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Am J Physiol Cell Physiol 292: C935-C944, 2007. First published September 27, 2006; doi:10.1152/ajpcell.00604.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Interactions between Na+ channels and Na+-HCO3– cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake

Scott K. Parks, Martin Tresguerres, and Greg G. Goss

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

Submitted 2 December 2005 ; accepted in final form 24 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pHi) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pHi from an NH4Cl-induced acidification in the absence of Na+. An apparent link between resting pHi and Na+-independent pHi recovery was made. We observed a unique pHi acidification event that was induced by extracellular Na+ addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na+-induced pHi acidification, whereas the minority (23%) demonstrated an alkalinization of pHi under the same circumstances. The focus of this study was placed on the Na+-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na+ uptake was responsible for the intracellular acidification. Further experiments revealed that pHi acidification could be abolished when Na+ was allowed entry into the cell, but the activity of an electrogenic Na+-HCO3– cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na+-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial Na+ uptake occurring via an apical Na+ channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells.

epithelial sodium channels; sodium-induced acidification; amiloride; phenamil; DIDS; acid-base; peanut lectin agglutinin binding; membrane potential; BCECF-AM; bis-oxonol; ion uptake

FISH LIVING IN A FRESHWATER environment are faced with a considerable challenge to maintain a high internal osmolarity in relation to their external environment. Consequently, the fish must spend a great deal of energy to actively uptake salts at the gill while preventing their passive loss to the environment. The gill is a complex epithelium that is responsible for directional transport of both Na+ and Cl– as well as whole body acid-base regulation. It comprises at least five different cell types (25), with the bulk of ion transport and acid-base regulation attributed to the activity of the mitochondria-rich (MR) cell. Currently accepted models of ion transport link Cl– uptake with HCO3– secretion, whereas Na+ uptake is coupled to H+ secretion via an apical Na+ channel electrogenically coupled to a V-type H+-ATPase (7, 13, 33, 35).

Goss et al. (12) first implicated direct MR cell involvement in whole body pH regulation. Because of the complex nature of the gill epithelium, it is difficult to ascribe specific function to a particular cell type. Our laboratory (9, 11, 37) has developed a technique to isolate MR cells from the gill epithelium and subsequently have described two functionally distinct MR cell subtypes. These cells can be separated based on whether or not they bind peanut lectin agglutinin (PNA+ and PNA–, respectively) (9). Further analysis has demonstrated an acid-activated phenamil and bafilomycin-sensitive Na+ influx in PNA– cells but not in PNA+ cells (37). However, the mechanisms involved in transepithelial Na+ transport remain unresolved.

The regulation of intracellular pH (pHi) is essential for the proper functioning of a number of cellular processes (26). At the same time, Cl– and Na+ uptakes across MR cells are directly linked to HCO3– and H+ secretion, respectively, as noted above. Therefore, examining the pHi regulatory characteristics of fish gill MR cells can provide information in four important areas: pHi regulation, systemic pH regulation, and cellular and systemic ion regulation. In fish gills, pHi regulation has been examined only in cultured epithelial pavement cells from rainbow trout (32, 47) and goldfish (41). However, direct characterization of MR cell pH regulation has not been reported.

The goal of this study was to investigate pHi regulation at the rainbow trout gill MR cells and to incorporate these findings into a working model for transepithelial ion and acid-base regulation. This study is the first to extensively investigate isolated and functionally identified gill MR cell pHi regulation. We report evidence to support our previous research that there are two functionally distinct MR cell populations. Differential pHi recovery responses were noted in Na+-free or Na+-containing medium, which correspond to different cell types in a mixed MR cell fraction. These findings, coupled with membrane potential experiments in this study, are an important step for the full characterization of MR cell subtypes and provide evidence for a new model for transepithelial Na+ transport in freshwater fish.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Adult rainbow trout (Oncorhyncus mykiss) were obtained from Alberta Trout Growers (Tofield, Alberta, Canada) and were maintained in flow through 450-liter fiberglass tanks filled with aerated and dechlorinated City of Edmonton tap water (hardness of 1.6 mmol/l as CaCO3, total alkalinity of 120 mg/l, pH 8.2). Water temperature in the tanks was maintained permanently at 15°C, and the photoperiod mimicked the natural pattern found in Edmonton, Alberta, Canada. Fish were fed once per day with dry commercial trout pellets. All experiments followed Canadian Council on Animal Care approved animal care protocol 215507.

Isolation of MR cells. Isolation of MR cells from the gill epithelium followed the techniques developed by Goss et al. (11). Trout were removed from the holding tanks and anesthetized by an overdose of tricane methanesulfonate (1 g/l) solution. After the pericardial cavity was opened and the pericardium removed, gills were perfused through the bulbous arteriosus with 50 ml of ice-cold, heparinized (15 mg) PBS (in mM: 137 NaCl, 2.7 KCl, 4.3 Na2PO4, 1.4 NaH2PO4; pH 7.8, 290 mOsm). Gill arches were then immediately excised from the fish, rinsed with dechlorinated tap water, and blotted lightly on paper towels before being placed in ice-cold PBS. Next, gill filaments were removed from the gill rakers in ~2- to 5-mm-wide sections and incubated three times (each 20 min) at 18°C in 5 ml of trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA; GIBCO, Burlington, ON, Canada). After each 20-min incubation, resultant cell suspensions were passed through a 96-µm nylon mesh filter into 5 ml of ice-cold FBS and rinsed through with PBS to inhibit trypsin activity. Final cell suspensions were washed twice with 50 ml of PBS and centrifuged (5 min, 1,500 g, 4°C). The resultant cellular pellets were resuspended in 3–5 ml of PBS and placed over a four-step Percoll gradient (2 ml, 1.09 g/ml; 2 ml, 1.06 g/ml; 2 ml, 1.05 g/ml; 3 ml, 1.03 g/ml) and centrifuged (45 min, 2,000 g, 4°C). The 1.09–1.06 g/ml Percoll interface has been found to contain a highly enriched population of MR cells, as demonstrated by positive staining for the vital mitochondrial dye 4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide and transmission electron microscopy (11). Therefore, cells from this interface were collected, washed in PBS, and used for all of the experiments in this study. The trypsin method of gill digestion was used because it improved adherence of MR cells to glass coverslips during perifusion experiments.

