Cytosolic pH Regulation Research Articles

Cytosolic and Acrosomal pH Regulation in Mammalian Sperm.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

C-di-AMP determines the hierarchical organization of bacterial RCK proteins

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.

Modulation of TRPM8 alters the phagocytic activity of microglia and induces changes in sub-cellular organelle functions

In this work, we investigated the presence and function of TRPM8, a non-selective and cold-sensitive Ca2+-permeable ion channel in the primary microglia cell as well as in microglia cell line BV2. We demonstrate that primary microglia as well as BV2 express TRPM8 endogenously. Both pharmacological activation or inhibition of TRPM8 causes enhanced uptake of bacterial particles at early time points of infection. In BV2, TRPM8 activation and/or LPS-signaling alters its surface expression and cytosolic ROS production. TRPM8 modulation in the absence and presence of LPS causes differential regulation of cytosolic pH and lysosomal pH. Notably, TRPM8 modulation also alters the correlation between lysosomal pH and cytosolic pH depending on TRPM8 modulation and the presence or absence of LPS. Collectively our data suggest that TRPM8 is involved in the regulation of subcellular organelle, i.e. mitochondrial and lysosomal functions. Data also suggest that primarily TRPM8 activation, but often deviation from endogenous TRPM8 function is linked with better innate immune function mediated by microglial cells. We suggest that TRPM8-mediated regulations of sub-cellular organelle functions are more context-dependent manner. Such understanding is relevant in the context of microglial cell functions and innate immunity.

Role of the Voltage-Gated Proton Channel Hv1 in Nervous Systems.

Hv1 is the only voltage-gated proton-selective channel in mammalian cells. It contains a conserved voltage-sensor domain, shared by a large class of voltage-gated ion channels, but lacks a pore domain. Its primary role is to extrude protons from the cytoplasm upon pH reduction and membrane depolarization. The best-known function of Hv1 is the regulation of cytosolic pH and the nicotinamide adenine dinucleotide phosphate oxidase-dependent production of reactive oxygen species. Accumulating evidence indicates that Hv1 is expressed in nervous systems, in addition to immune cells and others. Here, we summarize the molecular properties, distribution, and physiological functions of Hv1 in the peripheral and central nervous systems. We describe the recently discovered functions of Hv1 in various neurological diseases, including brain or spinal cord injury, ischemic stroke, demyelinating diseases, and pain. We also summarize the current advances in the discovery and application of Hv1-targeted small molecules in neurological diseases. Finally, we discuss the current limitations of our understanding of Hv1 and suggest future research directions.

Structural and Functional Diversity of Two ATP-Driven Plant Proton Pumps.

Two ATP-dependent proton pumps function in plant cells. Plasma membrane H+-ATPase (PM H+-ATPase) transfers protons from the cytoplasm to the apoplast, while vacuolar H+-ATPase (V-ATPase), located in tonoplasts and other endomembranes, is responsible for proton pumping into the organelle lumen. Both enzymes belong to two different families of proteins and, therefore, differ significantly in their structure and mechanism of action. The plasma membrane H+-ATPase is a member of the P-ATPases that undergo conformational changes, associated with two distinct E1 and E2 states, and autophosphorylation during the catalytic cycle. The vacuolar H+-ATPase represents rotary enzymes functioning as a molecular motor. The plant V-ATPase consists of thirteen different subunits organized into two subcomplexes, the peripheral V1 and the membrane-embedded V0, in which the stator and rotor parts have been distinguished. In contrast, the plant plasma membrane proton pump is a functional single polypeptide chain. However, when the enzyme is active, it transforms into a large twelve-protein complex of six H+-ATPase molecules and six 14-3-3 proteins. Despite these differences, both proton pumps can be regulated by the same mechanisms (such as reversible phosphorylation) and, in some processes, such as cytosolic pH regulation, may act in a coordinated way.

Regulation of Cytosolic pH: The Contributions of Plant Plasma Membrane H+-ATPases and Multiple Transporters.

Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H+ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H+ transport activity, namely, H+-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H+ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H+-ATPases mediate H+ extrusion, whereas most members of other six proteins mediate H+ influx, thus contributing to cytosolic pH homeostasis by directly modulating H+ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H+-coupled substrate influx, whereas other intra-family members facilitate H+-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H+-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H+ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H+ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.

