Transport characteristics of the apical anion exchanger of rabbit cortical collecting duct beta -cells

Cheryl Emmons

Departments of Internal Medicine, University of Cincinnati and Cincinnati Veterans Affairs Medical Center, Cincinnati, Ohio 45267-0585


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To functionally characterize transport properties of the apical anion exchanger of rabbit beta -intercalated cells, the mean change in anion exchange activity, dpHi/dt (where pHi is intracellular pH), was measured in response to lumen Cl- replacement with gluconate in perfused cortical collecting ducts (CCDs). beta -Cell apical anion exchange was not affected by 15-min exposure to 0.2 mM lumen DIDS in the presence of 115 mM Cl-. In contrast, apical anion exchange was significantly inhibited by 0.1 mM lumen DIDS in the absence of Cl-. beta -Cell apical anion exchange was unchanged by 15 mM maleic anhydride, 10 mM phenylglyoxal, 0.2 mM niflumic acid, 1 mM edecrin, 1 mM furosemide, 1 mM probenecid, or 0.1 mM diphenylamine-2-carboxylate. However, beta -cell apical anion exchange was inhibited by alpha -cyano-4-hydroxycinnamic acid, with an IC50 of 2.4 mM. Substitution of either sulfate or gluconate for lumen Cl- resulted in a similar rate of alkalinization. Conversely, pHi was unchanged by substitution of sulfate for lumen gluconate, confirming the lack of transport of sulfate on the beta -cell apical anion exchanger. Taken together, the results demonstrate a distinct "fingerprint" of the rabbit CCD beta -cell apical anion exchanger that is unlike that of other known anion exchangers.

intercalated cell; anion exchange; intracellular pH; acid-base transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CORTICAL SEGMENT of the kidney collecting duct (CCD) plays an important role in the maintenance of acid-base balance. CCD transepithelial bicarbonate transport is mediated by intercalated cells. beta -Intercalated cells are thought to effect transepithelial bicarbonate secretion via an apical Cl-/HCO-3 exchanger, whereas alpha -intercalated cells are thought to function in bicarbonate absorption via a basolateral Cl-/HCO-3 exchanger (27, 28). Three anion exchanger isoforms have been identified, with the prototype being AE1 (or band 3) of the erythrocyte (1, 19). Immunocytochemical studies have identified the basolateral anion exchanger of alpha -cells as AE1 (4, 27); however, the protein identity of the apical anion exchanger remains uncertain. No antibodies to any of the anion exchange isoforms have ever been demonstrated to bind to the CCD beta -cell apical membrane (4, 27). In addition, the apical anion exchanger of CCD beta -cells is not inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2-DIDS), a derivative of the stilbene drugs that inhibit all three anion exchange isoforms in micromolar concentrations (12). Similarly, CCD bicarbonate secretion is insensitive to stilbenes (26). A recent study of a transformed rabbit kidney beta -cell line suggested that the apical anion exchanger was AE1, the same protein as the basolateral exchanger (33). However, using primary beta -cell cultures, Fejes-Toth and co-workers (13) found that AE1 was expressed differentially in alpha - and beta -cells, suggesting that AE1 does not function as both the apical and the basolateral anion exchanger of intercalated cells. Such studies are hindered by the lack of any cell culture line with stable transport characteristics consistent with those demonstrated for beta -cells from in vitro perfusion experiments and the lack of markers for distinguishing alpha - and beta -cells, combined with the lack of concordance of such markers (i.e., apical peanut lectin binding for beta -cells) to functional properties of the cells. The present studies were done to functionally characterize transport properties of the apical anion exchanger of beta -cells of in vitro perfused rabbit CCDs relative to known properties of the three anion exchange isoforms, specifically with regard to 1) effect of Cl- on stilbene sensitivity, 2) sensitivity to nonstilbene inhibitors, and 3) transport of sulfate.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vitro tubule perfusion. Rabbit CCDs were isolated and perfused in vitro as described previously (11, 12). Male New Zealand White rabbits, 1.5-2 kg, with free access to rabbit chow (Agway Prolab, Syracuse, NY) and water were killed by cervical dislocation after pentobarbital anesthesia. The left kidney was removed, cut in coronal sections, and placed in room temperature solution 1 (see Table 1). CCDs ~0.4 mm in length were dissected from the outer one millimeter of cortex, with the superficial end starting at the most distal connecting tubule arcade into the collecting duct. The tubule was transferred to a 100-µl laminar flow chamber and cannulated, and the peritubular bathing solution was exchanged at a rate of 2 ml/min. For the experiments demonstrating the ability to measure intracellular pH (pHi) changes due to sulfate transport on AE1 in alpha -cells, outer medullary collecting ducts were dissected from the inner stripe (OMCDis).

