Ca2+-induced Ca2+ Release from the
Endoplasmic Reticulum Amplifies the Ca2+ Signal Mediated by
Activation of Voltage-gated L-type Ca2+ Channels in
Pancreatic
-Cells*
Raf
Lemmens,
Olof
Larsson,
Per-Olof
Berggren, and
Md. Shahidul
Islam
From the Rolf Luft Center for Diabetes Research, Department of
Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Institutet,
Karolinska Hospital, S-171 76 Stockholm, Sweden
Received for publication, October 17, 2000, and in revised form, December 18, 2000
 |
ABSTRACT |
Stimulus-secretion coupling in pancreatic
-cells involves membrane depolarization and Ca2+
entry through voltage-gated L-type Ca2+ channels, which is
one determinant of increases in the cytoplasmic free Ca2+
concentration ([Ca2+]i). We investigated how the
endoplasmic reticulum (ER)-associated Ca2+ apparatus
further modifies this Ca2+ signal. When fura-2-loaded mouse
-cells were depolarized by KCl in the presence of 3 mM
glucose, [Ca2+]i increased to a peak in two
phases. The second phase of the [Ca2+]i increase
was abolished when ER Ca2+ stores were depleted by
thapsigargin. The steady-state [Ca2+]i measured
at 300 s of depolarization was higher in control cells compared
with cells in which the ER Ca2+ pools were depleted. The
amount of Ca2+ presented to the cytoplasm during
depolarization as estimated from the integral of the increment in
[Ca2+]i over time
(
[Ca2+]i·dt) was ~30%
higher compared with that in the Ca2+ pool-depleted cells.
neo-thapsigargin, an inactive analog, did not affect
[Ca2+]i response. Using Sr2+ in the
extracellular medium and exploiting the differences in the fluorescence
properties of Ca2+- and Sr2+-bound fluo-3, we
found that the incoming Sr2+ triggered Ca2+
release from the ER. Depolarization-induced
[Ca2+]i response was not altered by U73122, an
inhibitor of phosphatidylinositol-specific phospholipase C, suggesting
that stimulation of the enzyme by Ca2+ is not essential for
amplification of Ca2+ signaling.
[Ca2+]i response was enhanced when cells were
depolarized in the presence of 3 mM glucose, forskolin, and
caffeine, suggesting involvement of ryanodine receptors in the
amplification process. Pretreatment with ryanodine (100 µM) diminished the second phase of the
depolarization-induced increase in [Ca2+]i. We
conclude that Ca2+ entry through L-type voltage-gated
Ca2+ channels triggers Ca2+ release from the ER
and that such a process amplifies depolarization-induced Ca2+ signaling in
-cells.
 |
INTRODUCTION |
Stimulation of pancreatic
-cells with glucose leads to membrane
depolarization and a consequent increase in the activity of the
voltage-gated L-type Ca2+ channels in the plasma membrane.
The resulting entry of Ca2+ through these channels is an
important determinant of the cytoplasmic free Ca2+
concentration
([Ca2+]i)1
necessary for insulin secretion. However,
-cells, like other cells,
also have highly structured Ca2+ stores like the
endoplasmic reticulum (ER). These stores are equipped with
Ca2+ pumps and Ca2+ release channels and are
thus potentially able to modulate depolarization-induced Ca2+ signaling in several ways (1). They may sequester some
of the Ca2+ entering through the voltage-gated
Ca2+ channels, or they may release additional
Ca2+ into the cytoplasm. The latter may be achieved by
different mechanisms, e.g. through Ca2+-mediated
activation of phosphatidylinositol-specific phospholipase C (PI-PLC)
and formation of inositol 1,4,5-trisphosphate
(Ins(1,4,5)P3) or direct gating of the intracellular
Ca2+ channels by the incoming Ca2+. The latter
phenomenon, called Ca2+-induced Ca2+ release
(CICR), is best known in heart muscle, where a small Ca2+
entry through the L-type voltage-gated Ca2+ channels
induces a much larger Ca2+ release through ryanodine
receptor Ca2+ channels. CICR, however, is not an exclusive
property of ryanodine receptors. The other family of intracellular
Ca2+ channels, i.e. the inositol
1,4,5-trisphosphate receptors (IP3Rs), as well as the
permeability transition pore of mitochondria are also capable of
mediating different forms of CICR (2, 3).
-Cells express both IP3Rs and ryanodine receptors, the
relative proportion of which appears to vary under different conditions (4-6). These Ca2+ channels are located on the ER membrane
that constitutes one anatomical Ca2+ pool with luminal
continuity (7). The type 3 isoform of the IP3R is the
predominant IP3R in ob/ob mouse
-cells,
whereas a ryanodine receptor resembling the type 2 ryanodine receptor
of heart is the main ryanodine receptor in these cells (6, 8, 9). Both
of these channels can be considered to be Ca2+-gated
Ca2+ channels, raising the possibility that
depolarization-induced Ca2+ signaling may be amplified by
Ca2+ release through these channels. CICR has been
considered in
-cells from indirect evidence (10); but the phenomenon
has not been demonstrated in situ, and its role in
amplification of Ca2+ signaling in these cells has not been
explored. Here we demonstrate that
-cells possess a mechanism for
amplification of depolarization-induced Ca2+ signaling by
Ca2+ release from the ER. The ER Ca2+ pools
modulate depolarization-induced changes in
[Ca2+]i by sequestering Ca2+ from the
cytoplasm as well as by releasing Ca2+ into the cytoplasm,
a process that is likely to involve CICR.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Fura-2/AM, fluo-3 free acid, and fluo-3/AM were
from Molecular Probes Europe (Leiden, The Netherlands). Thapsigargin
was from Life Technologies, Inc. and Calbiochem.
