Paradiso Anthony M, Coakley Raymond D, Boucher Richard C
Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248, USA.
J Physiol. 2003 Apr 1;548(Pt 1):203-18. doi: 10.1113/jphysiol.2002.034447. Epub 2003 Jan 31.
The polarized distribution of HCO3- transport was investigated in human nasal epithelial cells from normal and cystic fibrosis (CF) tissues. To test for HCO3- transport via conductive versus electroneutral Cl-/HCO3- exchange (anion exchange, AE) pathways, nasal cells were loaded with the pH probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and mounted in a bilateral perfusion chamber. In normal, but not CF, epithelia, replacing mucosal Cl- with gluconate caused intracellular pH (pHi) to increase, and the initial rates (Delta pH min-1) of this increase were modestly augmented (approximately 26 %) when normal cells were pretreated with forskolin (10 microM). Recovery from this alkaline shift was dependent on mucosal Cl-, was insensitive to the AE inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS; 1.5 mM), but was sensitive to the cystic fibrosis transmembrane conductance regulator (CFTR) channel inhibitor diphenylamine-2-carboxylate (DPC; 100 microM). In contrast, removal of serosal Cl- caused pHi to alkalinize in both normal and CF epithelia. Recovery from this alkaline challenge was dependent on serosal Cl- and blocked by H2DIDS. Additional studies showed that serosally applied Ba2+ (5.0 mM) in normal, but not CF, cells induced influx of HCO3- across the apical membrane that was reversibly blocked by mucosal DPC. In a final series of studies, normal and CF cells acutely alkaline loaded by replacing bilateral Krebs bicarbonate Ringer (KBR) with Hepes-buffered Ringer solution exhibited basolateral, but not apical, recovery from an alkaline challenge that was dependent on Cl-, independent of Na+ and blocked by H2DIDS. We conclude that: (1) normal, but not CF, nasal epithelia have a constitutively active DPC-sensitive HCO3- influx/efflux pathway across the apical membrane of cells, consistent with the movement of HCO3- via CFTR; and (2) both normal and CF nasal epithelia have Na+-independent, H2DIDS-sensitive AE at their basolateral domain.
研究了正常组织和囊性纤维化（CF）组织来源的人鼻上皮细胞中HCO₃⁻转运的极化分布。为了通过传导性与电中性Cl⁻/HCO₃⁻交换（阴离子交换，AE）途径检测HCO₃⁻转运，将鼻细胞用pH探针2',7'-双（羧乙基）-5（6）-羧基荧光素加载，并安装在双侧灌注室中。在正常上皮而非CF上皮中，用葡萄糖酸盐替代黏膜Cl⁻会导致细胞内pH（pHi）升高，当正常细胞用福斯可林（10 μM）预处理时，这种升高的初始速率（ΔpH min⁻¹）适度增加（约26%）。从这种碱性转变中的恢复依赖于黏膜Cl⁻，对AE抑制剂4,4'-二异硫氰酸二氢芪-2,2'-二磺酸（H₂DIDS；1.5 mM）不敏感，但对囊性纤维化跨膜电导调节因子（CFTR）通道抑制剂二苯胺-2-羧酸盐（DPC；100 μM）敏感。相反，去除浆膜Cl⁻会导致正常和CF上皮中的pHi碱化。从这种碱性刺激中的恢复依赖于浆膜Cl⁻并被H₂DIDS阻断。额外的研究表明，在正常细胞而非CF细胞中，浆膜施加Ba²⁺（5.0 mM）会诱导HCO₃⁻跨顶端膜内流，该内流被黏膜DPC可逆性阻断。在最后一系列研究中，通过用Hepes缓冲林格液替代双侧 Krebs 碳酸氢盐林格液（KBR）而急性碱化加载的正常和CF细胞，从碱性刺激中的恢复表现为基底外侧而非顶端恢复，该恢复依赖于Cl⁻，不依赖于Na⁺并被H₂DIDS阻断。我们得出结论：（1）正常鼻上皮而非CF鼻上皮在细胞顶端膜上有一条组成性激活的、对DPC敏感的HCO₃⁻内流/外流途径，这与通过CFTR的HCO₃⁻移动一致；（2）正常和CF鼻上皮在其基底外侧区域都有不依赖Na⁺、对H₂DIDS敏感的AE。
