Effect of reactive oxygen species on NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeation in Xenopus laevis oocytes

Marc Cougnon, Samia Benammou, Franck Brouillard, Philippe Hulin, and Gabrielle Planelles

Institut National de la Santé et de la Recherche Médicale Unité 467, Université Paris V, Faculté de Médecine Necker-Enfants Malades, 75730 Paris Cedex 15, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the effects of reactive oxygen species (ROS) on NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeation in Xenopus laevis oocytes, we used intracellular double-barreled microelectrodes to monitor the changes in membrane potential (Vm) and intracellular pH (pHi) induced by a 20 mM NH4Cl-containing solution. Under control conditions, NH4Cl exposure induced a large membrane depolarization (to Vm = 4.0 ± 1.5 mV; n = 21) and intracellular acidification [reaching a change in pHi (Delta pHi) of 0.59 ± 0.06 pH units in 12 min]; the initial rate of cell acidification (dpHi/dt) was 0.06 ± 0.01 pH units/min. Incubation of the oocytes in the presence of H2O2 or beta -amyloid protein had no marked effect on the NH4Cl-induced Delta pHi. By contrast, in the presence of photoactivated rose bengal (RB), tert-butyl-hydroxyperoxide (t-BHP), or xanthine/xanthine oxidase (X/XO), the same experimental maneuver induced significantly greater Delta pHi and dpHi/dt. These increases in Delta pHi and dpHi/dt were prevented by the ROS scavengers histidine and desferrioxamine, suggesting involvement of the reactive species 1Delta gO2 and ·OH. Using the voltage-clamp technique to identify the mechanism underlying the ROS-measured effects, we found that RB induced a large increase in the oocyte membrane conductance (Gm). This RB-induced Gm increase was prevented by 1 mM diphenylamine-2-carboxylate (DPC) and by a low Na+ concentration in the bath. We conclude that RB, t-BHP, and X/XO enhance NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx into the oocyte via activation of a DPC-sensitive nonselective cation conductance pathway.

ammonium ions; nonselective cationic conductance


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MAJOR BIOLOGICAL SOURCES of reactive oxygen species (ROS) are the electron transport systems of the mitochondrial membrane, the activity of cytoplasmic enzymes, and the cyclooxygenase pathway (15, 23, 34). Although they are produced physiologically and despite various cellular and extracellular defense systems (39), ROS seem to contribute to numerous pathophysiological states and diseases such as age-related alterations, inflammatory processes, ischemia-reperfusion damage, macular and neurological degeneration, DNA damage, programmed cell death, and carcinogenesis (1, 8, 29, 39). It is thus important to identify the multiple targets of oxidative threats. It can be expected, because of their lipid composition and the presence of proteins with SH groups, that the transport functions of cell membranes may be adversely affected by ROS and that ROS-induced damage may compromise cell homeostasis. Such interactions of ROS with various ionic transport systems of the cell membrane were recently reviewed (17). Our understanding of the effects of ROS on ionic transport pathways may further benefit from studies in Xenopus laevis oocytes functionally expressing heterologous channels (10, 30, 31, 35). Although numerous studies have been devoted to the properties of Xenopus oocytes (for reviews, see Refs. 9 and 36), little is known about the effects of ROS on the endogenous ionic pathways of this cell. However, a study focusing on the effects of ROS on K+ channels mentioned ROS activation of an endogenous nonselective cationic conductance of the oocyte membrane (10). We previously reported (6) that the oocyte membrane is endowed with at least two endogenous nonselective conductances. Both of these are physiologically active, influencing the resting membrane potential (6); one of them, nonselective cationic conductance (Gcat), allows NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx into the oocyte and is inhibited by diphenylamine-2-carboxylate (DPC) (5, 6).

The aim of the present study was to determine whether Gcat is sensitive to ROS. To this end, we used double-barreled selective microelectrodes to monitor membrane potential, membrane conductance, and intracellular pH (pHi) on NH4Cl exposure, under control conditions and in the presence of various ROS-generating systems. Results are consistent with the enhancement of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx in the presence of ROS, mediated by Gcat activation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Biological Material

Xenopus laevis (Centre National de la Recherche Scientifique, Montpellier, France) were kept in filtered water and fed twice a week with minced beef heart and live Chironoma. Anesthesia was achieved by brief immersion in cold water supplemented with 2 mM ethyl-n-aminobenzoate-methane sulfonate and was maintained by cooling the toad on ice during partial ovariectomy.

Ovarian fragments were incubated in an amphibian-adapted Ringer solution (see Artificial Solutions and Perfusion of Oocytes for composition) supplemented with 2 U/ml collagenase (1A; Sigma). After rinsing, stage V-VI oocytes were manually microdissected and defolliculated. Oocytes were stored at 18°C for up to 3 days in the Ringer solution supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin.

