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Proceedings of the Society for Experimental Biology and Medicine 225:211-220 (2000)
© 2000 Society for Experimental Biology and Medicine

Original Article

Characterization of the Calcium Signaling System in the Submandibular Cell Line SMG-C6

Xiao-Bing Liu, Xiuhua Sun, Ann-Christin Mörk2,, Michael W. J. Dodds, J. Ricardo Martinez3, and Guo H. Zhang1,


Secretory Cell Physiology Laboratory, Department of Pediatrics, University of Texas Health Science Center, San Antonio, Texas 78229–3900


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Establishment of salivary cell lines retaining normal morphological and physiological characteristics is important in the investigation of salivary cell function. A submandibular gland cell line, SMG-C6, has recently been established. In the present study, we characterized the phosphoinositide (PI)-Ca2+ signaling system in this cell line. Inositol 1,4,5-trisphosphate(1,4,5-IP3) formation, as well as Ca2+ storage, release, and influx in response to muscarinic, {alpha}1-adrenergic, P2Y-nucleotide, and cytokine receptor agonists were determined. Ca2+ release from intracellular stores was strongly stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA), epidermal growth factor (EGF), interleukin-6 (IL-6), and tumor necrosis factor-{alpha} (TNF{alpha}). Consistently, 1,4,5-IP3 formation was dramatically stimulated by ACh and ATP. ACh-stimulated cytosolic free Ca2+ concentration [Ca2+]i increase was inhibited by ryanodine, suggesting that the Ca2+-induced Ca2+ release mechanism is involved in the ACh-elicited Ca2+ release process. Furthermore, ACh and ATP partially discharged the IP3-sensitive Ca2+ store, and a subsequent exposure to thapsigargin (TG) induced further [Ca2+]i increase. However, exposure to TG depleted the store and a subsequent stimulation with ACh or ATP did not induce further [Ca2+]i increase, suggesting that ACh and ATP discharge the same storage site sensitive to TG. As in freshly isolated submandibular acinar cells, exposure to ionomycin and monensin following ACh or TG induced further [Ca2+]i increase, suggesting that IP3-insensitive stores exist in SMG-C6 cells. Ca2+ influx was activated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggesting the involvement of store-operated Ca2+ entry (SOCE) pathway. These results indicate that in SMG-C6 cells: (i) Ca2+ release is triggered by muscarinic and P2Y-nucleotide receptor agonists through formation of IP3; (ii) both the IP3-sensitive and -insensitive Ca2+ stores are present; and (iii) Ca2+ influx is mediated by the store-operated Ca2+ entry pathway. We conclude that Ca2+ regulation in SMG-C6 cells is similar to that in freshly isolated SMG acinar cells; therefore, this cell line represents an excellent SMG cell model in terms of intracellular Ca2+ signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The salivary gland is an excellent model for study of protein, fluid, and electrolyte secretion. Saliva secretion is a two-phase process. The primary saliva is produced by acinar cells and modified by the duct system through a variety of ion transport processes (1). Unlike many major organs, salivary glands are small and incapable of providing large amounts of cells for experimental use. Therefore, the establishment and characterization of immortalized salivary cell lines are of key importance for studying mechanisms of saliva formation and secretion in vitro. Although a number of immortalized ductal cell lines have been established and widely used in investigations of ion modification mechanisms in the duct system (2), very few immortalized salivary acinar cell lines are available. This is due primarily to the unique characteristics of acinar cells, which are highly differentiated so that their ability to maintain growth and differentiation, and especially maintenance of their acinar characteristics, is believed to be minimal.

Recently, several rat salivary cell lines, Par-C5 and C10 (PG), and SMG-C6 and C10, have been established using a plasmid containing a replication-defective simian virus (SV40) genome, and initial characterization suggests that they are of acinar origin (3, 4). These cell lines maintain the characteristics of acinar cells, including moderate amounts of secretory granules and cellular polarization. Furthermore, all these cell lines express ß-adrenergic receptors coupled to cAMP formation, and muscarinic and P2Y receptors coupled to the phosphoinositide (PI) signal transduction pathway (3, 4). Electrophysiological studies indicate that transcellular Cl- and Na+ movement is regulated by P2Y and muscarinic agonists in SMG-C6 (5) and Par-C10 cells (6). Recent studies (3-6) have suggested that these cell lines represent excellent cell models for studying fluid and ion transport processes. However, it remains unclear whether these cell lines have similar intracellular signaling systems to freshly isolated salivary cells. Therefore, the purpose of the present study was to characterize the primary signaling system regulating fluid and electrolyte secretion, the PI-Ca2+ system, in SMG-C6 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.
ACh, ATP, bovine serum albumin (BSA) (type V), EGTA, HEPES, ionomycin, NA, and ryanodine were from Sigma (St. Louis, MO). 2-Aminoethoxydiphenyl borate (2APB), IL-6, TG, and TNF-{alpha} were purchased from CalBiochem (La Jolla, CA). Basal Eagle Medium amino acids (BEM) were from GIBCO (Grand Island, NY). 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) and Fura-2/acetoxyl ester (AM) was from Molecular Probes (Eugene, OR). All other chemicals used were of the highest grade available.

