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INTRODUCTION |
Ca2+ signals regulate many cellular processes
including cell growth, fertilization, gene transcription, and apoptosis
(1). Increases in cytosolic Ca2+ levels are produced both
by Ca2+ released from internal stores and Ca2+
influxed from the extracellular space. A major pathway for
Ca2+ mobilization from internal stores is through inositol
1,4,5-trisphosphate receptors
(IP3R)1 after
stimulation of G-protein- or tyrosine kinase-coupled plasma membrane
receptors linked to phospholipase C (2-4). The decrease in the
Ca2+ content of the internal store then stimulates
Ca2+ entry through plasma membrane store-operated
Ca2+ channels (SOCs) by a process called store-operated
Ca2+ entry (SOCE) (5, 6). The mechanism by which a
reduction in the content of store Ca2+ results in opening
of SOCs remains unknown, but there are two major hypotheses. The
conformational coupling hypothesis suggests that there is direct
physical contact between IP3Rs and SOCs such that
conformational changes in the IP3R occurring upon
Ca2+ depletion of the internal store can affect the opening
of SOCs (7, 8). The diffusible messenger hypothesis suggests that the
Ca2+ store (endoplasmic reticulum) produces a diffusible
messenger that opens SOCs (9, 10).
Recently, a novel family of compounds called adenophostins (AdAs),
which are structurally distinct from IP3, have been
isolated from cultures of the fungus Penicillium
brevicompactum (11, 12). The AdAs are 10-100-fold more potent
than IP3 in opening IP3Rs (13) and are capable
of activating all three IP3R subtypes (13-15). Recently,
Hartzell et al. (16) and DeLisle et al. (17) have
shown that in Xenopus oocytes, low concentrations of AdA stimulate Ca2+-activated Cl
currents that are
activated by Ca2+ influx more than Cl
currents that are activated by Ca2+ released from stores.
Based on these observations, DeLisle et al. (17) suggested
that AdA may be capable of activating store-operated Ca2+
entry without first stimulating Ca2+ release from stores.
This is significant because it suggests that AdA may share structural
features with the putative diffusible Ca2+ entry signal
released by Ca2+-depleted endoplasmic reticulum.
Ca2+-activated Cl
currents have been used for
many years as real time indicators of sub-plasmalemmal Ca2+
in Xenopus oocytes (18-24), but clearly Cl
currents are only indirect indicators of Ca2+
concentration. Consequently, conclusions about cytosolic
Ca2+ concentration derived from these measurements are
subject to different interpretations. We have recently found that there
are two Ca2+-activated Cl
currents in the
oocyte that are selectively activated by Ca2+ released from
stores and by Ca2+ influx (24). The Ca2+
release-activated Cl
current
(ICl1-S) has an outwardly rectifying
steady-state current-voltage relationship, whereas the Ca2+
influx-activated Cl
current (ICl2)
has an inwardly rectifying steady-state current-voltage relationship
(24). This means that Ca2+-activated Cl
currents measured at constant negative membrane potentials, as was done
in the experiments of DeLisle et al. (17), are relatively insensitive indicators of Ca2+ released from stores. In our
experiments (16), we measured ICl1-S as an
outward current at positive membrane potentials and ICl2 as an inward current at negative membrane
potentials to more clearly differentiate between Ca2+
influx and Ca2+ release and to increase the sensitivity of
detection of Ca2+ release. Using this protocol,
IP3 activated both ICl1-S
("Ca2+ release") and ICl2
("Ca2+ influx"), but low concentrations of AdA often
activated only a tiny amount of ICl1-S, even
though ICl2 was robustly activated. But, because
we could not find a concentration of AdA which could activate
ICl2 without activating some small amount of
ICl1-S, we concluded that AdA did not activate
SOCE independently of Ca2+ release from stores. We
hypothesized that AdA activated relatively little
ICl1-S either because AdA released
Ca2+ from stores very slowly or that AdA released
Ca2+ from a subpopulation of stores which was tightly
coupled to SOCs.
