Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H⁺ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H⁺ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH_o) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH_i) and net H⁺ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH_i remained above 7.0 within a pH_o range of 4 to 6 and decreased below pH_o 4. Above pH_o 4, H⁺ extrusion increased correspondingly, with the maximum value at pH_o 5.5, and below pH_o 4, a net H⁺ influx was observed. Without glucose in the medium, the pH_i decreased, and a net H⁺ influx was observed below pH_o 5.5. A high correlation (R = 0.75 to 0.92) between the pH_i and net H⁺ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH_i homeostasis in L. monocytogenes subjected to acid stress.     

Cytosolic alkalinization mediated by abscisic Acid is necessary, but not sufficient, for abscisic Acid-induced gene expression in barley aleurone protoplasts

We investigated whether intracellular pH (pH_i) is a causal mediator in abscisic acid (ABA)-induced gene expression. We measured the change in pH_i by a “null-point” method during stimulation of barley (Hordeum vulgare cv Himalaya) aleurone protoplasts with ABA and found that ABA induces an increase in pH_i from 7.11 to 7.30 within 45 min after stimulation. This increase is inhibited by plasma membrane H⁺-ATPase inhibitors, which induce a decrease in pH_i, both in the presence and absence of ABA. This ABA-induced pH_i increase precedes the expression of RAB-16 mRNA, as measured by northern analysis. ABA-induced pH_i changes can be bypassed or clamped by addition of either the weak acids 5,5-dimethyl-2,4-oxazolidinedione and propionic acid, which decrease the pH_i, or the weak bases methylamine and ammonia, which increase the pH_i. Artificial pH_i increases or decreases induced by weak bases or weak acids, respectively, do not induce RAB-16 mRNA expression. Clamping of the pH_i at a high value with methylamine or ammonia treatment affected the ABA-induced increase of RAB-16 mRNA only slightly. However, inhibition of the ABA-induced pH_i increase with weak acid or proton pump inhibitor treatments strongly inhibited the ABA-induced RAB-16 mRNA expression. We conclude that, although the ABA-induced the pH_i increase is correlated with and even precedes the induction of RAB-16 mRNA expression and is an essential component of the transduction pathway leading from the hormone to gene expression, it is not sufficient to cause such expression.     

Altered proton extrusion in cells adapted to growth at low extracellular pH

Intracellular pH (pH_i) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pH_i in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pH_i relative to the extracellular pH (pH_e). To test the ability of low pH_e adapted cells to respond to a pH_i lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pH_i during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pH_e = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pH_i was consistently elevated in adapted cells as compared with cells grown at normal pH_e = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pH_i of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na⁺/H⁺ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl⁻/HCO⁻₃ exchange inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification. J. Cell. Physiol. 173:397–405, 1997. © 1997 Wiley-Liss, Inc.     

Transmembrane Proton Electrochemical Gradients in Dark Aerobic and Anaerobic Cells of the Cyanobacterium (Blue-Green Alga) Anacystis nidulans: Evidence for Respiratory Energy Transduction in the Plasma Membrane

The transmembrane proton electrochemical potential gradient Δμ_H+ in whole cells of Anacystis nidulans was measured in aerobic and anaerobic dark conditions using the distribution, between external medium and cell interior, of radioactively labeled weak acids (acetylsalicyclic acid, 5,5-dimethyloxazolidine-2,4-dione) or bases (imidazole, methylamine), and permeant ions (tetraphenylphosphonium cation, thiocyanate anion), as determined by flow dialysis. Alternatively, the movements across the plasma membrane of ΔpH-indicating atebrin or 9-aminoacridine, and of ΔΨ-indicating 8-anilino-l-naphthalenesulfonate were qualitatively followed by fluorescence measurements. Attempts were made to discriminate between the individual chemiosmotic gradients across the cytoplasmic (plasmalemma) and the intracytoplasmic (thylakoid) membranes. By use of the ionophores nigericin, monensin, and valinomycin, the components of the proton motive force, namely the proton concentration gradient ΔpH and the electric membrane potential ΔΨ were shown to be mutually exchangeable within the range of external pH values tested (3.2-11.0). Both components were depressed by the uncoupler carbonylcyanide m-chlorophenylhydrazone, though inhibition of ΔpH was much more pronounced than that of ΔΨ, notably in the alkaline pH₀ range. The total proton electrochemical gradient across the plasma membrane was significantly higher in aerobic than in anaerobic cells and increased markedly (i.e. became more negative) towards lower pH₀ values. This increase was paralleled by a similar increase in the rate of endogenous respiration of the cells. At the same time the ATPase inhibitor dicyclohexylcarbodiimide only slightly affected the proton motive force across the plasma membrane of aerobic cells. The results will be discussed in terms of a respiratorily competent plasma membrane in Anacystis nidulans.     

