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Am J Physiol Regul Integr Comp Physiol 291: R1490-R1498, 2006. First published June 8, 2006; doi:10.1152/ajpregu.00026.2006
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Hormonal and environmental regulation of epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss)

Arash Shahsavarani and Steve F. Perry

Department of Biology, Center for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, Canada

Submitted 11 January 2006 ; accepted in final form 5 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC mRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+] = 20–30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (~7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an ~100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. Immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells.

transient receptor potential vanilloid 6; hypercapnia; cortisol; hypercalcemia; soft water

UNLIKE OTHER VERTEBRATES that rely exclusively on dietary calcium to fulfill nutritional requirements, fish absorb a substantial component of their calcium needs from the surrounding water (4). The gill is considered to be the predominant site of Ca2+ uptake, although the skin may play a supplementary role (32). The current model for Ca2+ uptake across the fish gill (7, 8, 22, 27) proposes that the initial step in transepithelial uptake is the entry of Ca2+ into gill epithelial cells through apical membrane Ca2+ channels (31). The final step involves the movement of Ca2+ across the basolateral membrane by either Ca2+-ATPase-mediated active transport (9) or Na+/Ca2+ exchange (41). It is possible that by analogy to renal or intestinal Ca2+ uptake in mammals (1, 2, 15–17, 38, 39), the entry of Ca2+ through the apical Ca2+ channel is the rate-limiting step in branchial Ca2+ uptake. Therefore, modification in the kinetic properties or changes in the number of Ca2+ channels may underlie acute and chronic modifications of Ca2+ uptake rates across the gill.

The branchial epithelial Ca2+ channel (ECaC) has been cloned from several fish species, including pufferfish [Fugu rubripes (33); NCBI GenBank accession no. AY232821], zebrafish [Danio rerio (25); NCBI GenBank accession no. AY325807], and rainbow trout [Oncorhynchus mykiss (35); GenBank accession no. AY256348]. The ECaC belongs to the vanilloid subfamily of the transient receptor potential (TRP) superfamily (3, 40). Unlike in mammals, where two distinct forms of ECaC are encoded for by different genes (TRPV5 and TRPV6), there appears to be but a single gene in fish (33, 35). It has been suggested that fish ECaC and TRPV subfamilies diverged prior to a possible gene duplication giving rise to TRPV5 and TRPV6 (35). Although the site of Ca2+ uptake in freshwater fish is believed to be the chloride cell (also termed mitochondria-rich cell, MRC) (18, 23, 24, 27, 28, 32), ECaC appears to be ubiquitously expressed in all of the gill epithelial cell types, including pavement cells (PVCs) and chloride cells (35).

If Ca2+ entry through ECaC were indeed an important regulatory step in overall transepithelial Ca2+ flux, one would expect predictable changes in the levels of ECaC mRNA and protein in response to physiological cues known to modify branchial Ca2+ uptake rates. Thus, in the present study, it was predicted that conditions known to increase Ca2+ transport capacity, such as low environmental Ca2+ levels (32), cortisol treatment (6), or hypercapnia (21) would be associated with increased ECaC, expression. Hypercalcemia, a condition known to reduce branchial Ca2+ uptake (19, 30), on the other hand, would be expected to reduce ECaC expression. ECaC mRNA expression was monitored using real-time PCR, whereas protein levels were assessed using Western blot analysis and immunocytochemistry with a homologous polyclonal antibody.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care. Rainbow trout (O. mykiss) of both sexes were purchased from Linwood Acres Trout Farm (Campbellcroft, Ontario, Canada). The fish were held at the University of Ottawa in large fiberglass tanks supplied with flowing, aerated, and dechloraminated city water, maintained at 13°C on a 12:12-h light-dark photoperiod, and fed daily with a commercial trout diet. All procedures involving animals were carried out according to institutional guidelines, which are in accordance with those of the Canadian Council on Animal Care. The protocols used in this study were approved by the University of Ottawa Animal Care Committee in accordance with guidelines provided by the Canadian Council on Animal Care.