The purpose of this paper was to examine further the mechanisms of AdA
regulation of Ca2+-activated Cl
currents
using confocal scanning microscopy of oocytes loaded with fluorescent
Ca2+ indicators and two-microelectrode voltage clamp. Here
we show that activation of SOCE following injection of low
concentrations of AdA depends upon depletion of intracellular
Ca2+ stores. However, at low AdA concentrations the
kinetics of Ca2+ release from stores was >7-fold slower
than that observed with IP3. This slower mode of
Ca2+ release is apparently not effective in activating
ICl1-S. Therefore, different kinetics of
Ca2+ release can differentially affect Cl
current activation.
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EXPERIMENTAL PROCEDURES |
Isolation of Xenopus Oocytes--
Stage V-VI oocytes were
harvested from adult albino or normal Xenopus laevis females
(Xenopus I) as described by Dascal (18). Xenopus
were anesthetized by immersion in Tricaine (1.5 g/liter). Ovarian
follicles were removed and digested in normal Ringer with no added
calcium, containing 2 mg/ml collagenase type IA (Sigma Chemical Co.,
St. Louis, MO), for 2 h at room temperature. The oocytes were
extensively rinsed with normal Ringer, placed in L-15 medium (Life
Technologies, Inc., Gaithersburg, MD) and stored at 18 °C. Oocytes
were usually used within 1-5 days after isolation.
Imaging and Electrophysiological Methods--
Xenopusoocytes
were injected with 9 nl Ca-green-1 coupled to 70kd dextran (333 µM) for a final calculated oocyte concentration of ~3
µM, and voltage-clamped with two-microelectrodes using a GeneClamp 500 (Axon Instruments, Foster City, CA). Electrodes were
filled with 3 M KCl and had resistances of 1-4M
.
Oocyte resting potentials were between
20 mV and
50 mV. Typically, the membrane was held at 0 mV and stepped to +40 mV for 1.5 s every 15 s to monitor ICl1-S. Every 2.25 min, a 1.5-s duration pulse to
140 mV followed by a 1.5-s duration
pulse to +40 mV was given to monitor ICl2 and
ICl1-T, respectively. Images (256 × 256 pixels) were acquired 500 ms after the onset of each voltage pulse
using a Zeiss LSM 410 confocal box fitted to a Zeiss Axiovert 100TV
inverted microscope using a Zeiss 10× objective (0.5 numerical aperature). The confocal aperture was set at the maximal opening, resulting in a focal section 1267 × 1267 × 35 µm. Image
data was analyzed using the LSM 410 software or NIH image 1.60 on a Mac IIfx. Current data was analyzed on a Pentium PC using Origin 5.0 (Microcal Software, Northampton, MA). For plots of Ca2+
fluorescence, the fluorescence intensity of the entire confocal section
was averaged and expressed as a ratio of the background fluorescence
taken before IP3 injection. Experiments were performed at
room temperature (22-26 °C). Normal Ringer solution contained 123 mM NaCl, 2.5 mM KCl, 1.8 mM
CaCl2, 1.8 mM MgCl2, 10 mM HEPES, pH 7.4; Ca2+-free Ringer solution was
the same except that CaCl2 was omitted and,
MgCl2 was increased to 5 mM.
Oocytes were injected with IP3 using a Nanoject automatic
oocyte injector (Drummond Scientific Co., Broomall, PA). The injection pipette was pulled from glass capillary tubing in a manner similar to
the recording electrodes and then broken so that it had a beveled tip
with an inside diameter <20 µm. Solutions of IP3 or AdA
were prepared in Chelex resin-treated H2O. The
Ca2+ concentration in this solution was not buffered, but
injection of H2O produced no change in Ca-green
fluorescence or membrane current. Levels of the IP3R were
lowered by injection of 60 ng of the IP3R antisense primer
(AACTAGACATCTTGTCTGACATTGCTGCAG) one day before the experiment as
described by Kume et al. (25). The reverse sense primer
(CTGCAGCAATGTCAGACAAGATGTCTAGTT) was injected at the same level as a control.
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RESULTS |
Ca2+ Transient and Cl
Currents Activated
by High Concentrations of IP3--
The protocol used to
measure Ca2+-activated Cl
currents in
Xenopus oocytes in response to IP3 or AdA
injection while simultaneously measuring cytosolic Ca2+
with confocal microscopy and Ca-green dextran is shown in Fig. 1. About 30 min after injection of
Ca-green dextran, the oocytes were voltage-clamped at 0 mV and stepped
to +40 mV every 15 s to monitor ICl1-S.
