Hydrogen Peroxide Induces Intracellular Calcium Overload by
Activation of a Non-selective Cation Channel in an
Insulin-secreting Cell Line*
Paco S.
Herson
,
Kevin
Lee§,
Rob D.
Pinnock§,
John
Hughes§, and
Michael L. J.
Ashford
¶
From the
Department of Biomedical Sciences,
University of Aberdeen, Institute of Medical Sciences, Foresterhill,
Aberdeen AB25 2ZD, United Kingdom and the § Parke-Davis
Neuroscience Research Centre, Cambridge University, Forvie Site,
Robinson Way, Cambridge CB2 2QB, United Kingdom
 |
ABSTRACT |
Fura-2 fluorescence was used to investigate the
effects of H2O2 on
[Ca2+]i in the insulin-secreting cell line
CRI-G1. H2O2 (1-10 mM) caused a
biphasic increase in free [Ca2+]i, an initial
rise observed within 3 min and a second, much larger rise following a
30-min exposure. Extracellular calcium removal blocked the late, but
not the initial, rise in [Ca2+]i. Thapsigargin
did not affect either response to H2O2, but
activated capacitive calcium entry, an action abolished by 10 µM La3+. Simultaneous recordings of membrane
potential and [Ca2+]i demonstrated the same
biphasic [Ca2+]i response to
H2O2 and showed that the late increase in
[Ca2+]i coincided temporally with cell membrane
potential collapse. Buffering Ca2+i to low
nanomolar levels prevented both phases of increased [Ca2+]i and the
H2O2-induced depolarization. The
H2O2-induced late rise in
[Ca2+]i was prevented by extracellular
application of 100 µM La3+. La3+
(100 µM) inhibited the
H2O2-induced cation current and NAD-activated cation (NSNAD) channel activity in these cells.
H2O2 increased the NAD/NADH ratio in intact
CRI-G1 cells, consistent with increased cellular [NAD]. These data
suggest that H2O2 increases [NAD], which,
coupled with increased [Ca2+]i, activates
NSNAD channels, causing unregulated Ca2+ entry
and consequent cell death.
 |
INTRODUCTION |
Oxidative stress, through the production of oxygen metabolites,
particularly H2O2 and other reactive oxygen
species (free radicals), results in destruction of many cell types
through putative necrotic/apoptotic processes (1, 2). Furthermore,
excessive production of reactive oxygen species (e.g. via
mitochondrial oxidation) has been causally related in the etiology of
numerous degenerative disorders, including many age-related
neurodegenerative diseases such as Parkinson's disease, Huntington's
disease, and Alzheimer's disease (3-5). Although reactive oxygen
species have been implicated in cell death, the exact mechanism(s) are,
as yet, unclear. A favored hypothesis is that
H2O2 causes DNA strand breaks, leading to the
activation of nuclear poly(ADP-ribose) polymerase, which critically
depletes the cell of NAD, leading to eventual cell death (6). It has
also been postulated that H2O2 disrupts the
cell membrane integrity in a nonspecific manner through lipid
peroxidation (7). However, there is also a good correlation between
oxidative stress (H2O2 toxicity), induction of
reactive oxygen species, and an increase in intracellular
Ca2+ levels immediately preceding the final destructive
events (8).
Pancreatic beta cells have long been known to be particularly
susceptible to oxidative stress-induced destruction (9), making these
cells useful models for mechanistic studies. Indeed, alloxan, which is
toxic to pancreatic beta cells through the production of
H2O2 and ultimately the highly reactive
hydroxyl radical (·OH), (10-12), was observed to cause diabetes
mellitus in experimental animals over 50 years ago (13). This
susceptibility has been correlated with a reduced capacity to withstand
free radical attack through a limited cellular defense mechanism, as
pancreatic beta cells have been reported to be deficient in glutathione
peroxidase, catalase, and superoxide dismutase (14, 15) relative to
other tissues. Therefore, we have combined Ca2+ imaging and
electrophysiological recordings of an insulin-secreting cell line
(CRI-G1) to enable an investigation of the cellular consequences and
mechanisms underlying mammalian cell responses to oxidative stress.
Previously, it had been demonstrated, by intracellular recordings, that
exposure to alloxan causes irreversible depolarization of mouse
pancreatic beta cells (16). These initial observations have more
recently been substantiated using whole-cell recordings, which show
that alloxan and H2O2, through the production of reactive oxygen species, cause complete and irreversible
depolarization of CRI-G1 insulin-secreting cells (17). This study also
demonstrated that the H2O2-driven collapse of
the membrane potential is mediated by the opening of a previously
quiescent novel non-selective cation (NSNAD) channel.
Although activated by oxidative stress in intact cells, this
non-selective cation channel requires the presence of both
Ca2+ and NAD on the cytoplasmic aspect of excised patches
for channel activity to be observed (18, 19). Permeation studies
indicate that this channel has a significant conductance for divalent
cations, most notably Ca2+ (18); and therefore, oxidative
stress-induced activation of this channel would be expected to allow a
significant Ca2+ influx associated with the collapse of the
membrane potential.
We now report that exposure of CRI-G1 cells to concentrations of
H2O2 that activate NSNAD channels
causes a biphasic rise in [Ca2+]i. The second,
late rise in [Ca2+]i induced by
H2O2 reaches micromolar concentrations, indicating unregulated calcium influx. It is proposed that the second
rise in [Ca2+]i is caused by
H2O2-induced activation of NSNAD
channels, leading to concurrent depolarization and eventual cell death
through calcium overload. Some of these data have been reported
previously in preliminary form (20).