Role of Listeria monocytogenes σB in Survival of Lethal Acidic Conditions and in the Acquired Acid Tolerance Response

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σ^B, which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ΔsigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ΔsigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ΔsigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σ^B independent. σ^B-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σ^B reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σ^B does not appear to contribute to pH_i homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH_i buffering by the GAD system under the conditions examined in this study. In summary, a functional σ^B protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.     

Chaperone Stress 70 Protein (STCH) Binds and Regulates Two Acid/Base Transporters NBCe1-B and NHE1

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96–440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na⁺-dependent pH_i recovery from NH₄⁺ pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na⁺/H⁺ exchanger (NHE)-dependent pH_i recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca²⁺ ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pH_i through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pH_i regulation at times of cellular stress by enhancing the recovery from intracellular acidification.     

Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pH_i) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pH_i, allowing fast and noninvasive determination of pH_i that is ideally suited for direct analysis of individual bacterial cells present in complex environments.     

Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains

The effects of NaCl stress on cell area and intracellular pH (pH_i) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30–35%), it caused different pH_i changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pH_i homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pK_a (pK_a,i) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pK_a,i close to its pH_i homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pK_a,i to its pH_i homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pH_i were mainly due to hyperosmotic stress and not ionic stress.     

Quantitative Analysis of the Modes of Growth Inhibition by Weak Organic Acids in Saccharomyces cerevisiae

Weak organic acids are naturally occurring compounds that are commercially used as preservatives in the food and beverage industries. They extend the shelf life of food products by inhibiting microbial growth. There are a number of theories that explain the antifungal properties of these weak acids, but the exact mechanism is still unknown. We set out to quantitatively determine the contributions of various mechanisms of antifungal activity of these weak acids, as well as the mechanisms that yeast uses to counteract their effects. We analyzed the effects of four weak organic acids differing in lipophilicity (sorbic, benzoic, propionic, and acetic acids) on growth and intracellular pH (pH_i) in Saccharomyces cerevisiae. Although lipophilicity of the acids correlated with the rate of acidification of the cytosol, our data confirmed that not initial acidification, but rather the cell''s ability to restore pH_i, was a determinant for growth inhibition. This pH_i recovery in turn depended on the nature of the organic anion. We identified long-term acidification as the major cause of growth inhibition under acetic acid stress. Restoration of pH_i, and consequently growth rate, in the presence of this weak acid required the full activity of the plasma membrane ATPase Pma1p. Surprisingly, the proposed anion export pump Pdr12p was shown to play an important role in the ability of yeast cells to restore the pH_i upon lipophilic (sorbic and benzoic) acid stress, probably through a charge interaction of anion and proton transport.     

Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel,hHV1

Part of the “signature sequence” that defines the voltage-gated proton channel (H_V1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H_V1 genes. Replacing Trp²⁰⁷ in human H_V1 (hH_V1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, E_a, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp²⁰⁷ establishes the major energy barrier between closed and open hH_V1. Cation–π interaction between Trp²⁰⁷ and Arg²¹¹ evidently latches the channel closed. Trp²⁰⁷ mutants lost proton selectivity at pH_o >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H_V1 that is essential to its biological functions, was compromised. In the WT hH_V1, ΔpH-dependent gating is shown to saturate above pH_i or pH_o 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp²⁰⁷ mutants, ΔpH-dependent gating saturated at lower pH_o but not at lower pH_i. That Trp²⁰⁷ mutation selectively alters pH_o sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H_V1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pH_o and pH_i in H_V1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.     

