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1.
Gonzalez A Santofimia-Castaño P Rivera-Barreno R Salido GM 《Journal of physiology and biochemistry》2012,68(2):181-191
This work was designed in order to gain an insight on the mechanisms by which antioxidants prevent pancreatic disorders. We
have examined the properties of cinnamtannin B-1, which belongs to the class of polyphenols, against the effect of hydrogen
peroxide (H2O2) in mouse pancreatic acinar cells. We have studied Ca2+ mobilization, oxidative state, amylase secretion, and cell viability of cells treated with cinnamtannin B-1 in the presence
of various concentrations of H2O2. We found that H2O2 (0.1–100 μM) increased CM-H2DCFDA-derived fluorescence, reflecting an increase in oxidation. Cinnamtannin B-1 (10 μM) reduced H2O2-induced oxidation of CM-H2DCFDA. CCK-8 induced oxidation of CM-H2DCFDA in a similar way to low micromolar concentrations of H2O2, and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H2O2 induced a slow and progressive increase in intracellular free Ca2+ concentration ([Ca2+]c). Cinnamtannin B-1 reduced the effect of H2O2 on [Ca2+]c, but only at the lower concentrations of the oxidant. H2O2 inhibited amylase secretion in response to cholecystokinin, and cinnamtannin B-1 reduced the inhibitory action of H2O2 on enzyme secretion. Finally, H2O2 reduced cell viability, and the antioxidant protected acinar cells against H2O2. In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca2+ overload and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that
mediates pancreatitis. Our results support the beneficial effect of natural antioxidants in the therapy against oxidative
stress-derived deleterious effects on cellular physiology. 相似文献
2.
Previous studies indicate that the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca2+-dependent and attributed to the late Ca2+ rise. However, its role in fertilization still remains unclear. Simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase occurred at the time of peak current (tp) which corresponds to peak [Ca2+]i, suggesting that NO is not related to any other ionic changes besides [Ca2+]i. We measured O2 consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. Our results suggested NO increased O2 consumption. The fluorescence of reduced pyridine nucleotides, NAD(P)H was measured in controls and when the NO increase was eliminated by PTIO, a NO scavenger. Surprisingly, PTIO decreased the rate of the fluorescence change and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H2O2 and caused weak and high fertilization envelope (FE). Our results suggest that NO increase upregulates NAD(P)H and H2O2 production and consolidates FE hardening by H2O2. 相似文献
3.
This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+]o) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas. (Mol Cell Biochem 269: 165–173, 2005) 相似文献
4.
Salinity is among the environmental factors that affect plant growth and development and constrain agricultural productivity. Salinity stress triggers increases in cytosolic free Ca2+ concentration ([Ca2+]i) via Ca2+ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS). It is well established that ROS also triggers increases in [Ca2+]i. However, the relationship and interaction between salinity stress-induced [Ca2+]i increases and ROS-induced [Ca2+]i increases remain poorly understood. Using an aequorin-based Ca2+ imaging assay we have analyzed [Ca2+]i changes in response to NaCl and H2O2 treatments in Arabidopsis thaliana. We found that NaCl and H2O2 together induced larger increases in [Ca2+]i in Arabidopsis seedlings than either NaCl or H2O2 alone, suggesting an additive effect on [Ca2+]i increases. Following a pre-treatment with either NaCl or H2O2, the subsequent elevation of [Ca2+]i in response to a second treatment with either NaCl or H2O2 was significantly reduced. Furthermore, the NaCl pre-treatment suppressed the elevation of [Ca2+]i seen with a second NaCl treatment more than that seen with a second treatment of H2O2. A similar response was seen when the initial treatment was with H2O2; subsequent addition of H2O2 led to less of an increase in [Ca2+]i than did addition of NaCl. These results imply that NaCl-gated Ca2+ channels and H2O2-gated Ca2+ channels may differ, and also suggest that NaCl- and H2O2-evoked [Ca2+]i may reduce the potency of both NaCl and H2O2 in triggering [Ca2+]i increases, highlighting a feedback mechanism. Alternatively, NaCl and H2O2 may activate the same Ca2+ permeable channel, which is expressed in different types of cells and/or activated via different signaling pathways. 相似文献
5.
