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Staurosporine Mobilizes Ca²⁺ from Secretory Granules by Inhibiting Protein Kinase C in Rat Submandibular Acinar Cells

¹ Department of Oral Biology & Oral Science Research Center, BK21 Project for Medical Sciences, Yonsei University College of Dentistry, Shinchon-dong 134, Seodaemoon-gu, Seoul 120-752, Korea; and
² Department of Physiology and Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan;

Correspondence: ³ corresponding author, jeong{at}yumc.yonsei.ac.kr

	ABSTRACT

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Staurosporine was previously shown to mobilize Ca²⁺ from the thapsigargin-insensitive Ca²⁺ store in rat submandibular acinar cells. However, the nature of the store is not yet known. Therefore, in the present study, the staurosporine-releasable intracellular Ca²⁺ store was characterized. Staurosporine increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) after the inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store was depleted. Ionomycin caused only small increases in [Ca²⁺]_c after the depletion of the IP₃-sensitive Ca²⁺ store, whereas ionomycin+monensin caused large increases. However, ionomycin+monensin did not increase [Ca²⁺]_c when added after [Ca²⁺]_c was increased by staurosporine, indicating that the acidic Ca²⁺ store was the main source of Ca²⁺. The acidic Ca²⁺ store appeared to be associated with secretory granules, since ionomycin+monensin- and staurosporine-induced [Ca²⁺]_c increases were significantly reduced when the acinar cells were degranulated. The effect of staurosporine on [Ca²⁺]_c was mimicked by other protein kinase C inhibitors. Therefore, we conclude that staurosporine mobilizes Ca²⁺ from secretory granules, probably through the inhibition of protein kinase C in rat submandibular acinar cells.

Key Words: Secretory granules • staurosporine • submandibular acinar cells • calcium

	INTRODUCTION

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Fluid and electrolyte secretion from salivary acinar cells is mainly evoked by the stimulation of cholinergic, ₁-adrenergic, and substance P receptors (Case et al., 1986). The stimulation of these receptors increases cytosolic Ca²⁺ concentration ([Ca²⁺]_c), which opens Ca²⁺-sensitive Cl^- channels on the apical membrane, allowing for Cl^- efflux into the ductal lumen (Foskett, 1990). This provides a driving force for Na⁺ transport through the interstitial space from blood to the lumen, and the increase in the osmolarity causes water transport (Nauntofte, 1992). The increase in [Ca²⁺]_c is achieved by the mobilization of Ca²⁺ from intracellular Ca²⁺ stores and its influx across the plasma membrane (Putney, 1990). The Ca²⁺ mobilization from internal stores is triggered by the binding of inositol 1,4,5-trisphosphate (IP₃), which is produced from phosphatidylinositol 4,5-bisphosphate by the activation of phospholipase C (Berridge, 1993). The intracellular Ca²⁺ store, which is responsible for Ca²⁺ release in response to IP₃ binding, is thought to be located in the rough endoplasmic reticulum (ER).

Stimulation of phospholipase C also produces 1,2-diacylglycerol that activates protein kinase C, which has been implicated as a participant in Ca²⁺ homeostasis in various cell types, by providing negative feedback control of the intracellular signaling pathways (Nishizuka, 1988). Protein kinase C lowers [Ca²⁺]_c to prevent sustained elevation, either by inhibiting the hydrolysis of inositol phospholipids or by activating Ca²⁺ ATPase at the plasma membrane (Nishizuka, 1988).

We reported previously that a large amount of Ca²⁺ is released from thapsigargin-insensitive intracellular Ca²⁺ stores of rat submandibular acinar cells in response to staurosporine, a potent protein kinase C inhibitor (Shin et al., 1998). Since thapsigargin inhibits Ca²⁺ ATPase on the IP₃-sensitive Ca²⁺ store and thus depletes Ca²⁺ from the ER, the staurosporine-induced [Ca²⁺]_c increase is thought to result from Ca²⁺ mobilization from non-ER compartments. In secretory acinar cells, such as salivary acinar cells and pancreatic acinar cells, mitochondria and secretory granules have been suggested to be involved in intracellular Ca²⁺ homeostasis (Gerasimenko et al., 1996; Park et al., 2001). Therefore, we attempted to determine which subcellular compartment is responsible for the staurosporine-induced [Ca²⁺]_c increase, and whether this effect is mediated by the inhibition of protein kinase C. In this study, we found that secretory granules, an IP₃-insensitive, non-mitochondrial acidic Ca²⁺ store, were probably responsible for the staurosporine-induced [Ca²⁺]_c increase. Furthermore, other protein kinase C inhibitors, such as K252a, chelerythrine, H-7, and calphostin C, were also found to increase [Ca²⁺]_c, suggesting that the effect of staurosporine on the [Ca²⁺]_c increase might be mediated by the inhibition of protein kinase C.

