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Am J Physiol Renal Physiol 274: F1086-F1094, 1998;
0363-6127/98 $5.00
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Vol. 274, Issue 6, F1086-F1094, June 1998

Regulation of B-type intercalated cell apical anion exchange activity by CO2/HCOminus 3

Amy E. Milton and I. David Weiner

Division of Nephrology, Hypertension and Transplantation, University of Florida College of Medicine, and Gainesville Veterans Affairs Medical Center, Gainesville, Florida 32609

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The cortical collecting duct (CCD) B cell possesses an apical anion exchanger dissimilar to AE1, AE2, and AE3. The purpose of these studies was to characterize this transporter more fully by examining its regulation by CO2 and HCO-3. We measured intracellular pH (pHi) in single intercalated cells of in vitro microperfused CCD using the fluorescent, pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In the absence of extracellular CO2/HCO-3, luminal Cl- removal caused reversible intracellular alkalinization, identifying this transporter as a Cl-/base exchanger able to transport bases other than HCO-3. Adding extracellular CO2/HCO-3 decreased B cell pHi while simultaneously increasing Cl-/base exchange activity. Since intracellular acidification inhibits AE1, AE2, and AE3, we examined mechanisms other than pHi by which the stimulation occurred. These studies showed that B cell apical anion exchange activity was CO2 stimulated and carbonic anhydrase dependent. Moreover, the stimulation was independent of luminal bicarbonate, luminal pH or pHi, and changes in buffer capacity. We conclude that the B cell possesses an apical Cl-/base exchanger whose activity is regulated by CO2-stimulated, carbonic anhydrase-dependent cytoplasmic HCO-3 formation.

cortical collecting duct; intercalated cell; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

ANION EXCHANGERS play an important role in a wide variety of cell functions. These functions include intracellular pH (pHi), Cl- and volume regulation, transepithelial ion transport, and CO2 transport (reviewed in Refs. 1 and 22). At least three families of sodium-independent anion exchangers have been identified, AE1, AE2, and AE3 (1, 22). AE1, also known as band 3 protein, mediates bicarbonate transport in the red blood cell (43) and bicarbonate reabsorption in the renal collecting duct (16). AE2 is present in a wide variety of cell types, where it likely regulates pHi (5, 10, 20, 53). AE3 was first identified in brain neurons and the heart (23, 24), and it, too, appears to regulate pHi.

However, the cortical collecting duct (CCD) B-type intercalated cell (B cell) possesses an apical anion exchanger that is dissimilar to most known anion exchangers, including AE1, AE2, and AE3. Antibodies to AE1 localize to the collecting duct A-type intercalated cell basolateral membrane, but not to B cell apical membrane (2, 29). Disulfonic stilbenes inhibit most anion exchangers, including AE1 (6), AE2 (17, 18), and AE3 (28) and anion exchangers in other collecting duct cells (4, 26, 45, 47, 48, 51), but do not inhibit the B cell apical anion exchanger (13, 38, 39, 50). AE1, AE2, and AE3 activity are increased by intracellular alkalosis, enabling them to contribute to recovery from intracellular alkalosis (1, 18, 20, 23, 53). In contrast, the B cell apical anion exchanger does not acutely regulate pHi (47, 48). Thus the B cell apical Cl-/HCO-3 exchanger appears to be a unique anion exchanger isoform.