Amina El Jamali Anthony J. Valente Robert A. Clark 《Free radical biology & medicine》2010,48(6):798-810
The importance of H2O2 as a cellular signaling molecule has been demonstrated in a number of cell types and pathways. Here we explore a positive feedback mechanism of H2O2-mediated regulation of the phagocyte respiratory burst NADPH oxidase (NOX2). H2O2 induced a dose-dependent stimulation of superoxide production in human neutrophils, as well as in K562 leukemia cells overexpressing NOX2 system components. Stimulation was abrogated by the addition of catalase, the extracellular Ca2+ chelator BAPTA, the T-type Ca2+ channel inhibitor mibefradil, the PKCδ inhibitor rottlerin, or the c-Abl nonreceptor tyrosine kinase inhibitor imatinib mesylate or by overexpression of a dominant-negative form of c-Abl. H2O2 induced phosphorylation of tyrosine 311 on PKCδ and this activating phosphorylation was blocked by treatment with rottlerin, imatinib mesylate, or BAPTA. Rac GTPase activation in response to H2O2 was abrogated by BAPTA, imatinib mesylate, or rottlerin. In conclusion, H2O2 stimulates NOX2-mediated superoxide generation in neutrophils and K562/NOX2 cells via a signaling pathway involving Ca2+ influx and c-Abl tyrosine kinase acting upstream of PKCδ. This positive feedback regulatory pathway has important implications for amplifying the innate immune response and contributing to oxidative stress in inflammatory disorders. 相似文献
6.
Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca2+-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 °C to 37 °C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca2+ ([Ca2+]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe2+-accumulation following pretreatment with FeSO4. Thus intracellular Fe2+-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe2+-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe2+-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37 °C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe2+-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe2+-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2 channels by Fe2+ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe2+ is released by heme degradation under intracerebral hemorrhage. 相似文献
7.
Paola Llanos Ariel Contreras-Ferrat Genaro Barrientos Marco Valencia David Mears Cecilia Hidalgo 《PloS one》2015,10(6)
Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]). Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS. 相似文献
8.
Our previous results have demonstrated that both nitric oxide (NO) and hydrogen peroxide (H2O2) are involved in the promotion of adventitious root development in marigold (Tagetes erecta L.). However, not much is known about the intricate molecular network of adventitious root development triggered by NO and
H2O2. In this study, the involvement of calcium (Ca2+) and calmodulin (CaM) in NO- and H2O2-induced adventitious rooting in marigold was investigated. Exogenous Ca2+ was capable of promoting adventitious rooting, with a maximal biological response at 50 μM CaCl2. Ca2+ chelators and CaM antagonists prevented NO- and H2O2-induced adventitious rooting, indicating that both endogenous Ca2+ and CaM may play crucial roles in the adventitious rooting induced by NO and H2O2. NO and H2O2 treatments increased the endogenous content of Ca2+ and CaM, suggesting that NO and H2O2 enhanced adventitious rooting by stimulating the endogenous Ca2+ and CaM levels. Moreover, treatment with Ca2+ enhanced the endogenous levels of NO and H2O2. Additionally, Ca2+ might be involved as an upstream signaling molecule for CaM during NO- and H2O2-induced rooting. Altogether, the results suggest that both Ca2+ and CaM are two downstream signaling molecules in adventitious rooting induced by NO and H2O2. 相似文献
9.
NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene. 相似文献
10.
