Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.     

Native LDL‐induced oxidative stress in human proximal tubular cells: multiple players involved

Dyslipidemia is a well‐established condition proved to accelerate the progression of chronic kidney disease leading to tubulo‐interstitial injury. However, the molecular aspects of the dyslipidemia‐induced renal damage have not been fully clarified and in particular the role played by low‐density lipoproteins (LDLs). This study aimed to examine the effects of native non‐oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS‐induced ROS release mechanism. The LDL‐dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mtΔΨ, increased Ca²⁺ uptake and loss of cytochrome c. All the above LDL‐induced effects were completely abrogated by chelating extracellular Ca²⁺ as well as by inhibition of the Ca²⁺‐activated cytoplas‐mic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL‐induced intracellular redox unbalance is triggered by a Ca²⁺ inward flux‐dependent commencement of cPLA2 followed by activation of a lipid‐ and ROS‐based cross‐talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox‐ and Ca²⁺‐dependent dysfunctions leading to cell‐harming conditions. These findings may help to clarify the mechanism of dyslipidemia‐induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases.     

Glucose‐ and mannose‐induced stomatal closure is mediated by ROS production,Ca2+ and water channel in Vicia faba

Sugars act as vital signaling molecules that regulate plant growth, development and stress responses. However, the effects of sugars on stomatal movement have been unclear. In our study, we explored the effects of monosaccharides such as glucose and mannose on stomatal aperture. Here, we demonstrate that glucose and mannose trigger stomatal closure in a dose‐ and time‐dependent manner in epidermal peels of broad bean (Vicia faba). Pharmacological studies revealed that glucose‐ and mannose‐induced stomatal closure was almost completely inhibited by two reactive oxygen species (ROS) scavengers, catalase (CAT) and reduced glutathione (GSH), was significantly abolished by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), whereas they were hardly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM). Furthermore, glucose‐ and mannose‐induced stomatal closure was strongly inhibited by a Ca²⁺ channel blocker, LaCl₃, a Ca²⁺ chelator, ethyleneglycol‐bis(beta‐aminoethylether)‐N,N'‐tetraacetic acid (EGTA) and two water channel blockers, HgCl₂ and dimethyl sulfoxide (DMSO); whereas the inhibitory effects of the water channel blockers were essentially abolished by the reversing agent β‐mercaptoethanol (β‐ME). These results suggest that ROS production mainly via NADPH oxidases, Ca²⁺ and water channels are involved in glucose‐ and mannose‐induced stomatal closure.     

Plasma membrane Ca²+ transporters mediate virus-induced acquired resistance to oxidative stress

This paper reports the phenomenon of acquired cross‐tolerance to oxidative stress in plants and investigates the activity of specific Ca²⁺ transport systems mediating this phenomenon. Nicotiana benthamiana plants were infected with Potato virus X (PVX) and exposed to oxidative [either ultraviolet (UV‐C) or H₂O₂] stress. Plant adaptive responses were assessed by the combined application of a range of electrophysiological (non‐invasive microelectrode ion flux measurements), biochemical (Ca²⁺‐ and H⁺‐ATPase activity), imaging (fluorescence lifetime imaging measurements of changes in intracellular Ca²⁺ concentrations), pharmacological and cytological transmission electrone microscopy techniques. Virus‐infected plants had a better ability to control UV‐induced elevations in cytosolic‐free Ca²⁺ and prevent structural and functional damage of chloroplasts. Taken together, our results suggest a high degree of crosstalk between UV and pathogen‐induced oxidative stresses, and highlight the crucial role of Ca²⁺ efflux systems in acquired resistance to oxidative stress in plants.     

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca²⁺ and Na⁺ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac‐specific Sirt1 knockout (Sirt1^?/?). Sirt1^flox/flox mice were served as control. Sirt1^?/? mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca²⁺ transient amplitudes and sarcoplasmic reticulum (SR) Ca²⁺ stores, and increased SR Ca²⁺ leak were shown in the Sirt1^?/? mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca²⁺ current (I_{Ca, L}) was smaller in Sirt1^?/? myocytes, reverse‐mode Na⁺/Ca²⁺ exchanger (NCX) current was larger compared with those in control myocytes. Late Na⁺ current (I_{Na, L}) was enhanced in the Sirt1^?/? mice, alongside with elevated cytosolic Na⁺ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1^?/? mice. Sirt1^?/? cardiomyocytes showed down‐regulation of L‐type Ca²⁺ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), but up‐regulation of Ca²⁺/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca²⁺ and Na⁺ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.     