Inverted fluorescent microscopy. Acid-washed coverslips (no. 1 thickness, 15 mm round; Warner Instrument, Hamden, CT) were coated with 0.1% poly-L-lysine overnight and then rinsed with double-distilled water and 70% ethanol before each experiment. Aliquots of cell suspensions containing ~300,000 cells were added to 200 µl of 1% gentamicin sodium-containing buffer (in mM: 145 NaCl, 5 CaCl2, 1 MgCl2, 4 KCl, 15 HEPES; pH 7.8, 290 mOsm) with an additional 2 µl of both CaCl2 (1 M) and MgCl2 (1 M) to aid in cell attachment. Cellular suspensions were then placed on the prepared coverslips and kept undisturbed at 4°C for at least 1.5–2 h to allow for settlement and attachment of cells. We found that this length of time was sufficient to ensure cell attachment during perifusion experiments. Coverslips were then removed from the refrigerator, rinsed in Na+-containing buffer, and immediately exposed to 200 µl of Na+-containing buffer containing 2 µl of 5 mM, pH-sensitive BCECF-AM (50 µg in 16 µl DMSO and 20% pluronic acid). Incubation of the cells with BCECF-AM occurred for at least 45 min at a room temperature of 18°C. Next, coverslips were placed into a 70-µl imaging chamber (RC-20H; Warner Instrument) for perifusion experiments. MR cells on the coverslips were subjected to differential interference contrast microscopy (Nikon Eclipse TM-300) and fluorescence imaging (TE-FM epifluorescence attachment) with the use of an inverted microscope. The microscope was fitted with a xenon arc lamp (Lambda LS; Sutter Instruments, Novato, CA) to enable excitation of the BCECF-AM-loaded cells at wavelengths of 495 and 440 nm. Exposure time and the gain were adjusted at each trial to elicit adequate fluorescence. Images at both 440 and 495 nm were captured digitally on a mono 12-bit charge-coupled device camera (Retiga EXi; Burnaby, BC, Canada) every 5 s during the various perifusion experiments. Northern Eclipse version 6 software (Mississauga, ON, Canada) was used to compile the 495-to-440 nm ratios as an indication of the pHi levels.

Perifusion protocol. Solutions were added to the perifusion chamber using a six-input manifold (Mp-6; Warner Instrument) attached to gravity-feed, 60-ml syringes in syringe holder blocks equipped with pinch valves (VE-6; Warner Instrument) and controlled by VC-6 valve controllers (Warner Instrument). The perifusion rate was adjusted to ~0.5 ml/min. Cells were alkalinized and acidified by a 3-min ammonium chloride (20 mM NH4Cl) prepulse technique first described by Boron and De Weer (3). Cells were then allowed to recover from an acidification event under both Na+-free (in mM: 142.5 N-methyl-D-glucamine-Cl, 2.5 C5H14NO·HCO3–, 5 CaCl2, 1 MgCl2, 4 KCl, 15 HEPES; pH 7.8, 290 mOsm) and Na+-containing (in mM: 142.5 NaCl, 2.5 NaHCO3–, 5 CaCl2, 1 MgCl2, 4 KCl, 15 HEPES; pH 7.8, 290 mOsm) conditions. Other experimental protocols involved observing changes in pHi from the original resting state. This involved first cells to be exposed to Na+-free medium and then switching to a Na+-containing solution to cause a disturbance of pHi. After the cells were observed under control parameters, the same perifusion procedure would occur but with the addition of various drug treatments including amiloride (500 µM), phenamil (50 µM), and DIDS (1 mM). All of the solutions used were bubbled continuously with a gas mixture of 0.3% CO2 balanced with O2 throughout the experiments.

High-K+ solutions (in mM: 120 potassium gluconate, 20 KCl, 2 MgCl2, 20 HEPES) where adjusted to four separate pH values (~8.40, 7.80, 7.20, 6.60) and used for calibration of pHi at the end of each experiment. pHi and extracellular pH were equilibrated by the addition of the ionophore nigericin (5 µM) (5). The 495-to-440 nm ratios obtained at each calibration set point were then used to generate a regression equation for each cell. This equation was then extrapolated to the ratiometric data obtained over the course of the whole experiment, resulting in an internally calibrated pHi trace for each cell during the entire perifusion procedure.