《Cell calcium》2019
Hydrogen peroxide (H2O2) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H2O2 regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H2O2 on Ca2+ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca2+ signals and acetylcholine evoked Ca2+ increases were inhibited by H2O2. H2O2 inhibition of acetylcholine-evoked Ca2+ signals was reversed by catalase. H2O2 exerts its inhibition on the IP3 receptor as Ca2+ release evoked by photolysis of caged IP3 was supressed by H2O2. H2O2 suppression of IP3-evoked Ca2+ signalling may be mediated by mitochondria. H2O2 depolarized mitochondria membrane potential. Acetylcholine-evoked Ca2+ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP3-evoked Ca2+ release by H2O2 arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca2+ overload during IP3-linked Ca2+ signals by a H2O2 mediated negative feedback depolarization of the organelle and inhibition of IP3-evoked Ca2+ release. 相似文献
11.
Sujata Roychoudhury Sahl K Ghosh Tapati Chakraborti Sajal Chakraborti 《Molecular and cellular biochemistry》1996,159(2):95-103
We sought to investigate the mechanism(s) by which the oxidant H2O2 stimulates Ca2+ release from mitochondria of bovine pulmonary vascular smooth muscle tissue and to test the hypothesis that hydroxyl radical is involved in this phenomenon. Treatment of the smooth muscle tissue with 1 mM H2O2 dramatically stimulated hydroxyl radical generation as measured by methane (CH4) production by GLC using dimethylsulfoxide (DMSO) as the substrate. Pretreatment of the mitochondria with the hydroxyl radical scavanger dimethylthiourea (DMTU) prevented the increase in CH4 production caused by H2O2. In the absence of EGTA, H2O2 caused stimulation of Ca2+ release from mitochondria occurred with a lag time of about 4 min. Addition of EGTA to Ca2+ loaded mitochondria resulted an immediate loss of Ca2+ and that has been found to be augmented by H2O2. The release of Ca2+ by H2O2 did not appear to occur with concommitant increase in sucrose entry into, K+ release from, and swelling of mitochondria when the Ca2+ cycling was prevented by EGTA. These observations suggested that H2O2-mediated Ca2+ release from bovine pulmonary vascular smooth muscle tissue mitochondria occurred (i) through the involvement of hydroxyl radical; (ii) via specific pathway(s); and (iii) did not appear to happen primarily via nonspecific pore formation.Abbreviations H2O2
hydrogen peroxide
- OH·
hydroxyl radical
- t-buOOH
tert-butyl hydroperoxide
- CH4
methane
- GLC
gas liquid chromatography
- DMTU
dimethylthiourea
- EGTA
ethylene glycol bis(-aminoethyl ether)
- N
Ntetraacetic acid
- DMSO
dimethyl sulfoxide
- CH4
methane
- HBPS
Hank's buffered physiological saline
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
- MOPS
3-(N-morpholino)propane sulfonic acid
- Tris
tris (hydroxymethyl aminomethane) 相似文献
12.
A Mg2+-stimulated ATPase in platelet alpha-granule membranes: possible involvement in proton translocation 总被引:1,自引:0,他引:1
Isolated porcine platelet α granules display a Mg2+-stimulated ATPase activity. The enzyme is membrane bound and several criteria suggest that it is intrinsic to the α granules, rather than arising from contamination with other structures. Characterization of the ATPase revealed an apparent Km for ATP of 198 μm. Other nucleotides are also hydrolyzed by the enzyme, though at a slower rate. The enzyme has an absolute requirement for divalent cations, and both Mg2+ (apparent Km 0.93 mm) and Ca2+ (apparent Km 0.95 mm) can activate it. Maximal hydrolysis rates are higher with Mg2+ than with Ca2+. Micromolar Ca2+ in the presence of maximally stimulating Mg2+ concentrations produces a small additional enhancement of activity. The Mg2+ ATPase has a broad activity maximum between pH 6.5 and 8.5, and an activation energy of 11.8 Kcal/mol. Several independent observations suggest that the ATPase could be involved in H+ translocation across the granule membrane: (a) the activity is stimulated upon disrupting membrane continuity by either hypotonic lysis or addition of nondenaturing detergents; (b) proton ionophores enhance the activity in intact but not in disrupted α granules; (c) permeating anions stimulate the ATPase more than slowly permeant or impermeant ones; (d) addition of NH3 (as either NH4Cl or (NH4)2SO4) activates enzyme activity; (e) silicotungstate and disulfonic stilbene derivatives, which are inhibitors of other H+-transporting ATPases, also inhibit the α-granule enzyme. These findings are compared with the reported properties of H+ pumps of other storage and secretory organelles. 相似文献
13.