Effect of environmental Ca2+. Three aquaria (30 cm high x 30 cm wide x 61 cm long, 54-liter volume) were used with water temperature maintained at 13°C by using a recirculating water bath and a stainless-steel cooling coil. All sides of the aquaria were blackened to minimize visual stress. Three environmental Ca2+ levels were established (low = 20–30 nmol/l, normal = 200–300 nmol/l, high = 2–2.5 mmol/l). Ca2+ concentrations were adjusted using calcium nitrate as needed. Six fish [mean mass for all 18 fish: 97.4 ± 10.9 (SE) g] were placed into each environment for a period of 5 days. Water chemistry was monitored on a daily basis to ensure that Ca2+ levels were maintained within the target range. After the 5-day exposure, the animals were euthanized by a sharp blow to the head. The gill basket was quickly removed and gill filaments were isolated. Samples were immediately placed into liquid N2 and stored at –86°C until use. Tissue samples were also preserved for microscopy using standard procedures (see below). Water [Ca2+] was determined using flame emission spectrophotometry (model Spectra AA 250 Plus; Varian).

Effect of Ca2+ infusion. Benzocaine (ethyl-p-aminobenzoate; 2.4 x 10–4 mol/l) was used to anesthetize each fish (weighing between 180 and 260 g). The fish were then placed on a surgical table, where the gills were irrigated continuously with anesthetic solution. The dorsal aorta was cannulated (37) using a polyethylene cannula (PE-50; Clay-Adams). For recovery, fish were placed into individual opaque acrylic boxes provided with continuous flow of aerated fresh water (13° C) for 24 h before the commencement of the experiments.

After the recovery period, each fish was infused via the dorsal aorta cannula for a period of 24 h with a syringe pump (model 355: Sage Instruments). Fish were infused with either saline (140 mmol/l NaCl, pH 7.8) or Ca2+-enriched saline (0.01 mol/l CaCl2) at a rate of 1.0 ml/h for 24 h. At the conclusion of the experimental period, blood samples were collected for plasma Ca2+ measurements. Blood samples were immediately centrifuged (12,000 g for 1 min), and the plasma was collected and stored at –20°C until analysis. Plasma [Ca2+] was determined using flame emission spectrophotometry (model Spectra AA 250 Plus; Varian). Each animal was euthanized by a sharp blow to the head, and tissue samples were collected as previously described.

Effect of increased plasma cortisol levels. Fish (60–207 g) were anesthetized and implanted with 5 ml/kg of either cocoa butter (sham) or cocoa butter containing cortisol (22 mg of hydrocortisone 21-hemisuccinate per ml of cocoa butter). The fish were returned to holding tanks, where they were kept for 5 days without disturbance. After the experimental period, fish were quickly collected, euthanized, and sampled as previously described. Blood samples were also collected for cortisol measurements. These samples were centrifuged (12,000 g for 1 min), and the plasma was collected rapidly, frozen in liquid N2, and stored at –80°C until analysis with a commercial RIA kit (ICN Pharmaceuticals).

Effect of 48-h hypercapnia. Fish (205–327 g) were placed into individual opaque acrylic boxes with continuous flow of aerated fresh water (13°C) for 24 h before the start of the experiment. After this acclimation period, one group of fish was exposed to flowing water containing 1% CO2 in air (hypercapnic water PCO2 = 7.5 mmHg), while the other group was exposed to normally aerated water for 48 h. The desired level of hypercapnia was achieved by gassing a water equilibration column with appropriate mixtures of CO2 in air (Cameron gas mixer). The PCO2 of the water exiting the column was continuously monitored using a PCO2 electrode (Cameron Instruments) connected to a blood gas meter (Cameron Instruments). The PCO2 electrode was calibrated using solutions of water (13°C) equilibrated with mixtures of 0.5 or 1.0% CO2, achieved using the Cameron gas mixer. The final PCO2 of the water exiting the column was controlled by adjusting the flows of gas and water and the percentage of CO2 gassing the column. At the conclusion of the exposure period, fish were euthanized and samples were collected as previously described.

Real-time PCR analysis. Tissue samples were powdered under liquid N2 using a mortar and pestle. Tissue total RNA was extracted from 30 mg of tissue using Invitrogen TRIzol reagent. All procedures were followed as per the manufacturer’s instructions with the following modifications. No more than 30 mg of powdered tissue was used per 1 ml of TRIzol, and after the resuspension of the total RNA in 100 µl of nuclease-free water, the RNA was reextracted using 1 ml of TRIzol by repeating the entire procedure. The RNA was finally resuspended in 30 µl of nuclease-free water.

