Oxidative gating of water channels (aquaporins) in Chara by hydroxyl radicals

Hydroxyl radicals (*OH) as produced in the Fenton reaction (Fe²⁺ + H₂O₂ = Fe³⁺ + OH^– + *OH) have been used to reversibly inhibit aquaporins in the plasma membrane of internodes of Chara corallina. Compared to conventional agents such as HgCl₂, *OH proved to be more effective in blocking water channels and was less toxic to the cell. When internodes were treated for 30 min, cell hydraulic conductivity (Lp) decreased by 90% or even more. This effect was reversed within a few minutes after removing the radicals from the medium. In contrast to HgCl₂, radical treatment reduced membrane permeability of small lipophilic organic solutes (ethanol, acetone, 1‐propanol, and 2‐propanol) by only 24 to 52%, indicating some continued limited movement of these solutes across aquaporins. The biggest effect of *OH treatment on solute permeability was found for isotopic water (HDO), which largely used water channels to cross the membrane. Inhibition of aquaporins reduced the diffusional water permeability (P_d) by about 70%. For the organic test solutes, which mainly use the bilayer to cross the membrane, channel closure caused anomalous (negative) osmosis; that is, cells had negative reflection coefficients (σ_s) and were transiently swelling in a hypertonic medium. From the ratio of bulk (Lp or osmotic permeability coefficient, P_f) to diffusional (P_d) permeability of water, the number (N) of water molecules that align in water channels was estimated to be N = P_f/P_d = 46 (on average). Radical treatment decreased N from 46 to 11, a value still larger than unity, which would be expected for a membrane lacking pores. The gating of aquaporins by *OH radicals is discussed in terms of a direct action of the radicals when passing the pores or by an indirect action via the bilayer. The rapid recovery of inhibited channels may indicate an easy access of cytoplasmic antioxidants to closed water channels. As hydrogen peroxide is a major signalling substance during different biotic and abiotic stresses, the reversible closure of water channels by *OH (as produced from H₂O₂ in the apoplast in the presence of transition metals such as Fe²⁺ or Cu⁺) may be downstream of the H₂O₂ signalling. This may provide appropriate adjustments in water relations (hydraulic conductivity), and a common response to different kinds of stresses.     

Formation of Hydroxyl Radicals on Reaction of Hypochlorous Acid with Ferrocyanide, a Model IRON(II) Complex

Hypochlorous acid reacts with the model iron(II) complex, ferrocyanide (Fe(CN)₆^4-) in aqueous solution with the rate constant 220 ± 15 dm³ mol^-1 s^-1. Free hydroxyl radicals are formed in this reaction in 27% yield as shown by the hydroxylation of benzoate to give a product distribution identical to that of free (radiolytically generated) hydroxyl radicals. This reaction is three orders of magnitude faster than the analogous reaction involving hydrogen peroxide (the Fenton reaction), suggesting that the hypochlorous acid generated by activated neutrophils may be a source of hydroxyl radicals.     

衰老叶片和叶绿体中H_2O_2的累积与膜脂过氧化的关系

在自然衰老和ABA处理的叶片和叶绿体中活性氧H_2O_2均比对照明显增高。外加H_2O_2刺激水稻叶绿体膜脂过氧化作用。叶绿体的丙二醛含量随H_2O_2浓度、光照时间、光照强度及叶绿体完整性而变化。AsA、GSH、SOD、甘露醇和过氧化氢酶对外源H_2O_2引起的膜脂过氧化有缓解作用,Fe~(2+)有刺激作用。而H_2O_2对叶绿体过氧化损伤主要是转化为OH之故。     

Role of Hydroxyl Radicals in Escherzchza Colz Killing Induced by Hydrogen Peroxide

Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH -) generated by H₂O₂ and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H₂O₂ lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxarnine, did not affect the survival of cells exposed to 2.5mM H₂O₂. In addition cell vulnerability to the same H₂O₂ concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H₂O₂ higher than 10mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content.

It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.     