Artificial Solutions and Perfusion of Oocytes

Experiments were performed at room temperature. An oocyte was placed in a Plexiglas microchamber and was superfused for electrophysiological studies. The gravimetric delivery of various artificial solutions was controlled by a custom-made electronic switch made in our laboratory. The composition of the Ringer solution was (in mM) 85 NaCl, 1 KCl, 1 CaCl2, 1 MgCl2, and 5 Tris-aminomethane, buffered at pH 7.4 with NaOH. In the low-Na+ solution, four-fifths of the NaCl was replaced by 68 mM choline chloride. In the low-Cl- solution, NaCl was replaced by sodium gluconate and CaCl2 was raised to 3 mM (to compensate for Ca2+ chelation by gluconate salt). Membrane permeation to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was studied by exposing the oocytes to a solution containing 20 mM NH4Cl (at the expense of NaCl).

To investigate the effects of ROS, we added to the Ringer solution and the NH4Cl solution one of the following ROS-generating systems: rose bengal (RB; 0.1 µM), xanthine (X; 50 µM) + xanthine oxidase (XO; 50 mU/l), or tert-butyl-hydroxyperoxide (t-BHP; 500 µM). These compounds produce reactive species as follows (although not exhaustively, because of chain reactions, as referenced in Ref. 12): photoactivated RB produces singlet oxygen (1Delta gO2) and superoxide radical (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·), X/XO generates O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, and t-BHP generates O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and the hydroxyl radical (·OH). The oxygen metabolite hydrogen peroxide (H2O2) is not, strictly speaking, an active species; however, it can form ·OH and is reported to initiate free radical cytotoxicity, including cell toxicity induced by the beta -amyloid protein (3, 39). Thus we also tested the effects of H2O2 (500 µM) and of fragment 1-40 of the beta -amyloid protein (beta -A; 20 µM).

In the t-BHP, H2O2, and beta -A experimental series, the oocytes were preincubated in the presence of these compounds and then electrophysiological measurement was performed in their continuous presence; they were exposed to NH4Cl as soon as a stable puncture was assessed (2-5 min). In the X/XO series, the oocytes were impaled with a microelectrode during Ringer exposure before being perfused with X/XO-supplemented solutions. In the RB experimental series, the oocytes were impaled during Ringer perfusion and were then exposed in darkness to an RB-containing Ringer solution before being illuminated up to the end of the experiment; the NH4Cl-containing solution was introduced after 10 min of illumination. In a separate series, we used the 1Delta gO2 scavenger histidine (Hist; 1 mM) and the ·OH scavenger desferrioxamine (DF; 50 µM). Occasionally, we added transport system inhibitors to the artificial solutions: diphenylamine-2-carboxylate (DPC, 1 mM), 3'-5'-dichlorodiphenylamine-2-carboxylic acid (DCPPC, 100 µM), or gadolinium (Gd3+, 100 µM) was used to inhibit the oocyte nonselective cationic conductances (6); ouabain (100 µM), bumetanide (100 µM), or quinine (0.5 mM) was used to inhibit Na,K-ATPase, the Na-K-2Cl symport, and the quinine-sensitive K+ conductance, respectively (6).

All products were purchased from Sigma-France except beta -A (lot 518765; Bachem, France). DCDPC was a kind gift from Dr. H. J. Lang (Hoechst, Frankfurt, Germany).

Electrophysiological Measurements

Membrane potential and pHi measurements. Simultaneous measurements of membrane potential and pHi were performed with double-barreled pH-sensitive microelectrodes. The details of ion-sensitive microelectrode construction were reported previously (2). In the present study, the tip of the pH-sensitive barrel contained the Fluka H+ ionophore 95291, and its shank was filled with (in mM) 67 NaCl, 40 KH2PO4, and 23 NaOH. The shank of the conventional barrel was filled with 1 M KCl. The double-barreled microelectrode was gently beveled on a microgrinder (de Marco Eng, Geneva, Switzerland) and was then placed on a three-dimensional micromanipulator (MM 33; Narishige, Tokyo, Japan) and connected via Ag/AgCl electrodes to the input of an ultra-high-impedance electrometer (FD 223; WPI, Aston, UK). The electrical circuit was closed by a 1 M KCl agarose Ag/AgCl macroelectrode placed in the bath. On impalement of an oocyte, the conventional barrel of the microelectrode measured the transmembrane potential (Vm) and the pH-sensitive barrel measured the proton electrochemical potential (VH) across the cell membrane. The electrometer output displayed on a multichart recorder (Sefram; Servofram, Saint Etienne, France) gave both the Vm signal and the algebraic sum of VH - Vm.

pHi was obtained by the following relationship
pH<SUB>i</SUB> = pH<SUB>ref</SUB> − (<IT>V</IT><SUB>H</SUB> − <IT>V</IT><SUB>m</SUB>)/S (1)
where pHref is the pH of the control solution and S is the slope of the selective microelectrode, i.e., the chemical potential difference induced by a change of 1 pH unit. Selected microelectrodes had 50 mV <=  S <=  58 mV in the pH range of 7.4-6.4; we verified that S was not affected by the different compounds used in this study.