neo-thapsigargin was a generous gift from Drs. S. B. Christensen and M. Treiman (Royal Danish School of Pharmacy,
Copenhagen, Denmark). Ryanodine (98% pure) and ET-18-OCH3
(1-octadecyl-2-methyl-rac-glycero-3-phosphocholine) were
from Calbiochem. U73122
(1-[6-([(17
)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione) was a gift from Dr. J. E. Bleasdale (The Upjohn Co., Kalamazoo, MI). 2-Aminoethoxydiphenyl borate (also called diphenylboric acid 2-aminoethyl ester) was a gift from Dr. T. Maruyama (Ono Pharmaceutical Co., Minase Research Institute, Osaka, Japan) and was also bought from
Sigma. Neomycin sulfate was from the Upjohn Co. Cyclopiazonic acid and
2-nitro-4-carboxyphenyl N,N-diphenylcarbamate
were from Sigma. Xestospongin C was a gift from Dr. Kobayashi, Osaka
University, and was also bought from Calbiochem.
Isolation of Pancreatic Islets and Preparation of
-Cells--
Islets from fasting adult ob/ob mice from a
local colony were isolated by collagenase digestion and dispersed by
shaking in Ca2+- and Mg2+-deficient medium
(7). Cells were cultured on glass coverslips for 1-2 days in
RPMI 1640 medium supplemented with fetal calf serum (10%, v/v),
penicillin (100 IU/ml), and streptomycin (100 µg/ml).
Measurement of [Ca2+]i by
Microfluorometry--
Cells were incubated in RPMI 1640 medium
supplemented with 0.1% bovine serum albumin and 0.6 µM
fura-2/AM for 35 min at 37 °C. Cells were then incubated for an
additional 10 min in basal medium containing 125 mm NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.28 mM CaCl2, 25 mM HEPES, 3 mM glucose, and 0.1% bovine serum albumin (pH 7.4).
Coverslips were mounted as the bottom of a perifusion chamber on the
stage of an inverted epifluorescence microscope (Zeiss Axiovert 35M).
The superfusion chamber was designed to allow rapid exchange of fluids,
and complete exchange of fluids took place in <3 s. The stage was
thermostatically controlled to maintain a temperature of 37 °C in
the perifusate inside the chamber. The microscope was connected to a
SPEX Fluorolog-2 CM1T11I system for dual wavelength excitation
fluorometry. The excitation wavelengths generated by two monochromators
were directed to the cell by a dichroic mirror. The emitted light
selected by a 510-nm filter was monitored by a photomultiplier. The
excitation wavelengths were alternated at a frequency of 1 Hz, and the
duration of data collection at each wavelength was 0.33 s. The
emission at the excitation wavelength of 340 nm
(F340) and that of 380 nm
(F380) were used to calculate the fluorescence
ratio (R340/380). Single cells or small clusters
of cells, isolated optically by means of a diaphragm, were studied by
using a 40× 1.3 NA oil immersion objective (Zeiss, Plan Neofluar).
Background fluorescence was measured after quenching fura-2
fluorescence with manganese and was subtracted from the traces before
calculation of [Ca2+]i.
[Ca2+]i was calculated from
R340/380 according to Grynkiewicz et
al. (11). Maximum and minimum fluorescence ratios were
determined in separate experiments using thin films of external
standards containing 10 µM fura-2 and 2 M
sucrose in intracellular-like buffer containing 10 µM
fura-2 free acid and either 2 mM Ca2+ or no
Ca2+ in the presence of 2 mM EGTA. The
Kd for the Ca2+·fura-2 complex was
taken as 224 nM. To compensate for variations in output
light intensity from the two monochromators, all experiments included a
fluorescence ratio where both monochromators were set at 360 nm.
Fluo-3 Fluorescence Measurements--
For comparing the
fluorescence properties of Ca2+- or Sr2+-bound
fluo-3, we dissolved 1 µM fluo-3 free acid in buffer
containing 145 mM KCl and 20 mM HEPES (pH 7.2).
The buffer was then divided in to two portions: to one we added
SrCl2 (1 mM), and to the other CaCl2 (1 mM). Emission spectra were measured at
490 nm excitation using a SPEX Fluorolog-2 CM1T11I system. Scanning was
performed in a quartz cuvette containing a 1-ml solution at 20 °C.
Cells were loaded with fluo-3 by incubation with 0.5 µM
fluo-3/AM for 35 min, followed by incubation in basal medium for
another 10 min. Fluorescence was measured at 490 nm excitation, and
emitted light filtered by a 515-565-nm band-pass filter was recorded
by a photomultiplier.