	MATERIALS & METHODS

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Isolation of Acini from Rat Submandibular Glands
Submandibular salivary gland acini were isolated from male Sprague-Dawley rats, each weighing from 200 to 300 g, by the method described previously (Seo et al., 1999). Briefly, the rats were anesthetized with diethyl ether and killed by cardiac puncture. The submandibular salivary glands were removed and dissected free from the surrounding connective tissue and sublingual glands. The glands were minced and digested with 60 U/mL of collagenase for 1 hr in a HEPES-buffered physiological solution containing (in mM): NaCl 130, KCl 4.5, NaH₂PO₄ 1.0, MgSO₄ 1.0, CaCl₂ 1.5, HEPES-Na 10, HEPES free acid 10, D-glucose 10, Minimum Eagle’s Medium (MEM) amino acids (X1), 1% bovine serum albumin (BSA), and glutamine 2 (adjusted to pH 7.4, gassed with 100% O₂). The glands were mechanically dissociated by gentle pipetting at 20-minute intervals during digestion. The resulting suspensions of acini were filtered through a nylon mesh and washed twice.

To deplete secretory granules, isoproterenol (20 mg/kg) was given to the rats by intraperitoneal injection as described previously (Martinez et al., 1996), and the acini were obtained 2 hrs after the injection by the same method described above.

The project was approved by the Animal Ethics Committee of the Yonsei University Medical Center.

Fura-2 Loading and [Ca²⁺]_c Measurements
Acini were loaded with fura-2 by incubation with 2 µM acetoxymethyl ester of fura-2 (fura-2/AM) in HEPES-buffered solution for 30 min at room temperature under 100% O₂. They were then washed twice and re-suspended in a HCO₃^--buffered solution containing (in mM): NaCl 110, KCl 4.5, NaH₂PO₄ 1.0, MgSO₄ 1.0, CaCl₂ 1.5, NaHCO₃ 25, HEPES-Na 5, HEPES free acid 5, and D-glucose 10 (equilibrated with 95% O₂, 5% CO₂ to give a pH of 7.4). The cells became attached to the coverslip forming the base of the cell chamber. [Ca²⁺]_c was measured on the stage of an inverted microscope by spectrofluorometry (Photon Technology International, Brunswick, NJ, USA). Cells were exposed to excitation wavelengths of 340 nm and 380 nm at two-second intervals, and the emitted fluorescence was measured at 510 nm with a photomultiplier tube. The values of [Ca²⁺]_c were calculated according to the equation previously described (Grynkiewicz et al., 1985). To prepare the Ca²⁺-free solutions, we omitted Ca²⁺ from the perfusate and added 1 mM ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). All experiments were carried out at 37°C.

Results are presented as means + SEM. The statistical significance of differences between averaged data was assessed by unpaired Student’s t tests.

Materials
BSA, MEM amino acids, dimethyl sulfoxide (DMSO), staurosporine, chelerythrine, H-7, H-89, calphostin C, glutamine, thapsigargin, carbachol (CCh), ionomycin, monensin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), isoproterenol, EGTA, and collagenase (type IV) were purchased from Sigma (St. Louis, MO, USA). K252a was purchased from Calbiochem (San Diego, CA, USA). Fura-2/AM was obtained from Molecular Probes (Eugene, OR, USA) and was made up as a 2-mM stock solution in DMSO.