ICl1-S (current at the end of the +40 mV pulse, Fig. 1b) is an outward current at depolarizing potentials
that is activated quickly (~10 s) after IP3 injection by
Ca2+ released from intracellular stores (16, 24, 26). In
addition, once every 2.25 min, the oocyte was also stepped to
140 mV
to monitor ICl2 and then to +40 mV to monitor
ICl1-T. ICl2 (current at
the end of the
140 mV pulse, Fig. 1c) is an inward current that is activated by Ca2+ entry through SOCs driven by the
negative membrane potential. ICl1-T is a
transient outward current (peak outward current during the +40 mV
pulse, Fig. 1c) that was activated by a depolarizing pulse
preceded by a hyperpolarizing pulse to stimulate Ca2+
influx. The
140 mV pulse was given only once every 2.25 min to
minimize Ca2+ influx (and store refilling) during the
experiment. For a more detailed discussion of the Cl
currents see Hartzell and co-workers (24, 27, 28).

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Fig. 1.
Effect of large amounts of IP3 on
Cl currents and Ca2+ fluorescence in
Xenopus oocyte. The voltage protocol was designed
to minimize the amount of Ca2+ influx while still allowing
the visualization of the time-dependent activation of the
Cl channels after Ca2+ influx. The cell was
stepped to +40 mV for 1.5 s from a holding potential of 0 mV every
15 s for 9 consecutive episodes. In the 10th episode, the cell was
stepped to -140 mV then to +40 mV for 1.5 s each. Therefore,
every 10th pulse elicited ICl2 and
ICl1-T, whereas the intervening pulses elicited
ICl1-S. Cells were bathed in normal Ringer. The
oocyte was injected with Ca-green-1 coupled to 70-kDa dextran 30 min
before the experiment. The oocyte was voltage-clamped with two
microelectrodes, and 23 nl of 1 mM IP3 was
injected at the arrow. At the end of the experiment, the
oocyte was exposed to Ca2+-free Ringer containing 14 µM ionomycin (Ionom.) to assess the
Ca2+ content remaining in the stores. a, summary
of Cl current amplitudes before and after IP3
injection. Filled squares,
ICl1-S; open circles,
ICl2; and open triangles,
ICl1-T. b, current traces
corresponding to the +40 mV pulses labeled 1 and
2 in a. The voltage protocol used is shown at the
top. ICl1-S was measured as the outward current
at the end of the +40 mV pulse. c, current traces
corresponding to the 140 mV/+40 mV pulse combination labeled
3 and 4 in a. The voltage protocol
used is shown at the top. ICl2 was measured as
the inward current at the end of the 140 mV pulse.
ICl1-T was measured as the peak
time-dependent outward current during the +40 mV pulse
after the 140 mV pulse. d, Ca2+ fluorescence
measured simultaneously with the Cl currents.
Ca2+ levels were measured by Ca-green-1 fluorescence at +40
mV during the +40 mV pulses from the 0-mV holding potential
(FCa+40 (filled squares) and at -140 mV
(FCa-140 (open circles)) every 10th pulse.
Ca2+ fluorescence levels were measured from the entire
focal section and normalized to Ca2+-dependent
fluorescence before IP3 injection. At the end of the
experiment, the oocyte was exposed to 14 µM ionomycin in
calcium-free Ringer (Ionom./0 Ca2+) to
release any residual Ca2+ from stores (see Fig. 6). This
cell is representative of 13 cells.
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Fig. 1, a-c, shows the response of Cl
currents after injection of large amounts of IP3 (estimated
intra-oocyte concentration ~20 µM). When saturating
levels of IP3 were injected, ICl1-S
(filled squares) was activated immediately. As the stores
became depleted of Ca2+ and SOCE developed,
ICl1-T (open triangles) and
ICl2 (open circles) were activated.