Electrophysiological Recordings--
Voltage-gated
Ca2+ currents were recorded using the whole-cell mode of
the patch-clamp technique (12). Pipettes were prepared from
borosilicate glass capillary tubes, coated with Sylgard resin (Dow
Corning) near the tips, and fire-polished and had resistances of 2-6
mega-ohms. Cells were washed with a solution composed of 138 mM NaCl, 5.6 mM KCl, 1.2 mM
MgCl2, 10 mM CaCl2, 10 mM tetraethylammonium chloride, and 5 mM HEPES
(pH 7.4). The pipette solution contained 150 mM
N-methyl-D-glucamine, 110 mM HCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM EGTA, 3 mM MgATP, and 5 mM
HEPES (pH 7.15). N-Methyl-D-glucamine was
substituted for K+ in the pipette solution to prevent
outwardly directed K+ currents. Currents were recorded
using an Axopatch 200 amplifier (Axon Instruments, Inc., Foster City,
CA). Voltage steps were generated, digitized, and stored using the
program pClamp (Axon Instruments, Inc.) and Labmaster ADC (Scientific
Solutions). The current responses were filtered at 2 kHz. The pulse
protocol is given in the figure legends.
Statistical Analysis--
Numerical data are presented as
means ± S.E. Statistical significance was judged by Student's
t test for unpaired data.
 |
RESULTS |
-Cells perifused with basal medium containing 3 mM
glucose responded with an increase in the average
[Ca2+]i upon depolarization with 30 mM KCl (Fig. 1). Consistent with our previous reports (13), the [Ca2+]i
increase was dependent on the presence of extracellular Ca2+ and was abolished by nimodipine, a specific blocker of
the L-type voltage-gated Ca2+ channels. These results
suggest that in cultured mouse
-cells and under our experimental
conditions, depolarization-induced Ca2+ entry occurs
entirely through the L-type voltage-gated Ca2+ channels and
that depolarization per se does not induce detectable Ca2+ release from internal stores. Depolarization by KCl
yielded a rise in [Ca2+]i, the pattern of which
varied from cell to cell. In all cells, however,
[Ca2+]i increased in two phases, an initial rapid
phase followed by a slower increase to the peak (Fig. 1A).
On examination of a large number of cells, three patterns of
[Ca2+]i increase could be discerned (Fig. 1). The
most common was one where a rapid increase in
[Ca2+]i changed into a slower phase (Fig.
1A). In 6 out of 19 cells, the two phases were separated by
a transient dip in [Ca2+]i after the initial
rapid phase (Fig. 1B). In two other cells, the second phase
of the increase in [Ca2+]i consisted of a single
Ca2+ transient on elevated [Ca2+]i.
In some experiments, we used
-cells obtained from Wistar rats
instead of ob/ob mice, and the results were similar.

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Fig. 1.
Patterns of [Ca2+]i
changes in pancreatic -cells during sustained
depolarization. Fura-2-loaded cultured mouse -cells were
perifused with basal medium containing 3 mM glucose and
depolarized by rapid application of 30 mM KCl during the
period indicated by the horizontal bars. Following
depolarization, [Ca2+]i increased to a peak in
two phases. The pattern of [Ca2+]i increase shown
in A occurred in 11 out of 19 cells. In some cells
(B), the two phases were separated by a transient dip after
the first rapid phase of rise in [Ca2+]i (6 out
of 19 cells). In two cells, the depolarization-induced increase in
[Ca2+]i was accompanied by a Ca2+
transient on an elevated [Ca2+]i
(C).
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A biphasic increase in [Ca2+]i suggested the
possibility that multiple sources of Ca2+ may be involved
in depolarization-induced Ca2+ signaling. To test whether
the ER Ca2+ stores contributed to the
depolarization-induced changes in [Ca2+]i, we
examined [Ca2+]i responses in cells where the ER
Ca2+ stores were depleted by thapsigargin, a specific
inhibitor of SERCA (sarcoplasmic/endoplasmic
reticulum Ca2+-ATPases)
(14). Incubation of cells with 250 nM thapsigargin for 35 min depleted the ER Ca2+ pools as indicated by lack of
response to carbachol. The resting [Ca2+]i and
membrane potential in thapsigargin-treated cells were same as in the
controls (Figs. 2C and
3A), indicating that capacitative Ca2+ entry was
unlikely to confound results under these experimental conditions. In ER
Ca2+ pool-depleted cells, the [Ca2+]i
response upon depolarization differed from that in control cells with
regard to several aspects. In thapsigargin-treated cells, KCl increased
[Ca2+]i with a faster time course, and the decay
phase around the peak was also faster (Fig. 2C). The time
courses of increases in [Ca2+]i from the basal
level to the peak subsequent to depolarization in control and
thapsigargin-treated cells are illustrated in Fig. 2A. In
this figure, where data are presented as semilogarithmic plots, the
time course of the increase in [Ca2+]i in control
cells clearly deviates from a single exponential. Most strikingly,
thapsigargin treatment abolished the slower second phase of the
increase in [Ca2+]i seen in the control cells
(Fig. 2, A and C). [Ca2+]i
increased more rapidly in thapsigargin-treated cells than in control
cells. The maximum rates of increase in [Ca2+]i,
calculated as the differences between neighboring points, were 90 ± 14 and 45 ± 6 nM/s in the thapsigargin-treated and
control cells, respectively (p = 0.006, n = 28). The times required for
[Ca2+]i to rise from basal levels to the peaks
were 19 ± 4 and 85 ± 12 s in thapsigargin-treated and
control cells, respectively (p < 0.001, n = 30) (Fig. 2B). Depolarization-induced
peak [Ca2+]i was slightly higher in the
thapsigargin-treated cells, although the difference was not
statistically significant (485 ± 50 and 403 ± 30 nM in thapsigargin-treated and control cells, respectively;
n = 26). For comparison of
[Ca2+]i response in the two groups, we calculated
the integral of the increment in [Ca2+]i over
time (
[Ca2+]i·dt) as an
estimate of the amount of Ca2+ presented to the cytoplasm
during depolarization. A period of 300 s was chosen for
calculating the Ca2+ integral because during sustained
depolarization by KCl, [Ca2+]i returned to an
elevated steady-state level in most experiments by this time. The
steady-state [Ca2+]i measured at 300 s of
depolarization was significantly lower in thapsigargin-treated cells
(171 ± 16 and 233 ± 11 nM in
thapsigargin-treated and control cells, respectively; p = 0.003, n = 26). The time integral of the
[Ca2+]i increment during 300 s of
stimulation by KCl was ~30% higher in the control cells compared
with that in the Ca2+ pool-depleted cells
(p = 0.002, n = 26) (Fig.