Chilling‐induced Ca2+ overload enhances production of active oxygen species in maize (Zea mays L.) cultured cells: the effect of abscisic acid treatment

Chilling (4 °C) induced a prolonged high level of intracellular Ca²⁺ (Ca²⁺ overload) and lipid peroxidation in maize (Zea mays L. cv Black Mexican Sweet) cultured cells. However, such Ca²⁺ overload and enhanced lipid peroxidation were not seen in abscisic acid (ABA)‐treated cells, which had an improved chilling tolerance. A Ca²⁺ ionophore, A23187, caused Ca²⁺ overload in both ABA‐treated maize cells and the untreated control, whereas an enhanced lipid peroxidation was detected only in the control. The high level of active oxygen species (AOS) in the control during chilling at 4 °C could be reduced by the presence of lanthanum (La³⁺), a Ca²⁺ channel blocker, in the medium. Moreover, both the A23187‐induced lipid peroxidation and AOS production in the control could be reduced by extracellular EGTA, a Ca²⁺ chelator. Laser‐scanning confocal microscopy revealed that mitochondria were one of the major AOS sources under chilling and during A23187 treatment. In vitro assays showed that superoxide production in isolated maize mitochondria was enhanced by the presence of Ca²⁺. Findings suggest that chilling‐induced Ca²⁺ influx in the control triggers a marked generation of AOS, which in turn results in the enhanced lipid peroxidation. The ability of ABA‐treated cells to avoid the chilling‐induced Ca²⁺ influx may serve as a mechanism that prevents the chilling‐induced oxidative stress and thus results in less chilling injury.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

The nuclear transporter SAD2 plays a role in calcium‐ and H2O2‐mediated cell death in Arabidopsis

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca²⁺ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2‐5, which harbours a T‐DNA insertion in the 18^th exon of the importin beta‐like gene, SAD2. The H₂O₂‐induced increase in the [Ca²⁺]_cyt of the sad2‐5 mutant was greater than that of the wild type, and the sad2‐5 mutant showed clear cell death phenotypes and abnormal H₂O₂ accumulation under fumonisin‐B1 (FB1) treatment. CaCl₂ could enhance the FB1‐induced cell death of the sad2‐5 mutant, whereas lanthanum chloride (LaCl₃), a broad‐spectrum calcium channel blocker, could restore the FB1‐induced PCD phenotype of sad2‐5. The sad2‐5 fbr11‐1 double mutant exhibited the same FB1‐insensitive phenotype as fbr11‐1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium‐ and ROS‐mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.     

Inhibitors of Na+/Ca2+ exchanger prevent oxidant-induced intracellular Ca2+ increase and apoptosis in a human hepatoma cell line

Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca²⁺ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca²⁺ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2′,7′-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N′-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca²⁺ concentration, which was completely prevented by the antioxidants. An intracellular Ca²⁺ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca²⁺ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca²⁺ increase and induction of apoptosis were significantly inhibited by an extracellular Ca²⁺ chelator or Na⁺/Ca²⁺ exchanger blockers (bepridil and benzamil), whereas neither Ca²⁺ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca²⁺ through the activation of reverse mode of Na⁺/Ca²⁺ exchanger. However, tBOOH decreased intracellular Na⁺ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca²⁺ concentration by direct activation of the reverse mode of Na⁺/Ca>²⁺ exchanger, rather than indirect elevation of intracellular Na⁺ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca²⁺ may mediate this action of tBOOH. These results further suggest that Na⁺/Ca²⁺ exchanger may be a target for the management of oxidative hepatotoxicity.     

