The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO(3)(-):1 Na(+)

Abstract

The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO3-:Na+ stoichiometry is not larger then 2:However, there are no data available on either the reversal potential or the HCO3-:Na+ stoichiometry of pNBC1 in pancreatic cells. We studied pNBC1 function in mouse pancreatic duct cells. RT-PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na+ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS). Current-voltage relationships for the cotransporter, measured at three different Na+ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current-voltage relationships, all corresponded to a 2 HCO3-:1 Na+ stoichiometry. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO3-:1 Na+ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane.

Figure 1. RT-PCR amplification of mPEC1 cells reveals the message for pNBC1

Total RNA from mPEC1 cells or mouse pancreatic duct fragments was reversed transcribed using AMV reverse transcriptase (RT). Specific regions of pNBC1, kNBC1, NBC2 or NBC3 were amplified using primers as described in the Methods section. M, 1 kb ladder; lane 1 (mPEC1), pNBC1 primers, +RT; lane 2 (mPEC1), pNBC1 primers, -RT; lane 3 (mPEC1), kNBC1 primers, +RT; lane 4 (mouse kidney), kNBC1 primers, +RT; lane 5 (mouse kidney), kNBC1 primers, -RT; lane 6 (mPEC1), NBC2 primers, +RT; lane 7 (mouse testes), NBC2 primers, +RT; lane 8 (mouse testes), NBC2 primers, -RT; lane 9 (mPEC1), NBC3 primers, +RT; lane 10 (mouse heart), NBC3 primers, +RT; lane 11 (mouse heart), NBC3 primers, -RT; lane 12 (mouse pancreas), pNBC1 primers, +RT; lane 13 (mouse pancreas), pNBC1 primers, -RT.

Figure 2. Bicarbonate dependence of pNBC1 current

A 5-fold sodium concentration gradient was applied across the cell monolayer, which had been apically permeabilized with 20 μm amphotericin B, in the presence of CO₂/bicarbonate (A and B) by perfusing the basolateral compartment with solution 50Na and the apical compartment with solution 10Na. A, voltage pulse protocol used to collect I-V relationships in the absence (continuous line) and presence (dotted line) of 2 mm DNDS. Voltage was stepped from -100 to +100 mV in 10 mV steps. The current at each voltage was recorded for 5 s. B, I-V relationships in the absence (○) and presence (○) of DNDS obtained by averaging the current at each voltage over 5 s. C, I-V relationships in the absence (○) and presence (○) of DNDS for a 5-fold sodium concentration gradient but in the nominal absence of CO₂/bicarbonate.

Figure 3. Polarized expression of the basolateral marker Na⁺,K⁺-ATPase in apically permeabilized pancreatic duct cells

Cells were mounted in an Ussing chamber and the transepithelial voltage was clamped to zero. Cells were perfused initially with a solution containing K⁺ and Na⁺ (solution 4K in Table 1). At the indicated time the apical surface was permeabilized by adding 20 μm amphotericin B, which resulted in an increase in short-circuit current, I_SC. The increased I_SC following apical application of amphotericin B reflects the activity of the Na⁺,K⁺-ATPase, as demonstrated by a decrease in I_SC upon basolateral application of 1 mm of the enzyme’s inhibitor ouabain, or upon removal of K⁺ (not shown).

Figure 4. I-V relationships for pNBC1 in apically permeabilized monolayers

Cell membranes were permeabilized with 20 μm amphotericin B. ΔI is the DNDS-sensitive current. The potential of the basal compartment was taken as zero. The ratio of apical to basolateral Na⁺ concentration (AP/BL) was as follows: ▪, 10 mm/80 mm (solutions 10Na/80Na); ○, 10/50 (10Na/50Na); ▴, 10/20 (10Na/20Na); ⋄, 20/20 (20Na/20Na); ▵, 20/10; ○, 50/10; and □, 80/10. The reversal potentials for the different gradients were evaluated graphically from the intersection of the lines with the X-axis and are tabulated in Table 2.