Potassium depletion increases proton pump (H+-ATPase) activity in intercalated cells of cortical collecting duct

Randi B. Silver1, Sylvie Breton2, and Dennis Brown2

1 Department of Physiology and Biophysics, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York 10021; and 2 Program in Membrane Biology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129


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Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H+-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K+-depleted rats and that upregulation of tubular H+- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H+-ATPase activity was determined in individually identified ICs from control and chronically K+-depleted rats (9-14 days on a low-K+ diet) by monitoring K+- and Na+-independent H+ extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pHi) indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHi was determined by using ratiometric fluorescence imaging. The rate of pHi recovery in ICs in response to an acute acid load, a measure of plasma membrane H+-ATPase activity, was increased after K+ depletion to almost three times that of controls. Furthermore, the lag time before the start of pHi recovery after the cells were maximally acidified fell from 93.5 ± 13.7 s in controls to 24.5 ± 2.1 s in K+-depleted rats. In all ICs tested, Na+- and K+-independent pHi recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H+-ATPase. Analysis of the cell-to-cell variability in the rate of pHi recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K+-depleted rats. Immunocytochemical analysis of collecting ducts from control and K+-depleted rats showed that K+-depletion increased the number of ICs with tight apical H+ATPase staining and decreased the number of cells with diffuse or basolateral H+-ATPase staining. Taken together, these data indicate that chronic K+ depletion induces a marked increase in plasma membrane H+ATPase activity in individual ICs.

proton-adenosinetriphosphatase; bafilomycin; intracellular pH; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; immunocytochemistry


    INTRODUCTION
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IT IS WELL ESTABLISHED THAT HCO3- reabsorption increases in the cortical collecting duct (CCD) during chronic hypokalemia (13). Two possible mechanisms through which enhanced HCO3- reabsorption can occur in the CCD are via the vacuolar-type proton pump (H+-ATPase) and the H+/K+ ATPase (9), both of which are present in intercalated cells (ICs). Despite the presence of both of these transporters in ICs, the signals responsible for their regulation and their relative contributions to transepithelial proton transport and intracellular pH homeostasis are not well defined.

Functional studies using microperfused late distal tubules from control rats demonstrated active proton secretion (JHCO3), one-third of which was blocked by bafilomycin (27), a specific inhibitor of the H+-ATPase (6). Under conditions of K+ depletion, as a consequence of a low-K+ diet, JHCO3 almost doubles in this nephron segment (27). Much of the enhanced proton (H+) secretion associated with the K+- restricted diet was inhibited by Sch-28080, a specific blocker of the gastric H+-K+-ATPase (26). The contribution of bafilomycin-sensitive H+-ATPase to net acid secretion in tubules from K+-restricted rats was not studied. In microperfused terminal inner medullary collecting ducts from rats maintained on a low-K+ diet, only one-half of the measured total CO2 flux was sensitive to the H+-K+-ATPase inhibitors Sch-28080 and ouabain (25), leaving open the possibility that enhanced bafilomycin-sensitive H+-ATPase also contributes to the net acid flux under K+-depleted conditions. Recently, it was shown that electrogenic bafilomycin-sensitive H+ secretion is increased with chronic hypokalemia in microperfused distal rat nephron comprised of distal convoluted tubule, the connecting tubule, and the initial collecting tubule (3, 4). Furthermore, immunocytochemical localization of the proton translocating H+-ATPase showed that K+-deprivation resulted in an increase in the percentage of intercalated cells with H+-ATPase localized in the apical membrane (4). However, in this study the functional contribution of the ICs to net H+ secretion was not directly assessed.

The purpose of the present investigation was, therefore, to evaluate directly the bafilomycin sensitive H+-pumping activity in ICs of the CCD. H+-ATPase activity was measured in individual cells from control and K+-depleted rats by following the K+- and Na+-independent H+ extrusion rate of ICs in response to an acute acid load. Measurements were performed in ICs from split-open rat cortical collecting tubules (CCTs) in conjunction with ratiometric digital imaging techniques after the cells of the tubule were loaded with the pH-sensitive fluorophore 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The results revealed a marked increase in the rate of bafilomycin-sensitive pHi recovery in response to an acute acid load in cells from K+-depleted rats compared with ICs from control rats. Also, immunocytochemistry showed a significant increase in apical H+ATPase staining of ICs in the CCD from the K+-restricted rats. Taken together, these data indicate that chronic K+ depletion induces a marked increase in plasma membrane H+ATPase activity at the single IC level.