Injection of IP3 caused a large increase in
Ca2+ fluorescence at all potentials (Fig. 1d)
because of Ca2+ release from stores. Before the peak
fluorescence was reached, the fluorescence was the same at all
potentials, but afterward the fluorescence during the
140 mV pulse
became greater than the fluorescence during the +40 mV pulse. The
difference between the fluorescence at
140 mV and +40 mV is the
voltage-dependent Ca2+ fluorescence, which we
have shown is related to Ca2+ entry through SOCs (28).
Ca2+ Waves Stimulated by AdA Are Very
Slow--
Injection of large amounts of AdA (estimated intra-oocyte
concentration ~2 µM; note that AdA is 10-100 times
more potent than IP3 (13)) produced rather similar effects
on the Cl
currents to those produced by IP3
(Fig. 2, a-c). There was a striking difference, however, in the kinetics of the Ca2+
fluorescence change produced by IP3 and by AdA. The
Ca2+ fluorescence did not begin to increase for several min
and peaked ~8 min after injection of AdA (Fig. 2d). By
comparison, after IP3 injection, the Ca2+
fluorescence peaked in less than 2 min (Fig. 1d).

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Fig. 2.
Effect of large amounts of AdA on
Cl currents and Ca2+ fluorescence in
Xenopus oocyte. The conditions were identical to
Fig. 1 except that 23 nl of 100 µM AdA was injected at
the arrow. a, summary of Cl current
amplitudes before and after AdA injection. Filled squares,
ICl1-S; open circles, ICl2; and
open triangles, ICl1-T . b, current
traces corresponding to the +40 mV pulses labeled 1 and
2 in a. c, current traces
corresponding to the 140 mV/+40 mV pulse combination labeled
3 and 4 in a. d,
Ca2+ fluorescence during the +40 mV pulses from the 0 mV
holding potential (FCa+40 (filled squares) and
at -140 mV (FCa-140 (open circles)) every 10th
pulse. At the end of the experiment the oocyte was exposed to 14 µM ionomycin in calcium-free Ringer (Ionom./0
Ca2+) to release any residual Ca2+
from stores (see Fig. 6). Note that ICl-1T in
this cell does not completely inactivate when the cell is switched to
Ca2+-free Ringer, because not all of the Ca2+
in the bath had been removed by the time the pulse that stimulated
ICl1-T occurred. Typically, when the cell is
switched to Ca2+-free Ringer after AdA injection,
ICl1-T and ICl2
completely inactivated as after IP3 injection (Fig.
1a). This cell is representative of six cells.
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It may seem surprising in Fig. 2 that the Ca2+ wave peaked
so much more slowly than ICl1-S. It should be
noted that the 1-mm diameter oocyte is on the stage of an inverted
microscope and that the injection takes place at the top, whereas the
confocal image plane is <30 µm from the bottom. Cl
currents, which are measured from the entire surface of the oocyte, increase as soon as Ca2+ is released from stores near the
injection site. However, the slow increase in Ca2+
fluorescence partly reflects the very slow transit time of the Ca2+ wave from the injection site to the confocal image
plane ~1 mm away. There is some variability in the lag period between
AdA injection and the increase in Ca2+ fluorescence. This
variability is most likely related to the depth and position of the
injection pipette in the oocyte.
The Ca2+ waves induced by injection of smaller amounts of
AdA moved even more slowly. In Fig.
3a, typical traces of
Ca2+ fluorescence at +40 mV in response to injection of
large amounts of IP3 (~20 µM, filled
squares), large amounts of AdA (~2 µM, open
circles), and small amounts of AdA (~5 nM,
open triangles) are superimposed. In the case of low
concentrations of adenophostin, the time-to-peak of the
Ca2+ fluorescence was ~20 min. Fig. 3b shows
averages of the time-to-peak of the Ca2+ fluorescence to
these injections. The time-to-peak for large concentrations of AdA was
>2 times slower than for large concentrations of IP3, and
the time-to-peak for small concentrations of AdA was >7 times slower
than for large concentrations of IP3. It was not possible
to measure the time-to-peak for small IP3 concentrations because small IP3 concentrations produced oscillating
Ca2+ waves that exhibited no clear peak. The slowness of
the Ca2+ wave is illustrated in a different way in the
images in Fig. 3c. After injection of AdA, the spread of the
Ca2+ fluorescence is very slow relative to the spread of
the IP3-induced wave of Ca2+ release.

