Regulation of KCa
current by store-operated Ca2+
influx depends on internal Ca2+
release in HSG cells
Xibao
Liu1,
Eduardo
Rojas2, and
Indu S.
Ambudkar1
1 Secretory Physiology Section,
Gene Therapy and Therapeutics Branch, National Institute of Dental
Research, and 2 Laboratory of Cell
Biology and Biochemistry, National Institute of Diabetes and Digestive
and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
20892
 |
ABSTRACT |
This study examines the
Ca2+ influx-dependent regulation
of the Ca2+-activated
K+ channel
(KCa) in human submandibular
gland (HSG) cells. Carbachol (CCh) induced sustained increases in the
KCa current and cytosolic Ca2+ concentration
([Ca2+]i),
which were prevented by loading cells with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellular
Ca2+ and addition of
La3+ or
Gd3+, but not
Zn2+, inhibited the increases in
KCa current and
[Ca2+]i.
Ca2+ influx during refill (i.e.,
addition of Ca2+ to cells treated
with CCh and then atropine in
Ca2+-free medium) failed to evoke
increases in the KCa current but achieved internal Ca2+ store
refill. When refill was prevented by thapsigargin,
Ca2+ readdition induced rapid
activation of KCa. These data
provide further evidence that intracellular
Ca2+ accumulation provides tight
buffering of
[Ca2+]i
at the site of Ca2+ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggest
that the Ca2+ influx-dependent
regulation of the sustained KCa
current in CCh-stimulated HSG cells is mediated by the uptake of
Ca2+ into the internal
Ca2+ store and release via the
inositol 1,4,5-trisphosphate-sensitive channel.
calcium-activated potassium channel; store-operated calcium influx; salivary gland cells; muscarinic receptor
 |
INTRODUCTION |
INTRACELLULAR CALCIUM mobilization plays a central role
in coupling the activation of muscarinic receptors with the regulation of cellular function in a variety of cells, including exocrine gland
cells (2, 6, 7, 28-30). In exocrine gland cells, such as those
from salivary glands, muscarinic receptor stimulation leads to a
biphasic change in cytosolic Ca2+
concentration
([Ca2+]i),
with an initial rapid transient increase due to internal Ca2+ release and a lower more
sustained increase primarily due to Ca2+ influx (1, 10, 24, 26,
28-32, 34, 35). The increase in
[Ca2+]i
results in the activation of various ion channels; these include K+ and
Cl
channels, some
nonselective cation channels, and ion transporters such as the
Na+-K+-Cl
cotransporter (7, 14, 23, 28-30). These studies have
suggested that, although a transient activation of these ion channels
can be achieved by internal Ca2+
release, their sustained activation is dependent on
Ca2+ influx from the extracellular
medium. Ca2+ influx in exocrine
gland cells is primarily mediated via a store-operated Ca2+ influx pathway (24, 26, 31,
34) believed to be localized in the basolateral plasma membrane of
these cells (10, 22, 25, 26). Other
Ca2+ influx pathways might also
exist such as receptor-operated pathways (20) or nonspecific cation
channels (7, 28, 29).
Ca2+-activated
K+ channels
(KCa) have also been proposed to
be localized in the basolateral plasma membrane of exocrine gland
cells, and, because of the tight regulation by
[Ca2+]i,
the KCa activity has been used to
monitor the changes in
[Ca2+]i
in the subplasma membrane region (7, 10, 12, 14, 23, 28, 29).
The HSG cell line is a cloned cell line from the human submandibular
gland and has been widely used as a model to study receptor-mediated signaling and salivary gland pathology (15, 20, 27, 35). A numbers of
ion channels have been found in HSG cells, including a hypotonically
activated Cl
channel (17),
an outwardly rectifying Cl
channel (9), and a KCa (18). It
was suggested in an earlier report that activation of the muscarinic
receptor causes an increase in
[Ca2+]i
that in turn activates a KCa (18).
The channel was identified to be either of the large (BK) or
intermediate (IK) conductance type on the basis of its sensitivity to
charybdotoxin (ChTX) and quinine but relative insensitivity to
tetraethylammonium and apamin. Furthermore, simultaneous measurements
of intracellular Ca2+ and
K+ current demonstrated that
agonist-induced K+ current was
very tightly correlated with changes in
[Ca2+]i
in HSG cells (18). In general, these characteristics are largely
similar to those of K+ channels in
a variety of exocrine gland cells such as salivary and lachrymal, but
not rodent pancreatic, acinar cells. In addition, HSG cells also have
muscarinic receptor-stimulated
Ca2+ signaling mechanisms similar
to those seen in exocrine acinar cells. Stimulation of these cells with
the muscarinic agonist carbachol (CCh) induces a biphasic increase in
[Ca2+]i,
which is dependent on inositol 1,4,5-trisphosphate
(IP3)-induced intracellular
Ca2+ release and
Ca2+ influx. It was previously
reported that HSG cells have two types of
Ca2+ influx pathways: a large
component that is dependent on internal Ca2+ store depletion, i.e.,
store-operated Ca2+ influx, and a
relatively minor component that is dependent on muscarinic receptor
activation, likely via a G protein (20). Our studies showed that
CCh-stimulated
[Ca2+]i
elevation in thapsigargin (TG)-treated cells, i.e., via
store-independent Ca2+ influx
pathway, did not induce further hyperpolarization of HSG cells, i.e.,
via activation of the K+ channel.
On the basis of these data, we suggested that the sustained hyperpolarization in CCh-stimulated HSG cells is primarily regulated by
the store-operated Ca2+ influx
pathway.
In this study, we have examined the role of intracellular
Ca2+ release and store-operated
Ca2+ influx in the regulation of
the K+ channel in HSG cells by
CCh. By using the standard patch-clamp whole cell technique, we show
that the channel activation is dependent on CCh-stimulated
intracellular Ca2+ release, via
IP3-sensitive channels, and that
its sustained activation is determined by
Ca2+ influx, via the
store-operated Ca2+ influx
pathway. Importantly, we have examined
KCa activity during the
Ca2+ influx that occurs during
reloading of internal Ca2+ stores,
i.e., in the absence of internal
Ca2+ release. The results show
that Ca2+ influx alone cannot
support activation of the KCa
because of the rapid buffering of
[Ca2+]i
in the subplasma membrane region by the activity of the intracellular Ca2+ pump. Thus we suggest that
the Ca2+ influx-dependent
modulation of KCa activity in
CCh-stimulated HSG cells is not directly due to an elevation of
[Ca2+]i
at the site of Ca2+ influx but
rather is mediated via uptake of
Ca2+ into the intracellular
Ca2+ store and
IP3-dependent release.
 |
METHODS |
Cell culture.