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca²⁺] ([Ca²⁺]_i), and also on mechanisms that are independent of [Ca²⁺]_i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca²⁺]_i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca²⁺]_i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca²⁺ sensitivity of anion and inward‐rectifying K⁺ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca²⁺]_i elevations and clamping [Ca²⁺]_i to resting levels. The absence of [Ca²⁺]_i elevations was confirmed by ratiometric [Ca²⁺]_i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca²⁺]_i elevations above the physiological resting [Ca²⁺]_i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca²⁺]_i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca²⁺]_i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca²⁺ to activate S‐type anion channels and down‐regulate inward‐rectifying K⁺ channels, providing strong evidence for a Ca²⁺ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca²⁺]_i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca²⁺]_i sensitivity of stomatal closure mechanisms.     

Mitochondrial permeability transition pore is involved in oxidative burst and NETosis of human neutrophils

Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune and inflammatory diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating aberrant NET release. The initiation of NETosis after the recognition of pathogens by specific receptors is mediated by an increase in intracellular Ca²⁺ concentration, therefore, the use of Ca²⁺ ionophore A23187 can be considered a semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by NADPH oxidase, however, NETosis induced by Ca²⁺ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS).Using the mitochondria-targeted antioxidant SkQ1 and specific inhibitors of NADPH oxidase, we showed that both sources of ROS, mitochondria and NADPH oxidase, are involved in NETosis induced by A23187 in human neutrophils. In support of the critical role of mtROS, SkQ1-sensitive NETosis was demonstrated to be induced by A23187 in neutrophils from patients with chronic granulomatous disease (CGD). We assume that Ca²⁺-triggered mtROS production contributes to NETosis either directly (CGD neutrophils) or by stimulating NADPH oxidase. The opening of the mitochondrial permeability transition pore (mPTP) in neutrophils treated by A23187 was revealed using the electron transmission microscopy as a swelling of the mitochondrial matrix. Using specific inhibitors, we demonstrated that the mPTP is involved in mtROS production, NETosis, and the oxidative burst induced by A23187.     

Ca2+ Signaling and Cell Death Induced by Protriptyline in HepG2 Human Hepatoma Cells

The effect of protriptyline on Ca²⁺ physiology in human hepatoma is unclear. This study explored the effect of protriptyline on [Ca²⁺]_i and cytotoxicity in HepG2 human hepatoma cells. Protriptyline (50–150 μM) evoked [Ca²⁺]_i rises. The Ca²⁺ entry was inhibited by removal of Ca²⁺. Protriptyline‐induced Ca²⁺ entry was confirmed by Mn²⁺‐induced quench of fura‐2 fluorescence. Except nifedipine, econazole, SKF96365, GF109203X, and phorbol 12‐myristate 13 acetate did not inhibit Ca²⁺ entry. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 40% of protriptyline‐induced response. Treatment with protriptyline abolished BHQ‐induced response. Inhibition of phospholipase C (PLC) suppressed protriptyline‐evoked response by 70%. At 20–40 μM, protriptyline killed cells which was not reversed by the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester (BAPTA/AM). Together, in HepG2 cells, protriptyline induced [Ca²⁺]_i rises that involved Ca²⁺ entry through nifedipine‐sensitive Ca²⁺ channels and PLC‐dependent Ca²⁺ release from endoplasmic reticulum. Protriptyline induced Ca²⁺‐independent cell death.     

Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.     

Spermidine oxidase‐derived H2O2 regulates pollen plasma membrane hyperpolarization‐activated Ca2+‐permeable channels and pollen tube growth

Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca²⁺ concentration that accompanies pollen tube growth. Using the whole‐cell patch clamp and outside‐out single‐channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization‐activated Ca²⁺ current: the addition of Spd cannot induce the channel open probability increase in excised outside‐out patches, indicating that the effect of Spd in the induction of Ca²⁺ currents is exerted via a second messenger. This messenger is hydrogen peroxide (H₂O₂), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization‐activated Ca²⁺‐permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca²⁺‐permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal‐encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca²⁺‐permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca²⁺ influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd‐derived H₂O₂ signals Ca²⁺ influx, thereby regulating pollen tube growth.     

