PDF(Pubmed)
Abstract：
Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.
摘要：
在人类结肠细胞系和鼠肠中的研究表明存在Ca2激活的阴离子通道，大概是TMEM16a。严重囊性纤维化跨膜传导调节因子（CFTR）突变的患者是否有可能通过激活该替代途径分泌液体？类似于转运扩增/祖细胞（TA/PE）细胞的二维非分化结肠-肌成纤维细胞共培养物，以及类似近表面细胞的分化单层（DM）培养物，从健康对照（HL）和CFTR基因（PwCF）严重功能缺陷的患者中建立。还研究了F508del突变体和CFTR敲除(空)小鼠回肠和结肠粘膜。HLTA/PE单层对UTP(100µM)显示出稳健的短路电流响应(ΔIeq),forskolin(Fsk,10µM）和卡巴胆碱（CCH，100µM)，而ΔIeq在分化单层中小得多。选择性TMEM16a抑制剂Ani9（高达30µM）不会改变对管腔UTP的反应，显著降低Fsk诱导的ΔIeq，HLTA/PE结肠样单层中CCH诱导的ΔIeq显著增加。PwCFTA/PE和PwCF分化的单层显示可忽略的激动剂诱导的ΔIeq，没有显著的Ani9效应。当TMEM16a位于细胞内结构时，未检测到根尖膜中的染色。TMEM16a在人类结肠样细胞单层中高度表达，类似于结肠隐颈区的转运扩增细胞，从HL和PwCF。虽然它可能在调节激动剂诱导的CFTR介导的阴离子电流中起作用，它不位于根尖膜，它在囊性纤维化（CF）和健康人类结肠上皮中没有作为顶端阴离子通道的功能。