2D). In some experiments, we treated cells with a lower
concentration of thapsigargin (e.g. 50 nM). This
concentration of thapsigargin depleted ER Ca2+ pools almost
completely, but such depletion was achieved less consistently. However,
a qualitatively similar, although smaller effect on
[Ca2+]i response was seen also when cells were
treated with 50 nM thapsigargin. In some other experiments,
we treated cells with 10 µM cyclopiazonic acid, a
reversible inhibitor of SERCA, for 30 min and depolarized the cells
with KCl in the continued presence of the substance. The effects of
treatment with cyclopiazonic acid were similar to those obtained with
thapsigargin.

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Fig. 2.
Effect of depletion of ER Ca2+
pools on depolarization-induced [Ca2+]i
response. ER Ca2+ stores were depleted by incubating
the cells for 35 min with 250 nM thapsigargin. Cells were
depolarized by 30 mM KCl. A, the time course of
rise in [Ca2+]i from the basal to the peak level
upon depolarization is plotted on a semilogarithmic scale. Three traces
each for controls ( ) and thapsigargin (Tg)-treated
cells ( ) are illustrated. The traces are representative of 15 experiments with control cells and 15 experiments with
thapsigargin-treated cells. B, shown is the time taken for
the increase in [Ca2+]i from the basal level to
the peak in control and thapsigargin-treated cells. C,
changes in [Ca2+]i during 300 s of
depolarization by 30 mM KCl in control and
thapsigargin-treated cells are illustrated. Traces are representative
of 13 experiments done with thapsigargin-treated cells and 13 with
control cells. D, the time integral of the increase in
[Ca2+]i above the base line during 300 s of
depolarization by KCl was significantly higher in control cells
compared with thapsigargin-treated cells . Data are derived from 13 experiments in control cells and 13 thapsigargin-treated cells as
illustrated in C.
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We tested whether the reduction in depolarization-induced
[Ca2+]i response in thapsigargin-treated cells
could be due to inhibition of L-type voltage-gated Ca2+
channels by the compound, as has been reported in other cells (15). As
shown in Fig. 3A,
depolarization-induced whole-cell Ca2+ currents measured by
the patch-clamp technique in cells treated with a low concentration of
thapsigargin (i.e. 250 nM) were not significantly different from those recorded in controls. To test further if some other nonspecific effects of thapsigargin could account
for our results, we treated cells with an analog of the compound
epimerized at C-8 (neo-thapsigargin) (16). When used at low
concentrations, this analog of thapsigargin is virtually inactive as an
inhibitor of SERCA (17). As shown in Fig. 3B, neo-thapsigargin (250 nM for 35 min) did not
deplete the ER Ca2+ pools and also did not alter the
pattern of the depolarization-induced increase in
[Ca2+]i (cf. Fig. 1 and Fig.
2C). It was a possibility that thapsigargin treatment and
the consequent increase in [Ca2+]i caused
enhancement of plasma membrane Ca2+-ATPase activity, thus
accounting for the less pronounced [Ca2+]i
response seen in these cells (18). To test this, we increased
[Ca2+]i by activating the reverse mode of the
Na+/Ca2+ exchanger by substituting
Na+ with N-methyl-D-glucamine in the
perifusion medium. This maneuver caused a modest increase in
[Ca2+]i, the magnitude of which was not
significantly different in thapsigargin-treated cells compared with the
untreated controls (data not shown).

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Fig. 3.
Altered [Ca2+]i
response in thapsigargin-treated cells is not due to nonspecific
effects of the substance. A, treatment of cells with a
low concentration of thapsigargin (250 nM) for 35 min did
not alter the magnitude of voltage-dependent
Ca2+ currents through Ca2+ channels in the
plasma membrane. Cells were depolarized in 10-mV steps (100 ms) to
potentials between 60 and +50 mV from a holding potential of 70 mV.