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Cells from the insulin-secreting cell line
CRI-G1 were grown in Dulbecco's modified Eagle's medium containing
sodium pyruvate (0.01%) and glucose (0.1%) and supplemented with 10%
fetal calf serum and 1% (v/v) penicillin/streptomycin at 37 °C in a
humidified atmosphere of 95% air and 5% CO2. Cells were
passaged at 2-5-day intervals as described previously (21), plated
either onto glass coverslips or directly onto 3.5-cm Petri dishes at
dilutions that provided a subconfluent cell density (Falcon 3001), and
used 1-4 days after plating.
Electrophysiological Recording and Analysis--
Experiments
were performed using whole-cell current and voltage clamp and single
channel recording modes. Recording electrodes were pulled from
borosilicate glass capillaries and had resistances of 8-12 megaohms
for outside-out recordings and 2-6 megaohms for whole-cell experiments
when filled with electrolyte solution. Recordings were made using an
Axopatch-1D or List EPC-7 patch clamp amplifier. Data were recorded
onto digital audio tape and replayed for illustration onto a Gould TA
240 chart recorder. During current clamp experiments, hyperpolarizing
current pulses (50 pA and 0.2-s duration) were applied every 5 s
to monitor changes in input resistance. All voltage clamp experimental
protocols were generated, and the resultant data were stored using
PCLAMP6 (Axon Instruments, Inc.) and a Viglen PS/200 computer. In
whole-cell voltage clamp recording mode, the membrane potential was
held at
70 mV, and current-voltage relations were obtained by either applying 10-mV voltage steps of 200-ms duration with 100 ms between steps over the range
130 to
50 mV for
H2O2-activated currents or 10-mV steps from
40 to +40 mV every 5 s when investigating Ba2+
currents. The mean current amplitude of the final 50 ms of the voltage
clamp current response for each voltage jump was plotted against the
applied voltage to generate current-voltage relations. Typical values
for the series resistance during whole-cell recordings were 10-18
megaohms. All values in the text are expressed as mean ± S.E.
Statistical significance between data sets was determined using
unpaired Student's t test.
For outside-out patch experiments, the pipette solution contained 140 mM KCl, 5.02 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (pH 7.4), resulting in a free Ca2+
concentration of 50 µM; and the bathing medium consisted
of 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES (pH 7.2).
The pipette solution for whole-cell recordings contained 140 mM KCl, 0.6 mM MgCl2, and 10 mM HEPES (pH 7.2), resulting in a free Ca2+
concentration in the low nanomolar range, before stimulation with
H2O2, and allowing an increase in free
Ca2+ concentration during stimulation. Some whole-cell
recordings were performed with a pipette solution containing 140 mM KCl, 0.6 mM MgCl2, 10 mM EGTA, and 10 mM HEPES in order to clamp
intracellular Ca2+ in the low nanomolar range. The bath
solution consisted of normal saline (135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES (pH
7.4)). Additionally, when investigating voltage-dependent Ca2+ channel currents, the pipette contained 140 mM CsCl, 10 mM EGTA, 0.6 mM
MgCl2, 2.73 mM CaCl2, and 10 mM HEPES (pH 7.2), resulting in a free Ca2+
concentration of 100 nM; and the bath contained normal
saline supplemented with Ba2+ (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM BaCl2, and 10 mM HEPES (pH
7.4)).
Imaging Intracellular Calcium of Single Cells--
After washing
with normal saline supplemented with 3 mM glucose, the
plated cells were incubated with 2 µM fura-2/AM for
45-120 min at room temperature (22-25 °C) prior to experiments in
which [Ca2+]i was measured in intact cells.
Single coverslips were mounted in a chamber on top of an inverted
fluorescence microscope and perfused with normal saline plus 3 mM glucose. Measurements of [Ca2+]i
in individual cells were made from the fluorescence ratio (excitation
at 340/380 nm and emission at >510 nm for fura-2) using a specially
designed filter wheel assembly, incorporating a CCD camera, a
photomultiplier, and a suite of software (MAGICAL, Applied Imaging,
Sunderland, United Kingdom) that samples emission following excitation
at 340- and 380-nm wavelengths at 15-s intervals. [Ca2+]i was calculated from a calibration curve
using the equation [Ca2+]i = Kd·
·((R
Rmin )/(Rmax
R)), where Rmax, Rmin, and R are the maximum ratio,
minimum ratio, and measured ratio, respectively;
is minimum
380/maximum 380; and Kd represents the
Ca2+ binding affinity of fura-2.
Rmax, Rmin, and
were
determined from free standing solutions of 2 and 0 mM
Ca2+o (+5 mM EGTA) in HEPES buffer
solution as described previously (22, 23); these values did not vary
significantly from day to day. Zero extracellular Ca2+ was
obtained by the removal of CaCl2 from the normal saline as well as by the addition of 5 mM EGTA. All intact cell
imaging was performed on at least three separate cultures, and numbers quoted (n) represent the number of individual cells
analyzed. The intrinsic autofluorescence of cells, induced by the
presence of reduced pyridine nucleotides, was examined without loading with fura-2/AM. Significant changes in autofluorescence were observed using excitation at 360 nm and emission at >510 nm.