HSG cells were a gift from Dr. Mitsunobu Sato of the Second Department
of Oral and Maxillofacial Surgery, Tokushima University, Tokushima,
Japan. Cells were grown in Eagle's minimum essential medium with
Earle's balanced salt solution (Biofluids, Rockville, MD) with 5%
CO2 in air at 37°C in the
presence of 10% fetal calf serum, 2 mM
L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin (all from Biofluids). Cells
were fed three times a week and passaged when confluent. Cells were
passaged by detaching them from the tissue culture dish with 0.25%
trypsin-1.0 mM EDTA (Biofluids). A single cell suspension was reseeded
on coverslips, kept in a 35-mm culture dish (Corning), and cultured for
24 h before use.
Patch-clamp experiments.
The coverslips were cut to ~0.5 × 0.5 mm and placed
in a perfusion chamber (Warner Instrument, Hamden, CT). The perfusion rate, ~5 ml/min, was achieved by gravity-fed plastic tubes in a bath
solution that was continuously and simultaneously removed through a
vacuum line. Complete solution changes were obtained within 15 s. The
standard extracellular solution contained (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 0.1 EGTA, and 5 HEPES, pH 7.4. The pipette was filled with (in mM) 150 KCl, 2 MgCl2, 1 ATP, and 5 HEPES, pH 7.2. In some experiments, 150 mM KCl was replaced with 150 mM CsCl, and, in
others, either 10 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA) or 10-100 µM
IP3 was included in the pipette
solution.
Patch clamp in a whole cell configuration was performed at room
temperature on single HSG cells attached to coverslips using the
standard patch-clamp technique (13). Patch electrodes were made from
1.0-mm borosilicate glass tubing with filament (BF-100-50-10, Sutter Instrument, Novato, CA). The resistance of the pipette was
typically between 3 and 6 M
when filled. The chamber was grounded
with an Ag-AgCl pellet through a 150 mM NaCl-containing agar bridge.
Cell membrane and pipette capacitative transients were subtracted from
the records by the amplifier circuitry before sampling. Voltages were
not compensated for liquid junction potentials. Membrane currents were
measured with an Axopatch 200A amplifier in conjunction with pCLAMP 6.1 software and a Digidata 1200 analog-to-digital converter (Axon
Instruments, Foster City, CA). Whole cell
K+ currents were filtered at 2 kHz
(low-pass Bessel filter), sampled with an interval of 10 ms in a
gap-free mode, and recorded directly onto the hard drive of a Dell
Pentium computer from a holding potential of 0 mV, the
Cl
equilibrium potential,
for analysis. Digitized data were analyzed with the use of using pCLAMP
6.1 and Origin 4.1 (Microcal Software, Northampton, MA). In some
experiments, a holding potential of
85 mV, the
K+ equilibrium potential, was used
to test whether there was a CCh-induced inward current. In the
current-voltage
(I-V)
relationship experiments, the membrane potential was changed from
120 to +80 mV in a 20-mV step by generating square pulses of
2.56-s duration from a holding potential of
35 mV in a Clampex
module.
I-V
relationships were obtained from 10 µM CCh-induced peak currents. The
mean K+ current (total integrated
current induced by agonist application/total time of application) and
the amplitude of the current were measured using the Fetchan module.
The
I-V
relationship was calculated using the Clampfit module and exported to
the Origin 4.1 for further analysis.
Ca2+
measurements.
The fluorometric system used for intracellular
Ca2+ measurement using indo 1 (Molecular Probes, Eugene, OR) has been described previously (19).
Briefly, a single indo 1-loaded HSG cell was excited at 355 nm. The
fluorescence emissions at 410 and 485 nm (F410 and
F485, respectively) were measured
simultaneously using two photomultipliers. The output from each
photomultiplier was digitized at 2 Hz.
[Ca2+]i
was calculated with the use of the
F410 / F485
emission ratio by a custom-designed program using a calibration curve
based on different Ca2+ buffer
solutions. The iris diaphragm was set to a small field immediately
covering a single HSG cell so that the fluorescence was recorded from
one cell only.
All chemicals were obtained from Sigma Chemical (St. Louis, MO) except
tert-butylhydroxyquinone (BHQ),
apamin, ChTX, and IP3, which were
purchased from Calbiochem (La Jolla, CA), and BAPTA, which was obtained
from Molecular Probes. Data were statistically evaluated by using the
Student's t-test (two groups) or
ANOVA test (more than two groups). Data points (means ± SE) are
averages of the indicated number of experiments
(n).
 |
RESULTS |
CCh stimulation of KCa in HSG cells.
Stimulation of HSG cells with CCh induced an increase in the outward
current at a holding potential of 0 mV, the
Cl
equilibrium potential
(Fig.
1A),
in 94% of the cells. Oscillatory increases were observed at lower
concentrations of CCh (1-10 µM), whereas steady-state increases
in the current were obtained with higher concentrations (>100 µM).
Although the initial amplitude of the current in the same cell was
similar at all agonist concentrations (see Fig.
1A), the mean current increased
significantly with increasing CCh concentration, from 322 ± 123 pA
at 1 µM and 663 ± 175 pA at 10 µM to 1,161 ± 299 pA at 100 µM CCh (P < 0.05, n = 5). A concentration of 1 µM CCh
typically evoked baseline-separated oscillations with a mean frequency
of 3.7 ± 1.7 per minute (n = 5),
whereas 10 µM CCh induced either similar, fast baseline-separated oscillations (mean frequency of 6.6 ± 2.1 per minute,
n = 6; seen in one-half of the cells
tested) or slower oscillations, which were superimposed on a sustained
elevation of the current (as shown in Fig.
