The Physiological Role of Calmodulin in Rat Parotid Amylase Release

利用分散的大鼠腮腺细胞，观察了三种钙调素拮抗剂三氟拉嗪（TFP） W-7和W-5对淀粉酶释放和腺泡细胞结构的影响。TFP和W-7对异丙肾上腺素（ISO）或dbcamp刺激的淀粉酶释放均有较强的抑制作用，而弱钙调素拮抗剂W-5作用不大。即使在TFP和W-7存在的情况下，ISO也显著提高了环AMP水平。电子显微镜显示，用TFP或W-7处理腮腺细胞会导致管腔微绒毛和表面褶皱的丢失。当细胞在TFP或W-7存在的情况下被ISO刺激时，增大的管腔没有恢复到原来的大小，排出的分泌物质保留在管腔中。结构变化部分类似于细胞松弛素D（一种微丝破坏剂）引起的变化。W-5对腺泡细胞结构影响不大。Ca^<2+>预处理显著增强了ISO和DBcAMP诱导的淀粉酶释放。用Ca^<2+>螯合剂EGTA和BAPTA-AM预处理细胞内Ca^<2+>，减少了淀粉酶的释放。采用荧光Ca^<2+>指示剂fura-2检测ISO对胞质游离Ca^<2+>([Ca^<2+>]i)的影响。当浓度高达1mm时，ISO引起[Ca^<2+>]i的快速增加，而1mum ISO刺激最大淀粉酶释放，影响不大。由于α -肾上腺素受体拮抗剂酚妥拉明比β -肾上腺素受体拮抗剂更能阻断iso诱导的[Ca^<2+>]i的增加，因此[Ca^<2+>]i的增加是由于α -肾上腺素受体而不是β -肾上腺素受体的激活。本研究提示钙调素通过调控细胞骨架系统参与腮腺淀粉酶的胞吐。即使在[Ca^<2+>]i的静息水平下，钙调素也可能具有功能。

Using dispersed rat parotid cells, the effects of three calmodulin antagonists trifluoperazine (TFP), W-7 and W-5, on amylase release and acinar cell structure were examined. TFP and W-7 strongly inhibited both isoproterenol (ISO)- or DBcAMP-stimulated amylase release at a concentration of 50 or 100 muM, while W-5, a weak calmodulin antagonist, had a little effect. Cyclic AMP level was markedly elevated by ISO even in the presence of TFP and W-7.Electron micrographs demonstrated that treatment of parotid cells with either TFP or W-7 caused a loss of luminal microvilli and surface folds. When cells were stimulated by ISO in the presence of TFP or W-7, the enlarged lumina did not recover to their original size and the discharged secretory material was retained in the lumina. The structural changes were similar, in part, to those evoked by cytochalasin D, a microfilament disrupting agent. W-5 affected the acinar cell structure only a little.Amylase release induced by ISO or DBcAMP was significantly enhanced by pretreatment with Ca^<2+>. Depletion of intracellular Ca^<2+> by pre-treatment with the Ca^<2+>-chelators EGTA and BAPTA-AM reduced the amylase release.The effects of ISO on cytosolic free Ca^<2+>([Ca^<2+>]i)were examined using the fluorescent Ca^<2+>-indicator fura-2. At concentrations up to 1 mM, ISO caused a rapid increase in [Ca^<2+>]i, while 1 muM ISO, which stimulates the maximum amylase release, had little effect. Since the alpha-adrenoceptor antagonist phentolamine blocked the ISO-induced increasg+in [Ca^<2+>]i better than the beta-adrenoceptor antagonist, the increase in [Ca^<2+>]i is due to an activation of alpha-adrenoceptors rather than beta-adrenoceptors.This study suggests that calmodulin is involved in the exocytosis of parotid amylase through the regulation of the cytoskeletal system. Calmodulin may be functional even at resting levels of [Ca^<2+>]i.