Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na+/K+-ATPase
University of Florida, Department of Zoology, Box 118525, 223 Bartram Hall, Gainesville, FL 32611, USA
*e-mail: pmpierma{at}zoo.ufl.edu
Accepted July 2, 2001
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Summary |
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Key words: V-H+-ATPase, Na+/K+-ATPase, gills, elasmobranch, stingray, salinity, euryhaline, Dasyatis sabina, NaCl regulation, acidbase regulation.
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Introduction |
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V-H+-ATPase has been studied extensively in the mammalian renal collecting duct and turtle urinary bladder where immunocytochemical and ultrastructural research has demonstrated two populations of mitochondrion-rich intercalated cells that acidify or alkalinize the urine. Acidifying () intercalated cells are characterized by an apical cell membrane localization of the V-H+-ATPase, whereas alkalinizing (ß) intercalated cells express the V-H+-ATPase diffusely throughout their cytoplasm and on their basolateral membrane (Stetson and Steinmetz, 1985; Brown et al., 1988a; Brown et al., 1988b; Verlander et al., 1992; Verlander et al., 1994; Brown and Breton, 1996; Steinmetz et al., 1996; Brown and Breton, 2000). In mammals, this transporter is also important for acidification of the male reproductive tract and bone reabsorption by osteoclasts (Lee et al., 1996; Brown et al., 1997).
In aquatic vertebrates, V-H+-ATPase has been implicated in the energization of NaCl uptake. For example, in frog and toad skin it is well established that an apical V-H+-ATPase, localized to mitochondrion-rich cells, generates a membrane potential (inside negative) that drives Na+ entry into the epithelium via an apical Na+ channel (Harvey, 1992; Ehrenfeld and Klein, 1997), and can energize active Cl uptake through apical Cl/HCO3 exchangers (Larsen et al., 1996). Recently, the V-H+-ATPase has also been considered important for driving Na+ uptake across the gill epithelium of freshwater fishes. Similar to processes in frog skin, it was proposed that an apical V-H+-ATPase would generate a favorable electrical gradient to drive Na+ uptake through an apical Na+ channel (Lin and Randall, 1993; Lin et al., 1994; Sullivan et al., 1995).
This model of ion uptake in freshwater teleosts was supported by the results of Wilson et al. (Wilson et al., 2000), who found colocalization of the V-H+-ATPase with the epithelial Na+ channel (ENaC) on the apical membrane of pavement cells from the leading edge of tilapia gills (Oreochromis mossambicus). In rainbow trout (Oncorhynchus mykiss), V-H+-ATPase and ENaC were expressed in both pavement and chloride (Na+/K+-ATPase-rich) cells (Wilson et al., 2000). Other supporting evidence for the proposed role of V-H+-ATPase in freshwater teleost ion uptake includes bafilomycin inhibition of Na+ uptake in tilapia (O. mossambicus) and carp (Cyprinus carpio) (Fenwick et al., 1999), and decreased activity and localization of branchial V-H+-ATPase when freshwater rainbow trout are acclimated to sea water (Lin and Randall, 1993; Lin et al., 1994).
Since an apical V-H+-ATPase in freshwater teleost gills would directly pump protons into the environment, this transporter has also been hypothesized to play an important role in systemic acidbase balance. Experiments on rainbow trout have corroborated this hypothesis by demonstrating that gill V-H+-ATPase activity, immunoreactivity and mRNA expression all increase after exposure to environmental hypercapnia (Lin and Randall, 1993; Lin et al., 1994; Sullivan et al., 1995; Sullivan et al., 1996; Perry et al., 2000). Studies on branchial V-H+-ATPase in a true marine teleost have yet to be published, but the transporters role in acidbase regulation of seawater teleosts is assumed to be minimal, given the favorable Na+ gradient for Na+/H+ exchangers (Claiborne, 1998; Claiborne et al., 1999).