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Fig. 3.
Comparison of the velocity of
Ca2+ waves in response to IP3 and AdA
injection. a, Ca2+ fluorescence at +40 mV
in response to injection of 23 nl of 1 mM IP3
(filled circles), 23 nl of 100 µM AdA
(open circles), and 10 nl of 0.5 µM AdA
(open triangles). b, time-to-peak of
Ca2+ waves measured from time of injection of 23 nl of 1 mM IP3 (High IP3,
n = 6), 23 nl of 100 µM AdA (High
AdA, n = 5), and 10 nl of 0.5 µM AdA
(Low AdA, n = 7). The speed of the
Ca2+ release wave after IP3 or AdA injection
was estimated by calculating the time required for the
Ca2+-dependent fluorescence to reach its
maximal value. It was not possible to perform the same analysis on
cells injected with low levels of IP3, because in many
instances, such IP3 injections lead to repetitive
Ca2+ waves that oscillate and not a single wave that sweeps
through the entire oocytes as observed when the oocyte is injected with
high IP3 levels or AdA. The speed of the wave was
significantly slower (p < 0.006) between high
IP3 and AdA injections and between high and low AdA
injections. c, images of Ca-green-1 fluorescence in response
to injection of 23 nl of 1 mM IP3 (High
IP3) and 10 nl of 0.5 µM AdA (Low
AdA). The times in min at which the confocal images were taken are
indicated in the top left corner of each image. IP3 or AdA
were injected at time 0.
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These data confirm our earlier suggestion that AdA causes release of
Ca2+ from stores much more slowly than IP3
does. These findings support the idea that small concentrations of AdA
do not stimulate ICl1-S because slow release of
Ca2+ from stores does not elevate Ca2+ in the
vicinity of the Cl
channels to an activating level. This
could occur if efflux and/or local Ca2+ buffering removes
free Ca2+ as rapidly as it is released, so that an
effective Ca2+ concentration is not attained.
Small Concentrations of AdA Completely Deplete Ca2+
Stores--
Although Fig. 3 shows that low concentrations of AdA
release Ca2+ from stores, the question remains whether the
stimulation of Ca2+ entry by low concentrations of AdA is
because of depletion of stores. For example, the AdA-stimulated
Ca2+ release might be so slow that the stores refill. To
examine this question, we measured the effects of low concentrations of
AdA (~5 nM) that did not activate
ICl1-S on Ca2+ store depletion. Fig.
4 shows the results of a typical
experiment. Injection of 10 nl of 0.5 µM AdA did not
detectably stimulate ICl1-S (Fig. 4,
a-c), but both ICl1-T and
ICl2 developed robustly. ICl1-T and ICl2 were
dependent on extracellular Ca2+, and their activation
corresponded to the activation of SOCE (16). Ca2+
fluorescence began to increase about 5 min after AdA injection and
continued to increase for 20 min (Fig. 4d).
Voltage-dependent Ca2+ fluorescence (open
circles), which reflects SOCE, developed shortly after
Ca2+ release and remained at a high level for the duration
of the experiment. To test whether stores were depleted of
Ca2+, ionomycin in Ca2+-free Ringer was applied
to release Ca2+ from any remaining stores. Ionomycin had
only a very small effect on ICl1-S and had no
effect on the Ca2+ fluorescence at +40 mV. This showed that
the stores had been virtually completely depleted of Ca2+
by AdA.

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Fig. 4.
Effect of small amount of AdA on
Cl currents and Ca2+ fluorescence in
Xenopus oocyte. The conditions were identical to
Fig. 1 except that 10 nl of 0.5 µM AdA was injected at
the arrow. a, summary of Cl current
amplitudes before and after AdA injection. Filled squares,
ICl1-S; open circles,
ICl2; and open triangles,
ICl1-T. b, current traces
corresponding to the +40 mV pulses labeled 1 and
2 in a. c, current traces
corresponding to the 140 mV/+40 mV pulse combination labeled
3 and 4 in a and d.