We designed the current studies to characterize the B cell apical anion exchanger better by examining its regulation. We chose CO2/HCO-3 as a stimulus because it increases CCD HCO-3 secretion (38, 39), a known function of the B cell apical anion exchanger. We used in vitro microperfusion of rabbit CCD, measured pHi in single B cells, and quantified transporter activity as the rate of intracellular alkalinization following luminal Cl- removal. Our results show that CO2/HCO-3 stimulates anion exchange activity, that the activation requires CO2 but not extracellular bicarbonate, and that carbonic anhydrase activity is required. Thus these studies provide functional evidence that the B cell has an apical Cl-/base exchanger whose activity is regulated by intracellular bicarbonate formation.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Microperfusion. Standard techniques of in vitro microperfusion, as modified in this laboratory and previously reported in detail (50, 51), were used. Shelton Bunny Barn, Waverly Hall, GA, supplied 1.5- to 2-kg female New Zealand White rabbits for these studies. The solutions used were artificial solutions, and, unless otherwise mentioned, contained, in mM, 119.2 NaCl, 3 KCl, 25 HEPES, 2 KH2PO4, 1 sodium acetate, 1.2 CaCl2, 1 MgSO4, 5 alanine, and 8.3 glucose. HEPES-buffered solutions were titrated to pH 7.40 with tetramethylammonium-OH. Bicarbonate-containing solutions substituted NaHCO3 for HEPES. Chloride-free solutions substituted gluconate for chloride and increased total calcium to 4.0 mM to compensate for complexing with gluconate. CO2-containing solutions were bubbled with 95% O2-5% CO2, and did not contain HEPES. CO2-free solutions were bubbled with 100% O2. Osmolality was adjusted to 290 ± 7 mosmol/kgH2O with the principal salt. Table 1 summarizes the composition of the different solutions.

                              
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  		Table 1.   Solutions

pHi measurement. We measured B cell pHi using the fluorescent, pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as we have previously described in detail (46-50). Briefly, intercalated cells were loaded with BCECF by adding the acetoxymethyl ester of BCECF (BCECF-AM), 15 µM, to the luminal solution for ~5 min. This results in specific BCECF uptake by intercalated cells but not by the principal cell (46). At least 5 min were allowed after removing BCECF-AM before measuring basal pHi.

To measure pHi, a cell was excited at 490 and 440 nm, and emission was measured at 530 nm. Both the excitation and emission fields were limited to ~15 µm diameter, in which we centered a single intercalated cell. This technique allows pHi measurement in a single intercalated cell (46). Fluorescent emission intensity was measured ~100 times per minute and recorded on a personal computer using custom-written software. At the end of each experiment, we calibrated the fluorescence ratio to pHi in the range of 6.80 to 7.80 using the high K+-nigericin technique (44, 46). In preliminary experiments we found that BCECF reliably measured pHi to as high as pH 8.40 (data not shown).

Each experiment consisted of measurement of apical Cl-/base exchange activity, followed by changes in the luminal and/or peritubular solution, incubation for 30 min, and then a repeat measurement of apical Cl-/base exchange activity. We randomized the solution order to reduce time-dependent effects.

Intercalated cell type determination. We functionally identified a B cell as an intercalated cell with apical Cl-/base exchange activity (47). An intercalated cell without apical anion exchange activity was classified as an A-type intercalated cell and not included in this report. In some protocols, carbonic anhydrase inhibitors caused complete inhibition of apical Cl-/base exchange activity; the luminal and peritubular solutions were changed to CO2/HCO-3-containing solutions without inhibitors at the end of these experiments, and the cell was retested for apical Cl-/base exchange activity to ensure that it was a B cell.

Cl-/base exchange activity measurement. Apical Cl-/base exchange activity was measured as the rate of intracellular alkalinization following luminal chloride removal and the rate of intracellular acidification following luminal chloride return. The rate of pHi change was quantified using least-squares linear regression over a 6- to 15-s period when pHi change was linear.

When measuring Cl-/base exchange activity, the luminal solution was pumped at 1.7 ml/min through a pipette placed inside the perfusion pipette with its tip near the constriction of the perfusion pipette. Luminal solution that did not perfuse the tubule was drained via a line attached to rear of the perfusion pipette. A four-way valve (model HVD4-5; Hamilton, Reno, NV) was placed immediately before the pipette to allow rapid changes in the luminal solution. In preliminary studies, a 90% change of the luminal solution required ~3-4 s.

Buffer capacity measurement. Cells were acid loaded using a 5-min incubation with peritubular ammonium chloride, 40 mM (equimolar substitution for NaCl), followed by removal of ammonium chloride. We added ethylisopropylamiloride (EIPA), 1 µM, to the peritubular solution during the ammonium incubation and during the first 1-2 min following its removal to inhibit basolateral Na+/H+ exchange (47), so that we could measure accurately the extent of intracellular acidification. Buffer capacity (beta T) was calculated using the formula beta T = Delta [NH+4]i/Delta pHi, where [NH+4]i is the intracellular ammonium concentration.