V-H+-ATPase has been localized and/or measured in the gills of two marine elasmobranch species, Squalus acanthias and Raja erinacea (Kormanik et al., 1997; Wilson et al., 1997). In S. acanthias, Wilson et al. (Wilson et al., 1997) found V-H+-ATPase immunoreactivity in mitochondrion-rich cells of the gill interlamellar region, presumably within cytoplasmic tubulovesicles. Interestingly, we found that Na+/K+-ATPase was also localized to cells of the interlamellar region in gills from marine Atlantic stingrays (Dasyatis sabina) (Piermarini and Evans, 2000). Therefore, it is possible that V-H+-ATPase and Na+/K+-ATPase colocalize to the same branchial cell type in marine elasmobranchs.
The goals of this study were to examine the effects of environmental salinity on the expression of V-H+-ATPase in the gills of the Atlantic stingray and determine if V-H+-ATPase and Na+/K+-ATPase are expressed in the same cells. To date, V-H+-ATPase expression in the gills of an elasmobranch acclimated to different salinities has not been studied. In a previous study, we found that expression of Na+/K+-ATPase in the Atlantic stingray (Dasyatis sabina) was affected by environmental salinity (Piermarini and Evans, 2000). Gills from freshwater stingrays were characterized by a three- to fourfold higher activity and abundance of Na+/K+-ATPase relative to marine individuals. Since the V-H+-ATPase is considered important for ion uptake, we hypothesized a similar trend for this transporter.
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Materials and methods |
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Collection of gill tissue
Details of gill tissue collection for the immunoblotting and immunohistochemistry have been described (Piermarini and Evans, 2000). In brief, stingrays were perfused through the conus arteriosus with an elasmobranch Ringers solution (Forster et al., 1972), then gill filaments were trimmed off the arches and placed in fixative (3 % paraformaldehyde, 0.05 % glutaraldehyde, 0.05 % picric acid in 10 mmol l1 phosphate buffered saline, pH 7.3) for 24 h at 4°C. Additional filaments were snap-frozen in liquid nitrogen for immunoblot analysis and stored at 80°C until analyzed.
Anti- V-H+-ATPase B-subunit antibody
The antibody used in this study was developed by Filippova et al. (Filippova et al., 1998) and is a rabbit polyclonal antibody made against a 279-amino-acid region (residues 79357) of the V-H+-ATPase B-subunit from the insect Culex quinquefasciatus. This region of the insect B-subunit V-H+-ATPase shares 91 % amino acid identity with that published for teleost V-H+-ATPase B-subunits (Niederstatter and Pelster, 2000; Perry et al., 2000). The antibody was kindly provided by Dr William Harvey, Whitney Laboratory, University of Florida (with permission from Dr Sarjeet Gill, University of California at Riverside).
Immunoblotting of V-H+-ATPase B-subunit
Immunoblots were performed on polyvinylidene difluoride (PVDF) membranes (Bio-Rad) from a previous study (Piermarini and Evans, 2000), containing 20 µg of total gill membrane protein per lane. Details of tissue preparation, electrophoresis and blotting have been described (Piermarini and Evans, 2000). Since these PVDF membranes were previously used to detect Na+/K+-ATPase, it was necessary to strip the antibodies that were bound to the membrane.
To remove previous antibodies, PVDF membranes were soaked in 100 % methanol for 15 min and placed in a Strip-Buffer (62 mmol l1 Tris-base, 2 % sodium lauryl sulfate, 0.6 % ß-mercaptoethanol, pH 6.7) for 30 min at 60°C to strip previous primary and secondary antibodies off the PVDF. After stripping the PVDF, it was washed three times with dH2O (5 min each). The PVDF was reblocked with Blotto (Boehringer) for 1.5 h at 25°C, and then transferred to the primary antibody solution (polyclonal rabbit anti-insect V-H+-ATPase B-subunit diluted 1:10,000 in Blotto) and incubated overnight at 4°C.
