike earlier studies that monitored changes in (32)P-labeled cofilin, this Ab allowed us to monitor changes in the total mass of phosphorylated cofilin during neutrophil stimulation. Neutrophils stimulated with fMLP (1.0 microM) for 1.0 min exhibited a massive loss (> 85%) of phosphate from cofilin, which was blocked by an antagonist of phosphoinositide-specific phospholipase C (PI-PLC) (1.0 microM U73122). Products of PI-PLC, sn-1,2-diglyceride and inositol (1,4,5)-trisphosphate, are known to activate protein kinase C (PKC) and increase intracellular Ca(2+), respectively. Treatment of neutrophils with agents that selectively activate PKC [4beta-phorbol 12-myristate 13-acetate (PMA) ] or cellular Ca(2+) (ionophore A23187) also triggered dephosphorylation of cofilin. Both a nonspecific (100 nM staurosporine) and a highly selective antagonist of PKC (200 nM bisindolylmaleimide I) blocked dephosphorylation of cofilin in neutrophils stimulated with PMA but not with fMLP or ionophore A23187. The calmodulin (CaM) antagonists trifluoperazine (15 microM) and W-7 (50 microM) blocked dephosphorylation of cofilin in stimulated neutrophils whereas inactive/less-active analogs of these inhibitors (15 microM promethazine, 50 microM W-5) were substantially less effective. Calyculin A (40 nM), an antagonist of type 1 and 2A protein phosphatases, also triggered a massive dephosphorylation of cofilin in unstimulated neutrophils through a pathway that was insensitive to inhibitors of type 2B phosphatases. These data suggest that both PKC-dependent and independent pathways can trigger dephosphorylation of cofilin in neutrophils with the latter pathway predominating in fMLP-stimulated cells. These pathways may also contain CaM and a type 2C and/or novel phosphatase (e.g., slingshot). 相似文献
2.
We have studied activation-induced dephosphorylation of proteins in human neutrophils loaded with [32P]orthophosphate using two-dimensional gel electrophoresis and autoradiography. A major phosphoprotein of 20 kDa in resting neutrophils was markedly dephosphorylated upon activation of cells with chemotactic peptide or phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). Using a monoclonal anti-cofilin antibody, this phosphoprotein could be shown to be identical with cofilin, a protein implicated in actin filament remodeling. Signaling pathways leading to this dephosphorylation were further characterized. To define the role of PKC isoforms in cofilin dephosphorylation, we used different PKC inhibitors. Gö 6976 (10 μM), which inhibits preferentially PKC α and β, did not prevent PMA-induced dephosphorylation of cofilin, whereas Ro 31-8220 and CGP 41 251 (10 μM), which act also on Ca2+-independent PKC isoforms, almost completely suppressed this event. The lack of effect of Gö 6976 was not due to insufficient entry into the cells, as this drug suppressed PMA-induced increases in protein phosphorylation. Ca2+-independent PKC isoforms, rather than PKC α or β, may thus be involved in PMA-induced cofilin dephosphorylation. In contrast, Ro 31-8220 did not inhibit chemotactic peptide-induced cofilin dephosphorylation, suggesting here a PKC-independent pathway. The phosphatase inhibitor okadaic acid (1–2 μM) attenuated phosphorylation of cofilin in resting cells. This reduced level was not further attenuated by PMA. Phosphatases 1 and/or 2A may thus control cofilin phosphorylation in resting cells and contribute to PMA-induced cofilin dephosphorylation. Dephosphorylation of cofilin induced by PMA, chemotactic peptide, or okadaic acid was always accompanied by a shift of cofilin to the cell periphery into F-actin-rich areas. These findings suggest a role of cofilin in stimulus-dependent actin remodeling in motile neutrophils. 相似文献
3.
Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils. 相似文献
4.
Bejarano I Terrón MP Paredes SD Barriga C Rodríguez AB Pariente JA 《Molecular and cellular biochemistry》2007,296(1-2):77-84
We have studied the effect of exogenous administration of hydrogen peroxide (H2O2) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H2O2 evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar
to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was
reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator
dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H2O2 resulted in a increase in [Ca2+]
i
, indicating the release of calcium from intracellular stores. H2O2 abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive
calcium pools by fMLP was able to prevent H2O2-induced calcium release. We conclude that H2O2 induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process. 相似文献
5.
