Characterization of the calcium signaling system in the submandibular cellline SMG-C6

Establishment of salivary cell lines retaining normal morphological and phy siological characteristics is important in the investigation of salivary ce ll function. A submandibular gland cell line, SMG-C6, has recently been est ablished. 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 t o muscarinic, alpha (1)-adrenergic, P2Y-nucleotide, and cytokine receptor a gonists were determined. Ca2+ release from intracellular stores was strongl y stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA) , epidermal growth factor (EGF), interleukin-6 (IL-6), and tumor necrosis f actor-alpha (TNF alpha), Consistently, 1,4,5-IP3, formation was dramaticall y stimulated by ACh and ATP, ACh-stimulated cytosolic free Ca2+ concentrati on [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 C a2+ store, and a subsequent exposure to thapsigargin (TG) induced further [ Ca2+](i) increase. However, exposure to TG depleted the store and a subsequ ent 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, sugges ting that IP3-insensitive stores exist in SMG-C6 cells. Ca2+ influx was act ivated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggest ing the involvement of store-operated Ca2+ entry (SOCE) pathway. These resu lts indicate that in SMG-C6 cells: (i) Ca2+ release is triggered by muscari nic and P2Y-nucleotide receptor agonists through formation of IP3; (ii) bot h the IP3-sensitive and -insensitive Ca2+ stores are present; end (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 ce ll model in terms of intracellular Ca2+ signaling.