Combined Ca2+ Imaging and
Electrophysiology--
Methods for detecting
[Ca2+]i in cells while recording in the
whole-cell configuration were essentially the same as those used for
intact cells, except that the pipette solution contained 140 mM KCl, 0.6 mM MgCl2, 10 mM HEPES, and 50 µM fura-2 (pH 7.2), and in
some experiments, 10 mM EGTA was added to buffer intracellular Ca2+ to low nanomolar levels. The bath
solution consisted of normal saline (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 3 mM glucose (pH 7.4)). Under these conditions in the
cell-attached configuration, the fluorescence ratio indicated <10
nM free Ca2+i contamination of the
pipette solution from stock reagents. In these experiments, the cells
were viewed on an upright Zeiss Axioskop microscope with the
appropriate dichroic mirror (405 nm) and trinocular mounting to send
the emitted light to the photomultiplier tube (PHOCAL, Life Sciences
Resources, Basingstoke, United Kingdom).
Fura-2 and fura-2/AM were obtained from Molecular Probes, Inc.
(Eugene, OR). Thapsigargin was from Calbiochem (Badsoden,
Germany), and all other chemicals were from Sigma (Poole, United Kingdom).
All solution changes were achieved by superfusing the bath with a
gravity-feed system at a rate of ~10 ml/min, which allowed complete
bath exchange within 2 min. All experiments were performed at room
temperature (22-25 °C).
 |
RESULTS |
Intact CRI-G1 cells were loaded with fura-2/AM, and the effect of
H2O2 on the fura-2 ratio
([Ca2+]i) was monitored. The addition of either 1 or 10 mM H2O2 to the extracellular
medium resulted in a biphasic increase in [Ca2+]i
(Fig. 1A). Although the
[Ca2+]i response to 1 and 10 mM
H2O2 differed temporally (data not shown), the
magnitude of both the early and late phases of increased
[Ca2+]i was indistinguishable (p > 0.05). The initial rise occurred within 3 min, and the fura-2 ratio
increased from an initial resting level of 0.44 ± 0.01 (~57
nM) to 0.93 ± 0.02 (190 nM;
n = 72). The second, late response occurred ~30 min
later (~40 min for 1 mM H2O2) and
induced a rise to near-dye saturation (fura ratio > 2; >1
µM Ca2+i). Removal of extracellular
calcium by the addition of 5 mM EGTA decreased the initial
rise in [Ca2+]i (ratio rising from 0.40 ± 0.01 (48 nM) to 0.73 ± 0.02 (130 nM);
n = 52; p < 0.01) and completely
abolished the second rise induced by 10 mM
H2O2 (Fig. 1B). The re-addition of
extracellular calcium resulted in an immediate increase in
[Ca2+]i, again rising to near-dye saturation,
indicating that a calcium permeation pathway had been activated by the
H2O2 exposure. These results indicate that the
H2O2-induced early increase in intracellular
calcium levels results predominantly from mobilization of
Ca2+i from an intracellular source and that the
second, late increase in intracellular calcium levels is a result of
extracellular calcium influx.

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Fig. 1.
H2O2 causes a
biphasic rise in intracellular calcium levels. Shown are
fluorescence measurements of fura-2/AM-loaded CRI-G1 cells.
A, representative imaging experiment, from three cells (in
this and subsequent figures), illustrating the biphasic response caused
by the addition of 10 mM H2O2 to
the bathing medium. The initial rise occurred within 5 min, and a
second, massive rise (which reached near-dye saturation (>1
µM)) could be seen after ~30 min. B, a
separate experiment illustrating the effect of the addition of 10 mM H2O2 to the bathing medium in
the absence of extracellular calcium. Only the initial small sustained
rise in intracellular calcium levels occurred in the absence of
extracellular calcium. The re-addition of extracellular calcium
resulted in an immediate massive calcium increase (near-dye saturation
(>1 µM)). Note that exposure to
H2O2 in the absence of extracellular
Ca2+ was longer in duration than the time taken to reach a
maximal response in A.
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Previous studies examining the consequences of mammalian cell exposure
to reactive oxygen species have generally been inconclusive with
respect to the identity of the permeation pathway for the oxidative
stress-induced rise in [Ca2+]i consequent to
Ca2+ entry from the extracellular medium. There are reports
that implicate voltage-gated calcium channels (through the use of
organic Ca2+ channel blockers) as the source of calcium
influx (24, 25), whereas others indicate that such a pathway is
unlikely (26, 27). Even if voltage-gated calcium channels were not the
primary path, reactive oxygen species-derived depolarization of cells could activate these channels, which could then secondarily contribute to the increased intracellular calcium levels. To examine whether voltage-gated calcium channels contribute in any way to the
H2O2-induced increase in
[Ca2+]i in CRI-G1 cells, 100 µM
Cd2+ was applied to the extracellular environment of the
cells. This concentration of Cd2+ completely inhibits
voltage-gated calcium currents (n = 5) in this cell
line, an action partially reversible on washout of the Cd2+
(Fig. 2A). As further
verification that Cd2+ inhibits the function of
voltage-gated calcium channels in these cells, the increased
[Ca2+]i response associated with a
depolarization-induced (40 mM K+) calcium
influx was shown to be completely prevented by 100 µM Cd2+ (n = 12) (Fig. 2B).
However, in the presence of extracellular calcium, co-application of
100 µM Cd2+ and 10 mM
H2O2 failed to inhibit the
H2O2-induced rise in
[Ca2+]i, with the fura-2 ratio increasing to dye
saturation (n = 13) (Fig.