Chemicals. BCECF-AM was obtained from Molecular Probes (Eugene, OR) and stored frozen as a 30 mM stock solution in DMSO. EIPA was obtained from Research Biochemical and stored as a 1 mM solution in DMSO. t-Butyl-acetazolamide was graciously provided by Dr. Thomas H. Maren, University of Florida College of Medicine. All other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Statistics. Results are presented as means ± SE; n is reported as the number of cells studied. Statistical significance was analyzed using the paired Student's t-test and ANOVA, as appropriate. P < 0.05 was taken as evidence of statistical significance.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Apical Cl-/base exchange. The first studies examine whether the B cell apical anion exchanger transports base in the absence of exogenous CO2/HCO-3. Figure 1 shows a representative experiment. Luminal chloride removal caused intracellular alkalinization averaging 0.371 ± 0.070 pH units/min (P < 0.001 vs. zero, n = 25) in the absence of CO2/HCO-3. Returning luminal chloride caused pHi to decrease at a rate averaging -0.313 ± 0.074 pH units/min (P < 0.001 vs. zero, n = 25). These results indicate that the B cell apical anion exchanger transports base in the absence of exogenous CO2/HCO-3. Accordingly, we will refer to this transporter as a Cl-/base exchanger in the remainder of this report.


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  		Fig. 1.   Example of apical Cl-/base exchange activity in HEPES-buffered, CO2/HCO-3-free solutions. Luminal Cl- removal induces intracellular alkalinization, indicating base entry, while luminal Cl- return allows intracellular pH (pHi) to return to baseline, indicating Cl--dependent base exit.

Regulation by CO2/HCO-3. We next examined whether exogenous CO2/HCO-3 regulates apical Cl-/base exchange activity. These results are summarized in Fig. 2 and Table 2. Apical anion exchange activity averaged 0.204 ± 0.105 pH units/min (n = 4) in the absence of exogenous CO2/HCO-3 and was increased significantly to 2.844 ± 0.335 pH units/min in the presence of exogenous CO2/HCO-3 (P < 0.003 vs. without CO2/HCO-3, n = 4). Thus exogenous CO2/HCO-3 increases B cell apical Cl-/base exchange activity.


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  		Fig. 2.   Stimulation of B cell apical Cl-/base exchange activity by luminal and peritubular CO2/HCO-3. Rates of intracellular alkalinization following luminal Cl- removal. Lines connect paired measurements in individual cells (bullet , HEPES-buffered solution used first, then changed to CO2/HCO-3-buffered solution; open circle , CO2/HCO-3-buffered solutions used first, then changed to HEPES-buffered solution); square , mean ± SE.

                              
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  		Table 2.   Effects of CO2 and bicarbonate on pHi and apical anion exchange activity

One potential mechanism by which CO2/HCO-3 might regulate anion exchange activity is through changes in pHi. For example, AE1, AE2, and AE3 activity are increased by an increase in pHi (18, 20, 28, 53). However, cell alkalinization is unlikely to be the mechanism by which CO2/HCO-3 increased B cell apical anion exchange activity. As shown in Table 2, exogenous CO2/HCO-3 acidified the B cell while simultaneously increasing exchange activity. Thus factors other than intracellular alkalinization mediate CO2/HCO-3 stimulation of B cell apical anion exchange activity.

Effect of CO2. The high permeability of CO2 across plasma membranes means it can regulate a number of cellular processes. We next tested whether CO2 regulated B cell anion exchange activity by adding CO2 in the nominal absence of bicarbonate to the luminal fluid. Figure 3A and Table 2 summarize the results. Apical anion exchange activity averaged 0.454 ± 0.142 pH units/min in the absence of luminal CO2 (n = 8) and, in the presence of luminal CO2, was increased significantly to 2.578 ± 0.221 pH units/min (P < 0.001 vs. without luminal CO2, n = 8). Luminal CO2 increases B cell apical anion exchange activity.


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  		Fig. 3.   Effect of luminal CO2 on B cell apical Cl-/base exchange activity. A: effect of CO2-containing, nominally HCO-3-free luminal solution. B: effect of a CO2/HCO-3-containing luminal solution. The intracellular alkalinization rate following luminal Cl- removal is shown. A HEPES-buffered peritubular solution was used throughout the experiment. Lines connect measurements in individual cells (bullet , a HEPES-buffered solution was used first, then changed to a CO2/HCO-3- or CO2-buffered solution; open circle , a CO2/HCO-3- or CO2-buffered solution was used first, then changed to a HEPES-buffered solution); square , mean ± SE.