                              
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Table 1.   Composition of solutions

Solutions. Table 1 lists the compositions of the solutions used in this study. Solution 1 was Na+ free (tetramethylammonium replacement) and HEPES buffered and was used for dissection. Solution 2 was used for dye loading and, in combination with solution 3, for identification of anion exchange. These two solutions were Na+ free and HCO-3 buffered, differing only in the presence (solution 2) or absence of Cl- (equimolar gluconate replacement in solution 3). In solution 4, equimolar sulfate replaced Cl-. Solution 5, with addition of 10 µM nigericin, was used for calibration of pHi. The HCO-3-containing solutions were bubbled with 6.5% CO2 and 93.5% O2, whereas the HEPES-containing solutions were bubbled with 100% O2. Tetramethylammonium bicarbonate was made by bubbling tetramethylammonium hydroxide with 100% CO2. Glass tubing surrounded by heated water jackets connected the solution reservoirs and the perfusion chamber to minimize any CO2 or temperature loss. The pH of solutions 1, 2, 3, and 4 was adjusted to 7.40 with tetramethylammonium hydroxide or gluconic acid lactone, and all experiments were performed at 37°C.

pHi measurements/confocal imaging. To measure pHi, 5 µM of the acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) was added to the perfusate (solution 2) for 5 min, resulting in selective dye loading of only the intercalated cells (34). The fluorescent measurements were done with a dual-excitation laser-scanning inverted confocal fluorescent microscope, as previously described (11, 12). Briefly, a laser-scanning system (model MRC600; Bio-Rad, Hercules, CA) was coupled to the side port of a Diaphot inverted microscope (Nikon, Melville, NY). Excitation at 488 nm was provided by an argon laser (model 5424A; Ion Laser Technology, Salt Lake City, UT), and a helium-cadmium laser (model 4214NB; Liconix, Santa Clara, CA) was used for the 442-nm excitation. A ×40 fluorite objective (Nikon, NA 0.8) was used for all experiments. pHi was measured in single intercalated cells from an area of cytoplasm ~2-4 µm in diameter in up to three cells along a 250-µm length of tubule (SOM software, Bio-Rad) using gray scale image analysis. Tubule images were obtained using a zoom factor of 2.5, for a final magnification of ×800. The duration of laser exposure was limited to 1 s per ratio by a computer-driven electronic shutter (Vincent Associates, Rochester, NY). pHi measurements were taken every 5-10 s after a solution change. At the end of every experiment, an in vitro BCECF calibration curve was performed for each cell using nigericin and high-K+ solutions. Earlier studies using these solutions with a pH of 6.00, 6.30, 6.80, 7.35, and 7.80 for calibration points have shown that the 488/442 nm fluorescence excitation ratio-pHi relationship between 6.80 and 7.35 is linear in this experimental system (9). So for the present study, the pH of solution 5 was adjusted to only 7.35 and 6.80, values that bracketed the pHi changes occurring in the experiments, with KOH or HCl. The maximum decrease in the absolute intensity from the 442-nm excitation from the start to the end of any experiment was <10%.

Intercalated cell subtyping. Complexities exist in the functional phenotyping of rabbit CCD intercalated cells. By measuring pHi of CCD intercalated cells in response to sequential replacement of lumen and basolateral Cl- with gluconate, Emmons and Kurtz (12) first functionally identified rabbit outer CCD intercalated cells with both apical and basolateral anion exchange, noting that 57% of rabbit outer CCD intercalated cells had both apical and basolateral anion exchange (called gamma -cells), whereas 39% had exclusively apical anion exchange (beta -cells) and 4% had only basolateral anion exchange (alpha -cells). Concerned that intracellular Cl- depletion with such a protocol altered the ability to detect basolateral anion exchange, Weiner et al. (35) found that all female rabbit CCD intercalated cells with apical anion exchange also possessed basolateral anion exchange when the apical anion exchanger was immobilized by removal of both Cl- and HCO-3 simultaneously from the lumen. In contrast, in a preliminary report, Emmons (10) found 55% of intercalated cells from male CCDs were identified as possessing exclusively apical anion exchange with either a 0 Cl-, 25 mM HCO-3 or a 0 Cl-, 0 HCO-3 perfusate, whereas 45% cells demonstrated both apical anion exchange and basolateral with either perfusate, suggesting the existence of discrete beta - and gamma -subtypes in the male rabbit CCD at a given point in time.