The upper traces show examples of currents in the presence
and absence of thapsigargin. The lower graph shows compiled
data on the I-V relationship. B,
neo-thapsigargin, an inactive isomer of thapsigargin, did
not alter depolarization-induced [Ca2+]i
response. Cells were incubated for 35 min in 250 nM
neo-thapsigargin. Depolarization by KCl resulted in a
biphasic increase in [Ca2+ ]i as in control
cells. ER Ca2+ pools were not depleted by
neo-thapsigargin since carbachol (CCh) released
Ca2+ from the ER. Traces are representative of experiments
repeated at least three times. pF, picofarads.
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The results described above suggest the possibility that
Ca2+ entering through the L-type voltage-gated
Ca2+ channels might act as a trigger, giving rise to
further Ca2+ release from the ER. We intended to visualize
the trigger Ca2+ and the released Ca2+ more
directly. This could be done by using Sr2+ as a
Ca2+ surrogate and by exploiting the differences in the
fluorescence properties of Ca2+- and Sr2+-bound
fluo-3. We compared the fluorescence spectral properties of fluo-3 in
the presence of Ca2+ and Sr2+. Fig.
4A shows the emission spectra
of fluo-3 between 500- and 560-nm wavelengths in the presence of
saturating concentrations of Ca2+ or Sr2+. As
shown, fluo-3 yielded much less fluorescence when it bound Sr2+ compared with when it bound Ca2+. In the
experiments illustrated in Fig. 4 (B-D), we used fluo-3/AM instead of fura-2/AM to load the cells. In these experiments, we
omitted Ca2+ from the perifusion medium and instead added 1 mM Sr2+ to the medium. Under these conditions,
when the cells were depolarized by KCl, there was initially a modest
increase in fluorescence presumably due to Sr2+ entry and
the resulting increase in [Sr2+]i. Superimposed
on this fluorescence, there was a large but transient increase in
fluorescence, which was probably caused by Ca2+ release
from intracellular stores (Fig. 4B). To ascertain whether the Ca2+ transients triggered by Sr2+ were due
to Ca2+ release from the ER, we depleted the ER
Ca2+ stores with thapsigargin and repeated similar
experiments. As shown in Fig. 4C, in thapsigargin-treated
cells, despite depolarization-induced Sr2+ entry, there was
no Ca2+ spike as seen in the untreated cells.
Sr2+ was unable to trigger Ca2+ release also
when the common ER Ca2+ pools were depleted by carbachol
prior to depolarization by KCl (Fig. 4D).

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Fig. 4.
Differences in fluorescence properties of
fluo-3 complexes with Sr2+ and Ca2+
used to visualize Ca2+ release from ER. A,
shown are the fluorescence emission spectra of fluo-3 in the presence
of Ca2+ or Sr2+. Spectra were obtained in
HEPES/KCl buffer containing 1 µM fluo-3 free acid and 1 mM Ca2+ or Sr2+. The traces are
representative of experiments repeated twice. B, cells were
loaded with fluo-3 and perifused with extracellular medium containing 1 mM Sr2+ instead of Ca2+.
Depolarization of cells by 30 mM KCl in the presence of
extracellular Sr2+ resulted in an initial small increase in
fluorescence followed by large transient increases in fluorescence. The
latter is caused by Ca2+ released from the ER since it was
abolished when the ER Ca2+ pools were first depleted by
thapsigargin (Tg; C) or prior application of
carbachol (10 µM) (D). The traces are
representative of experiments repeated at least three times.
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These results suggest that during sustained depolarization and in
association with Ca2+ entry through the L-type
voltage-gated Ca2+ channels, there is a release of
Ca2+ from the ER. Such a Ca2+ release could be
mediated by several mechanisms. The first would involve activation of
PI-PLC by Ca2+ entering through the voltage-gated
Ca2+ channels, leading to formation of
Ins(1,4,5)P3 and the consequent activation of
IP3Rs. The second would involve direct gating of IP3Rs by Ca2+ in the presence of a basal or
slightly elevated level of the trisphosphate. The third would involve
activation of the ryanodine receptors by Ca2+. To determine
whether activation of PI-PLC by Ca2+ was involved, we tried
several inhibitors of the enzyme, e.g. neomycin, the ether
lipid ET-18-OCH3 (19), and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (20). However, we found
that these inhibitors themselves alter [Ca2+]i
homeostasis in
-cells by nonspecific mechanisms unrelated to the
inhibition of PI-PLC. Neomycin inhibited L-type voltage-gated Ca2+ channels as studied by the whole-cell mode of the
patch-clamp technique, an observation consistent with a previous report
(21). When applied to the resting
-cells, ET-18-OCH3 and
2-nitro-4-carboxyphenyl N,N-diphenylcarbamate
increased [Ca2+]i by unknown mechanisms. These
substances were therefore not suitable as PI-PLC inhibitors in intact
-cells. We then tested another commonly used inhibitor of PI-PLC,
U73122. This substance, despite reported nonspecific effects, appeared
suitable for inhibiting PI-PLC in
-cells. We perifused
-cells
with 10 µM U73122 for 300 s and then
depolarized the cell by KCl in the continued presence of the inhibitor.
U73122 itself did not affect [Ca2+]i in
-cells. As shown in Fig.
