Shin J-H, Son E J, Lee H S, Kim S J, Kim K, Choi J Y, Lee M G, Yoon J-H
The Airway Mucus Institute, Yonsei University College of Medicine, Seoul, Korea.
Acta Physiol (Oxf). 2007 Oct;191(2):99-110. doi: 10.1111/j.1748-1716.2007.01731.x. Epub 2007 Jul 17.
Anions have an important role in the regulation of airway surface liquid (ASL) volume, viscosity and pH. However, functional localization and regulation of anion exchangers (AEs) have not been clearly described. The aim of this study was to investigate the regulation of AE mRNA expression level in accordance with mucociliary differentiation and the functional expression of AEs cultured normal human nasal epithelial (NHNE) cells.
Nasal mucosal specimens from three patients are obtained and serially cultured cells are subjected to morphological examinations, RT-PCR, Western blot analysis and immunocytochemistry. AE activity is assessed by pHi measurements.
Expression of ciliated cells on the apical membrane and expression of MUC5AC, a marker of mucous differentiation, increased with time. AE2 and SLC26A4 mRNA expression decreased as mucociliary differentiation progressed, and AE4, SLC26A7 and SLC26A8 mRNA expression increased on the 14th and 28th day after confluence. Accordingly, AE4 protein expression also progressively increased. AE activity in 100 mM K(+) buffer solutions was nearly twofold higher than that in 5 mM K(+) buffer solutions. Moreover, only luminal AE activity increased about fourfold over the control in the presence of 5 microM forskolin. In the presence of 100 microM adenosine-5'-triphosphate (ATP) which evokes intracellular calcium signalling through activation of purinergic receptors, only luminal AE activity was again significantly increased. On the other hand, 500 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of most SLC4 and SLC26AE isoforms, nearly abolished AE activity in both luminal and basolateral membranes. We found that AE activity was affected by intracellular cAMP and calcium signalling in the luminal membrane and was DIDS-sensitive in both membranes of cultured NHNE cells.
Our findings through molecular and functional studies using cultured NHNE cells suggest that AEs may have an important role in the regulation of ASL.
阴离子在气道表面液体（ASL）的容量、黏度及pH调节中发挥重要作用。然而，阴离子交换蛋白（AEs）的功能定位和调节尚未得到清晰描述。本研究旨在探讨正常培养的人鼻上皮（NHNE）细胞中，AEs的mRNA表达水平随黏液纤毛分化的调节情况以及AEs的功能表达。
获取3例患者的鼻黏膜标本，对连续培养的细胞进行形态学检查、逆转录聚合酶链反应（RT-PCR）、蛋白质印迹分析及免疫细胞化学检测。通过测量细胞内pH（pHi）评估AE活性。
随着时间推移，顶端膜上纤毛细胞的表达以及黏液分化标志物MUC5AC的表达增加。随着黏液纤毛分化进展，AE2和SLC26A4 mRNA表达下降，而汇合后第14天和第28天，AE4、SLC26A7和SLC26A8 mRNA表达增加。相应地，AE4蛋白表达也逐渐增加。在100 mM K(+)缓冲液中的AE活性比在5 mM K(+)缓冲液中高出近两倍。此外，在存在5 μM福司可林的情况下，仅管腔AE活性比对照增加约四倍。在存在100 μM腺苷-5'-三磷酸（ATP）（通过激活嘌呤能受体引发细胞内钙信号传导）的情况下，仅管腔AE活性再次显著增加。另一方面，500 μM 4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）（大多数SLC4和SLC26AE亚型的抑制剂）几乎消除了管腔和基底外侧膜中的AE活性。我们发现，NHNE细胞培养物中管腔膜的AE活性受细胞内cAMP和钙信号传导影响，且在两种膜中均对DIDS敏感。
我们使用培养的NHNE细胞进行分子和功能研究的结果表明，AEs可能在ASL调节中发挥重要作用。
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