Oxidative stress decreases pHi and Na(+)/H(+) exchange and increases excitability of solitary complex neurons from rat brain slices

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH_i). pH_i was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH_i in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H₂O₂ decreased pH_i by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pH_i by 0.26 ± 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pH_i, causing a 0.42 ± 0.03 (n = 21) pH unit acidification. CT slowed pH_i recovery mediated by Na⁺/H⁺ exchange (NHE) from NH₄Cl-induced acidification by 53% (n = 20) in -buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH_i. These results indicate that oxidative stress 1) decreases pH_i in some SC neurons, 2) together with hypercapnia has an additive effect on pH_i, 3) partially inhibits NHE, and 4) directly affects excitability of CO₂/H⁺-chemosensitive SC neurons independently of pH_i changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction. hyperoxic hyperventilation; O₂ toxicity; pH regulation; brain stem; reactive oxygen species     

A computational analysis of central CO2 chemosensitivity in Helix aspersa

We created a single-compartment computer model of a CO₂ chemosensory neuron using differential equations adapted from the Hodgkin-Huxley model and measurements of currents in CO₂ chemosensory neurons from Helix aspersa. We incorporated into the model two inward currents, a sodium current and a calcium current, three outward potassium currents, an A-type current (I_KA), a delayed rectifier current (I_KDR), a calcium-activated potassium current (I_KCa), and a proton conductance found in invertebrate cells. All of the potassium channels were inhibited by reduced pH. We also included the pH regulatory process to mimic the effect of the sodium-hydrogen exchanger (NHE) described in these cells during hypercapnic stimulation. The model displayed chemosensory behavior (increased spike frequency during acid stimulation), and all three potassium channels participated in the chemosensory response and shaped the temporal characteristics of the response to acid stimulation. pH-dependent inhibition of I_KA initiated the response to CO₂, but hypercapnic inhibition of I_KDR and I_KCa affected the duration of the excitatory response to hypercapnia. The presence or absence of NHE activity altered the chemosensory response over time and demonstrated the inadvisability of effective intracellular pH (pH_i) regulation in cells designed to act as chemostats for acid-base regulation. The results of the model indicate that multiple channels contribute to CO₂ chemosensitivity, but the primary sensor is probably I_KA. pH_i may be a sufficient chemosensory stimulus, but it may not be a necessary stimulus: either pH_i or extracellular pH can be an effective stimuli if chemosensory neurons express appropriate pH-sensitive channels. The lack of pH_i regulation is a key feature determining the neuronal activity of chemosensory cells over time, and the balanced lack of pH_i regulation during hypercapnia probably depends on intracellular activation of pH_i regulation but extracellular inhibition of pH_i regulation. These general principles are applicable to all CO₂ chemosensory cells in vertebrate and invertebrate neurons. hypercapnia; potassium channels; computer modeling; central chemoreceptors     

Capacity for intracellular pH compensation during hypercapnia in white sturgeon primary liver cells

Fish, exposed to elevated water CO₂, experience a rapid elevation in blood CO₂ (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. White sturgeon, Acipenser transmontanus, are exceptionally CO₂ tolerant and can regulate tissue intracellular pH (pH_i) in the presence of a pronounced hypercapnic acidosis (preferential pH_i regulation). In this study, pH_i regulatory capacity of sturgeon liver cells in primary culture was examined to assess the suitability of employing this in vitro system to understand in vivo CO₂ tolerance in sturgeon. Using the pH-sensitive fluoroprobe BCECF, real-time changes in resting pH_i and rates of pH_i recovery were investigated during exposure to hypercapnia (3 and 6% CO₂) in the absence and presence of additional acid loads induced by (20 mM) ammonium prepulse. During short-term (10 min) exposure to hypercapnia (3 and 6% CO₂), sturgeon cells were acidified and no pH_i compensation was observed. However, when exposure to 6% CO₂ was extended to over 19 h, the CO₂-induced intracellular acidosis was partially compensated by a pH_i increase of over 0.2 pH unit despite the sustained extracellular acidosis, indicative of a capacity for preferential pH_i regulation in vitro. Since this capacity in sturgeon liver is present both in vivo and in vitro, the transmembrane transporters involved may be the same. Therefore, cell culture may be a suitable tool to identify the transporters (i.e., the cellular mechanisms underlying in vivo CO₂ tolerance) in white sturgeon and possibly in other hypercapnia-tolerant species.     