After primary antibody incubation, the PVDF was washed three times (15 min each) with Tris-buffered saline + 1 % Tween-20 (TTBS) with 5 % dry milk, then incubated with an alkaline-phosphatase-conjugated goat anti-rabbit IgG secondary antibody (Bio-Rad; diluted 1:3000 in Blotto) for 2 h at 25°C. The PVDF was then washed three times (15 min each) with TTBS, and a substrate solution (Bio-Rad Immun-Star ECL Kit) applied to the PVDF for 5 min at 25°C to initiate a luminescent signal. Binding of antibody was detected by exposing Hyperfilm-ECL imaging film (Amersham) to the PVDF membrane. Film was developed according to the manufacturers protocol. As a control, we incubated stripped membranes with normal rabbit or goat serum instead of the primary antibody and found no detectable signal.
Negatives were digitized into TIFF files using a UMAX flatbed scanner with transparency adapter, and analyzed using NIH Image version 1.61 (National Institutes of Health, USA). To quantify the relative abundance of the V-H+-ATPase B-subunit, we measured the intensity of the immunopositive bands using densitometry and standardized all measurements to the FW condition. Therefore, all intensity measurements of FW individuals were normalized to a value of 1.0.
Immunohistochemical localization of V-H+-ATPase B-subunit
Histological tissues used in this study were identical to those described previously (Piermarini and Evans, 2000). In brief, 6 µm serial sections of paraffin-embedded gill tissue were cut parallel to the long axis of the filament and placed on poly-L-lysine coated slides. Sections were deparaffinized in Hemo-De, hydrated in a graded ethanol series, and washed in 10 mmol l1 phosphate-buffered saline (PBS). A hydrophobic PAP-Pen (Electron Microscopy Suppliers) was used to draw circles around the tissue sections, and then 3 % H2O2 was placed on the sections for 30 min to inhibit endogenous peroxidase activity. Sections were also blocked with Biogenex Protein Block (BPB; normal goat serum with 1 % bovine serum albumin, 0.09 % NaN3 and 0.1 % Tween-20) for 20 min before application of the primary antibody.
The primary antibody (polyclonal rabbit anti-insect V-H+-ATPase B-subunit diluted 1:10,000 in BPB) was incubated on the sections overnight at 4°C. The antibody was rinsed off and the sections were washed in PBS for 5 min. The sections were then incubated with a biotinylated goat anti-rabbit IgG secondary antibody (Biogenex) and a horseradish peroxidase-labeled strepavidin solution (Biogenex) for 20 min each at 25°C. After washing with PBS for 5 min, antibody binding was visualized by applying the chromagen DAB (3,3'-diaminobenzidine tetra-hydrochloride; Biogenex) to the sections for 5 min at 25°C. No staining was detected when non-immune rabbit serum or BPB was used instead of primary antibody.
The number of immunopositive (V-H+-ATPase-rich) cells per gill lamella and per interlamellar region was counted to quantify the distribution of these cells. For each animal, three immunostained slides were chosen. On a section from each slide, the number of V-H+-ATPase-rich cells was counted on 30 randomly selected lamellae and interlamellar regions. Lengths of lamellae were also measured to standardize cell counts to lamellar length. Results are expressed as number of V-H+-ATPase-rich cells per 100 µm of lamella, per interlamellar region, and per 100 µm of lamella + interlamellar region (sum).
Double-labeling of V-H+-ATPase and Na+/K+-ATPase
To determine if V-H+-ATPase and Na+/K+-ATPase are expressed in the same cells, we used a double-labeling technique modified from the method of Verlander et al. (Verlander et al., 1996). Tissue sections for double-labeling were deparaffinized, hydrated, blocked and stained for V-H+-ATPase as described above. However, after being developed with the brown chromagen (DAB), the slides were rinsed in dH2O for 10 min and reblocked with BPB for 20 min. A mouse anti-chicken Na+/K+-ATPase antibody (monoclonal antibody a5 culture supernatant diluted 1:100 in normal goat serum) was then applied to the sections overnight at 4°C. The primary antibody, a5, developed by Dr Douglas Fambrough, was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA. Rinsing and developing was performed as described above, except a blue chromagen was used (Vector SG). Double-labeled sections were then observed to determine if V-H+-ATPase and Na+/K+-ATPase occurred in the same cells.
