Mitochondrial Ca2+ influx targets cardiolipin to disintegrate respiratory chain complex II for cell death induction

Massive Ca²⁺ influx into mitochondria is critically involved in cell death induction but it is unknown how this activates the organelle for cell destruction. Using multiple approaches including subcellular fractionation, FRET in intact cells, and in vitro reconstitutions, we show that mitochondrial Ca²⁺ influx prompts complex II of the respiratory chain to disintegrate, thereby releasing an enzymatically competent sub-complex that generates excessive reactive oxygen species (ROS) for cell death induction. This Ca²⁺-dependent dissociation of complex II is also observed in model membrane systems, but not when cardiolipin is replaced with a lipid devoid of Ca²⁺ binding. Cardiolipin is known to associate with complex II and upon Ca²⁺ binding coalesces into separate homotypic clusters. When complex II is deprived of this lipid, it disintegrates for ROS formation and cell death. Our results reveal Ca²⁺ binding to cardiolipin for complex II disintegration as a pivotal step for oxidative stress and cell death induction.Cellular calcium ion (Ca²⁺) overload is known to be of fundamental importance in pathological cell death induction for instance during brain ischemia, ischemia-reperfusion of the heart, and excitotoxicity of neurons.¹ Upon entering the cytosol from the extracellular space, Ca²⁺ ions accumulate in mitochondria at very high levels. An alternative route into mitochondria, observed during many scenarios of cell death, as well as when therapeutically induced by anticancer agents, is through Ca²⁺ release from the ER. After crossing the ER-mitochondrial junction, the ion is taken up by the mitochondrial calcium uniporter.^{2, 3} The close apposition of the two organelles ensures that a very high Ca²⁺ concentration can be reached in mitochondria.⁴ The direct target of mitochondrial Ca²⁺ influx for cell death induction, however, is unknown.Cells deficient in complex II of the respiratory chain become resistant to many cell death signals.⁵ The ability of this complex to produce deleterious amounts of reactive oxygen species (ROS) has been recognized.^{6, 7} Initial in vitro experiments using blue native gels indicated that during cell death, the sub-complex SDHA/SDHB, which remains enzymatically active,⁸ is specifically released from the membrane-anchoring SDHC and SDHD complex II subunits.⁹ It can then remove electrons from the substrate succinate and transfer them to molecular oxygen to generate ROS for cell death induction.^{5, 9}The principal lipid in the inner mitochondrial membrane that harbors the components of the respiratory chain, including complex II, is the diphosphatidylglycerol cardiolipin. This lipid is known to be involved in cell death, although its effects have been connected mostly with cellular sites different from its most prominent residence.^{10, 11, 12}In this study, we investigated whether excessive Ca²⁺ influx into mitochondria can affect on the integrity of complex II and activate this complex for cell death.     

Reduced mitochondrial Ca2+ transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase

Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear- and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A>G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca²⁺ homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A>G cells. This is due to a specific downregulation of mitochondrial Ca²⁺ uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca²⁺ homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient.Mitochondrial disorders include a wide range of pathological conditions characterized by defects in organelle homoeostasis and energy metabolism, in particular in the electron transport chain (ETC) complexes. They are mostly caused by mutations in nuclear- or mtDNA-encoded genes of the respiratory chain complexes leading to a variety of clinical manifestations, ranging from lesions in specific tissues, such as in Leber''s hereditary optic neuropathy, to complex multisystem syndromes, such as myoclonic epilepsy with ragged-red fibers, Leigh syndrome or the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS).^{1, 2} Despite the detailed knowledge of the molecular defects in these diseases, their pathogenesis remains poorly understood. The heterogeneity of signs and symptoms depends on the diversity of the genetic background and on patient-specific compensatory mechanisms. Several studies investigated the consequences of nuclear DNA mutations on intracellular organelle physiology and Ca²⁺ homeostasis.^{3, 4} Here we analyzed a cohort of patients with mutations in the mtDNA-encoded ND5 subunit of NADH dehydrogenase in order to correlate the clinical phenotype with relevant intracellular parameters involved in mitochondrial physiology, such as the rate of autophagy and mitophagy fluxes, mitochondrial Ca²⁺ dynamics, mitochondrial membrane potential and their functional relationship.Mitochondrial Ca²⁺ is a key regulator of organelle physiology, and impairment of cation homeostasis is a general feature of many pathological conditions, including mitochondrial diseases.⁵ In addition, Ca²⁺ uptake in this organelle has recently been demonstrated to be a fundamental regulator of autophagy.^{6, 7} Autophagy is involved in physiological organelle turnover and in the removal of damaged or non-functional mitochondria by autophagy (called ‘mitophagy'')^{8, 9, 10, 11} and is critical for organelle quality control. Given the pivotal role of mitochondrial Ca²⁺ in the adaptation of adenosine triphosphate (ATP) production to cellular energy demand, the recent identification of the channel responsible for Ca²⁺ entry into the organelle, the mitochondrial Ca²⁺ uniporter (MCU), is instrumental for the understanding of the regulation of mitochondrial Ca²⁺ transport in both physiological and pathological conditions. MCU was identified in 2011,^{12, 13} and in the following years, molecular insight on its complex regulatory mechanism was obtained. The pore region is composed of MCU, its isoform MCUb¹⁴ and essential MCU regulator (EMRE).¹⁵ The channel is gated by the Ca²⁺-sensitive proteins mitochondrial Ca²⁺ uptake 1 (MICU1) and MICU2^{16, 17, 18, 19} and further regulated by the SLC25A23 protein.²⁰ As to its cellular function, mitochondrial Ca²⁺ has been shown to stimulate ATP production by positive regulation of three key dehydrogenases of the tricarboxylic acid cycle²¹ and of the ETC.²² In parallel, unregulated and sustained organelle Ca²⁺ overload can also lead to the opening of the mitochondrial permeability transition pore,^{23, 24} with consequent dissipation of mitochondrial membrane potential (ΔΨ_mt), release of caspase cofactors and activation of the apoptotic cascade.⁵ Despite the significant molecular understanding of all these cellular processes, their role in the pathogenesis of mitochondrial diseases is still poorly understood. Here we investigated the interplay of these pathways and the possibility of their contribution to determine the severity of the pathology in a cellular model consisting of fibroblasts from patients carrying mutations in the mitochondrial ND5 gene.     

TRPC1-mediated Ca2+ entry is essential for the regulation of hypoxia and nutrient depletion-dependent autophagy

