Ca2+参与H2O2促进盐碱混合胁迫下燕麦种子的萌发和成苗

盐碱胁迫是制约作物高产优质的重要因素,Ca²⁺和H₂O₂作为信号分子参与作物逆境响应调节。为了解Ca²⁺是否参与H₂O₂对盐碱胁迫下植物种子萌发和成苗的调控,以燕麦（Avena nude）为试验材料,采用隶属函数分析方法,研究了胞外游离Ca²⁺螯合剂EGTA、质膜Ca²⁺通道阻断剂LaCl₃和液泡膜Ca²⁺释放抑制剂钌红（RR）与H₂O₂共处理对盐碱混合（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）胁迫下种子萌发和成苗的影响。结果表明,25~200 mmol·L^-1盐碱混合胁迫显著抑制燕麦的种子萌发和成苗,抑制程度随浓度提高而增强;0.001~2 mmol·L^-1 H₂O₂能够促进燕麦种子的萌发和成苗,且0.5 mmol·L^-1 H₂O₂可以显著缓解75 mmol·L^-1盐碱混合胁迫对燕麦种子萌发和成苗的抑制作用;而EGTA、LaCl₃和RR均能消减H₂O₂对盐碱混合胁迫下燕麦种子萌发和成苗的促进作用。表明Ca²⁺参与H₂O₂促进盐碱混合胁迫下燕麦种子萌发和成苗的信号转导过程。     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

油桃花芽破眠过程中H2O2代谢与Ca2+转运的关系

利用化学测定法分析高温、单氰胺和TDZ 3种破眠处理对“曙光”油桃休眠花芽H₂O₂代谢的主要影响,利用非损伤微测技术检测H₂O₂对休眠芽Ca²⁺转运的影响,研究H₂O₂在芽休眠解除过程中的调控作用.结果表明： 在深休眠时期,高温和单氰胺处理均能诱导芽内H₂O₂含量升高和过氧化氢酶(CAT)活性降低,并具有显著的破眠作用;TDZ对H₂O₂含量及CAT、过氧化物酶(POD)活性影响不大,破眠效果较差.休眠花芽原基组织钙通道活跃,对外源Ca²⁺呈吸收状态.外源H₂O₂可诱导休眠花芽原基组织Ca²⁺转运发生变化,低浓度H₂O₂降低Ca²⁺吸收速率,高浓度H₂O₂使组织对Ca²⁺的转运由吸收转变为释放.这表明休眠芽内H₂O₂信号和Ca²⁺信号相关联,通过诱导H₂O₂积累调控Ca²⁺信号可能在高温和单氰胺打破休眠的信号转导过程中起重要作用.     

Mechanism of the activation of arachidonic acid release by oxysterols in NRK 49F cells: Role of calcium

We previously demonstrated that oxysterols added to the culture medium of NRK 49F cells labelled with [¹⁴C] arachidonic acid potentiated arachidonic acid (AA) release and prostaglandin (PG) E₂ biosynthesis induced by the activation of these cells with fetal calf serum (FCS). In the absence of FCS, oxysterols had no effect on AA release. As phospholipase (Plase) A₂ activity is Ca²⁺-dependent, we investigated whether oxysterol potentiating effect on AA release was related to an effect of these compounds on cell Ca²⁺ concentration. In this paper, we show that the intensity of potentiation by oxysterol varies with the external cell Ca²⁺ concentration; when external Ca²⁺ is chelated by EGTA, the oxysterol effect persists, though it is decreased. The Ca²⁺ channel inhibitor nifedipine does not decrease the potentiating effect of 25-OH cholesterol, indicating that, if oxysterol favours Ca²⁺ entry into the cell, the nifedipine inhibited channel is not involved. At the usual concentration (5 μm/ml), oxysterols are not able to increase, mimmediately or after a short time of contact (90 min) the concentration of intracellular free Ca²⁺ ([Ca²⁺])_i measured by fluorescence of Quinn-2; at very high concentration of oxysterol (25 μm/ml), [Ca²⁺]_i only slightly increases (+30%). The liberation of AA induced by cell activation with the Ca²⁺ ionophore ionomycin is also potentiated by 25-OH cholesterol. All these observations are not in favour of a proper effect o oxysterols on cell Ca²⁺ level.     