The stimulation by luminal CO2 could be due to the CO2, or it could be due to the decreased pH, ~6.1, of a CO2-containing, nominally bicarbonate-free luminal solution. To differentiate these possibilities, we tested the effect of simultaneous luminal CO2 and HCO-3 addition, thereby avoiding changes in luminal pH. Table 2 and Fig. 3B summarize the results. Apical Cl-/base exchange activity averaged 0.350 ± 0.154 pH units/min in the absence of luminal CO2/HCO-3 and 2.954 ± 0.568 pH units/min in the presence of luminal CO2/HCO-3 (n = 4). Luminal CO2/HCO-3 significantly increased B cell apical Cl-/base exchange activity (P < 0.02).

CO2 addition increased B cell apical anion exchange activity to a similar extent, whether added alone, with luminal bicarbonate, or with both luminal bicarbonate and peritubular CO2/HCO-3 [P = not significant (NS) for difference by ANOVA]. These results strongly suggest that luminal CO2 itself increases apical anion exchange activity.

Effect of luminal bicarbonate. Because the solutions used in these protocols were at equilibrium, they also contained bicarbonate. For example, the CO2-containing, nominally bicarbonate-free luminal solution had a pH of ~6.1, indicating a bicarbonate content of ~ 1 mM. This raises the possibility that luminal bicarbonate mediates the increase in B cell apical anion exchange activity. Adding luminal bicarbonate could increase the rate of intracellular alkalinization with luminal chloride removal solely by increasing the gradient for base, e.g., bicarbonate, entry. To consider that possibility, we examined whether providing luminal bicarbonate, in the nominal absence of CO2, altered anion exchange activity.

First, we examined the effect of luminal bicarbonate when peritubular CO2/HCO-3 was absent. Figure 4A and Table 2 summarize these results. Under these conditions transporter activity averaged 0.247 ± 0.095 pH units/min (n = 5) in the absence of luminal bicarbonate and 0.192 ± 0.053 pH units/min (n = 5) in the presence of luminal bicarbonate. Luminal bicarbonate addition did not significantly alter apical Cl-/base exchange activity in the absence of peritubular CO2/HCO-3 (P = NS, n = 5).


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  		Fig. 4.   Effect of luminal HCO-3 on B cell apical Cl-/base exchange activity. A: with a HEPES-buffered peritubular solution. B: with a CO2/HCO-3-buffered peritubular solution. The intracellular alkalinization rate following luminal Cl- removal is shown. Lines connect measurements in individual cells (bullet , HEPES-buffered luminal solutions were used first, then changed to a HCO-3-buffered, nominally CO2-free luminal solutions; open circle , HCO-3-buffered, nominally CO2-free luminal solutions were used first, then changed to HEPES-buffered luminal solutions); square , mean ± SE.

We also examined the effect of luminal bicarbonate when peritubular CO2/HCO-3 was present. Table 2 and Fig. 4B summarize these results. Under these conditions, apical Cl-/base exchange activity averaged 0.731 ± 0.135 pH units/min in the absence of luminal bicarbonate (P < 0.01 vs. zero, n = 5). In the presence of luminal bicarbonate, activity averaged 1.058 ± 0.298 pH units/min (n = 5), a rate not significantly different from in its absence (P = NS). Adding luminal bicarbonate increases the luminal base content, and thus the gradient for base entry following luminal chloride removal, but does not regulate apical Cl-/base exchange activity. Thus exogenous CO2/HCO-3 appears to regulate B cell apical anion exchange activity through CO2-dependent effects.