Membrane potential experiments. Membrane potential (Vm) behavior was monitored with the fluorescent anionic dye bis-oxonol (Molecular Probes, Eugene, OR). The dye is lipophilic and increases or decreases in fluorescence on depolarization or hyperpolarization, respectively. Importantly, the negative charge on bis-oxonol prevents accumulation in mitochondria; therefore, the dye distributes across cell membranes according to the Vm, giving a reliable measurement of relative changes in Vm (29). Bis-oxonol fluorescence was measured with the inverted microscope as explained previously. Briefly, cells were loaded with the 5 µM bis-oxonol for 1 h before each experiment. Cell fluorescence was then monitored at 495-nm excitation and 530-nm emission as changes in the extracellular medium were made. Bis-oxonol (5 µM) was also placed in the experimental perifusion solutions to enable the uptake or extrusion of the dye according to Vm changes. Perifusion experiments followed the above protocol with simple switching between Na+-free condition and 145 mM Na+ in the extracellular medium. Longer exposure times were required to capture fluorescence compared with the pHi experiments. Consequently, images were captured once every 20 s to avoid excessive bleaching of the dye. Data are presented as change in fluorescence relative to initial fluorescent value for each individual cell.

Western blot analysis. Western blot analysis was done according to the technique described by Tresguerres et al. (45). Briefly, gill samples were immersed in liquid nitrogen, pulverized in a porcelain grinder, resuspended in 1:10 wt/vol of ice-cold homogenization buffer (250 mmol/l sucrose, 1 mmol/l EDTA, 30 mmol/l Tris, 100 mg/ml PMSF, and 2 mg/ml pepstatin; pH 7.4), and sonicated on ice for 20 s. Debris was removed by low-speed centrifugation (3,000 g, 10 min, 4°C), and the supernatant was kept as the whole gill homogenate and combined with 2x Laemmli buffer (24). Twenty micrograms of total protein, as estimated by bicinchoninic acid protein determination analysis (Pierce, Rockford, IL), were separated in a 7.5% polyacrylamide mini-gel (45 min at 180 V) and transferred to a nitrocellulose (NC) membrane (1 h at 100 V) using a wet transfer cell (Bio-Rad Laboratories, Hercules, CA). After the blocking procedure (5% chicken ovalbumin in 0.5 mol/l Tris-buffered saline with 0.1% Triton X-100; pH 8.0, overnight at 4°C), the NC membranes were incubated with the rabbit anti-rat kidney Na+-HCO3– cotransporter (rkNBC) antibody (42) with gentle agitation at 4°C overnight. After four washes of 15 min each with Tris-buffered saline-Triton X-100 (0.2%), the NC membrane was blocked for 15 min and then incubated with a fluorescent secondary goat anti-rabbit antibody at room temperature for 2 h. Bands were visualized by the Odyssey infrared imaging system and software (Li-Cor). NC membranes incubated without primary antibody served as the control and never showed positive immunoreactivity.

Immunohistochemistry. Immunohistochemistry with the anti-NBC (1:500 dilution) described above was carried out on the gill sections according to the procedure of Tresguerres et al. (45). Secondary antibody application and visualization were performed with the use of the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), following the manufacturer's directions. Sections were viewed by a Leica LMRXA compound microscope (Leica Microsystems, Wetzlar, Germany), and images were captured and digitalized with an Optronics MacroFire 1.0 digital camera and its associated software Picture Frame (Optronics, Goleta, CA). Micrograph brightness and contrast were adjusted with Adobe Photoshop 7.0 (Adobe Systems).

Analysis and statistics. After an experimental manipulation, analysis was performed on initial rates of pHi recovery. The initial change in pHi over change in time ({Delta}pHi/{Delta}t) was calculated for each cell under control and experimental conditions, and compiled results were compared by paired, two-tailed Student's t-test and one-way ANOVA with Bonferroni post hoc test for significance at the level of P < 0.05. {Delta}pHi/{Delta}t were analyzed over 30 or 60 s, following the invoked pHi change of interest. All summary data are presented as means ± SE. All experiments shown are representative of cells obtained from a minimum of two different fish. Total cell numbers and experimental trials are as presented. All statistical analysis was performed with GraphPad Prism version 3.0 software (San Diego, CA). Unless otherwise mentioned, the reagents used in this study were purchased from Sigma (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Viability and responsiveness of isolated fish gill MR cells. This is the first study involving pHi imaging on isolated MR cells; therefore, we needed to establish that the MR cells were viable and able to respond similarly to repeated exposures to acid-base disturbances over the course of the experiment. Figure 1 shows a representative trace of the MR cell's ability to encounter multiple acidification events induced by 20 mM NH4Cl exposure. Cells responded with consistent recovery patterns under repeated control experimental conditions. Viability of the cells over numerous pHi disturbances provided a framework to structure the rest of our experimental protocols. For each ensuing experiment, a consistent control was established before pharmacological testing occurred.


Figure 1
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  		Fig. 1. Representative trace demonstrating the ability of the isolated mitochondria-rich (MR) cells to withstand repeated acid-base disturbances. Cells were subjected to 2 consecutive 20 mM NH4Cl prepulse-induced acidifications and displayed the same behavior after initial and secondary acidifications. pHi, intracellular pH.

 
Effect of Na+-free medium on pHi recovery following an acid load: evidence for two distinct populations. In response to an intracellular acidification induced by 20 mM NH4Cl exposure, the mixed population of MR cells separated into two distinct pHi recovery patterns. One population of cells demonstrated an Na+-independent pHi recovery mechanism after acidification (Fig. 2A, trace a), whereas the other population of MR cells lacked pHi recovery in the absence of Na+ (Fig. 2A, trace b). During our data analysis, we noticed that the ability to recover from an acid load in the absence of Na+ correlated with the resting pHi of the cells involved. Comprehensive analysis of all cells tested found that ~84% of cells with a resting pHi >7.80 displayed an Na+-independent recovery behavior compared with 16% that did not (n = 175; Fig. 2B). In contrast, 73% of cells with a resting pHi <7.80 lacked a Na+-independent recovery compared with 23% that could recover in Na+-free conditions (n = 223; Fig. 2B). This suggested that there were at least two populations of cells with differential transport processes in the mixed MR cell population.