3A). The presence of
Cd2+ did produce a striking change in the time course of
the [Ca2+]i response in that the second phase of
the H2O2-induced increase in
[Ca2+]i occurred almost immediately on
application of the oxidative stress, with no obvious evidence of the
initial [Ca2+]i response (n = 20). This acceleration of the [Ca2+]i response is
possibly consistent with Cd2+ catalyzing the decomposition
of H2O2 to form reactive oxygen species (9).
The effects of Cd2+ on [Ca2+]i were
also examined in the absence of extracellular calcium, and
Cd2+ was demonstrated to have no effect on
[Ca2+]i per se and did not alter the
initial H2O2-induced rise in
[Ca2+]i (ratio rising from 0.37 ± 0.01 (42 nM) to 0.83 ± 0.06 (155 nM);
p > 0.05 compared with data in the absence of
Cd2+). However, re-addition of 1 mM
Ca2+-containing extracellular solution in the continued
presence of Cd2+ induced a rapid, large increase in
[Ca2+]i (n = 13) (Fig.
2B). Consequently, it appears highly unlikely that
activation of voltage-dependent calcium channels is
responsible for, or contributes significantly to, the
H2O2-induced second, late phase of increased
[Ca2+]i in CRI-G1 cells.

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Fig. 2.
Cd2+ inhibits
voltage-dependent Ba2+ currents and abolishes
depolarization-induced calcium influx. A, mean
current-voltage relation, from a cell at a holding potential of 70
mV, obtained over the voltage range 40 to +40 mV with 140 mM Cs+ in the pipette ( ) and the 1 mM extracellular Ca2+ replaced with 10 mM Ba2+. This current had a threshold for
activation of near 40 mV and was maximal at 0 mV. The addition of 100 µM Cd2+ ( ) to the bathing medium
completely abolished this current. Note the presence of a small
residual current at both hyperpolarized and depolarized potentials,
probably due to contamination by non-selective cation or anion
currents. Representative barium currents were observed at 10 mV; the
initial inward spike is due to activation of voltage-gated sodium
currents. The application of 100 µM Cd2+
resulted in complete and reversible inhibition of the barium current,
but had no effect on the sodium current. B, fluorescence
measurements of a single fura-2/AM-loaded CRI-G1 cell
illustrating the rise in [Ca2+]i caused by
depolarization elicited by 40 mM extracellular
K+. The additional presence of 100 µM
Cd2+ completely abolished the 40 mM
K+, depolarization-induced calcium increase.
Inhibition produced by Ca2+ was reversed upon removal
(data not shown).
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Fig. 3.
Cd2+ does not inhibit
H2O2-induced Ca2+ influx.
A, fluorescence measurements of fura-2/AM-loaded CRI-G1
cells showing a rapid and maximal increase in intracellular
Ca2+ levels caused by the application of 10 mM
H2O2 in the presence of 100 µM
Cd2+. Note the highly accelerated rate of the
[Ca2+] rise compared with Fig. 1A.
B, separate experiment illustrating the lack of effect of
100 µM Cd2+ on Ca2+ per
se and the effect of subsequent addition of 10 mM
H2O2 plus Cd2+ to the bathing
medium in the absence of extracellular calcium, which caused a small
sustained rise in intracellular calcium levels. The re-addition of
extracellular calcium in the continued presence of Cd2+
resulted in an immediate massive rise in Ca2+ levels, which
reached near-dye saturation (>1 µM).
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Another possible contributor to this second, large rise in
[Ca2+]i is by means of an intracellular
Ca2+-triggered capacitive entry system for Ca2+
(ICRAC) that has somehow become unregulated by
the presence of H2O2. The data described above
indicate that the initial phase of increased
[Ca2+]i in response to
H2O2 challenge is due to mobilization of
calcium from intracellular stores. Consequently, the possibility that
the calcium originates from microsomal stores was investigated using
thapsigargin, an endoplasmic reticulum Ca2+-ATPase
inhibitor that depletes intracellular Ca2+ stores by
blocking the uptake of calcium (28). The addition of 1 µM
thapsigargin to intact cells resulted in a rapid rise in
[Ca2+]i, which peaked and then declined, but did
not return to initial pre-thapsigargin levels (Fig.
4A), with the fura-2 ratio
increasing from 0.41 ± 0.01 (50 nM) to 0.83 ± 0.03 (155 nM) and then plateauing to a new level of
0.59 ± 0.02 (90 nM) (n = 32).
Following thapsigargin treatment, removal of extracellular calcium
decreased [Ca2+]i to a level below that seen in
control experiments, 0.32 ± 0.01 (32 nM;
p < 0.05). The subsequent addition of 10 mM H2O2 resulted in a response
indistinguishable from that of cells not exposed to thapsigargin (Fig.
1A), i.e. an initial sustained increase in
[Ca2+]i, with the fura-2 ratio rising from
0.32 ± 0.01 (32 nM) to 0.76 ± 0.02 (137 nM) (p > 0.05), followed by a second, larger rise following re-addition of extracellular Ca2+,
reaching near-dye saturation. It is therefore clear that
H2O2 mobilizes intracellular calcium from a
thapsigargin-insensitive source. Previous studies have demonstrated
that emptying of intracellular Ca2+ stores in pancreatic
beta cells triggers capacitive calcium entry through
ICRAC (29, 30) and that low (10-100
µM) concentrations of La3+ inhibit this
process (31). In the CRI-G1 cells, emptying of intracellular
Ca2+ stores with thapsigargin resulted in the activation of
ICRAC. This can be seen (Fig. 4, A
and B) by removal and re-addition of extracellular calcium,
which resulted in [Ca2+]i transients not observed
without exposure to thapsigargin (n = 32 and 24 with
and without thapsigargin, respectively). The induction of
[Ca2+]i transients indicative of
ICRAC activation in these cells was maximally
and reversibly inhibited by the presence of 10 µM
La3+ (Fig. 4B). In contrast, the presence of 10 µM La3+ did not abolish the influx of
extracellular Ca2+ (n = 13) induced by 10 mM H2O2, although there was an
~30% inhibition (Fig. 4B). However, application of 100 µM La3+ did eradicate the second
[Ca2+]i transient due to
H2O2-induced Ca2+ influx
(n = 24) (Fig. 4C), an action that was
reversible, as washout of the La3+ in the continued
presence of extracellular Ca2+ resulted in eventual massive
influx of Ca2+. Consequently, these data indicate that the
H2O2-induced Ca2+ influx is
pharmacologically distinct from the ICRAC
pathway in these cells and also exclude a nonspecific membrane
breakdown as mediator of Ca2+ entry.

