Eukaryotic initiation factor 2alpha kinase is a nitric oxide-responsive mercury sensor enzyme: potent inhibition of catalysis by the mercury cation and reversal by nitric oxide

The activity of one of the eukaryotic initiation factor 2alpha kinases, heme-regulated inhibitor (HRI), is modulated by heme binding. Here, we demonstrate for the first time that Hg2+ strongly inhibits the function of HRI (IC50=0.6 microM), and nitric oxide fully reverses this inhibition. Other divalent metal cations, such as Fe2+, Cu2+, Cd2+, Zn2+ and Pb2+, also significantly inhibit kinase activity with IC50 values of 1.9-8.5 microM. Notably, inhibition by cations other than Hg2+ is not reversed by nitric oxide. Our present data support dual roles of Hg2+ and nitric oxide in the regulation of protein synthesis during cell emergency states.     

胁迫时间与非毒性离子对重金属抑制拟南芥种子发芽及幼苗生长的影响

测定了Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺单一重金属胁迫对拟南芥种子发芽和幼苗生长的影响.结果表明,重金属对幼苗生长的毒性大于对种子发芽的毒性,以抑制种子发芽的IC₅₀为指标,4种重金属的毒性顺序为Hg²⁺＞Cd²⁺＞Pb²⁺/Cu²⁺,以幼苗生长为指标,则毒性顺序为:Cu²⁺＞Hg²⁺＞Cd²⁺/Pb²⁺,并随着胁迫时间延长,种子萌发率下降.此外,不同重金属在不同发芽时段对种子的毒性也不尽相同,Cd²⁺的毒性在种子吸水后的0～12 h大于12～24 h,而Hg²⁺毒性在12～24 h大于0～12 h,其中,种皮对减轻重金属毒性起着十分重要的作用.通过非毒性离子(Ca²⁺、Mg²⁺、K⁺、Na⁺)与重金属离子(Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺)交互作用对拟南芥种子发芽及幼苗生长效应的研究发现, mmol·L^-1的Ca²⁺、Mg²⁺、K⁺、Na⁺可以增强Hg²⁺对种子发芽的毒性,但对Cd²⁺的毒性却没有影响.对于幼苗来说,Ca²⁺、Mg²⁺、K⁺、Na⁺可以显著增强Hg²⁺的毒性,Ca²⁺可以缓解Cd²⁺的毒性,但却增加Cu²⁺的毒性,K⁺可以缓解Pb²⁺对幼苗的毒害作用.最后,本文对重金属的毒害机理进行了探讨.     

Omeprazole attenuates hyperoxic injury in H441 cells via the aryl hydrocarbon receptor

Hyperoxia contributes to the development of bronchopulmonary dysplasia in premature infants. Earlier we observed that aryl hydrocarbon receptor (AhR)-deficient mice are more susceptible to hyperoxic lung injury than AhR-sufficient mice, and this phenomenon was associated with a lack of expression of cytochrome P450 1A enzymes. Omeprazole, a proton pump inhibitor used in humans with gastric acid-related disorders, activates AhR in hepatocytes in vitro. However, the effects of omeprazole on AhR activation in the lungs and its impact on hyperoxia-induced reactive oxygen species (ROS) generation and inflammation are unknown. In this study, we tested the hypothesis that omeprazole attenuates hyperoxia-induced cytotoxicity, ROS generation, and expression of monocyte chemoattractant protein-1 (MCP-1) in human lung-derived H441 cells via AhR activation. Experimental groups included cells transfected with AhR small interfering RNA (siRNA). Hyperoxia resulted in significant increases in cytotoxicity, ROS generation, and MCP-1 production, which were significantly attenuated with the functional activation of AhR by omeprazole. The protective effects of omeprazole on cytotoxicity, ROS production, and MCP-1 production were lost in H441 cells whose AhR gene was silenced by AhR siRNA. These findings support the hypothesis that omeprazole protects against hyperoxic injury in vitro via AhR activation that is associated with decreased ROS generation and expression of MCP-1.     