In the present study, tubules were perfused and bathed in Na+-free, HCO-3-buffered solution (solution 2). The perfusate was then changed to a dye-free solution (solution 2) for 5 min before any measurements were recorded. All cells were initially subtyped according to the location of anion exchange. To functionally identify anion exchange, Cl- was replaced with gluconate in the perfusate (solution 3). Resultant intracellular alkalinization identified the presence of apical Na+-independent anion exchange (11, 12). After a new steady-state pHi was obtained (~1 min), Cl- was then replaced with gluconate in the bathing solution. Additional intracellular alkalinization identified the presence of basolateral Na+-independent anion exchange. beta -Cells were identified as those intercalated cells that demonstrated exclusively apical anion exchange. In the present study, beta -cells represented 48% of the outer CCD intercalated cells. Anion exchange activity was measured as the dpHi/dt, in the initial 30 s after lumen Cl- replacement with gluconate. All measurements of anion exchange activity were completed within 30 min of initiation of perfusion due to the time-dependent decay in HCO-3 transport by in vitro perfused rabbit CCDs (26).

Statistics. Results are reported as means ± SE. A paired t-test was used to compare results of anion exchange activity for the same cell in the protocols before and after exposure to a specific inhibitor and in the protocols designed to study sulfate transport. Statistical significance was accepted at the P <=  0.05 level.

Materials. Tetramethylammonium hydroxide was from Fisher. BCECF-AM was purchased from Molecular Probes. All other chemicals were from Sigma.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of stilbenes on apical anion exchange of rabbit CCD beta -intercalated cells. Prior studies had demonstrated a lack of effect of 0.5 mM H2-DIDS on the apical anion exchanger of rabbit CCD beta -cells (12). However, H2-DIDS is known to react more slowly with erythroid AE1 than DIDS (23). In addition, DIDS is the most potent stilbene antagonist of AE1 and is the stilbene derivative that has been most extensively studied for the three anion exchange isoforms (7). To determine the sensitivity of the apical anion exchanger of CCD beta -cells to DIDS, apical anion exchange activity was measured in single beta -intercalated cells in paired experiments before and after ~15 min addition of DIDS to the perfusate. Figure 1 shows a representative trace of this protocol with 0.2 mM lumen DIDS. In this tracing, baseline pHi was ~7.0. Replacement of lumen Cl- with gluconate at point A resulted in a brisk intracellular alkalinization. Neither replacement of bath Cl- with gluconate nor subsequent return of bath Cl- caused any further pHi change. Then, Cl- was returned to the lumen in addition to 0.2 mM lumen DIDS. After about 15 min, lumen Cl- was again replaced with gluconate in the ongoing presence of 0.2 mM DIDS (point B). An intracellular alkalinization similar to that in the absence of DIDS resulted. There was no difference in baseline pHi prior to the measurement of initial anion exchange (point A) between the groups of tubules (6.90 ± 0.06 in the 0 mM DIDS group, 6.94 ± 0.08 in the 0.1 mM DIDS group, and 6.88 ± 0.05 in the 0.2 mM DIDS group). Mean baseline anion exchange activity (dpHi/dt at point A) was no different between the groups of tubules (1.28 ± 0.08 pH/min in the 0 DIDS group, 1.22 ± 0.10 pH/min in the 0.1 mM DIDS group, and 1.16 ± 1.12 pH/min in the 0.2 mM DIDS group). There was also no difference in baseline pHi prior to the measurement of anion exchange activity in the absence (point A) and the presence of lumen DIDS (point B) in each group of tubules (data not shown). In the presence of Cl-, the mean percent inhibition of apical anion exchange was 5.0 ± 2.2% with 0.1 mM lumen DIDS (n = 15 cells, 6 CCDs) and 4.3 ± 2.8% with 0.2 mM lumen DIDS (n = 27 cells, 9 CCDs). These values were no different than the 4.7 ± 2.6% mean inhibition found in control experiments (n = 38 cells, 12 CCDs). Thus beta -cell apical anion exchange was not affected by either 0.1 or 0.2 mM lumen DIDS applied in the presence of 115 mM Cl-.


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Fig. 1.   Representative trace showing effect of 0.2 mM lumen DIDS exposure in presence (+) and absence (-) of lumen Cl- on apical anion exchange activity in a beta -cell. Rate of intracellular alkalinization in response to replacement of lumen Cl- (solution 2, Table 1) with gluconate (solution 3), used as a measure of apical anion exchange activity, is unchanged before (point A) and after (point B) 14-min perfusion with 0.2 mM lumen DIDS. In presence of 115 mM Cl-, 0.2 mM DIDS did not inhibit beta -cell apical anion exchange. pHi, intracellular pH.