Statistical analyses
Differences in mean number of V-H+-ATPase-rich cells were detected using a one-way analysis of variance (ANOVA), with a StudentNewmanKeuls post-hoc test. Differences in relative intensities of bands from immunoblots were detected using a KruskalWallis non-parametric ANOVA, with a KruskalWallis post-hoc test (Conover, 1980). All tests were 2-tailed and differences were considered significant if P<0.05.
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Results |
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Discussion |
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When freshwater stingrays were acclimated to sea water for 1 week, branchial V-H+-ATPase relative abundance decreased significantly (Fig. 2). This effect of salinity on branchial V-H+-ATPase expression has also been reported in a teleost (Oncorhynchus mykiss), where gill V-H+-ATPase activity and immunoreactivity decreased when freshwater trout were acclimated to sea water (Lin and Randall, 1993; Lin et al., 1994). A lower branchial V-H+-ATPase expression for the seawater-acclimated stingrays was expected, because active NaCl uptake would not be necessary in a seawater environment and passive Na+/H+ and Cl/HCO3 exchangers are thought to be responsible for acidbase extrusion in seawater fishes (Claiborne, 1998).
The immunohistochemical results of this study suggest that the higher overall abundance of V-H+-ATPase in freshwater stingray gills (Fig. 2) can be attributed to a greater number of V-H+-ATPase-rich cells found in the gill epithelium, especially on the lamellae (Fig. 3, Fig. 4). The dramatic decrease in the number of V-H+-ATPase-rich cells found on the lamellae in the seawater-acclimated and seawater stingrays may suggest that those cells have a specialized function for freshwater NaCl and acidbase balance. We have previously reported a similar effect of environmental salinity on the number and distribution of Na+/K+-ATPase-rich cells in the gills of the Atlantic stingray (Piermarini and Evans, 2000).
In freshwater stingrays, localization of V-H+-ATPase occurred diffusely throughout the cytoplasm and was associated with the basolateral membrane of relatively large cells (Fig. 5), presumably mitochondrion-rich (Wilson et al., 1997). It was expected that the V-H+-ATPase would be localized to the apical cell membrane of pavement cells and/or Na+/K+-ATPase-rich cells as it has been described in freshwater teleost species (Lin et al., 1994; Sullivan et al., 1995; Wilson et al., 2000). Basolateral localization of V-H+-ATPase is relatively rare in vertebrates and to date has only been described in ß-type intercalated cells of the mammalian collecting duct and turtle urinary bladder (Stetson and Steinmetz, 1985; Brown et al., 1988a; Brown et al., 1988b; Verlander et al., 1992; Brown and Breton, 1996). If the V-H+-ATPase-rich cells of freshwater stingray gills are analogous in function to ß-type intercalated cells then they would be involved with HCO3 excretion and Cl uptake via an apical Cl/HCO3 exchanger (Weiner and Hamm, 1990). This would be in contrast to freshwater teleost fishes in which Cl/HCO3 exchange is thought to occur in Na+/K+-ATPase-rich chloride cells (Sullivan et al., 1996; Wilson et al., 2000).
In the seawater-acclimated and seawater stingray gills, there appeared to be qualitative differences in the V-H+-ATPase labeling, such as stronger staining in the cytoplasm and less distinct staining along the basolateral membrane, relative to freshwater individuals (Fig. 5). Although ultrastructural studies would be required to quantify these qualitative differences, our findings are consistent with V-H+-ATPase regulation in other vertebrate tissues, in which recycling of the transporter between a cytoplasmic pool of vesicles and the plasma membrane has been demonstrated (Dixon et al., 1986; Stetson and Steinmetz, 1986; Verlander et al., 1992; Verlander et al., 1994; Brown and Breton, 2000). Therefore, the qualitative staining differences may indicate that seawater-acclimated and seawater stingrays have more V-H+-ATPase stored in cytoplasmic vesicles, and less transporter on the basolateral membrane, relative to freshwater stingrays. If the V-H+-ATPase-rich cells are functionally analogous to ß-intercalated cells (see above), then this trend would be expected, because Cl/HCO3 exchange in marine stingrays could be driven by the favorable gradient for Cl to enter the cells from sea water, rather than the active generation of a gradient by a V-H+-ATPase.