Properties of the Group IV phospholipase A2 family

The Group IV phospholipase A₂ family is comprised of six intracellular enzymes commonly called cytosolic phospholipase A₂ (cPLA₂) , cPLA₂β, cPLA₂γ, cPLA₂δ, cPLA₂ε and cPLA₂ζ. They are most homologous to phospholipase A and phospholipase B/lysophospholipases of filamentous fungi particularly in regions containing conserved residues involved in catalysis. However, a number of other serine acylhydrolases (patatin, Group VI PLA₂s, Pseudomonas aeruginosa ExoU and NTE) contain the Ser/Asp catalytic dyad characteristic of Group IV PLA₂s, and recent structural analysis of patatin has confirmed its structural similarity to cPLA₂. A characteristic of all these serine acylhydrolases is their ability to carry out multiple reactions to varying degrees (PLA₂, PLA₁, lysophospholipase and transacylase activities). cPLA₂, the most extensively studied Group IV PLA₂, is widely expressed in mammalian cells and mediates the production of functionally diverse lipid products in response to extracellular stimuli. It has PLA₂ and lysophospholipase activities and is the only PLA₂ that has specificity for phospholipid substrates containing arachidonic acid. Because of its role in initiating agonist-induced release of arachidonic acid for the production of eicosanoids, cPLA₂ activation is important in regulating normal and pathological processes in a variety of tissues. Current information available about the biochemical properties and tissue distribution of other Group IV PLA₂s suggests they may have distinct mechanisms of regulation and functional roles.     

Involvement of phospholipase A2 in norepinephrine release from synaptosomes isolated from rat cerebral cortex

Phospholipase A₂ added directly to superfused [³H]norepinephrine-labeled synaptosomes could cause the release of neurotransmitter molecules. Chloroquine and quinacrine, which block the action of phospholipase A₂, inhibited either the phospholipase A₂-stimulated or the high potassium-stimulated release of [³H]norepinephrine from synaptosomes. Only quinacrine blocked the high potassium-stimulated influx of Ca²⁺. It appears that during stimulation of synaptosomes, Ca²⁺ influx leads to the activation of phospholipase A₂, which in turn, hydrolyzes membrane phospholipids in situ. The formation of lysophospholipids may alter the microenvironment and the physicochemical properties of membranes, resulting in the release of neurotransmitter through exocytosis.     

AP-1 activation through endogenous H(2)O(2) generation by alveolar macrophages

Reactive oxygen species released during the respiratory burst are known to participate in cell signaling. Here we demonstrate that hydrogen peroxide produced by the respiratory burst activates AP-1 binding. Stimulation of the macrophage cell line NR8383 with respiratory burst agonists ADP and C5a increased AP-1 binding activity. Importantly, this increase in binding was blocked by catalase, confirming mediation by endogenous H₂O₂. Moreover, exogenously added H₂O₂ mimicked the agonists, and also activated AP-1. Antibodies revealed that the activated AP-1 complex is composed predominantly of c-Fos/c-Jun heterodimers. Treatment of the cells with ADP, C5a and H₂O₂ (100 μM) all increased the phosphorylation of c-Jun. c-Fos protein was increased in cells treated with C5a or high dose (200 μM) H₂O₂, but not in cells treated with ADP. The MEK inhibitor, PD98059, partially blocked the C5a-mediated increase in AP-1 binding. A novel membrane-permeable peptide inhibitor of JNK, JNKi, also inhibited AP-1 activation. Together these data suggest that C5a-mediated AP-1 activation requires both the activation of the ERK and JNK pathways, whereas activation of the JNK pathway is sufficient to increase AP-1 binding with ADP. Thus, AP-1 activation joins the list of pathways for which the respiratory burst signals downstream events in the macrophage.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H₂O₂) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H₂O₂ preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H₂O₂ stimulated divergent pathways, with caspases being activated only by H₂O₂. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H₂O₂, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H₂O₂. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

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

Characterization of phospholipase A2 and acyltransferase activities in squid (Loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions.

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.

The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.

Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

The role of H2O2 as a mediator of UVB-induced apoptosis in keratinocytes

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Previously, it has been reported that UVB-irradiation of keratinocytes leads to intracellular generation of hydrogen peroxide (H₂O₂) and that antioxidants can inhibit ROS-induced apoptosis. Although both UVB-irradiation and H₂O₂-incubation led to increased intracellular H₂O₂ levels, the antioxidants catalase and glutathione monoester (GME), inhibited apoptosis only when induced by H₂O₂, not by UVB. Furthermore, extracellular signal-regulated kinase (ERK), a prominent member of the mitogen-activated protein kinase (MAPK) family, was found to be activated by treatment with both UVB and H₂O₂. Inhibition of ERK phosphorylation by pre-treatment with PD98059 resulted in enhanced apoptosis after H₂O₂-exposure. However, no significant difference of apoptosis was observed between cells with and without inhibitor pre-treatment upon UVB-irradiation. DNA damage in the form of cyclobutane pyrimidine dimers was observed after exposure to UVB, but no photoproducts were found in H₂O₂-treated cells. These results suggest a ROS-independent pathway of UVB-induced apoptosis. Although UVB-irradiation causes moderate increase in H₂O₂, the generation of H₂O₂ does not contribute to the induction of apoptosis. Moreover, activation of ERK only blocks H₂O₂-dependent apoptosis but has no impact on UVB-induced apoptosis.     

Interaction of ovokinin(2-7) with vascular bradykinin 2 receptors

Intravenous administration of ovokinin(2–7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1–5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2–7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B₂ receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B₁ receptor antagonist [des-Arg¹⁰]-HOE140 (26.3±15.5%). Intracellular Ca²⁺ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2–7). The increases were abolished by HOE140 and unaffected by [des-Arg¹⁰]-HOE140. The elevation of intracellular Ca²⁺ by ovokinin(2–7) was dependent on Ca²⁺ entry from extracellular space as it was reduced in a Ca²⁺-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca²⁺ increase by the peptide. PA phosphohydrolase and phospholipase A₂ inhibitors significantly reduced the responses as well. Our results show that ovokinin(2–7) modulates cardiovascular activity by interacting with B₂ bradykinin receptors.     

水杨酸诱发的丹参悬浮培养细胞内H2O2迸发与其培养基碱化的关系

水杨酸(salicylic acid,SA)处理可诱导丹参悬浮培养细胞内H2O2产生及其培养基碱化。利用NADPH氧化酶抑制剂咪唑(imidazole,IMD)、H2O2淬灭剂二甲基硫脲(dimethylthiourea,DMTU)、质膜H+-ATPase抑制剂钒酸钠(Na3VO4)及激活剂壳梭孢菌素(fusicoccin,FC)处理丹参悬浮培养细胞,探讨SA诱导的H2O2迸发与培养基碱化之间的关系。结果表明,H2O2可促发培养基碱化,IMD和DMTU抑制SA诱发的培养基碱化,说明H2O2参与SA诱发的培养基碱化过程;SA抑制质膜H+-ATPase活性,Na3VO4引发培养基碱化并使H2O2迸发时间提前,FC处理逆转了SA诱导的培养基碱化及H2O2迸发,说明质膜H+-ATPase调控培养基pH值变化,培养基碱化促进了H2O2产生。因此,丹参悬浮培养细胞内H2O2水平与其培养基碱化程度之间相互关联、共同作用,协同响应SA的诱导。     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

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

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

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

Aging, cytochrome oxidase activity, and hydrogen peroxide release by mitochondria

The objective of this study was to explore the possible cause(s) underlying the previously observed, age-related increase in the rate of mitochondrial H₂O₂ release in the housefly. The hypothesis that an imbalance between different respiratory complexes may be a causal factor was tested. Cytochrome c oxidase activity was found to sharply decline in the latter part of the life span of the flies. Effects of different substrates and respiratory inhibitors were determined in order to ascertain if a decrease in cytochrome c oxidase activity could be responsible for the increased H₂O₂ release. H₂O₂ was measured spectrofluorometrically using horseradish peroxidase and p-hydrophenylacetate as an indicator. Neither NADH-linked substrates nor succinate caused a stimulation of H₂O₂ production. H₂O₂ release by mitochondria, inhibited with rotenone and antimycin A, was greatly increased upon supplementation with -glycerophosphate; however, the further addition of KCN or myxothiazol, to such preparations, caused a depression of H₂O₂ generation. In contrast, relatively low concentrations of KCN or myxothiazol were found to stimulate H₂O₂ release in insect mitochondria supplemented with -glycerophosphate and exposed to rotenone, but not antimycin A. Results are interpreted to suggest that partial inhibition of cytochrome c oxidase activity can lead to the stimulation of mictochondrial H₂O₂ production in the housefly at site(s) other than NADH dehydrogenase and ubisemiquinone/ cytochrome b region; a possible source may be glycerophosphate dehydrogenase.