Stilbenes are known to compete with Cl- for the external binding site of erythroid AE1 (7, 23). To determine whether the lack of DIDS sensitivity of the beta -cell apical anion exchanger was due, at least in part, to the presence of Cl-, additional experiments were performed with luminal DIDS exposure in the prolonged absence of Cl-. A representative trace of this protocol is seen in Fig. 2. After initial removal and return of lumen Cl- to identify apical anion exchange (point A), lumen Cl- was again replaced with gluconate but with either 0.1 or 0.2 mM DIDS added to the perfusate (point B). The tubule was perfused with DIDS in the prolonged absence of lumen Cl- for ~15 min. Then, Cl- was returned to the lumen, in the ongoing presence of DIDS. Finally, lumen Cl- was again replaced by gluconate to assess apical anion exchange activity (point C). In contrast to the lack of effect of DIDS on beta -cell apical anion exchange activity in the presence of Cl-, apical anion exchange activity was significantly decreased by lumen DIDS exposure in the absence of Cl-, as can be seen at point C in Fig. 2. There was no difference in baseline pHi prior to the measurement of initial anion exchange (point A) between the groups of tubules (6.88 ± 0.06 in the 0 DIDS group, 6.92 ± 0.05 in the 0.1 mM lumen DIDS group, and 6.90 ± 0.06 in the 0.2 mM lumen DIDS group). There was no difference in baseline anion exchange activity regardless of whether it was measured at either point A or point B for any tubule (data not shown). Mean baseline anion exchange (dpHi/dt at point A) was no different in the three groups of tubules (1.26 ± 0.10 pH/min in the 0 DIDS group, 1.28 ± 0.14 pH/min in the 0.1 mM DIDS group, and 1.18 ± 0.12 pH/min in the 0.2 mM DIDS group). There was also no difference in baseline pHi prior to measurement of anion exchange activity in the absence (points A and B) and presence (point C) of lumen DIDS for any group of tubules (data not shown).


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Fig. 2.   Representative trace showing effect of 0.2 mM lumen DIDS exposure in absence of lumen Cl- on apical anion exchange activity in a beta -cell. After initial measurement of apical anion exchange at point A (as described for Fig. 1 and in the text), 0.2 mM DIDS was added to a Cl--free luminal solution (solution 3) at point B, and perfusion continued for ~15 min. Remeasurement of apical anion exchange at point C revealed a significantly decreased rate of alkalinization, indicating inhibition of apical anion exchange by 0.2 mM DIDS in absence of Cl-.

Figure 3 summarizes the mean percent inhibition of beta -cell apical anion exchange due to lumen DIDS exposure in the absence of Cl-. In control experiments with this protocol, beta -cell apical anion exchange was not altered after a 15-min perfusion without lumen Cl- (n = 27 beta -cells, 9 CCDs). In contrast, after 0.1 mM lumen DIDS exposure in the absence of Cl-, there was a 36% inhibition of apical anion exchange (n = 8 beta -cells, 4 CCDs, P < 0.05 vs. control). Similarly, after 0.2 mM lumen DIDS exposure in the absence of Cl-, there was a 67% inhibition of apical anion exchange (n = 24 beta -cells from 9 CCDs, P < 0.001 vs. control). Thus, when applied in the absence of Cl-, both 0.1 and 0.2 mM lumen DIDS caused significant inhibition of beta -cell apical anion exchange.


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Fig. 3.   Mean percent inhibition of beta -cell apical anion exchange due to a 15-min exposure to lumen DIDS in absence of lumen Cl-. When applied in absence of Cl-, both 0.1 and 0.2 mM lumen DIDS caused significant inhibition of beta -cell apical anion exchange. * P < 0.05 vs. 0 DIDS; ** P < 0.001 vs. 0 DIDS; n indicates number of cells.

Effect of nonstilbene anion exchange inhibitors on the apical anion exchange of rabbit CCD beta -intercalated cells. To determine the sensitivity of the CCD beta -cell apical anion exchanger to other nonstilbene anion exchange inhibitors, apical anion exchange activity was measured in single beta -intercalated cells in paired experiments before and after ~15 min of luminal addition of known anion exchange inhibitors. An example of this protocol for 10 mM phenylglyoxal is shown in Fig. 4. At point A, baseline apical anion exchange was assessed by replacement of lumen Cl- with gluconate. Then Cl- was returned to the lumen with the addition of 10 mM phenylglyoxal for ~14 min. At point B, apical anion exchange was remeasured in the ongoing presence of phenylglyoxal. As is evident, there was no inhibition of apical anion exchange activity. Table 2 lists the different nonstilbene inhibitors tested in paired experiments with 15-min exposure to the particular inhibitor. In separate paired experiments, neither 15 mM maleic anhydride, 10 mM phenylglyoxal, 0.2 mM niflumic acid, 1 mM ethacrynic acid, 1 mM furosemide, 1 mM probenecid, nor 0.1 mM diphenylamine-2-carboxylate (DPC) inhibited beta -cell apical anion exchange activity.


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Fig. 4.   Representative trace showing effect of 10 mM lumen phenylglyoxal on apical anion exchange of a beta -cell. Apical anion exchange was unchanged before (point A) and after (point B) 12 min of perfusion with 10 mM phenylglyoxal.