Solutions.
Physiological salt solution (PSS) consisting of (in mM): 110 NaCl, 25 NaHCO3, 20 Hepes, 10 glucose, 5.4 KCl, 0.4 KH2PO4, 0.33 NaH2PO4, 1.2 CaCl2, 0.8 MgSO4, and 0.01% (w/v) BSA. Solution pH was adjusted to 7.4 after gassing with 95%O2/5% CO2 for 45 min. For Ca2+-free PSS, CaCl2 was omitted, and 30 µM BAPTA was added to adjust medium Ca2+ concentration [Ca2+]o to 100 nM. The nominally Ca2+-free medium contains 800 nM ionic Ca2+ (12). Using high-concentration (1 mM) EGTA to chelate Ca2+ induces depletion of intracellular Ca2+. Incubation of SMG-C6 cells in the Ca2+-free medium containing 1 mM EGTA for 3–5 min reduced the basal [Ca2+]i from 73 ± 4 nM (n = 19) to 26 ± 3 nM (n = 10; P < 0.001). Moreover, the EGTA-containing solution depleted the IP3-sensitive Ca2+ store, resulting in a reduction in Ca2+ release response. The [Ca2+]i increase stimulated by 10 µM ACh was 126 ± 15 nM (n = 10) following incubation in Ca2+-free medium containing 30 µM BAPTA and 24 ± 2 nM (n = 5; P < 0.001) in EGTA (1 mM)-containing medium for 5 min. Furthermore, an important advantage of using BAPTA as a Ca2+ chelator is that BAPTA, unlike EGTA, has a much (10,000-fold) higher affinity for Ca2+ than for Mg2+ (7). Thus, using BAPTA as a Ca2+ chelator will not affect Mg2+ concentration that is a critical factor for cell function including Ca2+ mobilization. Therefore, unless specifically indicated, most experiments in Ca2+-free medium were performed in a Ca2+-free medium containing 30 µM BAPTA.

Cell Culture.
The rat SMG cell line SMG-C6 established by Quissell et al. (3) was routinely grown at 37°C in a humidified 5% CO2 atmosphere on plastic tissue culture T-75 flasks in DMEM/F12 (1:1) medium containing 2.5% FBS, 20 nM sodium selenite, 5 µg/ml transferrin, 1.1 µM hydrocortisone, 0.1 µM retinoic acid, 2 nM T3, 8.4 ng/ml cholera toxin, 5 µg/ml insulin, 80 ng/ml EGF, 5 mM glutamine, 50 µg/ml gentamicin sulfate, 1 µg/ml amphotericin B, 100 µg/ml penicillin G, and 100 U/ml streptomycin and trace element mixture. The medium was changed twice weekly. Subculture was conducted by washing the cells with fresh, serum-free medium and adding 0.25% trypsin and 1 mM EDTA for 5 min. Trypsinized cells were counted, centrifuged, resuspended in fresh medium and seeded to new flasks. For the experiments, the confluent cells were trypsinized, rinsed twice with PSS, resuspended in fresh PSS containing 1% newborn bovine serum and 2% trypsin inhibitors, and incubated at 37°C for 2–3 hr before use. The cell viability was routinely tested by trypan blue exclusion.

Determination of [Ca2+]i.
[Ca2+]i was determined using the Ca2+ sensitive fluorescent probe Fura-2 as previously described (8, 9). Briefly, trypsinized SMG-C6 cells were incubated at 37°C in PSS containing 2% trypsin inhibitor for 2 hr and then loaded with Fura-2 by incubation with 2 µM Fura-2/AM for 20 min at 37°C in PSS containing 0.01% BSA. After loading, the cells were rinsed twice with PSS containing 0.01% BSA and kept at 37°C. For [Ca2+]i measurements, a 2-ml aliquot of Fura-2-loaded cells was quickly pelleted, resuspended in fresh medium (1.5 x 106 cells/ml) containing 0.01% BSA, and placed in a 4-ml cuvette. Fura-2 fluorescence was monitored with a PTI Deltascan fluorometer (PTI Inc., S. Brunswick, NJ). The excitation wavelengths used were 340 and 380 nm, and emission wavelength was 505 nm. Calibration of [Ca2+]i was performed for each measurement trace as previously described (8-10). Briefly, 1 mM CaCl2 and 50 µM ionomycin were sequentially added to obtain the limiting ratio for Ca2+ saturated form (Rmax) of Fura-2. Then, 0.0005% digitonin and 10 mM EGTA were sequentially added to obtain the limiting ratio for the unbound form (Rmin) of Fura-2. Fluorescence ratios of the 340/380 nm excitation and 505 nm emission were converted to [Ca2+]i according to Grynkiewicz et al. (11) using 224 nM as Kd of Fura-2 for Ca2+ at 37°C.

Measurement of Ca2+ Influx.
Ca2+ influx through SOCE activated by depletion of the IP3-sensitive intracellular store was measured by a method described previously (7, 13-14). Fura-2-loaded cells were stimulated with indicated reagents in Ca2+-free medium for the indicated time, and 1 mM Ca2+ was then added. The initial portion (first 15 sec) of [Ca2+]i changes after addition of Ca2+ was used to calculate Ca2+ influx rate (nM/min).

