pH Modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistiguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pH_int/pH_ext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2m). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pH_ext 5.8 (K _m =1.33vs. 1.70 m;V _max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect thatK _m, for carrier-mediated F transport. These data are consistent with similarK _i's for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H⁺ K _m (or OH^– IC₅₀) corresponding to pH 4.89. External pH affects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺) rather than competitive binding that is mutually exclusive.     

pH modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an outwardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistinguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pH_int/pH_ext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2 M). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at low pH (monovalent F^– predominates) and at least 2 H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pH_ext5.8 (K _m =1.33vs. 1.70 M;V _max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect theK _m for carrier-mediated F transport. These data are consistent with similarK _i ^' s for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H⁺ K _m (or OH^– lC₅₀) corresponding to pH 4.89. External pH effects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺),rather than competitive binding that is mutually exclusive.     

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.     

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO₃^?) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO₃^? by the surface‐bound enzyme carbonic anhydrase (CA_ext). Here, we examined other putative HCO₃^? uptake mechanisms in M. pyrifera under pH_T 9.00 (HCO₃^?: CO₂ = 940:1) and pH_T 7.65 (HCO₃^?: CO₂ = 51:1). Rates of photosynthesis, and internal CA (CA_int) and CA_ext activity were measured following the application of AZ which inhibits CA_ext, and DIDS which inhibits a different HCO₃^? uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO₃^? uptake by M. pyrifera is via an AE protein, regardless of the HCO₃^?: CO₂ ratio, with CA_ext making little contribution. Inhibiting the AE protein led to a 55%–65% decrease in photosynthetic rates. Inhibiting both the AE protein and CA_ext at pH_T 9.00 led to 80%–100% inhibition of photosynthesis, whereas at pH_T 7.65, passive CO₂ diffusion supported 33% of photosynthesis. CA_int was active at pH_T 7.65 and 9.00, and activity was always higher than CA_ext, because of its role in dehydrating HCO₃^? to supply CO₂ to RuBisCO. Interestingly, the main mechanism of HCO₃^? uptake in M. pyrifera was different than that in other Laminariales studied (CA_ext‐catalyzed reaction) and we suggest that species‐specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO₃^?:CO₂ due to ocean acidification.     

Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat

Enhanced Cl⁻ efflux during acidosis in plants is thought to play a role in cytosolic pH (pH_c) homeostasis by short-circuiting the current produced by the electrogenic H⁺ pump, thereby facilitating enhanced H⁺ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pH_c activates Cl⁻ efflux via two mechanisms. The first is a direct effect of pH_c on Cl⁻ efflux; the second mechanism comprises a pH_c-induced increase in affinity for cytosolic free Ca²⁺ ([Ca²⁺]_c), which also activates Cl⁻ efflux. Cl⁻ efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pH_c and [Ca²⁺]_c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl⁻ efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl⁻ efflux at 60% of the maximal level in a manner that was both pH_c and [Ca²⁺]_c independent. These findings imply that plasma membrane anion channels play a central role in pH_c regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction.     

HCO3?-independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase

The mechanisms of HCO₃⁻-independent intracellular pH (pH_i) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pH_i in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H⁺ pump by Cl⁻ removal or via the selective inhibitor bafilomycin had only a small effect upon resting pH_i and recovery following an acid load. In contrast, resting pH_i in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pH_i recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO₃⁻-independent pH_i regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.     

Adaptation of Denitrifying Populations to Low Soil pH

Natural denitrification rates and activities of denitrifying enzymes were measured in an agricultural soil which had a 20-year past history of low pH (pH ca. 4) due to fertilization with acid-generating ammonium salts. The soil adjacent to this site had been limed and had a pH of ca. 6.0. Natural denitrification rates of these areas were of similar magnitude: 158 ng of N g⁻¹ of soil day⁻¹ for the acid soil and 390 ng of N g⁻¹ of soil day⁻¹ at the neutral site. Estimates of in situ denitrifying enzyme activity were higher in the neutral soil, but substantial enzyme activity was also detected in the acid soil. Rates of nitrous oxide reduction were very low, even when NO₃⁻ and NO₂⁻ were undetectable, and were ca. 400 times lower than the rates of N₂O production from NO₃⁻. Denitrification rates measured in slurries of the acid and neutral soil showed distinctly different pH optima (pH 3.9 and pH 6.3) which were near the pH values of the two soils. This suggests that an acid-tolerant denitrifying population had been selected during the 20-year period of low pH.     