                              
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Table 2.   Inhibition of beta -cell apical anion exchange by nonstilbene agents

In contrast to the above agents that had no effect on beta -cell apical anion exchange, 6 mM alpha -cyano-4-hydroxycinnamic acid (CHC) almost totally inhibited apical anion exchange activity. Additional experiments with CHC found that the degree of inhibition of apical anion exchange for a given CHC concentration was no different after either a 1-min or a 15-min exposure to luminal CHC (data not shown). So, because of the nonspecificity of this inhibitor, a 1-min luminal CHC exposure was used in subsequent experiments to determine a dose-response curve. An example of this protocol is seen in Fig. 5. Baseline pHi in this beta -cell was ~6.95. At point A, lumen Cl- was replaced with equimolar gluconate, resulting in a brisk alkalinization, which returned to baseline with return of lumen Cl-. After a few minutes, 6 mM CHC was added to the perfusate for 1 min, and then, at point B, lumen Cl- was again replaced with gluconate in the ongoing presence of CHC. pHi was essentially unchanged with this maneuver. As is also seen in Fig. 5, apical anion exchange inhibition by CHC was reversible (point C). The mean percent inhibition of beta -cell apical anion exchange for various concentrations of CHC is listed in Table 2 and reveals an IC50 of 2.4 mM.


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Fig. 5.   Representative trace showing the effect of a 1-min exposure to 6 mM lumen alpha -cyano-4-hydroxycinnamic acid (CHC) on apical anion exchange of a beta -cell. Compared with initial apical anion exchange activity in this cell (point A), apical anion exchange is significantly inhibited after 1-min exposure to 6 mM luminal CHC (point B). This also demonstrates the reversibility of this inhibition of apical anion exchange after 5-min perfusion without CHC (point C).

Transport of sulfate on the beta -cell apical anion exchanger. One of the substrates band 3 is known to transport in addition to Cl- and HCO-3 is sulfate. In the first protocol designed to investigate the substitution of sulfate on the apical anion exchanger of CCD beta -cells, pHi was measured as lumen Cl- was acutely replaced by either equimolar gluconate or sulfate in random order. Figure 6 shows a representative tracing for this protocol. Baseline pHi was ~7.0. Replacement of lumen Cl- with equimolar sulfate (point A) resulted in a brisk alkalinization. Return of lumen Cl- resulted in return to a similar baseline pHi. Then, replacement of lumen Cl- with gluconate (point B) resulted in an alkalinization that was of a rate and magnitude similar to that which occurred with sulfate perfusion. Results of this protocol from 35 beta -cells are summarized in Table 3. Return of lumen Cl- between anion substitutions resulted in similar baseline pHi for both sulfate and gluconate (data not shown). Both the rate of pHi increase and the Delta pH were similar regardless of whether either gluconate or sulfate replaced lumen Cl-.


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Fig. 6.   Representative trace showing effect of replacement of lumen Cl- by equimolar sulfate (solution 4) or gluconate (solution 3) on beta -cell intracellular pH (pHi). Similar intracellular alkalinizations result from substitution of either sulfate (point A) or gluconate (point B) for luminal Cl-.

                              
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Table 3.   Effect of gluconate or sulfate substitution for lumen chloride on beta -cell pHi

In a second protocol designed to investigate transport of sulfate on the apical anion exchanger, lumen Cl- was initially replaced with gluconate. After a new steady-state pHi was reached, lumen gluconate was then replaced with either equimolar sulfate or chloride in a random order. Figure 7 demonstrates a typical tracing for this protocol. Baseline pHi was ~6.95. Replacement of lumen Cl- by gluconate resulted in a brisk alkalinization, with a new steady-state pHi of ~7.25. At point A, equimolar sulfate was substituted for gluconate. pHi was essentially unchanged over the next 8 min. However, substitution of Cl- for lumen sulfate (point B) resulted in a brisk acidification, back to the baseline pHi. Table 4 summarizes the results of this protocol for 41 beta -cells. Neither the rate of pHi change nor the Delta pH was significantly different from zero when sulfate replaced lumen gluconate. In contrast, brisk acidification was always seen when Cl- was returned to the lumen in place of gluconate. Taken together, these data indicate that sulfate is transported similarly to gluconate on the apical anion exchanger of rabbit CCD beta -cells.


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Fig. 7.   Representative trace showing effect of replacement of lumen gluconate (solution 3) by equimolar sulfate (solution 4) or Cl- (solution 2) on beta -cell pHi. At point A, beta -cell pHi is unchanged when sulfate is substituted for lumen gluconate; however, a brisk intracellular acidification occurs at point B when Cl- is substituted for lumen gluconate.