5A, U73122 did not inhibit the
biphasic increase in [Ca2+]i during KCl
depolarization (cf. Fig. 5B). Under these conditions, U73122 inhibited PI-PLC as evidenced by lack of [Ca2+]i response to carbachol in separate
experiments. These results indicate that amplification of
depolarization-induced Ca2+ signaling does not require
concomitant activation of PI-PLC by the incoming Ca2+. To
directly test if activation of IP3Rs by
Ins(1,4,5)P3 or Ca2+ was involved, we intended
to use xestospongin C and 2-aminoethoxydiphenyl borate,
membrane-permeable agents that have been reported to inhibit IP3Rs in neuronal cells (22). However, in control
experiments, we found that xestospongin C (20 µM) or
2-aminoethoxydiphenyl borate (up to 300 µM) (23) did not
inhibit Ca2+ release by Ins(1,4,5)P3-forming
agonists like carbachol or ADP
s in
-cells. These agents thus did
not appear suitable as membrane-permeable inhibitors of
IP3Rs in
-cells.

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Fig. 5.
Effect of a PI-PLC inhibitor (U73122) on the
depolarization-induced increase in [Ca2+]i
in -cells. Fura-2-loaded single -cells
were first perifused by 10 µM U73122 (A) or
0.1% Me2SO (DMSO; solvent for U73122)
(B) for 300 s. Cells were then depolarized by applying
KCl (30 mM) until [Ca2+]i increased
to its peak. In both cases, [Ca2+]i increased to
a peak in two phases. Data are representative of experiments repeated
at least three times with similar results.
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To test if ryanodine receptors were involved in amplifying
depolarization-induced Ca2+ signaling, we used ryanodine,
ruthenium red, and caffeine. We incubated cells with 100 µM ryanodine for 45 min. Such treatment is expected to
lock the ryanodine receptors in a subconductance state and thus deplete
the ryanodine-sensitive ER Ca2+ pools to a variable extent.
The effect of ryanodine treatment on depolarization-induced
Ca2+ response was small. When ryanodine-treated cells were
depolarized by KCl, the second phase of the
[Ca2+]i increase was diminished (Fig.
6B) as compared with that in
untreated controls (Fig. 6A). Consistent with this, the time
taken for [Ca2+]i to rise from the basal
level to the peak was significantly reduced in ryanodine-treated cells
compared with the untreated controls (52 ± 12 and 146 ± 27 s, respectively; p < 0.05, n = 7) (Fig. 6C). Low concentration of ruthenium red (10 µM), an inhibitor of ryanodine receptors, reduced
depolarization-induced [Ca2+]i response (Fig.
6D). We then examined whether agents that sensitize
ryanodine receptors can increase depolarization-induced Ca2+ response. Caffeine sensitizes ryanodine receptors to
Ca2+, and from previous studies, we know that in
situ activation of ryanodine receptors by caffeine requires
cAMP-dependent phosphorylation (5). When cells were
depolarized in the presence of 3 mM glucose and forskolin
(5 µM) plus caffeine (5 mM), a dramatic
increase in [Ca2+]i response in the form of
Ca2+ spikes was observed (Fig.
7A). Such an increased
Ca2+ response could be due to increased Ca2+
entry through L-type voltage-gated Ca2+ channels caused by
cAMP-dependent phosphorylation of the channel. But our
control experiments showed that this was not the case since forskolin
alone did not have a similar effect under these experimental conditions
(Fig. 7B).

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|
Fig. 6.
Effect of ryanodine and ruthenium red on
depolarization-induced increase in [Ca2+]i
in -cells. A, fura-2-loaded
single -cells were depolarized by 30 mM KCl during the
period indicated by the horizontal line. The trace shows a
control experiment with a typical biphasic increase in
[Ca2+]i to its peak. B, cells were
incubated for 45 min with 100 µM ryanodine and
depolarized by KCl. The second phase of the
[Ca2+]i increase was diminished by ryanodine
treatment. C, shown is the time taken for the increase in
[Ca2+]i from the basal level to the peak in
control and ryanodine-treated cells (p < 0.05, n = 7). D, [Ca2+]i
response was lower when cells were depolarized by KCl in the presence
of 10 µM ruthenium red (RR), a blocker of the
ryanodine receptor.
|
|

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|
Fig. 7.
Sensitization of the ryanodine receptor
enhances the depolarization-induced [Ca2+]i
increase in -cells. Fura-2-loaded
-cells were perifused with basal buffer containing 3 mM
glucose. A, cells were depolarized by KCl in the presence of
5 µM forskolin and 5 mM caffeine.
B, shown are the results of a control experiment in which
cells were depolarized by KCl in the presence of 5 µM
forskolin only. Note that the scales are different in A and
B. Traces are representative of at least three experiments
with similar results.
|
|
 |
DISCUSSION |
Plasma membrane depolarization and increase in
[Ca2+]i are two key events in stimulus-secretion
coupling of pancreatic
-cells. It is generally thought that
depolarization-induced [Ca2+]i increase in
-cells is solely due to Ca2+ entry through the
voltage-gated Ca2+ channels in the plasma membrane. In many
electrically excitable cells, however, Ca2+ entering
through the voltage-gated Ca2+ channels activates adjacent
intracellular Ca2+ channels (CICR). In such cells,
depolarization-induced [Ca2+]i increase is a
result of Ca2+ being presented to the cytoplasm through two
types of channels, i.e. the plasma membrane Ca2+
channels and the ER Ca2+ channels.