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO3 Cotransport

In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na⁺-driven influx of HCO₃ ⁻ causes the increase in intracellular pH (pH_i) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO₂/17 mM HCO₃ ⁻. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na⁺-driven Cl-HCO₃ exchanger, an electrogenic Na/HCO₃ cotransporter, or an electroneutral Na/HCO₃ cotransporter. To determine if the transporter is a Na⁺-driven Cl-HCO₃ exchanger, we depleted the cells of intracellular Cl⁻ by incubating them in a Cl⁻-free solution for an average of ∼11 min. We verified the depletion with the Cl⁻-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl⁻-depleted cells, the pH_i still increases after one or more exposures to CO₂/HCO₃ ⁻. Furthermore, the pH_i decrease elicited by external Na⁺ removal does not require external Cl⁻. Therefore, the transporter cannot be a Na⁺-driven Cl-HCO₃ exchanger. To determine if the transporter is an electrogenic Na/ HCO₃ cotransporter, we measured pH_i and plasma membrane voltage (V_m) while removing external Na⁺, in the presence/absence of CO₂/HCO₃ ⁻ and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO₂/HCO₃ ⁻ solutions contained 20% CO₂ and 68 mM HCO₃ ⁻, pH 7.3, to maximize the HCO₃ ⁻ flux. In pH_i experiments, removing external Na⁺ in the presence of CO₂/HCO₃ ⁻ elicited an equivalent HCO₃ ⁻ efflux of 281 μM s⁻¹. The HCO₃ ⁻ influx elicited by returning external Na⁺ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V_m change was 3.3 mV. Indeed, we found that removing external Na⁺ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO₂/ HCO₃ ⁻. Thus, the Na/HCO₃ cotransporter is electrogenic. Because a cotransporter with a Na⁺:HCO₃ ⁻ stoichiometry of 1:3 or higher would predict a net HCO₃ ⁻ efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO₃ cotransporter with a stoichiometry of 1:2.     

The Final Frontier of pH and the Undiscovered Country Beyond

The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K _w = [H⁺]×[OH⁻].We used K _w [in a reversed way] to calculate the number of undissociated H₂O molecules required by this equilibrium constant to yield at least one of its daughter ions, H⁺ or OH⁻ at a given pH. In this way we obtained a formula that relates pH to the minimal volume V_pH required to provide a physical meaning to K _w, (where N _A is Avogadro’s number). For example, at pH 7 (neutral at 25°C) V_pH = 16.6 aL. Any deviation from neutral pH results in a larger V_pH value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H⁺ ions at equilibrium, thus the definition of pH based on K _w is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H⁺ ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H⁺ ions as a general source for biochemical reactions. Consequences of this finding are discussed.     

Kinetics of ionic transport across frog skin: Two concentration-dependent processes

Summary Sodium and chloride influxes across the nonshort-circuited isolated skin ofRana esculenta were measured at widely varying external ionic concentrations.The curve describing sodium transport has two Michaelis-Menten components linked at an inflection point occurring at an external sodium concentration of about 7 meq. Chloride transport can also be represented by two saturating components. A possible explanation of these kinetics is discussed.At sodium concentrations lower than 4 meq it is possible to define a component of the sodium transport mechanism as having a high affinity for sodium and which is independent of the nature of the external anion. A high affinity for chloride of the chloride transport system functioning at low external concentrations is also found but is significantly different from that of sodium. These systems show the physiological characteristics of the countertransports (Na _ext ⁺ /H _int ⁺ ; Cl _ext ^– /HCO _3int ^– ) functioning at low external concentrations.At external concentrations higher than 4 meq a low affinity transporting system in which chloride and sodium are linked superimpose on the high affinity components.The physiological significance of these results is discussed.     

