Cytosolic pH Research Articles

Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.

Most carbonic anhydrases catalyse the reversible conversion of carbon dioxide to protons and bicarbonate, either as soluble cytosolic enzymes, in or at intracellular organelles, or at the extracellular face of the cell membrane as membrane-anchored proteins. Carbonic anhydrase isoform IX (CA IX), a membrane-bound enzyme with catalytic activity at the extracellular membrane surface, has come to prominence in recent years because of its association with hypoxic tissue, particularly tumours, often indicating poor prognosis. We have evaluated the catalytic activity of CA IX heterologously expressed in Xenopus laevis oocytes by measuring the amplitude and rate of cytosolic pH changes as well as pH changes at the outer membrane surface (pHs ) during addition and removal of 5% CO2 /25 mm HCO3-, and by mass spectrometry. Our results indicate both extracellular and intracellular catalytic activity of CA IX. Reduced rates of CO2 -dependent intracellular pH changes after knockdown of CA IX confirmed these findings in two breast cancer cell lines: MCF-7 and MDA-MB-231. Our results demonstrate a new function of CA IX that may be important in the search for therapeutic cancer drugs targeting CA IX.

Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin.

The K(+),H(+) ionophore and antibiotic nigericin has been shown to trigger apoptosis and is thus considered for the treatment of malignancy. Cellular mechanisms involved include induction of oxidative stress, which is known to activate erythrocytic Ca(2+)-permeable unselective cation channels leading to Ca(2+) entry, increase in cytosolic Ca(2+) activity ([Ca(2+)]i) and subsequent stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. This study explored whether and how nigericin induces eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3 fluorescence, pHi from BCECF fluorescence, ceramide abundance utilizing antibodies and reactive oxygen species (ROS) formation from DCFDA-dependent fluorescence. A 48-hr exposure of human erythrocytes to nigericin significantly increased the percentage of annexin-V-binding cells (0.1-10 nM), significantly decreased forward scatter (0.1-1 nM), significantly decreased cytosolic pH (0.1-1 nM) and significantly increased Fluo3 fluorescence (0.1-10 nM). Nigericin (1 nM) slightly, but significantly, increased ROS, but did not significantly modify ceramide abundance. The effect of nigericin on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca(2+). The nigericin-induced increase in [Ca(2+)]i and annexin V binding was again significantly blunted but not abolished by the Na(+)/H(+) exchanger inhibitor cariporide (10 μM). Nigericin triggers eryptosis, an effect paralleled by ROS formation, in part dependent on stimulation of Ca(2+) entry, and involving the cariporide-sensitive Na(+)/H(+) exchanger.

On a quest for stress tolerance genes: membrane transporters in sensing and adapting to hostile soils.

Abiotic stresses such as salinity, drought, and flooding severely limit food and fibre production and result in penalties of in excess of US$100 billion per annum to the agricultural sector. Improved abiotic stress tolerance to these environmental constraints via traditional or molecular breeding practices requires a good understanding of the physiological and molecular mechanisms behind roots sensing of hostile soils, as well as downstream signalling cascades to effectors mediating plant adaptive responses to the environment. In this review, we discuss some common mechanisms conferring plant tolerance to these three major abiotic stresses. Central to our discussion are: (i) the essentiality of membrane potential maintenance and ATP production/availability and its use for metabolic versus adaptive responses; (ii) reactive oxygen species and Ca(2+) 'signatures' mediating stress signalling; and (iii) cytosolic K(+) as the common denominator of plant adaptive responses. We discuss in detail how key plasma membrane and tonoplast transporters are regulated by various signalling molecules and processes observed in plants under stress conditions (e.g. changes in membrane potential; cytosolic pH and Ca(2+); reactive oxygen species; polyamines; abscisic acid) and how these stress-induced changes are related to expression and activity of specific ion transporters. The reported results are then discussed in the context of strategies for breeding crops with improved abiotic stress tolerance. We also discuss a classical trade-off between tolerance and yield, and possible avenues for resolving this dilemma.

Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells.

Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8-7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng ml(-1) LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH-sensitive indicator (pHrodo(®)) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker(®) Red DND-99 staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells.