1A). Higher concentrations of CCh
(100 µM to 1.0 mM) consistently induced a fast transient increase in
the outward current that was followed by lower steady-state current
(Fig. 1A). Figure
1B shows outward currents in a cell
that was stimulated repeatedly by 100 µM CCh; the interval between
stimulations was ~3 min. The differences in the mean amplitudes of
the first three stimulations are not significant
(P > 0.05, n = 4). The amplitudes of second and
third responses are 88.7 ± 8.1% and 71.6 ± 14.5% (n = 4), respectively, of the first
response. These data demonstrate that no rapid desensitization or
inactivation of the K+ channel
response occurs with repeated short exposure to CCh.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Carbachol (CCh)-induced outward currents.
A: concentration-dependent response of
CCh-induced Ca2+-activated
K+ channel
(KCa) current in human
submandibular gland (HSG) cells at a holding potential of 0 mV. CCh and
all other agents were continuously applied to the bath (indicated by
bars) at a rate of ~5 ml/min. This is a representative trace of
results obtained with >20 cells. B:
sequential stimulation of HSG cell. CCh (100 µM) was applied
repeatedly for 30 s (shown by bars), with an interval of ~3
min between applications in which the cell was washed by bath solution.
This is a representative trace of data from 7 different cells.
C:
1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic
acid (BAPTA), directly introduced into the cell via patch pipette, and
CCh (1-100 µM) were applied to the cell (indicated by
bars).
|
|
Previous reports demonstrated the presence of
KCa in HSG cells (18). We carried
out some initial studies to confirm these previous findings under our
experimental conditions, and the data are summarized here. The effects
of Ba2+ and
Cs+, potent inhibitors of
K+ channels, were examined.
Replacement of intracellular K+ by
Cs+ or internal administration of
Ba2+ (2 mM)
(n = 5, data not shown) completely
eliminated the CCh-induced responses. Furthermore, BAPTA (10 mM), a
Ca2+ chelator, was directly
introduced into the cytosol through the patch pipette. This treatment
completely abolished CCh-induced KCa at all concentrations of CCh
tested, from 1 to 100 µM (Fig. 1C,
compare with data in Fig. 1A).
Loading HSG cells with BAPTA also inhibited CCh-induced
[Ca2+]i
elevation (data not shown).
In addition, the
I-V
relationship was measured in unstimulated control HSG cells (Fig.
2A) and
CCh (10 µM)-stimulated cells (Fig.
2B). As shown in Fig.
2C, the current increased almost
linearly between
120 and 0 mV and reached a maximum between 0 and 20 mV. The reversal potential of the current was about
80
mV, which is close to the K+
equilibrium potential (
85 mV). When the holding potential was greater than +20 mV, the K+
currents became smaller, which is likely due to a decrease in the
driving force for Ca2+ influx
across the plasma membrane. Furthermore, consistent with the previous
report by Izutsu et al. (18), ChTX (50 nM), a large-conductance Ca2+-dependent
K+ channel inhibitor,
significantly reduced CCh-induced
K+ current to 12.9 ± 8.4%
(P < 0.05, n = 5), which was partially restored
to 45.7 ± 13.9% of the control when ChTX was removed (data not
shown). On the other hand, apamin, a small-conductance Ca2+-dependent
K+ channel inhibitor, did not have
any significant effect on CCh-induced outward current. These data
demonstrate that the CCh-induced outward current in HSG cells
maintained at 0 mV is mainly carried by
K+ via a ChTX-sensitive
Ca2+-dependent
K+ channel.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Current-voltage
(I-V)
relationship of CCh-induced peak
KCa.
A: control currents were recorded at
voltages of 120 to +80 mV with 20-mV steps from a holding
potential of 35 mV before application of CCh.
B: 10 µM CCh-induced peak current
was recorded under same conditions as control recording.
C:
I-V
relationship (in pA and mV, respectively) of peak current induced by 10 µM CCh (n = 6).
|
|
IP3- and TG-dependent stimulation of
KCa in HSG cells.
CCh-stimulated intracellular Ca2+
mobilization is mediated via increases in intracellular
IP3 and
IP3-induced release of
Ca2+ from internal
Ca2+ stores (1, 2, 15, 32). Thus
further experiments were carried out to test whether the
IP3-induced
Ca2+ release pathway is involved
in the CCh stimulation of KCa.
IP3, directly applied to HSG cells
via the patch pipette, typically caused oscillatory increases in
KCa at low concentrations of
IP3 (e.g., 10 µM, Fig.
3A) with
a frequency of 4.8 ± 1.6 oscillations/min (n = 5). At higher
IP3 concentrations (e.g., 100 µM), a relatively sustained increase in the current was induced that
appeared to be superimposed on an oscillatory current and ran down
within 2-3 min (Fig. 3B). The
mean current increased significantly with increasing
IP3 concentrations [from 662 ± 258 pA at 10 µM (n = 5) to
1,379 ± 424 pA at 100 µM
(P < 0.01, n = 9)]. It must be noted that
the responses induced by the dialysis of
IP3 were not as stable as that
induced by CCh, and 2 of 18 cells tested did not respond to
IP3 stimulation. However, the
pattern of currents induced by increasing concentrations of
IP3 was similar to that induced by
increasing concentrations of CCh, i.e., oscillations at relatively
lower concentrations and relatively sustained increases in the current
at higher concentrations. Importantly, addition of CCh during the
IP3-mediated response did not
alter the IP3-induced current
(data not shown) and addition of CCh after rundown of the
IP3-stimulated
KCa oscillations induced either a
very attenuated response (Fig. 3B)
or no response at all.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Inositol 1,4,5-trisphosphate
(IP3)-induced activation of
KCa in HSG cells.
A:
IP3 (10 µM) was applied to cell
at a holding potential of 0 mV through the patch pipette. Oscillations
of KCa were recorded in continuous
presence of IP3 (dashed line) and
were seen as soon as whole cell configuration was established. This is
a representative trace of data obtained from 5 different cells.
B:
IP3 (100 µM) evoked sustained
increases in KCa. Addition of CCh
in the presence of IP3 is shown by
bar. This is a representative trace of data from 11 cells.
|
|
We have previously reported that CCh stimulation of HSG cells induces a
Ca2+ influx that is dependent on
the depletion of internal Ca2+
stores, i.e., store-operated Ca2+
influx (20). To determine whether the store-dependent
Ca2+ influx regulates
KCa in HSG cells, cells were
treated with TG, an irreversible
Ca2+-ATPase inhibitor that
depletes intracellular Ca2+ stores
by inhibiting the Ca2+ pump, thus
activating the store-operated Ca2+
influx pathway (31). TG (1-10 µM) induced a biphasic increase in
K+ currents and attenuated a
subsequent response to CCh (Fig.