Ca2+ fluorescence during the +40 mV pulses from the 0-mV
holding potential (FCa+40 (filled squares)) and
at -140 mV (FCa-140 (open circles)) every 10th
pulse. At the end of the experiment, the oocyte was exposed to 14 µM ionomycin in calcium-free Ringer (Ionom./0
Ca2+) to release any residual Ca2+ from
stores (see Fig. 6). This cell is representative of eight cells.
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This result contrasts to that observed when small amounts of
IP3 were injected (Fig. 5).
Concentrations of IP3 that stimulated Ca2+
influx, as determined by the presence of voltage-dependent
Ca2+ fluorescence and activation of
ICl1-T and ICl2,
inevitably stimulated ICl1-S. In some cells, as
in Fig. 5, the increase in ICl1-S was not
accompanied by a significant increase in Ca2+ fluorescence,
because the IP3 effect was local and did not propagate into
the region of the oocyte that was imaged. Both
voltage-dependent Ca2+ fluorescence and
ICl1-T and ICl2
eventually declined to base line. Application of ionomycin at the end
of the experiment evoked a large increase in Ca2+
fluorescence and in ICl1-S, showing that the
stores were not completely depleted of Ca2+.

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Fig. 5.
Effect of small amounts of IP3 on
Cl currents and Ca2+ fluorescence in
Xenopus oocyte. The conditions were identical to
Fig. 1 except that 10 nl of 10 µM IP3 was
injected at the arrow. a, summary of Cl
current amplitudes before and after IP3 injection.
Filled squares, ICl1-S; open
circles, ICl2; and open
triangles, ICl1-T. b, current
traces corresponding to the +40 mV pulses labeled 1,
2, and 5 in a. c, current traces
corresponding to the 140 mV/+40 mV pulse combination labeled
3 and 4 in a. d,
Ca2+ fluorescence during the +40 mV pulses from the 0-mV
holding potential (FCa+40 (filled squares)) and
at -140 mV (FCa-140 (open circles)) every 10th
pulse. At the end of the experiment, the oocyte was exposed to 14 µM ionomycin in calcium-free Ringer (Ionom./0
Ca2+) to release any residual Ca2+ from stores
(see Fig. 6). This cell is representative of 14 cells.
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To obtain a more quantitative measure of the extent of store depletion
after injection of IP3 or AdA, we calculated the ratio of
ionomycin-induced Ca2+ release to IP3- or
AdA-induced Ca2+ release. This ratio gives a measure of the
level of residual Ca2+ in intracellular stores after
IP3R agonist injection. The results from these experiments
are shown in Fig. 6. Injections of high IP3, high AdA, or low AdA all left the stores largely
depleted of Ca2+. In contrast, low IP3
concentrations were less effective in depleting the stores. These data
show that concentrations of AdA that did not noticeably activate
ICl1-S were capable of depleting intracellular Ca2+ stores to similar levels as high concentrations of
IP3.

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Fig. 6.
The extent of store depletion after
IP3 or AdA injection. The relative Ca2+
content remaining in the internal stores after injection of
IP3 or AdA was assessed in Figs. 1, 2, 4, and 5 by exposure
of the oocyte to 14 µM ionomycin. The relative amount of
Ca2+ remaining in the store was expressed as the ratio of
the peak amplitude of the Ca2+ fluorescence at +40 mV
produced by ionomycin exposure to the peak Ca2+
fluorescence at +40 mV produced by IP3 or AdA injection.
Hi IP3, 23 nl of 1 mM IP3;
Hi AdA, 23 nl of 10 µM AdA; Lo
IP3, 10 nl of 10 µM IP3;
Lo AdA, 10 nl of 0.5 µM AdA. The
bars show mean ratio ±S.E. The number of cells used for
this analysis is indicated on top of each bar. The level of
Ca2+ in intracellular stores after high IP3,
high AdA, or low AdA treatments was not significantly different
(p > 0.32). However, all three treatments were
significantly different (p < 0.025) than injection of
low concentrations of IP3.
