Cinnamtannin B-1, a natural antioxidant that reduces the effects of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on CCK-8-evoked responses in mouse pancreatic acinar cells

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.     

Nitric oxide (NO) increase at fertilization in sea urchin eggs upregulates fertilization envelope hardening

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 (t_p) 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.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

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)     

Relationship between NaCl- and H2O2-Induced Cytosolic Ca2+ Increases in Response to Stress in Arabidopsis

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.     

Regulation of phagocyte NADPH oxidase by hydrogen peroxide through a Ca2+/c-Abl signaling pathway

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.     

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe2+ in human monocytic U937 cells

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.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

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.     

Ca<Superscript>2+</Superscript> and CaM are Involved in NO- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Adventitious Root Development in Marigold

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.     

The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH

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 K_0.5(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 K_0.5(Ca²⁺) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K_0.5(Ca²⁺) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K_0.5(Ca²⁺) values were determined at pH 6.8. Lower K_0.5(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.     

Hydrogen peroxide depolarizes mitochondria and inhibits IP3-evoked Ca2+ release in the endothelium of intact arteries

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.     

Role of hydroxyl radical in the oxidant H2O2-mediated Ca2+ release from pulmonary smooth muscle mitochondria

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)     

A Mg2+-stimulated ATPase in platelet alpha-granule membranes: possible involvement in proton translocation

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 K_m 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 K_m 0.93 mm) and Ca²⁺ (apparent K_m 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.     

Characterization of a Ca2+-stimulated lipid peroxidizing system in the sea urchin egg

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.     

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes

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 (I_Ca,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H₂O₂ induced an increase in I_Ca,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H₂O₂-induced LTF of I_Ca,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.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

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 K_m 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 K_m  0.25 μM, V_max  24 μmol P_i/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.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

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.     

Effects of aphids Myzus persicae on the changes of Ca2+ and H2O2 flux and enzyme activities in tobacco

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.     

Inositol 1,4,5-Trisphosphate Receptor in Chromaffin Secretory Granules and Its Relation to Chromogranins

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.     

Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation

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     

Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes

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.