The Role of Glycerol and Inorganic Ions in Osmoregulatory Responses of the Euryhaline Flagellate Chlamydomonas pulsatilla Wollenweber

The green euryhaline flagellate Chlamydomonas pulsatilla Wollenweber, isolated from a coastal marine environment, was grown exponentially over the salinity range of 10 to 200% artificial seawater (ASW). The cellular volume and aqueous space of the alga, measured by [¹⁴C] mannitol and ³H₂O tracer analyses of centrifuged cell pellets, ranged between 2.3 and 3.1 picoliters and between 1.5 and 2.1 picoliters, respectively. The nonaqueous space determined in those analyses (28-35%) was consistent with the cell composition of the alga. The glycerol content of the alga increased almost linearly with increasing salinity; its contribution to intracellular osmolality at 200% ASW was about 57%. The contribution of amino acids and soluble carbohydrates to the cell osmotic balance was small. Intracellular ion concentrations determined by analyzing centrifuged cell pellets of known [¹⁴C]mannitol space by atomic absorption spectrophotometry, and by neutron activation analyses of washed cells were similar. At 10% ASW, potassium and magnesium were the major cations, and chloride and phosphate were the major anions. The sodium and chloride content of the alga increased with increasing salinity; at 200% ASW the intracellular concentration of both sodium and chloride was about 400 millimolar. The intracellular osmolality (π_int) matched closely the external osmolality (π_ext) over the entire salinity range except at 10% ASW where π_int exceeded π_ext by 120 to 270 milliosmoles per kilogram H₂O.     

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).     

Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH_i), typically due to activation of Na⁺/H⁺ exchange (NHE). In the systemic vasculature, ET-1 increases pH_i, Na⁺/H⁺ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH_i and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH_i and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH_i and NHE activity induced by increasing concentrations of ET-1 (10⁻¹⁰ to 10⁻⁸ M). We found that application of exogenous ET-1 increased pH_i and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH_i or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH_i.     

Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage–sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H⁺. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl⁻, Br⁻, SCN⁻, and I⁻) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pH_e = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl⁻]_i under unlikely protonation conditions (pH_e = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H⁺ plays a minor role in dislodging the glutamate gate.     

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.     

Intracellular pH in rice and wheat root tips under hypoxic and anoxic conditions

The influence of anoxia and hypoxia on dynamic of intracellurar pH and ATP content in rice and wheat root tips was investigated with ³¹P-NMR spectroscopy. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. Anoxic wheat root tips were deficient in ADH induction, whereas increased activity of alcoholic fermentation enzymes took place in anoxic rice root tips, as well as in both species after hypoxic treatment. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt = 10^−pHcyt) for both hypoxic and anoxic conditions. In this addendum we want to focus the reader''s attention on the importance of adequate experimental design when hypoxia is under investigation and on some further perspectives of intracellular pH regulation in plants under anaerobic conditions.Key words: anoxia, hypoxia, rice, wheat, cytoplasmic pH regulation     

NO3 ?-induced pH Changes in Mammalian Cells : Evidence for an NO3?-H+ Cotransporter