On NH4Cl exposure, we measured the changes in Vm (Delta Vm) and the rate of cell acidification as a function of time (dpHi/dt). If cell buffering power and cell volume are constant, the measured increase in dpHi/dt reflects enhancement of initial NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx into the oocyte. After 12 min of NH4Cl exposure, we measured Delta pHi, the change of pHi from its initial value.

Voltage-clamp experiments. Voltage-clamp experiments were performed using two low-resistance (1-5 MOmega ) conventional microelectrodes filled with 3 M KCl. By means of a software program (pClamp, Axon Instrument, Dipsi, Chetillon, France), voltage steps lasting 5 s (±20 mV from resting Vm in the range -120 to +30 mV) were imposed by a clamp amplifier (Gene Clamp; Axon Instruments) to obtain current-voltage (I-V) curves.

Determination of Intrinsic Cell Buffering Power

The buffering power of a cell is a short-term regulatory mechanism for pHi, leading to attenuation, by release or consumption of protons, of the changes in pHi consecutive to an intracellular alkaline or acid load. This intrinsic property of the cell is not influenced by extracellular factors and is due to the presence of (various) cytoplasmic buffer systems that (as any buffer) have a stronger effect when pH is near the apparent dissociation constant (pK) of the cell buffer. As a corollary, the intrinsic cell buffering power (omega ) may vary depending on pHi changes. Thus, in this study, we performed a determination of omega  as a function of pHi. Determination of omega  has been detailed elsewhere (4). In the present study, determination of omega  in the oocyte was assessed by measuring pHi during exposure of oocytes to decreasing concentrations of a weak acid (acetic acid, ac) and its conjugate base (acetate, Ac-, at constant extracellular pH of 7.4. Sodium acetate-containing solutions (40, 20, 10, and 5 mM at the expense of NaCl concentration) were used. Delta pHi for each sodium acetate concentration step was measured. The concomitant change in intracellular Ac- concentration ([Ac-]i) was calculated with the equation
[Ac<SUP>−</SUP>]<SUB>i</SUB> = [ac]<SUB>i</SUB> × 10<SUP>pH<SUB>i</SUB>−pK′</SUP> (2)
where [ac]i is the intracellular concentration of ac (considered equal to its extracellular concentration), and pK' is the apparent dissociation constant for ac-Ac- (pK' = 4.76).

From the measured Delta pHi and from calculated [Ac-]i, omega  was then calculated at pHi intervals of 0.2 pH units, according to the following equation
&ohgr;=&Dgr;[Ac<SUP>−</SUP>]<SUB>i</SUB>/&Dgr;pH<SUB>i</SUB> (3)
Table 1 shows that in the 6.85 <=  pHi <=  7.45 range, omega  does not change.

                              
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Table 1.   Estimation of intrinsic cell buffering power in oocytes

Control of Oocyte Volume

All electrophysiological experiments were performed under stereomicroscopic control without observing any apparent change in the oocyte size. To further confirm that cell volume was not affected during our experiments, we performed an experimental series in which we continuously recorded the oocyte size as follows. Oocytes were deposited on a coverslip placed in a Plexiglas microchamber under an inverted microscope (IX 70 Olympus, Rungis, France). The oocyte size was monitored by a GEN 4 intensified charge-coupled device (CCD) camera (Princetown Inst. Roper Scientific, Evry, France) during exposure to various solutions; the perfusing system was as described above. In this series, cells were exposed to the Ringer solution and then to the NH4Cl-containing solution or to the Ringer solution and then to the RB-supplemented Ringer and to the RB-supplemented NH4Cl-containing solution, successively. Control of the experiments, image acquisition, and data analysis were handled by the software program Metafluor (Universal Imaging, Roper Scientific). The number of pixels was integrated as the surface (S) of the oocyte cross section. The relative change in volume (V) was calculated by the following equation
(S<SUB>P</SUB><IT>/S</IT><SUB>I</SUB>)<SUP>3<IT>/</IT>2</SUP><IT> = </IT>V<SUB>P</SUB>/V<SUB>I</SUB> (4)
where I refers to the initial Ringer perfusion and P to other perfusates.