Shanti S. Kappala Javier Espino Jose A. Pariente Ana B. Rodriguez Satyan Rajbhandari Arati Iyengar Keshore R. Bidasee Jaipaul Singh 《Molecular and cellular biochemistry》2014,387(1-2):251-260
Type 2 diabetic (T2DM) patients are immune-compromised having a higher susceptibility to infections and long-term complications in different parts of the body contributing to increased morbidity and mortality. A derangement in the homeostasis of intracellular free calcium concentration [Ca2+]i is known to be associated with several diseases in the body including T2DM. Both neutrophils and lymphocytes play active protective roles in host immune response to infection showing impairment in microbicidal functions including phagocytosis and chemotaxis which are calcium-dependent processes. This study evaluated the process of [Ca2+]i mobilization from both neutrophils and lymphocytes taken from blood of both T2DM patients and healthy age-matched control subjects investigating the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP), thapsigargin (TG), and hydrogen peroxide (H2O2) on [Ca2+]i homeostasis. This study employed isolated peripheral blood neutrophils and lymphocytes from 24 T2DM patients and 24 healthy volunteers. Either neutrophils or lymphocytes were stimulated separately with fMLP, TG, or H2O2. Induced changes in [Ca2+] in both neutrophils and lymphocytes were evaluated using spectrofluorometric methods. Stimulation of human neutrophils and lymphocytes with fMLP, TG, or H2O2 in the presence of [Ca2+]o resulted in significant decreases in [Ca2+]i mobilization from T2DM patients compared with healthy controls. These data indicate that neutrophils and lymphocytes from T2DM patients are less responsive to calcium mobilizing agents compared with granulocytes from healthy controls and this is possibly due to the hyperglycemia. The results suggest that agonist-evoked decrease in [Ca2+]i in immune cells might be one of the possible mechanisms of impaired immunity in diabetic patients. 相似文献
6.
Masaaki Satoh Hiroichi Asagami Dongchon Kang Shigeki Minakami Koichiro Takeshige 《Molecular and cellular biochemistry》1995,152(2):159-165
A chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced an acidification of cytosol by about 0.05 pH units in 30 sec followed by an alkalinization in human neutrophils. The quantitative contribution of acid production to the acidification was studied. The superoxide (O2
–) production stimulated by fMLP was not involved in the acidification because the production of acids in neutrophils from patients with chronic granulomatous disease who do not produce O2
–, was the same as that in normal neutrophils. The intracellular acidification was completely inhibited by deoxyglucose, suggesting that energy metabolism enhanced upon stimulation by fMLP might be the main source of the acidification. Although enhancement of the lactate formation by fMLP was 0.8 nmol/106 cells, which could lower intracellular pH by 0.08 pH units, the lactate production could not explain the initial acidification because the production of lactate started at 1 min after the stimulation while the intracellular acidification began immediately after the stimulation. Mitochondrial respiratory inhibitors such as KCN and rotenone had no effects on the fMLP-induced intracellular acidification. The fMLP-induced production of CO2 in 30 sec through the hexose monophosphate shunt was only 2.6 pmol/106 cells, which was calculated to decrease intracellular pH by only 0.0014. Thus, changes of energy metabolism induced by fMLP does not explain the acidification.Abbreviations fMLP
N-formyl-methionyl-leucyl-phenylalanine
- BCECF-AM
2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester
- PMA
phorbol 12-myristate 13-acetate
- CGD
chronic granulomatous disease
- HMP
hexose monophosphate
- pHi
intracellular pH 相似文献
7.
The correlation between an increased production of reactive oxygen species (ROS) and an enhanced calcium entry in primed neutrophils
stimulated with fMLP suggests that endogenous ROS could serve as an agonist to reinforce calcium signaling by positive feedback.
This work shows that exogenous H2O2 produced a rapid influx of Mn2+ and an increase of intracellular calcium. The H2O2 was insufficient to produce significant changes in the absence of extracellular calcium but addition of Ca2+ to H2O2-treated cells suspended in a free Ca2+/EGTA buffer resulted in a great increase in [Ca2+]i reflecting influx of Ca2+ across the cell membrane. The increase of intracellular calcium was inhibited by Ni2+, La3+, and hyperosmotic solutions of mannitol and other osmolytes. This raises the possibility that the secretion of H2O2 by activated neutrophils could act as an autocrine regulator of neutrophil function through the activation of calcium entry. 相似文献
8.