Figure 2
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  		Fig. 2. Na+-free recovery rates indicate 2 populations of MR cells. Cells were acidified with NH4Cl prepulses and allowed to recover in the absence of Na+. A: representative traces of cells that demonstrated an ability to recover from an acidification in Na+-free conditions (trace a) and those cells that were unable to recover pHi in the absence of Na+ (trace b). B: summary data demonstrating a link between Na+-free pHi recovery and the original resting pHi of the cell. The majority (84%) of MR cells with a resting pHi >7.8 (n = 175) displayed an Na+-independent pHi recovery mechanism compared with only a low percentage (23%) of cells with a resting pHi <7.8 (n = 223) demonstrating this same ability. Letters a and b correspond to the representative traces in A in reference to Na+-free recovery ability.

 
Novel behavior of one MR cell subtype: Na+-induced acidification. During experiments involving alteration of extracellular Na+ concentration ([Na+]e), we noticed a peculiar Na+-induced acidification event occurring in most of the MR cells. This occurred when the cells were switched from Na+-free to Na+-containing medium. All known transport mechanisms involving influx of Na+ predict an intracellular alkalinization. Therefore, we were interested in elucidating the mechanism underlying this peculiar pHi acidification. Consequently, experiments were performed by exposing the cells to Na+-free and Na+-containing medium in different combinations with no NH4Cl disturbances. We found that initial perifusion of Na+-free medium across the cells followed by a switch to Na+-containing medium resulted in an intracellular acidification in one population of MR cells (Fig. 3A, black trace). We observed that this event could be repeated in the same cells with a tendency toward a slight but significant decrease in the rate of acidification on the second exposure to Na+-containing medium after initial recovery in Na+-free medium ({Delta}pHi/{Delta}t of –0.120 ± 0.008 and –0.095 ± 0.010 pH units/min, respectively; Fig. 3B). Within the same set of experiments, there was a smaller percentage of MR cells that responded to this change with an Na+-induced alkalinization of pHi (Fig. 3A, gray trace). This event was also repeatable without a significant change in the pHi alkalinization rates between the primary and secondary Na+ exposure ({Delta}pHi/{Delta}t of 0.404 ± 0.072 and 0.374 ± 0.056 pH units/min, respectively; Fig. 3C). From this set of experiments and the ensuing pharmacology studies, we found that ~77% of cells from the mixed MR cell fraction demonstrated the Na+-induced pHi acidification behavior vs. 23% that exhibited the Na+-induced pHi alkalinization behavior (n = 227). These data support the finding of differential resting pHi and functional behavior as reported above as each subpopulation of MR cells segregates to either a low or high pHi after exposure to Na+.


Figure 3
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  		Fig. 3. Changes in pHi of MR cells induced by extracellular Na+. Cells were initially exposed to Na+-free medium and then switched to 145 mM Na+ solution. A: sample traces demonstrating that 1 population of MR cells (black trace) responds to the change from Na+-free to Na+-containing medium with an acidification, whereas the other population (gray trace) exhibits an alkalinization in the same conditions. B: summary data showing the repeatability of the rate of Na+-induced pHi change ({Delta}pHi/{Delta}t) in this population of MR cells. There was a slight but significant reduction in the rate of the second Na+-induced acidification compared with the control (*P < 0.05, paired t-test, n = 37 cells, 6 separate experiments). C: summary data showing the repeatability of the rate of Na+-induced alkalinization in this population of MR cells. There was no significant difference between the two rates of Na+-induced alkalinization (P > 0.05, paired t-test, n = 18 cells, 6 separate experiments).

 
Na+ induced acidification: involvement of Na+ channels. In this study, we have specifically focused our attention on understanding the MR cell population exhibiting an Na+-induced acidification because, to our knowledge, this has not been reported before in any system. Within this experimental protocol, we then began a series of pharmacological profiling experiments to determine the mechanism(s) of Na+-induced acidification. In each experiment, cells were initially challenged with increased [Na+]e to allow for identification of the specific cell type. The effect of subsequent pharmacological compounds was then examined in cells exhibiting an Na+-induced acidification. Amiloride, an inhibitor of both Na+/H+ exchangers (NHEs) and epithelial Na+ channels (eNaC) (23), effectively removed the acidification originally caused by Na+ introduction and replaced it with a slight alkalinizing trend (Fig. 4A). The effect of amiloride was statistically significant with a {Delta}pHi/{Delta}t of –0.145 ± 0.013 pH units/min induced on control exposure to Na+-containing medium after Na+-free exposure compared with a {Delta}pHi/{Delta}t of 0.062 ± 0.016 pH units/min when the same cells were exposed to Na+ in the presence of amiloride (500 µM; Fig. 4B). Next, we tested phenamil, a 5'-substituted derivative of amiloride known to block eNaCs at micromolar concentrations (23) with no known effects on NHEs, even at much higher concentrations. After the same experimental protocol, Na+-induced acidification was eradicated in the presence of 50 µM phenamil (Fig. 5A). We also observed that acidification would occur again in the presence of Na+ when phenamil was removed, indicating effective and reversible washout of the drug (Fig. 5A). Both the control Na+-induced acidification ({Delta}pHi/{Delta}t of –0.139 ± 0.009 pH units/min) and the washout Na+-induced acidification ({Delta}pHi/{Delta}t of –0.138 ± 0.023 pH units/min) were significantly different from the pHi change induced by Na+ in the presence of 50 µM phenamil ({Delta}pHi/{Delta}t of 0.057 ± 0.010 pH units/min) but not significantly different from each other (Fig. 5B). Finally, application of amiloride under steady-state Na+-free conditions did not result in a change in pHi.