Statistics

Results are expressed as means ± SE, with n = number of oocytes or N = number of observations. All series were performed on oocytes provided from at least three different X. laevis donors. Unless otherwise stated, the statistical analyses were performed with the two-tailed paired or unpaired Student's t-test; differences were considered significant for a P value <0.05.


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ABSTRACT
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METHODS
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Effects of NH4Cl Under Control Conditions

We first ran a series of control experiments (n = 21) to check NH4Cl effects on Vm and pHi. As shown in Fig. 1 and summarized in Table 2, exposure to the NH4Cl-containing solution induced an immediate membrane depolarization and slow acidification: Vm became slightly positive and then stabilized or slightly repolarized. In 13 oocytes, the acidification was preceded by a very slight alkalinization of ~0.02 pH units. After 12 min of NH4Cl exposure, Delta pHi was 0.59 ± 0.06 pH units (Table 2). The maximal NH4Cl-induced cell acidification was assessed in 17 oocytes: it reached 0.75 ± 0.07 pH units after 20 min of NH4Cl exposure.


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Fig. 1.   Effect of NH4Cl on membrane potential (Vm) and intracellular pH (pHi) under control conditions. Tracing was obtained with a double-barreled pH-sensitive microelectrode. Introduction of the microelectrode into the cell yields a negative deflection of the electrical potential. Timing of perfusion is indicated by horizontal bars below the graph [open bar, Ringer solution; filled bar, NH4Cl (20 mM)]. A: pHi; note the slight intracellular alkalinization (indicated by arrow) on NH4Cl exposure and the acid rebound on NH4Cl withdrawal. B: Vm.


                              
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Table 2.   Effect of NH4Cl on membrane potential, and pHi under control conditions or in presence of RB

On NH4Cl withdrawal, membrane potential rapidly repolarized and pHi slowly returned toward its resting value. In 18 oocytes, this cell realkalinization was preceded by an acid rebound (0.06 ± 0.01 pH units), which was absent in 3 oocytes. Complete return to the baseline pHi value took >1 h; in most cases, the recording did not last this long.

In a separate series (n = 8), we looked for change in oocyte volume during this experimental procedure. On NH4Cl exposure, as well as on NH4Cl withdrawal, no relative change in volume occurred (0.12 ± 0.1% and -0.03 ± 0.1%).

Effects of NH4Cl in Presence of Rose Bengal

To study the effects of ROS on NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeation, the oocytes were exposed to an NH4Cl-containing solution in the presence of RB while Vm and pHi were measured. In a first series (n = 14), we investigated the effects of RB exposure. In the dark, addition of RB to the Ringer solution did not significantly modify Vm or pHi (P = 0.2). Photoactivation of RB (obtained by illumination of oocytes) induced a membrane depolarization but had no effect on baseline pHi (Fig. 2). As shown in Table 2, Vm did not reach positive values on NH4Cl exposure, whereas it did do so in the control series. The NH4Cl-induced Delta Vm was lower than in the control series but was associated with an enhanced dpHi/dt and a larger Delta pHi. The maximal cell acidification, measured after 20 min of NH4Cl exposure, was also increased [1.19 ± 0.12 (n = 9) vs. 0.75 ± 0.07 pH units in control series (n = 17); P < 0.05]. In contrast with the control series, NH4Cl exposure in the presence of RB did not induce an initial cell alkalinization; on NH4Cl withdrawal, an acid rebound (0.07 ± 0.01 pH units) was noticed in 11 of 14 oocytes.


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Fig. 2.   Effect of NH4Cl on Vm and pHi in the presence of rose bengal. Tracing was obtained with a double-barreled pH-sensitive microelectrode. Timing of perfusion is indicated by horizontal bars below the graph [open bar, Ringer solution; hatched bars, Ringer solution supplemented with rose bengal in darkness (heavy hatching) or during illumination (light hatching); filled bar, NH4Cl (20 mM)]. A: pHi; note the acid rebound on NH4Cl withdrawal. B: Vm.

In a separate series (n = 7), we checked the effects of this protocol on cell volume. As regards the initial volume (i.e., during Ringer perfusion), no relative change occurred during successive exposures to RB (0.15 ± 0.1%), exposures to photoactivated RB (0.18 ± 0.1%), during RB + NH4Cl exposure (0.23 ± 0.1%), or on NH4Cl withdrawal (0.13 ± 0.1%).