Takashi Mitsuyama Koichiro Takeshige Takashi Furuno Takuo Tanaka Kouko Hidaka Masayoshi Abe Nobuyuki Hara 《Molecular and cellular biochemistry》1995,145(1):19-24
Intact human neutrophils produced superoxide (O2
–) by the stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) even when the extracellular Ca2+ was absent (0.56±0.13 nmol/min per 106 cells). The production by fMLP was enhanced more than twice in the presence of the extracellular Ca2+. Moreover, the O2
– production by fMLP in the presence of extracellular Ca2+ was enhanced nearly three times by the treatment of cells with H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). The enhancement was not observed when the extracellular Ca2+ was depleted from the reaction mixture. In addition, H-89 did not enhance fMLP-induced O2
– production of electropermeabilized neutrophils in which the intracellular Ca2+ concentration was fixed to about 100 nM. These observations suggest that not only Ca2+ influx but the inhibition of PKA is necessary for the maximum O2
– production by fMLP and that the O2
– production is partially suppressed by the activation of PKA induced by fMLP. 相似文献
9.
Cellular stimuli generate reactive oxygen species (ROS) via the local action of NADPH oxidases (Nox) to modulate cytoskeletal organization and cell migration through unknown mechanisms. Cofilin is a major regulator of cellular actin dynamics whose activity is controlled by phosphorylation/dephosphorylation at Ser3. Here we show that Slingshot-1L (SSH-1L), a selective cofilin regulatory phosphatase, is involved in H2O2-induced cofilin dephosphorylation and activation. SSH-1L is activated by its release from a regulatory complex with 14-3-3ζ protein through the redox-mediated oxidation of 14-3-3ζ by H2O2. The ROS-dependent activation of the SSH-1L-cofilin pathway stimulates the SSH-1L–dependent formation of cofilin-actin rods in cofilin-GFP–expressing HeLa cells. Similarly, the formation of endogenous ROS stimulated by angiotensin II (AngII) also activates the SSH-1L-cofilin pathway via oxidation of 14-3-3ζ to increase AngII-induced membrane ruffling and cell motility. These results suggest that the formation of ROS by NADPH oxidases engages a SSH-1L-cofilin pathway to regulate cytoskeletal organization and cell migration. 相似文献
10.
The fMLP receptor of peritoneal macrophages stimulated by fMLP graftedliposomes as ligand, was analysed and compared with respective controlsfor its ability to promote killing of intracellular Leishmaniaparasites. fMLP grafted liposomes show greater efficacy in killingintracellular L. donovani (MHOM/IN/1983/AG83) parasites in a timedependent manner than free fMLP. fMLP grafted liposomes also releasemore active oxygen intermediates and reactive nitrogen intermediates(O–
2, H2O2, NO) than free fMLP. The key enzymes PKCand PTK for the respiratory burst and nitric oxide generation werefound to be important in this fMLP receptor mediated signaling processas the enzyme specific inhibitors viz. staurosporine, genistein andAG126 suppressed the leishmanicidal effect of fMLP graftedliposomes. The above findings suggest that the fMLP receptorof macrophages activates PKC and PTK mediated signalling thatis responsible for the intracellular parasite killing. 相似文献
11.
Kobayashi T Ogawa Y Watanabe Y Furuya M Kataoka S Garcia del Saz E Tsunawaki S Dinauer MC Seguchi H 《Histochemistry and cell biology》2004,122(4):323-332
Macrophages produce superoxide (O2–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O2– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H2DCFDA), O2– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H2DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O2–-producing cells, but not in O2–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H2O2 dismutated from O2–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA. 相似文献
12.