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Fig. 4.
Thapsigargin does not affect the
H2O2-induced Ca2+
response. A, shown are fluorescence measurements of
fura-2/AM-loaded CRI-G1 cells illustrating the biphasic rise in
intracellular Ca2+ levels initiated by 1 µM
thapsigargin (Thaps). Following thapsigargin treatment,
removal of extracellular Ca2+ decreased
[Ca2+]i to a level below control values. The
re-addition of extracellular Ca2+ caused a biphasic
Ca2+ response essentially identical to that caused by
thapsigargin. This is due to capacitive calcium entry. The subsequent
addition of 10 mM H2O2 in the
absence of extracellular Ca2+ caused a sustained rise in
[Ca2+]i indistinguishable from controls. The
re-addition of extracellular calcium resulted in an immediate massive
rise in Ca2+ levels, which reached near-dye saturation (>1
µM). B, shown is a separate experiment
illustrating the complete and reversible inhibition of
thapsigargin-induced capacitive calcium entry by 10 µM
La3+. In contrast, 10 µM La3+ did
not prevent the H2O2-induced Ca2+
influx seen upon re-addition of extracellular Ca2+.
C, in cells not treated with thapsigargin,
H2O2 induced the initial rise in
Ca2+ levels in the absence of extracellular
Ca2+, but re-addition of extracellular Ca2+ in
the presence of 100 µM La3+ prevented the
immediate second, large [Ca2+] increase. Removal of the
La3+ in the presence of extracellular calcium allowed the
late phase of calcium entry to be re-established.
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The identity of the Ca2+ entry pathway responsible for the
second, late increase in [Ca2+]i induced by
H2O2 in CRI-G1 cells was investigated using whole-cell current clamp recordings with 50 µM fura-2 in
the intracellular solution in order to monitor simultaneously membrane
potential and the levels of free intracellular calcium. After
initiation of the whole-cell recording mode, the cell interior was
dialyzed with an ATP-free solution, which resulted in cell
hyperpolarization (Fig. 5A,
upper trace). The membrane potential changed from an initial
resting value of approximately
40 mV to a new stable potential of
74 ± 1.8 mV (n = 6). Concomitant with the
hyperpolarization was a decrease in cell input resistance. This
hyperpolarization and decreased input resistance are due to the opening
of ATP-sensitive K+ (KATP) channels as internal
ATP is washed out of the cell (17, 32). Following stabilization of the
membrane potential and input resistance, application of 10 mM H2O2 caused a sustained increase in [Ca2+]i (Fig. 5A, lower
trace), which preceded any effect on membrane potential. This
increase occurred within 3 min (2.6 ± 0.2 min; n = 6), and the fura-2 ratio rose from 0.45 ± 0.02 (46 nM) to 0.87 ± 0.08 (150 nM). There was
then a further delay of ~10-12 min before a second phase of
increased [Ca2+]i was observed (fura-2 ratio
rising from 0.87 ± 0.08 to 1.94 ± 0.16 (>600
nM)) (Fig. 5A, lower trace). These
[Ca2+]i responses to 10 mM
H2O2 almost exactly recapitulated those
obtained in intact cells. The decreased delay for the second phase of
increased [Ca2+]i during whole-cell recordings in
comparison with intact cells is likely due to dialysis of the cells'
natural antioxidant protective mechanisms (e.g.
glutathione). The second, late phase of increased
[Ca2+]i coincided with the appearance of a slow,
irreversible depolarization to ~0 mV. This
H2O2-induced depolarization is consistent with
our previous report on the action of H2O2 in
this cell line (17).

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Fig. 5.
H2O2 depolarizes
CRI-G1 cells as well as causes a biphasic calcium response. Shown
are the results from the simultaneous measurement of fura-2
fluorescence and whole-cell current clamp recording of the membrane
potential of CRI-G1 cells. A, application of 10 mM H2O2 induces a biphasic rise in
intracellular calcium levels (lower trace), with the second,
larger rise coinciding exactly with cellular depolarization
(upper trace). B, simultaneous measurement of
fura-2 fluorescence and cell membrane potential with nominally
calcium-free electrode solution (10 mM EGTA). Application
of 10 mM H2O2 had no effect on
either intracellular calcium or cell membrane potential. Note the
initial hyperpolarization of the cell membrane potential on initiation
of the whole-cell recording configuration (due to intracellular
dialysis of ATP). The downward deflections of the membrane potential
traces in A and B were caused by hyperpolarizing
current pulses (50 pA and 0.2-s duration) applied every 5 s to
monitor input resistance changes.