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Animals and Tubule Preparation

Pathogen-free Sprague-Dawley rats of both sexes (Charles River Laboratories, Kingston, NY), weighing between 75 and 150 g, were used for these experiments. Rats were fed either a normal diet (Purina Formulab 5008; Na+ content 0.28%, K+ content 0.11%) or a K+-deficient diet that was otherwise nutritionally balanced (Harlan Teklad 170550; Na+ content 0.4%) for 9-14 days.

CCTs were prepared and mounted in a perfusion chamber as previously described (19). Rats were killed by cervical dislocation, the kidneys were removed, and tubules were dissected free and opened to form a flat epithelium. Sometimes, two tubules were used from each animal. Solutions were gravity fed into a manually operated six-port Hamilton valve. The solution leaving the valve passed directly into a miniature water-jacketed glass coil (Radnoti Glass Technology, Monrovia, CA) for temperature regulation. The warmed solution entered the experimental chamber, which was mounted on the stage of an inverted epifluorescence microscope (Nikon Diaphot). The temperature of the superfusate in the chamber was maintained at 37°C.

Solutions

Tubules were superfused with HEPES-buffered solutions (Table 1) as described previously (19). A 1 mM solution of bafilomycin A1 (LC Laboratories, New Bedford, MA; Alexis, San Diego, CA) was prepared in dimethyl sulfoxide, and then diluted 1:10,000 to give a final concentration of 100 nM in the experimental superfusates. All chemicals were obtained from Sigma Chemical unless otherwise stated. Nigericin (Molecular Probes, Eugene, OR) was added to K+-Ringer solutions (solutions A and B) from a 10 mM stock (3 parts ethanol:1 part dimethyl formamide) to give a final concentration of 10 µM. Individual vials (50 µg) of the acetoxymethyl derivative of BCECF-acetyoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, OR) were stored dry at 0°C and reconstituted in dimethyl sulfoxide (at a concentration of 10 mM) for each experiment. The final loading concentration of dye was 5 µM in Na+-Ringer solution.

                              
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Table 1.   Composition of solutions

Fluorescence Measurements of Intracellular pH (pHi) With BCECF

Equipment. The basic components of the experimental apparatus have been described previously (18, 19) and consist of the following: an inverted epifluorescence microscope (Nikon Diaphot) equipped with a 75-W xenon lamp, Nikon CF Fluor ×100/1.3-na oil-immersion objective, two Metal Tek filter wheels back to back, a computer controllable excitation light shutter, and a cooled charge-coupled device camera (Princeton Instruments ) with a frame transfer chip (EEV-37) and 12-bit readout. The emitted fluorescence signal is relayed as real-time continuous output to an IBM PC/AT-compatible clone, and the image pairs were collected on a Sierra Pinnacle Micro optical disk drive (1.3 Gbytes). The imaging work station is controlled by using the Metafluor software package (Universal Imaging). Quantitative image pairs at 490- and 440-nm excitation with emission at 520 nm were obtained every 15 s for the duration of the experiment. The fluorescence excitation was shuttered off except during the brief periods required to record an image. To correct for intrinsic autofluorescence and background, images were obtained by using the experimental acquisition configuration on the split-opened portion of tubule before loading of the dye. These background images were subtracted from the corresponding images of cell fluorescence, and these corrected ratios were used for data analysis.

BCECF-loading and identification of intercalated cells. Split-open tubules were loaded in the experimental chamber with BCECF (5 µM) from both the basolateral and luminal sides at room temperature for 15 min, after which they were superfused with Na+-Ringer solution (solution 1) at 37°C for at least 15 min before the start of the experiment. Calibration of the emitted signal from each cell in the tubule was performed at the end of each experiment. Extracellular pH was varied from 6.8 to 7.8 (solutions A and B) in the presence of the K+/H+ exchanger nigericin (10 µM) in 145 mM K+ according to the method of Thomas et al. (24) and as described previously in this preparation (19). Cells in the experimental field of view were analyzed singularly and independently from their neighbors, as previously described (18, 19). ICs were differentiated from principal cells by both their visual appearance under transmitted light and by their much greater loading of BCECF, as previously described (19, 20, 28). Because our measurements revealed a normal distribution of pHi recovery rates after an acid load in the IC-population under control conditions, no attempt was made to distinguish alpha -type from beta -type intercalated cells for the functional portion of this study. Values from all intercalated cells measured were grouped together for statistical analysis.