                              
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Table 4.   Effect of chloride or sulfate substitution for lumen gluconate on beta -cell pHi

Although sulfate is thought to have the same binding affinity for erythroid AE1 as Cl-, it is transported at a rate ~3 orders of magnitude slower than Cl- (16). To make certain that the reason for the apparent lack of sulfate transport on the apical anion exchanger of beta -cells was not due to inability of the experimental system used for these studies to detect sulfate-induced pHi changes, experiments were done to measure pHi of single alpha -cells (known to have basolateral AE1) from rabbit OMCDis in response to basolateral ionic changes. For each OMCDis intercalated cell, the absence of an apical anion exchanger in the cell was initially demonstrated by the lack of any pHi change resulting from replacement of lumen Cl- with gluconate. Then, pHi of single OMCDis alpha -cells was measured as bath Cl- was acutely replaced by either equimolar gluconate or sulfate in random order. Replacement of bath Cl- with equimolar gluconate resulted in an alkalinization at a rate of 1.53 ± 0.19 pH/min in 14 alpha -cells from 6 OMCDis, similar to that seen for CCD beta -cells. However, in contrast to the results in CCD beta -cells (Table 3; Fig. 6), after return of bath Cl- and restoration of baseline pHi, replacement of bath Cl- with equimolar sulfate in the same cells caused an intracellular acidification at a rate of -1.01 ± 0.20 pH/min.

For erythroid AE1, sulfate transport by the basolateral anion exchanger occurs by sulfate-proton cotransport (16). Although the mode of sulfate transport by kidney AE1 has not yet been proved, given the presence of E681 (the proton binding site for sulfate-proton cotransport in human red cells) in kidney AE1, it is very likely that sulfate-proton cotransport occurs in the kidney also. When bath Cl- is replaced by equimolar sulfate, the ion concentration gradients acutely change, with a 25 mM Cl- concentration gradient from the cell to the bath and an ~115 mM sulfate concentration gradient from the bath into the cell. Thus the intracellular acidification can be explained by Cl- leaving the cell down its concentration gradient in exchange for sulfate entry down its concentration gradient from the bath into the cell.

To confirm the ability to identify sulfate-induced pHi changes in alpha -cells, separate experiments were done where bath Cl- was initially changed to equimolar gluconate, resulting in alkalinization. After establishment of a new steady-state pHi, the bath solution was changed to either equimolar Cl- or sulfate, in a random order. OMCDis alpha -cell pHi decreased at a rate of -1.45 ± 0.14 pH/min in 11 cells from 4 tubules in response to Cl- substitution for bath gluconate, not different from the response demonstrated in CCD beta -cells. However, in contrast to the response in CCD beta -cells (Table 4; Fig. 7), OMCDis alpha -cell pHi decreased at a rate of -1.12 ± 0.10 pH/min when sulfate was substituted for bath gluconate in the same 11 cells.

One technical point worthy of note concerns the differences in effective osmotic coefficients between anions and the possibility that increased bath osmolarity with sulfate substitution altered cell volume. That cell volume did not change in the experiments with bath sulfate substitution was evidenced by the lack of any directional change in the 442-nm signal (within the overall <10% decrease that occurs over the time course of any experiment due to bleaching and dye loss). In addition, neither cell area based on two-dimensional tracings of individual cell outlines from saved images taken along the longitudinal axis of the tubule 1 min after each ion substitution nor the inner or outer diameters of the tubule from the same images were altered in either bath chloride, gluconate, or sulfate (data not shown). Finally, intracellular dye uptake in the cells was homogeneous, so it is unlikely that the measured signal came mostly from an intracellular osmotically isolated compartment. This indicates that no volume changes occurred with the bath anion substitutions.

Thus the pHi changes seen in the bath sulfate substitution protocols identify functional sulfate transport on the basolateral surface of OMCDis alpha -cells. This could be accomplished by exchange of intracellular chloride for extracellular sulfate, but might be masked by exchange of intracellular bicarbonate for extracellular sulfate. These could well be mediated by AE1. However, another sulfate/anion exchanger might also be contributing, since at pH 7.0, erythroid AE1 has never been observed to transport sulfate so rapidly in comparison to chloride.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There are at least three members of the AE anion exchanger gene family. AE1, the prototype anion exchanger, comprises ~25% of the protein mass of the erythrocyte membrane and has also been identified as the basolateral anion exchanger of CCD alpha -intercalated cells. In addition, two nonerythroid anion exchange isoforms (AE2 and AE3) have been identified. AE2 has been shown to be a basolateral anion exchanger in non-alpha -intercalated cells, and at least one other AE has been reported in the kidney in abstract publications (2, 3). Although the three isoforms have ~85% amino acid sequence homology of their transmembrane domains, each has a distinctive pattern of tissue expression, and differences in both transport function and regulation between the isoforms have also been demonstrated (1, 19). The present studies were done to functionally "fingerprint" transport properties of the apical anion exchanger of beta -intercalated cells of in vitro perfused rabbit CCDs relative to known properties of the three anion exchange isoforms, specifically with regard to 1) effect of Cl- on stilbene sensitivity, 2) sensitivity to nonstilbene inhibitors, and 3) transport of sulfate.