In another series (n = 17), we observed that the effects of photoactivated RB were largely prevented by adding 1 mM Hist (a 1Delta gO2 scavenger) to the RB-containing solution. As shown in the representative tracing in Fig. 3, the decrease in resting Vm on RB + Hist illumination was delayed compared with that observed with RB alone (see Fig. 2): after 10 min of illumination of the RB + Hist solution the Vm loss was 7.5 ± 1.8 mV, whereas after 10 min of illuminated RB alone it was significantly higher (14. 3 ± 2.4 mV, P < 0.05). On NH4Cl exposure in the presence of RB + Hist, Vm depolarized to 1.2 ± 1.5 mV, a value not significantly different from that in the control series (4.0 ± 1.5; n = 21; P = 0.2). Moreover, in the presence of RB + Hist, the NH4Cl-induced Delta pHi and dpHi/dt were 0.76 ± 0.06 pH units and 0.06 ± 0.01 pH unit/min, respectively, both values being significantly lower than with RB alone (1.13 ± 0.09 pH units and 0.12 ± 0.01 pH unit/min; n = 14; P < 0.05).


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Fig. 3.   Effect of NH4Cl on Vm and pHi in the presence of rose bengal and histidine. Tracing was obtained with a double-barreled pH-sensitive microelectrode. Timing of perfusion is indicated by horizontal bars below the graph [open bar, Ringer solution; hatched bars, Ringer solution supplemented with rose bengal and histidine in darkness (heavy hatching) or during illumination (light hatching); filled bar, NH4Cl (20 mM)]. A: pHi. B: Vm.

Activation of Nonselective Cationic Conductance by Rose Bengal

We previously reported (6) that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeation into oocytes occurs via several pathways. Because the RB-induced increase in NH4Cl-induced dpHi/dt was consistent with the activation of a NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-permeant pathway by ROS, we wished to identify this transport system.

We first investigated whether Na,K-ATPase, the Na-K-2Cl symport, or a K+ conductance might be involved in the observed increase in dpHi/dt. The lack of effect of their respective inhibitors argues against a role for any of these. Adding ouabain, bumetanide, and quinine to the artificial solutions failed to prevent the increase in NH4Cl-induced dpHi/dt in the presence of photoactivated RB [dpHi/dt = 0.13 ± 0.05 pH unit/min (n = 8) in presence of RB + NH4Cl + inhibitors vs. 0.12 ± 0.01 pH unit/min (n = 14) in the presence of RB + NH4Cl].

We next checked to see whether RB activates Gcat, a major pathway for NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx into the oocyte (6). To this end, we performed a series of voltage-clamp experiments. Figure 4A shows typical oocyte I-V curves obtained during perfusion with Ringer and during exposure to RB; similar results were obtained in seven oocytes. Clearly, perfusion with RB leads to a large increase of oocyte membrane conductance, but only in the presence of light. Figure 4B shows that DPC induced a strong inhibition of the induced Gm increase (n = 5). The reversal potential (Erev) of the DPC-sensitive current fell between -10 and 0 mV. DCPPC (n = 3) had a mild inhibitory effect on the RB-induced Gm increase (results not shown); Gd3+ (n = 3) was ineffective (results not shown).


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Fig. 4.   Oocyte current-voltage (I-V) curves. Typical I-V curves obtained by means of 2-electrode voltage clamp are shown. A: open circle , control condition; , addition of rose bengal in darkness; , illuminated rose bengal. B: open circle , control condition; black-triangle, addition of rose bengal in darkness and in the presence of diphenylamine-2-carboxylate (DPC); triangle , illumination of rose bengal in the presence of DPC; , illuminated rose bengal (DPC withdrawal).

To discriminate between a RB-induced activation of Gcat or of a Cl- conductance (GCl), I-V curves of oocytes exposed to photoactivated RB were determined with either a low-Na+ (n = 13) or a low-Cl- (n = 8) perfusate. Figure 5 shows that reducing the Na+ concentration in the bath prevented the RB-induced increase of Gm (Fig. 5A), whereas the low-Cl- perfusate did not (Fig. 5B). Together, these results are consistent with RB-induced activation of a DPC-sensitive Gcat.


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Fig. 5.   Oocyte I-V curves. A: open circle , control condition; black-triangle, illuminated rose bengal under low-Na+ condition. B: open circle , control condition; black-down-triangle , illuminated rose bengal under low-Cl- condition.

