Characterization of H+ and HCO3- transporters in CFPAC-1 human pancreatic duct cells

Figure 1 Buffering capacity of CFPAC-1 human pancreatic duct cells at different pH_i values. Polarized CFPAC-1 cells were exposed to varying concentrations of NH₄Cl (0–30 mmol/L), while Na⁺ and HCO₃^– were omitted from the bath solution to block the Na⁺ and HCO₃^–-dependent pH regulatory mechanisms. β_i was estimated by the Henderson–Hasselbalch equation (n=50). The total buffering capacity (β_total) was calculated as β_total=β_i+β_HCO3^–, where β_HCO3^– is the buffering capacity of the HCO₃^–/CO₂ system. β_HCO3^–=2.3×[HCO₃^–]_i. [HCO₃^–]_i is the intracellular concentration of HCO₃^–.

Figure 2 Functional polarities of CFPAC-1 cells. CFPAC-1 cells were grown to confluence on permeable supports, loaded with the pH-sensitive fluorescent dye BCECF-AM (2 µmol/L) and mounted into a perfusion chamber, which allowed the simultaneous perfusion of different solutions to the basolateral and apical membranes. The figure shows representative pH_i traces demonstrating cell polarity. The administration of standard HCO₃^–/CO₂ solution to the apical and the basolateral sides of the cells showed a marked difference in response (n = 12–20).

Figure 3 Basolateral HCO₃^– uptake – effects of Cl^– and/or Na⁺ withdrawal. The figure shows representative pH_i traces. A: The rate of HCO₃^– uptake was significantly decreased in Cl-free conditions compared to standard conditions (J(B) = 73.1 ± 7.2% and 100 ± 8.5%, respectively, n = 5); B: The administration of basolateral HCO₃^–/CO₂ in Na⁺-free conditions (n = 6) greatly decreased ∆pH_i and J(B) compared to standard conditions, suggesting that a large proportion of the HCO₃^– uptake was due to a Na⁺-sensitive process, most likely NBC; C: Interestingly, the decrease in the rate of HCO₃^– uptake was obviously ameliorated in the absence of both Na⁺ and Cl^– (n  =  6) compared to the Na⁺-free condition.

Figure 4 Basolateral HCO₃^– uptake – changes in (A) ∆pH_i and (B) J(B) caused by the withdrawal of Cl^– and/or Na⁺. SE of the control groups (standard, black bar) is somewhat different for each of the respective ion-withdrawal groups. The SEs depicted on the standard bars are the maximum values observed. ^aP<0.05 and ^bP<0.01 vs control group ^dP<0.01 vs Cl^–-free group and/or ^fP<0.01 vs Na⁺-free group.

Figure 5 Basolateral HCO₃^– uptake – effects of H₂-DIDS, acetazolamide and varying extracellular K⁺ concentration. The figure shows representative pH_i traces. A: The anion transport inhibitor 600 µmol/L H₂-DIDS (added to the basolateral membrane of cells for 2 min before and during the administration of basolateral HCO₃^–/CO₂, dashed line, n=6) significantly decreased the extent of alkalinization (∆pH_i) and rate of J(B) (n=6); B: Varying extracellular [K⁺] caused significant differences in ∆pH_i and J(B) (n=9–11); C: The application of the membrane-permeable carbonic anhydrase inhibitor acetazolamide (100 µmol/L, n = 6) significantly decreased the overall ∆pH_i and J(B). HS: HEPES.

Figure 6 Basolateral HCO₃^– uptake – changes in (A) ∆pH_i (B) J(B) caused by application of H₂-DIDS, acetazolamide, ethoxyzolamide, STP and varying extracellular K⁺ concentration. Data are shown as mean ± SE (n = 6–11). SE of the control groups (standard, black bar) is somewhat different for each of the respective ion-withdrawal groups. The SE depicted on the control bar was the maximum values observed. ^aP<0.05 and ^bP<0.01 vs control group or ^cP<0.05 vs K⁺-free group. ATZ: acetazolamide; ETZ: ethoxyzolamide; STP: 1-N-(4-sulfamoylphenylethyl)-2,4,6-trimethylpyridine perchlorate. The SE depicted on the standard bars was the maximum values observed.

Figure 7 Localization of Cl^–/HCO₃^– exchangers in CFPAC-1 cells. The figure shows representative pH_i traces (n = 6). The localization of Cl^–/HCO₃^– exchangers was performed by measuring the effect of changes in Cl^– gradient across the luminal or basolateral membranes on pH_i. In the presence of HCO₃^–, the removal of Cl^– from the apical membrane did not result in changes of pH_i. The removal of Cl^– from the basolateral membrane caused a ∆pH_i of 0.08 ± 0.01 and a J(B) of 4.37 ± 0.46 mmol/L B/min. Furthermore, the basolateral administration of 250 µmol/L H₂-DIDS completely blocked pH_i changes caused by the withdrawal of Cl^–.

Figure 8 Localization of Na⁺/H⁺ exchangers in CFPAC-1 cells. The figure shows representative pH_i traces. A: After exposure to NH₄⁺ in the absence of Na⁺ on both sides of the duct cells, pH_i reduced to 6.73 ± 0.03 (n = 12), and stabilized at this level. We could not detect any active H⁺-pumps in CFPAC-1 cells, since pH_i did not increase in the absence of extracellular Na⁺. Upon addition of Na⁺ to the apical or the basolateral side prompted the pH_i to recover towards the resting values (n = 6-10); B: The administration of 300 µmol/L amiloride (dashed line) to the Na⁺-free (from 2 min before the addition of Na⁺) and Na⁺-containing HEPES solutions prevented the recovery of pH_i in the presence of Na⁺ (n = 6). Removal of amiloride resulted in the recovery of pH_i to resting values.

Figure 9 Expression of pNBC1, AE2, NHE1, and GAPDH mRNA in CFPAC-1 cells. Mw: molecular weight ladder (bp indicates number of base pairs).

Figure 10 H⁺ and HCO₃^– transport mechanisms in CFPAC-1 human pancreatic duct cells. The basolateral membrane expresses Na⁺/HCO₃^– co-transporters, Na⁺/H⁺ exchangers and Cl^–/HCO₃^– exchangers plus an unidentified Na⁺-independent HCO₃^– entry pathway. These transporters facilitate the rapid flux of HCO₃^– ions into the cell. K⁺ channels are also likely to be expressed on the basolateral membrane because altering extracellular K⁺ concentration affects HCO₃^– influx. The apical membrane expresses Na⁺/H⁺ exchangers, but not CFTR and Cl^–/HCO₃^– exchangers found in normal pancreatic duct cells. The apical membrane is an effective barrier to HCO₃^– influx from the lumen, but is freely permeable to CO₂. CA: Carbonic anhydrase.