汞、镉、铜污染对鱼草细胞膜系统的毒害作用

通过生理生化反应测定、激光共聚焦扫描显微镜及透射电镜观察等实验手段,研究了汞（Hg²⁺）、镉（Cd²⁺）、铜（Cu2⁺）对高等水生植物鱼草(Cabomba caroliniana A. Gray)细胞膜系统的毒害作用.结果表明：在3种重金属离子作用下,鱼草叶细胞活性氧（ROS）与丙二醛（MDA）含量上升,保护酶系统活性紊乱,膜脂过氧化程度加剧;质膜受损,膜透性增加,质壁分离;叶绿体膨胀至解体,类囊体膜上的光合色素光激发过程受阻,平均自发荧光强度降低;线粒体嵴突膨胀、减少,膜破损;核膜破裂.Hg²⁺、Cd²⁺、Cu²⁺对鱼草细胞膜系统的影响存在着一定的剂量效应关系.膜系统的稳定性在植物抗重金属胁迫的过程中起着关键性的作用.鱼草对Hg²⁺污染较为敏感,致死浓度为0.3～0.5 mg·L^-1,而对Cd²⁺、Cu²⁺具有较强的抗性,可用作生物防治中的抗性植物.     

Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species

Background and Aims

Uptake of heavy metals by plant root cells depends on electro-physiological parameters of the plasma membrane. In this study, responses of the plasma membrane in root cells were analysed where early reactions to the metal ion-induced stress are localized. Three different Arabidopsis species with diverse strategies of their adaptation to heavy metals were compared: sensitive Arabidopsis thaliana and tolerant A. halleri and A. arenosa.

Methods

Plants of A. thaliana Col-0 ecotype and plants of A. arenosa and A. halleri originating from natural metallicolous populations were exposed to high concentrations of Zn²⁺. Plants were tested for root growth rate, cellular tolerance, plant morphology and cell death in the root apex. In addition, the membrane potential (E_M) of mature cortical root cells and changes in the pH of the liquid culture media were measured.

Key Results

Primary roots of A. halleri and A. arenosa plants grew significantly better at increased Zn²⁺ concentrations than A. thaliana plants. Elevated Zn²⁺ concentrations in the culture medium induced rapid changes in E_M. The reaction was species-specific and concentration-dependent. Arabidopsis halleri revealed the highest insensitivity of the plasma membrane and the highest survival rate under prolonged treatment with extra-high concentrations. Plants were able to effectively adjust the pH in the control, but much less at Zn²⁺-induced lower pH.

Conclusions

The results indicate a similar mode of early reaction to Zn²⁺, but with different extent in tolerant and sensitive species of Arabidopsis. The sensitivity of A. thaliana and a high tolerance of A. halleri and A. arenosa were demonstrated. Plasma membrane depolarization was lowest in the hyperaccumulator A. halleri and highest in A. thaliana. This indicates that rapid membrane voltage changes are an excellent tool to monitor the effects of heavy metals.     

Influence of heavy metal ions on the electrophysical properties of Anacystis nidulans and Escherichia coli cells]

The influence of heavy metal ions (Ag+, Cu2+, Cd2+, Pb2+, Mn2+, Zn2+, Gd3+, 1 microM-1 mM) on Anacystis nidulans and Escherichia coli cells has been studied by means of electrophoresis and electro-orientation spectroscopy methods. It has been shown that changes of cell electrophoretic mobility (EM) and low-frequency (20 Hz) electro-orientation effect (EOE) observed with the increase of metal cation concentration characterize the adsorption of these ions on surface layers of cell envelopes. The degree and the character of these changes depend on cation valency and the initial value of cell EM. At the same time different changes of EM and EOE as a result of the multivalent cation adsorption allows to conclude that in that case the anisotropy of the cell surface increases. Cell damages were determined by changes in high-frequency EOE of cells which indicated the disturbance of barrier properties of their cytoplasmic membrane. Toxic effects of Ag+, Cu2+, Cd2+ ions on cells of both species and of Pb2+ on E.coli cells were observed. By toxic effects on the cytoplasmic membrane these ions could be placed in the order: for A.nidulans cells--Ag+ greater than Cu2+ greater than Cd2+; for E.coli cells Ag+ greater than Cu2+ greater than Cd2+ greater than Pb2+. Higher toxicity of heavy metals on E.coli cells seems to be connected with the more negative charge of deep layers of the cell surface.     