Figure 4
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  		Fig. 4. Effect of 500 µM amiloride on the Na+-induced acidification following Na+-free exposure at resting pHi. A: representative trace showing the effective removal of Na+-induced acidification in the presence of 500 µM amiloride. The acidification event was replaced with a trend toward alkalinization. B: summary data of the significant difference between the rates of pHi change ({Delta}pHi/{Delta}t) caused by the control Na+ introduction and the Na+ exposure in the presence of 500 µM amiloride (*P < 0.05, paired t-test, n = 21 cells, 2 separate experiments).

 

Figure 5
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  		Fig. 5. Effect of 50 µM phenamil on the Na+-induced acidification following Na+-free exposure at resting pHi. A: representative trace showing the effective removal of Na+-induced acidification in the presence of 50 µM phenamil. The effect of phenamil was reversed by washout with Na+-containing solution. B: summary data showing that both the control Na+ and washout Na+ exposure caused the MR cells to acidify at a rate that was significantly different from the slight alkalinization rate caused by Na+ exposure in the presence of 50 µM phenamil. No significant difference was noted between the acidification rate caused by the control Na+ introduction and the washout Na+ exposure (P < 0.05, repeated-measures one-way ANOVA, Bonferroni post hoc test, n = 36 cells, 4 separate experiments). Letters a and b indicate significant differences.

 
Na+-induced acidification: involvement of NBC. One possibility for observing a pHi acidification is via the extrusion of HCO3–. The NBC operating in export mode has been shown to function in this manner by transporting three HCO3– molecules for each Na+ (43). Expression studies of the renal NBC exhibited a reduction in pHi when extracellular Na+ was removed from frog oocytes (40). This acidification of pHi was attributed to Na+ moving down the gradient created and an excess of HCO3– being carried with it. Therefore, we examined the effect of DIDS, a known inhibitor of NBCs (4, 39, 40), on the Na+-induced acidification event. DIDS (1 mM) caused essentially the same effect as amiloride and phenamil by removing the Na+-induced acidification (Fig. 6A). The control Na+-induced acidification ({Delta}pHi/{Delta}t of –0.121 ± 0.007 pH units/min) was abolished in the presence of 1 mM DIDS ({Delta}pHi/{Delta}t of 0.038 ± 0.016 pH units/min) with a slight alkalinization noted (Fig. 6B). We were unable to wash out the DIDS because it tends to bind irreversibly. Instead, we observed a continuation of the same trend with Na+ washout compared with Na+ in the presence of DIDS (Fig. 6A). Application of DIDS under steady-state Na+-free conditions did not result in a change in pHi.


Figure 6
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  		Fig. 6. Effect of 1 mM DIDS on the Na+-induced acidification following Na+-free exposure at resting pHi. A: representative trace showing the effective removal of Na+-induced acidification in the presence of 1 mM DIDS. The effect of DIDS was not reversed by washout with Na+-containing solution. B: summary data showing that the control Na+ exposure caused the MR cells to acidify at a rate that was significantly different from the slight alkalinization rate caused by Na+ exposure in the presence of 1 mM DIDS (*P < 0.05, paired t-test, n = 80 cells, 8 separate experiments).

 
Next, we examined the role of carbonic anhydrase (CA) in providing the required HCO3– molecules for the electrogenic NBC activity. Experiments were performed as above with 500 µM acetazolamide, a level similar to that demonstrated to block gill CA activity (10). We observed that inhibition of CA acted to effectively replace the Na+-induced acidification with an alkalinization, which was larger than that observed for either amiloride, phenamil, or DIDS treatment (Fig. 7A). The control Na+-induced acidification ({Delta}pHi/{Delta}t of –0.192 ± 0.023 pH units/min) compared with the presence of 500 µM acetazolamide ({Delta}pHi/{Delta}t of 0.180 ± 0.037 pH units/min), as shown in Fig. 7B.


Figure 7
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  		Fig. 7. Effect of 500 µM acetazolamide (ACZ) on the Na+-induced acidification following Na+-free exposure at resting pHi. A: representative trace showing the effective removal of Na+-induced acidification in the presence of 500 µM acetazolamide. The effect of 500 µM acetazolamide was reversed by washout with Na+-free solution. B: summary data showing that the control Na+ exposure caused the MR cells to acidify at a rate that was significantly different from the effect of Na+ exposure in the presence of 500 µM acetazolamide (*P < 0.05, paired t-test, n = 23 cells, 10 separate experiments).

 
Changes in Vm: involvement of electrogenic NBC. If an electrogenic NBC was involved in the Na+-induced acidification event, it would be accompanied by distinct changes in Vm of the MR cells. A hallmark feature of an electrogenic NBC acting in efflux mode is the induction of a depolarization of Vm (40). By loading the MR cells with bis-oxonol and exposing them to single cell imaging, we again observed two distinct Vm behaviors. The large majority of cells (79%, n = 111) demonstrated a distinct and repeatable depolarization of Vm after introduction of 145 mM Na+ following Na+-free exposure (Fig. 8, black trace). Conversely, a smaller percentage of cells (21%, n = 29) demonstrated a hyperpolarization in response to the same experimental manipulation (Fig. 8, gray trace). The relative percentages of Na+-induced depolarization (79%) match well with the relative proportion of cells demonstrating the Na+-induced acidification (77%) (Fig. 3).


