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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Methods
Results
Discussion
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-gamma 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