Reverse transcription was performed using the Stratascript reverse transcriptase kit (Stratagene). Complementary DNA was synthesized as per the kit manufacturer’s instructions with the following changes. Final reaction volume was adjusted to 12.5 µl, and 0.5 µg of total RNA was used with 0.15 µg of random hexamer primers.

Real-time PCR was performed using a MX 4000 Multiplex quantitative PCR system (Stratagene) with Brilliant SYBR Green QPCR master mix (Stratagene) as per the manufacturer’s instructions with the following modifications. The total reaction volume was reduced to 25 µl; 0.5 µl of cDNA template was used, and primer concentrations were 0.150 nmol/l for each primer. All primers (see below) were designed and optimized for the following PCR reaction conditions: 15 min at 95°C, 45 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. At the end of each run, a dissociation curve was established to determine the purity of the amplicons in each reaction. Those samples exhibiting more than one dissociation peak (indicative of multiple products) were eliminated. Control samples (diluted RNA samples) were examined at random to test for the presence of genomic DNA contamination. Primers for real-time PCR were as follows: ECaC-QPCR1 forward, 5'-GGACCCTTCCATGTCATTCTTATT-3'; ECaC-QPCR2 reverse, 5'-ACAGCCATGACAACTGTTTCC-3'; beta-actin forward, 5'-CCAACAGATGTGGATCAGCAA-3'; beta-actin reverse, 5'-GGTGGCACAGAGCTGAAG GGTA-3'.

Tissue preservation and immunocytochemistry. Gill filaments were quickly removed from freshly dissected gill arches collected after an experiment. The filaments were then placed in ice-cold 4% paraformaldehyde (pH 7.4) and kept at 4°C overnight. The filaments were then transferred to phosphate-buffered saline (PBS) containing 15% sucrose for 2 h at 4°C and, finally, transferred to PBS containing 30% sucrose for at least 2 h before sectioning. Tissue samples were embedded in Shandon Cryomatrix embedding medium (Fisher), and thin sections (10 µm) were prepared using a Leica CM 1850 cryostat at –18°C. Sections were placed on SuperFrost++ (Fisher Scientific) microscope slides, air-dried for 10 min, and stored at –20°C until use.

Sections were incubated in situ (3 x 5 min) with a blocking buffer containing 2% normal goat serum, 0.1 mol/l PBS, 0.9% Triton-X, 1% gelatin, and 2% BSA. They were then incubated for 2 h at room temperature, in a humidified chamber, with one of two primary antibodies diluted in the blocking buffer: {alpha}5, a mouse monoclonal antibody against the {alpha}1-subunit of chicken Na+/K+-ATPase (1:100; University of Iowa Hybridoma Bank) or trout ECaC (1:200). For ECaC, custom polyclonal antibodies were raised in rabbit (Abgent, San Diego, CA) against an 18-amino acid region (SQFRFRLQNRKGWKEMLD) of rainbow trout ECaC protein. This region corresponded to amino acids 18 through 36 (see Ref. 35). For negative controls, sections were incubated with blocking buffer lacking primary antibodies, with preimmune serum (ECaC), or with antibodies preabsorbed with excess peptide antigen (ECaC). The {alpha}5 antibody has been used in numerous previous studies to localize Na+/K+-ATPase in fish tissues (e.g., Ref. 43). The slides were then washed (3 x 5 min) in 0.1 mol/l PBS. For double-immunofluorescence staining, the trout anti-rabbit ECaC was detected with a 1:400 dilution of Alexa 488-coupled goat anti-rabbit IgG (Fisher), and {alpha}5 was detected with a 1:400 dilution of Alexa 546-coupled goat anti-mouse IgG (Fisher). Slides were incubated in a humid chamber for 1 h at room temperature. The slides were then washed (3 x 5 min) in 0.1 mol/l PBS and mounted with a mounting medium (Vector Laboratories) containing 4',6'-diamidino-2-phenylindole (DAPI) to stain nuclei.