Measurement of 1,4,5-IP3.
1,4,5-IP3 formation was determined as previously described (13, 15, 16) using a radioimmunoassay kit (Amersham, Arlington Heights, IL). Briefly, cells were incubated in PSS at 37°C, and stimulated with ACh or ATP for 0–5 min. The reaction was terminated by adding an equal volume of 1 M ice-cold trichloroacetic acid. Samples were left on ice for 15 min, then centrifuged at 6000 rpm in a microfuge at 4°C for 15 min. Trichloroacetic acid was removed by extracting with diethyl ether four times. Samples were then neutralized to pH 7.0 with 0.5 M NaHCO3, and 1,4,5-IP3 concentration was determined according to the procedure recommended by Amersham.

Data Presentation and Statistics.
All results are presented as means ± SEM of separate determinations using different cell preparations. Comparisons were made using Student t test or the analysis of variance followed by the Newman-Keuls test. P-values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
[Ca2+]i Increase in Response to Stimuli.
The basal level of [Ca2+]i in unstimulated SMG-C6 cells was 82 ± 4 nM (n = 37) with a range of 50 nM to 120 nM. Stimulation with 10 µM ACh elicited a sharp initial increase in [Ca2+]i. The peak increase was 160 ± 11 nM (n = 8) in the presence of extracellular Ca2+ (from 87 ± 11 nM to 247 ± 4 nM) and 142 ± 19 nM (n = 6) in Ca2+-free medium (from 63 ± 8 nM to 204 ± 25 nM) (Fig. 1A)Go. The sustained increase was 56 ± 5 (n = 8) in the presence of external Ca2+ and 11 ± 3 nM (n = 6) in Ca2+-free medium (Fig. 1A)Go.



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  		Figure 1.  [Ca2+]i response to acetylcholine, ATP, norepinephrine, and thapsigargin. SMG-C6 cells were loaded with Fura-2 in physiological salt solution at 37°C, and [Ca2+]i was determined in the presence (+Ca2+) or absence (- Ca2+) of 1.2 mM Ca2+. At the time indicated by arrows, 10 µM acetylcholine (ACh), 2 mm ATP (ATP), 10 µM norepinephrine (NA), or 3 µM thapsigargin (TG) was added. Traces are representative of at least five separate experiments.

 
Stimulation with 2 mM ATP, a P2Y-nucleotide receptor agonist, triggered a rapid and large increase in [Ca2+]i. The initial increase was 462 ± 27 nM (n = 5) in the presence of extracellular Ca2+ (from 70 ± 9 nM to 532 ± 63 nM) and 327 ± 24 nM (n = 5) in Ca2+-free medium (from 60 ± 7 nM to 387 ± 28 nM) (Fig. 1B)Go. The sustained [Ca2+]i increase after ATP was 49 ± 4 nM (n = 5) in the presence of external Ca2+ and 21 ± 2 nM in Ca2+-free medium (Fig. 1B)Go. To elucidate which subtype of P2Y-receptors mediates this response to ATP, [Ca2+]i mobilization stimulated by ATP, UTP, and ADP were compared. The initial [Ca2+]i increases were identical in response to ATP (100%) and UTP (108 ± 1%, n = 5; P > 0.05). However, ADP did not induce a substantial response (6.7 ± 0.5%, n = 4). These results suggest that the ATP-induced Ca2+ response is likely mediated by P2Y2 receptors.

On the other hand, stimulation of {alpha}1-adrenergic receptors with 10µM NA did not result in a substantial increase in [Ca2+]i. The initial increase was only 34 ± 5nM in PSS (n= 5), and the sustained increase was 31 ± 4nM. In Ca2+-free medium, the NA-induced increase was minimal. The initial increase was 16 ± 4 nM, and the sustained increase was 8 ± 2 nM (n = 5) (Fig. 1C)Go.

As shown in Figure 1DGo, exposure of SMG-C6 cells to the endoplasmic Ca2+-ATPase inhibitor TG (3 µM) induced a gradual increase in [Ca2+]i. The peak increase was 184 ± 19 nM (n = 5), and the sustained increase at 5 min was 67 ± 8 nM (n = 5) in PSS. In Ca2+-free medium, a similar peak increase was observed (174 ± 25 nM, n = 5), but the sustained increase was much smaller (29 ± 4 nM, n = 5) (Fig. 2)Go. Exposure to 100 nM EGF, 200 ng/ml of IL-6 or 50 ng/ml of TNF-{alpha} did not induce a notable [Ca2+]i increase (Fig.2)Go.



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  		Figure 2.  Initial [Ca2+]i increase in response to various agonists. SMG-C6 cells were loaded with Fura-2, and [Ca2+]i was measured in physiological salt solution containing 1.2 mM Ca2+. [Ca2+]i increase was triggered by addition of 2 mM ATP (ATP), 10 µM acetylcholine (ACh), 10 µM norepinephrine (NA), 3 µM thapsigargin (TG), 100 ng/ml of epidermal growth factor (EGF), tumor necrosis factor-{alpha} (TNF-{alpha} or interleukin-6 (IL-6). Values are means ± SEM of at least five separate experiments.

 
To test whether ATP and ACh stimulate different intracellular signaling systems that mediate the Ca2+ mobilization responses, Fura-2-loaded SMG-C6 cells were simultaneously stimulated with 10 µM ACh and 2 mM ATP in Ca2+-free medium. The initial increase in [Ca2+]i in response to this combined stimulation was 203 ± 10 nM (n = 5), which was greater than the increase in response to ACh, but significantly smaller than ATP-stimulated increase (P < 0.002), suggesting that the same signaling system mediates the responses to muscarinic and P2Y agonists.