水杨酸调节决明根系铝诱导的氧化胁迫

水杨酸(Salicylicacid,SA)在调节生物和非生物胁迫,诱导植物氧化胁迫中起着重要的作用,但对铝诱导的氧化胁迫的调节作用尚不清楚。本文研究了SA对决明(CassiatoraL.)根系铝诱导的H2O2和O2-含量变化,包括抗氧化酶活性以及细胞质膜过氧化胁迫变化的影响。介质中20mmol/L铝处理增加质膜透性,导致MDA含量上升及根尖细胞Evansblue染色加重(测定细胞死亡),而外源供给5mmol/LSA能缓解铝诱导的氧化胁迫。SA处理能明显降低根尖H2O2和O2-的含量,但两者含量与CAT、APX和GR的活性变化没有相关性,而与POD活性增加有关。水杨酸诱导H2O2含量的下降与抑制O2-积累和SOD活性有关。结果表明,SA可能激活一条由H2O2介导的、依赖于POD的抗氧化机制来缓解脂质的过氧化作用。     

水杨酸调节决明根系铝诱导的氧化胁迫

水杨酸(Salicylic acid,SA)在调节生物和非生物胁迫,诱导植物氧化胁迫中起着重要的作用,但对铝诱导的氧化胁迫的调节作用尚不清楚.本文研究了SA对决明(Cassiatora L.)根系铝诱导的H2O2和O2-含量变化,包括抗氧化酶活性以及细胞质膜过氧化胁迫变化的影响.介质中20 μmol/L铝处理增加质膜透性,导致MDA含量上升及根尖细胞Evans blue染色加重(测定细胞死亡),而外源供给5 μmol/L SA能缓解铝诱导的氧化胁迫.SA处理能明显降低根尖H2O2和O2-的含量,但两者含量与CAT、APX和GR的活性变化没有相关性,而与POD活性增加有关.水杨酸诱导H2O2含量的下降与抑制O2积累和SOD活性有关.结果表明,SA可能激活一条由H2O2介导的、依赖于POD的抗氧化机制来缓解脂质的过氧化作用.     

Gating of water channels (aquaporins) in cortical cells of young corn roots by mechanical stimuli (pressure pulses): effects of ABA and of HgCl2

Hydraulic properties (half-time of water exchange, T1/2, and hydraulic conductivity, Lp; T1/2 approximately 1/Lp) of individual cells in the cortex of young corn roots were measured using a cell pressure probe for up to 6 h to avoid variations between cells. When pulses of turgor pressure of different size were imposed, T1/2 (Lp) responded differently depending on the size. Pulses of smaller than 0.1 MPa, which induced a small proportional water flow, caused no changes in T1/2 (Lp). Medium-sized pulses of between 0.1 and 0.2 MPa caused an increase in T1/2 (decrease in Lp) by a factor of 4 to 23. The effects caused by medium-sized pulses were reversible within 5-20 min. When larger pulses of more than 0.2 MPa were employed, changes were not reversible within 1-3 h, but could be reversed within 30 min in the presence of 500 nM of the stress hormone ABA. Cells with a short T1/2 responded to the aquaporin blocker mercuric chloride (HgCl2). The treatment had no effect on cells which exhibited long T1/2 following a mechanical inhibition by the large-pulse treatment. Step decreases in pressure resulted in the same inhibition as step increases. Hence, the treatment did not cause a stretch-inhibition of water channels and was independent of the directions of both pressure changes and water flows induced by them. It is concluded that inhibition is caused by the absolute value of intensities of water flow within the channels, which increased in proportion to the size of step changes in pressure. Probable mechanisms by which the mechanical stimuli are perceived are (i) the input of kinetic energy to the channel constriction (NPA motif of aquaporin) which may cause a conformational change of the channel protein (energy-input model) or (ii) the creation of tensions at the constriction analogous to Bernoulli's principle for macroscopic pores (cohesion-tension model). Estimated rates of water flow within the pores were a few hundred micro m s-1, which is too small to create sufficient tension. They were much smaller than those proposed for AQP1. Based on literature data of single-channel permeability of AQP1, a per channel energy input of 200 kBxT (kB=Boltzmann constant) was estimated for the energy-input model. This should be sufficient to initiate changes of protein conformation and an inactivation of channels. The data indicate different closed states which differ in the amount of distortion and the rates at which they relax back to the open state.     