Autophagy is a cellular catabolic process needed for the degradation and recycling of protein aggregates and damaged organelles. Although Ca²⁺ is suggested to have an important role in cell survival, the ion channel(s) involved in autophagy have not been identified. Here we demonstrate that increase in intracellular Ca²⁺ via transient receptor potential canonical channel-1 (TRPC1) regulates autophagy, thereby preventing cell death in two morphologically distinct cells lines. The addition of DMOG or DFO, a cell permeable hypoxia-mimetic agents, or serum starvation, induces autophagy in both epithelial and neuronal cells. The induction of autophagy increases Ca²⁺ entry via the TRPC1 channel, which was inhibited by the addition of 2APB and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Importantly, TRPC1-mediated Ca²⁺ entry resulted in increased expression of autophagic markers that prevented cell death. Furthermore, hypoxia-mediated autophagy also increased TRPC1, but not STIM1 or Orai1, expression. Silencing of TRPC1 or inhibition of autophagy by 3-methyladenine, but not TRPC3, attenuated hypoxia-induced increase in intracellular Ca²⁺ influx, decreased autophagy, and increased cell death. Furthermore, the primary salivary gland cells isolated from mice exposed to hypoxic conditions also showed increased expression of TRPC1 as well as increase in Ca²⁺ entry along with increased expression of autophagic markers. Altogether, we provide evidence for the involvement of Ca²⁺ influx via TRPC1 in regulating autophagy to protect against cell death.Autophagy is a cellular process responsible for the delivery of proteins or organelles to lysosomes for its degradation. Autophagy participates not only in maintaining cellular homeostasis, but also promotes cell survival during cellular stress situations.^{1, 2} The stress conditions including nutrient starvation, hypoxia conditions, invading microbes, and tumor formation, have been shown to induce autophagy that allows cell survival in these stressful or pathological situations.¹ In addition, autophagy also recycles existing cytoplasmic components to generate the molecules that are required to sustain the most vital cellular functions.³ Till date, three forms of autophagy have been identified, which are designated as chaperone-mediated autophagy, microautophagy, and macroautophagy.⁴ Although the precise mechanism as to how autophagy is initiated is not well understood, many of the genes first identified in yeast that are involved in autophagy have orthologs in other eukaryotes including human homologs.^{5, 6} The presence of similar genes in all organisms suggests that autophagy might be a phenomenon that is evolutionally conserved that is essential for cell survival. In addition, since autophagy delivers a fresh pool of amino acids and other essential molecules to the cell, initiation of autophagy is highly beneficial particularly during nutritional stress situations or tissue remodeling during development and embryogenesis.⁶ Consequently, impaired or altered autophagy is often implicated in several pathologies, like neurodegenerative disorders and cancer,^{7, 8, 9} which again highlight its importance.Ca²⁺ has a vital role in the regulation of a large number of cellular processes such as cell proliferation, survival, migration, invasion, motility, and apoptosis.^{10, 11} To perform functions on such a broad spectrum, the cells have evolved multiple mechanisms regulating cellular Ca²⁺ levels, mainly by regulating the function of various Ca²⁺ channels present in different locations. Mitochondrial, ER, lysosomal, and cytosolic Ca²⁺ levels are regulated by Ca²⁺ permeable ion channels localized either on the membranes of the intracellular organelles or on the plasma membrane.¹⁰ The Ca²⁺ permeable channels, including families of TRPCs, Orais, voltage-gated, two-pore, mitochondrial Ca²⁺ uniporter, IP₃, and ryanodine receptors have all been identified to contribute towards changes in intracellular Ca²⁺ ([Ca²⁺]_i).^{10, 12, 13, 14} Channels of the TRPCs and Orai families have been related to several Ca²⁺-dependent physiological processes in various cell types, ranging from cell proliferation to contractility, to apoptosis under both physiological and pathological conditions.¹² Moreover, it has been suggested that intracellular Ca²⁺ is one of the key regulators of autophagy;¹⁵ however, the possible role of Ca²⁺ in autophagy is still inconclusive. Many reports also suggest that Ca²⁺ inhibits autophagy,^{16, 17, 18} whereas others have indicated a stimulatory role for Ca²⁺ towards autophagy.^{19, 20, 21} Furthermore, the identity of the major Ca²⁺ channel(s) involved in autophagy is not known. Members of the TRPC family have been suggested as mediators of Ca²⁺ entry into cells. Activation of the G-protein (G_q/11–PLC pathway) leads to the generation of second messenger IP₃.^{10, 22} IP₃ binds to the IP₃R, which initiates Ca²⁺ release from the ER stores, thereby facilitating stromal interacting molecule-1 (STIM1) to rearrange and activate Ca²⁺ entry via the store-operated channels.²² Two families of proteins (TRPCs and Orais) have been identified as potential candidates for SOC-mediated Ca²⁺ entry.^{12, 22} However, their role in autophagy has not yet been determined. Thus, here we investigated the role of Ca²⁺ entry channels (TRPCs and Orais) in autophagy and show that both hypoxia-mimetic and nutrient depression induces autophagy in two different cell lines. Furthermore, our data indicates that autophagy was dependent on TRPC1-mediated increase in intracellular Ca²⁺ levels, suggesting that TRPC1 has an important role in regulating autophagy and inhibiting cell death.     

A Signaling Pathway Linking Nitric Oxide Production to Heterotrimeric G Protein and Hydrogen Peroxide Regulates Extracellular Calmodulin Induction of Stomatal Closure in Arabidopsis