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To determine whether the later large increase in
[Ca2+]i induced by H2O2
is dependent upon the initial increase in [Ca2+]i, simultaneous recordings of membrane
potential and [Ca2+]i were made with 10 mM EGTA included in the intracellular pipette solution to
clamp the [Ca2+]i to low nanomolar levels. As
illustrated in Fig. 5B, the inclusion of 10 mM
EGTA in the pipette clamped the [Ca2+]i below the
resting level (0.26 ± 0.01; <1 nM), and this did not
change after application of 10 mM
H2O2. Furthermore, although the reduction in
intracellular calcium had no effect on the hyperpolarization induced by
the washout of the internal ATP (the mean cell hyperpolarization was to
75 ± 2.4 mV; n = 4), it completely prevented
the H2O2-induced depolarization (Fig. 5B, lower trace).
Clearly, activation of the late phase of increased
[Ca2+]i by H2O2 is
dependent upon the initial rise in calcium levels, is through a
specific permeation pathway, and is consequent to the depolarization of
the cells. Previous studies have demonstrated that the
H2O2-induced depolarization of CRI-G1 cells is
due to the opening of a novel non-selective cation conductance
(NSNAD channel), which also allows permeation of
Ca2+ (17, 18). Activation of this channel by
H2O2 is shown in Fig.
6, where cell-attached single channel
recordings from CRI-G1 cells were performed. Following application of
H2O2 (4.4 mM), there was a delay of
~9-10 min before the appearance of single channel currents (Fig.
6A). This current was characterized by a linear
current-voltage relation with a conductance of 70 pS 1 and a reversal
potential of 0 mV (Fig. 6, B and C). Excision of
the patch into the inside-out configuration resulted in loss of channel
activity, which could only be recovered by application of NAD to the
cytoplasmic aspect of the membrane patch (Fig. 6C), identifying the channel as NSNAD (17). We also examined the likelihood that this channel is responsible for the late phase of
Ca2+ entry induced by H2O2 by
determining the sensitivity of this conducting pathway to inhibition by
La3+. Following attainment of the whole-cell recording
configuration (Fig. 7A), the
conductance of the cell was small (1.46 ± 0.54 nS;
n = 3), with a reversal potential of
42.3 ± 4.0 mV (n = 3), which is close to the cell resting membrane
potential of approximately
40 mV. After dialysis with the ATP-free
pipette solution, the conductance increased to 18.2 ± 2.80 nS,
with a reversal potential of
70 ± 3.6 mV, which is attributed
to activation of KATP channels. In contrast, subsequent to
the H2O2-induced depolarization of CRI-G1
cells, voltage clamp recordings demonstrated the activation of a
non-selective cation current characterized by a linear slope conductance (Fig. 7A) of 4.6 ± 1.1 nS with a
concurrent loss of KATP current, indicated by a shift in
the reversal potential to 5.7 ± 8.5 mV (n = 3).
Application of 100 µM La3+ caused a 50-80%
inhibition of the H2O2-activated current,
decreasing the slope conductance to 2.1 ± 0.1 nS with only a
slight shift in the reversal potential (to
1 mV). The inhibition of
this current by La3+ was partially reversed after a >5-min
wash, with the slope conductance increasing to a value of 3.7 ± 0.2 nS (n = 3) (Fig. 7A). The inhibitory effect of La3+ was also determined on single channel
currents activated by 1 mM NAD+ in the presence
of 50 µM Ca2+ in isolated outside-out
membrane patches. In all outside-out patches examined, the addition of
100 µM La3+ to the extracellular membrane
aspect caused complete cessation of channel activity (Fig.
7B), an action not reversible even with prolonged washing
(n = 6).

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Fig. 6.
Activation of single channel currents by
H2O2. A, representative
cell-attached recording, with 40 mV applied pipette potential,
illustrating the appearance of single channel activity 9-10 min
following exposure of the cell to 4.4 mM
H2O2. B, single channel
current-voltage relations obtained from the same patch either
cell-attached ( ) or after the patch had been excised and the channel
re-activated by 500 µM internal NAD ( ). C,
the single channel currents observed in this experiment. Note the
similarity (amplitude of currents and the long open duration) between
the channel activated by H2O2 and that
activated by NAD in the inside-out patch configuration.
|
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Fig. 7.
La3+ inhibits
H2O2-activated whole-cell current and single
NSNAD channel activity. A, current-voltage
relations obtained from a voltage-clamped cell, over the voltage range
130 to 50 mV, from a holding potential of 70 mV prior to and
following H2O2-induced (10 mM) cell
depolarization. Initially, the current was linear with a reversal
potential near 40 mV ( ), and following dialysis of the cell with
an ATP-free solution ( ), the current dramatically increased, and the
reversal potential shifted more negative, indicating activation of the
KATP current. In the presence of
H2O2, the KATP current was
abolished, and there was an inward current characterized by a linear
current-voltage relation ( ), which, on application of 100 µM external La3+, was significantly reduced
( ), an action only partially reversed on washout of La3+
( ). Representative families of currents were generated in the same
experiment. B, representative continuous outside-out
recording illustrating the irreversible inhibition of NAD-activated
(NSNAD) single channel activity by 100 µM
external La3+. This experiment was performed on an
outside-out patch with 1 mM internal NAD and 50 µM free Ca2+ in the pipette at a membrane
potential of 40 mV.
|
|
The data presented above are consistent with the notion that
H2O2 induces the release of Ca2+
from an as yet undefined intracellular Ca2+ store, which
yields the first phase of increased [Ca2+]i, and
this is obligatory for the subsequent activation of a substantial
Ca2+ influx through NSNAD channels, producing
the second phase of increased [Ca2+]i. The
activity of the NSNAD channel in isolated patches is
Ca2+-dependent, but the presence of
intracellular Ca2+ per se is not sufficient to
sustain channel activity; there is also an absolute requirement for
intracellular NAD. Therefore, the effect of
H2O2 exposure on pyridine nucleotide levels was examined in intact cells utilizing the autofluorescence properties of
these nucleotides (30, 33). Application of 10 mM
H2O2 caused a rapid (within 2-3 min) but
transient decrease in NAD(P)H autofluorescence both in the absence and
presence of extracellular calcium (n = 24;
p > 0.05) (Fig. 8).