One distinguishing characteristic of the three anion exchange isoforms is their sensitivity to stilbene inhibitors. Stilbenes can cause inhibition of an anion exchanger either reversibly by competing with extracellular anions for the external anion binding site or irreversibly by binding covalently to one of two conserved lysines. Thus the particular stilbene agent used, as well as its concentration and the duration of exposure, the presence of other anions, and the temperature affect the degree of inhibition of anion exchange (7, 16, 23). DIDS is the most potent stilbene antagonist of AE1 (7). Of the three isoforms, the erythroid AE1 isoform demonstrates the highest degree of inhibition due to DIDS, with an IC50 of 0.08 µM in the presence of Cl- (7). Although still sensitive in the micromolar range, the nonerythroid anion exchangers require higher DIDS concentrations for inhibition. Humphreys and coworkers (15) found an IC50 of 13 µM in 131 mM Cl solutions, but 0.53 µM in 0 Cl solutions for murine AE2 expressed in oocytes. Similarly, He and coworkers (14) found an IC50 of DIDS of 4 µM for rat AE2 expressed in SF9 cells in 0 Cl medium. In contrast, Lee and co-workers (21) found a DIDS IC50 of 142 µM for mouse AE2 expressed in human 293 cells. It is noteworthy that the IC50 for Cl- flux on the anion exchanger in K562 cells, known to express AE2 mRNA, is 1.5 µM (20). The reasons for the higher AE2 inhibitory DIDS concentration demonstrated in the study by Lee et al. (21) is not clear but may be reflective of the different expression systems used in the two studies, either due to differences in membrane composition of the specific expression system or differences in the surface density of the expressed anion exchanger. For murine AE3, Lee and co-workers (21) found the DIDS IC50 was 0.43 µM in 0 Cl solutions at 37°C for this isoform expressed in human 293 cells.

The present studies demonstrate that the apical anion exchanger of rabbit CCD beta -cells demonstrates significantly less sensitivity to DIDS than is known for the three anion exchange isoforms. It should be noted that many of the studies in erythrocytes or of expressed anion exchangers involve significantly longer exposures to the inhibitors than used in the present study. In the present studies, the exposure to DIDS was limited to 15 min because of the time required to load the cells with BCECF and complete the baseline anion exchange measurement coupled to the known time-dependent decay in CCD bicarbonate transport, and likely reflects a combination of reversible and irreversible interactions. In the presence of Cl-, 0.2 mM DIDS had no effect on the beta -cell apical anion exchanger, whereas in the absence of Cl-, the 0.2 mM DIDS significantly inhibited the beta -cell apical exchanger. It is unlikely that depletion of intracellular Cl- is responsible for the effect of Cl- removal on DIDS sensitivity, given the similar alkalinization seen in the control tubules with this protocol without DIDS. Although the higher concentrations of DIDS required to inhibit the beta -cell apical exchanger might reflect differences in membrane composition of the beta -cell, this characteristic could also be due to structural differences in the stilbene-binding site compared with the known anion exchange isoforms.

In addition to stilbenes, several other agents inhibit anion exchange at relatively low concentrations (16). Of the three anion exchange isoforms, nonstilbene inhibitor potency has been studied most extensively for AE1. Motais and Cousin (24) found an instantaneous and reversible inhibition of ox erythrocyte anion exchange to ethacrynic acid with an IC50 of 7 µM. Cousin and Motais (8) found that niflumic acid caused an instantaneous, reversible, and noncompetitive inhibition with an IC50 of 0.6 µM in human erythrocytes. Obaid et al. (25) showed that 15 mM maleic anhydride, an agent known to react with amino groups of proteins, caused a 40% decrease in Cl flux in human erythrocytes. Motais and Cousin (24) found an IC50 of 40 µM to probenecid in ox erythrocytes. Brazy and Gunn (6) found a rapid inhibition of human erythrocyte anion exchange by furosemide, with an IC50 of 150 µM. Carbantchnik and Greger (7) reported an IC50 of 70 µM for DPC for anion exchange in human erythrocytes. Although phenylglyoxal is usually thought of as an arginine-specific reagent, this directed inhibition occurs under an alkaline extracellular pH and is competitive with Cl-. In conditions different from these, phenylglyoxal can also cause a reversible non-arginine-specific inhibition and has been shown to inhibition Cl- self-exchange in human erythrocytes with an IC50 of 2 mM, studied at pH 7.2 (17). In contrast, under the perfusion conditions in this study, beta -cell apical anion exchange was not altered by 1 mM ethacrynic acid, 0.2 mM niflumic acid, 15 mM maleic anhydride, 1 mM probenecid, 1 mM furosemide, 0.1 mM DPC, or 10 mM phenylglyoxal.