The influence of chlorsulfuron herbicide on GABA metabolism and oxidative damage in lentil (Lens culinaris Medik) and wheat (Triticum aestivum L.) seedlings

Abiotic stresses like salinity, drought, cold, heat, nutrient deficiency, and nutrient toxicity act as a major threats for crops quality and productivity. Herbicides are large category of chemical classes that affect various metabolic pathways and energy cascade in plants. γ-aminobutyric acid (GABA) is proposed to be critically involved in nitrogen metabolism, cytosolic pH regulation, and protection against oxidative imbalance in response to a wide range of abiotic stresses in plants. The objectives of this study were to determine the influence of chlorsulfuron herbicide on GABA metabolism and oxidative damage in two wheat (Triticumaestivum L.) cultivars (Hurani 75 and Um Qayes) and two lentil cultivars (Jordan 1 and Jordan 2) with respect to seed germination, GABA and malondialdehyde (MDA) levels accumulation, transcription of glutamate decarboxylase gene (GAD) mRNA level, total proteins, and carbohydrates contents. A significant increase in GABA, MDA and GAD mRNA level, proteins, and carbohydrates contents under chlorsulfuron treatments in the wheat and lentil cultivars was found in this study. The significant increase in GAD gene expression indicates the increase in GABA level under chlorsulfuron treatments. Hurani 75 and Jordan 2 cultivars showed significant decrease in protein level at high concentration of chlorsulfuron, whereas Um Qayes and Jordan 1 cultivars showed more tolerance under the same treatments. This study indicates that GABA metabolism might be one of multiple integrated signaling and metabolic pathways of herbicide stress adaptation and tolerance in wheat and lentil seedlings. This suggests that GABA molecule may act as a protective and metabolic signaling molecule in carbohydrates and proteins metabolism in plants under herbicidal treatments.

Aquaporins in Plants.

Aquaporins are membrane channels that facilitate the transport of water and small neutral molecules across biological membranes of most living organisms. In plants, aquaporins occur as multiple isoforms reflecting a high diversity of cellular localizations, transport selectivity, and regulation properties. Plant aquaporins are localized in the plasma membrane, endoplasmic reticulum, vacuoles, plastids and, in some species, in membrane compartments interacting with symbiotic organisms. Plant aquaporins can transport various physiological substrates in addition to water. Of particular relevance for plants is the transport of dissolved gases such as carbon dioxide and ammonia or metalloids such as boron and silicon. Structure-function studies are developed to address the molecular and cellular mechanisms of plant aquaporin gating and subcellular trafficking. Phosphorylation plays a central role in these two processes. These mechanisms allow aquaporin regulation in response to signaling intermediates such as cytosolic pH and calcium, and reactive oxygen species. Combined genetic and physiological approaches are now integrating this knowledge, showing that aquaporins play key roles in hydraulic regulation in roots and leaves, during drought but also in response to stimuli as diverse as flooding, nutrient availability, temperature, or light. A general hydraulic control of plant tissue expansion by aquaporins is emerging, and their role in key developmental processes (seed germination, emergence of lateral roots) has been established. Plants with genetically altered aquaporin functions are now tested for their ability to improve plant tolerance to stresses. In conclusion, research on aquaporins delineates ever expanding fields in plant integrative biology thereby establishing their crucial role in plants.

Pathogenesis of Adrenal Aldosterone-Producing Adenomas Carrying Mutations of the Na(+)/K(+)-ATPase.

Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism, leading to secondary hypertension. Somatic mutations in the gene for the α1 subunit of the Na(+)/K(+)-ATPase were found in about 6% of APAs. APA-related α1 subunit of the Na(+)/K(+)-ATPase mutations lead to a loss of the pump function of the Na(+)/K(+)-ATPase, which is believed to result in membrane depolarization and Ca(2+)-dependent stimulation of aldosterone synthesis in adrenal cells. In addition, H(+) and Na(+) leak currents via the mutant Na(+)/K(+)-ATPase were suggested to contribute to the phenotype. The aim of this study was to investigate the cellular pathophysiology of adenoma-associated Na(+)/K(+)-ATPase mutants (L104R, V332G, G99R) in adrenocortical NCI-H295R cells. The expression of these Na(+)/K(+)-ATPase mutants depolarized adrenal cells and stimulated aldosterone secretion. However, an increase of basal cytosolic Ca(2+) levels in Na(+)/K(+)-ATPase mutant cells was not detectable, and stimulation with high extracellular K(+) hardly increased Ca(2+) levels in cells expressing L104R and V332G mutant Na(+)/K(+)-ATPase. Cytosolic pH measurements revealed an acidification of L104R and V332G mutant cells, despite an increased activity of the Na(+)/H(+) exchanger. The possible contribution of cellular acidification to the hypersecretion of aldosterone was supported by the observation that aldosterone secretion of normal adrenocortical cells was stimulated by acetate-induced acidification. Taken together, mutations of the Na(+)/K(+)-ATPase depolarize adrenocortical cells, disturb the K(+) sensitivity, and lower intracellular pH but, surprisingly, do not induce an overt increase of intracellular Ca(2+). Probably, the autonomous aldosterone secretion is caused by the concerted action of several pathological signaling pathways and incomplete cellular compensation.

The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH

NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.

Carbonic anhydrase IX inhibition is an effective strategy for osteosarcoma treatment

Objective: Hypoxia-inducible factor 1, a regulator of CA IX activity, is often overexpressed in human osteosarcoma (OS) but not in normal tissues, and its expression levels correlate with prognosis. In this study, we investigated the therapeutic potential of newly synthesized CA IX sulfonamide inhibitors in OS.Methods: CA IX expression was evaluated in OS cell lines and bone marrow stromal cells (BMSC). After treatment with CA IX inhibitors, cell proliferation, apoptosis, cell cycle, extracellular and cytosolic pH changes were evaluated both in vitro and in mouse OS xenografts.Results: CA IX expression levels were significantly higher in OS than in BMSC. Accordingly, CA IX inhibitor 3 induced remarkable cytotoxicity on OS cells without affecting BMSC proliferation. This activity was increased under hypoxia, and was mediated by cell cycle arrest and by the modulation of cytosolic and extracellular pH. In vivo, CA IX inhibitor 3 reduced tumor growth by inducing significant necrosis.Conclusions: Our results provide a strong rationale for the clinical use of the newly synthesized CA IX inhibitor 3 in human OS.

Toward a biophysical understanding of the salt stress response of individual plant cells

We present and explore a kinetic model of ion transport across and between the membranes of an isolated plant cell with an emphasis on the cell's response to salt (Na+) stress. The vacuole, cytoplasm and apoplast are treated as concentric regions separated by tonoplast and plasma membranes. The model includes the transport of Na+, K+, Cl− and H+ across both membranes via primary active proton pumps, secondary active antiporters and symporters, as well as passive ion channels. In addition, water transport is included, allowing us to investigate both the osmotic and ionic components of salt stress. The model's predictions of steady state and transient cytosolic pH and Na+ concentrations were found to be quantitatively comparable to measured experimental values. Through an extensive simulation study we have identified and characterized scenarios in which individual transport processes (H+ pumps, Na+/H+ antiporters and channels involved in the transport of Na+) and their combinations have major effects on the level of Na+ in each of the cell compartments. This systematic study emulates the effects of overexpressing and inhibiting transporter genes by genetic modification and hence we have compared our simulations with observations from experiments conducted on transgenic plants. The simulations suggest that overexpressing tonoplast Na+/H+ antiporter genes and tonoplast H+ pump genes lead to an increase in the storage of Na+ in the vacuole (helping the cell to maintain water uptake under salt stress), with only a transient influence on the cytoplasmic Na+ concentration. The model predicts effects of varying the expression of transporter genes (individually or in combination) which have yet to be investigated in experiments. For example, our findings indicate that simultaneously overexpressing plasma membrane and tonoplast Na+/H+ antiporter genes would lead to improvements in both ionic and osmotic stress tolerance. The results demonstrate the importance of simultaneously modelling the transport of Na+ across both the tonoplast and plasma membrane, a task not undertaken previously.

