Characterization of H(+) and HCO(3)(-) transporters in CFPAC-1 human pancreatic duct cells.

摘要

AIM:To characterize H(+) and HCO(3)(-) transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the DeltaF508 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 HCO(3)(-)/CO(2) at the apical and basolateral membranes of CFPAC-1 cells. Na(+)/ HCO(3)(-) co-transporters (NBCs) and Cl(-)/ HCO(3)(-) exchangers (AEs) were present on the basolateral membrane, and Na(+)/H(+) exchangers (NHEs) on both the apical and basolateral membranes of the cells. Basolateral HCO(3)(-) uptake was sensitive to variations of extracellular K(+) concentration, the membrane permeable carbonic anhydrase (CA) inhibitors acetazolamide (100 mumol/L) and ethoxyzolamide (100 mumol/L), and was partially inhibited by H(2)-DIDS (600 mumol/L). The membrane-impermeable CA inhibitor 1-N-(4-sulfamoylphenylethyl)-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO(3)(-) 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 mumol/L forskolin did not significantly influence Cl(-)/ HCO(3)(-) exchange on the apical and basolateral membranes. The administration of 250 mumol/L H(2)-DIDS significantly inhibited the basolateral AE. Amiloride (300 mumol/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(-)/ HCO(3)(-) exchanger activity, CFPAC-1 cells express similar H(+) and HCO(3)(-) transporters to those observed in native animal tissue.