4A). BHQ, a reversible Ca2+-ATPase
inhibitor, produced effects similar to TG; however, in this case, the
response could be recovered by washing off BHQ from the cells
(n = 7, data not shown). These data
suggest that CCh, IP3, and TG (or
BHQ) stimulate KCa via internal
Ca2+ store depletion. Figure
4B shows that TG treatment induced a transient increase in the KCa
current in cells perfused with a Ca2+-free medium. However, the
current was rapidly increased when Ca2+ was reintroduced into the
medium. These data clearly show that the store-operated
Ca2+ influx pathway can regulate
KCa.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Activation of KCa by thagsigargin
(TG) in HSG cells. A: cell exposed to
TG (10 µM, indicated by dashed line) after a control response to CCh
(100 µM, indicated by first solid bar) was recorded. Response to CCh
in the presence of TG was also recorded (indicated by second solid
bar). B: cells were treated with TG
(10 µM, dashed line) in a
Ca2+-free medium (solid line),
following which Ca2+ was
reintroduced into the medium. Traces are representative of data from 10 cells.
|
|
Effect of extracellular
Ca2+ on the
regulation of the KCa current in
CCh-stimulated HSG cells.
As discussed above, activation of the muscarinic receptor in HSG cells
induces a biphasic increase in
[Ca2+]i:
an initial rapid transient increase and a subsequent lower sustained
elevation (15, 20, 35). The initial elevation of
Ca2+ is due to intracellular
Ca2+ release from
IP3-sensitive
Ca2+ stores, whereas the sustained
elevation is dependent on Ca2+
influx from extracellular medium. Consistent with these previous discoveries, Fig.
5A shows
the CCh-stimulated biphasic
[Ca2+]i
increase in a single HSG cell loaded with indo 1. A concentration of
100 µM CCh induced a rapid transient increase followed by a sustained
elevation of Ca2+. The resting and
peak levels of
[Ca2+]i
following addition of 100 µM CCh were 138 ± 8.5 nM
(n = 8) and 375 ± 59 nM
(n = 8). The sustained elevation of
[Ca2+]i
was dependent on Ca2+ influx,
since removal of extracellular
Ca2+ reduced
[Ca2+]i
to the resting level and reintroduction of extracellular
Ca2+ restored sustained
[Ca2+]i.
The pattern of CCh-induced increases in the
KCa current was similar to that of
[Ca2+]i
(Fig. 5B). The sustained
KCa current was decreased to
resting levels when external Ca2+
was removed and recovered when
Ca2+ was reintroduced into the
medium. These data, together with the effect of BAPTA (Fig.
1C), demonstrate that the
K+ current reflects, and is
dependent on, the underlying changes in
[Ca2+]i
induced following CCh stimulation of the cells.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
CCh-induced increases in cytosolic
Ca2+ concentration
([Ca2+]i)
and KCa in HSG cells.
A: intracellular
Ca2+ was determined by monitoring
indo 1 fluorescence. CCh was added to cell (indicated by bar) after a
basal level of
[Ca2+]i
was recorded. During the CCh-induced sustained elevation of
[Ca2+]i,
extracellular Ca2+ was removed
(indicated by bar) and reintroduced.
B:
KCa was measured using a similar
experimental paradigm as in Fig. 4A in
a different cell from the same cell preparation. These are
representative traces of data obtained with 12 cells.
|
|
Consistent with the above discussion, the initial activation of the
K+ channel by CCh was not affected
by the removal of external Ca2+
(Fig.
6A). The
amplitude of the transient increase in the CCh-induced K+ current in a
Ca2+-free medium (second and third
responses) was not significantly different from that induced in the
presence of extracellular Ca2+
(82.1.8 ± 8.1% and 70.8 ± 13.7%, respectively, of the first
response, P > 0.05, n = 4). The differences in amplitudes
induced by repeated CCh stimulation in these experiments and in control
experiments shown in Fig. 1B were not
significant. Note that in these experiments Ca2+ was removed from the medium
after removal of CCh, which allows refill of internal
Ca2+ stores. When
Ca2+ was removed before CCh (Fig.
6B), a condition in which refill of
internal Ca2+ stores does not
occur, the current induced by subsequent simulation of the cells with
CCh was greatly reduced to 20.7 ± 6.8% of the control response
(P < 0.05, n = 5). However, when
Ca2+ was reintroduced, the stores
refilled and the CCh-induced response was restored to 65.1 ± 5.1%
(Fig. 5C). These data suggest that CCh stimulation of the K+ current
is dependent on internal Ca2+
release. However, because the refill status of the internal
Ca2+ store is dependent on
Ca2+ influx, the initial
activation of KCa is also
dependent, indirectly, on store-operated
Ca2+ influx.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Role of intracellular Ca2+ release
in CCh-stimulated KCa activity in
HSG cells. A: a similar protocol to
that in Fig. 1B was applied except
that Ca2+-free medium was perfused
before and during the second or third exposure to CCh (indicated by the
corresponding bars). B: experimental
protocol similar to that in the Fig. 4 was employed. Control currents
were recorded before extracellular
Ca2+ was removed (indicated by
dashed line). Note that Ca2+-free
buffer plus 1 mM EGTA was perfused to the bath before CCh was
removed.
|
|
Ca2+
influx-dependent regulation of KCa in HSG
cells.
The role of Ca2+ influx in
CCh-stimulated oscillations of the
KCa current was next examined.
Removal of external Ca2+ abolished
CCh-induced sustained oscillations of
KCa (Fig.