1,4,5-IP3 Formation in Response to ACh and ATP.
To examine the relationship between 1,4,5-IP3 formation and initial [Ca2+]i increase, the time-course of ACh- and ATP-stimulated 1,4,5-IP3 formation was measured. As shown in Figure 3, 1,4,5GoGoGoGo-IP3 content before stimulation was 1.3 ± 0.5 pmoles/106 cells (n = 4). Stimulation with 10 µM ACh or 2 mM ATP induced a similar time-dependent increase. At 15 sec post-ACh stimulation, the cellular 1,4,5-IP3 concentration was 14.5 ± 0.5 pmoles/106 cells (n = 3), an 11-fold increase. The ATP-stimulated increase in 1,4,5-IP3 was significantly greater (20.2 ± 2.0 pmoles/106 cells (n= 3) (P < 0.05 vs. that stimulated by ACh)). Thereafter, 1,4,5-IP3 concentrations were maintained at a stable level, ~ 6–8-fold higher than the unstimulated level (Fig.3)Go.



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  		Figure 3.  1,4,5-IP3 formation in response to acetylcholine and ATP. SMG-C6 cells were incubated in physiological salt solution (PSS) at 37°C and stimulated with 10 µM acetylcholine (ACh) or 2 mM ATP (ATP) for 0, 0.25, 0.5, 1, 3, and 5 min. Values are means ± SEM of 3–5 separate experiments using different cell preparations. *P < 0.05 vs ACh-stimulated 1,4,5-IP3 formation.

 


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  		Figure 4.  Effects of 2APB and ryanodine on acetylcholine-stimulated [Ca2+]i increase. SMG-C6 cells were loaded with Fura-2, and [Ca2+]i was determined in Ca2+-free medium. Cells were preincubated with (A) DMSO for 3 min (CTL), (B) 100 µM 2APB for 3 min, or (C) 200 µM ryanodine for 10 min. At the time indicated by arrows, 10 µM acetylcholine (ACh) was added. Traces are representative of at least four separate experiments using different cell preparations.

 


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  		Figure 5.  Ca2+ release induced by sequential exposure to acetylcholine, thapsigargin, and ATP. SMG-C6 cells were loaded with Fura-2, and [Ca2+]i was determined in Ca2+-free medium containing 30 µM BAPTA. At the time indicated by arrows, 10 µM acetylcholine (ACh), 2 mM ATP (ATP), and 3 thapsigargin (TG) were added. Traces are representative of at least five separate experiments using different cell preparations.

 
To determine whether the combination of ACh and ATP stimuli induces a potentiation of 1,4,5-IP3 formation, cells were simultaneously stimulated with 10 µM ACh and 2 mM ATP, as described above. The formation of 1,4,5-IP3 in response to both ACh and ATP was 12.8 ± 0.8 pmoles/106 cells (n = 3) at 30 sec poststimulation (not shown), identical to the responses stimulated by these agonists separately. These results suggest that both muscarinic and P2Y nucleotide receptors are coupled to the same intracellular signaling pathway.

Ca2+ Release Mechanism.
It has been suggested that IP3-triggered Ca2+ release is mediated by two types of receptor channels, the IP3R channel, and the ryanodine receptor (RyR) channel through the process known as Ca2+-induced Ca2+ release (CICR) in pancreatic (17) and salivary cells (18, 19). To elucidate whether the release of IP3-sensitive Ca2+ stores in SMG-C6 cells is through these mechanisms, we examined Ca2+ mobilization in response to ACh in the presence of 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP3R-mediated Ca2+ release (20), or RyR inhibitor ryanodine (18, 21). As shown in Figure 4Go, ACh (10 µM) stimulation of SMG-C6 cells preincubated with vehicle, dimethylsulfoxide (DMSO), in Ca2+-free medium elicited a rapid initial [Ca2+]i increase (127 ± 9 nM, n = 6) (Fig. 4A)Go. Pretreatment with 100 µM 2APB for 3 min prevented the ACh-induced [Ca2+]i increase (27 ± 5 nM, n = 4; P < 0.001) (Fig. 4B)Go. Interestingly, preincubation with 200 µM ryanodine for 10 min also dramatically inhibited the ACh-stimulated initial [Ca2+]i increase (38 ± 3 nM, n = 4; P < 0.001) (Fig. 4C)Go. These results suggest that both RyR and the CICR mechanism are involved in the process of discharging the IP3-sensitive Ca2+ store.