-Cells possess the
requisite molecular components required for mediating CICR (5, 10).
However, the issue of whether ER Ca2+ stores and the
associated Ca2+ channels participate in the
depolarization-induced Ca2+ signaling in intact
-cells
has remained an open question. Thapsigargin, a tool for studying
involvement of ER Ca2+ stores in cellular processes, has
made it possible to address this question (24). Using this approach, we
now demonstrate that the ER Ca2+ stores modulate
depolarization-induced Ca2+ signaling in two ways. In our
study, the rate of increase in [Ca2+]i following
depolarization was significantly faster in thapsigargin-treated cells
compared with controls. This suggests that following Ca2+
entry through the voltage-gated Ca2+ channels, the ER
Ca2+-ATPase immediately participates in removing
Ca2+ from the cytoplasm into the ER. This rapid
sequestration of Ca2+ into the ER can retard the rate of
increase in [Ca2+]i and limit the peak
[Ca2+]i during depolarization. Such effects may
have implications in the prevention of short-term and long-term toxic
effects of [Ca2+]i on
-cells. On the other
hand, Ca2+ uptake into the ER may have important
consequences in loading the latter compartment. This can ensure
generation of a larger Ca2+ flux from the ER, thereby
prolonging the Ca2+ transient and amplifying
[Ca2+]i-dependent signaling (25).
The other important finding of our study is that depolarization-induced
Ca2+ signaling in
-cells is amplified by release of the
ion from the intracellular stores. The Ca2+ stores that
participate in this process are the ones associated with the ER. This
is evident from the fact that during depolarization, the steady-state
[Ca2+]i as well as the integral of the increment
in [Ca2+]i over time were significantly higher in
control cells compared with cells in which the ER Ca2+
pools were depleted by thapsigargin. The time integral of
[Ca2+]i response was higher in the control cells
compared with the thapsigargin-treated cells, despite the fact that, in the latter, the ER Ca2+ pump was inhibited, and ER could
not contribute to the removal of Ca2+ entering into the
cytoplasm through the voltage-gated Ca2+ channels. To
demonstrate more directly that Ca2+ entering through the
voltage-gated Ca2+ channels can activate ER
Ca2+ channels in
-cells, we substituted Sr2+
for Ca2+ in the extracellular medium. The rationale for
this approach is that Sr2+ can enter through the
voltage-gated Ca2+ channels and can activate ryanodine
receptors (26). But whereas Ca2+ yields large fluorescence
upon binding to fluo-3, Sr2+ does not (Fig. 4A).
Using this approach, we could demonstrate that Sr2+,
entering through the voltage-gated Ca2+ channels, induces
release of Ca2+ from the ER. Thus, our results show that
during depolarization, the net increase in
[Ca2+]i in
-cells is a result of
Ca2+ entry through voltage-gated Ca2+ channels
and Ca2+ release from the ER, suggesting that the two
processes may be coupled. The succession of two events, i.e.
Ca2+ uptake into and release from the ER, may thus play a
role in modulating the magnitude and spatiotemporal aspects of
depolarization-induced [Ca2+]i signaling in
-cells.
The mechanisms that couple Ca2+ entry through the
voltage-gated L-type Ca2+ channels to Ca2+
release from the ER may involve multiple ER Ca2+ channels.
One view is that depolarization and the resulting increase in
[Ca2+]i activate PI-PLC, leading to generation of
Ins(1,4,5)P3, which by itself activates IP3Rs,
causing a further increase in [Ca2+]i. This view
originates from the facts that a minimal level of Ca2+ is
essential for activation of PI-PLC (27, 28) and that Ca2+
increases PI-PLC activity in mouse islet homogenates (28). In this
view, an increase in the concentration of Ins(1,4,5)P3 (as
opposed to an increase in [Ca2+]) is the predominant
mechanism that couples Ca2+ entry to Ca2+
release. However, the relative roles played by any postulated increase
in the concentration of Ins(1,4,5)P3 versus an
increase in [Ca2+] in this coupling process merit closer
examination. In intact cells, KCl depolarization leads to little or
only a modest and transient (~30 s) increase in PI-PLC activity
(29-32). This is particularly true for mouse islets, which express low
levels of Ca2+-sensitive PI-PLC isoforms such as PI-PLC
1
(29, 33). Moreover, studies that have demonstrated increased PI-PLC
activity in islets during KCl depolarization were performed using
suspensions of islets in static incubations. Under such conditions,
depolarization-induced Ca2+-dependent exocytosis
leads to accumulation of components of secretory granules,
e.g. ATP, in the medium, which may stimulate PI-PLC by
activating specific receptors. Stimulation of PI-PLC activity seen in
these assays may thus be partly due to autocrine or paracrine stimulation of receptors linked to PI-PLC. Thus, these protocols are
not suitable to test whether depolarization-induced increases in
[Ca2+]i per se can directly stimulate
PI-PLC in intact
-cells in the absence of ligand binding. Studies in
other cells demonstrate that a physiological increase in
[Ca2+]i does not directly activate PI-PLC (34),
but may potentiate activation of the enzyme when PI-PLC-linked
receptors are occupied by appropriate ligands (35). In our experiments, in which we used single mouse
-cells and a system for rapid washout of medium by continuous perifusion, the likelihood of autocrine or
paracrine interactions was minimal. It is therefore likely that, under
our experimental conditions, a KCl-induced rise in [Ca2+]i increased cellular
Ins(1,4,5)P3 level only minimally. The molecular mechanisms
involved in the opening of IP3Rs are complex. In addition
to Ins(1,4,5)P3, Ca2+ plays a fundamental role
in this process. From studies on Ins(1,4,5)P3-induced Ca2+ release in different cells, it is known that
Ca2+ and Ins(1,4,5)P3 are coactivators of the
IP3R (36), i.e. the receptor must bind both of
these agonists before the associated channel opens (37). From these
considerations, it appears likely that Ca2+ entering
through the voltage-gated Ca2+ channels can directly
trigger IP3R, apparently in the context of minimal
elevation of the trisphosphate. In this scenario, the IP3R
is viewed to operate mainly as a CICR channel. In our experiments, U73122, an inhibitor of PI-PLC, did not reduce amplification of
depolarization-induced Ca2+ signaling. This observation
lends support to the notion that activation of PI-PLC by
Ca2+ is not essential for the amplification of
depolarization-induced Ca2+ signaling.