Neutrophils stimulated with 4 beta-phorbol 12-myristate 13-acetate (PMA) release large quantities of superoxide (O2-) and exhibit phosphorylation of two proteins with molecular masses of 47(p47) and 49 kDa (p49). Addition of inhibitors of protein kinases (e.g. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) to these cells after stimulation with PMA results in the loss of 32P from these proteins and a rapid cessation of O2- release (e.g. Heyworth, P. G., and Badwey, J. A. (1990) Biochim. Biophys. Acta 1052, 299-305). In this paper we report that antagonists of type 1 and 2A protein phosphatases (okadaic acid, calyculin A) prevented both the loss of 32P from p47 and the termination of O2- release in stimulated neutrophils treated with H-7. Calyculin A also caused a remarkable hyperphosphorylation of a number of proteins in neutrophils and increased O2- release from these cells in response to a suboptimal amount of PMA. Enzymes present in both the soluble and particulate fractions of neutrophils catalyzed the near complete dephosphorylation of 32P-labeled p47 and p49 bound to Immobilon-P membranes. Dephosphorylation of these blotted phosphoproteins occurred at physiological rates and was inhibited by okadaic acid and calyculin A. These data strongly suggest that p47 undergoes a continual cycle of phosphorylation and dephosphorylation throughout the period of O2- release when PMA is the stimulus. Moreover, we show that antagonists of type 1 and 2A protein phosphatases block dephosphorylation of p47 both in vivo and in vitro, indicating that these enzymes may modulate O2- release under certain circumstances. 相似文献
13.
Isao Uno 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,631(1):59-69
A phosphorylated regulatory subunit of cyclic AMP-dependent protein kinase (type II) was purified to homogeneity from inorganic [32P]phosphate-injected rats.A new method of measuring the phosphorylation reaction was developed. It was found that this regulatory subunit was phosphorylated in cells and comprised 60, 82 and 55% of the total regulatory subunit in brain, heart and liver cytosol fractions from rats, respectively.Dephosphorylation was stimulated by cyclic nucleotides. The Ka values for cyclic AMP and cyclic IMP were 0.30 and 1.0 μM, respectively. Purified phosphoprotein phosphatase could dephosphorylate the regulatory subunit and this reaction was also stimulated by cyclic nucleotides with similar Ka values. The inhibitors of phosphoprotein phosphatase, NaF and ZnCl2, protected against dephosphorylation unless ADP or cyclic AMP were present. 相似文献
14.
The effects of pentoxifylline (PTX) on spontaneous pseudopod formation in neutrophils in response to the tripeptide formyl-Met-Leu-Phe
(fMLP), endotoxin, human complement C5a, and leukotriene B4 (LTB4) were examined in autologous plasma. Unseparated supernatant leukocyte suspensions from fresh heparinized venous human blood
were incubated with PTX (0-5 mM) for 25 min and then stimulated for 5–25 min within a range of concentrations of fMLP, endotoxin,
complement C5a, and LTB4. The cell suspensions were fixed with glutaraldehyde and stained with crystal violet in acetic acid; the percentage of neutrophils
with pseudopods was determined under high-resolution light microscope. The results show that PTX significantly decreases formation
of pseudopods in the presence of all four stimulators. The mechanism of pseudopod suppression appears to be independent of
the adenosine receptor. PTX and its analogues, HWA 138 and HWA 448, decreased pseudopod formation by similar amounts when
stimulated with 10−8M fMLP. These results suggest that PTX may improve microvascular perfusion and attenuate neutrophilmediated injury by reducing
the degree of neutrophil pseudopod formation in free suspension and microvascular entrapment. 相似文献
15.
Nagaishi K Adachi R Kawanishi T Yamaguchi T Kasahara T Hayakawa T Suzuki K 《Journal of biochemistry》1999,125(5):891-898
We studied the roles of cofilin, an actin-binding phosphoprotein, in superoxide production of neutrophil-like HL-60 cells triggered by opsonized zymosan (OZ). OZ caused dephosphorylation of cofilin as well as a transient increase of F-actin. Both reactions were complete within 30 s. Okadaic acid (OA) magnified the OZ-triggered O2--production 3.3-fold at 1 microM, but inhibited it completely at 5 microM. We used these critical concentrations to study the effects of OA on changes in phosphorylation and intracellular localization of cofilin. The OZ-induced dephosphorylation of cofilin was inhibited by 5 microM OA but not by 1 microM OA. Subcellular fractionation and immunoblotting revealed that 1 microM OA increased cofilin on the phagosomal membranous fraction but 5 microM OA decreased it. At 1 microM, OA increased translocation of p47phox to membranes, which may explain in part the enhancing effect of 1 microM OA. Confocal laser scanning microscopy showed that: (i) Cofilin diffused throughout the cytosol of resting cells, but accumulated at the plasma membranes forming phagocytic vesicles in activated cells. (ii) At 1 microM, OA had little effect on the OZ-evoked translocation of cofilin, whereas 5 microM OA suppressed it completely. (iii) OA alone, which could not trigger the phagocytic respiratory burst, did not cause any change in the distribution of cofilin at such concentrations. Furthermore, in a superoxide-producing cell-free system employing membranous and cytosolic fractions, affinity-purified anti-cofilin antibody showed an enhancing effect. These results suggest that cofilin participates in the superoxide production of the OZ-activated phagocytes through dephosphorylation and translocation. The roles of cofilin in the activated leukocytes will be discussed. 相似文献
16.
