Am. J. Respir. Cell Mol. Biol., Volume 18, Number 1, January, 1998 32-42

Neutrophil Elastase Promotes Rapid Exocytosis in Human Airway Gland Cells by Producing Cytosolic Ca²⁺ Oscillations

Michaël Maizieres, Hervé Kaplan, Jean Marc Millot, Noël Bonnet, Michel Manfait, Edith Puchelle, and Jacky Jacquot

INSERM U.314, Reims Cedex, France; and Laboratoire de Spectroscopie Biomoléculaire, UFR de Pharmacie, Université de Reims, IFR 53, Reims Cedex, France

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca²⁺ concentration [Ca²⁺]_i can promote different exocytotic responses, we monitored dynamic changes in [Ca²⁺]_i and secretory granule (SG) exocytosis in 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 neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca²⁺]_i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 µM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 µM histamine (10% decrease) or 100 µM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca²⁺]_i induced by histamine (1.0-100 µM) and ATP (10-100 µM), HNE (0.01-1 µM) generated asynchronous oscillations in [Ca²⁺]_i over the first 8-min period. Depletion of internal Ca²⁺ stores with thapsigargin (500 nM) induced 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 extracellular 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 extracellular Ca²⁺ and [Ca²⁺]_i oscillations rather than the absolute level of [Ca²⁺]_i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca²⁺]_i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

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