Levin Marc H, Sullivan Shannon, Nielson Dennis, Yang Baoxue, Finkbeiner Walter E, Verkman A S
Department of Medicine, University of California, San Francisco, California 94143-0521, USA.
J Biol Chem. 2006 Sep 1;281(35):25803-12. doi: 10.1074/jbc.M604332200. Epub 2006 Jul 7.
Recent data indicate the clinical benefit of nebulized hypertonic saline in cystic fibrosis lung disease, with a proposed mechanism involving sustained increase in airway surface liquid volume. To account for the paradoxical observation that amiloride suppresses the beneficial effect of hypertonic saline, it has been previously concluded (Donaldson, S. H., Bennett, W. D., Zeman, K. L., Knowles, M. R., Tarran, R., and Boucher, R. C. (2006) N. Engl. J. Med. 354, 241-250) that amiloride-inhibitable aquaporin (AQP) water channels in airway epithelia modulate airway surface liquid volume. Here, we have characterized water permeability and amiloride effects in well differentiated, primary cultures of human airway epithelial cells, stably transfected Fisher rat thyroid epithelial cells expressing individual airway/lung AQPs, and perfused mouse lung. We found high transepithelial water permeability (P(f), 54 +/- 5 microm/s) in airway epithelial cells that was weakly temperature-dependent and inhibited by >90% by reduced pH in the basal membrane-facing solution. Reverse transcription-PCR and immunofluorescence suggested the involvement of AQPs 3, 4, and 5 in high airway water permeability. Experiments using several sensitive measurement methods indicated that amiloride does not inhibit water permeability in non-cystic fibrosis (non-CF) or CF airway epithelia, AQP-transfected Fisher rat thyroid cells, or intact lung. Our data provide evidence against the mechanism proposed by Donaldson et al. to account for the effects of amiloride and hypertonic saline in CF lung disease, indicating the need to identify alternate mechanisms.
近期数据表明雾化高渗盐水对囊性纤维化肺病具有临床益处，其推测机制涉及气道表面液体量的持续增加。为了解释氨氯地平抑制高渗盐水有益作用这一矛盾现象，此前已有研究得出结论（唐纳森，S.H.，贝内特，W.D.，泽曼，K.L.，诺尔斯，M.R.，塔兰，R.，以及鲍彻，R.C.（2006年）《新英格兰医学杂志》354卷，241 - 250页），即气道上皮细胞中氨氯地平可抑制的水通道蛋白（AQP）水通道调节气道表面液体量。在此，我们对人气道上皮细胞的高分化原代培养物、稳定转染表达单个气道/肺AQP的Fisher大鼠甲状腺上皮细胞以及灌注的小鼠肺中的水通透性和氨氯地平作用进行了表征。我们发现气道上皮细胞具有高跨上皮水通透性（P(f)，54±5微米/秒），其对温度的依赖性较弱，且在面向基底膜的溶液中pH降低时，水通透性被抑制>90%。逆转录 - PCR和免疫荧光表明AQP 3、4和5参与了高气道水通透性。使用多种灵敏测量方法的实验表明，氨氯地平在非囊性纤维化（非CF）或CF气道上皮、AQP转染的Fisher大鼠甲状腺细胞或完整肺中均不抑制水通透性。我们的数据提供了证据，反驳了唐纳森等人提出的关于氨氯地平及高渗盐水在CF肺病中作用机制的观点，表明需要确定其他机制。
