Delmotte Philippe, Sanderson Michael J
Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue, North Worcester, MA 01655, USA.
Am J Respir Cell Mol Biol. 2006 Jul;35(1):110-7. doi: 10.1165/rcmb.2005-0417OC. Epub 2006 Feb 16.
Ciliary beat frequency (CBF) is a key factor in the defense of the airways, and ATP can stimulate CBF by increasing intracellular calcium concentration ([Ca2+]i). However, the regulatory effects of ATP have been mainly studied in cultured or isolated epithelial cells from the large cartilaginous airways. The aim of this study was to evaluate the regulation of CBF in small airways of lung slices that are representative of in vivo tissue. Mice lungs were inflated with agarose and cut into thin slices with a vibratome. CBF in the small bronchioles was observed with differential interference contrast microscopy and quantified using high-speed digital imaging (at 240 images s(-1)). We found that the in situ organization of the ciliated cells was well preserved and that their CBF was high. We verified the fidelity of our recording system by analyzing rapid changes in CBF in response to temperature. However, we found that ATP had no effect on CBF, despite the fact that the [Ca2+]i, measured with confocal fluorescence imaging, was increased. Ionomycin and purinergic or beta-adrenergic agonists also failed to increase CBF. Similar results were obtained in outgrowths of cells cultured from lung slices. By contrast, ATP increased the slower CBF of outgrowths of ciliated cells cultured from tracheal rings. Therefore, we conclude that CBF in intrapulmonary airways of mice is maintained at a maximum rate and cannot be further increased by agonist stimulation. These conditions would ensure that mucociliary clearance is constantly active to provide continuous airway protection.
纤毛摆动频率（CBF）是气道防御的关键因素，ATP可通过增加细胞内钙浓度（[Ca2+]i）来刺激CBF。然而，ATP的调节作用主要是在来自大软骨气道的培养或分离上皮细胞中进行研究的。本研究的目的是评估在代表体内组织的肺切片小气道中CBF的调节情况。用琼脂糖对小鼠肺进行充气，并用振动切片机切成薄片。用微分干涉对比显微镜观察小支气管中的CBF，并使用高速数字成像（每秒240帧图像）进行量化。我们发现纤毛细胞的原位组织结构保存良好，其CBF较高。我们通过分析CBF对温度的快速变化来验证记录系统的准确性。然而，我们发现尽管用共聚焦荧光成像测量的[Ca2+]i增加了，但ATP对CBF没有影响。离子霉素以及嘌呤能或β-肾上腺素能激动剂也未能增加CBF。从肺切片培养的细胞生长物中也得到了类似的结果。相比之下，ATP增加了从气管环培养的纤毛细胞生长物中较慢的CBF。因此，我们得出结论，小鼠肺内气道中的CBF维持在最大速率，激动剂刺激不能进一步增加。这些情况将确保黏液纤毛清除持续活跃，以提供持续的气道保护。