Ca2+ Stores.
To examine whether the ACh-, ATP-, and TG-induced initial [Ca2+]i increases are due to Ca2+ release from the same Ca2+ storage site, the IP3-sensitive store, we sequentially stimulated the cells with ACh and TG, ACh and ATP, as well as ATP and TG in Ca2+-free medium. As shown in Figure 5AGo, after stimulation with 10 µM ACh, a subsequent exposure to 3 µM TG induced another Ca2+ release peak. On the other hand, exposure to TG first resulted in a large Ca2+ release, and subsequent stimulation with ACh did not induce a substantial further release (Fig. 5B)Go, indicating that the Ca2+ store was almost depleted by TG. In a similar pattern, after stimulation with ACh, subsequent stimulation with ATP elicited another Ca2+ release peak (Fig. 5C)Go. However, stimulation in reverse order (i.e., with ATP first) triggered a large Ca2+ release, and subsequent ACh stimulation only induced a small further release (Fig. 5D)Go, suggesting that ACh at this concentration (10 µM) did not deplete the store, and ATP (2 mM) induced further release from the store. Interestingly, whereas stimulation with ATP triggered a large release, a subsequent exposure to TG still induced a considerable additional [Ca2+]i increase (Fig. 5E)Go. Furthermore, when cells were exposed to TG first, the subsequent ATP stimulation did not induce any significant further [Ca2+]i increase (Fig. 5F)Go. These results suggest either that the TG-sensitive store is larger than the ATP-sensitive one, or that ATP alone cannot deplete the store.

Previous studies (11, 22, 23) have demonstrated that freshly isolated rat SMG acinar cells contain a granule-associated Ca2+ store that is insensitive to IP3 but releasable by ionomycin plus monensin. To examine whether SMG-C6 cells have a similar IP3-insensitive Ca2+ store, the cells were sequentially exposed to TG, ionomycin, and monensin in regular Ca2+-free medium (containing 30 µM BAPTA). As shown in Figure 6AGo, after the ACh-stimulated [Ca2+]i increase, 1 µM ionomycin induced another large [Ca2+]i peak (321 ± 7 nM, n = 5) followed by a third increase in response to monensin treatment (185 ± 16 nM, n = 5). Similarly, after TG exposure, ionomycin elicited another [Ca2+]i increase (183 ± 20 nM, n = 4), and monensin triggered a further increase (171 ± 16 nM, n = 5) (Fig. 6B)Go in Ca2+-free medium, suggesting that IP3-insensitive Ca2+ stores are present in these cells.



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  		Figure 6.  Ca2+ release induced by sequential exposure to acetylcholine or thapsigargin, ionomycin, and monensin. SMG-C6 cells were loaded with Fura-2, and [Ca2+]i was monitored at 37°C in Ca2+-free medium containing 30 µM BAPTA or 1 mM EGTA (+EGTA). Cells were sequentially exposed to (A) 10 µM acetylcholine (ACh), 1 µM ionomycin (Iono), and 10 µM monensin (Mon), or (B) 3 µM thapsigargin (TG), 1 µM ionomycin (Iono), and 10 µM Monensin (Mon) at the time indicated by the arrows. Traces are representative of at least five separate measurements using different cell preparations.

 
To rule out the possibility that the extracellular Ca2+ (100 nM) in this medium may partially contribute to the [Ca2+]i increases in response to ionomycin and monensin, the same measurements were conducted in Ca2+-free medium containing 1 mM EGTA. As shown in Figure 6Go, EGTA-containing medium reduced the ACh-induced [Ca2+]i increase (40 ± 5 nM, n = 5; P < 0.01). The ionomycin- and monensin-induced [Ca2+]i increases were identical to those in BAPTA-containing medium (Fig. 6A)Go. Similarly, the TG-induced [Ca2+]i increase was significantly smaller (81 ± 7 nM, n = 5; P < 0.002), and the ionomycin- and monensin-induced [Ca2+]i increases were comparable to those in BAPTA-containing medium. These results suggest that EGTA (1 mM)-containing medium depletes the IP3-sensitive Ca2+ store, and that the [Ca2+]i increases in response to ionomycin and monensin are due to Ca2+ release from intracellular Ca2+ stores, and are not derived from Ca2+ influx.

Ca2+ Influx.
As shown in Figure 7Go, addition of Ca2+ after incubation of unstimulated SMG-C6 cells in Ca2+-free medium for 5 min induced a small [Ca2+]i increase mediated by Ca2+ influx. The initial Ca2+ influx rate was 56 ± 7 nM/min (n = 5) (Figs. 7A & 7B)Go. This Ca2+ influx was significantly stimulated by 10 µM ACh (196 ± 21 nM/min, n = 10; P < 0.001) (Figs. 7A & 7B)Go. Stimulation with 2 mM ATP also induced a significant increase in Ca2+ influx (192 ± 28 nM/min, n = 5; P < 0.001) (Fig. 7B)Go. Exposure to 3 µM TG induced a slightly larger Ca2+ influx (220 ± 7 nM/min, n = 5) (Figs. 7A & 7B)Go. However, stimulation with NA did not activate Ca2+ influx (54 ± 12 nM/min, n = 5) (Fig. 7B)Go, which was consistent with the Ca2+ release response. These results suggest that Ca2+ influx activated by ACh, ATP, and TG is through the SOCE pathway. To test whether Ca2+ influx activated by ATP, ACh, and TG is through the same entry pathway, we measured Ca2+ influx in the cells stimulated with combinations of ACh and TG, ACh and ATP, or ATP and TG. As shown in Figure 7BGo, combining these stimuli did not alter the Ca2+ influx rate (ACh + TG: 227 ± 20 nM/min, n = 5; ATP + ACh, 215 ± 7 nM/min, n = 5; ATP + TG, 219 ± 27 nM/min, n = 5), suggesting that the same entry pathway mediates the Ca2+ influxes.



