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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;

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).

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.

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


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