Extracellular calmodulin (ExtCaM) regulates stomatal movement by eliciting a cascade of intracellular signaling events including heterotrimeric G protein, hydrogen peroxide (H₂O₂), and Ca²⁺. However, the ExtCaM-mediated guard cell signaling pathway remains poorly understood. In this report, we show that Arabidopsis (Arabidopsis thaliana) NITRIC OXIDE ASSOCIATED1 (AtNOA1)-dependent nitric oxide (NO) accumulation plays a crucial role in ExtCaM-induced stomatal closure. ExtCaM triggered a significant increase in NO levels associated with stomatal closure in the wild type, but both effects were abolished in the Atnoa1 mutant. Furthermore, we found that ExtCaM-mediated NO generation is regulated by GPA1, the Gα-subunit of heterotrimeric G protein. The ExtCaM-dependent NO accumulation was nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGα). In addition, cGα Atnoa1 and gpa1-2 Atnoa1 double mutants exhibited a similar response as did Atnoa1. The defect in gpa1 was rescued by overexpression of AtNOA1. Finally, we demonstrated that G protein activation of NO production depends on H₂O₂. Reduced H₂O₂ levels in guard cells blocked the stomatal response of cGα lines, whereas exogenously applied H₂O₂ rescued the defect in ExtCaM-mediated stomatal closure in gpa1 mutants. Moreover, the atrbohD/F mutant, which lacks the NADPH oxidase activity in guard cells, had impaired NO generation in response to ExtCaM, and H₂O₂-induced stomatal closure and NO accumulation were greatly impaired in Atnoa1. These findings have established a signaling pathway leading to ExtCaM-induced stomatal closure, which involves GPA1-dependent activation of H₂O₂ production and subsequent AtNOA1-dependent NO accumulation.Plant guard cells control opening and closure of the stomata in response to phytohormones (e.g. abscisic acid [ABA]) and various environmental signals such as light and temperature, thereby regulating gas exchange for photosynthesis and water status via transpiration (Schroeder et al., 2001). Cytosolic calcium ([Ca²⁺]_i) has been shown to be a key second messenger that changes in response to multiple stimuli in guard cells (McAinsh et al., 1995; Grabov and Blatt, 1998; Wood et al., 2000). A large proportion of Ca²⁺ is localized in extracellular space. It has been shown that external Ca²⁺ concentration ([Ca²⁺]_o) promotes stomatal closure and induces oscillation in [Ca²⁺]_i in guard cells (MacRobbie, 1992; McAinsh et al., 1995; Allen et al., 2001). However, how the guard cells perceive [Ca²⁺]_o concentration and convert [Ca²⁺]_o changes into [Ca²⁺]_i changes was not understood until a calcium-sensing receptor (CAS) in the plasma membrane of guard cells in Arabidopsis (Arabidopsis thaliana) was identified (Han et al., 2003). The external Ca²⁺ (Ca²⁺_o)-induced [Ca²⁺]_i increase is abolished in CAS antisense lines (Han et al., 2003). Both [Ca²⁺]_o and [Ca²⁺]_i show diurnal oscillation that is determined by stomatal conductance, whereas the amplitude of [Ca²⁺]_i oscillation is reduced in CAS antisense lines (Tang et al., 2007). The reduced amplitude of [Ca²⁺]_i diurnal oscillation in response to Ca²⁺_o treatment suggests the potential existence of other [Ca²⁺]_o sensor(s) that may transmit [Ca²⁺]_o information into the [Ca²⁺]_i response in coordination with CAS. Extracellular calmodulin (ExtCaM) could be such an additional [Ca²⁺]_o sensor.Calmodulin is a well-known Ca²⁺ sensor that is activated upon binding of Ca²⁺. It has been shown that calmodulin exists not only intracellularly but also extracellularly in many plant species (Biro et al., 1984; Sun et al., 1994, 1995; Cui et al., 2005). ExtCaM has been implicated in several important biological functions, such as the promotion of cell proliferation, pollen germination, and tube growth (Sun et al., 1994, 1995; Ma and Sun, 1997; Ma et al., 1999; Cui et al., 2005; Shang et al., 2005). ExtCaM is found in the cell wall of guard cells in Vicia faba and in the epidermis of Arabidopsis by immunogold labeling/electron microscopy and western-blot analyses, respectively, and the endogenous CaM in the extracellular space has been shown to regulate stomatal movements (Chen et al., 2003; Xiao et al., 2004). Under natural conditions, once the activity of ExtCaM has been inhibited by its membrane-impermeable antagonist W7-agrose or CaM antibody, stomatal opening under light is enhanced and stomatal closure in darkness is inhibited in V. faba and Arabidopsis (Chen et al., 2003; Xiao et al., 2004). [Ca²⁺]_i and cytosolic hydrogen peroxide (H₂O₂) changes, two events involved in ExtCaM-regulated stomatal movement (Chen et al., 2004), are likely regulated by light/darkness (Chen and Gallie, 2004; Tang et al., 2007), suggesting that ExtCaM plays an important physiological role in the regulation of stomatal diurnal rhythm. Calmodulin-binding proteins have been found in the protoplast of suspension-cultured Arabidopsis cells, supporting the idea that ExtCaM functions as a peptide-signaling molecule (Cui et al., 2005). Furthermore, ExtCaM triggers [Ca²⁺]_i elevation in guard cells of V. faba and Arabidopsis and in lily (Lilium daviddi) pollen (Chen et al., 2004; Xiao et al., 2004; Shang et al., 2005). These observations support the notion that ExtCaM could be a potential [Ca²⁺]_o sensor for external calcium, and this external calcium sensing could subsequently regulate the [Ca²⁺]_i level through a signaling cascade.It is interesting that ExtCaM and ABA induce some parallel changes in second messengers in guard cell signaling. Our previous studies show that ExtCaM induces [Ca²⁺]_i increase and H₂O₂ generation through the Gα-subunit (GPA1) of a heterotrimeric G protein, and increased H₂O₂ further elevates [Ca²⁺]_i (Chen et al., 2004). G protein, Ca²⁺, and H₂O₂ are well-known second messengers in ABA-induced guard cell signaling (McAinsh et al., 1995; Grabov and Blatt, 1998; Pei et al., 2000; Wang et al., 2001; Zhang et al., 2001; Liu et al., 2007). However, the signaling cascade triggered by ExtCaM in guard cells is poorly understood. New ABA signaling components in guard cells could provide a clue in the study of the molecular mechanism of ExtCaM guard cell signaling.Recently, nitric oxide (NO) has been shown to serve as an important signal molecule involved in many aspects of developmental processes, including floral transition, root growth, root gravitropism, adventitious root formation, xylogenesis, seed germination, and orientation of pollen tube growth (Beligni and Lamattina, 2000; Pagnussat et al., 2002; He et al., 2004; Prado et al., 2004; Gabaldón et al., 2005; Stohr and Stremlau, 2006). Increasing evidence points to a role for NO as an essential component in ABA signaling in guard cells (Garcia-Mata and Lamattina, 2001, 2002; Neill et al., 2002). It has been shown that nitrate reductase (NR) reduces nitrite to NO, and the nia1, nia2 NR-deficient mutant in Arabidopsis showed reduced ABA induction of stomatal closure (Desikan et al., 2002; Bright et al., 2006). Although animal nitric oxide synthase (NOS) activity has been detected in plants and inhibitors of mammalian NOS impair NO production in plants (Barroso et al., 1999; Corpas et al., 2001), the gene(s) encoding NOS in plants is still not clear. AtNOS1 in Arabidopsis was initially reported to encode a protein containing NOS activity (Guo et al., 2003). However, recent studies have raised critical questions regarding the nature of AtNOS1 and suggested that AtNOS1 appears not to encode a NOS (Crawford et al., 2006; Zemojtel et al., 2006). However, the originally described Atnos1 mutant is deficient in NO accumulation (Crawford et al., 2006). Consequently, AtNOS1 was renamed AtNOA1 (for NITRIC OXIDE ASSOCIATED1; Crawford et al., 2006). Therefore, the Atnoa1 mutant provides a useful tool for dissecting the function of NO in plants. At present, the molecules that regulate NO generation in ABA-mediated guard cell signaling are not clear. Evidence suggests that H₂O₂, a second messenger important for the regulation of many developmental processes and stomatal movement (Pei et al., 2000; Zhang et al., 2001; Coelho et al., 2002; Demidchik et al., 2003; Kwak et al., 2003), regulates NO generation in guard cells (Lum et al., 2002; He et al., 2005; Bright et al., 2006).Given the parallel signaling events induced by ABA and ExtCaM, we investigated whether NO is involved in the regulation of ExtCaM-induced stomatal closure in Arabidopsis and whether it is linked to G protein and H₂O₂, two key regulators of both ExtCaM and ABA regulation of stomatal movements. Using Arabidopsis mutants (e.g. GPA1 null mutants, the NO-producing mutant Atnoa1, and the guard cell H₂O₂ synthetic enzymatic mutant atrbohD/F) combined with pharmacological analysis, we present compelling evidence to establish a linear functional relationship between Gα, H₂O₂, and NO in ExtCaM guard cell signaling.     

Role of extracellular calcium and mitochondrial oxygen species in psychosine-induced oligodendrocyte cell death