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Thus, we conclude that AdA stimulates SOCE as a consequence of
depletion of internal Ca2+ stores and not by some direct
effect on SOCs. Furthermore, previous conclusions, based on
Ca2+-activated Cl
current activation, which
suggested that low concentrations of AdA stimulate SOCE without
releasing Ca2+ from stores (17), can be explained by the
observation that slow release of Ca2+ from stores is often
insufficient to activate ICl1-S.
Effect of AdA on SOCE Requires Active IP3R--
If
this conclusion is correct, the effects of AdA on SOCE should depend on
the ability of the IP3R to release Ca2+. Thus,
treatments that suppress IP3R function should inhibit the
effects of AdA injections. We suppressed IP3R function
either by injecting the competitive inhibitor heparin (Fig.
7) or by reducing IP3R
expression by injection of antisense oligonucleotides to the
Xenopus IP3R (Fig.
8).

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Fig. 7.
Heparin blocks both IP3-
and AdA-dependent store depletion.
a-b,cells were injected with 10 nl of 0.5 µM
AdA alone (a; n = 5) or preinjected with 92 nl of 100 mg/ml heparin before AdA injection (b;
n = 6). c-d, cells were injected with 10 nl
of 50 µM IP3 alone (c;
n = 5) or preinjected with 92 nl of 100 mg/ml heparin
before IP3 injection (d; n = 3).
The site of injection is indicated by the arrow.
Cl currents are measured as described in Fig. 1.
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Fig. 8.
Lowering IP3R levels blocks AdA
and IP3 induced SOCE. Oocytes were injected
with 60 ng of sense (a and c) or antisense
(b and d) IP3R oligonucleotides (25)
and incubated for 1-2 days. Oocytes were injected with 10 nl of 0.5 µM AdA (a and b) or with 23 nl of
10 µM IP3 (c and d) as
indicated by the arrow in each panel.
Filled squares, ICl1-S; open
circles, ICl2; and open
triangles, ICl1-T. n = 6-7
oocytes/panel.
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Injection of heparin to block the IP3R significantly
reduced ICl2 and ICl1-T
currents induced by small AdA injections (Fig. 7, a-b). In
a similar fashion, heparin blocked the Cl
current
response induced by IP3 (Fig. 7, c-d). Reducing
IP3R levels by antisense oligonucleotides as described
previously by others (25, 29) also reduced the effects of
IP3 and AdA treatments on ICl1-T and
ICl2 (Fig. 8). The effects of antisense
treatment were less pronounced than the effects of heparin, but it was
clear that antisense had a significant effect. Note that although
antisense treatment inhibited ICl2 and
ICl1-T in response to IP3 injection, there was no decrease in levels of ICl1-S (Fig.
8d). Actually, ICl1-S was slightly
potentiated as compared with sense-injected cells (Fig. 8c).
This observation could be explained if one assumes there are two
distinct subpopulations of IP3 receptors with differential turnover rates of the IP3R. If we postulate the existence
of subsets of stores, one close to the ICl1-S
Cl
channels containing IP3Rs with a very slow
turnover rate and a second located further from the Cl
channels containing an IP3R population that turns over
rapidly, then injection of antisense IP3 oligonucleotides
will reduce the levels of IP3Rs in the latter subset
faster, resulting in sufficient Ca2+ release after
IP3 injection to activate ICl1-S but
insufficient release from most of the stores to induce significant SOCE.
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DISCUSSION |
In many cell types, release of Ca2+ from endoplasmic
reticulum stores stimulates Ca2+ influx into the cytosol
from the extracellular space through SOCs by a process termed SOCE. The
mechanisms by which release of Ca2+ from stores stimulates
SOCE is unknown, but one hypothesis states that the endoplasmic
reticulum releases a diffusible chemical messenger that opens SOCs. The
search for such a calcium influx factor has so far not been very
fruitful, and the putative calcium influx factors that have been
discovered have not found universal acceptance (9, 10). When it was
suggested that AdA could stimulate Ca2+ influx without
stimulating Ca2+ release from stores (17), some hope was
raised that clues to the structure of calcium influx factors would be
learned from AdA. The suggestion that AdA could stimulate
Ca2+ entry without depleting Ca2+ from stores
was based on the observation that low concentrations of AdA did not
stimulate Ca2+-activated Cl
currents in the
absence of extracellular Ca2+ and therefore did not release
Ca2+ from stores but did stimulate
Ca2+-activated Cl
currents in the presence of
Ca2+ influx. The present studies using Ca2+
imaging demonstrate, however, that even very low amounts of AdA (calculated oocyte concentration ~5 nM) released
Ca2+ from stores. Under these conditions, even though
ICl1-S was not activated, the stores were
completely depleted of Ca2+ as demonstrated by the
inability of ionomycin to increase Ca2+ fluorescence. We
believe that the Ca2+ released from stores by low
concentrations of AdA is unable to activate
ICl1-S because of its significantly slower rate
of Ca2+ release (~7 times slower than
IP3).