Effect of carbonic anhydrase inhibition. CO2 is rapidly permeable across cell membranes and can regulate cellular processes through a number of mechanisms. One mechanism is through carbonic anhydrase-dependent intracellular bicarbonate production. To test this possibility, we examined the effect of carbonic anhydrase inhibitors on apical Cl-/base exchange activity. Peritubular acetazolamide, 100 µM, a membrane-permeant carbonic anhydrase inhibitor, was used in the first set of studies. Figure 5A and Table 3 summarize the results. Apical Cl-/base exchange activity was completely inhibited both in the absence and presence of exogenous CO2/HCO-3 (dpHi/dt = -0.022 ± 0.023 and -0.010 ± 0.159 pH units/min, respectively, P = NS vs. zero for each, P = NS for stimulation by CO2/HCO-3, n = 6). An acetazolamide-sensitive process regulates both basal and CO2/HCO-3-stimulated apical Cl-/base exchange activity.


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  		Fig. 5.   Effect of carbonic anhydrase inhibitors on B cell apical Cl-/base exchange activity in presence and absence of bilateral CO2/HCO-3. Inhibitors were added only to the peritubular solution. A: acetazolamide, 100 µM. B: ethoxyzolamide, 10 µM. C: t-butyl-acetazolamide, 100 µM. The intracellular alkalinization rate following luminal Cl- removal is shown. Lines connect paired measurements in individual cells (bullet , HEPES-buffered solution used first, then changed to CO2/HCO-3-buffered solution; open circle , CO2/HCO-3-buffered solutions used first, then changed to HEPES-buffered solution); square , mean ± SE. Standard error bars are not shown when smaller than the size of the symbol.

                              
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  		Table 3.   Effect of carbonic anhydrase inhibitors on pHi and apical anion exchange activity

To confirm these results, we tested the effect of a second carbonic anhydrase inhibitor, ethoxyzolamide. These results are summarized in Fig. 5B and Table 3. Addition of ethoxyzolamide, 10 µM, to the peritubular solution completely inhibited apical anion exchange activity both in the absence and the presence of exogenous CO2/HCO-3 (dpHi/dt = -0.012 ± 0.012 and -0.026 ± 0.018 pH units/min, respectively, P = NS vs. zero for each, P = NS for stimulation by CO2/HCO-3, n = 4). Thus two different carbonic anhydrase inhibitors block both basal and CO2/HCO-3-stimulated anion exchange activity.

The effects of ethoxyzolamide and acetazolamide could be related to either carbonic anhydrase inhibition or structurally related, carbonic anhydrase-independent effects. To differentiate between these possibilities, we used an acetazolamide analog that does not inhibit carbonic anhydrase, t-butyl-acetazolamide, 100 µM (30). Figure 5C and Table 3 summarize these results. Apical Cl-/base exchange activity averaged 0.408 ± 0.329 pH units/min (n = 4) in the absence of extracellular CO2/HCO-3 and was increased significantly to 3.687 ± 0.432 pH units/min in the presence of CO2/HCO-3 (P < 0.02 vs. without exogenous CO2/HCO-3, n = 4).

Acetazolamide and ethoxyzolamide appear to inhibit B cell apical anion exchange activity by inhibiting carbonic anhydrase, not through structurally related, carbonic anhydrase-independent effects. The finding of CO2-stimulated, carbonic anhydrase-dependent anion exchange activity regulation indicates functionally that intracellular bicarbonate formation regulates B cell apical anion exchange activity.

Effect of pHi. As discussed before, CO2/HCO-3 stimulated anion exchange activity despite decreasing pHi. When taking into consideration all of the protocols in this report, we found pHi did not have a statistically significant effect on anion exchange activity (P = NS by ANOVA). Thus it is unlikely that pHi is a major, independent regulator of B cell apical anion exchange activity.

Effect of CO2 and bicarbonate on B cell buffer capacity. When using dpHi/dt to compare transport rates under different conditions, one must determine whether the experimental conditions alter buffer capacity (37). Table 4 summarizes the measurement of B cell buffer capacity under different conditions. CO2 and bicarbonate, whether added to the luminal or peritubular solutions, did not significantly alter B cell buffer capacity (P = NS by ANOVA). Changes in buffer capacity do not explain the differences in transporter activity.

                              
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  		Table 4.   Effect of CO2 and bicarbonate on buffer capacity

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

This study examines B cell apical anion exchange activity regulation by CO2/HCO-3 and reveals several new findings. The primary findings are that exogenous CO2/HCO-3 increases B cell apical anion exchange activity and that this stimulation is mediated through intracellular bicarbonate formation. In addition, the current study identifies that the B cell apical anion exchanger should be identified as a Cl-/base exchanger, because it transport bases other than bicarbonate.