Plant Nitrate Reductases Regulate Nitric Oxide Production and Nitrogen-Fixing Metabolism During the Medicago truncatula–Sinorhizobium meliloti Symbiosis

Nitrate reductase (NR) is the first enzyme of the nitrogen reduction pathway in plants, leading to the production of ammonia. However, in the nitrogen-fixing symbiosis between legumes and rhizobia, atmospheric nitrogen (N2) is directly reduced to ammonia by the bacterial nitrogenase, which questions the role of NR in symbiosis. Next to that, NR is the best-characterized source of nitric oxide (NO) in plants, and NO is known to be produced during the symbiosis. In the present study, we first surveyed the three NR genes (MtNR1, MtNR2, and MtNR3) present in the Medicago truncatula genome and addressed their expression, activity, and potential involvement in NO production during the symbiosis between M. truncatula and Sinorhizobium meliloti. Our results show that MtNR1 and MtNR2 gene expression and activity are correlated with NO production throughout the symbiotic process and that MtNR1 is particularly involved in NO production in mature nodules. Moreover, NRs are involved together with the mitochondrial electron transfer chain in NO production throughout the symbiotic process and energy regeneration in N2-fixing nodules. Using an in vivo NMR spectrometric approach, we show that, in mature nodules, NRs participate also in the regulation of energy state, cytosolic pH, carbon and nitrogen metabolism under both normoxia and hypoxia. These data point to the importance of NR activity for the N2-fixing symbiosis and provide a first explanation of its role in this process.

Water Electrolysis Carried Out on Microelectrodes to Obtain New Insights into the Regulation of Cytosolic pH

AbstractIn this study, the use of electrochemistry in cell biology is extended by showing that electrochemically generated microscale pH gradients can be used to gain new insights into the regulation of cytosolic pH of normal and cancer cells. The developed procedure involves positioning a carbon fiber microelectrode into the extracellular space of adherently growing cells and setting its potential to values suitable for electrooxidation or electroreduction of water. While the electrooxidation of water decreases the pH of the solution surrounding the microelectrode (because it produces hydronium ions), the electroreduction of water increases the pH of the same solution (because it produces hydroxide ions). Fluorescence microscopy is then used to observe the impact of the electrochemically generated microscale pH gradient on the cytosolic pH of cells loaded with a fluorescent pH sensor. The obtained results indicate that electrochemically induced acid stress affects the cytosolic pH of normal cells significantly faster than that of cancer cells while electrochemically induced alkaline stress appears to have very limited impact on the cytosolic pH of both cell types. In comparison to classic experiments concerning the regulation of cytosolic pH using perfusion chambers, the developed, electrochemistry‐based, approach has the advantages of a better spatial and temporal resolution and elimination of the flow‐induced shear stress.

Plant Endomembrane Dynamics: Studies of K+/H+ Antiporters Provide Insights on the Effects of pH and Ion Homeostasis.

Plants remodel their cells through the dynamic endomembrane system. Intracellular pH is important for membrane trafficking, but the determinants of pH homeostasis are poorly defined in plants. Electrogenic proton (H+) pumps depend on counter-ion fluxes to establish transmembrane pH gradients at the plasma membrane and endomembranes. Vacuolar-type H+-ATPase-mediated acidification of the trans-Golgi network is crucial for secretion and membrane recycling. Pump and counter-ion fluxes are unlikely to fine-tune pH; rather, alkali cation/H+ antiporters, which can alter pH and/or cation homeostasis locally and transiently, are prime candidates. Plants have a large family of predicted cation/H+ exchangers (CHX) of obscure function, in addition to the well-studied K+(Na+)/H+ exchangers (NHX). Here, we review the regulation of cytosolic and vacuolar pH, highlighting the similarities and distinctions of NHX and CHX members. In planta, alkalinization of the trans-Golgi network or vacuole by NHXs promotes membrane trafficking, endocytosis, cell expansion, and growth. CHXs localize to endomembranes and/or the plasma membrane and contribute to male fertility, pollen tube guidance, pollen wall construction, stomatal opening, and, in soybean (Glycine max), tolerance to salt stress. Three-dimensional structural models and mutagenesis of Arabidopsis (Arabidopsis thaliana) genes have allowed us to infer that AtCHX17 and AtNHX1 share a global architecture and a translocation core like bacterial Na+/H+ antiporters. Yet, the presence of distinct residues suggests that some CHXs differ from NHXs in pH sensing and electrogenicity. How H+ pumps, counter-ion fluxes, and cation/H+ antiporters are linked with signaling and membrane trafficking to remodel membranes and cell walls awaits further investigation.