The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems
chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine
on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition
conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC50 values not exceeding 4.6 μmol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold
higher alkaloid concentrations. At concentrations below 10 μmol/L, sanguinarine and chelerythrine prevented phosphorylation
of ∼80 kDa protein and sequestered ∼60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired
PMA-stimulated translocation of autophosphorylated PKCα/βII isoenzymes, but both alkaloids induced dephosphorylation of the
turn motif in PKCα/βII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation.
Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of ∼70 kDa protein by PDK1. We
conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather
than by inhibition of PKC activity. 相似文献
17.
《Insect Biochemistry》1990,20(1):29-36
An in vitro analysis of endogenous dephosphorylation of a particulate-associated cAMP-dependent 34 kDa phosphoprotein from the brains of Manduca sexta larvae revealed the presence of phosphatase activity in the same fraction. The rate of dephosphorylation is stimulated by the polyamine spermine, is markedly inhibited by vanadate and Zn2+, and proceeds in the absence of divalent cations. The purified protein inhibitor of phosphatase 1, inhibitor-2, inhibits dephosphorylation in a dose dependent manner. The calmodulin antagonists trifluoperazine and calmidazolium also block dephosphorylation, suggesting the presence of phosphatase 2-B. This study suggests the possible co-localization of a particulate associated cAMP-dependent protein kinase and associated phosphatases with their phosphorylated protein substrates in the insect brain. 相似文献
18.
Serge Ostrovidov Patricia Franck Josette Capiaumont Brigitte Dousset Francine Belleville 《In vitro cellular & developmental biology. Animal》1998,34(3):259-264
Summary The effect of low concentrations of hydrogen peroxide (H2O2) (5 × 10−7−9.5 × 10−7
M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell
growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H2O2 (8 × 10−7
M) but H2O2 concentration of 7 × 10−6
M and above increased cell death. H2O2 stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 × 10−7 or 1 × 10−5
M H2O2 was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino
acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control
cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine
123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content
of cells in 8 × 10−7
M H2O2 was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123)
and H2O2 or peroxides (detected with DCFH-DA) by cells incubated with 1 × 10−5
M H2O2 which increased with increasing H2O2 until cell death. 相似文献
19.
Calcium signal-induced cofilin dephosphorylation is mediated by Slingshot via calcineurin 总被引:11,自引:0,他引:11
Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a member of a SSH family of protein phosphatases, in mediating Ca2+-induced cofilin dephosphorylation. The Ca2+ ionophore A23187 and Ca2+-mobilizing agonists, ATP and histamine, induced SSH1L activation and cofilin dephosphorylation in cultured cells. A23187- or histamine-induced SSH1L activation and cofilin dephosphorylation were blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca2+-induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolished A23187- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and increased the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings, we suggest that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L. 相似文献
20.
In the absence of added Fe2+, the ATPase activity of isolatedSchizosaccharomyces pombe plasma membranes (5–7 μmolP
i per mg protein per min) is moderately inhibited by H2O2 in a concentration-dependent manner. Sizable inactivation occurs only at 50–80 mmol/L H2O2. The process, probably a direct oxidative action of H2O2 on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the
radical scavengers mannitol and Tris, and involves a decrease of both theK
m of the enzyme for ATP and theV of ATP splitting. On exposing the membranes to the Fenton reagent (50 μmol/L Fe2+ +20 mmol/L H2O2), which causes a fast production of HO− radicals, the ATPase is 50–60% inactivated and 90% of added Fe2+ is oxidized to Fe3+ within 1 min. The inactivation occurs only when Fe2+ is added before H2O2 and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO− radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine
implies that the process requires the presence of Fe2+ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe2+ for H2O2, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide
radical that gives rise to delayed HO− production. 相似文献