Effects of NH4Cl in Presence of Other ROS-Generating Systems

Finally, we tested the effects of other ROS-generating systems on Vm and pHi. When using t-BHP as the ROS-generating system (n = 15), we observed in preliminary experiments that an incubation time >15 min with this compound induced both cell acidification and membrane depolarization. The resting Vm values attained, but not the resting pHi values, were positively correlated with the duration of incubation (Fig. 6). As with the RB series, NH4Cl-induced Delta pHi and dpHi/dt were significantly higher than their control values (Table 3) and no initial alkalinization of the cell was noted on NH4Cl exposure. In a separate series (n = 6), we added 50 µM DF (an ·OH scavenger) to the t-BHP solutions. Part of the Vm loss observed with t-BHP alone was prevented: resting Vm was -42 mV for the shortest incubation (50 min) and -9 mV for the longest incubation (180 min), with a mean value of -19.2 ± 4.9 mV. However, the decrease in baseline pHi was not prevented by the presence of DF (pHi = 7.21 ± 0.07). During NH4Cl exposure, Vm was -1.8 ± 2.8 mV (not different from control value); Delta pHi was 0.39 ± 0.09 pH units and dpHi/dt was 0.04 ± 0.01 pH unit/min, i.e., both were significantly lower than with t-BHP alone (0.8 ± 0.1 pH units and 0.11 ± 0.01 pH unit/min; n = 15; P < 0.05).


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Fig. 6.   Effect of incubation time with tert-butyl-hydroxyperoxide on Vm and on pHi. , Vm (mV) values; the relationship between Vm and incubating time is given by the solid line (y = 0.155x - 30.88; R2 = 0.79; P < 0.05). , pHi values; the relationship between pHi and incubating time is indicated by the dashed line (y = -3.72 × 10-4 x + 7.18; R2 = 0.056; P = 0.4).


                              
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Table 3.   Effect of NH4Cl on Vm and pHi under control conditions or in presence of ROS-generating systems

In contrast to the effect of t-BHP, addition of X/XO to the artificial solutions did not alter resting Vm or pHi. In this experimental series (n = 6), NH4Cl-induced Delta pHi and dpHi/dt were nonetheless significantly higher than the control values (Table 3). As with the RB and t-BHP series, initial alkalinization on NH4Cl exposure was not observed.

We next investigated the effects of H2O2. Preliminary experiments showed that addition of H2O2 to the perfusate had no effects on Vm or pHi or on NH4Cl-induced Delta Vm, Delta pHi, or dpHi/dt. Even after 90 min of incubation in the presence of H2O2 (n = 8), the only significant effect on any of these parameters was that the NH4Cl-induced Delta Vm was lower than in the control series (Table 3).

Finally, we tested the effect of incubating the oocytes with beta -A. After the longest incubation (24 h, n = 12), the only significant change was cell acidification (Table 3). Together, these results show that, as observed in the presence of RB, NH4Cl-induced dpHi/dt is enhanced in the presence of t-BHP and X/XO.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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NH4Cl-Induced pHi Changes Under Control Conditions

In most cells, extracellular NH4Cl induces a rapid cell alkalinization followed by a partial reacidification. This pHi pattern is due to a higher membrane permeability to NH3 than to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (22): the initial alkalinization on NH4Cl exposure reflects NH3 influx and its protonation within the cell; the secondary reacidification reflects a delayed NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx followed by the partial dissociation of ammonium ions into NH3 and H+. However, a few cell types do not exhibit the NH4Cl-induced pHi pattern described above, because they are endowed with unusual NH3 and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> membrane permeabilities. Colonic crypt cells show no change in pHi during luminal NH4Cl perfusion because their apical membrane is impermeable to both NH3 and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (28). Glial cells from bee retina exhibit a very transient initial cell alkalinization on NH4Cl exposure and then rapidly acidify below baseline pHi because they are endowed with cation-Cl- cotransporters (18). The presence of Na-K(NH<UP><SUB>4</SUB><SUP>+</SUP></UP>)-2Cl cotransport confers high NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeability to the apical membrane of medullary thick ascending limb cells, and because of the lack of NH4Cl-induced initial alkalinization, these cells have been considered impermeable to NH3 (13).