Inhibition of phorbol ester binding and protein kinase C activity by heavy metals

Other laboratories have reported biphasic effects of heavy metals on protein kinase C activity: stimulation followed by inhibition at higher concentrations. We demonstrate that these earlier findings most likely resulted from a combination of the effect of the heavy metals to liberate Ca2+ from Ca2+-EGTA buffer systems and the direct inhibitory effects of the metals on protein kinase C. Simulations of such interactions substantiate this conclusion. When soluble protein kinase C is prepared without the addition of Ca2+ or chelator, heavy metals (Cd2+, Cu2+, Hg2+, Zn2+, in the 10 microM range) inhibit the activity of, and the binding of regulatory ligands to, protein kinase C. Heavy metals inhibit the extent of [3H]phorbol dibutyrate binding without affecting the affinity of the interaction, an inhibition that is not surmounted by excess phospholipid. Heavy metals also inhibit the phospholipid-dependent catalytic activity of protein kinase C in a manner that excess phosphatidylserine can overcome. The inhibition of enzyme activity by heavy metals cannot be surmounted by excess Ca2+ or Mg2+. The inhibitory effects of heavy metals are not confined to protein kinase C. Heavy metals also inhibit cyclic AMP binding to cyclic AMP-dependent protein kinase and the catalytic activity of that kinase, but in a distinctly different pattern.     

重金属离子对猪红细胞膜Ca~(2+)-ATP酶的作用

猪红细胞膜Ca~(2+)-ATP酶是一种钙调蛋白(CaM)依赖酶,其活力又依赖巯基的完整性。实验应用Ca~(2+)-ATP酶这一模型体系观察到重金属离子,Pb~(2+)、Cd~(2+)和Hg~(2+)都能替代Ca~(2+),激活CaM,从而激活Ca~(2+)-ATP酶;其最大刺激活力分别为85％、80％和30％,半刺激浓度分别为32、27和0.7μmol/L。当三种重金属离子的浓度增加时,则与Ca~(2+)-ATP酶的巯基结合,抑制酶的活力,Pb2~(2+)、Cd~(2+)和Hg~(2+)的半抑制浓度分别为370、440和2μmol/L。抑制作用为渐进性过程,而刺激作用为即时效应。抑制作用可为巯基化物,特别是二巯基化物所逆转。研究结果提示,CaM可能是重金属中毒最初作用的靶分子,而重金属中毒不仅使CaM“开关”失灵,还可能导致细胞内Ca~(2+)的调节全面失控。     

龙胜县丘陵山区土壤与罗汉果重金属含量及潜在生态危害评价——以宝赠村为例

桂林市龙胜县作为罗汉果的三大主产区之一,种植区土壤重金属含量及罗汉果质量影响到该区罗汉果产业的健康发展.为探索龙胜县丘陵山区典型贫困村罗汉果园的安全性,该文研究了宝赠村典型罗汉果园土壤及罗汉果果实中砷、铜、锌、铅、镉、铬、汞7种重金属含量,并采用Hankanson指数法分析了其潜在生态风险.结果表明:(1)龙胜丘陵山区...     

Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.     

Interference of heavy metal cations with fluorescent Ca2+ probes does not affect Ca2+ measurements in living cells

In studies about the effects of heavy metals on intracellular Ca2+, the use of fluorescent probes is debated, as metal cations are known to affect the probe signal. In this study, spectrofluorimetric experiments in free solution, using Fluo-3 and Fura-2, showed that Zn2+ and Cd2+ enhanced the probe signal, Cu2+ quenched it, and Hg2+ had no effect. Addition of GSH prevented most of these effects, suggesting the occurrence of a similar protective role in living cells. Digital imaging of living mussel haemocytes loaded with Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 microM Zn2+ had no effect. In particular, Cd2+ produced the strongest probe signal rise in free solution, but the lowest fluorescence increase in cells. Probe calibration yielded [Ca2+]i values characteristic of resting levels in control and Zn2+-exposed cells, and, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy metals in living cells are completely different from those obtained in free solution, indicating that fluorescent probes can be a suitable tool to record the effects of heavy metals on [Ca2+]i.     

Growth suppression of Leydig TM3 cells mediated by aryl hydrocarbon receptor

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental toxicity in reproductive organs. To elucidate the function of AhR, we generated stable transformants of TM3 cells overexpressing wild-type aryl hydrocarbon receptor (AhR) or its mutants which carried mutations in nuclear localization signal or nuclear export signal. In the presence of 3-methylcholanthrene (MC), proliferation of the cells transfected with wild-type AhR was completely suppressed, whereas cells expressing AhR mutants proliferated in a manner equivalent to control TM3 cells, suggesting AhR-dependent growth inhibition. The suppression was associated with up-regulation of cyclin-dependent kinase inhibitor p21^Cip1, which was abolished by pretreatment with actinomycin D. A p38 MAPK specific inhibitor, SB203580, blocked the increase of p21^Cip1 mRNA in response to MC. Treatment with indigo, another AhR ligand, failed to increase of p21^Cip1 mRNA, although up-regulation of mRNA for CYP1A1 was observed. These data suggest AhR in Leydig cells mediates growth inhibition by inducing p21^Cip1.     