	RESULTS

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Staurosporine Induced a [Ca²⁺]_c Increase by Mobilizing Ca²⁺ from a Non-ER Compartment
Cells were first stimulated with CCh, and perfusate Ca²⁺ was removed in the continued presence of CCh to deplete Ca²⁺ from the ER, the IP₃-sensitive intracellular Ca²⁺ store. As shown in Fig. 1A, stimulation of acini with 10 µM CCh led to an immediate rise in [Ca²⁺]_c from 44.4 ⁺ 3.8 nM to 159.5 ⁺ 13.1 nM (n = 9), followed by a slow decrease. Withdrawal of Ca²⁺ from the perfusate resulted in a rapid decrease in [Ca²⁺]_c to the basal level. However, the subsequent addition of 200 nM staurosporine to the perfusate induced a gradual increase in [Ca²⁺]_c, which reached a peak value of 170.4 ⁺ 39.1 nM (n = 9) approximately 60 min after the addition of staurosporine. In some cases, a rapid decrease of individual fluorescence intensities at 340 and 380 nm was observed some time after [Ca²⁺]_c reached a maximum level, suggesting that prolonged elevation of [Ca²⁺]_c by staurosporine stimulation was toxic to the cells (data not shown). Without the addition of staurosporine, [Ca²⁺]_c remained unchanged (n = 4, Fig. 1B). These results indicated that 200 nM staurosporine caused Ca²⁺ release from a non-ER compartment in rat submandibular acinar cells.

View larger version (18K): [in this window] [in a new window]   Figure 1. Staurosporine released Ca2+ from the IP3-independent Ca2+ store in rat submandibular acinar cells. (A) After the application of 10 µM carbachol, perfusate Ca2+ was removed, and 200 nM staurosporine was added in the continued presence of carbachol (n = 9). (B) As a control experiment, [Ca2+]c was measured without the addition of staurosporine (n = 4). Fura-2-loaded cells were stimulated at the times indicated by the bars.

View larger version (22K): [in this window] [in a new window]   Figure 2. Non-mitochondrial, acidic compartment played a major role in the staurosporine-induced [Ca2+]c increase in rat submandibular acinar cells. Fura-2-loaded cells were stimulated with 10 µM carbachol, and perfusate Ca2+ was removed. Cells were then exposed to 100 nM ionomycin (n = 5; A) or 5 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; n = 4; B). Ionomycin and FCCP induced only small increases in [Ca2+]c, indicating that mitochondria did not contain sufficient Ca2+ to support staurosporine-induced [Ca2+]c increases. When cells were exposed to 10 µM monensin in the presence of 100 nM ionomycin, there was a large increase in [Ca2+]c (n = 9; C), suggesting that the acidic subcellular compartment contained a large amount of Ca2+. (D) Cells were stimulated with 10 µM carbachol, and perfusate Ca2+ was removed. Cells were then exposed to 200 nM staurosporine, causing the [Ca2+]c increase. When [Ca2+]c reached the maximum, 100 nM ionomycin was added, resulting in a decrease in [Ca2+]c to the basal level. Addition of 10 µM monensin in the presence of ionomycin did not induce an increase in [Ca2+]c, indicating that staurosproine released Ca2+ from the acidic compartment in rat submandibular acinar cells (n = 4).

To investigate whether this acidic compartment was responsible for the staurosporine-induced [Ca²⁺]_c increase, we treated cells with both ionomycin and monensin after [Ca²⁺]_c increase had reached a maximum level in response to staurosporine. As shown in Fig. 2D, 100 nM ionomycin caused a decrease in [Ca²⁺]_c toward the baseline, which was probably due to the rapid efflux of Ca²⁺. After [Ca²⁺]_c had plateaued, 10 µM monensin was added. In contrast to the large increase in [Ca²⁺]_c in response to ionomycin+monensin as shown in Fig. 2C, there was no change in [Ca²⁺]_c (n = 4). Furthermore, the addition of staurosporine after the treatment with ionomycin+monensin did not induce [Ca²⁺]_c increases (data not shown). These results suggest that staurosporine mobilizes Ca²⁺ from the Ca²⁺ store, which is sensitive to ionomycin+monensin, i.e., from the acidic compartments, such as the secretory granules.