A cohesion/tension mechanism explains the gating of water channels (aquaporins) in Chara internodes by high concentration

Isolated internodes of Chara corallina have been used to study the gating of aquaporins (water channels) in the presence of high concentrations of osmotic solutes of different size (molecular weight). Osmolytes were acetone and three glycol ethers: ethylene glycol monomethyl ether (EGMME), diethylene glycol monomethyl ether (DEGMME), and triethylene glycol monoethyl ether (TEGMEE). The 'osmotic efficiency' of osmolytes was quite different. Their reflection coefficients ranged between 0.15 (acetone), 0.59 (EGMME), 0.78 (DEGMME), and 0.80 (TEGMEE). Bulk water permeability (Lp) and diffusive permeabilities (Ps) of heavy water (HDO), hydrogen peroxide (H2O2), acetone, and glycol ethers (EGMME, DEGMME, and TEGMEE) were measured using a cell pressure probe. Cells were treated with different concentrations of osmotic solutes of up to 800 mM ( approximately 2.0 MPa of osmotic pressure). Inhibition of aquaporin activity increased with both increasing concentration and size of solutes (reflection coefficients). As cell Lp decreased, Ps increased, indicating that water and solutes used different passages across the plasma membrane. Similar to earlier findings of an osmotic gating of ion channels, a cohesion/tension model of the gating of water channels in Chara internodes by high concentration is proposed. According to the model, tensions (negative pressures) within water channels affected the open/closed state by changing the free energy between states and favoured a distorted/collapsed rather than the open state. They should have differed depending on the concentration and size of solutes that are more or less excluded from aquaporins. The bigger the solute, the lower was the concentration required to induce a reversible closure of aquaporins, as predicted by the model.     

Effect of amino acids on X-ray-induced hydrogen peroxide and hydroxyl radical formation in water and 8-oxoguanine in DNA

Generation of hydrogen peroxide and hydroxyl radicals in L-amino acid solutions in phosphate buffer, pH 7.4, under X-ray irradiation was determined by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system and using the fluorescent probe coumarin-3-carboxylic acid, respectively. Amino acids are divided into three groups according to their effect on the hydrogen peroxide formation under irradiation: those decreasing yield of H2O2, having no effect, and increasing its yield. All studied amino acids at 1 mM concentration decrease the yield of hydroxyl radicals in solution under X-ray irradiation. However, the highest effect is observed in the order: Cys > His > Phe = Met = Trp > Tyr. At Cys, Tyr, and His concentrations close to physiological, the yield of hydroxyl radicals decreases significantly. Immunoenzyme analysis using monoclonal antibodies to 8-oxoguanine (8-oxo-7,8-dihydroguanine) was applied to study the effect of amino acids with the most pronounced antioxidant properties (Cys, Met, Tyr, Trp, Phe, His, Lys, Arg, Pro) on 8-oxoguanine formation in vitro under X-ray irradiation. It is shown that amino acids decrease the content of 8-oxoguanine in DNA. These amino acids within DNA-binding proteins may protect intracellular DNA against oxidative damage caused by formation of reactive oxygen species in conditions of moderate oxidative stress.     

