Fundamental research for an inhalational therapy using hyposmotic solution to stimulate airway ciliary motility

采用视频增强对比显微镜（VEC）测量大鼠气管纤毛细胞在薄片制备中的纤毛搏动频率（CBF）。乙酰胆碱（ACh）或ATP通过细胞内Ca^<2+>浓度（[Ca^<2+>]_i）以剂量依赖的方式增加CBF。适当的低渗透应激（-40mosM）增强了疼痛刺激的气管纤毛细胞CBF的增加。本研究表明，舒明（一种嘌呤能受体抑制剂）和apyrase（一种ATP酶/ADPase）消除了乙酰胆碱刺激的CBF增加的低渗透增强作用，ATP增强了乙酰胆碱刺激的[Ca^<2+>] i升高和CBF增加。低渗透胁迫（-40mosM）瞬间增加了[Ca^<2+>]_i，增强了ach刺激的[Ca^<2+>]_i的增加。在无Ca^<2+>的条件下，未检测到乙酰胆碱刺激的CBF增加的低渗透增强。这些观察结果表明，低渗透应激刺激ATP从气管释放。释放的ATP可诱导乙酰胆碱刺激的气管纤毛细胞[Ca^<2+>]_i和CBF进一步升高，这可能是由嘌呤能受体介导的。另一方面，与气管不同，在远端气道纤毛细胞（肺片）中，ACh或ATP没有增加CBF和[Ca^<2+>]_i，尽管不含Ca^<2+>的溶液降低了CBF，而离子霉素（5μM）或thapsigarin （2μ m）增加了CBF。特布他林（10μM）是一种选择性β_2-肾上腺素能激动剂，可增加气管和远端气道纤毛细胞的CBF。这些观察结果表明，Ca^<2+>-通过远端气道纤毛细胞嘌呤能受体或毒蕈碱受体的动员机制可能与气管纤毛细胞的动员机制不同。因此，CBF的增加在大鼠气管和远端气道上皮中受到不同的调节。

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) or ATP increased CBF mediated via intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in a dose-dependent manner. An adequate hypo-osmotic stress (-40mosM) potentiated ACh-stimulated CBF increase in tracheal ciliary cells. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP potentiates ACh-stimulated rises in [Ca^<2+>]_i and CBF increase. A hypo-osmotic stress (-40mosM) transiently increased [Ca^<2+>]_i and potentiated the ACh-stimulated [Ca^<2+>]_i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca^<2+>-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca^<2+>]_i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors. On the other hand, in distal airway ciliary cells (lung slice) unlike trachea, ACh or ATP did not increase CBF and [Ca^<2+>]_i, although a Ca^<2+>-free solution decreased CBF, and ionomycin (5μM) or thapsigargin (2μM) increased it. Terbutaline (10μM), a selective β_2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca^<2+>-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. Thus, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.