Specimens were observed and photographed using a Zeiss Axiophot light microscope and a Hamamatsu C5985 chilled charge-coupled device camera. Images were captured using the Metamorph v4.01 imaging system.

Western blot analysis. Proteins were prepared from frozen tissues by homogenization on ice in 1 ml of extraction buffer containing 50 mM Tris·HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS, and protease inhibitor cocktail (Roche). The samples were incubated on ice for 10 min and briefly sonicated to break up any DNA that might have been extracted. The samples were centrifuged at 14,000 g for 10 min at 4°C, and the supernatants were stored at –80°C before use. Protein concentrations were determined using a microbicinchoninic acid protein assay (Pierce) with BSA as standard. Samples (50 µg of protein) were size-fractionated by reducing SDS-PAGE using 7% separating and 5% stacking polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). After transfer, each membrane was blocked for 1 h in PBST (1x PBS, 0.1% Tween 20)-5% milk and probed with a dilution of 1:3,000 rabbit anti-trout ECaC overnight at 4°C. The membranes were then probed for 1 h at room temperature with 1:4,000 goat anti-rabbit antibody (Pierce). After each exposure to antibody, the membranes were washed 3 x 5 min in PBST. The specific bands were detected using enhanced chemiluminescence (ECL; SuperSignal West Pico chemiluminescent substrate; Pierce), and blots were exposed to Kodak X-Omat, blue XB-1 film (Fisher). The protein size marker used was obtained from Fermentas Life Sciences. To demonstrate specificity of the trout ECaC antibody, we combined primary antiserum with excess (20 µg) of the peptide against which the antibody was raised. Additional negative controls included incubating blots with blocking buffer lacking antibodies or with preimmune serum.

To assess for equal loading, we stripped blots with Re-Blot Plus mild stripping solution (Chemicon). The blot was incubated in 1x stripping solution for 20 min at room temperature and then rinsed for 10 min in PBST. After rinsing, the blot was then blocked twice in 5% PBST-milk for 10 min each. The blot was then probed with an anti-beta-tubulin antibody (1:1,000; Sigma-Aldrich Canada) for 1 h at 37°C. The blot was then incubated in anti-mouse Ig, horseradish peroxidase (1:5,000) for 1 h at room temperature. After additional washings, the proteins were visualized using ECL as described above.

The density of the antigenic bands was determined by scanning the films and then analyzing the digital images using commercial software (Quantity One v4.1.1). The results are presented as the ratio of ECaC to tubulin band density.

Statistical analysis. Statistical analysis was performed using SigmaStat (version 2.03; SPSS, Chicago, IL). One-way analysis of variance was used to determine the effect of environmental Ca2+ and cortisol implant on ECaC expression. In all other experiments, the Student’s t-test was used; significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of ECaC in the gill. Figure 1 depicts the typical pattern of ECaC expression in the trout gill epithelium that was observed throughout the course of this study. ECaC was localized to the apical membranes of lamellar PVCs, as well as to cells expressing Na+/K+-ATPase (presumed to be MRCs). Interestingly, not all of the cells expressing Na+/K+-ATPase coexpressed ECaC (Fig. 1A). Preabsorption of the primary antibody with excess peptide antigen eliminated nearly all fluorescence signal (Fig. 1B). On a Western blot (Fig. 1C), the ECaC antibody recognized a single immunoreactive band at 90 kDa; the band was not observed after preabsorption with peptide antigen.


Figure 1
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  		Fig. 1. Colocalization of epithelial Ca2+ channel (ECaC), Na+/K+-ATPase, and nuclei in rainbow trout (Oncorhynchus mykiss) gill. The cell nuclei appear as blue, ECaC appears as green, and Na+/K+-ATPase appears as red. Yellow represents colocalization of ECaC and Na+/K+-ATPase. A: representative image of gill lamellae; note that most Na+/K+-ATPase-positive cells exhibit apical ECaC immunoreactivity (asterisks). In addition, some cells appeared to exclusively express ECaC (arrows) or Na+/K+-ATPase (arrowheads), whereas other cells appeared to lack either protein. B: representative image of a gill section preabsorbed with ECaC peptide antigen (blocking peptide). Scale bars in A and B represent 20 µm. C: representative Western blot showing the presence of a single immunoreactive band at 90 kDa in trout gill (lane 1) that was not observed when the antibody was preabsorbed with excess blocking peptide (lane 2).