7A), which were recovered when Ca2+ was
reintroduced to the medium. Similarly, the sustained oscillations and
steady-state increases in KCa
induced by introducing IP3 in the
patch pipettes were also inhibited by removal of extracellular Ca2+
(n = 6, data not shown). To more
directly demonstrate the involvement of
Ca2+ influx,
La3+ (1 mM), which is an effective
Ca2+ channel antagonist and
blocker of Ca2+ influx in a wide
variety of nonexcitable cells including salivary gland cells (26, 30),
was introduced into the cell medium. CCh-induced sustained oscillations
in KCa were first decreased and
then abolished in the continued presence of
La3+. The current recovered once
La3+ was removed from the medium
(Fig. 7B). Sustained elevation of [Ca2+]i
in CCh-stimulated HSG cells was also blocked by addition of La3+ to the cell medium (data not
shown). These data suggest that Ca2+ influx regulates the
sustained activation of KCa in
CCh-treated HSG cells. As mentioned above, internal
Ca2+ store refill is achieved by
Ca2+ influx via the
store-dependent pathway. Thus, to further demonstrate that
La3+ blocks
KCa by inhibiting
Ca2+ influx, the effect of
La3+ on the refill of internal
Ca2+ stores was examined (Fig.
8A). The
cells were first stimulated with CCh, and then
La3+ was added before removal of
CCh. Cells were then restimulated with CCh in the continued presence of
La3+. The amplitude of the second
response to CCh was significantly reduced to 15.1 ± 5.1% of that
in the control response (P < 0.01, n = 6). The inhibition was partially
recovered to 37.6 ± 9.6% when
La3+ was washed out.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of Ca2+ influx on
CCh-induced sustained oscillations of
KCa.
A: current was measured in continued
presence of 10 µM CCh (dashed line). Perfusion with
Ca2+-free medium and EGTA is shown
by the bar. B: experimental conditions
were similar to those in A. Cell was
continuously perfused with medium containing CCh (10 µM), indicated
by dashed line. Addition of 1 mM
La3+ to the bath is also indicated
(solid bar).
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 8.
La3+ and
Gd3+, but not
Zn2+, block
Ca2+ influx-dependent regulation
of KCa. Same protocol as described
in Fig. 1B was used. Perfusion with
CCh-containing medium is shown by solid bars. Addition of the test
cations (1 mM) is shown by dashed lines, that is, with
La3+
(A),
Gd3+
(B), and
Zn2+
(C). Data are representative of
results obtained with 16 cells.
|
|
We used the KCa current to further
characterize the Ca2+ influx
pathway. Gd3+ has been reported to
block stretch-activated and nonspecific cation channels (5, 33), and
Zn2+ has been shown to inhibit the
internal Ca2+ release-activated
Ca2+ current
(ICRAC) in mast
cells and T lymphocytes (2, 6, 8).
Zn2+ also inhibits store-operated
Ca2+ influx in salivary gland
cells (4, 11, 22). The effects of these cations on
KCa are shown in Fig. 8. The
amplitude of CCh-induced KCa in
the presence of Gd3+ was
significantly decreased to 25.9 ± 7.4%
(P < 0.01, n = 5) of the control
current, and this reduction was also partially restored when
Gd3+ was removed (to 46.7 ± 10.2%, Fig. 8B). In general,
La3+ and
Gd3+ mimicked the effects of
extracellular Ca2+ removal on the
CCh-induced activation and sustained increases in
KCa. However,
Zn2+ did not inhibit CCh-induced
KCa in HSG cells (Fig.
8C). The mean amplitude of
CCh-induced current in the presence of
Zn2+ was 88.6 ± 6.9% of that
in the control (P > 0.05, n = 5). In aggregate, these results
indicate that 1)
Ca2+ influx is necessary for
maintaining CCh-induced sustained oscillations and steady-state
increases in KCa,
2)
Ca2+ influx, via internal
Ca2+ store refill, also determines
the initial activation of KCa by CCh, and 3)
Ca2+ influx is mediated via a
La3+- and
Gd3+-sensitive, but
Zn2+-insensitive, pathway. We have
also measured the effect of Gd3+
and Zn2+ on CCh-induced elevation
in
[Ca2+]i
and have observed that Gd3+, but
not Zn2+, is similar to
La3+ in blocking the sustained
elevation of
[Ca2+]i
(data not shown).
Regulation of KCa by
Ca2+ influx is
dependent on internal
Ca2+ release in
HSG cells.
The data presented above demonstrate that the sustained
KCa current in CCh-stimulated HSG
cells is primarily regulated by Ca2+ influx. To examine the effect
of Ca2+ influx in the absence of
internal Ca2+ release, the
KCa current was measured during
refill of internal Ca2+ stores.
Cells were first stimulated with CCh in a
Ca2+-free medium, and atropine was
then added to terminate the muscarinic receptor-mediated signaling
(i.e., IP3-dependent intracellular release was inactivated). Reintroduction of
Ca2+ in the cell medium did not
induce any change in KCa (Fig.
9B, also
see trace in Fig. 6, A and
B). However, under these conditions, Ca2+ influx did occur, resulting
in the refill of internal Ca2+
stores. This is shown by the response to a subsequent addition of TG
that was larger than that obtained in cells in which the internal
stores were not allowed to fully refill (compare data in Fig.
9B with Fig.
9A; in
A, TG was added ~1 min after
perfusion with CCh-containing medium was stopped). In the absence of
atropine, the IP3-mediated
Ca2+ release pathway remains
activated, and in this case readdition of
Ca2+ to the medium induced rapid
activation of KCa. Subsequent
removal of Ca2+ from the medium
and addition of TG induced a small increase in [Ca2+]i
due to release of Ca2+ from
partially refilled stores or from stores not mobilized by CCh.
Intracellular Ca2+ accumulation
has been suggested to strongly buffer
Ca2+ in the subplasma membrane
region in exocrine acinar cells (21, 25). Furthermore, previous studies
have indicated that refill of internal
Ca2+ stores is achieved without
significant increases in
[Ca2+]i
(24, 26, 31, 34). The data in Fig. 9 are consistent with these previous
findings and indicate that the KCa
activity monitors
[Ca2+]i
in the region of Ca2+ influx. To
examine the role of intracellular
Ca2+ pump activity on the
regulation of KCa, cells were
treated with CCh, followed by atropine and then TG (i.e.,
Ca2+ influx activated but
IP3 receptor and
Ca2+ pump inhibited).
Reintroduction of Ca2+ into the
cell medium induced rapid activation of
KCa (Fig.