Addition of calcium chloride to an egg homogenate of Strongylocentrotus purpuratus stimulates O2 consumption which is not inhibited by millimolar cyanide. Results strongly suggest that Ca2+-stimulated O2 consumption is at least partially the result of polyunsaturated fatty acid oxidation. First, addition of arachidonic acid (AA), or other polyunsaturated fatty acids, to the homogenate enhance Ca2+-stimulated O2 consumption; this enhancement, by AA, being coupled to its oxidation to a hydroxy fatty acid. Second, calcium stimulates a lipase activity in the homogenate that is capable of releasing free fatty acids. Third, Ca2+-stimulated O2 consumption and AA oxidation have virtually identical calcium requirements and pH optima. The sequence of events then is that upon calcium addition to the homogenate, lipase activity is increased which liberates free fatty acids. At the same time calcium also activates a polyunsaturated fatty acid oxygenase, possibly lipoxygenase, that converts the free fatty acids to hydroxy fatty acids. The possible physiological importance of this reaction is underscored by the high affinity for Ca2+ [approximately 10(-7)M], an ion known to increase above the required levels at fertilization. The pH activity profile also suggests possible physiological modulation because a pH change of 6.8 increasing to 7.2, as suggested to occur after fertilization, yields almost a twofold increase in O2 consumption. Egg homogenates from many other invertebrate species have the ability to oxidize AA in a Ca2+-dependent fashion. For the investigated species, the presence of Ca2+-stimulated O2 consumption and AA oxidation correlates with the presence of cyanide insensitive respiration in the intact egg. 相似文献
14.
Song YH Choi E Park SH Lee SH Cho H Ho WK Ryu SY 《Free radical biology & medicine》2011,51(9):1708-1716
Oxidative stress remodels Ca2+ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca2+/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca2+ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca2+ current (ICa,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H2O2 induced an increase in ICa,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H2O2-induced LTF of ICa,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca2+ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H2O2 via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia. 相似文献
15.
The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca2+. At free Mg2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg2+ -independent, but is strongly stimulated by submicromolar Ca2+ concentrations Km 0.25 μM, Vmax 24 μmol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely. 相似文献
16.
Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2 (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca2+-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease
activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2
(TIMP-2) indicating that the Ca2+-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies
of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding
tissue inhibitor of metalloprotease in the membrane.
In addition to increasing the Ca2+ATPase activity, H2O2 also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability
to degrade14C-gelatin. The protease activity and the Ca2+ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H2O2 was due to reactive oxidant species(es). Both basal and H2O2 stimulated MMP-2 activity and Ca2+ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α2-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H2O2 increased MMP-2 activity and that subsequently stimulated Ca2+ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of
MMP-2 or H2O2 to the smooth muscle membrane suspension caused submaximal increase in Ca2+ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca2+ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H2O2 augments further the Ca2+ATPase activity caused by the respective low doses of either H2O2 or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca2+ATPase activity in the membrane caused by the combined treatment of MMP-2 and H2O2. 相似文献
17.