Cellular autofluorescence decreased by 17.4 ± 1.8% within the
first 5 min and then gradually returned to control levels during the
remainder of the experiment (1 h). This indicates that
H2O2 causes an initial oxidation of pyridine nucleotides, resulting in an increase in the NAD/NADH ratio, indicating a transient increase in NAD levels. Such a change in NAD combined with
the initial rise in [Ca2+]i could conspire to
create an environment conducive to NSNAD channel
activation.

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Fig. 8.
Effect of H2O2 on
CRI-G1 autofluorescence. The application of 10 mM
H2O2 caused a transient decrease in NAD(P)H
autofluorescence in the presence (A) or absence
(B) of extracellular Ca2+.
|
|
 |
DISCUSSION |
Previous studies have demonstrated that
H2O2, most probably through the generation of
reactive oxygen species, causes an increase in intracellular calcium
levels that precedes, if not causes, cell death in a wide variety of
cell types, including cardiac (27) and smooth muscle cells (26),
pancreatic acinar cells (34), alveolar macrophages (35), and central
nervous system neurons (36). Here, we describe the effects of
H2O2 on [Ca2+]i in the
insulin-secreting cell line CRI-G1 and clearly demonstrate that
H2O2 also disrupts calcium homeostasis in these cells. There is no clear consensus in the literature indicating the
likely mechanism by which H2O2 causes an
increase in [Ca2+]i; suggestions that have been
promulgated include influx through voltage-dependent
calcium channels (25, 35), nonspecific changes in membrane calcium
permeability (37, 38), alteration in Na+-Ca2+
exchange (39), or changes in calcium release from intracellular stores
(40, 41).
The data presented herein indicate that both release of calcium from an
intracellular source and influx of extracellular calcium contribute to
the rise in free [Ca2+]i induced by
H2O2 in CRI-G1 cells. In the absence of extracellular calcium, H2O2 caused a
significant increase in [Ca2+]i, which began
within 2-3 min, reached a plateau quickly, and was sustained at the
elevated level for as long as the extracellular calcium was absent. The
subsequent re-addition of extracellular Ca2+ resulted in a
rapid increase in [Ca2+]i. On the basis of these
data, it is concluded that H2O2 initially
stimulated release of calcium from an intracellular source and a later
influx of extracellular Ca2+. Additional studies indicated
that depletion of the intracellular calcium stores with thapsigargin
had no effect on the magnitude of the [Ca2+]i
increase elicited by H2O2 in the absence of
extracellular calcium, indicating that H2O2
induced release of calcium from a thapsigargin-insensitive source. This
effectively negates the possibility that H2O2
stimulated calcium release from either inositol 1,4,5-trisphosphate-sensitive or calcium-induced endoplasmic reticulum stores. The possibility that other intracellular organelles, in particular the nucleus (41) or mitochondria (42, 43), are responsible
for the initial calcium response requires investigation.
Simultaneous measurements of membrane potential and
[Ca2+]i were made on single CRI-G1 cells, and
regardless of the intracellular dialysis caused by the whole-cell
recording configuration, H2O2 caused a biphasic
increase in [Ca2+]i, with the second, larger rise
coinciding temporally with cellular depolarization. The depolarization
elicited by H2O2 is not simply due to a
reduction in KATP channel current in these cells, as in
separate cell-attached recordings, it was demonstrated that the
collapse of the membrane potential is associated with the induction of
a separate conducting pathway (see below). This result indicates that
the initial rise in [Ca2+]i activates a
calcium-dependent process that leads to both cell
depolarization and, as a consequence, a second rise in
[Ca2+]i. Further substantiation of this
hypothesis was obtained by chelating intracellular calcium to low
nanomolar levels, an action that prevented both phases of the
Ca2+ response and the H2O2-induced
depolarization. The source of this later, larger increase in
[Ca2+]i is clearly from the extracellular milieu,
and consequently, a number of conducting pathways were considered as
possible contributors.
Activation of voltage-gated calcium channels has been implicated in the
autoimmune-associated destruction of pancreatic beta cells (44) and has
been suggested to contribute to cellular response to oxidative stress
(25, 35, 45). Therefore, a possible contribution to the observed
effects of H2O2 from this permeation pathway
was investigated in CRI-G1 cells using the inorganic calcium channel
blocker Cd2+. A Cd2+ concentration (100 µM) that maximally inhibited the voltage-gated calcium
current in these cells was shown to completely inhibit the
Ca2+ influx induced by depolarization. However, this
concentration of Cd2+ had no effect on the magnitude (but
did accelerate the response) of the
H2O2-induced rise in
[Ca2+]i, indicating that
voltage-dependent calcium channels are not involved in the
H2O2-induced late phase of calcium influx observed in these cells. Another possible calcium permeation pathway, in this case linked to intracellular calcium stores, is the capacitive calcium entry (ICRAC), a non-selective cation
current present in pancreatic beta cells and activated by emptying of
intracellular stores (30, 46, 47). Depletion of intracellular
Ca2+ stores in CRI-G1 cells with thapsigargin resulted in
the activation of a calcium influx pathway, indicative of the presence
of ICRAC in these cells.