Phe356 in the yeast Ca2+ channel component Mid1 is a key residue for viability after exposure to alpha-factor

The yeast Mid1 protein is an integral membrane protein required for the viability of differentiated cells and Ca2+ influx induced by mating pheromone. Our previous study has identified a loss-of-function mutation, F356S. The F356S mutant is completely unable to maintain viability, but still has Ca2+ accumulation activity near the wild-type level. Here we further examined in detail the F356S mutation to unravel the function of Phe356. After exposure to the pheromone, the F356S mutant was not fully rescued by high extracellular Ca2+, like the mid1 null mutant, suggesting that Phe356 and Mid1 itself are also required for viability maintenance mechanism that does not involve Ca2+ signalling. Substitutions of hydrophilic amino acids for Phe356 caused lethality and low Ca2+ accumulation, but those of hydrophobic amino acids did not. Substitutions of small amino acids for Phe356 caused a significantly reduced viability, but did not affect Ca2+ accumulation. We suggest that the hydrophobicity of the Phe356 residue is important for both viability maintenance and Ca2+ uptake, and that its size for viability maintenance.     

Single nucleotide polymorphism analysis and functional characterization of the human Ah receptor (AhR) gene promoter

The aryl hydrocarbon receptor (AhR) mediates biological and toxicological actions of e.g., halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Although much is known about the biochemical and molecular mechanisms of AhR action, little is known about the control of the expression of the AhR gene itself. Therefore, we aimed at the identification and characterization of regions important for constitutive AhR gene expression. First, we screened 2.6 kb of the 5(')-flanking region of the AhR gene in 91 healthy Caucasian volunteers for naturally occurring genetic variants. Seven variants were detected. However, they do not seem to influence AhR gene expression in lymphocytes. Using a 2.7 kb AhR promoter luciferase reporter gene construct and various deletion constructs, a putative regulatory region was identified and characterized further by electrophoretic mobility shift assays and site-directed mutagenesis. These investigations were confirmed by cotransfection experiments in Drosophila SL2 cells. The obtained results prove an involvement of Sp1 in AhR gene regulation.     

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.     

Cd2+胁迫对银芽柳PSⅡ叶绿素荧光光响应曲线的影响

以盆栽银芽柳为材料,利用MINI-IMAGING-PAM荧光成像测定系统,研究了Cd²⁺胁迫下叶片叶绿素荧光参数的变化及其光响应曲线。结果表明,初始荧光Fo与最大荧光Fm随着Cd²⁺浓度的增大而呈现先升后降的趋势,Fo与Fm在200 mg/LCd²⁺处理4周时达到最高值,400 mg/LCd²⁺处理则显著下降;PSⅡ最大光化学效率(Fv/Fm)与PSⅡ潜在光化学效率(Fv/Fo)显著受 Cd²⁺胁迫抑制,但随Cd²⁺浓度的增加呈先降后升的变化趋势。Cd²⁺胁迫下各叶绿素荧光参数的光响应结果表明,PSⅡ实际光量子效率Y(Ⅱ)、荧光淬灭系数(qP)随光化光强度的增加呈下降趋势,而同光强下高浓度Cd²⁺ 使Y(Ⅱ)与(qP) 显著降低;PSⅡ调节性能量耗散的量子产额Y(NPQ)、非光化学淬灭系数(qN)与表观电子传递速率(ETR)则随着光强增加呈上升趋势,同光强下高浓度Cd²⁺处理显著提高Y(NPQ)、qN 与ETR。Cd²⁺胁迫下,PSⅡ非调节性能量耗散的量子产额Y(NO)稳定在较低水平,同光强下Y(NO)随Cd²⁺浓度增加略有提高。说明,银芽柳通过调节PSⅡ反应中心开放程度与活性,对Cd²⁺胁迫表现出较强的耐性,高浓度Cd²⁺胁迫导致PSⅡ反应中心关闭或不可逆失活,表现出光抑制。     

A store-operated calcium channel in Drosophila S2 cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ > Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.     

Inhibition of rabbit reticulocyte lysate protein synthesis by heavy metal ions involves the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.