Multiple targets of chemosensitive signaling in locus coeruleus neurons: role of K+ and Ca2+ channels

Westudied chemosensitive signaling in locus coeruleus (LC) neurons usingboth perforated and whole cell patch techniques. Upon inhibition offast Na⁺ spikes by tetrodotoxin (TTX), hypercapnic acidosis[HA; 15% CO₂, extracellular pH (pH_o) 6.8]induced small, slow spikes. These spikes were inhibited byCo²⁺ or nifedipine and were attributed to activation ofL-type Ca²⁺ channels by HA. Upon inhibition of bothNa⁺ and Ca²⁺ spikes, HA resulted in a membranedepolarization of 3.52 ± 0.61 mV (n = 17) thatwas reduced by tetraethylammonium (TEA) (1.49 ± 0.70 mV,n = 7; P < 0.05) and absent(0.97 ± 0.73 mV, n = 7; P < 0.001) upon exposure to isohydric hypercapnia (IH; 15%CO₂, 77 mM HCO, pH_o 7.45).Either HA or IH, but not 50 mM Na-propionate, activatedCa²⁺ channels. Inhibition of L-type Ca²⁺channels by nifedipine reduced HA-induced increased firing rate andeliminated IH-induced increased firing rate. We conclude that chemosensitive signals (e.g., HA or IH) have multiple targets in LCneurons, including TEA-sensitive K⁺ channels andTWIK-related acid-sensitive K⁺ (TASK) channels.Furthermore, HA and IH activate L-type Ca²⁺ channels, andthis activation is part of chemosensitive signaling in LC neurons.

     

Effect of hypercapnia on intracellular pH regulation in a rainbow trout hepatoma cell line,RTH 149

Fish exposed to elevated water CO₂ experience a rapid increase in blood CO₂ levels (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. While the mechanisms associated with extracellular pH regulation have been well explored, much less is known about intracellular pH (pH_i) regulation. There is great interest in developing non-animal models for research. One such model is the rainbow trout hepatoma cell line (RTH 149), which has been used to study a wide range of topics; however, no studies have investigated its potential use in pH_i regulation. Employing the pH-sensitive fluoroprobe BCECF, the present study examined pH_i regulation in RTH 149 under normocapnia and during extracellular acidification induced by either elevated CO₂ or 1 M HCl. During exposure to hypercapnia, RTH 149 cells were acidified without recovery as long as the elevated CO₂ was maintained. In addition, rates of pH_i recovery from NH₄Cl-induced acidosis were significantly lower in cells exposed to hypercapnia or HCl compared to that in normocapnic cells, indicating that elevated CO₂ indirectly impeded pH_i recovery through a reduction in pH_e and/or pH_i. Moreover, pH_i regulation in RTH 149 was EIPA-sensitive, suggesting that an NHE may be involved. Overall, RTH 149 may have the potential for identifying transporters likely to play a role in pH_i regulation in fish. However, it should not be used as a complete replacement for in vivo studies, especially to quantify acid–base regulatory ability at whole animal level, since RTH 149 appeared to have enhanced pH_i recovery rates relative to primary hepatocytes.     

CO2 chemosensitivity in Helix aspersa: three potassium currents mediate pH-sensitive neuronal spike timing

Elevated levels of carbon dioxide increase lung ventilation in Helix aspersa. The hypercapnic response originates from a discrete respiratory chemosensory region in the dorsal subesophageal ganglia that contains CO₂-sensitive neurons. We tested the hypothesis that pH-dependent inhibition of potassium channels in neurons in this region mediated the chemosensory response to CO₂. Cells isolated from the dorsal subesophageal ganglia retained CO₂ chemosensitivity and exhibited membrane depolarization and/or an increase in input resistance during an acid challenge. Isolated somata expressed two voltage-dependent potassium channels, an A-type and a delayed-rectifier-type channel (I_KA and I_KDR). Both conductances were inhibited during hypercapnia. The pattern of voltage dependence indicated that I_KA was affected by extracellular or intracellular pH, but the activity of I_KDR was modulated by extracellular pH only. Application of inhibitors of either channel mimicked many of the effects of acidification in isolated cells and neurons in situ. We also detected evidence of a pH-sensitive calcium-activated potassium channel (I_KCa) in neurons in situ. The results of these studies support the hypothesis that I_KA initiates the chemosensory response, and I_KDR and I_KCa prolong the period of activation of CO₂-sensitive neurons. Thus multiple potassium channels are inhibited by acidosis, and the combined effect of pH-dependent inhibition of these channels enhances neuronal excitability and mediates CO₂ chemosensory responses in H. aspersa. We did not find a single "chemosensory channel," and the chemosensitive channels that we did find were not unique in any way that we could detect. The protein "machinery" of CO₂ chemosensitivity is probably widespread among neurons, and the selection process whereby a neuron acts or does not act as a respiratory CO₂ chemosensor probably depends on the resting membrane potential and synaptic connectivity. carbon dioxide     