Figure 8
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  		Fig. 8. Changes in membrane potential (Vm) of MR cells as indicated by the voltage sensitive dye bis-oxonol. Changes in Vm are represented as changes in relative fluorescent units (RFU), with an increase in fluorescence being indicative of membrane depolarization. Black trace is representative of MR cells that demonstrated a repeatable Na+-induced depolarization of Vm on switching from Na+ free solution to 145 mM Na+ (79%, n = 111/140, 14 separate experiments). Gray trace shows that the minority of MR cells demonstrated a repeatable Na+-induced hyperpolarization of Vm under identical experimental conditions (21%, n = 29/140).

 
To further support our contention that an electrogenic NBC functions in export mode in our system, we tested the effect of DIDS on the Na+-induced depolarization. We observed that DIDS was able to effectively remove the depolarizing effect of Na+ or replace it with a hyperpolarizing trend in the majority of cells where depolarization occurred (75%, n = 28; Fig. 9).


Figure 9
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  		Fig. 9. Representative trace showing changes in membrane potential (Vm) of MR cells in the presence of Na+ and 1 mM DIDS. Changes in Vm are indicated by the voltage-sensitive dye bis-oxonol as indicated by RFU. DIDS (1 mM) was effective at removing the Na+-induced depolarization in 75% of the MR cells that were demonstrated to originally show this behavior (n = 21/28, 5 separate experiments).

 
NBC immunoreactivity. The anti-rkNBC antibody recognized a distinct band (~110 kDa) from gill homogenates as estimated from Western blot analysis (Fig. 10A). Immunohistochemistry in the trailing edge of the gill filament revealed distinct positive staining in cells located on the lamellae (Fig. 10B). The cellular staining pattern was concentrated in the basolateral region, and the apical region was mostly devoid of signal. Some cells exhibited a diffuse cytoplasmic staining pattern; however, this could be attributed to the extensive branching of the basolateral membrane.


Figure 10
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  		Fig. 10. Immunoreactivity of the anti-rat kidney Na+-HCO3– cotransporter (rkNBC1) in the trout gill. A: Western blot showing a distinct band of ~110 kDa from whole gill homogenates. B: immunohistochemistry of the trailing edge of the gill filament exhibiting specific cellular staining of NBC. Notice that positive cells are found mostly on the lamellae and that the cellular staining pattern is concentrated in the basolateral region with the apical area being devoid of signal in most cells. Cytoplasmic staining patterns are likely due to the extensive branching of the tubular system of the basolateral membrane in these cells. Bar = 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Freshwater fish gill MR cells perform transepithelial Na+ uptake from an extremely dilute environment (<1 mM Na+), which requires unique physiological solutions to overcome the unfavorable gradient. Here, we propose a new model for transepithelial Na+ uptake at the freshwater fish gill (Fig. 11) based on our pHi assessments and Vm imaging of isolated MR cells. Our data clearly demonstrate two functionally distinct MR cell subtypes and support the previously reported findings from our laboratory (9, 37). There is a clear separation in resting pHi between the two MR cell populations. Potentially, a lower resting pHi would be advantageous for proton extrusion and a higher pHi would be advantageous for base extrusion. However, to test this hypothesis, observation of the resting pHi of MR cells in an intact gill filament is required.


Figure 11
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  		Fig. 11. Freshwater transepithelial Na+ uptake metabolon in gill non-peanut lectin agglutinin binding (PNA–) MR cells. Note how the tubular system allows for the close proximity of the apical (A) Na+ channel and H+-ATPase with the basolateral (B) NBC. The electrogenic NBC transfer is represented as "3?" HCO3– transported with 1 Na+ due to thermodynamic analysis but inability to directly ascribe the stoichiometry. Sites of pharmacological inhibition in our study are indicated by stop arrows. The proposed Vm is indicated, and +/ – signs are used to help demonstrate this point.

 
We were initially investigating how changes in [Na+]e would affect pHi regulation. Surprisingly, the majority of MR cells exhibited a unique Na+-induced acidification, a puzzling behavior that to our knowledge has never been reported. Typically, in all other cells, switching from Na+-free to Na+-containing medium would activate either NHEs, which would drive Na+ into the cell countered by H+ extrusion (31), or activate inward-directed HCO3– cotransport via NBCs (39). Therefore, analyzing the data in terms of a single transport process could seemingly only result in an Na+-induced pHi alkalinization. To our knowledge, there is no specific Na+-coupled H+ import mechanism that could explain our results. Therefore, we had to account for the behavior by implicating an HCO3– export mechanism.

It is important to remember that a specific function of fish gill MR cells is transepithelial Na+ uptake from dilute freshwater (6). Thus it is possible that the observed Na+-induced acidification represents the coordinated response of transporters located at both the apical and basolateral membranes. We propose a model whereby Na+ enters the cell via an apical transporter (phenamil-sensitive Na+ channel) and subsequently leaves the cell via a basolateral transporter (DIDS-sensitive electrogenic NBC) (Fig. 11).

The present models for freshwater Na+ uptake in MR cells involve Na+ entry via apical eNaCs that are electrogenically coupled to a V-type H+-ATPase (6, 13, 33, 35). In vivo application of the selective inhibitor of V-type H+-ATPase bafilomycin to the water greatly reduced whole body Na+ influx in both tilapia and carp (7). Similar experiments with phenamil, a selective inhibitor of eNaCs (1, 23), resulted in comparable reductions of Na+ influx in rainbow trout (17). Although this is compelling evidence for Na+ entry through an eNaC in the gill MR cells, cloning of an eNaC homologue from fish gill has been unsuccessful thus far. Consequently, Na+ entry will only be attributed to a phenamil-sensitive Na+ channel for the rest of this paper without regard to its molecular identity.

