Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis
Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis
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DOI：
10.1074/jbc.m809161200
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发表时间：
2009-03-20
影响因子：
4.8
通讯作者：
Verkman, A. S.
中科院分区：
文献类型：
--
作者：
Haggie, Peter M.;Verkman, A. S.
CFTR突变导致囊性纤维化（CF）肺部疾病的机制仍不确定。Teichgraber等人最近报道了CF肺中神经酰胺的增加，这被认为是由于气道上皮细胞中溶酶体酸化缺陷和随后的pH依赖性神经酰胺代谢酶的损伤引起的（Teichgraber，V.，Ulrich，M.，Endlich，N.，Reithmuller，J.，Wilker，B.，Conceicao Ce Olivereira-Munding，C.，货车Heeckeren，A. M.，巴尔，M. L.，von Kurthy，G.，Schmid，K. W.的，韦勒，M.，Tummler，B.，Lang，F.，Grassme，H.，Doring，G.，和Gulbins，E.（2008）Nat.Med.14，382-391）。在这里，我们测量了几个CFTR表达和缺陷的细胞系，新鲜分离的气道上皮细胞从非CF和CF小鼠和人类，以及分化良好的原代培养的人非CF和CF气道上皮细胞的溶酶体pH值。使用荧光pH指示剂通过比率成像测量溶酶体pH，该指示剂由与俄勒冈州绿色488缀合的40-kDa右旋糖酐和四甲基罗丹明组成。在所有细胞类型中，溶酶体pH类似于4.5，不受噻唑烷酮CFTR抑制剂CFTRinh-172的影响，并且在巴弗洛霉素抑制液泡质子泵后增加至类似于6.5。非CF与CF人或小鼠气道上皮细胞的溶酶体pH值无显著差异。我们的结果提供了直接证据，反驳了Teichgraber等人的结论，即溶酶体酸化是CFTR依赖性的、CF受损的或导致神经酰胺累积的。因此，需要其他机制来解释CF气道中神经酰胺的增加。非CFTR机制，如ClC型氯离子通道，可能参与维持细胞器酸化过程中的电中性。
The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgraber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgraber, V., Ulrich, M., Endlich, N., Reithmuller, J., Wilker, B., Conceicao Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kurthy, G., Schmid, K. W., Weller, M., Tummler, B., Lang, F., Grassme, H., Doring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was similar to 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTRinh-172, and increased to similar to 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgraber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.