Immunocytochemistry

Tissue preparation. Control or K+-depleted Sprague-Dawley rats were anesthetized with pentobarbital sodium (Nembutal; 0.1 ml of a 50 mg · ml solution-1 · 100 g body wt-1), and kidneys were fixed by perfusion through the left ventricle with a fixative containing 4% paraformaldehyde, 10 mM sodium periodate, 70 mM lysine [modified periodate-lysine-paraformaldehyde (PLP)], and 5% sucrose, as previously described (7). After a 5-min perfusion, kidneys were removed, sliced, and fixed by immersion for a further 6 h before rinsing and storage in PBS (10 mM sodium phosphate buffer containing 0.9% NaCl, pH 7.4). For preparation of 5-µm sections, tissues were cryoprotected by immersion in 30% sucrose for at least 1 h before sectioning with a Reichert Frigocut microtome using disposable knives.

Immunostaining procedure. Tissue sections (5 µm) picked up on Fisher Superfrost Plus slides were rinsed for 10 min in PBS and then treated with 1% SDS for 5 min. This step increases antigenicity in frozen sections of PLP-fixed tissues, as previously described (8). After three more rinses (5 min each) in PBS to remove the SDS, sections were incubated for 20 min in PBS/1% BSA to block nonspecific background staining. An affinity-purified primary anti-H+-ATPase antibody prepared in rabbit against the 11 COOH-terminal amino acids of the 56-kDa B1-subunit of the bovine H+-ATPase ("kidney" isoform) (14) was applied for 2 h at room temperature at a dilution of 1:100. After washing 2 × 5 min in high-salt PBS (PBS containing 2.7% NaCl) to reduce nonspecific staining, and a further 1 × 5 min in normal PBS, secondary goat anti-rabbit IgG (diluted 1:60) coupled to FITC (Jackson Immunologicals) was applied for 1 h. After further washing as above, sections were mounted in Vectashield antifading solution (Vector Laboratories, Burlingame, CA) diluted 1:1 in 0.1 M Tris · HCl, pH 8.0.

Sections were examined by using a Bio-Rad Radiance 2000 confocal microscope. Digital images were imported into Adobe Photoshop (4.0) and printed on a Tektronix Phaser 440 dye sublimation color printer.

Serum Analysis

Blood samples were obtained from some animals via the dorsal aorta and analyzed for Na+, K+, and Cl- by a commercial laboratory.

Statistics

Results are expressed as means ± SE, where n refers to the number of cells analyzed individually, unless otherwise noted. Different IC subtypes have been described in CCDs, but no apparent and consistent heterogeneity in the rate of pHi recovery from an acid load that would be suggestive of a differential response between alpha - and beta -ICs, was found in the present experiments. Therefore, data from all ICs examined for each experimental group were pooled for final statistical analysis. In addition, the numbers of animals and tubules used in each protocol are indicated in the figure legends. The NH4Cl pulse protocol was performed only once in each tubule, followed by the in situ nigericin calibration. Significant differences were determined by ANOVA. Significance was asserted if P < 0.05.


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Effects of a Low-K+ Diet on Acid-Base and Electrolyte Balance

Plasma K+, Na+, and Cl- values were measured in three control rats and six low-K+ rats and are presented in Table 2. Rats maintained on a low-K+ diet were hypokalemic compared with control animals (P < 0.0001).