Among the IP3Rs, ob/ob
-cells express mainly
the type 3 isoform (IP3R-3) (6). This isoform of
IP3R is 10 times less sensitive to the trisphosphate
compared with IP3R-1 and even less sensitive than
IP3R-2 (38). Given that Ins(1,4,5)P3 and
Ca2+ are co-agonists of IP3Rs and that
IP3R-3 is only poorly sensitive to the trisphosphate alone,
it is likely that, under physiological conditions, the role of
Ca2+ as a trigger of IP3R-3 is of crucial
importance. In other words, IP3R-3 can operate as a CICR
channel much like the ryanodine receptors, albeit requiring assistance
from a basal or minimally elevated level of the trisphosphate. Of the
three ryanodine receptors,
-cells appear to express only the type 2 isoform, which typically mediates CICR in heart (5, 6, 9). The level of
expression of ryanodine receptors in our ob/ob
-cells is
low compared with that in heart (5). Despite this,
depolarization-induced [Ca2+]i response was
dramatically enhanced by caffeine when the xanthine drug was applied
together with a cAMP-elevating agent, forskolin. It may be noted that
this enhancement could not be attributed to cAMP-dependent
phosphorylation of L-type voltage-gated Ca2+ channels since
forskolin alone did not have significant effects on
[Ca2+]i response. cAMP-dependent
phosphorylation is required for in situ activation of
ryanodine receptors in
-cells (5). Protein kinase A phosphorylation
relieves the inhibition of the ryanodine receptor by intracellular
Mg2+ and promotes dissociation of FKBP12.6, events that
facilitate channel activation (39, 40). These results provide good
evidence that type 2 ryanodine receptors participate in amplification
of depolarization-induced Ca2+ signaling in
-cells, but
do not exclude the roles of IP3Rs in the process. Since
Ca2+ can gate both of these channels, it is likely that
both types of channels participate in mediating CICR. The fact that
ruthenium red and high concentrations of ryanodine only partially
blocked the second phase of depolarization-induced
[Ca2+]i response further supports that both
ryanodine receptors and IP3Rs may be involved in the process.
We demonstrate that in addition to the voltage-gated Ca2+
channels in the plasma membrane, the ER Ca2+ stores
participate in generating depolarization-induced Ca2+
signaling in
-cells. The latter does not act only as a
Ca2+ sink, but rather plays important roles in amplifying
the depolarization-induced Ca2+ signaling by CICR. We have
demonstrated amplification of Ca2+ signaling in cultured
-cells by using EGTA-based Ca2+ indicators that act as
mobile buffers and that have the potential to interfere with the
process of CICR. It is likely that the amplification process is more
pronounced in vivo. Such amplified Ca2+
signaling may have physiological relevance in mediating cellular processes that require high [Ca2+]i.
 |
FOOTNOTES |
*
This work was supported in part by Swedish Medical Research
Council Grants K200-32X-13469-01A, 72X-09890, 72X-00034, 72XS-12708, and 72X-09891; Swedish Natural Science Research Council Grant U-AA/ST
11616-302l; the Novo Nordisk Foundation; the Swedish Diabetes Association; the Swedish Society of Medicine; and the Karolinska Institutet.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.
Recipient of a career development award from the Juvenile Diabetes
Research Foundation. To whom correspondence should be addressed. Tel.
and Fax: 46-8-6731832; E-mail: Shahidul.Islam@molmed.ki.se.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M009463200
 |
ABBREVIATIONS |
The abbreviations used are:
[Ca2+]i, cytoplasmic free Ca2+
concentration;
ER, endoplasmic reticulum;
PI-PLC, phosphatidylinositol-specific phospholipase C;
Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate;
CICR, Ca2+-induced Ca2+ release;
IP3R, inositol 1,4,5-trisphosphate receptor;
AM, acetoxymethyl ester;
ADP
S, adenosine 5'-O-(2-thiodiphosphate).
 |
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