The physiological role and the control mechanism of Ca^<2+>-mobilization in stimulation-secretion coupling in parotid gland

Carbachol （CCh）是一种胆碱能激动剂，可增加大鼠腮腺腺泡细胞的胞浆游离钙浓度（[Ca^<2+>]_i）和淀粉酶释放，并呈剂量依赖性。用细胞内Ca^<2+>拮抗剂TMB-8或细胞内Ca^<2+>螯合剂BAPTA处理腺泡细胞，可强烈减弱CCh引起的[Ca^<2+>]_i的增加，但不显著抑制淀粉酶的释放。在细胞悬液中联合添加Ca^<2+>离子载体离子霉素和微粒体atp酶抑制剂thapsigargin可显著增加[Ca^<2+>]_i，但对淀粉酶释放的影响远小于CCh。当细胞悬液中加入ATP时，观察到[Ca^<2+>]_i迅速升高。这种[Ca^<2+>]_i反应不太可能是由PI击穿介导的，因为ATP对IP_3的形成几乎没有影响。尽管[Ca^<2+>]_i明显增加，但淀粉酶释放不受细胞外ATP的诱导。蛋白激酶C激活剂PMA刺激淀粉酶释放的量与CCh诱导的相似。Staurosporine，一种蛋白激酶C抑制剂，同样抑制cch和tpa诱导的淀粉酶释放。这些结果表明，[Ca^<2+>]_i的增加在毒蕈碱刺激下的淀粉酶释放中并不起重要作用。淀粉酶的释放可能主要由蛋白激酶c的激活介导，另一方面，用TMB-8或BAPTA处理细胞强烈抑制cch诱导的K^+释放。离子和ThG的联合添加引起K^+的显著释放，但PMA不影响基础K^+释放或增强cch诱导的K^+释放。这些结果表明，cch诱导的K^+释放是由[Ca^<2+>]_i的快速增加介导的，而与蛋白激酶C的激活无关

Carbachol (CCh), a cholinergic agonist, increased both cytosolic free calcium concentration ([Ca^<2+>]_i) and amylase release in rat parotid acinar cells in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca^<2+> antagonist TMB-8 or the intracellular Ca^<2+> chelator BAPTA strongly attenuated the increase in [Ca^<2+>]_i evoked by CCh, but did not significantly suppress amylase release. A combined addition of the Ca^<2+> ionophore ionomycin and the microsomal ATPase inhibitor thapsigargin to cell suspension caused a noticeable increase in [Ca^<2+>]_i, but the effect on amylase release was much smaller than that of CCh. When ATP was added to cell suspension, a rapid elevation of [Ca^<2+>]_i was observed. This [Ca^<2+>]_i response is unlikely to be mediated by PI breakdown, because ATP had little or no effect on IP_3 formation. Despite the marked increase in [Ca^<2+>]_i, amylase release was not induced by extracellular ATP. The protein kinase C activator PMA stimulated amylase release in quantities similar to those induced by CCh. Staurosporine, a protein kinase C inhibitor, similarly inhibited both the CCh-and TPA-induced amylase release. These results suggest that an increase in [Ca^<2+>]_i does not play an essential role in amylase release by muscarinic stimulation. The amylase release may be primarily mediated by activation of protein kinase c.On the other hand, treatment of cells with TMB-8 or BAPTA strongly suppressed the CCh-induced K^+ release. A combined addition of Iono and ThG caused a marked release of K^+, but PMA did not affect basal K^+ release or potentiate the CCh-induced K^+ release. These results indicate that the CCh-induced K^+ release is mediated by a rapid increase in [Ca^<2+>]_i but is not associated with activation of protein kinase C