How do the kinetics of Ca2+ release from stores determine
the response of the Cl
channels? It has been suggested
that ICl1-S responds to the rate-of-change of
cytosolic Ca2+ (20) because the peak activation of
ICl1-S corresponds to the maximum rate of change
of cytosolic Ca2+ and because the amplitude of
ICl1-S does not correlate with the steady-state
levels of cytosolic Ca2+. However, we have shown (27) that
the turn-off of ICl1-S is not explained by
inactivation of the current as previously suggested (20). Furthermore,
we have found that the Ca2+ concentration measured by
cytosolic Ca2+ dyes (such as Ca2+-green
dextran) does not reflect the concentration of Ca2+ just
below the plasma membrane (measured by lipophilic Ca2+ dyes
such as Ca-green C18) (28). We have presented evidence that the
subplasmalemmal Ca2+ concentration changes much more
quickly than does the Ca2+ concentration deeper in the
cytosol because plasma membrane Ca2+ efflux systems can
rapidly clear Ca2+ from the subplasmalemmal space.
Consequently, we would predict that the subplasmalemmal
Ca2+ concentration would depend on the relative rates of
Ca2+ release from stores and cytosolic Ca2+
buffering and Ca2+ efflux from the oocyte. If
Ca2+ release is slow, the concentration of Ca2+
in the subplasmalemmal space may not rise sufficiently to activate Ca2+-activated Cl
channels.
The different kinetics of Ca2+ release produced by AdA and
IP3 are probably related to differences between AdA and
IP3 activation of IP3Rs. First, the apparent
diffusion coefficient of AdA or IP3 in the cytosol will
depend on the fraction of molecules (
) that are bound to the
IP3R at any one time (Dobs = D/
). Because AdA has a 100-fold higher affinity for the
IP3R than IP3 does, AdA diffusion will be
slower because a larger fraction of the total AdA (compared with
IP3) will be bound to IP3Rs. Second, AdA
exhibits a higher cooperativity in activating IP3Rs than
IP3 does. Hirota et al. 1995 (13) have shown
that IP3 has a Hill coefficient of 1.8 for Ca2+
release by the type 1 IP3R, whereas the Hill coefficient
for AdA was 3.9. This implies that at least 2 molecules of
IP3 and 4 molecules of AdA are needed to open an
IP3R. This factor will also contribute to the slow movement
of the Ca2+ release wave in response to small amounts of
AdA. Accordingly, the elementary Ca2+ release events
("Ca2+ puffs") induced by AdA have been shown by
Marchant and Parker (30) to be smaller and faster than those induced by
IP3. Because Ca2+ waves are initiated by the
summation of Ca2+ puffs, the smaller and faster puffs
induced by AdA may contribute to the slower propagation of the AdA
wave. However, the mechanisms by which AdA releases Ca2+
from stores remains to be fully elucidated.
Although low concentrations of AdA evoke a slow release of
Ca2+ from stores and little or no
ICl1-S, high concentrations release Ca2+ only about 2-fold more slowly than IP3 and
also evoke significant ICl1-S. This finding that
AdA activates different ICl1-S responses depending upon the kinetics of Ca2+ release from
intracellular stores is interesting because it provides another example
of how the temporal features of a Ca2+ signal contribute to
its physiological consequences. Different receptors can induce
different Ca2+ release kinetics depending on factors
including spatial localization of the receptor and/or
IP3-sensitive stores or the activation of different PLC
isoforms (31-36). These different release kinetics can then play an
important role in determining which effectors are activated.