Globoid cell leukodystrophy (GLD) is a metabolic disease caused by mutations in the galactocerebrosidase (GALC) gene. GALC is a lysosomal enzyme whose function is to degrade galacto-lipids, including galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of function causes progressive intracellular accumulation of PSY. It is widely held that PSY is the main trigger for the degeneration of myelinating cells and progressive white-matter loss. However, still little is known about the molecular mechanisms by which PSY imparts toxicity. Here, we address the role of calcium dynamics during PSY-induced cell death. Using the human oligodendrocyte cell line MO3.13, we report that cell death by PSY is accompanied by robust cytosolic and mitochondrial calcium (Ca²⁺) elevations, and by mitochondrial reactive oxygen species (ROS) production. Importantly, we demonstrate that the reduction of extracellular calcium content by the chelating agent ethylenediaminetetraacetic acid can decrease intra-mitochondrial ROS production and enhance cell viability. Antioxidant administration also reduces mitochondrial ROS production and cell loss, but this treatment does not synergize with Ca²⁺ chelation. Our results disclose novel intracellular pathways involved in PSY-induced death that may be exploited for therapeutic purposes to delay GLD onset and/or slow down its progression.Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a childhood leukodystrophy triggered by mutations in the galactocerebrosidase (GALC) gene; the physio-pathological hallmarks of GLD are progressive demyelination, reactive astrocytosis and microgliosis.¹ GALC is a lysosomal enzyme essential for the normal catabolism of galacto-lipids, including galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of function causes progressive accumulation of PSY, a cytotoxic metabolite that has been assumed as the main cause for GLD pathogenesis.² PSY leads to Schwann cell and oligodendrocyte death, but still little is known about the molecular mechanisms by which PSY imparts toxicity. It has been demonstrated that PSY accumulates in cell membrane raft micro-domains, disrupting their architecture³ and inhibiting protein kinase C translocation to the plasma membrane.⁴ Recently, increased raft clustering was also reported in cultured dorsal root ganglion neurons prepared from the GLD murine model (i.e., the Twitcher mouse), and this was associated with the dysregulation of tyrosine kinase receptor A membrane recruitment and ligand-tyrosine kinase receptor A activated endocytosis.⁵ PSY induces p53-mediated apoptotic cell death,⁶ tumor necrosis factor-related apoptosis,^{6, 7} activation of secretory phospholipase A2,⁸ cytochrome C release from mitochondria and apoptosis activation via the caspase-9 pathway.⁹ Moreover, several authors found that peroxisomal β-oxidation was significantly inhibited and very long-chain fatty acid levels and reactive oxygen species (ROS) production were increased in PSY-treated cells.^{10, 11}Calcium (Ca²⁺) is an essential ion for cell life, acting as a key second messenger in almost all cellular functions. It is well established that Ca²⁺ is one of the main second messengers involved in apoptotic cell death in neurons and in other cell types; sustained cytosolic Ca²⁺ increase can activate apoptosis.¹² This can originate from extracellular influx or by release from intracellular stores like the endoplasmic reticulum.¹³ Importantly, mitochondria are also involved in Ca²⁺ homeostasis.¹⁴ Mitochondrial Ca²⁺ in basal conditions is maintained at low concentrations, but mitochondria are organelles that can take up high Ca²⁺ concentrations; indeed, different stimuli, such as nutrients, hormones or neurotransmitters that increase the cytoplasmic Ca²⁺ content also induce intra-mitochondrial Ca²⁺ increase.¹⁵ If this increase is relevant, ROS production increases and this is associated with mitochondrial membrane destruction, release of cytochrome C and apoptosis induction.¹⁶ During this process, pro-apoptotic Bcl2 family of proteins plays a crucial role by regulating the intracellular/mitochondrial Ca²⁺ content, and by inducing mitochondrial permeabilization, the essential step for cytochrome C release and caspase activation.^{12, 17}It has been reported that some sphingolipid metabolites, such as ceramides and sphingosine, can play a crucial role in many steps of apoptosis induction as regulators of some Bcl2 family proteins, by increasing intracellular Ca²⁺ levels and inducing mitochondrial stress.¹⁸ However, these mechanisms have never been explored during PSY-induced cell death.In this article, we report on the role of intracellular Ca²⁺ dynamics during PSY-induced cell death in vitro. Using the human oligodendrocyte cell line MO3.13 and fluorescent probes, we measured Ca²⁺ variations in cytoplasm and mitochondria upon PSY administration until cell death. Moreover, we studied oxidative stress production in mitochondria by flow cytometry and time-lapse confocal fluorescence microscopy. Finally, in order to rescue cell viability in presence of PSY, we investigated the use of Ca²⁺ chelation in the extracellular medium, and its possible synergic effect with antioxidant treatment.     

The zinc sensing receptor,ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn²⁺ are not well understood. Here, we determined a role of the colonocytic Zn²⁺ sensing receptor, ZnR/GPR39, in mediating Zn²⁺-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn²⁺-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn²⁺ by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.The intestinal epithelial barrier, located at the interface between the body and the digestive system lumen, facilitates electrolyte and nutrient absorption while protecting against permeation of antigenic, toxic or infectious materials.¹ Stressful conditions in the intestinal lumen require rapid and continuous renewal of the epithelial layer,² which occur through tightly regulated and balanced proliferation, migration and differentiation processes.³Zn²⁺ deficiency leads to a reduction of epithelial cell proliferation rate, thus limiting renewal of gastrointestinal mucosa, but it also impairs barrier function, increases permeability and enhances cell death.^{4, 5} Zn²⁺ supplementation reverses these processes and restores integrity of colon epithelium.^{5, 6, 7} Importantly, Zn²⁺ supplementation reduces diarrheal disease activity index^{8, 9} and is effective in decreasing severity of histological and clinical scores in in vivo models of ulcerative colitis.^{10, 11} Despite these observations, the signaling pathways linking Zn²⁺ to intestinal epithelial function or integrity are poorly understood.We identified a Zn²⁺ sensing receptor, ZnR, which links between changes in extracellular Zn²⁺ and major intracellular signaling pathways. Functional studies indicated that ZnR is a Gq-coupled receptor, which triggers inositol 1,4,5-trisphosphate (IP3)-dependent release of intracellular Ca²⁺,¹² leading to activation of mitogen-activated protein (MAP) and phosphoinositide 3 (PI3) kinase pathways.^{13, 14} Recent studies showed that the Zn²⁺-dependent Ca²⁺ rise is mediated by the G-protein-coupled receptor GPR39, in colonocytes.¹⁵ ZnR/GPR39 mediates recovery from acidic pH by upregulation of Na⁺/H⁺ exchange in colonocytic and keratinocytic cell lines as well as in native colon epithelial cells, demonstrating the important role of this receptor in epithelial physiology.^{13, 15, 16} Finally, ZnR/GPR39 enhances colonocytes survival from butyrate induced stress.¹⁵ Notably, GPR39 knockout (KO) mice show symptoms of Zn²⁺ deficiency: accelerated gastric emptying and increased fecal secretion.¹⁷ However how Zn²⁺ or ZnR/GPR39 signaling promote colon epithelial function is not well understood. Here we show that ZnR/GPR39 regulates extracellular Zn²⁺-dependent proliferation and differentiation processes in colonocytes. Furthermore, we show that ZnR/GPR39 is essential for expression and localization of tight junction proteins in vitro and in vivo, and thereby regulates the formation of the colon epithelial barrier.     