In the present study, we first examined in a control series the effects of 20 mM NH4Cl on pHi of Xenopus laevis oocytes. The mean baseline pHi was 7.4, i.e., a slightly more alkaline value than previously reported by our group (6, 7), perhaps because of seasonal variations (9, 36). In agreement with previous results from our laboratory (6, 7) and other laboratories (5), we observed in the present study that exposure of oocytes to 20 mM NH4Cl induces a deep cell acidification. This cell acidification is consistent with massive NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry into the oocyte. Careful examination of pHi tracings of the control series revealed that, on NH4Cl exposure, 13 oocytes exhibited a tiny initial alkalinization, consistent with NH3 permeation. In eight oocytes from the control series, the lack of initial alkalinization may suggest that they were impermeable to NH3. However, this suggestion is not supported in five of these cells, in which an acid rebound occurred on NH4Cl withdrawal: an acid rebound is a qualitative mirror image of initial alkalinization, missing because it was overwhelmed by the acidifying process of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx and by the increase in cell buffering power afforded by intracellular NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Finally, in the control series, three oocytes presented neither initial alkalinization on NH4Cl exposure nor acid rebound on NH4Cl withdrawal, consistent with an undetectable NH3 permeability in these cells. At present, we have no explanation for these discrepancies in NH3 permeability of the oocyte membrane. First, no macroscopic difference was observed among the cells: all studied oocytes were selected at stages V-VI. Second, the initial alkalinization was not a microelectrode capacitive artifact (difference in resistances between the 2 microelectrode barrels may result in apparent transient alkalinization when a large depolarization occurs), because recordings with a single microelectrode also showed both types of behavior, depending only on the punctured oocyte. Third, the baseline pHi of oocytes without initial cell alkalinization (or without acid rebound) was not different from the baseline pHi of the other oocytes, whereas in glial cells the amplitude of the initial alkalinization was related to the value of resting pHi (18). Thus we can only speculate that differences in lipid composition and/or in the expression level of an endogenous transport system (permeable to ammoniac gas) account for the difference in NH3 permeability among oocytes. A better understanding of the oocyte membrane's gas permeability is important because these cells are of particular interest in studying permeation through functionally expressed channels (19, 20).

Effects of ROS on Vm, pHi, and NH4Cl-Induced pHi Changes

From the literature, it appears that the effect of ROS on pHi depends on the nature of the reactive species and of the tissue. In phagocytes, the superoxide radical (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) raises pHi (26) and, conversely, a rise in pHi enhances the production of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· (27). In C6 glioma cells (33) and in H9c2 cardiac cells (37) the hydroxyl radical (·OH), produced from H2O2 breakdown by the Fenton reaction, induces cell acidosis, but in rat hepatocytes neither H2O2 nor t-BHP affects resting pHi (24). Our results show that in oocytes, peroxides have various effects on resting pHi: t-BHP leads to cell acidification, and so does beta -A, which is reported to induce H2O2 formation (3), but H2O2 itself has no effect on pHi. Of course, the cellular effects of beta -A involved more than just H2O2 production (25), which may explain why we observed different effects of beta -A and H2O2. Another possibility is that we simply missed an H2O2 effect because of an insufficient H2O2 concentration. However, H2O2 concentrations (25-50 µM) much lower than those used in the present study (500 µM) were sufficient to affect the function of Kv3.3 and Kv3.4 Shaker K+ channels expressed in oocytes (35). We also observed that peroxides had different effects on resting Vm: t-BHP induced a decrease in Vm, whereas beta -A and H2O2 did not affect its value. From these observations, the ROS-induced decrease in resting pHi and the ROS-induced decrease in resting Vm do not seem to be related (as supported by Fig. 6). The reason why a NH4Cl-induced dpHi/dt increase was not obtained with every ROS-generating system may be the susceptibility of amino acid(s) in Gcat sequence to the different reactive species. It was proposed that given positions of cysteine, histidine, or methionine explain the sensitivity of Kv3.3 and Kv3.4 to H2O2 or the sensitivity of Kv1.4 and Kv3.4 to RB (10, 35).

To investigate the effect of ROS on NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx in oocytes, we analyzed pHi changes during NH4Cl exposure. The NH4Cl-induced cell acidification (Delta pHi) reflects a net proton excess subsequent to intracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> dissociation and to the H+ shuttle that occurs during long-term exposure to NH4Cl. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx, Phi NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, is manifested as the product Phi NH<UP><SUB>4</SUB><SUP>+</SUP></UP> = (dpHi/dt) · omega  · V. To see whether the measured increases in dpHi/dt directly reflect the enhancement of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx into the oocyte, in other words, whether the term omega  · V was constant, we monitored the oocyte size and measured the oocyte buffering power. This latter determination was performed in the range of 6.85 <=  pHi<= 7.45 (because cell acidosis was observed in the t-BHP and beta -A series; see Table 3) and constitutes, to our knowledge, the first report of omega  as a function of pHi in oocytes. No change in relative volume was observed, and omega  was found constant in the range of resting pHi measured in the present study. Together, these results allow us to firmly conclude that there was an increased NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx in the RB, X/XO, and t-BHP series. The lack of transient cell alkalinization in these experimental series is also consistent with this conclusion.

In summary, during RB, X/XO, and t-BHP exposure, a NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-permeant membrane pathway is activated. The prevention of this activation by Hist and DF suggests involvement of 1Delta gO2 and ·OH in this effect.

