Picher Maryse, Burch Lauranell H, Boucher Richard C
Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina, 7010 Thurston-Bowles Building, Chapel Hill, NC 27599, USA.
J Biol Chem. 2004 May 7;279(19):20234-41. doi: 10.1074/jbc.M400305200. Epub 2004 Mar 1.
Extracellular nucleotides are among the most potent mediators of mucociliary clearance (MCC) in human lungs. However, clinical trials revealed that aerosolized nucleotides provide only a transient improvement of MCC to patients diagnosed with cystic fibrosis (CF). In this study, we identified the mechanism that eliminates extracellular nucleotides from human airways. Polarized primary cultures of human bronchial epithelial cells were impermeable to extracellular nucleotides but rapidly dephosphorylated ATP into ADP, AMP, and adenosine. The half-life of a therapeutic ATP concentration (0.1 mm) was approximately 20 s within the periciliary liquid layer. The mucosal epithelial surface eliminated P2 receptor agonists (ATP = UTP > ADP > UDP) at 3-fold higher rates than the serosal surface. We also showed that mucosal (not serosal) ectoATPase activity increases toward areas most susceptible to airway obstruction (nose < bronchi << bronchioles). Bronchial cultures from patients with CF, primary ciliary dyskinesia, or alpha1-antitrypsin deficiency exhibited 3-fold higher mucosal (not serosal) ectoATPase activity than normal cultures. Time course experiments indicated that CF enhances ATP elimination and adenosine accumulation on the mucosal surface. Furthermore, nonspecific alkaline phosphatase was identified as the major regulator of airway nucleotide concentrations in CF, primary ciliary dyskinesia, and alpha1-antitrypsin deficiency. The ectoAT-Pase activity and mRNA expression of mucosally restricted nonspecific alkaline phosphatase were 3-fold higher on bronchial cultures from these patients than from healthy subjects. This study demonstrates that the duration of nucleotide-mediated MCC is limited by epithelial ectonucleotidases throughout human airways, with the efficiency of this mechanism enhanced in chronic inflammatory lung diseases, including CF.
细胞外核苷酸是人类肺部黏液纤毛清除（MCC）最有效的介质之一。然而，临床试验表明，雾化核苷酸仅能使被诊断为囊性纤维化（CF）的患者的MCC得到短暂改善。在本研究中，我们确定了从人类气道清除细胞外核苷酸的机制。人支气管上皮细胞的极化原代培养物对细胞外核苷酸是不可渗透的，但能迅速将ATP去磷酸化为ADP、AMP和腺苷。在纤毛周围液体层中，治疗性ATP浓度（0.1 mM）的半衰期约为20秒。黏膜上皮表面清除P2受体激动剂（ATP = UTP > ADP > UDP）的速率比浆膜表面高3倍。我们还表明，黏膜（而非浆膜）外切ATP酶活性朝着最易发生气道阻塞的区域增加（鼻腔 < 支气管 << 细支气管）。CF、原发性纤毛运动障碍或α1抗胰蛋白酶缺乏症患者的支气管培养物显示，其黏膜（而非浆膜）外切ATP酶活性比正常培养物高3倍。时间进程实验表明，CF增强了ATP在黏膜表面的清除和腺苷的积累。此外，非特异性碱性磷酸酶被确定为CF、原发性纤毛运动障碍和α1抗胰蛋白酶缺乏症患者气道核苷酸浓度的主要调节因子。这些患者支气管培养物中黏膜限制性非特异性碱性磷酸酶的外切ATP酶活性和mRNA表达比健康受试者高3倍。本研究表明，核苷酸介导的MCC持续时间受到整个人类气道上皮外核苷酸酶的限制，在包括CF在内的慢性炎症性肺病中，这种机制的效率会增强。