Cellular mechanisms involved in CO(2) and acid signaling in chemosensitive neurons

An increase in CO₂/H⁺ is a major stimulus for increased ventilation and is sensed by specialized brain stem neurons called central chemosensitive neurons. These neurons appear to be spread among numerous brain stem regions, and neurons from different regions have different levels of chemosensitivity. Early studies implicated changes of pH as playing a role in chemosensitive signaling, most likely by inhibiting a K⁺ channel, depolarizing chemosensitive neurons, and thereby increasing their firing rate. Considerable progress has been made over the past decade in understanding the cellular mechanisms of chemosensitive signaling using reduced preparations. Recent evidence has pointed to an important role of changes of intracellular pH in the response of central chemosensitive neurons to increased CO₂/H⁺ levels. The signaling mechanisms for chemosensitivity may also involve changes of extracellular pH, intracellular Ca²⁺, gap junctions, oxidative stress, glial cells, bicarbonate, CO₂, and neurotransmitters. The normal target for these signals is generally believed to be a K⁺ channel, although it is likely that many K⁺ channels as well as Ca²⁺ channels are involved as targets of chemosensitive signals. The results of studies of cellular signaling in central chemosensitive neurons are compared with results in other CO₂- and/or H⁺-sensitive cells, including peripheral chemoreceptors (carotid body glomus cells), invertebrate central chemoreceptors, avian intrapulmonary chemoreceptors, acid-sensitive taste receptor cells on the tongue, and pain-sensitive nociceptors. A multiple factors model is proposed for central chemosensitive neurons in which multiple signals that affect multiple ion channel targets result in the final neuronal response to changes in CO₂/H⁺. hypercapnia; brain stem; ventilation; peripheral chemoreceptor; glia; gap junction; glomus; channel; calcium; potassium; carbonic anhydrase; taste receptor; nociception     

Decrease in rat taste receptor cell intracellular pH is the proximate stimulus in sour taste transduction

Taste receptor cells (TRCs)respond to acid stimulation, initiating perception of sour taste.Paradoxically, the pH of weak acidic stimuli correlates poorly with theperception of their sourness. A fundamental issue surrounding sourtaste reception is the identity of the sour stimulus. We tested thehypothesis that acids induce sour taste perception by penetratingplasma membranes as H⁺ ions or as undissociated moleculesand decreasing the intracellular pH (pH_i) of TRCs. Our datasuggest that taste nerve responses to weak acids (acetic acid andCO₂) are independent of stimulus pH but strongly correlatewith the intracellular acidification of polarized TRCs. Taste nerveresponses to CO₂ were voltage sensitive and were blockedwith MK-417, a specific blocker of carbonic anhydrase. Strong acids(HCl) decrease pH_i in a subset of TRCs that contain apathway for H⁺ entry. Both the apical membrane and theparacellular shunt pathway restrict H⁺ entry such that alarge decrease in apical pH is translated into a relatively smallchange in TRC pH_i within the physiological range. Weconclude that a decrease in TRC pH_i is the proximate stimulus in rat sour taste transduction.

     

Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH

Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pH_i) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl^–/Cl^– and Cl^–/HCO₃^– exchange are inhibited by acid pH_i or extracellular pH (pH_o). We have investigated the importance to pH sensitivity of the eight histidine (His) residues within the AE2 COOH-terminal transmembrane domain (TMD). Wild-type mouse AE2-mediated Cl^–/Cl^– exchange, measured as DIDS-sensitive ³⁶Cl^– efflux from Xenopus oocytes, was experimentally altered by varying pH_i at constant pH_o or varying pH_o. Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal ³⁶Cl^– efflux at pH_o 7.4 and acid shifted the pH_o vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl^– transport activity with retention of pH_o sensitivity. In contrast to the effects of DEPC on wild-type AE2, pH_o sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pH_i, pH_i sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pH_i and pH_o. pH regulation; histidine residues; Cl^–/HCO₃^– exchange     

Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH(2)-terminal cytoplasmic domain