PH regulation in glycosomes of procyclic form Trypanosoma brucei

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells

Background/Aims: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H⁺ ions, which is in part accomplished by Na⁺/H⁺ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi), NHE1 transcript levels, NHE1 protein abundance, Na⁺/H⁺ exchanger activity, and lactate release. Methods: Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM) treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pH_i utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na⁺/H⁺ exchanger activity from Na⁺ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS) from 2’,7’-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm)/ fluorometric enzymatic assay. Results: A 48 hour treatment with Ellagic acid (20 µM) significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na⁺/H⁺ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. Conclusion: Ellagic acid (20µM) markedly down-regulates ROS formation and NHE1 expression leading to decreased Na⁺/H⁺ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those effects presumably contribute to reprogramming and growth inhibition of tumor 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.

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.

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.

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

The SLC9 gene family encodes Na(+)/H(+) exchangers (NHEs). These transmembrane proteins transport ions across lipid bilayers in a diverse array of species from prokaryotes to eukaryotes, including plants, fungi, and animals. They utilize the electrochemical gradient of one ion to transport another ion against its electrochemical gradient. Currently, 13 evolutionarily conserved NHE isoforms are known in mammals [22, 46, 128]. The SLC9 gene family (solute carrier classification of transporters: www.bioparadigms.org) is divided into three subgroups [46]. The SLC9A subgroup encompasses plasmalemmal isoforms NHE1-5 (SLC9A1-5) and the predominantly intracellular isoforms NHE6-9 (SLC9A6-9). The SLC9B subgroup consists of two recently cloned isoforms, NHA1 and NHA2 (SLC9B1 and SLC9B2, respectively). The SLC9C subgroup consist of a sperm specific plasmalemmal NHE (SLC9C1) and a putative NHE, SLC9C2, for which there is currently no functional data [46]. NHEs participate in the regulation of cytosolic and organellar pH as well as cell volume. In the intestine and kidney, NHEs are critical for transepithelial movement of Na(+) and HCO3(-) and thus for whole body volume and acid-base homeostasis [46]. Mutations in the NHE6 or NHE9 genes cause neurological disease in humans and are currently the only NHEs directly linked to human disease. However, it is becoming increasingly apparent that members of this gene family contribute to the pathophysiology of multiple human diseases.

Human Voltage-Gated Proton Channel Hv1: A New Potential Biomarker for Diagnosis and Prognosis of Colorectal Cancer

Solid tumors exist in a hypoxic microenvironment, and possess high-glycolytic metabolites. To avoid the acidosis, tumor cells must exhibit a dynamic cytosolic pH regulation mechanism(s). The voltage-gated proton channel Hv1 mediates NADPH oxidase function by compensating cellular loss of electrons with protons. Here, we showed for the first time, that Hv1 expression is increased in colorectal tumor tissues and cell lines, associated with poor prognosis. Immunohistochemistry showed that Hv1 is strongly expressed in adenocarcinomas but not or lowly expressed in normal colorectal or hyperplastic polyps. Hv1 expression in colorectal cancer is significantly associated with the tumor size, tumor classification, lymph node status, clinical stage and p53 status. High Hv1 expression is associated significantly with shorter overall and recurrence-free survival. Furthermore, real-time RT-PCR and immunocytochemistry showed that Hv1 is highly expressed in colorectal cancer cell lines, SW620, HT29, LS174T and Colo205, but not in SW480. Inhibitions of Hv1 expression and activity in the highly metastatic SW620 cells by small interfering RNA (siRNA) and Zn2+ respectively, markedly decrease the cell invasion and migration, restraint proton extrusion and the intracellular pH recovery. Our results suggest that Hv1 may be used as a potential biomarker for diagnosis and prognosis of colorectal carcinoma, and a potential target for anticancer drugs in colorectal cancer therapy.

Effect of Azathioprine on Na+/H+ Exchanger Activity in Dendritic Cells

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na⁺/H⁺ exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na⁺/H⁺ exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH_i) was estimated utilizing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na⁺/H⁺ exchanger activity from the Na⁺-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 µg/ml) led to a transient increase of Na⁺/H⁺ exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 µM) did not significantly alter the Na⁺/H⁺ exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na⁺/H⁺ exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na⁺/H⁺ exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.