Augmentation of NAD+ by NQO1 attenuates cisplatin-mediated hearing impairment

Cisplatin (cis-diaminedichloroplatinum-II) is an extensively used chemotherapeutic agent, and one of its most adverse effects is ototoxicity. A number of studies have demonstrated that these effects are related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD⁺) has emerged as a key regulator of cellular energy metabolism and homeostasis. Here, we demonstrate for the first time that, in cisplatin-mediated ototoxicity, the levels and activities of SIRT1 are suppressed by the reduction of intracellular NAD⁺ levels. We provide evidence that the decrease in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) transferase (PARP)-1 activation and microRNA-34a through p53 activation aggravates cisplatin-mediated ototoxicity. Moreover, we show that the induction of cellular NAD⁺ levels using β-lapachone (β-Lap), whose intracellular target is NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD⁺ levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity.Cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic agent extensively used to treat a variety of solid tumors in the head and neck, bladder, lung, ovaries, testicles, and uterus.¹ However, progressive irreversible side effects of cisplatin, including nephrotoxicity and ototoxicity, greatly impair the patient''s quality of life and frequently result in the need to lower the dosage during treatment or discontinuation of the treatment. Cisplatin ototoxicity primarily occurs in the cochlea and is generally caused by apoptotic damage to the outer hair cells (OHCs), spiral ganglion cells, and the marginal cells of the stria vascularis. In recent years, studies have demonstrated that cisplatin ototoxicity is also closely related to the damage of cochlear tissue by increased production of reactive oxygen species (ROS) and accompanied by the depletion of antioxidant substances and increased lipid peroxidation.^{2, 3} ROSs, particularly the hydroxyl radical, have a critical role in cisplatin-induced p53 activation through DNA damage.⁴ Although it is not easy to differentiate the cause from the consequence, a positive feedback loop between inflammatory cytokines and oxidative stress that worsen the cochlear damage is considered as one of the major mechanisms that facilitate cisplatin-induced hearing impairment.⁵ Interestingly, p53 and NF-κB have been described as key mediators of cisplatin-induced toxicity because of their involvement in oxidative stress, DNA damage, and inflammation through a mutual feedback process of ‘cause and effect.''^{6, 7} In addition, activities of p53 and NF-κB could be regulated by post-translational modifications, including phosphorylation and acetylation. Recent studies have reported that acetylated p53 and NF-κB are correlated with cisplatin-induced toxicity. Furthermore, acetylation of p53 and NF-κB is critically involved in cisplatin-induced renal injury.^{8, 9}Cellular nicotinamide adenine dinucleotide (NAD⁺) and NADH levels have been shown to be important mediators of energy metabolism and cellular homeostasis.^{10, 11} As NAD⁺ acts as a cofactor for various enzymes, including sirtuins (SIRTs), poly(ADP-ribose) transferases (PARPs), and cyclic ADP (cADP)-ribose synthases,^{12, 13} the regulation of NAD⁺ level may have therapeutic benefits through its effect on NAD⁺-dependent enzymes. SIRTs, NAD⁺-dependent protein deacetylases, are present as seven homologs of Sir2 (SIRT1-7) that show differential subcellular localizations in mammals.¹¹ Among these, nuclear SIRT1 is activated under energy stress conditions, such as fasting, exercise, or low glucose availability. ¹⁴ SIRT1 has a key role in metabolism, development, stress response, neurogenesis, hormone responses, and apoptosis^{15, 16} by deacetylation of substrates, such as NF-κB, FOXO, p53, and histones.^{17, 18, 19}PARPs, the most abundant ADP-ribosyl transferases, also use NAD⁺ to generate large amounts of poly(ADP-ribose) (PAR), which facilitate the recruitment of DNA repair factors. In particular, PARP-1 is a DNA damage sensor that can be activated in response to DNA damage by various pathophysiological conditions, including oxidative stress and inflammatory injury. However, excessive hyperactivation of PARP-1 causes the depletion of intracellular NAD⁺ and ATP levels, which eventually leads to cell death.^{20, 21} PARP-1 activation is also known as one of the important pathogenic mechanisms in cisplatin-induced toxicity.^{22, 23}A cytosolic antioxidant flavoprotein NADH:quinone oxidoreductase 1 (NQO1) catalyzes the reduction of quinones to hydroquinones by utilizing NADH as an electron donor, which consequently increases intracellular NAD⁺ levels.^{24, 25} In addition, accumulation evidence suggests that NQO1 has a role in other biological activities, including anti-inflammatory processes, scavenging of superoxide anion radicals, and stabilization of p53 and other tumor suppressor proteins.^{26, 27, 28} Several substrates of NQO1 enzyme, including mitomycin C, RH1, AZQ, Coenzyme Q10, and idebenone, have been identified,^{29, 30} of which β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione; β-Lap) is recently well studied as a substrate of NQO1.^{31, 32} β-Lap was first isolated from the bark of the lapacho tree and reported to inhibit tumor cell line growth.³³ However, recent reports indicate that the conversion of NADH to NAD⁺ by NQO1 and β-Lap has beneficial effects on several characteristics of metabolic syndrome, for example, prevention of health decline in aged mice, amelioration of obesity or hypertension, prevention of arterial restenosis, and protection against salt-induced renal injury.^{34, 35, 36, 37, 38} Furthermore, we recently have demonstrated that conversion of NADH to NAD+ by NQO1 and β-Lap suppresses cisplatin-induced acute kidney injury by downregulating potential damage mediators such as oxidative stress and inflammatory responses.⁹Although a link between NAD⁺-dependent molecular events and cellular metabolism is evident, it remains unclear whether modulation of NAD⁺ levels has an impact on cisplatin-induced hearing impairment. Therefore, herein we investigated the role of NAD⁺ metabolism on cisplatin-induced cochlear dysfunction, and the effect of increased levels of intracellular NAD⁺ facilitated by β-Lap on cisplatin-induced hearing impairment with a particular interest in NAD⁺-dependent enzymatic pathways including SIRTs and PARPs.     

Balancing functions of annexin A6 maintain equilibrium between hypertrophy and apoptosis in cardiomyocytes

Pathological cardiac hypertrophy is a major risk factor associated with heart failure, a state concomitant with increased cell death. However, the mechanism governing progression of hypertrophy to apoptosis at the single-cell level remains elusive. Here, we demonstrate annexin A6 (Anxa6), a calcium (Ca²⁺)-dependent phospholipid-binding protein critically regulates the transition of chronic hypertrophied cardiomyocytes to apoptosis. Treatment of the H9c2(2-1) cardiomyocytes with hypertrophic agonists upregulates and relocalizes Anxa6 with increased cytosolic punctate appearance. Live cell imaging revealed that chronic exposure to hypertrophic agonists such as phenylephrine (PE) compromises the mitochondrial membrane potential (ΔΨ_m) and morphological dynamics. Such chronic hypertrophic induction also activated the caspases 9 and 3 and induced cleavage of the poly-(ADP-ribose) polymerase 1 (Parp1), which are the typical downstream events in the mitochondrial pathways of apoptosis. An increased rate of apoptosis was evident in the hypertrophied cardiomyocytes after 48–72 h of treatment with the hypertrophic agonists. Anxa6 was progressively associated with the mitochondrial fraction under chronic hypertrophic stimulation, and Anxa6 knockdown severely abrogated mitochondrial network and dynamics. Ectopically expressed Anxa6 protected the mitochondrial morphology and dynamics under PE treatment, and also increased the cellular susceptibility to apoptosis. Biochemical analysis showed that Anxa6 interacts with Parp1 and its 89 kDa cleaved product in a Ca²⁺-dependent manner through the N-terminal residues (1–28). Furthermore, expression of Anxa6^S13E, a mutant dominant negative with respect to Parp1 binding, served as an enhancer of mitochondrial dynamics, even under chronic PE treatment. Chemical inhibition of Parp1 activity released the cellular vulnerability to apoptosis in Anxa6-expressing stable cell lines, thereby shifting the equilibrium away from cell death. Taken together, the present study depicts a dual regulatory function of Anxa6 that is crucial for balancing hypertrophy with apoptosis in cardiomyocytes.Complex machineries govern the life and death decisions in mammalian cells through a dynamic equilibrium, which is essential for physiological homeostasis.¹ Such equilibrium is critical for cardiac myocytes because of their terminally differentiated states and low proliferative capacities. Stress response in cardiomyocytes often involves a switch between survival and cell death pathways.^{2, 3, 4} Cardiomyocyte hypertrophy is an adaptive response to stress, which may turn maladaptive and fatal,⁵ as evident in cardiovascular disorders that leads to heart failure.⁶ Hypertrophied phenotypes are also associated with a balance between cell growth and programmed cell death.⁷ These processes are aided by several patrolling proteins, which sense and operate to ameliorate the anomalies.^{8, 9} Understanding the dynamics of such signaling events is vital for the development of novel therapeutic strategies.Anxa6 belongs to the annexin family of calcium (Ca²⁺)/phospholipid-binding proteins.¹⁰ A major cardiac annexin,¹¹ Anxa6 has diverse functions ranging from handling intracellular Ca²⁺ signaling, cholesterol transport,¹² Ras inactivation¹³ and vesicular traffic.¹⁴ Anxa6 mostly functions as an intracellular scaffold.¹⁵ Although mice with targeted depletion of the Anxa6 gene remain viable,¹⁶ functional redundancies within the annexin family have been proposed to compensate for the loss of Anxa6 function.^{17, 18} A 10-fold overexpression of Anxa6 targeted to the heart developed cardiomyopathies in mice, whereas cardiomyocytes from Anxa6-knockout mice exhibited increased contractility and altered Ca²⁺ turnover.^{19, 20} Such contradictory findings may indicate participation of Anxa6 in counterbalancing signaling mechanisms. Moreover, end-stage heart failures have been reported to be associated with downregulation of Anxa6, and, in general, Anxa6 has compensatory roles in chronic pathological conditions.^{20, 21, 22} However, the function of differential Anxa6 expression or dynamics in chronic cardiomyocyte hypertrophy is poorly understood.We have reported the interactions of Anxa6 with the sarcomeric α-actinin and its role in cardiomyocyte contractility.²³ Recently, we have characterized a role of Anxa6 in the antihypertrophic signaling via the regulation of atrial natriuretic peptide (ANP) secretion.²⁴ The mechanistic spectrum of Anxa6 in the earlier study was limited to a short-term (24 h) exposure of H9c2 cardiomyocytes to the α1-adrenergic receptor agonist phenylephrine (PE). The dynamics of Anxa6 within this small window yielded valuable insight into the spatiotemporal regulation of hypertrophic signaling. Here, we extended the study to understand the dynamics of Anxa6 under chronic hypertrophic conditions. The mechanodeficient H9c2(2-1) cardiomyocyte line has been instrumental in our study to rule out the contributions of Anxa6 towards contractility,²³ owing to its multidimensional scaffold activity and functional compensations.^{17, 18} The H9c2 cardiomyocytes have been extensively characterized and ARE an established animal origin-free model for studying signal-transduction pathways in cardiomyocytes, including hypertrophy.^{25, 26}Adrenergic stimulation is crucial in compensatory and pathological cardiac hypertrophy, an early state that may proceed towards heart failure.²⁷ Cardiac hypertrophy at advanced stages (chronic) is associated with mitochondrial dysfunction, which also contributes to cardiac decompensation.²⁸ To explore the temporal events under chronic hypertrophy, we analyzed the effects of adrenergic induction on mitochondrial membrane potential (ΔΨm) and morphological dynamics, parameters that are directly correlated with mitochondrial dysfunction and programmed cell death.^{29, 30, 31} Anxa6 has been reported to be associated with mitochondria in some cell types.^{17, 32, 33} In the present study, we aim to understand the functions of Anxa6 under chronic hypertrophic conditions that may progress towards apoptosis.     