                              
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Table 2.   Plasma electrolyte values

Bafilomycin-Sensitive, Na+- and K+-Independent pHi Recovery in Response to an Acute Acid Load

Control rats. H+-ATPase function was assayed as the rate of Na+- and K+- independent intracellular alkalinization in response to an acute pulse of 10 mM NH4+. Figure 1A is a representative trace of this response as observed in an individual IC from a control rat tubule. In this and all subsequent traces, the experimentally determined ratios have been converted to pHi as described in MATERIALS AND METHODS. Ratio image pairs were generated every 15 s and are represented by the data points from the IC in this experimental trace. After the steady-state pHi in Na HEPES-buffered solution (Table 1, solution 1) was monitored, an acute acidosis was induced in the cells by superfusing the tubule with a solution containing 10 mM NH4Cl, in the absence of extracellular Na+ and K+, with these cations replaced in the solution with N-methyl-D-glucamine (Table 1, solution 2). The superfusate was then changed to a Na+-, K+-, and NH4Cl-free solution (Table 1, solution 3), resulting in an abrupt intracellular acidosis that, as shown in Fig. 1A, brought the pHi down to 6.3. Within 90 s of reaching the nadir pHi, the pHi began to increase steadily presumably due to H+ extrusion via the H+-ATPase. The initial H+ extrusion rate measured in this cell (Delta pHi/Delta t), as calculated from the slope indicated by the dashed line in Fig. 1A, is 0.07 pHi U/min, which eventually leveled off to a pHi of 6.7. 


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Fig. 1.   A: K+- and Na+-independent intracellular pH (pHi) recovery after acute exposure to an NH4Cl acid pulse in an intercalated cell (IC) from a split-open tubule of a control rat in the absence of extracellular K+ and Na+. Y-axis represents the pHi as determined from the intracellular calibration of the dye in this tubule. The tubule was initially superfused with Na+-Ringer solution (NaR; solution 1, Table 1) and then changed to 10 mM NH4Cl (solution 2, Table 1). Acute exposure to NH4Cl resulted in acidification after its removal. On removal of the NH4, Na+, and K+, the pHi fell from an initial value of 7.00 to 6.30. Within 90 s, the pHi started to increase, with intracellular alkalinization occurring at a rate of 0.07 pHi U/min. The slope or rate of the K+- and Na+-independent pHi recovery was calculated at the beginning of the recovery process as illustrated in the trace. B: effect of bafilomycin. Y-axis represents the pHi as determined from the intracellular calibration of the dye in this tubule. The tubule was initially superfused with solution 1 (Table 1) and then changed to solution 2 (Table 1). Bafilomycin A1 (100 nM) was present in the superfusate from the NH4Cl until the end of the protocol as shown. Addition of the blocker prevented the K+- and Na+-independent H+ efflux observed in Fig. 1A, demonstrating H+-ATPase activity is responsible for the pHi recovery.

To be certain that this observed K+- and Na+-independent intracellular alkalinization was due to H+-pump activity, the same acid pulse protocol described above was performed but with the addition of the H+-ATPase inhibitor bafilomycin at a concentration (100 nM) known to inhibit H+ pump activity in rat distal tubules (4, 27) and CCDs (19). A representative trace of the pHi recovery response to an acute acidosis in the presence of bafilomycin, from a single IC from a control split-open CCT, is shown in Fig. 1B. In the presence of bafilomycin, the K+- and Na+-independent pHi recovery response was virtually abolished compared with the representative trace in Fig. 1A. Taken together, these results indicate that the IC-specific H+ATPase is responsible for the pHi recovery induced by the NH4Cl acid pulse and observed under control conditions in these cells.

K+ deficiency. To test the hypothesis that K+ depletion stimulates acid secretion via increased H+- ATPase activity in ICs, the above acid pulse protocol performed in control ICs was carried out in tubule preparations from low-K+ rats. Figure 2 is a representative response of an individual IC from a tubule of a K+-restricted rat. On removal of the NH4Cl pulse, the pHi fell to ~6.6 from an initial value of ~7.5. Within 15 s of the NH4Cl pulse, the pHi started to increase in this cell at an initial rate of 0.20 pHi U/min, bringing the pHi back to the pre-acid load initial value. This rate of intracellular alkalinization was much greater than that observed in the control IC of Fig. 1A. The mean rate of Na+- and K+-independent pHi recovery for all of the ICs from low-K+ rats was 0.22 ± 0.02 pH U/min, (n = 44 ICs, 6 tubules, 5 rats), which is almost threefold greater (P < 0.0001) than the mean rate measured in control ICs (0.08 ± 0.01 pH U/min, n = 57 ICs, 8 tubules, 6 rats) (Fig. 3).


