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Original Article

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

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

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, 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- (TNF). 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.

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