9C). Thus the activation of
KCa by
Ca2+ influx alone is achieved only
when internal Ca2+ release is
activated (CCh or TG treated) or internal
Ca2+ accumulation is inhibited (TG
treated, also see Fig. 10).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
Ca2+ influx-dependent regulation
of KCa in CCh-stimulated HSG cells
is mediated via internal Ca2+
release. A:
KCa regulation by
Ca2+ influx in CCh-stimulated
cells. Cells were stimulated with CCh in a
Ca2+-free medium. Addition and
removal of 1 mM Ca2+ are
indicated. Status of internal Ca2+
stores was checked by addition of TG.
B: effect of
Ca2+ influx on
KCa during refill of internal
Ca2+ stores. Additions of CCh,
atropine, Ca2+, and TG are
indicated. C:
Ca2+ influx-dependent regulation
of KCa following inhibition of
intracellular Ca2+ pump. Additions
of CCh, TG, and Ca2+ are
indicated. Data represent 3-5 experiments in each condition.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 10.
Model for regulation of KCa by
Ca2+ influx in CCh-stimulated HSG
cells. See DISCUSSION for details.
|
|
 |
DISCUSSION |
The data presented describe the
[Ca2+]i-dependent
regulation of a large-conductance
KCa in CCh-stimulated HSG cells.
The data demonstrate that there is a strong association between the
increase in
[Ca2+]i
and the increase in the K+ current
in HSG cells stimulated with CCh. We have shown that the initial
increase in KCa is dependent on
the initial elevation of
[Ca2+]i,
which is due to the release of
Ca2+ from intracellular stores.
This activation is mimicked by agents that induce release of
Ca2+ from intracellular stores,
such as IP3, TG, and BHQ.
Furthermore, consistent with previous
[Ca2+]i
measurements, the initial amplitude of
KCa is not altered by removal of
extracellular Ca2+. However, under
conditions in which the internal
Ca2+ store is depleted, CCh
activation of KCa is decreased,
i.e., in cells treated with IP3,
TG, or BHQ or when internal Ca2+
store refill is prevented by removal of external
Ca2+ or addition of
La3+ or
Gd3+. Importantly, our data show
that Ca2+ influx is required for
CCh-induced sustained oscillations and steady-state increases in the
K+ current in HSG cells. Removal
of extracellular Ca2+ reduced the
sustained elevation of
[Ca2+]i,
resulting in a corresponding decrease in
KCa (Figs. 4, 7, and 9). These
data are consistent with several reports showing that the sustained,
oscillatory, or steady-state increases in [Ca2+]i
in a number of cells, including salivary gland cells, require Ca2+ influx (10, 15, 18, 20, 23,
24, 26, 32). Several different
Ca2+ influx pathways have been
proposed to be present in nonexcitable cells, including store-operated
(capacitative) Ca2+ entry, second
messenger (i.e., IP3)-operated
Ca2+ entry, and receptor-operated
Ca2+ entry (1, 2, 6, 20, 31). We
have previously reported the presence of store-operated
Ca2+ entry in HSG cells. In
addition, we had also reported a small Ca2+ entry component that appeared
to be independent of the store status and was regulated by the
muscarinic receptor, either directly or via a G protein (20). In the
present study, CCh did not stimulate further increases in
KCa in TG- or BHQ-stimulated
cells, suggesting that only the store-operated
Ca2+ influx pathway is primarily
involved in sustaining the K+
current in HSG cells. This is consistent with our earlier results showing membrane potential changes in CCh-stimulated HSG cell by using
membrane potential-sensitive fluorescent dyes (20).
The molecular mechanism involved in mediating
Ca2+ influx in nonexcitable cells
is not yet known. However, it has been reported recently that the
store-operated Ca2+ entry pathway,
where depletion of intracellular
Ca2+ stores stimulates
Ca2+ influx across the plasma
membrane, is mediated via an
ICRAC channel (2,
6, 8, 16). Electrophysiological studies with mast cells and T
lymphocytes have shown that
ICRAC has a very
low conductance: ~1,000-fold lower than the conductance of classical
voltage-sensitive Ca2+ channels
(6, 8). ICRAC is
activated by various stimuli, such as the
Ca2+-mobilizing agonists (e.g.,
CCh) or second messengers (e.g.,
IP3) or the inhibitors of the
Ca2+ pump (e.g., BHQ or TG). It is
highly Ca2+ selective and is
strongly inhibited by La3+ or low
concentrations of Zn2+ and by high
[Ca2+]i.
Although we have not shown direct measurements of the
Ca2+ influx current in HSG cells
here, we have shown that the sustained activation of
KCa, which reflects a sustained
elevation of
[Ca2+]i,
is induced by stimulation of the cells with CCh, BHQ, or TG or by
introduction of IP3 into the
cells. These results are similar to our previously reported data in
which Ca2+ entry into fura
2-loaded HSG cells was measured. Furthermore, we have also shown here
that 1) the sustained activation of
KCa is dependent on extracellular
Ca2+, i.e., on
Ca2+ influx, and is blocked by
La3+ and
Gd3+, but not by
Zn2+, and that
2) the inhibition of
KCa by the divalent cations is due
to the inhibition of Ca2+ influx.
Zn2+ has been reported to
effectively block
ICRAC in RBL mast
cells. Thus the Ca2+ influx
pathway in HSG cells does not appear to show typical characteristics of
ICRAC. On the
other hand, Gd3+, which blocks
Ca2+ influx into HSG cells (data
not shown), has been used extensively to block stretch-activated and
voltage-gated cation channels (5, 33). More recently, it has been shown
to block cation influx mediated by the
Trp gene product, which has been
proposed as a candidate protein for the store-operated
Ca2+ influx activity (3). However,
further studies are required to fully describe the electrophysiological
characteristics of the Ca2+ influx
pathway in HSG cells.
The involvement of store-operated
Ca2+ influx in CCh-dependent
regulation of KCa in HSG cells is
demonstrated by the following. 1) TG
and BHQ mimic CCh-induced increases in
KCa conductance and attenuate the
response induced by CCh and vice versa.
2) Inhibition of
Ca2+ influx prevents initial
activation of KCa by preventing
refill of internal Ca2+ store(s).