In seawater stingrays, V-H+-ATPase-rich cells were only found on the interlamellar region of the gills, which corroborates the results of Wilson et al. (Wilson et al., 1997) who localized V-H+-ATPase in the gills of a seawater elasmobranch (Squalus acanthias) using an antibody to the A-subunit. Wilson et al. (Wilson et al., 1997) reported a cytoplasmic localization for V-H+-ATPase in mitochondrion-rich cells, and suggested it was stored in tubulovesicles that may be recruited to the apical membrane under conditions of acidosis (similar to -intercalated cells). However, we hypothesize that these vesicles would be recruited to the basolateral membrane and function similar to a ß-type intercalated cell. This would imply that another cell type and transporter are involved with acid excretion (see below).
Since branchial V-H+-ATPase (this study) and Na+/K+-ATPase (Piermarini and Evans, 2000) abundance and distribution were affected similarly by salinity, we were interested in determining whether these two transporters are localized to the same cells. Results from double-labeling gills for V-H+-ATPase and Na+/K+-ATPase demonstrated that these two transporters are in separate cells, regardless of environmental salinity (Fig. 6). This is important because it suggests there may be two types of ionocytes in the gill epithelium of elasmobranch fishes. Previous studies have suggested the elasmobranch gill epithelium contains two types of mitochondrion-rich cells, based on the appearance of two distinct apical cell membrane morphologies (Laurent and Dunel, 1980; Crespo, 1982). Our study provides the first immunohistochemical evidence for two mitochondrion-rich cell populations in the elasmobranch gill.
The finding of separate V-H+-ATPase-rich and Na+/K+-ATPase-rich cells also has important functional implications. For example, this separation may indicate that Cl uptake/HCO3 excretion and Na+ uptake/H+ excretion occur in V-H+-ATPase-rich and Na+/K+-ATPase-rich cells, respectively. Segregation of Cl and Na+ uptake (and HCO3 and H+ excretion) is known to occur in the mammalian collecting duct and turtle urinary bladder; ß-type intercalated cells express basolateral V-H+-ATPase, which helps drive HCO3 excretion and Cl uptake via an apical Cl/HCO3 exchanger (Stetson et al., 1985; Stetson and Steinmetz, 1985; Stetson and Steinmetz, 1986; Brown et al., 1988a; Verlander et al., 1992), -type intercalated cells express apical V-H+-ATPase that drives H+ excretion and HCO3 reabsorption via a band-3 Cl/HCO3 exchanger (Stetson and Steinmetz, 1985; Stetson and Steinmetz, 1986; Brown et al., 1988a; Verlander et al., 1988), and principal cells express basolateral Na+/K+-ATPase that helps drive Na+ uptake via an apical ENaC (Kashgarian et al., 1985; Alvarez de la Rosa et al., 2000). We propose that the V-H+-ATPase-rich cells in the stingray gill are functionally analogous to ß-type intercalated cells and are the putative sites of Cl uptake and HCO3 excretion. In contrast, we propose that the Na+/K+-ATPase-rich cells are a functional amalgam of
-type intercalated cells and principal cells. We hypothesize that Na+/K+-ATPase-rich cells excrete H+ and absorb Na+, but use different apical mechanisms than the mammalian collecting duct and turtle urinary bladder, such as an apical NHE isoform that has been colocalized to Na+/K+-ATPase-rich cells in the gills of marine elasmobranchs (Sue Edwards, Georgia Southern University, personal communication). Our hypothetical model of branchial NaCl and acidbase transport in the Atlantic stingray is presented in Fig. 7.
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Acknowledgments |
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