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Fig. 2.   K+- and Na+-independent pHi recovery after acute exposure to an NH4Cl acid pulse in an IC from a split-open tubule of K+-depleted rat. Y-axis represents the pHi as determined from the intracellular calibration of the dye in this tubule. The tubule was initially superfused with solution 1 (Table 1) and then changed to solution 2 (Table 1). Acute exposure to NH4Cl resulted in acidification after its removal. On removal of the NH4, Na+, and K+, the pHi fell to ~6.6 from the initial value of 7.5. Within 15 s, the pHi started to increase in this cell at an initial rate of 0.20 pHi U/min and returned the pHi back to the initial pHi of 7.5.



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Fig. 3.   Comparison of H+-ATPase activity (Delta pHi/Delta t) in the absence and presence of bafilomycin in control ICs and K+-depleted ICs. Values are means ± SE. The K+- and Na+-independent Delta pHi/Delta t (pH U/min) rate is compared in ICs from control rats and in ICs from K+-depleted rats with and without bafilomycin (100 µM) added to the superfusate (*** P < 0.0001).

Bafilomycin significantly inhibited the K+- and Na+- independent intracellular alkalinization rates in control ICs (to 0.02 ± 0.02 pH U/min, n = 12 ICs, 3 tubules, 3 rats; P < 0.05) and in ICs from low-K+ rats (to 0.02 ± 0.01 pHi U/min n = 16 ICs, 3 tubules, 3 rats). These results demonstrate that chronic K+ restriction stimulates the activity of bafilomycin-sensitive H+-ATPase in ICs of the CCD.

Another difference between the responses of the control ICs and the K+-depleted ICs was the magnitude of the pHi recovery response as evidenced by a comparison of Figs. 1A and 2. As shown in Fig. 2, the pHi in the IC from the K+-restricted tubule leveled off to the initial pHi value of ~7.5 whereas in the control IC the pHi recovery was partial, returning to 6.7. Generally, the pHi recovery in response to the imposed acidosis was more complete in the ICs from the K+-depleted rats compared with control ICs (Fig. 4), but this difference was not statistically significant.


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Fig. 4.   Graph of pHi values measured before and after the NH4Cl pulse in ICs from K+-depleted rats (A) and control rats (B). The baseline pHi refers to the pHi measured at the beginning of the experimental protocol in HEPES-buffered NaR solution. The nadir pHi corresponds to the lowest value reached on removal of the acid. The final pHi value shown represents the final value reached in the IC in the absence of extracellular Na+ and K+.

To determine whether H+-ATPase activity is influenced by pHi, and accounts for the difference in rates seen in control and low-K+ ICs, we compared the pHi values measured during three different parts of the experimental protocol. The three points represented in Fig. 4 are the following: initial pHi (in HEPES-buffered Na+-Ringer solution; solution 1, Table 1); the nadir pHi reached on removal of the NH4Cl solution; and the final steady-state pHi reached. There was no difference in the initial pHi measured in the control ICs (7.58 ± 0.07) and low-K+ ICs (7.50 ± 0.07). The nadir pHi reached on removal of the NH4Cl pulse was also similar between the two groups (6.40 ± 0.04 for control vs. 6.47 ± 0.04 for low-K+ ICs). In a comparison of the final pHi reached in the absence of extracellular K+ and Na+, the cells from low-K+ rats tended to be more alkaline than the control ICs (7.11 ± 0.11 vs. 6.92 ± 0.07, respectively), but, as indicated above, this difference was not statistically significant. It appears that factors other than pHi are influencing the activity of the H+ pump under conditions of chronic K+ restriction.

To determine whether the changes in H+-ATPase function observed at the single IC level reflect changes in the CCD IC population as a whole, the cell-to-cell variability in the rate of the pHi recovery response was analyzed. When individual pHi recovery values were plotted, there was a clear cell-to-cell variability in the pHi recovery response in the group of control ICs and the group of ICs from K+-depleted rats. This variability in the pHi recovery response is shown in Fig. 5, where the number of cells within each group (K+-depleted and control) is plotted against pHi recovery rate, with the rates binned in increments of 0.10 pHi U/min. As shown in Fig. 5A, all of the 44 ICs from K+-depleted rats showed an H+-ATPase-dependent recovery from the acid load. The mean pHi recovery response was higher in these cells (0.22 ± 0.02 pHi U/min) compared with control ICs, and the response ranged from 0.02 to 0.55 pHi U/min. The variability in the pHi recovery response for the 57 control ICs studied is shown in Fig. 5B. In the control ICs, 10 of the 57 ICs studied showed no H+-ATPase-dependent pHi recovery from the acute acid load, and the response ranged from 0 to 0.38 pHi U/min, with a mean value of 0.08 ± 0.01 pHi U/min.