《Journal of Plant Interactions》2013,8(1):883-888
In order to understand the continuous defense reactions of host plants against insect attack, a tobacco variety G140 was infested by tobacco aphid Myzus persicae for 2 h to 5 d. The changes of transmembrane ionic fluxes (Ca2+) and hydrogen peroxide (H2O2) were detected by the technique of noninvasive micro-test and their relationship was further studied. It was found that H2O2 accumulation depended on Ca2+ influx. Ca2+ flux exhibited a strong influx at all infestation periods by aphids, while H2O2 showed an efflux behavior. The slight variation tendency of Ca2+ influx and H2O2 efflux was consistent. The activities of the corresponding defense proteins, peroxidase (POD) and catalase (CAT) enzyme, were enhanced to respond to the insect attacks, much higher than those tobacco in control. The Ca2+ influx and H2O2 efflux, as well as the activities of POD and CAT enzymes, were increased in a long period of aphid feeding. It indicated that a continuous physiological response of tobacco to aphid infestation could be initiated and lasted for a long time. 相似文献
18.
The inositol 1,4,5-trisphosphate (IP3)-mediated intracellular Ca2+ releases in secretory cells play vital roles in controlling not only the intracellular Ca2+ concentrations but also the Ca2+-dependent exocytotic processes. Of intracellular organelles that release Ca2+ in response to IP3, secretory granules stand out as the most prominent organelle and are responsible for the majority of IP3-dependent Ca2+ releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP3-mediated Ca2+ release as well as the presence of the IP3 receptor (IP3R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP3R isoforms, and 58–69% of total cellular IP3R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2–4 mM) of chromogranins
and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin
II are high-capacity, low-affinity Ca2+ binding proteins, binding 30–93 mol of Ca2+/mol of protein with dissociation constants of 1.5–4.0 mM. Due to this high Ca2+ storage properties of chromogranins secretory granules contain ~40 mM Ca2+. Furthermore, chromogranins A and B directly interact with the IP3Rs and modulate the IP3R/Ca2+ channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively.
Coupled chromogranins change the IP3R/Ca2+ channels to a more ordered, release-ready state, whereby making the IP3R/Ca2+ channels significantly more sensitive to IP3. 相似文献
19.
Roger F. Castilho Paulo C. Carvalho-Alves Anibal E. Vercesi Sérgio T. Ferreira 《Molecular and cellular biochemistry》1996,159(2):105-114
The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca2+ transporting ATPase which carries out active Ca2+ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe2+ + H2O2 HO· + OH–+ Fe3+) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0–1.5 MM H2O2 plus 50 M Fe2+ and 6 mM ascorbate. Ca2+ uptake carried out by the Ca2+-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca2+ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca2+ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of 45Ca2+ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca2+-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca2+-ATPase. Electrophoretic analysis of oxidized Ca2+-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca2+-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca2+-pump may be related to aminoacid oxidation and fragmentation of the protein.Abbreviations AcP
acetylphosphate
- BHT
butylhydroxytoluene
- DTT
dithiothreitol
- Hepes
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid
- SDS
sodium dodecyl sulfate
- SDS-PAGE
polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
- SR
sarcoplasmic reticulum
- SRV
sarcoplasmic reticulum vesicles
- TBA
thiobarbituric acid
- TBARS
thiobarbituric acid-reactive substances
- TFP
trifluoperazine 相似文献
20.
Tara L. Lysechko Samuel M. S. Cheung Hanne L. Ostergaard 《The Journal of biological chemistry》2010,285(41):31174-31184
Pyk2 was identified as a Ca2+-dependent kinase, however, the regulation of Pyk2 by Ca2+ in T cells remains controversial. We found that Ca2+ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca2+ dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the calmodulin inhibitor W7, and we detected no Ca2+-regulated association between Pyk2 and calmodulin. Ca2+-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca2+-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H2O2 scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H2O2 induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca2+ mobilization induces the production of reactive oxygen species, which in turn activate the Erk pathway, leading to Src-family kinase-dependent Pyk2 phosphorylation. Our data demonstrate that Pyk2 is not a Ca2+-dependent kinase in T cells but instead, increased intracellular Ca2+ induces Pyk2 phosphorylation through production of reactive oxygen species. These findings are consistent with the possibility that Pyk2 acts as an early sensor of numerous extracellular signals that trigger a Ca2+ flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events. 相似文献