The Cytosolic pH of Individual Saccharomyces cerevisiae Cells Is a Key Factor in Acetic Acid Tolerance.

It was shown recently that individual cells of an isogenic Saccharomyces cerevisiae population show variability in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the population level. In the current study, we investigated whether cell-to-cell variability in acetic acid tolerance could be explained by the observed differences in the cytosolic pHs of individual cells immediately before exposure to the acid. Results obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic pH drop after exposure to the acid. Moreover, only cells with a low initial cytosolic pH, which experienced a less severe drop in cytosolic pH, were able to proliferate. A similar correlation between initial cytosolic pH and cytosolic pH drop was also observed in the more acid-tolerant strain MUCL 11987-9. Interestingly, a fraction of cells in the MUCL 11987-9 population showed initial cytosolic pH values below the minimal cytosolic pH detected in cells of the strain CEN.PK113-7D; consequently, these cells experienced less severe drops in cytosolic pH. Although this might explain in part the difference between the two strains with regard to the number of cells that resumed proliferation, it was observed that all cells from strain MUCL 11987-9 were able to proliferate, independently of their initial cytosolic pH. Therefore, other factors must also be involved in the greater ability of MUCL 11987-9 cells to endure strong drops in cytosolic pH.

Novel Paraoxonase 2-Dependent Mechanism Mediating the Biological Effects of the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxo-Dodecanoyl)-L-Homoserine Lactone.

Pseudomonas aeruginosa produces N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12), a crucial signaling molecule that elicits diverse biological responses in host cells thought to subvert immune defenses. The mechanism mediating many of these responses remains unknown. The intracellular lactonase paraoxonase 2 (PON2) hydrolyzes and inactivates 3OC12 and is therefore considered a component of host cells that attenuates 3OC12-mediated responses. Here, we demonstrate in cell lines and in primary human bronchial epithelial cells that 3OC12 is rapidly hydrolyzed intracellularly by PON2 to 3OC12 acid, which becomes trapped and accumulates within the cells. Subcellularly, 3OC12 acid accumulated within the mitochondria, a compartment where PON2 is localized. Treatment with 3OC12 caused a rapid PON2-dependent cytosolic and mitochondrial pH decrease, calcium release, and phosphorylation of stress signaling kinases. The results indicate a novel, PON2-dependent intracellular acidification mechanism by which 3OC12 can mediate its biological effects. Thus, PON2 is a central regulator of host cell responses to 3OC12, acting to decrease the availability of 3OC12 for receptor-mediated effects and acting to promote effects, such as calcium release and stress signaling, via intracellular acidification.

Cell cycle progression is regulated by intertwined redox oscillators.

The different phases of the eukaryotic cell cycle are exceptionally well-preserved phenomena. DNA decompaction, RNA and protein synthesis (in late G1 phase) followed by DNA replication (in S phase) and lipid synthesis (in G2 phase) occur after resting cells (in G0) are committed to proliferate. The G1 phase of the cell cycle is characterized by an increase in the glycolytic metabolism, sustained by high NAD+/NADH ratio. A transient cytosolic acidification occurs, probably due to lactic acid synthesis or ATP hydrolysis, followed by cytosolic alkalinization. A hyperpolarized transmembrane potential is also observed, as result of sodium/potassium pump (NaK-ATPase) activity. During progression of the cell cycle, the Pentose Phosphate Pathway (PPP) is activated by increased NADP+/NADPH ratio, converting glucose 6-phosphate to nucleotide precursors. Then, nucleic acid synthesis and DNA replication occur in S phase. Along with S phase, unpublished results show a cytosolic acidification, probably the result of glutaminolysis occurring during this phase. In G2 phase there is a decrease in NADPH concentration (used for membrane lipid synthesis) and a cytoplasmic alkalinization occurs. Mitochondria hyperfusion matches the cytosolic acidification at late G1/S transition and then triggers ATP synthesis by oxidative phosphorylation. We hypothesize here that the cytosolic pH may coordinate mitochondrial activity and thus the different redox cycles, which in turn control the cell metabolism.