 
Effect of environmental Ca2+. Real-time PCR analysis of rainbow trout gill tissue from fish exposed to low (20–40 nmol/l), normal (200–300 nmol/l), or high (2–2.5 mmol/l) environmental Ca2+ levels revealed a significant 10-fold increase in ECaC mRNA expression in fish exposed to low Ca2+ compared with the control fish (Fig. 2A). ECaC protein levels also were significantly increased in the fish exposed to low levels of Ca2+ but unaltered in the animals kept in high-Ca2+ water (Fig. 2A). Immunocytochemical analysis of gill sections indicated increased amounts of ECaC protein at the tips of the lamellae with an apparent increase in Na+/K+-ATPase-rich cell population at the base of the lamellae (Fig. 2B). Although not quantified, there also appeared to be a reduction in the intensity and extent of ECaC fluorescence and in the numbers of Na+/K+-ATPase-enriched cells in the fish kept in high-Ca2+ water (Fig. 2D).


Figure 2
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  		Fig. 2. Effects of 5-day exposure to low (20–40 nmol/l), normal (200–300 nmol/l), or high (2–2.5 mmol/l) environmental Ca2+ levels on ECaC mRNA and protein expression or distribution of ECaC and Na+/K+-ATPase immunoreactivity in rainbow trout gills. A: results for mRNA (n = 6 for each treatment) are presented relative to beta-actin expression within the given sample. Densitometry results for protein (n = 5 for controls, n = 6 for low Ca2+) were normalized to the reference protein, beta-tubulin. All data are expressed as means ± SE. *P < 0.05, significant difference from the control (normal Ca2+) group (1-way ANOVA). B–F: colocalization of ECaC, Na+/K+-ATPase, and nuclei after 5-day exposure to low (20–40 nmol/l; B), normal (200–300 nmol/l; C), and high (2–2.5 mmol/l; D) environmental Ca2+ levels. The cell nuclei appear as blue, ECaC appears as green, and Na+/K+-ATPase appears as red. Yellow represents colocalization of ECaC and Na+/K+-ATPase. All photos were taken with the same exposure times and adjusted identically for contrast and brightness. However, the image in B is the superposition of 2 pictures in which the intensity of green signal was significantly reduced in the image at right (distal filaments) to produce a more uniform pattern of fluorescence intensity. Inset at top right is shown enlarged in E and F. E: an enlarged and enhanced image of the tip of a lamella in which the cells expressing high levels of ECaC appear to be enlarged. F: the same image digitally adjusted to more clearly show the stained nuclei. Inset in C depicts a section in which ECaC primary antibody was omitted.

 
Effect of intravascular Ca2+ infusion. Fish infused with CaCl2 for 24 h exhibited a 2.5-fold increase in plasma Ca2+ levels (from 3.31 ± 0.24 to 8.11 ± 0.74 mmol/l). Gene expression analysis revealed a significant decrease in ECaC mRNA expression after 24 h of infusion, yet protein levels were unchanged (Fig. 3).


Figure 3
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  		Fig. 3. Effects of 24 h of intra-arterial infusion of CaCl2 on ECaC mRNA and protein expression in rainbow trout gills. Results for mRNA (n = 4 for each treatment) are presented relative to beta-actin expression within the same sample. Densitometry results for protein (n = 4 for each treatment) were normalized to the reference protein, beta-tubulin. All data are expressed as means ± SE. *P < 0.05, significant difference from the control (infused with 140 mmol/l NaCl) group (Student’s t-test). NS, not significant.

 
Effect of cortisol treatment. Fish treated with cortisol implants exhibited an ~50-fold increase in plasma cortisol levels compared with the control (untreated) fish (116.2 ± 32.2 vs. 2.3 ± 0.9 ng/ml). The sham-treated fish (cocoa butter only) also demonstrated high plasma cortisol levels (94.4 ± 19.0 ng/ml), and thus the data from these fish were not included in the analysis. The fish given cortisol implants displayed significant threefold increases in relative ECaC mRNA expression and protein levels in relation to the control fish (Fig. 4A). Qualitatively, the gills of cortisol-treated fish possessed greater numbers of Na+/K+-ATPase-rich cells, especially on lamellae; the majority of these cells appeared to express apical ECaC (Fig. 4B).


