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.     

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     

Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes

Continuous measurements of cytoplasmic pH (pH_c) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (7–33 ? 0–12, standard conditions)changes no more than 0.1 pH_c, per pH_o-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pH_cand hyperpolarize, while weak bases alkalize pH_c and depolarizethe cells, (iii) 1.0 mol M,³ NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm^–3 CCCP has no significant effect on pH_c, if added atpH 9.6 or 7.2, but acidifies pH_c by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pH_c, while K⁺, Na⁺, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pH_c with an optimum of about50 mol m^–3 H⁺pH_c-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H⁺-cotransport(e.g. H⁺/Cl^–) rather than H⁺-uniport, is no threat topH_c. The proton export pump, although itself reacting to changesin pH_c, influences pH_c only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pH_c control in Sinapis, while the interlocked H⁺-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis     

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.

     

Regulation of the Cytoplasmic pH of Chara corallina in the Absence of External Ca2+: Its Significance in Relation to the Activity and Control of the H+ Pump

Effects of removal of external Ca²⁺ on the cytoplasmic pH (pH_c)of Chara corallina have been measured with the weak acid 5,5-dimethyl-oxazolidine-2,4-dione(DMO) as a function of external pH (pH₀) and of the externalconcentration of K⁺. Removal of Ca²⁺ always decreased pH_c whenpH₀ was below about 6.0; the decrease was about 0.2–0.4units at pH₀ 5.0, increasing to about 0.5 units at pH₀ 4.3.When pH₀ was 6.0 or higher the removal of Ca²⁺ had little orno effect on pH_c. This situation was not altered by changingthe concentration of K⁺, though in some experiments at pH₀ 5.0–5.2there was a slight decrease in pH₀ (about 0.2 units) when K⁺was increased from 0.2 to 2.0 mol m^–3, an effect apparentlyreversed when K⁺ was higher (5.0 or 10.0 mol m^–3). Theresults suggest that H⁺ transport continues in the absence ofexternal Ca²⁺, despite previous suggestions to the contrary,and that the H⁺ pump does not necessarily run near thermodynamicequilibrium with its chemical driving reaction. They indicate,rather, that the H⁺ pump is under kinetic control and providefurther evidence for the inadequacy of present models for theoperation of the H⁺ pump in charophyte cells, especially inrelation to its proposed role in regulating pH_c. Key words: Chara corallina, Cytoplasmic pH, Calcium     

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     

Phosphate Uptake in the Cyanobacterium Synechococcus R-2 PCC 7942

Phosphate uptake rates in Synechococcus R-2 in BG-11 media (anitrate-based medium, not phosphate limited) were measured usingcells grown semi-continuously and in continuous culture. Netuptake of phosphate is proportional to external concentration.Growing cells at pH_o 10 have a net uptake rate of about 600pmol m^–2 s^–1 phosphate, but the isotopic flux for³²P phosphate was about 4 nmol m^–2 s^–1. There appearsto be a constitutive over-capacity for phosphate uptake. TheK_m and V_max, of the saturable component were not significantlydifferent at pH_o 7.5 and 10, hence the transport system probablyrecognizes both H₂PO^–₄and HPO^2–₄. The intracellularinorganic phosphate concentration is about 3 to 10 mol m^–3,but there is an intracellular polyphosphate store of about 400mol m^–3. Intracellular inorganic phosphate is 25 to 50kJ mol^–1 from electrochemical equilibrium in both thelight and dark and at pH_o 7.5 and 10. Phosphate uptake is veryslow in the dark ( 100 pmol m^–2 s^–1) and is light-activated(pH_o 7.51.3 nmol m^–2 s^–1, pH_o 10600 pmol m^–2s^–1). Uptake has an irreversible requirement for Mg²⁺in the medium. Uptake in the light is strongly Na⁺-dependent.Phosphate uptake was negatively electrogenic (net negative chargetaken up when transporting phosphate) at pH_o 7.5, but positivelyelectrogenic at pH_o 10. This seems to exclude a sodium motiveforce driven mechanism. An ATP-driven phosphate uptake mechanismneeds to have a stoichiometry of one phosphate taken up perATP (1 PO_{4 in}/ATP) to be thermodynamically possible under allthe conditions tested in the present study. (Received June 16, 1997; Accepted September 4, 1997)     