Influence of Dexamethasone on Na+/H+ Exchanger Activity in Dendritic Cells

Glucocorticoids regulate the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. Glucocorticoids influence the function of other cell types by modulating the activity of the Na⁺/H⁺exchanger (NHE), a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether dexamethasone influences Na⁺/H⁺ exchanger activity in DCs. The DCs were isolated from mouse bone marrow, cell volume was estimated from forward scatter in FACS analysis, cytosolic pH (pH_i) utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺ dependent realkalinization after an ammonium pulse. Treatment with the glucocorticoid dexamethasone (100 nM; 1, 4, 16 and 24h) significantly decreased pH_i (≧4 h) and gradually increased Na⁺/H⁺ exchanger activity (=16 h). The stimulation of Na⁺/H⁺ exchanger activity by dexamethasone was virtually abrogated by glucocorticoid receptor blocker mefiprestone (1 µM) and NHE3 inhibitor dimethyl amiloride (5 µM), but not prevented by NHE1 inhibitor cariporide (10 µM). Dexamethasone treatment significantly increased SGK1 mRNA levels. Stimulation of Na⁺/H⁺ exchanger activity by dexamethasone was blunted in DCs lacking SGK1. Dexamethasone treatment did not significantly alter ROS formation but significantly decreased the forward scatter. Exposure of DCs to lipopolysacharide (LPS, 1 µg/ml) led to a transient increase followed by a decline of Na⁺/H⁺ exchanger activity and to enhanced forward scatter as well as ROS formation, all effects significantly blunted in the presence of dexamethasone (100 nM). In conclusion, glucocorticoid treatment decreased pH_i and cell volume, effects paralleled by upregulation of Na⁺/H⁺ exchanger activity in DCs. Moreover, glucocorticoids blunted the stimulation of Na⁺/H⁺ exchanger activity, cell swelling and ROS formation following LPS treatment.

Effect of Bacterial Lipopolysaccharide on Na+/H+ Exchanger Activity in Dendritic Cells

The function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, is stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS). In macrophages, ROS formation is paralleled by activation of the Na⁺/H⁺ exchanger, a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether LPS influence Na⁺/H⁺ exchanger activity in DCs. The DCs were isolated from murine bone marrow, cell volume was estimated from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, apoptosis from annexin V binding, cytosolic pH (pH_i) from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺ dependent realkalinization following an ammonium pulse. Exposure of DCs to LPS (1 µg/ml) led to a transient increase of Na⁺/H⁺ exchanger activity. Moreover, LPS increased forward scatter and ROS formation and decreased apoptosis. The NHE1 inhibitor cariporide (10 µM) virtually abrogated Na⁺/H⁺ exchanger activity, inhibited LPS-induced cell swelling, blunted LPS-induced ROS formation and reversed the antiapoptotic effect of LPS. Na⁺/H⁺ exchanger activity was stimulated by oxidative stress and LPS induced stimulation of NHE activity was abolished in presence of ROS chelators (Tempol, Tiron and Vitamin C). In conclusion, LPS treatment transiently upregulates the Na⁺/H⁺ exchanger in DCs, an effect required for the effects of LPS on DC survival, cell volume and ROS formation.

PH regulation in an acidophilic green alga – a quantitative analysis

Short-term cytosolic pH regulation has three components: H(+) binding by buffering groups; H(+) transport out of the cytosol; and H(+) transport into the vacuole. To understand the large differences plants show in their tolerance to acidic environments, these three components were quantified in the acidophilic unicellular green alga Eremosphaera viridis. Intracellular pH was recorded using ion-selective microelectrodes, whereas constant doses of weak acid were applied over different time intervals. A mathematical model was developed that describes the recorded cytosolic pH changes. Recordings of cytosolic K(+) and Na(+) activities, and application of anion channel inhibitors, revealed which ion fluxes electrically compensate H(+) transport. The cytosolic buffer capacity was beta = 30 mM. Acidification resulted in a substantial and constant H(+) efflux that was probably driven by the plasmalemma H(+)-ATPase, and a proportional pH regulation caused by H(+) pumped into the vacuole. Under severe cytosolic acidification (> or = 1 pH) more than 50% of the ATP synthesized was used for H(+) pumping. While H(+) influx into the vacuole was compensated by cation release, H(+) efflux out of the cell was compensated by anion efflux. The data presented here give a complete and quantitative picture of the ion fluxes during acid loading in an acidophilic green plant cell.