Fused cells of frog proximal tubule: II. Voltage-dependent intracellular pH

Summary Experiments were performed in intact proximal tubules of the doubly perfused kidney and in fused proximal tubule cells ofRaha esculenta to evaluate the dependence of intracellular pH (pH_i) on cell membrane potential applying pH-sensitive and conventional microelectrodes. In proximal tubules an increase of the K^– concentration in the peritubular perfusate from 3 to 15 mmol/liter decreased the peritubular cell membrane potential from –55±2 to –38±1 mV paralleled by an increase of pH _i , from 7.54±0.02 to 7.66±0.02. The stilbene derivative DIDS hyperpolarized the cell membrane potential from –57 ± 2 to –71 ±4 mV and led to a significant increase of the K^–-induced cell membrane depolarization, but prevented the K^–-induced intracellular alkalinization. Fused proximal tubule cells were impaled by three microelectrodes simultaneously and cell voltage was clamped stepwise while pH _i changes were monitored. Cell membrane hyperpolarization acidified the cell cytoplasm in a linear relationship. This voltage-induced intracellular acidification was reduced to about one-third when HCO₃ ions were omitted from the extracellular medium. We conclude that in proximal tubule cells pH _i depends on cell voltage due to the rheogenicity of the HCO ₃ ^– transport system.     

Relationship between Acid Tolerance, Cytoplasmic pH, and ATP and H+-ATPase Levels in Chemostat Cultures of Lactococcus lactis

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pH_o). A decrease in either the dilution rate or the pH_o led to a decrease in the cytoplasmic pH (pH_i) of the cells, and similar levels of acid tolerance were observed at any specific pH_i irrespective of whether the pH_i resulted from manipulation of the growth rate, manipulation of the pH_o, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pH_o of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pH_o of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pH_i. The rate of ATP formation was not correlated with pH_i, but a good correlation between the cellular level of H⁺-ATPase and pH_i was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pH_i was established. Each pH_i below 6.6 was characterized by unique levels of ATR, H⁺-ATPase, and ATP. High levels of H⁺-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H⁺-ATPase is one of the ATR proteins induced by acid pH_i through growth at an acid pH_o or a slow growth rate.     

The acid tolerance response of Bacillus cereus ATCC14579 is dependent on culture pH, growth rate and intracellular pH

The food pathogen Bacillus cereus is likely to encounter acidic environments (i) in food when organic acids are added for preservation purposes, and (ii) during the stomachal transit of aliments. In order to characterise the acid stress response of B. cereus ATCC14579, cells were grown in chemostat at different pH values (pH_o from 9.0 to 5.5) and different growth rates (μ from 0.1 to 0.8 h⁻¹), and were submitted to acid shock at pH 4.0. Cells grown at low pH_o were adapted to acid media and induced a significant acid tolerance response (ATR). The ATR induced was modulated by both pH_o and μ, and the μ effect was more marked at pH_o 5.5. Intracellular pH (pH_i) was affected by both pH_o and μ. At a pH_o above 6, the pH_i decreased with the decrease of pH_o and the increase of μ. At pH_o 5.5, pH_i was higher compared to pH_o 6.0, suggesting that mechanisms of pH_i homeostasis were induced. The acid survival of B. cereus required protein neo-synthesis and the capacity of cells to maintain their pH_i and ΔpH (pH_i - pH_o). Haemolysin BL and non-haemolytic enterotoxin production were both influenced by pH_o and μ.     

The leak mode of type II Na+-Pi cotransporters

Na⁺-coupled phosphate cotransporters of the SLC34 gene family catalyze the movement of inorganic phosphate (P_i) across epithelia by using the free energy of the downhill electrochemical Na⁺ gradient across the luminal membrane. Electrogenic (NaPi-IIa/b) and electroneutral (NaPi-IIc) isoforms prefer divalent P_i and show strict Na⁺:P_i stoichiometries of 3:1 and 2:1, respectively. For electrogenic cotransport, one charge is translocated per transport cycle. When NaPi-IIa or NaPi-IIb are expressed in Xenopus oocytes, application of the Pi transport inhibitor phosphonoformic acid (PFA) blocks a leak current that is not detectable in the electroneutral isoform. In this review, we present the experimental evidence that this transport-independent leak originates from a Na⁺-dependent uniport carrier mode intrinsic to NaPi-IIa/b isoforms. Our findings, based on the characteristics of the PFA-inhibitable leak measured from wild-type and mutant constructs, can be incorporated into an alternating access class model in which the leak and cotransport modes are mutually exclusive and share common kinetic partial reactions.     