The role of aquaporins in root water uptake

The capacity of roots to take up water is determined in part by the resistance of living tissues to radial water flow. Both the apoplastic and cell-to-cell paths mediate water transport in these tissues but the contribution of cell membranes to the latter path has long been difficult to estimate. Aquaporins are water channel proteins that are expressed in various membrane compartments of plant cells, including the plasma and vacuolar membranes. Plant aquaporins are encoded by a large multigene family, with 35 members in Arabidopsis thaliana, and many of these aquaporins show a cell-specific expression pattern in the root. Mercury acts as an efficient blocker of most aquaporins and has been used to demonstrate the significant contribution of water channels to overall root water transport. Aquaporin-rich membranes may be needed to facilitate intense water flow across root tissues and may represent critical points where an efficient and spatially restricted control of water uptake can be exerted. Roots, in particular, show a remarkable capacity to alter their water permeability over the short term (i.e. in a few hours to less than 2-3 d) in response to many stimuli, such as day/night cycles, nutrient deficiency or stress. Recent data suggest that these rapid changes can be mostly accounted for by changes in cell membrane permeability and are mediated by aquaporins. Although the processes that allow perception of environmental changes by root cells and subsequent aquaporin regulation are nearly unknown, the study of root aquaporins provides an interesting model to understand the regulation of water transport in plants and sheds light on the basic mechanisms of water uptake by roots.     

The effect of prior exercise on ex vivo induction of heme oxygenase-1 in human lymphocytes

It was postulated that prior demanding exercise would suppress the induction of the oxidant-responsive protein Heme Oxygenase-1 (HO-1) in mononuclear cells following subsequent ex vivo H₂O₂ treatment. Eight male subjects completed two trials in a randomized order (one rest and one exercise) and ex vivo HO-1 protein induction was determined following H₂O₂ treatment in lymphocytes and monocytes before and after each trial using a newly developed and reproducible assay. Lymphocytes obtained 2 h post-exercise showed a modest reduction in HO-1 protein expression in response to ex vivo treatment with H₂O₂ (p<0.05). The plasma concentration of the HO-1 suppressor α1-antitrypsin increased immediately post-exercise (p<0.05) and it is tentatively suggested that this may explain the modest transient reduction in ex vivo HO-1 protein induction in lymphocytes in response to an independent oxidant challenge following a prior bout of demanding exercise.     

Hydroxyl radicals mediate injury to endothelium-dependent relaxation in diabetic rat

The purpose of this study was to determine the radical species which mediates the toxic effects of exogenous oxygenderived free radicals on endothelial function of chronic diabetic rat aorta. Endothelium-dependent relaxation to acetylcholine was impaired in diabetic vessels. Exposure to the exogenous free radical generating system of xanthine plus xanthine oxidase selectively impaired endothelium-dependent relaxation to acetylcholine in control and diabetic aorta with relaxations essentially abolished in diabetic aorta. The loss of relaxation to acetylcholine in diabetic aorta was prevented or attenuated by pretreatment with catalase, dimethylthiourea or desferrioxamine, but not by mannitol or superoxide dismutase. These results suggest that hydroxyl radicals play an important role in the endothelial injury produced by oxygen-derived free radicals in chronic diabetic rat aorta. Furthermore, the site of the injury is likely due to intracellular generation of hydroxyl radicals.     

紫杉烷类化合物的抗氧化应激作用(英文)

为探讨紫杉烷类化合物的抗氧化应激作用,选择适当浓度双氧水处理神经母细胞瘤细胞(SK-N-SH)建立氧化应激细胞模型,用自红豆杉科的植物(Taxus cuspidate)中分离纯化的四种紫杉烷类化合物Taxine B、7-Deacetyl-taxine B、5-Cinnamoyloxy-taxin B、7,10-Diacetyl-2′-deoxyl-taxine A干预过氧化应激模型细胞,用四甲基偶氮唑盐(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,MTT)微量酶反应比色法筛选具有神经保护活性的化合物,Hoechst荧光染色观察细胞核形态改变,DCFH-DA检测细胞内活性氧含量.发现氧化应激模型细胞活力明显减低,应用7-Deacetyl-taxine B、5-Cinnamoyloxy-taxin B后细胞活力显著提高,细胞核形态改变明显减轻,细胞内活性氧含量明显减少.结果表明7-Deacetyl-taxine B、5-Cinnamoyloxy-taxin B可以减少细胞内氧自由基含量,减轻细胞核损伤,提高细胞活力.     