The effect of NO₃ ⁻ on intracellular pH (pH_i) was assessed microfluorimetrically in mammalian cells in culture. In cells of human, hamster, and murine origin addition of extracellular NO₃ ⁻ induced an intracellular acidification. This acidification was eliminated when the cytosolic pH was clamped using ionophores or by perfusing the cytosol with highly buffered solutions using patch-pipettes, ruling out spectroscopic artifacts. The NO₃ ⁻- induced pH change was not due to modulation of Na⁺/H⁺ exchange, since it was also observed in Na⁺/H⁺ antiport-deficient mutants. Though NO₃ ⁻ is known to inhibit vacuolar-type (V) H⁺-ATPases, this effect was not responsible for the acidification since it persisted in the presence of the potent V-ATPase inhibitor bafilomycin A₁. NO₃ ⁻/HCO₃ ⁻ exchange as the underlying mechanism was ruled out because acidification occurred despite nominal removal of HCO₃ ⁻, despite inhibition of the anion exchanger with disulfonic stilbenes and in HEK 293 cells, which seemingly lack anion exchangers (Lee, B.S., R.B. Gunn, and R.R. Kopito. 1991. J. Biol. Chem. 266:11448– 11454). Accumulation of intracellular NO₃ ⁻, measured by the Greiss method after reduction to NO₂ ⁻, indicated that the anion is translocated into the cells along with the movement of acid equivalents. The simplest model to explain these observations is the cotransport of NO₃ ⁻ with H⁺ (or the equivalent counter-transport of NO₃ ⁻ for OH⁻). The transporter appears to be bi-directional, operating in the forward as well as reverse directions. A rough estimate of the fluxes of NO₃ ⁻ and acid equivalents suggests a one-to-one stoichiometry. Accordingly, the rate of transport was unaffected by sizable changes in transmembrane potential. The cytosolic acidification was a saturable function of the extracellular concentration of NO₃ ⁻ and was accentuated by acidification of the extracellular space. The putative NO₃ ⁻-H⁺ cotransport was inhibited markedly by ethacrynic acid and by α-cyano-4-hydroxycinnamate, but only marginally by 4,4′-diisothiocyanostilbene-2,2′ disulfonate or by p-chloromercuribenzene sulfonate. The transporter responsible for NO₃ ⁻-induced pH changes in mammalian cells may be related, though not identical, to the NO₃ ⁻-H⁺ cotransporter described in Arabidopsis and Aspergillus. The mammalian cotransporter may be important in eliminating the products of NO metabolism, particularly in cells that generate vast amounts of this messenger. By cotransporting NO₃ ⁻ with H⁺ the cells would additionally eliminate acid equivalents from activated cells that are metabolizing actively, without added energetic investment and with minimal disruption of the transmembrane potential, inasmuch as the cotransporter is likely electroneutral.     

The Ca2+-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes,consistent with a 2 Ca2+/nH+ exchange mechanism

The Ca²⁺-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca²⁺ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol. 104:147–163). Cytoplasmic pH (pH_cyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca²⁺ to 2.5 μM and monitoring active Ca²⁺ extrusion into a Ca²⁺-free medium. The pump was shown to perturb pH_cyt, to not respond to changes in membrane potential and to respond to imposed changes in pH_cyt in a manner consistent with the Ca²⁺ pump acting as a 2 Ca²⁺/nH⁺ exchanger. (i) Raising the external pH (pH_ext) from 7.40 to 7.60 lowers the V_max of the pump in basal condition (V_max,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pH_ext to 7.13 raised V_max,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG⁺) medium, the pump operation against high [Ca²⁺]_cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pH_cyt down to 6.23 reduces the V_max,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na⁺ raises the V_max,1 (basal state withK _m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (V_max,2=280±28 μM/min;K _m,2=502±36 nM). (vi) Transient elevation of pH_cyt by NH₄Cl at high [Ca²⁺]_cyt activates the pump eightfold (V_max,2≥671±350 μM/min). The large activation by alkaline pH_cyt at high [Ca²⁺]_cyt can be explained by Ca²⁺-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol. 130:63–82) and by increased Ca²⁺ affinity of calmodulin at high pH.     

Anaerobic Sulfide Oxidation with Nitrate by a Freshwater Beggiatoa Enrichment Culture

A lithotrophic freshwater Beggiatoa strain was enriched in O₂-H₂S gradient tubes to investigate its ability to oxidize sulfide with NO₃⁻ as an alternative electron acceptor. The gradient tubes contained different NO₃⁻ concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O₂, H₂S, pH, and NO₃⁻ were determined with microsensors. The more NO₃⁻ that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO₃⁻ to oxidize sulfide at depths below the depth that O₂ penetrated. In the presence of NO₃⁻ Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO₃⁻ was added. These thick mats spatially separated O₂ and sulfide but not NO₃⁻ and sulfide, and therefore NO₃⁻ must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O₂ flux and a twofold-higher sulfide flux into the NO₃⁻-exposed mats compared to the fluxes for controls without NO₃⁻. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S⁰ with NO₃⁻ as the electron acceptor.