Mycobacterium tuberculosis ESAT-6 is a leukocidin causing Ca2+ influx,necrosis and neutrophil extracellular trap formation

Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca²⁺ overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca²⁺ activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca²⁺, stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a leading source of mortality by infectious disease, with one-third of the world''s population infected, 8.6 million new cases of TB and 1.3 million deaths annually.¹ The fundamental feature of TB transmission is the generation of a pulmonary tubercle lesion that contains a cuff of immune cells surrounding a necrotic core laden with extracellular bacteria. This lesion may ‘cavitate'' into the airways of the lung releasing the bacteria to allow transmission via the respiratory route. The essential contribution of macrophage and neutrophil cell death to the generation of this pathology has been recognised in many studies; however, notably in a comprehensive study of TB lesion development, Medlar² observed that the polymorphonuclear cells were attracted to lesions following the death of mononuclear cells and that the bulk of necrotic tissue in human caseating tubercle lesions represented dead polymorphonuclear cells.We now know that the bacterium has evolved mechanisms to regulate the mode and timing of macrophage cell death.^{3, 4, 5, 6, 7} After initial infection into the lungs, it is supposed that the bacterium is phagocytosed by alveolar macrophages which migrate into the interstitium of the lung.⁸ The bacterium is able to replicate intracellularly in the macrophage, inhibiting apoptosis until at a certain bacterial load it induces necrosis of the macrophage.⁹ The ensuing inflammation attracts monocytes and neutrophils from post-capillary venules that engulf the released bacteria, and thus sequential rounds of replication and inflammation enable the generation of the tubercle lesion. However, although we are beginning to understand the mechanisms of macrophage cell death control,^{4, 5, 6} we know very little about how M. tuberculosis modulates neutrophil death.It is also clear that in some circumstances neutrophils have an antimycobacterial capacity,^10,11 which may be mediated by the direct generation of reactive oxygen species (ROS) or by apoptosis of the infected neutrophils and subsequent efferocytosis of the apoptotic body combined with ROS-dependent killing.^12,13 Additionally, neutrophil apoptosis has been linked to effective generation of adaptive immunity in M. tuberculosis infection.¹⁴ However, to counter this, M. tuberculosis has been recently shown to inhibit neutrophil apoptosis¹⁴ and furthermore, has been observed to induce necrosis.^11,12 Interestingly, neutrophil necrosis only occurs on exposure to virulent strains which express the region of difference 1 (RD1) which encodes a type VII secretion system (ESX) that secretes proteins including the abundant early secretory antigen-6 (ESAT-6).^{12,15, 16, 17} Thus the bacterial induction of pro-inflammatory neutrophil necrosis may have dual benefit to the pathogen by removing the antimicrobial threat of the neutrophil while simultaneously facilitating the generation of the necrotic cavitating lesions that drive TB transmission.The mechanism of necrosis in neutrophils can be varied and controlled. The most recent to be described is ‘NETosis'',^18,19 whereby death of the neutrophil results in formation of a structure made of DNA with a histone backbone which contains neutrophil elastase, myeloperoxidase (MPO) and metalloproteinases. These ‘traps'' are known to be produced in vivo and associate with bacteria in some infections.^{18,20, 21, 22} Importantly they have been shown to be produced by M. tuberculosis infected neutrophils in vitro²³ although with no bactericidal activity. As well as ‘NETosis'' there are also other described mechanisms of neutrophil death, one of which is ‘secondary necrosis''. In vitro aging, without any stimuli, results in the necrosis of neutrophils that have undergone apoptosis (termed secondary necrosis). Previous investigations have shown that neutrophils that have externalised phosphatidylserine, and are therefore ‘apoptotic'', can undergo secondary necrosis caused by a Ca²⁺ influx leading to the activation of a subtype of Ca²⁺ activated protease, calpain.²⁴ This we termed Ca²⁺ Induced Necrosis (CAIN). In the present study, we elucidate the molecular events that link the RD1 encoded ESX-1 type VII secretion system with neutrophil necrosis. We chose to investigate the ESAT-6 protein, which is secreted by ESX-1, because it interacts with lipid membranes and is thought to be pore-forming,^{25, 26, 27, 28} thus it has the potential to influence intracellular Ca²⁺. Furthermore, calpain has been shown previously to be active in M.bovis infections that were dependent on the RD1 locus.²⁹ ESAT-6 has also been shown to have cytotoxic effects to pneumocytes³⁰ and T lymphocytes.³¹ Therefore, this study aimed to determine if ESAT-6 had a leukocidin action, and if so, whether this was dependent on intracellular flux of Ca²⁺, activation of calpain, and further, if this resulted in the formation of neutrophil extracellular traps (NETs).     

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.     