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  		Figure 7.  Ca2+ influx activated by acetylcholine, ATP, norepinephrine, and thapsigargin. SMG-C6 cells were loaded with Fura-2, and [Ca2+]i was determined in Ca2+-free medium. Cells were exposed to 2 mM ATP (ATP), 10 µM acetylcholine (ACh), 10 µM norepinephrine (NA), 3 µM thapsigargin (TG), or a combination of ACh and TG (ACh + TG), ATP and ACh (ATP + ACh), or ATP and TG (ATP + TG) for 5 min, and 1 mM Ca2+ was added to initiate Ca2+ influx. The same volume of vehicle was added in unstimulated control (CTL) cells. Values are means ± SEM of at least five separate measurements using different cell preparations. (A) Representatives of Ca2+ influx. (B) The initial rates of Ca2+ influx. *** P < 0.001 vs unstimulated cells.

 
Since Ca2+ influx through the SOCE pathway is activated by depletion of the IP3-sensitive store, we further examined the effects of inhibiting Ca2+ release from this store. Cells were pretreated with 100 µM 2APB or 200 µM ryanodine to block Ca2+ release, then stimulated with 10 µM ACh. Both inhibitors significantly attenuated the ACh-elicited Ca2+ release (Fig. 8A)Go. The rate (Fig. 8B)Go and magnitude (Fig. 8C)Go of Ca2+ influx were also proportionally reduced, indicating that the Ca2+ influx is through the SOCE pathway.



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  		Figure 8.  Effects of 2APB and ryanodine on acetylcholine-activated Ca2+ influx. SMG-C6 cells were loaded with Fura-2, and [Ca2+]i was determined in Ca2+-free medium. Cells were preincubated with DMSO for 3 min (CTL), 100 µM 2APB for 3 min, or 200 µM ryanodine for 10 min, then stimulated with 10 µM ACh. One mM Ca2+ was added after stimulation for 5 min. (A) The initial [Ca2+]i increase, (B) Ca2+ influx rate, and (C) magnitude of [Ca2+]i peak after addition of Ca2+ ({Delta}Plateau) were measured. Values are means ± SEM of at least four separate determinations using different cell preparations. ** and ***, P < 0.01 and 0.001 vs control cells.

 
SOCE is sensitive to La3+ (24-26). Therefore, the effect of La3+ on ACh-, ATP-, and TG-activated Ca2+ influx was examined. As shown in Figure 9Go, 0.2 mM La3+ significantly reduced ACh-, ATP-, and TG-stimulated Ca2+ influx. The initial influx rates were 42 ± 16 nM/min (n = 5; P < 0.001), 7 ± 4 nM/min (n = 5; P < 0.001) and 108 ± 8 (n = 4; P < 0.001) in ACh-, ATP-, and TG-stimulated cells, respectively (Fig. 9)Go.



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  		Figure 9.  Effect of La3+ on Ca2+ influx activated by acetylcholine, ATP, or thapsigargin. SMG-C6 cells were loaded with Fura-2, and [Ca2+]i was measured in Ca2+-free medium. Cells were exposed to 10 µM acetylcholine (ACh), 2 mM ATP (ATP), or 3 µM thapsigargin (TG) for 4 min, and 0.2 mM LaCl3 (+La3+) or vehicle (–La3+) was added. At 5 min postaddition of ACh, ATP, or TG, 1 mM Ca2+ was added to initiate Ca2+ influx. Values are means ± SEM of at least four separate determinations using different cell preparations. *** P < 0.001 vs without La3+ (-La3+).

 
To examine the relationship between the filling state of the IP3-sensitive Ca2+ store and Ca2+ influx, we characterized the TG-induced Ca2+ release and influx. As shown in Figure 10Go, exposure to TG resulted in a concentration-dependent initial [Ca2+]i increase (EC50= 6 nM TG). Ca2+ influx rate and the magnitude of [Ca2+]i increase ({Delta}Plateau) were also TG concentration-dependent (both EC50= 3 nM TG). Furthermore, the same values of EC20 (1 nM TG) were obtained for initial [Ca2+]i increase (Ca2+ release), influx rate, and {Delta}Plateau. However, the values of EC80 were 31 nM TG for [Ca2+]i increase, 8 nM TG for both influx rate and {Delta}Plateau (Fig. 10)Go. These results suggest that Ca2+ influx is initiated by reduction of the IP3-sensitive Ca2+ store, but the maximal influx is probably not dependent on complete depletion of the store.



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  		Figure 10.  The concentration-response relationship of the initial [Ca2+]i increase, the rate and magnitude of Ca2+ influx activated by thapsigargin. SMG-C6 cells were loaded with Fura-2, and [Ca2+]i was measured in Ca2+-free medium. Cells were exposed to 0.1, 0.3, 1, 3, 10, 30 100, 300, 1000, and 3000 nM TG for 5 min, and 1 mM Ca2+ was then added to initiate Ca2+ influx. The net increase in [Ca2+]i ({Delta}[Ca2+]i), initial influx rate ({Delta}Rate), and the magnitude of the [Ca2+]i rise after addition of Ca2+ ({Delta}Plateau) were calculated and plotted. Values are means ± SEM of four separate experiments. Curves are produced by sigmoid (Boltzman) fit.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study clearly show that regulation of Ca2+ mobilization occurs primarily through muscarinic and P2Y-nucleotide (but not {alpha}1) receptors in SMG-C6 cells. Stimulation of muscarinic and P2Y receptors elicited a rapid Ca2+ release from the IP3-sensitive store, and depletion of this store in turn activated Ca2+ influx via the SOCE pathway. These results suggest that this cell line retains the physiological Ca2+ mobilizing pathway (i.e., plasma membrane muscarinic), and nucleotide receptor agonist stimulation elicits 1,4,5-IP3 formation and Ca2+ release, which in turn activates Ca2+ influx through SOCE.