Figure 4
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  		Fig. 4. Effect of increased plasma cortisol concentrations on ECaC mRNA and protein expression or distribution of ECaC and Na+/K+-ATPase immunoreactivity in rainbow trout gills. Animals were implanted for 5 days with cocoa butter containing cortisol and compared with fish containing no implant. A: results for mRNA (n = 7 for each treatment) are presented relative to beta-actin expression within the same sample. Densitometry results for protein (n = 7 for each treatment) were normalized to the reference protein, beta-tubulin. All data are expressed as means ± SE. *P < 0.05, significant difference from the control group (Student’s t-test). B: colocalization of ECaC, Na+/K+-ATPase, and nuclei in fish with no cortisol implant. C: colocalization of ECaC, Na+/K+-ATPase, and nuclei in fish after 5 days of treatment with cortisol implant. The cell nuclei appear as blue, ECaC appears as green, and Na+/K+-ATPase appears as red. Yellow represents colocalization of ECaC and Na+/K+-ATPase. All photos were taken with the same exposure times and adjusted identically for contrast and brightness.

 
Effect of hypercapnia. Exposure of fish to hypercapnia (~7.5 mmHg) for a period of 48 h significantly increased ECaC mRNA expression ~100-fold (Fig. 5A), whereas ECaC protein levels were increased only 2.1-fold. Immunocytochemical analysis of gill sections revealed a striking increase in the intensity of apical ECaC expression after hypercapnia on both Na+/K+-enriched MRCs and lamellar PVCs (Fig. 6, B and C).


Figure 5
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  		Fig. 5. Effects of 48 h of hypercapnia (n = 6) on ECaC mRNA or protein expression or distribution of ECaC and Na+/K+-ATPase immunoreactivity in rainbow trout gills. A: results for mRNA (n = 6 for each treatment) are presented relative to beta-actin expression within the same sample. Densitometry results for protein (n = 4 for each treatment) were normalized to the reference protein, beta-tubulin. All data are expressed as means ± SE. *P < 0.05, significant difference from the control group (Student’s t-test). B: colocalization of ECaC, Na+/K+-ATPase, and nuclei. The cell nuclei appear as blue, ECaC appears as green, and Na+/K+-ATPase appears as red. Yellow represents colocalization of ECaC and Na+/K+-ATPase.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The goal of this study was to provide indirect evidence that ECaC is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC mRNA with real-time PCR and ECaC protein with Western blots and immunocytochemistry in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity.

The results of real-time PCR analyses clearly demonstrated that the levels of ECaC mRNA varied in direct relation to the Ca2+-transporting capacity of the gill, increasing in fish exposed to low environmental Ca2+ level, elevated plasma cortisol concentration, or hypercapnia and decreasing in fish experiencing experimentally induced hypercalcemia. Except for the fish experiencing hypercalcemia, there was a matching change in ECaC protein levels associated with the mRNA changes. The changes in protein levels were not always of the same magnitude as the mRNA changes. The results of the immunocytochemistry, although not quantitative, suggest that the altered levels of ECaC detected in this study reflected modifications of ECaC protein within both chloride cells and PVCs.

Low environmental Ca2+ levels. After exposure of trout to low environmental Ca2+ concentrations for 5 days, ECaC gene expression increased ~10-fold, whereas protein levels were increased 2.5-fold. Adult zebrafish, exposed to similar experimental conditions, exhibited a similar response in ECaC gene expression (Shahsavarani A and Perry SF, unpublished data). In addition, Pan et al. (25) recently demonstrated that exposure of zebrafish embryos to water containing low levels of Ca2+ (0.02 mmol/l) caused an increase in ECaC mRNA expression in gills and skin. Thus ECaC clearly is being affected (directly or indirectly) by environmental Ca2+ levels, and this suggests that the increased capacity of the trout gill (32) or zebrafish embryo (25) to absorb Ca2+ after exposure to soft water may indeed reflect increased numbers of apical membrane Ca2+ channels.