Activation of Ca2+-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells

The effects ofcyanide (CN) on whole cell current measured with the perforated-patchmethod were studied in adrenal medullary cells. Application of CNproduced initially inward and then outward currents at 52 mV ormore negative. As the membrane potential was hyperpolarized, amplitudeand latency of the outward current (I_o) by CNbecame small and long, respectively. A decrease in the externalNa⁺ concentration did not affectthe latency for CN-inducedI_o but enhancedthe amplitude markedly. The CNI_o reversedpolarity at 85 mV, close to the Nernst potential forK⁺, and was suppressed by theK⁺ channel blockers curare andapamin but not by glibenclamide, suggesting thatI_o is due to theactivation of Ca²⁺-dependentK⁺ channels. Consistent with thisnotion, the Ca²⁺-mobilizingagents, muscarine and caffeine, also producedI_o. Exposure toCN in a Ca²⁺-deficient medium for4 min abolished caffeine- or muscarine-induced I_o withoutdevelopment ofI_o, and additionof Ca²⁺ to the CN-containingsolution inducedI_o. We concludethat exposure to CN producesCa²⁺-dependentK⁺ currents in an externalCa²⁺-dependent manner, probablyvia facilitation of Ca²⁺ influx.

     

Effect of Temperature and External pH on the Cytoplasmic pH of Chara corallina

Cytoplasmic pH (pH_c) in Chara corallina was measured (from [¹⁴C]stribution)as a function of external pH (pH₀)and temperature. With pH₀near 7, pH_c at 25?C is 7.80; pH_cincreases by 0.005 pH units?C^–1 temperature decrease, i.e. pH_c at 5 ?C is 7.90. WithpH? near 5.5, the increase in pH_c with decreasing temperatureis 0.015 units ?C^–1 between 25 and 15?C, but 0.005 units?C^–1 between 15 and 5?C. This implies a more precise regulationof pH_c with variations in pH_o at 5 or 15 ?C compared with 25?C. The observed dp H_c/dT is generally smaller than the –0.017units ?C^–1 needed to maintain a constant H⁺/OH^–1,or a constant fractional ionization of histidine in protein,with variation in temperature. It is closer to that needed tomaintain the fractional ionization of phosphorylated compoundsor of CO₂–HCO₃^– The value of dpH_c/dT has importantimplications for several regulatory aspects of cell metabolism.These include (all as a function of temperature) the rates ofenzyme reactions, the _H+ at the plasmalemma(and hence the energy available for cotransport processes),and the mechanism for pH_c regulation by the control of bidirectionalH⁺ fluxes at the plasmalemma.     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

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.

     

Intracellular pH Regulation in the Giant-Celled Marine Alga Chaetomorpha darwinii

The vacuolar pH (pH_v) and the cytoplasmic pH (pH_c) of the marinegiant-celled green alga Chaetomorpha darwinii were measuredby pH microelectrode techniques on extracted vacuolar sap, andby the [^I4C]DMO distribution method respectively. Equilibrationof DMO occurred with a half-time of about 2 h, with an apparentP_DMO of 3.6 x 10^–5 cm s^–1, but the vacuolar concentrationof free, undissociated DMO was always less than the externalconcentration. The explanation offered for freshwater giant-celledalgae of net DMO^– leakage across the plasmalemma cannotapply to Chaetomorpha darwinii, since electrically-driven DMO^–exit from the cytoplasm should be similar across the plasmalemmaand the tonoplast in these cells with large, vacuole-positivepotential differences across the tonoplast. pHc was accordinglycomputed assuming either tonoplast or plasmalemma equilibrationof DMO, with correction for DMO metabolism within the cell.pH_c was 8.0–8.3 in the light in artificial seawater (pH_oabout 8.0), was some 0.5 units lower in the dark, and was slightlylower with an external pH of 7. Vacuolar pH was 6.5–6.9,without consistent effects of illumination or of external pHof 7 rather than 8. While µ_H+ at the tonoplast was similarto that in giant-celled freshwater algae (although with a greatercontribution from relative to pH), µ_H+ at the plasmalemmawas less than 8 kJ mol^–1, i.e. less than one-third ofthe value in freshwater green algae. µ_Na+ was some 13kJ mol^–1 at the plasmalemma. The possibility that theprimary active transport process at the plasmalemma of Chaetomorphadarwinii (and certain other marine algae) is Na⁺ efflux ratherthan H⁺ efflux is discussed.     