ICRAC is known to be inhibited by low
concentrations (<100 µM) of extracellular metal ions,
particularly La3+ (31, 48). In CRI-G1 cells, the capacitive
calcium entry pathway was completely blocked by the application of low
(10 µM) concentrations of La3+. However, this
concentration of La3+ had only a small inhibitory effect on
the H2O2-induced late increase in
[Ca2+]i, indicating that the calcium influx
activated by H2O2 is distinct from that
activated by emptying of intracellular Ca2+ stores.
Although low concentrations of La3+ failed to block the
H2O2-induced calcium influx, a higher
concentration of La3+ (100 µM) completely
abolished the calcium influx. This also indicates that although the
calcium influx is independent of both ICRAC and
voltage-dependent calcium channels, it cannot be explained by nonspecific membrane degradation.
Recently, it has been demonstrated that application of
H2O2 to CRI-G1 cells evokes the activation of a
calcium-dependent non-selective cation (NSNAD)
channel, which results in the complete and irreversible collapse of the
cell membrane potential (17). The NSNAD channel is
permeable to calcium (18), and therefore, the possibility that the
second, larger rise in [Ca2+]i resulted from
influx of calcium through the NSNAD channel was examined.
Dual calcium imaging and whole-cell current clamp recordings support
the contention that the activation of the NSNAD channel by
H2O2 is causally related to the late increase in [Ca2+]i. There is an excellent temporal
coincidence between the cellular depolarization and the late phase of
Ca2+ influx caused by H2O2, and
removal of extracellular Ca2+ prevents the depolarization.
Although H2O2 also inhibits KATP currents (most likely due to Ca2+ entry initiating
KATP current rundown/inactivation) concomitant with the
appearance of the non-selective cation current (see Fig. 7A), the overriding influence for cell depolarization is the
activation of the NSNAD channel as evidenced by the
cell-attached recordings. Furthermore, strong evidence for the
NSNAD channel providing the depolarizing driving force is
provided by the inhibition of the H2O2-induced
Ca2+ influx by 100 µM La3+, a
concentration of La3+ that was also observed to inhibit the
H2O2-activated macroscopic current as well as
the NAD-activated single channel currents. In the absence of a specific
inhibitor of the NSNAD channel, we cannot exclude other
explanations for the above results, but activation of this permeation
pathway appears to be the most parsimonious interpretation. It should
be noted that the NSNAD channel appears to be a separate
conductance pathway from that activated by incretin hormones and
maitotoxin, which has the characteristics of a small conductance (30 pS), calcium-activated, non-selective cation channel (49).
The diabetogenic compound alloxan and H2O2 have
been postulated to destroy pancreatic beta cells through DNA damage,
which consequently activates nuclear poly(ADP-ribose) synthase, leading to critical depletion of cellular NAD pools (6). This appears to
contradict the mechanism proposed above, as cellular NAD is presumably
required to activate the NSNAD channel. However, the autofluorescence results indicate that H2O2
causes an immediate oxidation of NAD(P)H, resulting in an increased
NAD/NADH ratio, which then declines again within 1 h. Therefore,
although the final outcome of H2O2 exposure may
well be NAD depletion, the acute effect is an increase in NAD levels,
which, coupled with a rise in [Ca2+]i, may well
be sufficient to activate the NSNAD channel, causing the
cell to depolarize, to flood with calcium, and ultimately to die.
Consequently, we propose that H2O2 causes a
loss of intracellular calcium homeostasis in CRI-G1 cells by inducing a
biphasic rise in [Ca2+]i, with the first phase
caused by mobilization of intracellular calcium and the second, larger
phase due to influx from the extracellular medium. The source of
intracellular calcium is not the inositol 1,4,5-trisphosphate- or
cyclic ADP-ribose-sensitive endoplasmic reticulum stores, and its
identity is as yet elusive. However, the second phase of increased
[Ca2+]i is consistent with the influx of
extracellular calcium permeating through the NSNAD channel.
This represents a novel calcium influx pathway activated by oxidative
stress. The wider implications of this are unclear as yet, but there
have been many studies in which a large influx in calcium has been
observed following oxidative stress, but in which the influx pathway
has not been identified. For example, Bielefeldt et al. (26)
have recently shown that intestinal smooth muscle cells exposed to
H2O2 produce a biphasic rise in
[Ca2+]i, reminiscent of the data presented
within. Additionally, their response was insensitive to both depletion
of intracellular Ca2+ stores and inhibition of
voltage-dependent calcium channels. Similarly, the human
astrocytoma cell line UC11MG has been shown to respond with a biphasic
rise in [Ca2+]i to exposure to
H2O2, which was also insensitive to calcium
channel blockers (50). Therefore, it is possible that these and other
unattributed Ca2+ influxes resulting from cell
death-inducing stimuli are caused by the activation of the
NSNAD channel or by a similar non-selective cation channel.
 |
FOOTNOTES |
*
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.
¶
To whom correspondence should be addressed. Tel.:
44-1224-273055; Fax: 44-1224-273019; E-mail:
mike.ashford{at}abdn.ac.uk.
The abbreviation used is:
S, siemens.
 |
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