Phenamil sensitivity in our study supports an apical Na+ channel as the route of entry for Na+ into the gill. This complements a recent paper from our laboratory (37) demonstrating phenamil-sensitive Na+ uptake in only the PNA– MR cell fraction. This fraction is reported to be ~80% of the total mixed MR cell population (11), and this is in agreement with the present study in which we found that ~77% of our cells displayed the phenamil-sensitive, Na+-induced acidification. Together, this strongly suggests that a component of the Na+-induced pHi acidification observed in this study is via an apical Na+ channel in PNA– MR cells.

The mechanism for basolateral efflux of Na+ has typically been assumed to occur via Na+-K+ ATPase, which is highly expressed in MR cells (6, 28, 35). However, exit of Na+ via Na+-K+-ATPase would not result in any pHi changes. Our study is unique in that we provide functional evidence for an alternate route of Na+ efflux. We propose that a basolateral NBC is coupled with the apical Na+ channel for transepithelial Na+ movement complemented by the action of the Na+-K+ ATPase to maintain a low [Na+]i, thereby allowing for uptake from the dilute medium. In our experiments, [Na+]e is kept at 145 mM, which is much higher than normal environmental Na+ concentration in freshwater (<1 mM). This would tend to drive the Na+ influx via the apical Na+ channel very strongly. It is not technically feasible for us to experimentally determine the relative importance of either NBC or Na+-K+-ATPase in the exit of Na+ under physiological conditions where environmental Na+ is very low (<1 mM). However, our observed results suggest that NBC efflux is directly linked to Na+ influx via a phenamil-sensitive Na+ channel, which is the physiological mechanism of entry in intact freshwater fish (6, 17).

Perry et al. (34) successfully cloned an NBC from rainbow trout that was homologous to the electrogenic NBC1. Furthermore, they observed a large increase in NBC1 mRNA at the gill in response to a hypercapnia-induced respiratory acidosis that was also associated with a transient increase in branchial V-type H+-ATPase mRNA levels. In addition, acid infusions in rainbow trout were shown to cause an increase in Na+ influx (14), whereas elevations of plasma HCO3– by infusion of NaHCO3 is known to decrease Na+ influx (15). These data support our proposal that a basolateral electrogenic NBC could be utilized for transepithelial Na+ uptake in vivo.

NBC activity was first described in 1983 to take place on the basolateral membrane of the proximal tubule in the salamander Ambystoma tigrinum (2). However, it was not until recently (40) that this transporter (NBC1) was successfully cloned and subsequently localized to the basolateral membrane of the proximal tubule (27, 42). This breakthrough enabled further understanding of the NBC in a variety of other animals and tissues. An electrogenic NBC1 was recently cloned from the Japanese dace (Tribolodon hakonensis) gill and was localized to the basolateral membrane of MR cells by immunohistochemistry (21). Interestingly, NBC1 was only detected in a subset of MR cells from the Japanese dace gill, aligning with our findings of two functionally distinct MR cell subtypes. Similarly, we demonstrate specific immunoreactivity in a subset of cells on the gill lamellae. Cellular staining for rkNBC1 was basolateral or "cytosolic," similar to the pattern found previously (21) and in accordance with a basolateral localization in the cell in our model.

Functional studies of the cloned proximal tubule NBC1 demonstrated a DIDS-sensitive pHi acidification and Vm depolarization when acting in efflux mode (40). The stoichiometry of this transporter appears to be tissue specific, with one Na+ being transferred for three HCO3– in the proximal tubule of the kidney vs. 1:2 in other parts of the body (43). Our Vm data strongly support the involvement of an electrogenic NBC because we see appropriate changes in response to our ion-substitution experiments. The introduction of Na+ induced a depolarization in virtually the same percentage of cells that exhibited an Na+-induced intracellular acidification (79% vs. 77%). The sensitivity of both events to DIDS further implicates electrogenic NBC involvement in the transepithelial Na+ movement in these MR cells.

For an NBC to function in export mode, a hyperpolarized Vm is absolutely required to have a net driving force for HCO3– export. Unfortunately, we were unable to directly calibrate the resting Vm of the cells due to technical difficulties with the dye. However, we can roughly calculate, based on external and internal ion (Na+ and HCO3–) levels, the reversal potential, which can give valid estimates of the required Vm to drive the transporter in export mode. Although measurements of [Na+]i in gill MR cells are needed to complete transport models, they are difficult to obtain. The reported values for [Na+]i of 55 mM (46) and 62 mM (30) must be interpreted with caution because of a number of confounding variables as discussed by Morgan et al. (30). Comparatively, in cells from the frog skin epithelium, [Na+]i levels have been measured at 6–17 mM (20, 38). The fish gill is believed to mimic frog skin with respect to Na+ uptake in MR cells; therefore, previous reports of gill MR cells [Na+]i are likely overestimated.

The theoretical reversal potential (Erev) is the point at which no flux would occur through the NBC in efflux mode and can be approximated by the following formula (18):

Formula
where R is the gas constant, T is the absolute temperature in kelvins, and F is the Faraday constant.