The role ofintracellular pH (pH_i) in regulation of AE2 function inXenopus oocytes remains unclear. We therefore compared AE2-mediated ³⁶Cl efflux fromXenopus oocytes during imposed variation of extracellular pH(pH_o) or variation of pH_i at constantpH_o. Wild-type AE2-mediated ³⁶Clefflux displayed a steep pH_o vs. activity curve, withpH_o(50) = 6.91 ± 0.04. SequentialNH₂-terminal deletion of amino acid residues in tworegions, between amino acids 328 and 347 or between amino acids 391 and510, shifted pH_o(50) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocytepH_i at constant pH_o and were shown to beneither substrates nor inhibitors of AE2-mediated Cltransport. At constant pH_o, AE2 was inhibited byintracellular acidification and activated by intracellularalkalinization. Our data define structure-function relationships withinthe AE2 NH₂-terminal cytoplasmic domain, which demonstratesdistinct structural requirements for AE2 regulation by intracellularand extracellular protons.

     

pH heterogeneity at intracellular and extracellular plasma membrane sites in HT29-C1 cell monolayers

In the colonic mucosa, short-chain fatty acids changeintracellular pH (pH_i) and extracellular pH(pH_e). In this report, confocal microscopy anddual-emission ratio imaging of carboxyseminaphthorhodofluor-1 were usedfor direct evaluation of pH_i and pH_e in asimple model epithelium, HT29-C1 cells. Live cell imaging along theapical-to-basal axis of filter-grown cells allowed simultaneousmeasurement of pH in the aqueous environment near the apical membrane,the lateral membrane, and the basal membrane. Subapical cytoplasmreported the largest changes in pH_i after isosmoticaddition of 130 mM propionate or 30 mM NH₄Cl. In restingcells and cells with an imposed acid load, lateral membranes hadpH_i values intermediate between the relatively acidicsubapical region (pH 6.3-6.9) and the relatively alkaline basalpole of the cells (pH 7.4-7.1). Transcellular pH_igradients were diminished or eliminated during an induced alkalineload. Propionate differentially altered pH_e near the apicalmembrane, in lateral intracellular spaces between adjacent cells, andnear the basal membrane. Luminal or serosal propionate causedalkalinization of the cis compartment (where propionate wasadded) but acidification of the trans compartment only inresponse to luminal propionate. Addition of NH₄Cl produced qualitatively opposite pH_e excursions. The microscopicvalues of pH_i and pH_e can explain a portion ofthe selective activation of polarized Na/H exchangers observed inHT29-C1 cells in the presence of transepithelial propionate gradients.

     

Acid-induced responses in hamster chorda tympani and intracellular pH tracking by taste receptor cells

HCl- and NaCl-induced hamster chorda tympani nerve responseswere recorded during voltage clamp of the lingual receptive field. Voltage perturbations did not influence responses to HCl. In contrast, responses to NaCl were decreased by submucosal-positive and increased by submucosal-negative voltage clamp. Responses to HCl were insensitive to the Na⁺ channel blockers,amiloride and benzamil, and to methylisobutylamiloride (MIA), anNa⁺/H⁺exchange blocker. Responses to NaCl were unaffected by MIA but weresuppressed by benzamil. Microfluorometric and imaging techniques wereused to monitor the relationship between external pH(pH_o) and the intracellular pH(pH_i) of fungiform papilla tastereceptor cells (TRCs) following2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein loading.TRC pH_i responded rapidly andmonotonically to changes in pH_o.This response was unaffected byNa⁺ removal or the presence ofamiloride, benzamil, or MIA. The neural records and the data fromisolated TRCs suggest that the principal transduction pathway for acidtaste in hamster is similar to that in rat. This may involve themonitoring of changes in TRC pH_i mediated through amiloride-insensitiveH⁺ transport across TRC membranes.This is an example of cell monitoring of environmental pH through pHtracking, i.e., a linear change inpH_i in response to a change inpH_o, as has been proposed for carotid bodies. In taste, the H⁺transport sites may be concentrated on the basolateral membranes ofTRCs and, therefore, are responsive to an attenuatedH⁺ concentration from diffusion ofacids across the tight junctions.

