Maizieres M, Kaplan H, Millot J M, Bonnet N, Manfait M, Puchelle E, Jacquot J
INSERM U.314, and Laboratoire de Spectroscopie Biomoléculaire, UFR de Pharmacie, Université de Reims, France.
Am J Respir Cell Mol Biol. 1998 Jan;18(1):32-42. doi: 10.1165/ajrcmb.18.1.2841.
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 microM) 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 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) 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.
人类气道腺细胞中负责调节黏液胞吐作用的分子和离子机制仍不清楚。为了确定细胞内游离钙离子浓度[Ca2+]i的动态变化是否能促进不同的胞吐反应，我们监测了单个人类气管黏膜下浆液腺（HTG）细胞中[Ca2+]i和分泌颗粒（SG）胞吐作用的动态变化。这些变化是细胞暴露于与气道炎症和疾病相关的三种不同促分泌素后产生的反应：人类中性粒细胞弹性蛋白酶（HNE）、组胺和ATP。使用定量紫外激光显微荧光测定法，用Indo-1/AM测定单细胞中[Ca2+]i的动态变化。通过对SG脱颗粒的荧光视频显微镜观察和ELISA方法，在单细胞中测量SG胞吐作用的速率。与细胞暴露于100 microM组胺（降低10%）或100 microM ATP（降低6%）后观察到的情况相比，HTG细胞暴露于低浓度HNE（1.0 microM）在最初8分钟刺激期内导致SG胞吐作用速率较高（初始喹吖因荧光降低52%）。与组胺（1.0 - 100 microM）和ATP（10 - 100 microM）诱导的[Ca2+]i快速短暂升高相反，HNE（0.01 - 1 microM）在最初8分钟内使[Ca2+]i产生异步振荡。用毒胡萝卜素（500 nM）耗尽细胞内钙库，在添加每种促分泌素后，观察到的[Ca2+]i升高显著降低（P < 0.01），但对SG胞吐反应影响不大。有趣的是，去除细胞外钙（+5 mM EGTA）显著降低（P < 0.01）了暴露于促分泌素后[Ca2+]i的升高和SG胞吐作用的速率。我们还证明，细胞外钙的内流和[Ca2+]i振荡而非[Ca2+]i的绝对水平调节气道腺细胞对HNE胞吐反应的快速起始和程度。综上所述，这些结果提供了有力证据，表明[Ca2+]i是人类气道腺细胞胞吐作用调节过程中的关键细胞内信使。