The first major finding of this study is that extracellular CO2/HCO-3 increases B cell apical Cl-/base exchange activity. Similar results have been reported for Cl-/base exchangers in a number of cells types (21, 35), including AE2 (20). However, extracellular CO2/HCO-3 does not universally increase Cl-/base exchange activity (25). The observation that CO2/HCO-3 increases B cell apical anion exchange activity is consistent with previous studies examining the entire CCD. CO2/HCO-3 increases net CCD bicarbonate secretion (33, 42), which reflects changes in both bicarbonate secretion and proton secretion, and it increases Cl- self-exchange (33, 42), which measures both B cell apical Cl-/Cl- self-exchange and basolateral Cl- exit. The current study identifies that at least part of the stimulation of net bicarbonate secretion and Cl- self-exchange by exogenous CO2/HCO-3 is due to increased B cell apical anion exchange activity.

The increased activity of this transporter in response to CO2/HCO-3 appears to be due to intracellular bicarbonate formation. Transport activity is stimulated by CO2 and requires carbonic anhydrase activity. CO2 is highly permeable across cell membranes and can regulate a number of cellular processes, including intracellular bicarbonate formation. Acetazolamide and ethoxyzolamide are potent and specific inhibitors of carbonic anhydrase and thus CO2-stimulated intracellular bicarbonate formation (31). Acetazolamide may inhibit some anion exchangers through carbonic anhydrase-independent effects (8, 12), raising the possibility that this may occur in the B cell. The similar findings with two carbonic anhydrase inhibitors, but not with an inactive, structural analog, make this possibility unlikely but, of course, cannot completely exclude it.

The finding that carbonic anhydrase inhibitors decreased Cl-/base exchange activity in the absence of exogenous CO2/HCO-3 probably reflects inhibition of endogenously produced CO2-dependent intracellular bicarbonate formation. The CCD both generates CO2 (19, 27) and secretes bicarbonate in the absence of exogenous CO2/HCO-3 (38, 39). Furthermore, cells generate CO2 in large part through mitochondrial respiration, and the B cell is characterized by a mitochondria-rich cytoplasm (29). These considerations show that carbonic anhydrase activity is necessary for CO2 to stimulate B cell anion exchange activity and provide functional evidence that intracellular bicarbonate regulates B cell apical anion exchange activity.

Previous studies have shown that CO2/HCO-3 increases CCD Cl- self-exchange (33, 42), an alternative mode for the B cell apical anion exchanger. Moreover, this effect appears to be mediated through intracellular bicarbonate (33). However, the effect of CO2/HCO-3 on Cl- self-exchange appears to be mediated through effects on the basolateral Cl- channel (32). The current study is consistent with these findings and indicates that CO2/HCO-3, acting through intracellular bicarbonate, regulates both the apical anion exchanger and the basolateral Cl- channel.

The effect of intracellular bicarbonate on AE1, AE2, and AE3 is less well defined. AE1 and AE2 are stimulated by intracellular alkalinization, enabling them to contribute to pHi regulation (53). However, this appears to be mediated by intracellular protons (53) and does not require intracellular bicarbonate. Intracellular alkalinization increases AE3 activity, but this has been shown only in CO2/HCO-3-containing solutions, so it is impossible to determine whether the regulation is due to intracellular bicarbonate or protons (28). The effect of intracellular bicarbonate on AE1 activity is unclear, as different studies suggest it both inhibits and stimulates AE1 activity (7, 9, 14, 15, 52). Determination of whether intracellular bicarbonate regulates the activity of other anion exchangers, including AE2 and AE3, will require further study.

This study also shows that the B cell apical anion exchanger transports bases other than bicarbonate. Several other anion exchangers, including AE1 and AE2, also transport bases other than bicarbonate (34, 36, 40, 41). These finding are consistent with a preliminary report by Emmons and Kurtz (11). However, transport of bases other than bicarbonate is not a universal feature of anion exchangers (36).