     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles

Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pH_i, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, ‘bulk pH values’, pH_o, were measured by a combination electrode. While pH_i = pH_o for neutral, pH_i < pH_o for anionic and pH_i > pH_o for cationic liposomes prepared in 5.0 · 10^?3 M phosphate buffers. pK_a values for the ionization of pyranine were 7.22 ± 0.04 and 6.00 ± 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-d-α-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calculated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Glucose activates H(+)-ATPase in kidney epithelial cells

The vacuolar H⁺-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H⁺-ATPase remain largely unknown. The present study examines the effect of glucose on H⁺-ATPase activity in the pig kidney epithelial cell line LLC-PK₁. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH₃/NH₄⁺ prepulse, and rates of intracellular pH (pH_i) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K⁺ free to eliminate Na⁺/H⁺ exchange and H⁺-K⁺-ATPase activity. After NH₃/NH₄⁺-induced acidification, LLC-PK₁ cells had a significant pH_i recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH_i recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH_i recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H⁺-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification. proton secretion; glycolysis; intracellular pH; concanamycin A     

Regulation of the Cytoplasmic pH of Chara corallina: Response to Changes in External pH

Effects of external pH (pH_o) on the cytoplasmic pH (pH_c) ofChara corallina have been measured with the weak acid 5, 5-dimethyloxazolidine-2,4-dione (DMO) following standardized pretreatment of cells insolutions at pHo 4.5, 6.3 and 8.3. Irrespective of pH_c duringpretreatment, pH_o responded to pHo during the experimental periodsof 150–180 min or (in one experiment) 90–110 min.There were increases or decreases of about 0.5 in pHo when cellswere transferred from pH_o 4.5 to 8.3 or vice versa. In the darkpH_c was 0.2–0.3 units lower than the corresponding valuein the light. The results are discussed in relation to the factorsinvolved in the regulation of pH_c in C. corallina, which maybegin to break down below about pH_o4.5, as indicated by relativelylarge decreases in pH_c at low pH_o. Key words: Chara corallina, Cytoplasmic pH, External pH, DMO     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.

     

pH-dependent modulation of Kv1.3 inactivation: role of His399

The Kv1.3 K⁺ channel lacks N-type inactivation, but during prolonged depolarized periods it inactivates via the slow (P/C type) mechanism. It bears a titratable histidine residue in position 399 (equivalent of Shaker 449), a site known to influence the rate of slow inactivation. As opposed to several other voltage-gated K⁺ channels, slow inactivation of Kv1.3 is slowed when extracellular pH (pH_o) is lowered under physiological conditions. Our findings are as follows. First, when His399 was mutated to a lysine, arginine, leucine, valine or tyrosine, extracellular acidification (pH 5.5) accelerated inactivation reminiscent of other Kv channels. Second, inactivation of the wild-type channel was accelerated by low pH_o when the ionic strength of the external solution was raised. Inactivation of the H399K mutant was also accelerated by high ionic strength at pH 7.35 but not the inactivation of H399L. Third, after the external application of blocking barium ions, recovery of the wild-type current during washout was slower in low pH_o. Fourth, the dissociation rate of Ba²⁺ was pH insensitive for both H399K and H399L. Furthermore, Ba²⁺ dissociation rates were equal for H399K and the wild type at pH 5.5 and were equal for H399L and the wild type at pH 7.35. These observations support a model in which the electric field of the protonated histidines creates a potential barrier for potassium ions just outside the external mouth of the pore that hinders their exit from the binding site controlling inactivation. In Kv1.3, this effect overrides the generally observed speeding of slow inactivation when pH_o is reduced. extracellular pH; potassium channel; histidine; barium; high ionic strength     

Role of Na(+)/H(+) exchanger during O(2) deprivation in mouse CA1 neurons

To determine the role ofmembrane transporters in intracellular pH (pH_i) regulationunder conditions of low microenvironmental O₂, we monitoredpH_i in isolated single CA1 neurons using the fluorescentindicator carboxyseminaphthorhodafluor-1 and confocal microscopy. Aftertotal O₂ deprivation or anoxia (PO₂ 0 Torr), a large increase in pH_i was seen in CA1neurons in HEPES buffer, but a drop in pH_i, albeit small,was observed in the presence of HCO. Ionicsubstitution and pharmacological experiments showed that the largeanoxia-induced pH_i increase in HEPES buffer was totallyNa⁺ dependent and was blocked by HOE-694, stronglysuggesting the activation of the Na⁺/H⁺exchanger (NHE). Also, this pH_i increase in HEPES bufferwas significantly smaller in Na⁺/H⁺ exchangerisoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons,demonstrating that NHE1 is responsible for part of the pH_iincrease following anoxia. Both chelerythrine and H-89 partly blocked,and H-7 totally eliminated, this anoxia-induced pH_iincrease in the absence of HCO. We conclude that1) O₂ deprivation activatesNa⁺/H⁺ exchange by enhancing protein kinaseactivity and 2) membrane proteins, such as NHE, activelyparticipate in regulating pH_i during low-O₂states in neurons.