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Fig. 5.   Histogram showing variability of pHi recovery rates in K+-depleted (A) and control ICs (B). The ordinate represents the no. of cells, and the abscissa the pHi recovery rates that have been binned in increments of 0.10 pH U/min. A: variability of the response of 44 ICs from K+-depleted rats. B: the response of 57 ICs from control rats.

Immunocytochemistry

Because the functional results presented above do not take into account in which membrane of the IC the H+-ATPase resides, we performed immunocytochemistry in the two groups of rats using H+-ATPase antibodies. As previously described (1) heavily stained ICs alternated with unstained cells in connecting segments and collecting ducts. In control rat kidneys, the apical, basolateral, and diffuse patterns of H+-ATPase staining that characterize different IC subtypes were readily distinguished (Fig. 6A). As shown in Fig. 6A, many of the stained cells have basolateral, bipolar, or diffuse staining, and some have unique apical staining. A very different staining pattern was observed in tubules from K+-deprived rats, as shown in Fig. 6B. In collecting ducts from low-K+ rats, the basolateral staining pattern was seen much less frequently, and apical staining predominated.


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Fig. 6.   Immunocytochemical staining using antibodies against the 56-kDa H+-ATPase B1-subunit in control (A) and K+-depleted (B) rat kidney cortex. In collecting ducts from control rats, cells with basolateral and diffuse/bipolar staining are frequently found (arrows), and some cells with tight apical staining can also be seen (arrowhead). In collecting ducts from K+-depleted rats, cells with basolateral and diffuse/bipolar staining (arrow) were much less frequent whereas cells with tight apical staining were common (arrowheads). Bars = 10 µm.


    DISCUSSION
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ABSTRACT
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MATERIALS AND METHODS
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The BCECF-loaded, split-tubule preparation was used in combination with dual excitation digital imaging to study H+-ATPase activity in individual ICs in response to an imposed acidosis. ICs were visually identified under epi-illumination by their much brighter appearance after BCECF loading compared with the neighboring principal cells, as previously described (19). Our results indicate that maintaining rats on a low-K+ diet stimulates bafilomycin-sensitive H+-pump activity in the plasma membrane of ICs to a level that is almost three times greater than that observed in ICs from control rats. In addition, the lag time before the start of the intracellular alkalinization process, once the nadir pHi had been reached, was much shorter in the ICs from the K+-depleted CCDs (24.5 ± 2.1 s, n = 30 ICs) compared with control ICs (93.5 ± 13.7 s, n = 35 ICs). This more rapid onset of alkalinization may indicate that the ICs from CCDs of low-K+ rats are poised to deal with an intracellular acid load more efficiently than control cells. This is consistent with the hypothesis that the H+-ATPase content of the plasma membrane may be initially greater in ICs from K+-depleted rats. In contrast, ICs from control rats may have to mobilize more pumps from an intracellular vesicular pool to the cell surface before significant proton extrusion can occur, a process that has been shown to occur after both acute (16) and chronic systemic acidosis (5), as well as after exposure of isolated collecting ducts and turtle bladder to basolateral CO2 (11, 17, 23).

It is generally accepted that the electroneutral, gastric-like H+-K+-ATPase is also functional in the CCD. At the molecular level, mRNA expression of the alpha -subunit of the gastric pump is seen in the cells of the CCD under K+-replete conditions and is enhanced with K+-depletion (22). Functionally, a Sch-28080-sensitive gastric-like H+-K+-ATPase has been identified at the individual IC level in rabbit and rat CCD (19, 20) and in microperfused rabbit CCD (29, 30) and rat distal tubule (15, 27) with K+depletion. These results suggest that the ICs of the CCD possess functional H+-ATPase and H+-K+-ATPase under conditions of chronic K+depletion. The relative contributions of both ATPases to net acid secretion under hypokalemic conditions remain to be elucidated.