Secretory Granules were the Source of Ca²⁺ for the Staurosporine-induced [Ca²⁺]_c Increase
To examine whether the secretory granules represent the acidic compartment in rat submandibular acinar cells, we obtained degranulated cells (see MATERIALS & METHODS) and stimulated them with ionomycin+monensin or staurosporine after Ca²⁺ was depleted from the IP₃-sensitive Ca²⁺ store in the absence of perfusate Ca²⁺. The magnitude of the ionomycin (100 nM) + monensin (10 µM)-induced [Ca²⁺]_c increase in degranulated cells was 22.3 ⁺ 3.7 (n = 5; Fig. 3A), which was significantly smaller compared with the value obtained in control acinar cells (p < 0.01). In addition to this, the 200-nM staurosporine-induced [Ca²⁺]_c increase was also significantly reduced in degranulated cells (37.4 ⁺ 4.7, n = 5, p < 0.01; Fig. 3B). These results suggest that secretory granules are responsible for the ionomycin+monensin- and staurosporine-induced [Ca²⁺]_c increases in rat submandibular acinar cells.

View larger version (18K): [in this window] [in a new window]   Figure 3. The acidic, staurosporine-releasable Ca2+ store was the secretory granules in rat submandibular acinar cells. The rats were treated with isoproterenol (20 mg/kg, i.p.), and the acinar cells were obtained 2 hrs later by means of a method described in MATERIALS & METHODS. Fura-2-loaded cells were stimulated with 10 µM carbachol, and the perfusate Ca2+ was removed. Cells were then treated with 100 nM ionomycin, followed by 10 µM monensin (A) or 200 nM staurosporine (B) at the times indicated by the bars. The sizes of [Ca2+]c increases induced by ionomycin+monensin and staurosporine observed in the degranulated cells were significantly smaller compared with those observed in control cells (Figs. 2C, 1A). Each trace is representative of five independent experiments.

View larger version (17K): [in this window] [in a new window]   Figure 4. Protein kinase C inhibitors, but not protein kinase A inhibitor, induced qualitatively similar increases in [Ca2+]c in rat submandibular acinar cells. Fura-2-loaded cells were stimulated with 10 µM carbachol, and perfusate Ca2+ was removed. Cells were then treated with 1 µM K252a (n = 3; A), 25 µM chelerythrine (n = 3; B), 400 µM H-7 (n = 3; C), 500 nM calphostin C (n = 5; D), and 1 µM H-89 (n = 4; E) at the times indicated by the bars.

	DISCUSSION

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Next we tested whether the IP₃-insensitive acidic Ca²⁺ store was responsible for the [Ca²⁺]_c increase induced by staurosporine. It has been well-established that ionomycin is not able to discharge acidic Ca²⁺ stores unless the pH gradient across the membrane of the acidic Ca²⁺ store has collapsed (Pressman and Fahim, 1982). Therefore, in the present study, we used monensin, a Na⁺/H⁺ exchanger, to "collapse" the pH gradient and found that ionomycin in combination with monensin released a large amount of Ca²⁺.

Having confirmed that the acidic Ca²⁺ store contained a large amount of Ca²⁺, we examined whether the acidic Ca²⁺ store was responsible for the staurosporine-induced [Ca²⁺]_c increase. If staurosporine releases Ca²⁺ from the acidic Ca²⁺ store, then the application of ionomycin+monensin would not increase [Ca²⁺]_c after staurosporine had depleted this store. Indeed, in our study, ionomycin+monensin did not increase [Ca²⁺]_c after the staurosporine-sensitive Ca²⁺ store was depleted, suggesting that staurosporine mobilized Ca²⁺ from the ionomycin+monensin-sensitive acidic Ca²⁺ store.

The subcellular localization of the acidic Ca²⁺ store responsible for the staurosporine-induced [Ca²⁺]_c increase was further examined. Previous reports have suggested that, among the acidic compartments, such as the lysosomal/endosomal compartment, the secretory granules, and the trans-Golgi network, the secretory granules are the major Ca²⁺ reservoir in most secretory cells, including rat submandibular acinar cells (Nicaise et al., 1992; Martinez et al., 1996; Scheenen et al., 1998). In support of this, analysis of our data showed that the [Ca²⁺]_c increase induced by ionomycin+monensin was greatly reduced in degranulated cells, suggesting that the secretory granules were the major acidic Ca²⁺ store in rat submandibular acinar cells. Furthermore, we also showed that the size of the staurosporine-induced [Ca²⁺]_c increase was significantly smaller in the degranulated cells. Therefore, the increase in [Ca²⁺]_c in response to staurosporine observed in our study was probably due to the release of Ca²⁺ from secretory granules.

