NEUTROPHIL ELASTASE PROMOTES RAPID EXOCYTOSIS IN HUMAN AIRWAY GLAND-CELLS BY PRODUCING CYTOSOLIC CA2+ OSCILLATIONS

The molecular and ionic mechanisms responsible for the regulation of m ucus exocytosis in human airway gland cells remain poorly defined, To determine whether dynamic changes of intracellular free Ca2+ concentra tion [Ca2+](i) can promote different exocytotic responses, we monitore d dynamic changes in [Ca2+](i) and secretory granule (SG) exocytosis i n individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human n eutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2](i) from single cells were determined with Indo-1/AM using quantitati ve UV laser microspectrofluorometry. The rate of SC exocytosis was mea sured in single cells by fluorescence videomicroscopy of SG degranulat ion and by the ELISA method. Exposure of HTC cells to a low concentrat ion of HNE (1.0 mu M) caused a high rate of SC exocytosis (52% decreas e in the initial quinacrine fluorescence) during the first S-min stimu lation period compared with that observed following exposure of tile c ells to 100 mu M histamine (10% decrease) or 100 mu M ATP (6% decrease ). In contrast to a rapid and transient rise in [Ca2+](i) induced by h istamine (1.0-100 mu M) and ATP (10-100 mu M), HNE (0.01-1 mu M) gener ated asynchronous oscillations in [Ca2+](i) over the first S-min perio d, Depletion of internal Ca2+ stores with thapsigargin (500 nM) induce d a significant reduction (P < 0.01) in the observed increases in [Ca2 +](i) upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extr acellular Ca2+(+5 mM EGTA) significantly reduced (P < 0.01) both the [ Ca2+](i) increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellul ar Ca2+ and [Ca2+](i) oscillations rather than the absolute level of [ Ca2+](i) regulate the rapid onset and extent of exocytotic responses t o HNE in airway gland cells. Taken together, these results provide str ong evidence that [Ca2+](i) is a critical intracellular messenger in t he regulation of exocytosis process in human airway gland cells.