Our study demonstrates an increased H+ secretion via membrane-associated H+-ATPase in ICs from K+-depleted rats. These results are in agreement with previous work by Bailey et al. (3, 4) showing that K+ depletion induced an increase in electrogenic H+-ATPase activity in the rat distal tubule perfused in vivo. These findings at first appear to differ from the enzymatic results of others measuring H+- ATPase activity in individually microdissected CCDs from control and K+-depleted rats (9, 10). The enzymatic studies showed there was no effect of chronic K+ depletion on H+-ATPase activity in rat collecting ducts (9, 10). However, the apparent discrepancy may just reflect differences in what is actually being measured. The biochemical assay used for assessing H+-ATPase activity is performed on permeabilized microdissected nephron segments and represents the total pool of enzyme, consisting of the relative amounts of intracellular (vesicular) plus the plasma membrane H+-ATPase activity. Our measurements as well as those of Bailey (3, 4) represent functional plasma membrane-bound H+-ATPase only. Our immunocytochemical results support the concept that the increase in H+-ATPase activity measured at the individual cell level represents movement of H+-ATPase from an intracellular vesicular pool to the plasma membrane. This suggests that K+ depletion results in a redistribution of preexisting pumps and not synthesis of new pumps, which is consistent with the enzymatic findings.

The signal responsible for the enhanced H+- ATPase activity in individual ICs is not yet known. Although it has been suggested that plasma aldosterone levels modulate H+-ATPase activity (9), chronic hypokalemia is characterized by low plasma aldosterone levels (4). It has also been speculated that hypokalemic intracellular acidosis may contribute to the increased insertion of H+-ATPase into apical membrane (4); however, under conditions of our study the initial pHi measured in HEPES-buffered solutions was not different between the control and the hypokalemic ICs (Fig. 4). In addition, it appears that the buffering capacity of the ICs is similar between the control and K+-depleted groups in that the nadir pHi reached on removal of the NH4Cl pulse is also comparable between the two groups. We cannot exclude the possibility, however, that an in vivo cell acidosis plays a role in increased insertion of H+-ATPase into the plasma membrane.

Under conditions of low K+, the distribution of H+-ATPase in ICs was considerably modified compared with cells from control rats. In particular, cells with basolateral, bipolar, or diffuse staining were not as abundant, and more cells with a tight apical band of staining were found in K+-depleted rats. Although it is generally accepted that all alpha -type ICs have apical H+-ATPase (either as a tight band or a diffuse subapical staining), some beta -type ICs (identified by the absence of basolateral AE1 staining) can also have tight apical H+-ATPase staining (2, 12). Therefore, the increased abundance of apically stained IC found in the present study could result from an increased apical polarization of H+-ATPase in alpha -type ICs and/or a redistribution of H+-ATPase to the apical pole of beta -type ICs. Further studies will be required to resolve this issue.

The analysis of the cell-to-cell variability in the pHi response rates (Fig. 5) provides insight into the functional heterogeneity of the IC population under control and K+-depleted conditions. These data support the immunocytochemical results that K+-depletion stimulates insertion of H+-ATPase into the plasma membrane, because no IC failed to recover from an acid load. Presumably, the control ICs that showed little or no pHi recovery were those in which proton pumps were mainly intracellular.

In conclusion, this investigation demonstrates enhanced H+-ATPase activity at the single IC level, as evidenced by the functional data and confirmed by the immunocytochemical results. The implication of our finding is that H+-ATPase residing in the apical membrane of ICs is actively contributing to the increased proton secretion associated with chronic hypokalemia, often leading to systemic metabolic alkalosis. This upregulation may contribute to the increased secretory H+ transport that occurs under low-K+ conditions, and it could also reflect a cellular response involved in maintaining intracellular ion and pH homeostasis in this pathophysiological state.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45828 and the Underhill and Wild Wings Foundations (R. B. Silver) and DK-42956 (D. Brown and S. Breton).


    FOOTNOTES

Address for reprint requests and other correspondence: R. B. Silver, Dept. of Physiology and Biophysics, Joan and Sanford I. Weill Medical College of Cornell Univ., 1300 York Ave., New York, NY 10021 (E-mail: rbsilve{at}mail.med.cornell.edu).

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.

Received 4 October 1999; accepted in final form 9 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 279(1):F195-F202
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