Inner voltage clamping. A method for studying interactions among hydrophobic ions in a lipid bilayer

Ketterer, et al. (1971) have suggested that a combination of electrostatic and chemical interactions may cause hydrophobic ions absorbed within a bilayer lipid membrane to reside in two potential wells, each close to a membrane surface. The resulting two planes of charges would define three regions of membrane dielectric: two identical outer regions each between a plane of absorbed charges and the plane of closest approach of ions in the aqueous phase; and the inner region between the two planes of adsorbed charges. The theory describing charge translocation across the inner region is based on a simple three-capacitor model. A significant theoretical conclusion is that the difference between the voltage across the inner region, V_i, and the voltage across the entire membrane, V_m, is directly proportional to the amount of charge that has flowed in a voltage clamp experiment. We demonstrate that we can construct an “inner voltage clamp” that can maintain, with positive feedback, a constant inner voltage, V_i. The manifestation of proper feedback is that the clamp current (after a voltage step) will exhibit pure (i.e., single time-constant) exponential decay, because the voltage dependent rate constants governing translocation will be independent of time. The “pureness” of the exponential is maximized when the standard deviation of the least-square fit of the appropriate exponential equation to the experimental data is minimized. The concomitant feedback is directly related to the capacitances of the inner and outer membrane regions, C_i and C_o.

Experimental results with tetraphenylborate ion adsorbed in bacterial phosphatidylethanolamine/n-decane bilayers indicate C_i ~ 5 · 10^-7F/cm² and C_o ≈ 5 · 10^-5F/cm².

     

Determination of ΔpH in cholinergic synaptic vesicles: Its effect on storage and release of acetylcholine

The interior of purified cholinergic Torpedo vesicles is acidic, pH_in = 5.2 at external pH = 7.4. The internal pH changes linearily as a function of external pH yielding ΔpH = 2.0 and 2.5 at pH_out = 6.3 and 9.1 respectively. The proton translocator carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) + the ionophore valinomycin dissipate the proton gradient across the vesicular membrane and concurrently induce acetylcholine release from vesicles suspended in K⁺ buffer. The effect of FCCP + valinomycin is not sensitive to external pH values between 6.3 and 9.1 and is diminished at lower external pH. The possible role of intravesicular pH and of the proton electrochemical gradient in the storage of acetylcholine within cholinergic vesicles is discussed.     

The H+-ATPase in the Plasma Membrane of Saccharomyces cerevisiae Is Activated during Growth Latency in Octanoic Acid-Supplemented Medium Accompanying the Decrease in Intracellular pH and Cell Viability

Saccharomyces cerevisiae plasma membrane H⁺-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pH_i). The cell population used as inoculum exhibited a significant heterogeneity of pH_i, and the fall of pH_i correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.     

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : I. Role of HCO3?

We studied the regulation of intracellular pH (pH_i) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH_i. Intrinsic buffering power (β_I) was 10.5 mM (pH unit)⁻¹ at pH_i 7.0, and decreased linearly with pH_i; the best-fit line to the data had a slope of −10.0 mM (pH unit)⁻². In the absence of HCO₃ ⁻, pH_i recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH_i 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH_i. Acid extrusion was inhibited 68% (pH_i 6.23) by substituting Li⁺ for Na⁺ in the bath solution. Switching from a CO₂/HCO₃ ⁻-free to a CO₂/HCO₃ ⁻-containing bath solution caused mean steady state pH_i to increase from 6.82 to 6.90, due to a Na⁺-driven HCO₃ ⁻ transporter. The HCO₃ ⁻-induced pH_i increase was unaffected by amiloride, but was inhibited 75% (pH_i 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pH_i 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO₂/HCO₃ ⁻-induced pH_i increase was blocked when external Na⁺ was replaced with N-methyl-d-glucammonium (NMDG⁺). In the presence of HCO₃ ⁻, the Na⁺-driven HCO₃ ⁻ transporter contributed to the pH_i recovery from an acid load. For example, HCO₃ ⁻ shifted the plot of acid-extrusion rate vs. pH_i by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO₃ ⁻ increased acid extrusion 3.8-fold (pH_i 6.20). When astrocytes were acid loaded in amiloride, with Li⁺ as the major cation, HCO₃ ⁻ failed to elicit a substantial increase in pH_i. Thus, Li⁺ does not appear to substitute well for Na⁺ on the HCO₃ ⁻ transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na⁺-driven HCO₃ ⁻ transporter are the predominant acid extruders in astrocytes.     