The results of previous studies (23, 24) provided evidence that gill MRCs are involved in Ca2+ uptake. On the basis of these and other indirect or correlative studies (18, 26, 28, 32), a model was constructed (27) in which the MRC was implicated as the principal (potentially exclusive) cell type responsible for Ca2+ uptake. In the present study, however, immunocytochemical analysis of gill cross sections in fish exposed to soft water failed to demonstrate strong colocalization of Na+/K+-ATPase (representative of MRCs) and ECaC-positive cells (Fig. 2). Under control conditions, ECaC appeared to be widely distributed to both PVCs and a subset of MRCs. A similar conclusion was reached by Shahsavarani et al. (35) upon examination of both gill sections and cultured cells. During exposure to soft water, there appeared to be a shift in the cell types expressing ECaC. Specifically, ECaC appeared to be highly expressed in a subpopulation of enlarged cells located at the tips of lamellae (Fig. 2B). This regional increase in ECaC expression in fish exposed to a low Ca2+ environment suggests the presence of a previously unidentified cell type that may be playing a significant role in Ca2+ uptake.

Exposure of trout to the varied levels of environmental Ca2+ used in the present study could potentially lead to transient or longer-term alterations in plasma Ca2+ levels. Therefore, the changes in ECaC mRNA expression observed in this study could reflect sensing of ambient and/or internal Ca2+. The presence of a Ca2+-sensing receptor (CaR) has been demonstrated in the trout gill (34), and thus it is conceivable that CaR is involved in the sensing of low ambient Ca2+ levels and the initiation of events leading to increased transcription of ECaC. Furthermore, it is well established that increased levels of internal Ca2+ can be sensed and can lead to downstream effects to reduce branchial Ca2+ uptake (19). Clearly, further work is required to elucidate the relative importance of external versus internal changes in Ca2+ in promoting transcriptional changes to ECaC expression.

Various studies have clearly demonstrated an increase in MRC surface area (through cellular enlargement, as well as cellular proliferation) with exposure to soft water (14, 32). Similar results were noted in the present study (compare Fig. 2, B and C). The more important observation, however, was that the MRCs in the soft water fish did not display any obvious increase in ECaC expression.

Although the levels of ECaC mRNA and protein were increased in soft water fish, there was no reduction in ECaC mRNA or protein associated with exposing fish to Ca2+-enriched water. It has been established previously (32) that maximal Ca2+ transport capacity (Jmax) is reduced in trout exposed to high ambient Ca2+. Thus the absence of any regulation of ECaC mRNA (and presumably protein) in these fish suggests that the reduced Ca2+ uptake reflects nontranscriptional control of ECaC or modulation of another component of the overall transepithelial Ca2+ absorption process [basolateral plasma membrane Ca2+-ATPase (PMCA) or Na+/Ca2+ exchange].

Hypercalcemia. Infusing fish with Ca2+-enriched saline for 24 h resulted in a marked elevation of plasma Ca2+ levels and a concomitant reduction in ECaC mRNA levels. Because ambient Ca2+ concentration was unchanged, the results provide strong evidence for an internal Ca2+-sensing mechanism linked to transcriptional control of ECaC. This mechanism may be similar to the one proposed to initiate the release of the hypocalcemic hormone stanniocalcin (STC), in which the CaR is thought to be involved (34). The release of STC from the corpuscles of Stannius during acute hypercalcemia is a critical mechanism leading to rapid reductions in the rate of branchial Ca2+ uptake (5, 19, 30, 42) thought to involve modification of Ca2+ conductance through existing Ca2+ channels. The results of the present study demonstrate that chronic hypercalcemia may lower Ca2+ uptake by an additional mechanism, a reduction in the number of ECaCs. However, because the reduced levels of ECaC mRNA were not accompanied by a reduction in ECaC protein levels, it is possible that a longer period of hypercalcemia is required for transcriptional changes to significantly impact protein levels.