Modulation of the GABA(A)-gated chloride channel by reactive oxygen species

The accumulation of reactive oxygen species during cellular injury leads to oxidative stress. This can have profound effects on ionic homeostasis and neuronal transmission. Gamma-aminobutyric acid (GABA) neurotransmission is sensitive to reactive oxygen species, but most studies have indicated that this is due to alterations in GABA release. Here, we determined whether reactive oxygen species can alter GABA(A) receptor-gated Cl- channels in the adult hippocampus. First, we measured the effects of hydrogen peroxide on intracellular Cl- using UV laser scanning confocal microscopy and the Cl(-)-sensitive probe, 6-methoxy-N-ethylquinolium iodide (MEQ). Superfusion of adult rat hippocampal slices with hydrogen peroxide for 10 min decreased MEQ fluorescence (elevation in [Cl-]i) significantly in area CA1 pyramidal cell soma. Alterations in [Cl-]i were prevented by the vitamin E analog Trolox, an antioxidant that scavenges free radicals. After exposure of slices to hydrogen peroxide, the ability of the GABA agonist muscimol to increase [Cl-]i was attenuated. To determine if GABA(A) receptors were sensitive to oxidative insults, the effect of hydrogen peroxide on the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to GABA-gated Cl- channels was measured using receptor autoradiography and homogenate binding assays. Hydrogen peroxide inhibited [35S]TBPS binding in a regionally selective manner, with the greatest inhibition in cerebral cortex, hippocampus and striatum, areas vulnerable to oxidative stress. Similarly, xanthine and xanthine oxidase, which generate superoxide radicals, reduced [35S]TBPS binding in these regions. The effect of hydrogen peroxide on [35S]TBPS binding was non-competitive and was prevented by Trolox and the iron chelator, deferoxamine. We conclude that reactive oxygen species may compromise GABA(A)-mediated neuronal inhibition via interaction with pre and postsynaptic sites. A reduction in GABA(A)-gated Cl- channel function during periods of oxidative stress may contribute to the development of neuronal damage.     

Adaptation of the Phytopathogenic Fungus Fusarium decemcellulare to Oxidative Stress

The adaptive response of the phytopathogenic fungus Fusarium decemcellulare to the oxidative stress induced by hydrogen peroxide and juglone (5-hydroxy-1,4-naphthoquinone) was studied. At concentrations higher than 1 mM, H₂O₂ and juglone completely inhibited the growth of the fungus. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.25 mM) and juglone (0.1 mM) led to the development of a resistance to high concentrations of these oxidants. The stationary-phase cells were found to be more resistant to the oxidants than the logarithmic-phase cells. The adaptation of fungal cells to H₂O₂ and juglone was associated with an increase in the activity of cellular catalase and superoxide dismutase, the main enzymes involved in the defense against oxidative stress.     

Mercurial-sensitive water transport in barley roots

An isolated barley root was partitioned into the apical and basal part across the partition wall of the double-chamber osmometer. Transroot water movement was induced by subjecting the apical part to a sorbitol solution, while the basal part with the cut end was in artificial pond water. The rate of transroot osmosis was first low but enhanced by two means, infilitration of roots by pressurization and repetition of osmosis. Both effects acted additively. The radial hydraulic conductivity (Lp_r) was calculated by dividing the initial flow rate with the surface area of the apical part of the root, to which sorbitol was applied, and the osmotic gradient between the apical and basal part of the root. Lp_r which was first 0.02–0.04 pm s⁻¹ Pa⁻¹ increased up to 0.25–0.4 pm s⁻¹ Pa⁻¹ after enhancement. Enhancement is assumed to be caused by an increase of the area of the plasma membrane which is avallable to osmotic water movement. The increased Lp_r is in the same order of magnitude as the hydraulic conductivity (Lp) of epidermal and cortical cells of barley roots obtained by Steudie and Jeschke (1983). HgCl₂, a potent inhibitor of water channels, suppressed Lp_r of non-infiltrated and infiltrated roots down to 17% and 8% of control values, respectively. A high sensitivity of Lp_r to HgCl₂ suggests that water channels constitute the most conductive pathway for osmotic radial water movement in barley roots.