Regulation of Ca2+ signals in a parotid cell line Par-C5

The Ca2+ signaling system in an established immortalized rat parotid acinar cell line, Par-C5, was examined using the Ca2+-sensitive fluorescent indicator fura-2 and by measuring inositol 1,4,5-trisphosphate (IP3) formation. Agonist-induced increase in intracellular Ca2+ ([Ca2+]i) by mobilization of intracellular stores and influx across the cell membrane was stimulated by acetylcholine (ACh) and ATP, whereas noradrenaline-(NA)-induced a small [Ca2+]i increase mediated primarily by release from intracellular Ca2+ stores. [Ca2+]i increase by ACh and ATP was meditated through the phosphoinositide signal pathway since both agonists significantly increased 1,4,5-IP3 formation and Ca2+ mobilization was abolished by the phospholipase C inhibitor U73122. In Ca2+-free medium, ACh or ATP discharged the IP3-sensitive Ca2+ store and essentially abolished subsequent [Ca2+]i response to thapsigargin (TG). Exposure to ionomycin and monensin after TG induced a further mobilization of Ca2+, suggesting IP3-insensitive stores are present. Furthermore, depletion of IP3-sensitive Ca2+ stores by TG, ACh and ATP enhanced plasmalemmal Ca2+-entry pathways. Exposure to tumor necrosis factor-α (TNF-α), a cytokine associated with lymphocyte invasion of salivary epithelial cells in autoimmune disorders, significantly reduced ACh-stimulated Ca2+ mobilization. TNF-α inhibitory effect on Ca2+ mobilization was not directly due to an interaction on muscarinic receptors since ACh-induced 1,4,5-IP3 formation was not altered. These results in the Par-C5 cell line indicate 1) [Ca2+]i is regulated by muscarinic and P2Y-nucleotide receptors and partly by α1-adrenergic receptors; 2) IP3-sensitive and -insensitive Ca2+ stores exist; 3) Ca2+ influx activated by ACh, ATP or TG is mediated by the store-operated Ca2+ entry pathway; and 4) muscarinic agonist-stimulated Ca2+ mobilization is altered by the cytokine TNF-α.