Effect of Ammonium on Dark-CO2 Fixation and on Cytosolic and Vacuolar pH Values in Eremosphaera viridis de Bary (Chlorococcales)

At constant external [CO₂], rates of dark-CO₂ fixation of theunicellular green alga Eremosphaera viridis were drasticallyincreased (up to 40-fold) by addition of ammonium (NH₃+ NH₄⁺)at external pH values (pH₀) between 6.0 and 8.0. The cytosolicpH was monitored under identical conditions by micro-pH-electrodemeasurements, and cytosolic and vacuolar pH by the ³¹P-NMR technique.Addition of ammonium (5.0 mol m^– pH₀ 7.0) caused a rapidand transient acidification of the cytosol during the first4 min. Thereafter, the cytosolic pH remained constant at itsoriginal value. A rather constant cytosolic pH was also confirmedby ³¹P-NMR measurements, which, in addition, indicated a slowalkalization of the vacuole (about 0.5 units within 30 min afteraddition of ammonium). Since the dramatic stimulation of dark-CO₂ fixation by ammoniumis not mediated by an alkalization of the cytosol, nor by directammonium effects on phosphoenolpyruvate carboxylase (PEPC, E.C.4.1.1.31 [EC] ), the role of vacuolar alkalization as a possible triggerfor the stimulation of PEP-carboxylase is discussed. Key words: Cytosolic pH, dark-CO₂ fixation, pH-regulation, vacuolar pH     

Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel

Rabbit and human ClC-2GCl channels are voltagesensitive and activated by protein kinase A and low extracellular pH.The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2GCl channel and to determinewhich amino acid residues play a role in this acid activation. Channelopen probability(P_o) at ±80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H⁺concentration (that is, extracellular pH,pH_trans), with anapparent acidic dissociation constant of pH 4.95 ± 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increasedP_o threefold atpH_trans 7.4, at which the channelnormally exhibits lowP_o. Withextracellular pH reduction (protonation) or amidation, increasedP_o was due to asignificant increase in open time constants and a significant decreasein closed time constants of the channel gating, and this effect wasinsensitive to applied voltage. With the use of site-directedmutagenesis, the extracellular region EELE (amino acids416-419) was identified as the pH sensor and amino acid Glu-419was found to play the key or predominant role in activation of theClC-2G Cl channel byextracellular acid.

     

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.

     

Membrane Potential Measurement of Protoplasts Isolated from Vigna mungo Hypocotyl Using a Fluorescent Probe, diS-C3-(5)

Membrane potentials of protoplasts isolated from Vigna mungohypocotyl segments were measured using the fluorescent probediS-C₃-(5). The fluorescence intensity changed in response tothe external K⁺ concentration. Membrane potential was estimatedto be inside negative (–85?8 mV at 0.1 mM KCl) from theNernst equation for K₊. The membrane potential was not affectedby DCCD (50 µM) or low temperature (5?C). Addition of0.5 mM Ca₂₊ to the protoplast suspension markedly depolarizedthe membrane potential, and subsequent EDTA treatment repolarizedit to the initial level. The Ca₂₊ effect on the membrane potentialmay be due to change in the permeability ratio of Cl^–to K₊. (Received December 16, 1986; Accepted April 22, 1987)     

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     

Light-Induced Changes in Cytosolic pH in Leaf Cells of Egeria densa: Measurements with pH-Sensitive Microelectrodes

Light-induced changes of cytosolic pH (pH_c) and the plasmalemmapotential (E_m) in dark-adapted leaf cells of the aquatic plant,Egeria densa were measured simultaneously with double-barreledpH-sensitive microelectrodes. Upon illumination, pH_c increasedtransiently and then decreased to a level that was lower thanthe original value, while the plasmalemma was greatly hyperpolarizedafter an initial small depolarization. DCMU inhibited the light-inducedchanges in both pH_c and E_m. DCMU acted without directly inhibitingthe electrogenic proton pump in the plasmalemma since a decreasein pH_c caused by treatment with butyrate (H⁺-loading) hyperpolarizedthe plasmalemma in DCMU-pretreated cells. N.N-Dicyclohexylcarbodiimide(DCCD) also inhibited the light-induced changes in both pH_cand E_m. This result may be explained by direct inhibition ofthe proton pump in the plasmalemma by DCCD since the decreasein pH_c caused by butyrate did not induce membrane hyperpolarizationin DCCD-treated leaf cells. Fusicoccin induced membrane hyperpolarizationand slight acidification of the cytosol. DCCD inhibited thefusicoccin-induced changes in both pH_c and E_m. The mechanismof the light-induced changes in pH_c is discussed in relationto activities of the proton pump in the plasmalemma and photosynthesis. (Received January 10, 1994; Accepted June 9, 1994)     

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     

Control of Passive Permeability in the Chara Plasmalemma

Conductance to K⁺ alters as a function of membrane potential(_m). Conductance to H⁺ (or OH^–) changes with externalpH (pH^o) This conductance change can be modulated by alteringcytoplasmic pH or external K⁺ concentration, both of which alsoalter _m. We suggest a role for H⁺ conductance in regulatingcytoplasmic pH above pH^o 7.0.