Synthesis and bioevaluation of heterocyclic derivatives of Cleistanthin-A

The vacuolar H+-ATPase (V-ATPase) was proposed as a key target for new strategies in cancer treatment recently. We have synthesized a novel class of derivatives of Cleistanthin-A bearing heterocyclic moieties. Most of these compounds displayed potent antiproliferative effects on four cancer cells at submicromolar concentration, and they have no cytotoxicity on normal WRL-68 cells at 200nM. The most potent compound 3a has been shown to inhibit the activity of vacuolar H+-ATPase at submicromolar concentration, and it could also significantly decrease the cytosolic pH values in HepG2 cells. The current findings provide valuable insights for future development of novel V-ATPase inhibitors as anticancer agents.

Involvement of Ca2+in Vacuole Degradation Caused by a Rapid Temperature Decrease inSaintpauliaPalisade Cells: A Case of Gene Expression Analysis in a Specialized Small Tissue

Saintpaulia (African violet) leaves are known to be damaged by a rapid temperature decrease when cold water is applied to the leaf surface; the injury is ascribed to the chloroplast damage caused by the cytosolic pH decrease following the degradation of the vacuolar membrane in the palisade cells. In this report, we present evidence for the involvement of Ca(2+) in facilitating the collapse of the vacuolar membrane and in turn in the temperature sensitivity of Saintpaulia leaves. In the presence of a Ca(2+) chelator (EGTA) or certain Ca(2+) channel inhibitors (Gd(3+) or La(3+)) but not others (verapamil or nifedipine), the pH of the vacuole, monitored through BCECF (2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein) fluorescence, did not increase in response to a rapid temperature drop. These pharmacological observations are consistent with the involvement of mechanosensitive Ca(2+) channels in the collapse of the vacuolar membrane. The high level of expression of an MCA- (Arabidopsis mechanosensitive Ca(2+) channel) like gene, a likely candidate for a mechanosensitive Ca(2+) channel(s) in plant cells, was confirmed in the palisade tissue in Saintpaulia leaves by using a newly developed method of gene expression analysis for the specialized small tissues.

LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

ObjectiveLeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H+ and lactate. H+ extrusion is in part accomplished by Na+/H+ exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. MethodsNHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pHi estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. ResultsA 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na+/H+ exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). ConclusionsLeftyA markedly down-regulates NHE1 expression, Na+/H+ exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition.

TM9SF4 is a novel V-ATPase-interacting protein that modulates tumor pH alterations associated with drug resistance and invasiveness of colon cancer cells.

An inverted pH gradient across the cell membranes is a typical feature of malignant cancer cells that are characterized by extracellular acidosis and cytosol alkalization. These dysregulations are able to create a unique milieu that favors tumor progression, metastasis and chemo/immune-resistance traits of solid tumors. A key event mediating tumor cell pH alterations is an aberrant activation of ion channels and proton pumps such as (H+)-vacuolar-ATPase (V-ATPase). TM9SF4 is a poorly characterized transmembrane protein that we have recently shown to be related to cannibal behavior of metastatic melanoma cells. Here, we demonstrate that TM9SF4 represents a novel V-ATPase-associated protein involved in V-ATPase activation. We have observed in HCT116 and SW480 colon cancer cell lines that TM9SF4 interacts with the ATP6V1H subunit of the V-ATPase V1 sector. Suppression of TM9SF4 with small interfering RNAs strongly reduces assembly of V-ATPase V0/V1 sectors, thus reversing tumor pH gradient with a decrease of cytosolic pH, alkalization of intracellular vesicles and a reduction of extracellular acidity. Such effects are associated with a significant inhibition of the invasive behavior of colon cancer cells and with an increased sensitivity to the cytotoxic effects of 5-fluorouracil. Our study shows for the first time the important role of TM9SF4 in the aberrant constitutive activation of the V-ATPase, and the development of a malignant phenotype, supporting the potential use of TM9SF4 as a target for future anticancer therapies.

