Characterization of H^+ and HCO_3^- transporters in CFPAC-1 human pancreatic duct cells

AIM： To characterize H^＋ and HCO3^- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the AF508 CFTR mutation. METHODS： CFPAC-1 cells were seeded at high density onto permeable supports and grown to confluence. The cells were loaded with the pH-sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of the basolateral and apical membranes. Transmembrane base flux was calculated from the changes in intracellular pH and the buffering capacity of the cells. RESULTS： Our results showed differential permeability to HCO3^＋/CO2 at the apical and basolateral membranes of CFPAC-1 cells. Na^＋/HCO3^- co-transporters （NBCs） and Cl^-/HCO3^- exchangers （AEs） were present on the basolateral membrane, and Na＋/H＋ exchangers （NHEs） on both the apical and basolateral membranes of the cells. Basolateral HCO3 uptake was sensitive to variations of extracellular K^＋ concentration, the membrane permeable carbonic anhydrase （CA） inhibitors acetazolamide （100 μmol/L） and ethoxyzolamide （100 μmol/L）, and was partially inhibited by H2-DIDS （600 μmol/L）. The membrane-impermeable CA inhibitor 1-N-（4-sulfamoylphenylethyl）-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO3^- uptake.The basolateral AE had a much higher activity than that in the apical membrane, whereas there was no such difference with the NHE under resting conditions. Also, 10 μmol/L forskolin did not significantly influence Cl^-/HCO3^- exchange on the apical and basolateral membranes. The administration of 250 μmol/L H2-DIDS significantly inhibited the basolateral AE. Amiloride （300 μmol/L） completely inhibited NHEs on both membranes of the cells. RT-PCR revealed the expression of pNBC1, AE2, and NHE1 mRNA. CONCLUSION： These data suggest that apart from the lack of CFTR and apical Cl^-/HCO3^- exchanger activity, CFPAC-1 cells express similar H^＋ and HCO3^- transporters to those observed in native animal tissue.

瞄准：在极化的 CFPAC-1 描绘 H+ 和 HCO3- 运输 ers 人的胰腺的管房间，它与 DeltaF508 CFTR 变化从一个膀胱的纤维变性病人被导出。方法：CFPAC-1 房间在高密度被播种到可渗透的支持上并且成长到融合。房间与 pH 敏感的荧光灯的染料 BCECF 被装载，并且增长了进一个灌注房间，它允许 basolateral 和顶端的膜的同时的灌注。Transmembrane 基础流动在细胞内部的 pH 和房间的缓冲能力从变化被计算。结果：我们的结果显示出微分渗透到 HCO3-/CO2 在顶端并且 CFPAC-1 房间的 basolateral 膜。Na+/HCO3- co-transporters (NBC ) 和 Cl-/HCO3- exchangers (AE ) 在两个上在 basolateral 膜，和 Na+/H+ exchangers (NHE ) 上是在场的顶端并且房间的 basolateral 膜。Basolateral HCO3- 举起对细胞外的 K+ 集中的变化敏感，膜可渗透的碳酸酐酶(CA ) 禁止者乙酰唑胺(100 micromol/L ) 和 ethoxyzolamide (100 micromol/L ) ，并且被 H2do (600 micromol/L ) 部分禁止。膜透不过的 CA 禁止者 1-N-(4-sulfamoylphenylethyl )-2,4,6-trimethylpyridine 高氯酸盐没在 HCO3- 举起上有任何效果。basolateral AE 在顶端的膜比那举办了一项高得多的活动，而没有有在放松条件下面的 NHE 的如此的差别。另外， 10 micromol/L forskolin 显著地没影响 Cl-/HCO3- 交换在上顶端并且 basolateral 膜。250 micromol/L H2do 的管理显著地禁止了 basolateral AE。Amiloride (300 micromol/L ) 完全在房间的两膜上禁止了 NHE。RT-PCR 揭示了 pNBC1， AE2，和 NHE1 mRNA 的表示。结论：这些数据建议除了 CFTR 和顶端的 Cl-/HCO3- exchanger 活动的缺乏， CFPAC-1 细胞表示类似的 H+ 和 HCO3- 运输 ers 到在本国的动物织物观察的那些。