Elevated plasma cortisol levels. Plasma cortisol levels have been shown to increase in fish exposed to soft water (6, 29, 32, 36). Because of the hypercalcemic action of cortisol (6, 32), this response presumably helps to maintain Ca2+ homeostasis in soft water environments. The hypercalcemic actions of cortisol have been attributed to increased branchial Ca2+ uptake associated with MRC proliferation (20) and increased activity of the basolateral PMCA (6). In this study, we have presented evidence for an additional mechanism underlying the hypercalcemic effects of cortisol that involves a transcriptional increase in ECaC. The increased levels of ECaC appeared to be a direct result of MRC proliferation. Thus, although the apparent expression of ECaC per MRC did not change, the number of MRCs expressing ECaC was increased by cortisol treatment. It is possible, therefore, that the increased rates of Ca2+ uptake observed (6) after cortisol treatment in vivo reflect the combined effects of increased PMCA activity and a greater number of apical membrane Ca2+ channels.

Hypercapnia. Exposure of trout or bullhead (Ictalurus nebulosus) to hypercapnia has been shown to cause a remodeling of the gill epithelium, whereby the surface area of MRCs exposed to the water is markedly reduced due to their apparent covering by neighboring PVCs (10–13). Assuming that the MRCs are the predominant sites of Ca2+ uptake, one would expect hypercapnia exposure to cause a reduction in the rate of Ca2+ uptake, because MRC apical surface area is being reduced. Indeed, this prediction was tested by MacKenzie and Perry (21), who exposed rainbow trout to hypercapnia while monitoring branchial and renal Ca2+ fluxes. Despite a 68% reduction in the surface area of exposed MRCs, the hypercapnic trout in that study (21) actually exhibited a significant increase in the rate of Ca2+ uptake due to an increase in Jmax. The increase in Jmax for Ca2+ uptake was not associated with any changes PMCA, because the ATP-dependent Ca2+-transporting capacity of basolateral membrane vesicles was unaffected by hypercapnia. Instead, it was suggested that the increased rates of Ca2+ uptake in hypercapnic fish might reflect modification of apical membrane Ca2+ channels. The results of the present study provide compelling evidence that the mechanism underlying the increased rates of Ca2+ uptake observed by MacKenzie and Perry (21) in hypercapnic trout is transcriptional upregulation of branchial ECaC. It is possible that the number of Ca2+ channels on MRC apical membranes is being increased to enhance the transporting capacities of cells still exposed to the water. Alternatively, the numbers of channels may be increasing on PVCs to compensate for the loss of exposed MRC surface area. Given the widespread cellular localization of ECaC (e.g., Fig. 1) and the likelihood that both MRCs and PVCs are involved in Ca2+ uptake (35), it is probable that ECaC expression is being increased in both cell types during hypercapnia. Although it is not possible to distinguish between exposed and covered MRCs from light micrographs, the results of the immunocytochemistry suggest that ECaC expression is increasing in a subset of both MRCs and PVCs.

Perspectives

The results of this study provide indirect evidence that the passage of Ca2+ through the gill ECaC is a regulated step controlling the overall flux of Ca2+ across the gill of rainbow trout and, presumably, other teleost species. Thus the Ca2+-transport capacity of the gill can be altered, based on need, by the transcriptional adjustment of ECaC protein levels. The results also suggest that ECaC is expressed in a variety of gill epithelial cells (see also Ref. 35) and not restricted to MRCs as previous models had suggested. This observation is in apparent conflict with the widely held view that the MRC is the exclusive site of branchial Ca2+ uptake in fish. However, because transcellular Ca2+ uptake is assured by the combined actions of Ca2+ entry across the apical membrane and its exit across basolateral membranes, it is possible that only the MRCs possess ample machinery for the exit step. Clearly, there is a need to reevaluate the roles of the PVCs and MRCs in Ca2+ uptake.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Sciences and Engineering Research Council of Canada Discovery and Research Tools grants (to S. F. Perry). Financial support to A. Shahsavarani was provided by Ontario Graduate Scholarship and Ontario Graduate Scholarship in Science and Technology grants.


   ACKNOWLEDGMENTS
 
We are grateful to Brian McNeill for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Perry, Dept. of Biology, Center for Advanced Research in Environmental Genomics, Univ. of Ottawa, 10 Marie Curie, Ottawa, Ontario, K1N 6N5 Canada (e-mail sfperry{at}science.uottawa.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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