Melatonin protects rat cerebellar granule cells against electromagnetic field‐induced increases in Na+ currents through intracellular Ca2+ release

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low‐frequency electromagnetic field (ELF‐EMF)‐induced Na_v activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Na_v current (I_Na) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced I_Na increase. This inhibitory effect of MT is mimicked by an MT₂ receptor agonist and was eliminated by an MT₂ receptor antagonist. The Na_v channel steady‐state activation curve was significantly shifted towards hyperpolarization by ELF‐EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF‐EMF. ELF‐EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK‐7 did not reduce the ELF‐EMF‐induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF‐EMF‐induced Na_v activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca²⁺ level, but it significantly elevated the intracellular Ca²⁺ level evoked by the high K⁺ stimulation in cerebellar GC that were either exposed or not exposed to ELF‐EMF. In the presence of ruthenium red, a ryanodine‐sensitive receptor blocker, the MT‐induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal I_Na that result from ELF‐EMF exposure through Ca²⁺ influx‐induced Ca²⁺ release.     

Indole-3-acetic acid-induced oxidative burst and an increase in cytosolic calcium ion concentration in rice suspension culture

Indole-3-acetic acid (IAA) is the major natural auxin involved in the regulation of a variety of growth and developmental processes such as division, elongation, and polarity determination in growing plant cells. It has been shown that dividing and/or elongating plant cells accompanies the generation of reactive oxygen species (ROS) and a number of reports have suggested that hormonal actions can be mediated by ROS through ROS-mediated opening of ion channels. Here, we surveyed the link between the action of IAA, oxidative burst, and calcium channel activation in a transgenic cells of rice expressing aequorin in the cytosol. Application of IAA to the cells induced a rapid and transient generation of superoxide which was followed by a transient increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). The IAA-induced [Ca²⁺]_c elevation was inhibited by Ca²⁺ channel blockers and a Ca²⁺ chelator. Furthermore, ROS scavengers effectively blocked the action of IAA on [Ca²⁺]_c elevation.     

Advanced oxidation protein products induce pre‐osteoblast apoptosis through a nicotinamide adenine dinucleotide phosphate oxidase‐dependent,mitogen‐activated protein kinases‐mediated intrinsic apoptosis pathway

Osteoblast apoptosis contributes to age‐related bone loss. Advanced oxidation protein products (AOPPs) are recognized as the markers of oxidative stress and potent inducers of apoptosis. We have demonstrated that AOPP accumulation was correlated with age‐related bone loss. However, the effect of AOPPs on the osteoblast apoptosis still remains unknown. Exposure of osteoblastic MC3T3‐E1 cells to AOPPs caused the excessive generation of reactive oxygen species (ROS) by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Increased ROS induced phosphorylation of mitogen‐activated protein kinases (MAPKs), which subsequently triggered intrinsic apoptosis pathway by inducing mitochondrial dysfunction, endoplasmic reticulum stress, and Ca²⁺ overload and eventually leads to apoptosis. Chronic AOPP loading in aged Sprague‐Dawley rats induced osteoblast apoptosis and activated NADPH oxidase signaling cascade, in combination with accelerated bone loss and deteriorated bone microstructure. Our study suggests that AOPPs induce osteoblast apoptosis by the NADPH oxidase‐dependent, MAPK‐mediated intrinsic apoptosis pathway.     

灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。     

Electrophysiological localization of blue light sensory sites in etiolated dicotyledon seedlings

Changes induced by blue light (BL) in ion fluxes and pH in etiolated seedlings of dicotyledons (Cucumis sativus L., Phaseolus lunatus L., Pisum sativum L.) were studied using a non‐invasive ion‐selective microelectrode technique (MIFE). BL induced significant Ca²⁺ uptake within the first 5 min after onset in upper parts of the whole cucumber seedling. To locate the sites of the BL perception, studies on excised segments were performed. The data obtained demonstrated that the observed Ca²⁺ uptake is associated with the very top of the hypocotyl hook of cucumber and butter bean, and with the whole epicotyl of pea, although the response was stronger for the upper parts of the epicotyl. Addition of Gd³⁺ and La³⁺ into the bathing solution completely prevented Ca²⁺ influx, implicating the involvement of Ca²⁺ channels in the regulation of Ca²⁺ transport during BL exposure. BL‐induced changes in H+ and Ca²⁺ fluxes were not likely to be linked to the one mechanism, as they were separated spatially and pharmacologically.