Intracellular Carbonic Anhydrase Activity Sensitizes Cancer Cell pH Signaling to Dynamic Changes in CO2 Partial Pressure

Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO₂, HCO₃⁻ and H⁺. Intracellular CA (CA_i) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CA_i activity, measured as cytoplasmic CO₂ hydration rate (k_f), ranged from high in colorectal HCT116 (∼2 s⁻¹), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous k_f = 0.18 s⁻¹) in cervical HeLa and breast MDA-MB-468 cells. CA_i activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CA_i catalysis was not obligatory for supporting acid extrusion by H⁺ efflux or HCO₃⁻ influx, nor for maintaining intracellular pH (pH_i) uniformity. However, in the absence of CA_i activity, acid loading from a highly alkaline pH_i was rate-limited by HCO₃⁻ supply from spontaneous CO₂ hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO₂ partial pressure (pCO₂) that disturb cytoplasmic CO₂-HCO₃⁻-H⁺ equilibrium. In cancer cells with high CA_i activity, extracellular pCO₂ fluctuations evoked faster and larger pH_i oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca²⁺] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pH_i of cells with low CA_i activity was less responsive to pCO₂ fluctuations. Such low pass filtering would “buffer” cancer cell pH_i from non-steady-state extracellular pCO₂. Thus, CA_i activity determines the coupling between pCO₂ (a function of tumor perfusion) and pH_i (a potent modulator of cancer cell physiology).     

The mycotoxin ochratoxin A deranges pH homeostasis in Madin-Darby canine kidney cells

Ochratoxin A (OTA) is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Added to the culture medium, OTA transforms MDCK cells in a manner similar to exposure to alkaline stress. By means of video-imaging and microelectrode techniques, we investigated whether OTA (1 mol/liter) affects intracellular pH (pH.), Cl^– (Cl _i ^– ) or cell volume of MDCK cells acutely exposed to normal (pH_norm=7.4) and alkaline (pH_alk=7.7) conditions. At pH_norm, OTA increased Cl _i ^– by 2.6±0.4 mmol/liter (n=14, P<0.05) but had no effect on pH _i . At pH_alk, application of OTA increased Cl _i ^– by 8.6±2.6 mmol/liter (n=10, P< 0.05) and raised pH _i by 0.11±0.03 (n= 8, P<0.05). The Cl^–HCO ₃ ^– exchange inhibitor DNDS (4,4-dinitro-stilbene-2, 2-disulfonate; 10 mol/liter) eliminated the OTA-induced changes of pH _i and Cl _i ^– . OTA did not affect cell volume under both pH_norm and pH_alk conditions.We conclude that the OTA-induced blockade of plasma membrane anion conductance increases Cl _i ^– without changing cell volume. The driving force of plasma membrane Cl^–/HCO ₃ ^– exchange dissipates, leading to a rise of pH _i when cells are exposed to an acute alkaline load. Thus, OTA interferes with pH _i and Cl _i ^– homeostasis leading to morphological and functional alterations in MDCK cells.The work was supported by the Deutsche Forschungsgemeinschaft (DFG, Si 170/7-1).We thank the Zeiss Company (Oberkochen, Germany) for providing the Attofluor video-imaging system for the intracellular Ca²⁺ measurements.This study was carried out with the technical assistance of Sigrid Mildenberger and Ruth Freudinger.