Impact of phenanthrene exposure on activities of nitrate reductase, phosphoenolpyruvate carboxylase, vacuolar H+-pyrophosphatase and plasma membrane H+-ATPase in roots of soybean, wheat and carrot

Crops differ in uptake of polycyclic aromatic hydrocarbons (PAHs). However, the physiological mechanism underlying the differences in PAH uptake among crop species is unclear. As PAH transport by H+-coupled symporters may affect cytosolic pH, and intracellular pH is precisely regulated for metabolism, four enzymes linked with cytosolic pH regulation in root cells were determined at different concentrations of phenanthrene (PHE) and pHs. Activity of nitrate reductase (NRase) is stimulated by PHE and follows the order: wheat>carrot>soybean. The optimum pH of NRase is 7.5 in wheat and carrot, while 6.5 in soybean. The activity of phosphoenolpyruvate carboxylase (PEPCase) is generally decreased by PHE and always lower in soybean than in wheat and carrot. PEPCase peaks its maximum activity at pH 7.0 and wheat shows the highest value with high PHE (1.2mgL−1) at pHs of 7.0 and 8.0 in comparison with carrot and soybean. Vacuolar H+-pyrophosphatase (V-H+-PPase) is promoted by PHE in wheat and carrot, while it does not change significantly in soybean (P>0.05). V-H+-PPase reaches its maximum value at pH 7.5 and follows the order of wheat>carrot>soybean at alkaline medium with high PHE. Plasma membrane (PM) H+-ATPase is enhanced by K+ and follows the order: wheat>soybean>carrot. The orders of activity of these enzymes are not consistent with that of PHE uptake, indicating their various functions. However, in soybean, PM H+-ATPase activity under high PHE and the uptake of PHE are always higher than those in wheat and carrot, suggesting that PM H+-ATPase plays an essential role in PHE uptake. Therefore, the changes of PM H+-ATPase activity seem to be a direct consequence responding to PHE stress and those of NRase, PEPCase and V-H+-PPase might be the downstream accidents regulated by cytosoilc pH. Our study provides insight into the roles of these enzymes in non-photosynthetic tissues and supports their importance in the physiological responses to PHE stress among different crops.

Self-incompatibility-induced programmed cell death in field poppy pollen involves dramatic acidification of the incompatible pollen tube cytosol.

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]cyt of the pollen tubes in vivo, we show that [pH]cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]cyt and its effect on several components in the context of SI-induced events and PCD.

Regulation of Na+/H+ Exchanger in Dendritic Cells by Akt1

Background/Aims: Dendritic cells (DCs), antigen-presenting cells critically important for primary immune response and establishment of immunological memory, are activated by bacterial lipopolysaccharides (LPS) resulting in stimulation of Na⁺/H⁺ exchanger, ROS formation and migration. The effects are dependent on phosphoinositide 3 (PI3) kinase and paralleled by Akt phosphorylation. The present study explored the contribution of the Akt isoform Akt1. Methods: Cytosolic pH (pH_i) (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence), Na⁺/H⁺ exchanger activity (Na⁺ dependent realkalinization after an ammonium pulse), cell volume (forward scatter in FACS analysis), and ROS production (2′,7′-dichlorodihydrofluorescein diacetate [DCFDA] fluorescence) were determined in DCs isolated from bone marrow of mice lacking functional Akt1/PKBα (akt1^-/-) and their wild type littermates (akt1^+/+). Results: Forward scatter was lower in akt1^-/- than in akt1^+/+ DCs, whereas pH_i, Na⁺/H⁺ exchanger activity and ROS formation were less in untreated akt1^-/- and akt1^+/+ DCs. Exposure of DCs to LPS was followed by increase of forward scatter and ROS formation to a similar extent in akt1^-/- and in akt1^+/+ DCs. A 4 hours treatment with either LPS (1µg/ml) or tert-butylhydroperoxide (tBOOH, 5 µM) significantly stimulated Na⁺/H⁺ exchanger activity in both genotypes, effects, however, significantly blunted in akt1^-/- DCs. Conclusion: The present observations demonstrate that Akt1 is required for the full stimulation of Na⁺/H⁺ exchanger activity by LPS or oxidative stress in dendritic cells.