Heme oxygenase-1 protects against Alzheimer's amyloid-β1-42-induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer''s disease, catabolises heme to biliverdin, Fe²⁺ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediates cellular K⁺ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ_1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrP^c, and rat primary hippocampal neurons. Aβ_1-42 (containing protofibrils) caused a concentration-dependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrP^c-expressing cells showing greater susceptibility to Aβ_1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ_1-42. The CO-donor CORM-2 protected cells against Aβ_1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ_1-42 treatment suggesting that K⁺ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca²⁺ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K⁺ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent.Amongst the earliest of events leading to neuronal loss in Alzheimer''s disease (AD) is the loss of functional synapses,^{1, 2, 3} apparent long before deposition of amyloid β peptide (Aβ)-containing plaques.⁴ Although other parts of the neurone (e.g. the axon or soma) appear intact, their health at this early stage of disease progression is not clear. However, neurones ultimately die in AD and there is clear evidence that numerous events indicative of apoptosis occur even at early stages of disease progression.^{5, 6, 7, 8} Thus, targeting of apoptotic mechanisms may be of therapeutic value in AD as well as in other neurodegenerative disorders. Furthermore, apoptosis is established as a mechanism of neuronal loss following other types of pathological stresses including ischemia associated with stroke,⁹ which can predispose individuals to the development of AD.^{10, 11, 12}Apoptosis is strongly influenced by intracellular K⁺ levels¹³ which regulate caspase activation, mitochondrial membrane potential and volume, osmolarity and cell volume.^{13, 14} K⁺ loss via K⁺ channels is a key early stage in apoptosis,^{15, 16, 17, 18, 19} and K⁺ channel inhibitors can protect against apoptosis triggered by numerous insults including oxidative stress.^{20, 21} Evidence suggests a particularly important role for the voltage-gated channel Kv2.1 in this process: expression of dominant negative Kv2.1 constructs (thus lacking functional Kv2.1 channels) protects against oxidant-induced apoptosis, and over-expression of Kv2.1 increases susceptibility to apoptosis.^{22, 23} Pro-apoptotic agents cause a rapid increase in the surface expression of Kv2.1 channels,²⁴ but whether or not this occurs in AD remains to be determined. Alternative pathways recently reported to promote cell death include activation of the AMP-dependent protein kinase (AMP kinase) which can act either as a Tau kinase²⁵ or to inhibit the mTOR pathway²⁶ and thus contribute to neurodegeneration.Heme oxygenases (HO) are enzymes widely distributed throughout the body. In the central nervous system, HO-2 is constitutively expressed in neurones and astrocytes, while HO-1 is inducible in both cell types.^{27, 28, 29, 30} Both HO-1 and HO-2 break down heme to liberate biliverdin, ferrous iron (Fe²⁺) and carbon monoxide (CO). This catalysis is of biological significance since it is crucial to iron and bile metabolism, and also generates a highly effective antioxidant in bilirubin (from biliverdin via bilirubin reductase). Numerous stimuli can induce HO-1 gene expression,³¹ including oxidative stress³² and Aβ peptides.³³ Importantly, HO-1 is strikingly up-regulated in AD patients, a finding considered indicative of oxidative stress.^{27, 34, 35} Induction of HO-1 is clearly a neuroprotective response (although in some cases can exert detrimental effects²⁷). However, there is growing evidence that CO can be neuroprotective, for example against the damage of focal ischemia.³⁶ Our recent studies have demonstrated that CO provides protection against oxidant-induced apoptosis by selectively inhibiting Kv2.1.^{23, 37} In the present study, we have investigated whether HO-1, or its product CO, can provide protection against Aβ-induced toxicity in the human neuroblastoma, SH-SY5Y, and in rat primary hippocampal neurones, and whether this involves regulation of K⁺ channels. We show that both HO-1 and CO protect cells against the toxicity of protofibrillar Aβ_1-42 but that protection does not arise from inhibition of apoptosis-associated K⁺ efflux, but rather by inhibition of AMPK activation.     

Cerium oxide nanoparticles protect against Aβ-induced mitochondrial fragmentation and neuronal cell death

Evidence indicates that nitrosative stress and mitochondrial dysfunction participate in the pathogenesis of Alzheimer''s disease (AD). Amyloid beta (Aβ) and peroxynitrite induce mitochondrial fragmentation and neuronal cell death by abnormal activation of dynamin-related protein 1 (DRP1), a large GTPase that regulates mitochondrial fission. The exact mechanisms of mitochondrial fragmentation and DRP1 overactivation in AD remain unknown; however, DRP1 serine 616 (S616) phosphorylation is likely involved. Although it is clear that nitrosative stress caused by peroxynitrite has a role in AD, effective antioxidant therapies are lacking. Cerium oxide nanoparticles, or nanoceria, switch between their Ce³⁺ and Ce⁴⁺ states and are able to scavenge superoxide anions, hydrogen peroxide and peroxynitrite. Therefore, nanoceria might protect against neurodegeneration. Here we report that nanoceria are internalized by neurons and accumulate at the mitochondrial outer membrane and plasma membrane. Furthermore, nanoceria reduce levels of reactive nitrogen species and protein tyrosine nitration in neurons exposed to peroxynitrite. Importantly, nanoceria reduce endogenous peroxynitrite and Aβ-induced mitochondrial fragmentation, DRP1 S616 hyperphosphorylation and neuronal cell death.Nitric oxide (NO) is a neurotransmitter and neuromodulator required for learning and memory.¹ NO is generated by NO synthases, a group of enzymes that produce NO from L-arginine. In addition to its normal role in physiology, NO is implicated in pathophysiology. When overproduced, NO combines with superoxide anions (O₂·⁻), byproducts of aerobic metabolism and mitochondrial oxidative phosphorylation, to form peroxynitrite anions (ONOO⁻) that are highly reactive and neurotoxic. Accumulation of these reactive oxygen species (ROS) and reactive nitrogen species (RNS), known as oxidative and nitrosative stress, respectively, is a common feature of aging, neurodegeneration and Alzheimer''s disease (AD).¹Nitrosative stress caused by peroxynitrite has a critical role in the etiology and pathogenesis of AD.^{2, 3, 4, 5, 6, 7} Peroxynitrite is implicated in the formation of the two hallmarks of AD, Aβ aggregates and neurofibrillary tangles containing hyperphosphorylated Tau protein.^{1, 4, 7} In addition, peroxynitrite promotes the nitrotyrosination of presenilin 1, the catalytic subunit of the γ-secretase complex, which shifts production of Aβ to amyloid beta (Aβ)42 and increases the Aβ42/Aβ40 ratio, ultimately resulting in an increased propensity for aggregation and neurotoxicity.⁵ Furthermore, nitration of Aβ tyrosine 10 enhances its aggregation.⁶ Peroxynitrite can also modify enzymes, such as triosephosphate isomerase,⁴ and activate kinases, including Jun amino-terminal kinase and p38 mitogen-activated protein kinase, which enhance neuronal cell death.^{8, 9} Moreover, peroxynitrite can trigger the release of free metals such as Zn²⁺ from intracellular stores with consequent inhibition of mitochondrial function and enhancement of neuronal cell death.^{10, 11, 12} Finally, peroxynitrite can irreversibly inhibit complexes I and IV of the mitochondrial respiratory chain.^{11, 13}Because mitochondria have a critical role in neurons as energy producers to fuel vital processes such as synaptic transmission and axonal transport,¹⁴ and mitochondrial dysfunction is a well-documented and early event in AD,¹⁵ it is important to consider how peroxynitrite and nitrosative stress affect mitochondria. Although the ultimate cause of mitochondrial dysfunction in AD remains unclear, an imbalance in mitochondrial fission and fusion is one possibility.^{1, 14, 16, 17, 18} Notably, peroxynitrite, N-methyl D-aspartate (NMDA) receptor activation and Aβ can induce mitochondrial fragmentation by activating mitochondrial fission and/or inhibiting fusion.¹⁶ Mitochondrial fission and fusion is regulated by large GTPases of the dynamin family, including dynamin-related protein 1 (DRP1) that is required for mitochondrial division,¹⁹ and inhibition of mitochondrial division by overexpression of the GTPase-defective DRP1^K38A mutant provides protection against peroxynitrite-, NMDA- and Aβ-induced mitochondrial fragmentation and neuronal cell death.¹⁶The exact mechanism of peroxynitrite-induced mitochondrial fragmentation remains unclear. A recent report suggested that S-nitrosylation of DRP1 at cysteine 644 increases DRP1 activity and is the cause of peroxynitrite-induced mitochondrial fragmentation in AD;²⁰ however, the work remains controversial, suggesting that alternative pathways might be involved.²¹ For example, peroxynitrite also causes rapid DRP1 S616 phosphorylation that promotes its translocation to mitochondria and organelle division.^{21, 22} In mitotic cells, DRP1 S616 phosphorylation is mediated by Cdk1/cyclinB1 and synchronizes mitochondrial division with cell division.²³ Interestingly, DRP1 is S616 hyperphosphorylated in AD brains, suggesting that this event might contribute to mitochondrial fragmentation in the disease.^{21, 22} A recent report indicates that Cdk5/p35 is responsible for DRP1 S616 phosphorylation,²⁴ and notably aberrant Cdk5/p35/p25 signaling is associated with AD pathogenesis.²⁵ Thus, we explored here the possible role of DRP1 S616 hyperphosphorylation in Aβ- and peroxynitrite-mediated mitochondrial fragmentation.Under normal conditions, accumulated mitochondrial superoxide anions and hydrogen peroxide (H₂O₂) can be neutralized by superoxide dismutase (SOD) and catalase. Nitrosative stress in aging and AD might be explained by a loss of antioxidant enzymes. Previous studies suggest that expression of SOD subtypes is decreased in the human AD brain.^{26, 27} Furthermore, SOD1 deletion in a mouse model of AD increased the burden of amyloid plaques.²⁶ By contrast, overexpression of SOD2 in a mouse model of AD decreased the Aβ42/Aβ40 ratio and alleviated memory deficits.^{28, 29} There is currently a lack of antioxidants that can effectively quench superoxide anions, H₂O₂ or peroxynitrite and provide lasting effects. Cerium is a rare earth element and cerium oxide (CeO₂) nanoparticles, or nanoceria, shuttle between their 3+ or 4+ states. Oxidation of Ce⁴⁺ to Ce³⁺ causes oxygen vacancies and defects on the surface of the crystalline lattice structure of the nanoparticles, generating a cage for redox reactions to occur.³⁰ Accordingly, nanoceria mimic the catalytic activities of antioxidant enzymes, such as SOD^{31, 32} and catalase,³³ and are able to neutralize peroxynitrite.³⁴ Because of these antioxidant properties, we hypothesized that nanoceria could detoxify peroxynitrite and protect against Aβ-induced DRP1 S616 hyperphosphorylation, mitochondrial fragmentation and neuronal cell death.     