The physiological significance of intragranular Ca²⁺ has not been fully elucidated. High Ca²⁺ concentrations in acidic compartments have long been suggested to play a role in packaging and processing of intravesicular content and in the regulation of the exocytosis process in many cell types, including salivary acinar cells (Lucy and Ahkong, 1986; Lang and Schleef, 1996; Scheenen et al., 1998). In addition, the presence of IP₃ receptors on the granular membrane, and thus the involvement of secretory granules in [Ca²⁺]_c regulation, has also been suggested in pancreatic acinar cells (Gerasimenko et al., 1996) but remains controversial (Muallem and Lee, 1997; Yule et al., 1997). In the present study, prolonged stimulation of cells with CCh, an IP₃-generating agonist, did not deplete Ca²⁺ from secretory granules, suggesting that secretory granules in rat submandibular acinar cells do not possess functional IP₃ receptors and thus do not seem to participate in the regulation of [Ca²⁺]_c in this cell type.

The mechanisms by which Ca²⁺ is transported and accumulated in the secretory granules are still unknown. The Ca²⁺-proton exchanger driven by the low pH inside the granules and the ATPase-dependent Ca²⁺ uptake system have been reported to be responsible for the maintenance of the high Ca²⁺ concentration in the secretory granules, although the exact nature of these systems has not been identified (Porter et al., 1991; Nicaise et al., 1992). In the present study, we provide evidence that protein kinase C might have a role in the maintenance of the intragranular Ca²⁺ concentration. If the activity of the transporters responsible for Ca²⁺ uptake into the granules is maintained by protein kinase C, then inhibition of the kinase may lead to Ca²⁺ leakage into cytosol. Since the exchange rate of Ca²⁺ across the granular membrane is slow (Pozzan et al., 1994), the inhibition of the Ca²⁺ uptake system will lose Ca²⁺ into cytosol slowly, as observed in this study. Furthermore, the presence of protein kinase C activities and kinase substrates was reported in rat parotid gland secretory granule membrane and was suggested to contribute to secretion (Dowd et al., 1987). Therefore, it might be possible that protein kinase C is involved in the maintenance of the Ca²⁺ transport activity and thus the maintenance of a high Ca²⁺ concentration in the secretory granules, which plays an important role in the exocytosis of the granules.

Staurosporine is one of the most potent inhibitors of protein kinase C, and it has been reported to increase [Ca²⁺]_c in many cell types by inhibiting protein kinase C (King and Rittenhouse, 1989; Winkler et al., 1990; Xu et al., 1993). However, staurosporine is not specific, and it has also been shown to increase [Ca²⁺]_c by mechanisms that are independent of protein kinase C (Wong et al., 1992; Turkson et al., 1994; Tojyo et al., 1995). Therefore, in this study, we used various protein kinase C inhibitors—such as K252a and chelerythrine, which inhibit the catalytic domain of protein kinase C, and H-7 and calphostin C, which inhibit the regulatory domain—to examine whether the staurosporine-induced [Ca²⁺]_c increase resulted from the inhibition of protein kinase C. All of the inhibitors induced qualitatively similar changes in [Ca²⁺]_c, although the magnitudes and the time courses of [Ca²⁺]_c increases were not identical. In contrast to the protein kinase C inhibitors, H-89, an inhibitor of protein kinase A, did not cause an increase in [Ca²⁺]_c. Analysis of these data suggests that the inhibition of protein kinase C might be responsible for the staurosporine-induced Ca²⁺ release from secretory granules.

We therefore conclude that staurosporine mobilizes Ca²⁺ from secretory granules, probably by the inhibition of protein kinase C in rat submandibular acinar cells.

	ACKNOWLEDGMENTS

 
This study was supported by a grant of the Korea Health 21 R&D Project (01-PJ5-PG3-20507-0098), Ministry of Health & Welfare, Republic of Korea.

	FOOTNOTES

 
^* authors contributing equally to this work;

Received for publication November 7, 2001. Revision received August 19, 2002. Accepted for publication September 10, 2002.

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Journal of Dental Research, Vol. 81, No. 11, 788-793 (2002)
DOI: 10.1177/154405910208101113


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