Decline in stimulus responsiveness of secretory granules in salivary glands with age

Abstract

Secretory granules produced by salivary acinar cells accumulate if secretory stimulation is suppressed. Aged and deteriorated secretory granules can cause tissue damage because of abnormal secretion and/or intracellular leakage. To elucidate the deterioration process, we characterized the changes in the stimulus responsiveness of secretory granules using HaloTag technology. We established a system in which HaloTag-fused cystatin D, a salivary protein, was transported to the secretory granules of rat parotid acinar cells in primary culture. HaloTags can be labeled with cell-permeable ligands conjugated to fluorescent dyes in living cells. To observe the new and old secretory granules, the cells were labeled with two HaloTag ligands conjugated to different fluorescent dyes at different times. We measured the secretion rates of newly synthesized and old HaloTag-fused proteins in the absence and presence of isoproterenol, a β-adrenergic agonist, at 3 and 6 h after initial labeling. Sequential labeling of HaloTag-fused proteins with two different dyes enabled the discrimination between new and old secretory granules. The secretory responsiveness of the proteins synthesized within 3 h to isoproterenol was higher than that of proteins synthesized earlier. However, there was no significant difference in the responsiveness between the new and old proteins at 6 h after initial labeling. New secretory granules have a higher sensitivity to stimulants than older ones and that their response declines over time.