Downs Charles A, Kreiner Lisa H, Johnson Nicholle M, Brown Lou Ann, Helms My N
1 Nell Hodgson Woodruff School of Nursing.
Am J Respir Cell Mol Biol. 2015 Jan;52(1):75-87. doi: 10.1165/rcmb.2014-0002OC.
The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91(phox) signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 ± 3.03 μg versus 2.48 ± 0.53 μg), lower sRAGE (1,205 ± 292 pg/ml versus 1,910 ± 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47(phox) and increased gp91(phox)-dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91(phox) abrogated the response.
晚期糖基化终末产物受体（RAGE）是免疫球蛋白家族的多配体成员，可能在肺液体平衡调节中起关键作用。我们对吸烟者和非吸烟者支气管肺泡灌洗液中的可溶性RAGE（sRAGE）、一种诱饵异构体以及晚期糖基化终末产物（AGEs）进行了定量，并检验了AGEs通过蛋白激酶C（PKC）-gp91（phox）信号传导至上皮钠通道（ENaC）来调节肺液体平衡的假说。吸烟者的人支气管肺泡灌洗样本显示AGEs增加（9.02±3.03μg对2.48±0.53μg），sRAGE降低（1205±292pg/ml对1910±263pg/ml），上皮衬液体积降低（97±14ml对133±17ml）。sRAGE水平在非吸烟者中不能预测ELF体积；然而，在吸烟者中，较高的sRAGE水平预示着较高的ELF体积。对大鼠肺泡Ⅰ型上皮细胞的单通道膜片钳分析显示，AGEs使以通道数量（N）与开放概率（Po）的乘积（NPo）衡量的ENaC活性从0.19±0.08增加至0.83±0.22（P = 0.017），随后添加4-羟基-2,2,6,6-四甲基哌啶-N-氧基使ENaC的NPo降低至0.15±0.07（P = 0.01）。在Ⅱ型细胞中，人AGEs使ENaC的NPo从0.12±0.05增加至0.53±0.16（P = 0.025），添加4-羟基-2,2,6,6-四甲基哌啶-N-氧基使ENaC的NPo降低至0.10±0.03（P = 0.013）。使用分子和生化技术，我们观察到抑制RAGE和PKC活性可减弱AGE诱导的ENaC激活。AGEs诱导p47（phox）磷酸化并增加gp91（phox）依赖性活性氧生成，RAGE或PKC抑制可消除该反应。最后，气管内注入AGEs可促进肺液体清除，而同时抑制RAGE、PKC和gp91（phox）可消除该反应。
