cAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3- cotransporter pNBC1 in human embryonic kidney (HEK293) cells

Abstract

Background: The renal (kNBC1) and intestinal (pNBC1) electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct.

Results: Na+- and HCO3-dependent pHi recovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO2/HCO3-. Forskolin (10(-5) M) had no effect in untransfected cells, but inhibited the pHi recovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10(-4) M), the pHi recovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na+/HCO3- cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue.

Conclusion: Regulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.

Figure 1

RT-PCR in untransfected HEK293 cells (HEK293), HEK293 cells transiently transfected with pNBC1 (+pNBC1), as well as human kidney and colon samples using kNBC1- and pNBC1-specific primers (see methods section). While neither isoform was detected in untransfected HEK293 cells, a pNBC1 fragment of the expected size (612 bp) was amplified from transfected HEK293 cells and human colon. kNBC1 was exclusively detected in human kidney samples (expected PCR product size: 489 bp). H₂O indicates the reaction where water was used as a template.

Figure 2

Confocal images of HEK cells transiently transfected with GFP-pNBC1. Panel (A) shows the GFP staining of transfected cells and the weak autofluorescence of untransfected cells. After placing regions of interest covering most of the cytosol, cells were in situ loaded with the pH-sensitive dye BCECF (B), and the time course of the intracellular pH was recorded. C: To determine the optimal calibration procedure for the pH range of interest (for calculation of the recovery rates at the reference pH_i, see Fig. 3B), a calibration curve was constructed using high K⁺/nigericin solution to clamp intracellular pH at different levels. As expected, its slope is less steep at low pH_i values and steeper in the linear range (n = 6). To minimize the error during the calibration procedure within the pH range of interest, the calibration points 6.2 and 7.0 were chosen. Since the ratio values varied considerably between the experiments, each individual region was calibrated after the respective experiment subsequently.

Figure 3

Starting pH_i, reference pH_i, and representative pH_i tracings from pNBC1-transfected and untransfected HEK293 cells. A, B: Box and whisker diagram of the starting- and reference pH_i values from pNBC1-transfected and untransfected cells. Steady-state pH_i was significantly higher in transfected cells, pointing to increased baseline Na⁺/HCO₃^- cotransport activity (*: p < 0.001, Student's t-test for paired samples, n = 7). The reference pH_i after acidification was, however, not different between the two. C: After imposing an acid load using the "NH₄-prepulse" method, cells acidified to a similar extent. Upon Na⁺ readdition, most untransfected cells showed no significant recovery, while pH_i increased rapidly in transfected cells (representative tracings). It should be noted that our 2-point calibration approach is optimized for the low pH_i values at which transporter activity is measured, and that the higher pH_i values are therefore overestimated.

Figure 4

pH_i recovery from an acid load in untransfected (Ctr) and transfected cells (pNBC1, mock) in the absence and presence of CO₂/HCO₃^-. A: in O₂/HEPES buffer, no significant difference in the pH_i recovery rate in untransfected and pNBC1-transfected cells was noted (n = 8). B: In the presence of CO₂/HCO₃^-, however, pH_i was accelerated 5-fold in transfected vs. untransfected HEK293 cells (*: p < 0.05, Student's t-test for paired samples, n = 7), indicating successful functional transfection of the pNBC1 protein. C, D: in mock-transfected vs. untransfected cells on the same slide, pH_i recovery rates were relatively low, and no significant differences were noted between mock-transfected and untransfected, or between the absence and presence of CO₂/HCO₃^-, respectively (n = 6–8).

Figure 5

Effect of cAMP-dependent and cholinergic stimulation on pH_i recovery in untransfected and pNBC1-transfected HEK293 cells. A: Forskolin did not elicit any significant changes in pH_i recovery in untransfected cells (n = 6). Again, transfected cells displayed a significantly faster pH_i recovery under control conditions than their untransfected counterparts (*: p < 0.05 vs. untransfected control, Student's t-test for paired samples, n = 6). However, forskolin strongly inhibited pH_i recovery in pNBC1-transfected HEK293 cells (**: p < 0.01 vs. unstimulated control, Student's t-test for unpaired samples, n = 7). B: Carbachol caused a slight acceleration of pH_i recovery in untransfected cells, which might be due to effects on endogenous Na⁺/H⁺ exchange or on Na⁺/HCO₃^- exchanger isoforms other than pNBC1 or kNBC1 (*: p < 0.05 vs. unstimulated control, Student's t-test for unpaired samples, n = 6). pNBC1 transfection again led to a significantly faster pH_i recovery (**: p < 0.05 vs. untransfected control, Student's t-test for paired samples, n = 8). However, carbachol did not cause any additional significant changes to pH_i recovery in transfected cells (ns: not significant, n = 8).