Involvement of Gcat in RB-induced Increase in NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> Influx

NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx into oocytes is mainly mediated by a Gcat that is physiologically active under control conditions and is further activated in the presence of NH4Cl, but the influx may also occur via Na,K-ATPase, the Na-K-2Cl symport, and a quinine-sensitive K+ conductance (6); these latter transport systems are not mainly involved in the ROS-induced increase in dpHi/dt, because their inhibitors did not modify it. On the other hand, results listed in Tables 2 and 3 show that, in the presence of several ROS-generating systems including the presence of photoactivated RB, resting Vm is depolarized from its control level. Theoretically, membrane depolarization may result from inhibition of Na,K-ATPase or reduction of the K+ conductance, in which case a decrease in oocyte Gm would be expected. Membrane depolarization can also result from activation of a conductance to an ion whose equilibrium potential (E) is more positive than the resting Vm, in which case an increase in Gm would be expected. This latter possibility is supported by the increase in slope conductance observed in the presence of photoactivated RB (Fig. 4A). This Gm increase was prevented by DPC (Fig. 4B), an inhibitor of GCl and of Gcat (11). Because the Erev of the DPC-sensitive current cannot serve to discriminate with accuracy between an effect on GCl or Gcat (both ECl and Ecat being near Erev, see Ref. 6), we performed separate series of I-V curves under low-Cl- and low-Na+ conditions (Fig. 5). The external reduction of bath Na+ concentration prevented the RB effect on Gm, whereas reduction of bath Cl- did not. Together, these results are consistent with an RB-induced activation of Gcat.

The activation of Gcat is also consistent with the Vm and NH4Cl-induced Delta Vm values reported in this study (Tables 2 and 3). The activation of a conductance permeable to cationic species tends to displace resting Vm toward Ecat and thus to a less polarized value. Consequently, further activation of Gcat induces Delta Vm of lower amplitudes than in control conditions.

We previously established (6) that Gcat is inhibited by DPC and to a lesser extent by DCDPC, but not by Gd3+. A similar profile of Gcat inhibition was observed in the voltage-clamp experiments of the present study. The activation of a nonselective cationic conductance by oxidative stress has been reported in vascular endothelial cells, although pharmacological inhibition was not investigated (16). The same study reported that a 1-h incubation with t-BHP induced a membrane depolarization of calf endothelial cells to the value of -4 mV, i.e., a value near Ecat, and the authors concluded that this change in resting Vm reflects the activation of a nonselective cationic conductance (16). Interestingly, very similar results were obtained in our study with the longest incubations of oocytes in the presence of t-BHP (Fig. 6): cells were depolarized to a slightly negative Vm value. A slight negativity of Vm was also reached on NH4Cl exposure in the presence of several ROS-generating systems (see Tables 2 and 3), consistent with a nearly full activation of Gcat in the presence of ROS + NH4Cl (Gcat overwhelming other partial membrane conductances, thus bringing Vm near to the value of Ecat). Under control conditions, by comparison, Vm depolarized all the way to positive voltages on NH4Cl exposure. This result is consistent with a previous study from our group (6) showing that NH4Cl induced the concomitant activation of Gcat and of other membrane conductances, including a putative Na+ conductance; indeed, a Na+ conductance induced by depolarization was also recently reported by others (21). From the above results, we conclude that the decrease in oocyte resting Vm in the presence of ROS is consecutive to the activation of a DPC-sensitive Gcat. This activation is subsequently reinforced by the presence of extracellular NH4Cl and induces an increase in NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx into the oocyte, as reflected by a dpHi/dt enhancement.

In summary, our study provides insights into ROS effects on the endogenous properties of the Xenopus oocyte membrane, which are important because this cell is a particularly interesting model for the study of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and NH3 membrane transport (20) because of its high permeability to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and low NH3 permeability (6). The consequences of ROS effects on the physiological function of the oocyte are still speculative. It is known that ROS have deleterious effects in male germinal cells (14) but play a beneficial role in oocyte maturation (32). Because NH4Cl exposure mimics the fertilization potential that prevents polyspermia in amphibian oocytes, further studies should focus on the effects of ROS during oocyte activation and fertilization. At present, our results show that in Xenopus oocytes, ROS may activate a nonselective cationic conductance and may induce a decrease in resting pHi. These observations should be taken into account in studies dealing with ROS effects on ionic transport systems after their functional expression in Xenopus laevis oocytes.


    ACKNOWLEDGEMENTS

We are indebted to Mireille Blonde for technical assistance and to Dr. S. Randall Thomas for help in the writing of this manuscript. We are especially indebted to Dr. Takis Anagnostopoulos, who launched us in the direction of ROS effects.


    FOOTNOTES

Address for reprint requests and other correspondence: G. Planelles, Inserm U 467, Université Paris V, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France (E-mail: planelle{at}necker.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 6, 2002;10.1152/ajpcell.00410.2001

Received 22 August 2001; accepted in final form 1 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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