The subtype of the P2Y-nucleotide receptor responsible for the ATP-induced Ca2+ release is probably P2Y2 since ATP and UTP induce identical Ca2+ releases, and ADP and UDP were not effective over the similar concentration range (3 and the present study). P2-nucleotide receptors consist of two major receptor classes, P2X and P2Y receptors (27). At least 7 subtypes of P2X receptors have been identified (i.e., P2X1 to P2X7). Some of these receptors are ligand-gated nonselective cation channels, such as P2X4 and P2X7, which lead to Ca2+ influx through these cation channels. The Ca2+ mobilization in response to ATP in the present study was not mediated by P2X receptors since ATP caused a dramatic increase in 1,4,5-IP3 formation and a large Ca2+ release from intracellular Ca2+ stores. P2Y receptors are a large family consisting of at least 5 subtypes, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 (27). Stimulation of these subtypes elicits 1,4,5-IP3 production and Ca2+ mobilization. However, only P2Y2 receptors respond to ATP and UTP equally; P2Y1 and P2Y11 do not respond to UTP, and P2Y4 shows a larger response to UTP than to ATP. Uniquely, UDP is a better agonist of the P2Y4 subtype than UTP and ATP. SMG-C6 cells responded to ATP and UTP equally, but showed a much smaller response to UDP (3) or ADP (3 and the present study), strongly suggesting that the Ca2+ mobilization in these cells is mediated by P2Y2 receptors.

Salivary glands express P2-nucleotide receptors and these receptors participate in the regulation of gland function, such as ion transport (28). However, whether salivary cells physiologically express P2Y receptors is much less clear. In immortalized salivary cell lines, P2Y2 receptors are extensively expressed (3, 4, 10, 29, 30). In contrast, freshly isolated SMG acinar cells of adult rats did not show a substantial Ca2+ mobilizing response to UTP stimulation, but culturing these cells for 3 hr to 6 days induced significant increases in the Ca2+ response (31). Furthermore, unilateral ligation of the main excretory duct of rat SMG glands also induced expression of P2Y2 receptors. The Ca2+ mobilization response to the P2Y2 agonist UTP was moderately increased (31). We also observed that SMG acinar cells freshly isolated from adult rats did not show a Ca2+ mobilization when stimulated with ATP, but SMG cells from newborn rats responded to ATP, showing a moderate (63% ± 9%) [Ca2+]i increase (Zhang and Martinez, unpublished data). These studies suggest that expression of P2Y receptors may be associated with development and regeneration after tissue damage (31). Further investigation on this issue is indicated.

The present study also showed that stimulation of {alpha}1-receptors with NA did not induce a substantial Ca2+ mobilization in SMG-C6 cells, consistent with the report by Quissell etal. (3), who found that total IP (IP+ IP2+ IP3) formation and Ca2+ increase in response to the {alpha}1-agonists epinephrine and phenylephrine were minimal. This may be due to the lack of the receptor in these cells as reported in most other immortalized salivary cell lines (2). The {alpha}1-receptor appears less likely to be functionally expressed after immortalization than other receptors. Moreover, the coupling between {alpha}1-receptors and the PI signaling system in SMG acinar cells remains controversial. In freshly isolated SMG acinar cells, {alpha}1-receptors may not play a critical role in regulating the Ca2+ signal. It has been reported that parotid gland (PG) acinar cells respond strongly to {alpha}1-agonists, manifesting large increases in IP3 formation and Ca2+ mobilization (32, 33), although Soltoff etal. (34) observed that stimulation of rat PG acinar cells with 10 µM phenylephrine only slightly (35%) increased [Ca2+]i. In freshly isolated SMG acinar cells, 1,4,5-IP3 formation and Ca2+ release from intracellular stores in response to {alpha}1-receptor stimulation are relatively small (35). Similarly, the immortalized PG cell line Par-C5 retains {alpha}1-receptors, manifesting a moderate [Ca2+]i increase in response to NA stimulation (Zhang and Martinez, unpublished data). Elucidation of the mechanism mediating this reduced receptor expression following immortalization and the different expression between SMG and PG acinar cells would be of great interest.

The underlying mechanism mediating IP3-activated Ca2+ release in salivary cells remains unclear. Wakui et al. (17) suggested a two-store model to explain this process in pancreatic cells. According to this model, 1,4,5-IP3 first triggers Ca2+ release from the IP3-sensitive store, and the Ca2+ released from this store activates RyR channels in the second store leading to further Ca2+ release. The results in the present study demonstrated that muscarinic- or P2Y2 agonist–stimulated Ca2+ release involves two types of receptors, IP3R and RyR. This is evidenced by the findings that ACh-stimulated Ca2+ release was significantly inhibited not only by 2APB, but also by ryanodine. These results suggest that 1,4,5-IP3 first elicits Ca2+ release, and the increase in [Ca2+]i activates RyR, leading to further Ca2+release. Nevertheless, whether these receptors are localized at the same store or two separate stores needs further investigation.

