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
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ABSTRACT |
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To investigate the
effects of reactive oxygen species (ROS) on NHpHi) 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
-amyloid protein had no
marked effect on the NH4Cl-induced
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
pHi and
dpHi/dt. These increases in
pHi
and dpHi/dt were prevented by the ROS scavengers
histidine and desferrioxamine, suggesting involvement of the reactive
species 1
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
ammonium ions; nonselective cationic conductance
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INTRODUCTION |
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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
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
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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-ClTo 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
(1gO2) and superoxide radical
(O
-amyloid protein (3, 39). Thus we also tested the
effects of H2O2 (500 µM) and of fragment
1-40 of the
-amyloid protein (
-A; 20 µM).
In the t-BHP, H2O2, and -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
1
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 -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.
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(1) |
Voltage-clamp experiments.
Voltage-clamp experiments were performed using two low-resistance
(1-5 M) 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 (
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(2) |
From the measured pHi and from calculated
[Ac
]i,
was then calculated at
pHi intervals of 0.2 pH units, according to the following
equation
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(3) |
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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
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(4) |
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. ![]() |
RESULTS |
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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,
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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
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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 1gO2 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
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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Activation of Nonselective Cationic Conductance by Rose Bengal
We previously reported (6) that NHWe 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 NH10 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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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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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
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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 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 Vm,
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
Vm was lower than in the control series
(Table 3).
Finally, we tested the effect of incubating the oocytes with -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.
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DISCUSSION |
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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 NHIn 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
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 (OTo investigate the effect of ROS on NHpHi) reflects a net proton excess subsequent to
intracellular NH
NH
NH
· V.
To see whether the measured increases in dpHi/dt
directly reflect the enhancement of NH
· 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
-A series; see Table 3) and
constitutes, to our knowledge, the first report of
as a function of
pHi in oocytes. No change in relative volume was observed,
and
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
In summary, during RB, X/XO, and t-BHP exposure, a
NHgO2 and ·OH in this effect.
Involvement of Gcat in RB-induced Increase in
NH
The activation of Gcat is also consistent with
the Vm and NH4Cl-induced
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
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
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
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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