Several factors require consideration when interpreting this study. First, direct assessment of base transport requires consideration of cell volume and buffer capacity, in addition to the rate of pHi change (37). In no protocol did CO2 and/or HCO-3 measurably change buffer capacity. This was surprising, because total buffer capacity (beta T) is the sum of intrinsic buffer capacity (beta i) and the bicarbonate buffer capacity (beta HCO3) (37). Since beta HCO3 is proportional to the intracellular bicarbonate concentration (37), the lack of change in beta T suggests B cell beta i may, in the pHi range studied, vary in the opposite direction as beta HCO3. Using the measured rate of pHi change and buffer capacity, as well as an estimate of B cell volume calculated from CCD inner and outer diameter (19.5 and 37.25 µM, n = 4, respectively),1 base transport caused by luminal Cl- removal in the presence of CO2/HCO-3 averaged 5.3 × 10-13 eq · cell-1 · min-1. In no protocol did CO2 and/or bicarbonate visually change B cell size. Although small changes in cell volume could have occurred in response to CO2 or bicarbonate, there would have been needed an ~10-fold increase in B cell volume to account for the change in dpHi/dt that occurred in response to CO2/HCO-3 removal. No such change in cell volume was apparent. As a result, pHi changes appear to reflect changes in base transport.

The CO2-equilibrated, nominally bicarbonate-free solutions contain ~1 mM bicarbonate, raising the possibility that effects seen with CO2 addition were due to the added bicarbonate, not to the CO2. However, this is not likely to be the case. Luminal bicarbonate addition, in the nominal absence of CO2, did not increase apical Cl-/base exchange activity significantly. Although there was a tendency for a nonstatistically significant increase, transport activity remained significantly less than with the CO2-containing luminal solutions despite an ~25-fold higher bicarbonate concentration. Thus it is unlikely that the bicarbonate content of CO2-equilibrated solutions explains the changes in apical Cl-/base exchange activity that were seen.

Another consideration is that changes in luminal pH in some protocols may have had independent effects on apical anion exchange activity. Again, this is not likely to have materially altered the conclusions in this study. Extracellular pH changes generally produce parallel changes in anion exchanger activity (53), yet changing luminal pH had no consistent effect on B cell apical anion exchange activity. Thus it is unlikely that the luminal pH changes in some protocols had a significant effect on this study's conclusions.

Finally, basal pHi in many protocols was relatively high. This may result from overestimation of actual pHi by the high potassium-nigericin calibration technique (3). Alternatively, it may be due to inhibition of apical base transport in combination with continued proton secretion by H+-ATPase and H+-K+-ATPase. The high pHi values in some protocols raise the possibility that there might be a pHi threshold effect where the transporter is abruptly inhibited. Although we cannot exclude such a possibility, we are unaware of examples where this has occurred. More important is that the apparently high basal pHi did not interfere with measuring anion exchange activity, as our preliminary studies showed that BCECF accurately measured pHi to as high as 8.40.

In summary, B cell luminal bicarbonate secretion is mediated by an apical Cl-/base exchanger able to transport bases other than bicarbonate. CO2/HCO-3 increases B cell apical Cl-/base exchange activity. This process is due to CO2 and requires carbonic anhydrase activity, and it appears to be mediated by intracellular bicarbonate formation.

    ACKNOWLEDGEMENTS

We thank Drs. R. Tyler Miller and Charles S. Wingo for helpful discussions. We also appreciate the technical assistance of April R. Starker and the secretarial assistance of Gina Cowsert.

    FOOTNOTES

Funds from National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45788 and a Merit Review Grant from the Department of Veterans Affairs supported these studies.

1 This calculation assumes that the CCD can be modeled as a tubular structure, that ~500 cells are present per millimeter tubule length, and that principal and intercalated cell volume are similar. Using these assumptions, intercalated cell volume (Vic) can be calculated as Vic = pi  · [(do/2)2 - (di/2)2] · (1 mm/500 cells), where do and di are outer and inner diameters, respectively.

Address for reprint requests: I. David Weiner, Division of Nephrology, Hypertension and Transplantation, Univ. of Florida College of Medicine, P.O. Box 100224, Gainesville, FL 32610.

Received 27 August 1997; accepted in final form 19 February 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Alper, S. L. The band 3-related anion exchanger (AE) gene family. Annu. Rev. Physiol. 53: 549-564, 1991[Medline].

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