Therefore, a Vm slightly more hyperpolarized than the calculated reversal potential would be required to drive the transporter in export mode. In this formula, n refers to the transport ratio of HCO3– to Na+ or simply the stoichiometry (either 1, 2, or 3). Using a conservative estimate of [Na+]i of 10 mM, measured [Na+]e of 145 mM, intracellular HCO3– concentration ([HCO3–]i) of 2 mM (16), and extracellular HCO3– concentration ([HCO3–]e) of 7 mM (15) would result in a reversal potential of –82 mV when the NBC is operating with a stoichiometry of 1 Na+ to 3 HCO3–. This means that the MR cells would only be required to maintain a Vm of less than –82 mV for the NBC to function in efflux mode, which is not beyond reasonable predictions. However, if the stoichiometry is 1 Na+ to 2 HCO3–, the reversal potential then becomes –133 mV, a much more hyperpolarized value. If intracellular HCO3– concentration is raised, this will shift the reversal potential to a more depolarized value and therefore promote the transport in efflux mode. Furthermore, if we lower [Na+]i to a very low value (2 mM) to help in apical entry from dilute freshwater, the reversal potential of the 1:3 NBC using these values is only –103 mV, again a feasible value. Therefore, the combination of NBC (1 Na+ to 3 HCO3–) and a reasonably hyperpolarized Vm all support the transport mechanism in our proposed model. Interestingly, the only other place where the NBC functions in efflux mode is the mammalian proximal tubule (for review, see Ref. 43), an epithelium also specialized for transepithelial Na+ transport. The filtrate from which Na+ reabsorption occurs contains much higher [Na+]e than is found in freshwater, but the extrusion occurs at the basolateral membrane where the [Na+]e is ~140 mM, supporting our findings that NBC activity can function in export mode under these conditions.

Previous pHi imaging experiments on trout hepatocytes have demonstrated electrogenic NBC activity (8). The putative NBC was proposed to act in influx mode, thus enabling pHi recovery from acidosis. The stoichiometry, although not determined, was likely 1 Na+ to 2 HCO3– because of its role in HCO3– influx (4). In our study, DIDS-sensitive acidification and depolarization indicate the presence of an NBC working in efflux mode, suggesting that trout express functionally different NBCs in a tissue-specific manner, as shown previously in mammalian systems (43).

Structural analysis has revealed that there is extensive branching of the basolateral membrane of MR cells into an elaborate tubular system (36) that is in very close contact with the apical membrane. This creates the potential for specialized microenvironments that can help to overcome otherwise energetically unfavorable processes. We propose that one mechanism for transcellular Na+ uptake from freshwater is facilitated via a close structural relationship of both apical Na+ channels and basolateral NBCs acting in efflux mode. However, it is unknown whether ultrastructural characteristics such as the tubular system of the MR cell are maintained during cell isolation performed in this study. Yet, our present data imply that the MR cell ultrastructure is at least partially maintained during the isolation procedure to produce the cellular behaviors observed in this study. Importantly, the tubular system has been known to become reestablished with the basolateral membrane in isolated MR cells that form aggregates (36).

Overcoming the gradient for Na+ extrusion via an NBC would be facilitated by the action of CA, which has been localized in MR cells in a variety of fish, including rainbow trout (22). Abolishment of the Na+-induced acidification with CA inhibition via acetazolamide in our study supports the involvement of CA in this process. Interestingly, the magnitude of the alkalinization after acetazolamide treatment was larger than that noticed using amiloride, phenamil, or DIDS. Because [HCO3–]i is the strongest determinant in the equation for the reversal potential of the NBC, lowering [HCO3–]i via acetazolamide will shift the reversal potential to more negative values, which would favor HCO3– influx and exacerbate the alkalinization.

The mammalian electrogenic NBC1 is known to bind CA II on its COOH-terminal domain (19, 39), although this has not yet been demonstrated in fish. In our model, CA would produce HCO3– within the proximity of the NBC to drive Na+ efflux across the basolateral membrane despite the unfavorable Na+ concentration gradient. This is supported in the mouse proximal convoluted tubule cell line mPCT1296(d) transfected with kidney NBC1 where inhibition of CA with acetazolamide leads to a 65% reduction in current in HCO3– efflux mode only (19). These points illustrate how multiple proteins can act as a functional unit for transepithelial Na+ movement. Our suggestion of a coordinated activity between an Na+ channel, NBC, and CA follows the concept of a metabolon described in biochemical context as a group of proteins acting together to accomplish one metabolic function (44). Therefore, we propose to refer to our model as the freshwater Na+ uptake metabolon.

In summary, we have provided evidence for a new model of transepithelial Na+ uptake in the freshwater fish gill. We support previous research that Na+ influx into the trout is via an apical, phenamil-sensitive Na+ channel. We propose that one mode of Na+ efflux from the MR cell is directly coupled to a basolateral NBC in contrast to the prevailing assumption that the basolateral Na+-K+-ATPase is the only route for Na+ efflux. Furthermore, we demonstrate that this transport occurs in one subtype of MR cell present on the gill. Our novel finding opens up new avenues for elucidating the cellular and molecular mechanism of transepithelial ion movement in this model epithelium.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was funded by a National Sciences and Engineering Research Council (NSERC) Discovery grant, a NSERC Research Tools and Instrument grant, an Alberta Heritage Foundation for Medical Research, and a Canada Foundation for Innovation grant to G. G. Goss, and an Izaak Walton Killam Memorial Scholarship to M. Tresguerres.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. K. Parks, Dept. of Biological Sciences, Univ. of Alberta, Edmonton, Alberta, T5G 2E9, Canada (e-mail: skparks{at}ualberta.ca)

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. Section 1734 solely to indicate this fact.


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 GRANTS
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