Less is known about the effects of nonstilbene inhibitors on the AE2 and AE3 isoforms. Most information about nonstilbene inhibitor potencies for AE2 comes from the work of Humphreys and coworkers (15), who measured 36Cl- influx mediated by the murine isoform expressed in oocytes. In separate studies, 25 µM niflumic acid, 500 µM probenecid, and 1 mM CHC caused only minimal nonsignificant decreases in 36Cl- flux, and 50 µM dipyridamole was without effect (15). Whether the higher inhibitor concentrations used in the present study would have altered AE2 transport is not known. Unfortunately, the nonstilbene inhibitors have not been studied for the AE3 isoform.

Although the physiological function of the anion exchange proteins is to effect Cl-/HCO-3 exchange, they can transport other anions, including sulfate. Sulfate is thought to have the same binding affinity for erythroid AE1 as Cl- but is transported at a rate ~3 orders of magnitude slower than Cl- (16). Although the sulfate flux is slower than that of Cl- on AE1, given the rapidity of the AE1-mediated Cl- flux, it is possible to measure an erythrocyte sulfate flux that is greater than baseline sulfate permeability, and the potency of inhibitors has been demonstrated to be practically identical for both sulfate and Cl- tracer flux on AE1 (16). Sulfate transport has been demonstrated in AE2 and AE3 expression systems (29). One difficulty in comparing substrate fluxes between experiments is that the relative fluxes are affected by specific conditions of the experiments, such as temperature, anion concentrations, and pH. Nonetheless, the present studies were unable to identify any transport of sulfate on the beta -cell apical anion exchanger over that of gluconate. This lack of demonstrable transport of sulfate in beta -cells is consistent with the finding of Boyer and Burg (5) who noted that replacement of lumen chloride with sulfate in perfused rabbit CCDs inhibited unidirectional bicarbonate secretion.

Apart from the three anion exchange isoforms, there are two anion exchangers that bear particular similarities to the beta -cell transporter. First, the turtle bladder, thought to be the homolog of the mammalian collecting duct, possesses an apical anion exchanger that is thought to mediate bicarbonate secretion. Neither the molecular nor the protein identity of the turtle bladder apical anion exchanger is known at present. It is interesting that, similar to the lack of transport of sulfate on the beta -cell apical anion exchanger described in the present studies, substitution of sulfate for Cl- caused the bicarbonate flux to decrease to 13% in the turtle bladder (18). However, unlike the characteristics demonstrated for the beta -cell apical anion exchanger presented here, bicarbonate flux in turtle bladders was unchanged by the presence of 9 mM CHC, but decreased slightly (14%) but significantly with 1 mM furosemide (14). In further contrast to the beta -cell apical anion exchanger, bicarbonate flux in the turtle bladder was significantly inhibited (32%) by 100 µM DIDS in the presence of 102 mM Cl- (32). Some substrate differences appear to exist between the turtle bladder and the beta -cell apical anion exchanger as well. Substitution of Br- for Cl- caused the turtle bladder bicarbonate flux to decrease to less than 25% of control flux (18). In contrast, a study of halide transport on the beta -cell apical anion exchanger was unable to distinguish Br- transport from that of Cl- (11).

Neutrophils possess an anion exchanger that is relatively insensitive to DIDS. The neutrophil anion exchanger can be inhibited by CHC, with a Ki of 9 mM, but is insensitive to 1 mM furosemide and 1 mM ethacrynic acid (30). Interestingly, the neutrophil anion exchanger, similar to the beta -cell apical anion exchanger, does not transport sulfate. However, there are some apparent substrate differences between the neutrophil and the beta -cell transporters. Whereas the neutrophil anion exchanger transports F- with greater affinity than I-, no transport of F- could be detected on the beta -cell apical anion exchanger (11, 30). As for the beta -cell apical anion exchanger, the molecular/protein identity of the neutrophil anion exchanger is also not known.

The transport "fingerprints" of the beta -cell apical anion exchanger described here are distinct from those demonstrated for any of the three anion exchange isoforms or from those described for anion exchange transporters in other tissues. Yet unrecognized features of the beta -cell apical anion exchanger must account for these distinctive transport characteristics.


    ACKNOWLEDGEMENTS

This work was supported by a Veterans Affairs Career Development Research Award and a Veterans Affairs Merit Review Award.


    FOOTNOTES

Portions of this work were published in abstract form (J. Am. Soc. Nephrol. 5: 252, 1994; and J. Am. Soc. Nephrol. 6: 308, 1995).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. Emmons, Univ. of Cincinnati, PO Box 670585, Cincinnati, OH 45267-0585.

Received 2 March 1998; accepted in final form 22 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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