Our data also suggest that SMG-C6 cells, similar to freshly isolated acinar cells, have both IP3-sensitive and -insensitive Ca2+ stores. The former is located primarily in the endoplasmic reticulum (ER) and releases Ca2+ through IP3-sensitive channels operating as IP3R (36, 37). However, the location of the IP3-insensitive Ca2+ stores is less clear. Several organelles have been suggested to store and release Ca2+, including secretory granules (11, 22, 23). Ca2+ stored in secretory granules is not discharged by receptor stimulation, or ionomycin, probably because the granules are acidic. It is well established that ionomycin is unable to discharge acidic Ca2+ stores unless the pH gradient is collapsed by monensin (38-40). Our results clearly showed that ionomycin induced a large Ca2+ release following depletion of the IP3-sensitive store with TG. Furthermore, addition of monensin following ionomycin resulted in another release of Ca2+, probably from secretory granules. These results indicate that Ca2+ stores in immortalized SMG-C6 cells are similar to those in freshly isolated SMG acinar cells.

In SMG acinar cells, depletion of the IP3-sensitive store activates Ca2+ influx through SOCE. The present study demonstrated that Ca2+ influx in response to ACh, ATP, or TG in immortalized SMG-C6 cells is also mediated by the same mechanism as in freshly isolated SMG acinar cells. This conclusion is confirmed by the results showing that ACh-, ATP-, or TG-stimulated Ca2+ influx was dramatically inhibited by the SOCE pathway blocker La3+. It is noteworthy that both ACh- and ATP-induced Ca2+ influxes were slightly smaller (13% and 11%, respectively) than the TG-activated influx. This is consistent with the Ca2+ release elicited by these reagents. Since TG induced a complete discharge of the IP3-sensitive store, the rate of Ca2+ influx was proportionally larger. Furthermore, combination of these stimuli (i.e., ACh + TG, ACh + ATP, or ATP + TG) did not induce a larger Ca2+ influx than TG alone, suggesting that the Ca2+ influx rate reached its maximum by TG treatment. These results also provided further evidence for the same pathway mediating ACh-, ATP-, and TG-stimulated Ca2+ influx.

It has been widely recognized that the opening of the SOCE pathway or channel is controlled by the filling state of the IP3-sensitive Ca2+ store. The data of the present study suggested that the initiation of Ca2+ influx is signaled by discharging the IP3-sensitive store as the same EC20 (1 nM TG) was observed for Ca2+ release (reflected by the initial [Ca2+]i increase), Ca2+ influx rate, and the magnitude of [Ca2+]i plateau after addition of Ca2+ (Fig. 10)Go. However, the maximal Ca2+ influx does not require complete depletion of the IP3-sensitive store. This was evidenced by different EC80 values for Ca2+ release, influx rate, and the magnitude ({Delta}Plateau). EC80 for both Ca2+ influx rate and magnitude were 8 nM TG, but the EC80 for Ca2+ release was 31 nM TG. Furthermore, the maximal influx rate and magnitude were reached by less than 100 nM TG, but the maximal Ca2+ release (complete depletion of the store) needed 1000 nM TG. Two possibilities may underlie this phenomenon. One is that reduction in stored Ca2+ by 50% or more produces the maximal signal for Ca2+ influx. Therefore, partial emptying of the store is sufficient for the maximal Ca2+ entry. Another possibility is that TG discharges not only the IP3-sensitive store, but also some IP3-insensitive pools, and the release of Ca2+ from these pools is much slower than discharging the IP3-sensitive store. This is quite possible since the mechanism of Ca2+ release by TG is inhibition of Ca2+-ATPase. The prerequisite for this mechanism is that Ca2+ sequestration in these pools must have the same type of Ca2+-ATPase as in the endoplasmic reticulum (the IP3-sensitive store). In this case, the dose-response curve for TG-induced Ca2+ release may be extended to the right side, implying the requirement for higher concentrations to reach the maximal release. Further investigations are needed to delineate these mechanisms.

In summary, the present study clearly shows that the Ca2+ signaling system in SMG-C6 cells is similar to that in freshly isolated SMG acinar cells, including 1,4,5-IP3 formation, Ca2+ storage, release, and influx in response to muscarinic agonists. However, expression of P2Y2 receptors and the lack of Ca2+ mobilization response to {alpha}1-agonists are the major differences between this cell line and freshly isolated cells. Nevertheless, the immortalized SMG-C6 cell line is an excellent cell model for studying the phosphoinositide-Ca2+ signaling system in SMG cells.


   Footnotes
 
This work was supported by NIH grant DE09270.

1 To whom requests for reprints should be addressed at the Secretory Cell Physiology Laboratory, Department of Pediatrics, Mail Code 7827, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229–3900. E-mail: Zhangg{at}UTHSCSA.edu Back

2 Present Address: AstraZeneca R&D Södertälje, Health Economics & Outcomes Research, 85 Södertäje, Sweden. Back

3 Present Address: Division of Extramural Research, NIDCR, National Institutes of Health, Bethesda, MD 20892. Back


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication February 14, 2000. Accepted for publication July 19, 2000.




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