NaCl-Induced Alternations of Cellular and Tissue Ion Fluxes in Roots of Salt-Resistant and Salt-Sensitive Poplar Species

Using the scanning ion-selective electrode technique, fluxes of H⁺, Na⁺, and Cl⁻ were investigated in roots and derived protoplasts of salt-tolerant Populus euphratica and salt-sensitive Populus popularis 35-44 (P. popularis). Compared to P. popularis, P. euphratica roots exhibited a higher capacity to extrude Na⁺ after a short-term exposure to 50 mm NaCl (24 h) and a long term in a saline environment of 100 mm NaCl (15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na⁺ efflux and a correspondingly increased H⁺ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na⁺/H⁺ exchange in root tissues and cells was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H⁺-ATPase inhibitor). These results indicate that the Na⁺ extrusion in stressed P. euphratica roots is the result of an active Na⁺/H⁺ antiport across the plasma membrane. In comparison, the Na⁺/H⁺ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na⁺ at both the tissue and cellular levels. Moreover, salt-treated P. euphratica roots retained a higher capacity for Cl⁻ exclusion than P. popularis, especially during a long term in high salinity. The pattern of NaCl-induced fluxes of H⁺, Na⁺, and Cl⁻ differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.Soil salinity causes increasingly agricultural and environmental problems on a worldwide scale, especially in arid areas. When plant roots are subjected to saline environments with high NaCl content, external Na⁺ and Cl⁻ establish a large electrochemical gradient favoring the passive entry of salt ions through a variety of cation and anion channels and/or transporters in the plasma membrane (PM; Blumwald et al., 2000; Hasegawa et al., 2000; White and Broadley, 2001; Roberts, 2006; Demidchik and Maathuis, 2007). The entry and accumulation of toxic ions lead to disruption of ion homeostasis and finally cause secondary stress, e.g. oxidative bursts (Zhu, 2001, 2003). Accordingly, the maintenance of low salt concentration in the cytosol is of great importance for salt adaptation of plants (Greenway and Munns, 1980; Munns and Tester, 2008).Active Na⁺ extrusion to the apoplast or external environment is essential for sustaining Na⁺ homeostasis in the cytosol (Blumwald et al., 2000; Tester and Davenport, 2003; Zhu, 2003; Apse and Blumwald, 2007). PM Na⁺/H⁺ antiporters have been widely considered to play a crucial role in active Na⁺ extrusion under saline conditions (Shi et al., 2000, 2002; Qiu et al., 2002; Martínez-Atienza et al., 2007). NaCl-induced activity of PM Na⁺/H⁺ antiporter has been reported in crop species, tomato (Solanum lycopersicum; Wilson and Shannon, 1995), Arabidopsis (Arabidopsis thaliana; Qiu et al., 2002, 2003), and rice (Oryza sativa; Martínez-Atienza et al., 2007). Furthermore, overexpression of the Na⁺/H⁺ antiporter gene AtSOS1 decreases the accumulation of Na⁺ in transgenic Arabidopsis under NaCl stress (Shi et al., 2003). These PM Na⁺/H⁺ antiporters depend on electrochemical H⁺ gradients, which are generated by PM H⁺-ATPase (Blumwald et al., 2000; Zhu, 2003). Using an ion-selective microelectrode, Shabala and a colleague suggested the involvement of PM H⁺-ATPase in the Na⁺/H⁺ antiport according to H⁺ kinetics on salt shock (Shabala, 2000; Shabala and Newman, 2000). Therefore, the NaCl-induced H⁺ pumping is fundamental to Na⁺/H⁺ exchange and salinity tolerance (Ayala et al., 1996; Vitart et al., 2001; Chen et al., 2007; Gévaudant et al., 2007). However, the active Na⁺/H⁺ antiport across PM and the contribution to salt exclusion have been rarely investigated in tree species.Munns and Tester (2008) claimed that Cl⁻ toxicity is more important than Na⁺ toxicity in some woody species, e.g. citrus. Similarly, we have noticed that the inability to restrict Cl⁻ uptake contributes to the NaCl-induced salt damage in salt-sensitive poplar (Populus spp.) species, in addition to toxicity of excess Na⁺ (Chen et al., 2001, 2002, 2003). The differences in Cl⁻ tolerance exhibited by plants are usually related to the ability to restrict Cl⁻ transport to the aerial part (Greenway and Munns, 1980; White and Broadley, 2001). Excluding Cl⁻ from the xylem seems to be an effective mechanism for lotus to cope with the interactive effect of salt and water logging (Teakle et al., 2007). An influx of Cl⁻, immediately after addition of NaCl, was observed in bean (Vicia faba) mesophyll tissue (Shabala, 2000). The Cl⁻ flux response to salt shock is helpful to reveal the rapid adjustments of plants to salinity. However, Cl⁻ fluxes in salt-adapted roots, which are necessary to clarify plant adaptations to long durations of salinity, have not been examined.Populus euphratica has been widely considered as a model plant to elucidate physiological and molecular mechanisms of salt tolerance in woody species (Chen et al., 2001, 2002, 2003; Gu et al., 2004; Ottow et al., 2005a, 2005b; Junghans et al., 2006; Wang et al., 2007, 2008; Wu et al., 2007; Zhang et al., 2007). Comparative studies have shown that salt-stressed P. euphratica seedlings accumulate less Na⁺ and Cl⁻ in root and shoot tissues than salt-sensitive poplar species (Chen et al., 2001, 2002). It is suggested that the greater capacity to exclude NaCl in P. euphratica is likely the result of salt uptake and transport restriction in roots (Chen et al., 2002, 2003). However, this needs further investigations, e.g. by electrophysiology, to clarify.In this study, we used a noninvasive ion flux technique to measure the tissue and cellular fluxes of H⁺, Na⁺, and Cl⁻ in roots of the salt-tolerant P. euphratica and salt-sensitive P. popularis 35-44. The aim was to compare the NaCl-induced alternations of ion fluxes in poplar species differing in salt tolerance. Furthermore, we examined the effects of pH, salt shock, and PM transport inhibitors on Na⁺ and H⁺ fluxes in root-derived protoplasts of the salt-tolerant species, P. euphratica, which exhibited an evident Na⁺ exclusion under saline conditions.