3) Inhibition of
Ca2+ influx prevents sustained
activation of KCa due to loss of
sustained [Ca2+]i
elevation. Our model for the regulation of
KCa by
Ca2+ influx in HSG cells is shown
in Fig. 10. We have shown that
Ca2+ influx alone, in the absence
of internal Ca2+ release (i.e.,
during refill of internal Ca2+
stores), does not activate KCa
(Fig. 10B, see data in Fig.
9B). When the intracellular
Ca2+ accumulation is inhibited,
KCa is activated by
Ca2+ influx (Fig.
10C, see data in Fig.
9C). These data clearly indicate that the intracellular Ca2+ store
membrane and the plasma membrane are likely to be in close proximity,
consistent with previous studies (10, 25). However, presently we cannot
rule out the possibility that other
Ca2+ stores may be present that
are not closely situated to the plasma membrane and thus likely not
involved in the regulation of KCa activity.
In aggregate, the data presented above suggest that the
[Ca2+]i
increase in the region of Ca2+
influx appears to be strongly buffered by intracellular
Ca2+ accumulation and that there
is minimal diffusion of Ca2+ from
this region under conditions when
Ca2+ influx is activated. Such
buffering has been recently suggested in pancreatic cells, where it was
shown that influx of Ca2+ induced
refill of internal Ca2+ stores
without giving rise to elevations in
[Ca2+]i,
unless the intracellular Ca2+
accumulation was inhibited by TG (Ref. 25, also see
footnote1).
The present studies provide further evidence for this buffering by
using the KCa activity as a
readout for subplasma membrane changes in
[Ca2+]i.
The data (see Fig. 9A) indicate
that, when the IP3-dependent Ca2+ release pathway in the
internal Ca2+ store is activated,
Ca2+ entering the cell via the
Ca2+ influx pathway in the plasma
membrane can reach the K+ channel
and activate it. We suggest that, in CCh-stimulated HSG cells, this is
mediated by the uptake of Ca2+
into the internal store and release via the
IP3-sensitive channel, without
significant accumulation in the store (Fig.
10A). An assumption in our model is
that IP3-dependent release of
Ca2+ from the store does not
induce a change in the Ca2+ pump
activity (decrease) or in the diffusion (increase) of
Ca2+ from the site of influx. An
important question that arises from the above model is why the cell
would expend considerable energy to pump
Ca2+ into the store while the
IP3-sensitive release channel is
activated. A possible explanation is that such a mechanism allows the
cell to direct localized release of
Ca2+ and also prevents significant
increase in
[Ca2+]i
at the site of influx. Localized sites for intracellular
Ca2+ uptake and release have
recently been proposed in salivary and pancreatic acinar cells (21,
25). Further studies will be required to determine whether the
subcellular localization of the
Ca2+ influx protein(s), the
K+ channel, the
IP3 receptor, and the internal
Ca2+ pump in the sub-plasma
membrane region of the HSG cell determine the regulation of
KCa by
[Ca2+]i.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Bruce J. Baum for his encouragement and support during the
course of this work. We also thank the Scientific Director, National
Institute of Dental Research, for financial assistance toward purchase
of the equipment and for providing the space.
 |
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. §1734 solely to indicate this fact.
1
While this paper was under review, Mogami et
al. (25a) reported very similar results in pancreatic acinar cells by
measuring the Ca2+-activated
Cl
current. The model
proposed by these authors is similar to that proposed by us (see Fig.
10), with the exception that the sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) pump activity
is increased when internal Ca2+
stores are depleted and decreased on store refill. This is not inconsistent with the assumption in our model that the SERCA pump activity is not decreased when the internal
Ca2+ store is depleted. Increased
activity of the SERCA pump will be even more efficient in reducing the
Ca2+ concentration near the site
of Ca2+ influx and thus in
limiting the diffusion of Ca2+
from this region.
Address for reprint requests: I. Ambudkar, Bldg. 10, Rm. 1N-113,
National Institutes of Health, Bethesda, MD 20892.
Received 12 January 1998; accepted in final form 13 May 1998.
 |
REFERENCES |
1.
Ambudkar, I. S.,
Y. Hiramatsu,
T. Lockwich,
and
B. J. Baum.
Activation and regulation of Ca2+ entry in rat parotid gland acinar cells.
Crit. Rev. Oral Biol. Med.
4:
4221-4255,
1993.
2.
Berridge, M. J.
Capacitative calcium entry.
Biochem. J.
312:
1-11,
1995[Medline].
3.
Birnbaumer, L.,
X. Zhu,
M. Jiang,
G. Boulay,
M. Peyton,
B. Vannier,
D. Brown,
D. Platano,
H. Sadeghi,
E. Stephani,
and
M. Birnbaumer.
On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp protein.
Proc. Natl. Acad. Sci. USA
26:
15195-15202,
1996.
4.
Chauthaiwale, J. V.,
T. Lockwich,
and
I. S. Ambudkar.
Characteristics of a low affinity Ca2+ influx component in rat parotid basolateral plasma membranes.
J. Membr. Biol.
162:
139-145,
1998[Medline].
5.
Chen, Y.,
S. M. Simasko,
J. Niggel,
W. J. Sigurdson,
and
F. Sachs.
Calcium uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1790-C1798,
1996[Abstract/Free Full Text].
6.
Clapham, D. E.
Calcium signaling.
Cell
80:
259-268,
1995[Medline].
7.
Cook, D. I.,
M. L. Roberts,
E. W. Van Lennep,
and
J. A. Young.
Secretion by major salivary glands.
In: Physiology of the Gastrointestinal Tract, edited by L. Johnson,
J. Cristensen,
M. Jackson,
E. Jacobson,
and J. Walsh. New York: Raven, 1994, p. 1065-1107.
8.
Fasolato, C.,
B. Innocenti,
and
T. Pozzan.
Receptor-activated Ca2+ influx: how many mechanisms for how many channels.
Trends Pharmacol. Sci.
15:
77-83,
1994[Medline].
9.
Fatherazi, S.,
K. I. Izutsu,
R. B. Wellner,
and
C. M. Belton.
Hypotonically activated chloride current in HSG cells.
J. Membr. Biol.
142:
181-193,
1994[Medline].
10.
Foskett, J. K.,
P. J. Gunter-Smith,
J. E. Melvin,
and
R. J. Turner.
Physiological localization of an agonist-sensitive pool of Ca2+ in parotid acinar cells.
Proc. Natl. Acad. Sci. USA
86:
167-171,
1989[Abstract].
11.
Foskett, J.,
and
D. C. P. Wong.
[Ca2+]i inhibition of Ca2+ release-activated Ca2+ influx underlies agonist- and thapsigargin-induced [Ca2+]i oscillations in salivary acinar cells.
J. Biol. Chem.
269:
31525-31532,
1994[Abstract/Free Full Text].
12.
Gallacher, D. V.,
and
A. P. Morris.
The receptor-regulated Ca2+ influx in mouse submandibular acinar cells is sodium dependent: a patch clamp study.
J. Physiol. (Lond.)
384:
119-130,
1987[Abstract].
13.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
14.
Hayashi, T.,
P. Poronnik,
J. A. Young,
and
D. I. Cook.
The ACh-evoked, Ca2+-activated whole-cell K+ current in mouse mandibular secretory cells. Whole-cell and fluorescence studies.
J. Membr. Biol.
152:
253-259,
1996[Medline].
15.
He, X. J.,
X. Z. Wu,
R. B. Wellner,
and
B. J. Baum.
Muscarinic receptor regulation of Ca2+ mobilization in a human salivary cell line.
Pflügers Arch.
413:
505-510,
1989[Medline].
16.
Hoth, M.
Depletion of intracellular calcium stores activates an outward potassium current in mast and RBL-1 cells that is correlated with CRAC channels activation.
FEBS Lett.
390:
285-288,
1996[Medline].
17.
Ishikawa, T.,
and
D. I. Cook.
Characterization of an outwardly rectifying chloride channel in a human submandibular gland duct cell line (HSG).
Pflügers Arch.
427:
203-209,
1994[Medline].
18.
Izutsu, K. T.,
S. Fatherazi,
and
R. B. Wellner.
Characteristics and regulation of a muscarinically activated K current in HSG-PA cells.
Am. J. Physiol.
266 (Cell Physiol. 35):
C58-C66,
1994[Abstract/Free Full Text].
19.
Jaimovich, E.,
and
E. Rojas.
Intracellular Ca2+ transient induced by high external K+ and tetracaine in cultured rat myotubes.
Cell Calcium
15:
356-368,
1994[Medline].
20.
Kaplan, M. D.,
S. E. Taylor,
and
I. S. Ambudkar.
G-protein and capacitatively regulated Ca2+ entry pathways are activated by muscarinic receptor stimulation in a human submandibular ductal cell line.
Pflügers Arch.
428:
439-445,
1994[Medline].
21.
Lee, M. G.,
X. Xu,
W. Zeng,
J. Diaz,
T. H. Kuo,
F. Wuytack,
L. Racymackers,
and
S. Muallem.
Polarized expression of Ca2+ pumps in pancreatic and salivary gland cells. Role in initiation and propagation of [Ca2+]i waves.
J. Biol. Chem.
272:
15771-15776,
1997[Abstract/Free Full Text].
22.
Lockwich, T.,
J. Chauthaiwale,
A. V. Ambudkar,
and
I. S. Ambudkar.
Reconstitution of a passive Ca2+-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells.
J. Membr. Biol.
148:
277-285,
1995[Medline].
23.
Martin, S. C.,
and
T. J. Shuttleworth.
Muscarinic-receptor activation stimulates oscillations in K+ and Cl
currents which are acutely dependent on extracellular Ca2+ in avian salt gland cells.
Pflügers Arch.
426:
231-238,
1994[Medline].
24.
Mertz, L.,
B. J. Baum,
and
I. S. Ambudkar.
Refill status of the agonist-sensitive calcium pool regulates Mn2+ influx in parotid acini,
J. Biol. Chem.
265:
15010-15114,
1990[Abstract/Free Full Text].
25.
Mogami, H.,
K. Nakano,
A. V. Tepikin,
and
O. H. Petersen.
Ca2+ flow via tunnels in polarized cells: recharging of apical Ca2+ stores by focal Ca2+ entry through basal membrane patch.
Cell
88:
49-55,
1997[Medline].
25a.
Mogami, H.,
A. V Tepikin,
and
O. H. Petersen.
Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen.
EMBO J.
17:
435-442,
1998[Abstract/Free Full Text].
26.
Muallem, S.
Calcium transport pathways of pancreatic acinar cells.
Annu. Rev. Physiol.
51:
83-105,
1989[Medline].
27.
Patton, L.,
and
R. B. Wellner.
Biology of the Salivary Glands, edited by K. Dobrosielski-Vergona. Salem, MA: CRC, 1993, p. 319-342.
28.
Petersen, O. H.
Stimulus-secretion coupling: cytoplasmic Ca2+ signals and control of ion channels in exocrine acinar cells.
J. Physiol. (Lond.)
448:
1-51,
1992[Medline].
29.
Petersen, O. H.,
and
D. V. Gallacher.
Electrophysiology of pancreatic and salivary acinar cells.
Annu. Rev. Physiol.
50:
65-80,
1988[Medline].
30.
Putney, J. W., Jr.
Identification of cellular activation mechanisms associated with salivary secretion.
Annu. Rev. Physiol.
48:
75-88,
1986[Medline].
31.
Putney, J. W., Jr.
Capacitative calcium entry revisited.
Cell Calcium
11:
611-624,
1990[Medline].
32.
Putney, J. W., Jr.,
and
G. S. Bird.
The inositol phosphate-calcium signaling system in non-excitable cells.
Endocr. Rev.
14:
610-631,
1993[Medline].
33.
Ross, P. E.,
and
D. E. Calahan.
Calcium influx pathways mediated by swelling or stores depletion in mouse thymocytes.
J. Gen. Physiol.
106:
415-445,
1995[Abstract].
34.
Takemura, H.,
and
J. W. Putney, Jr.
Capacitative calcium entry in parotid acinar cells.
Biochem. J.
258:
409-412,
1989[Medline].
35.
Wu, A. J.,
Z. J. Chen,
B. J. Baum,
and
I. S. Ambudkar.
Interferon-
induced persistent depletion of internal Ca2+ stores in a human salivary gland cell line.
Am. J. Physiol.
270 (Cell Physiol. 39):
C514-C521,
1996[Abstract/Free Full Text].
Am J Physiol Cell Physiol 275(2):C571-C580
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society