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cotransporter abundance and location in Atlantic salmon: effects of
seawater and smolting
Conte Anadromous Fish Research Center, Biological Resources Division, United States Geological Survey, Turners Falls 01376, and Organismic and Evolutionary Biology Program, University of Massachusetts, Amherst, Massachusetts 01003
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Na+-K+-2Cl
cotransporter abundance and location was examined in the gills of
Atlantic salmon (Salmo salar) during seawater acclimation and smolting. Western blots revealed three bands centered at 285, 160, and 120 kDa. The Na+-K+-2Cl
cotransporter was colocalized with
Na+-K+-ATPase to chloride cells on both the
primary filament and secondary lamellae. Parr acclimated to 30 parts
per thousand seawater had increased gill
Na+-K+-2Cl cotransporter
abundance, large and numerous
Na+-K+-2Cl cotransporter
immunoreactive chloride cells on the primary filament, and reduced
numbers on the secondary lamellae. Gill
Na+-K+-2Cl cotransporter levels
were low in presmolts (February) and increased 3.3-fold in smolts
(May), coincident with elevated seawater tolerance. Cotransporter
levels decreased below presmolt values in postsmolts in freshwater
(June). The size and number of immunoreactive chloride cells on the
primary filament increased threefold during smolting and decreased in
postsmolts. Gill Na+-K+-ATPase activity and
Na+-K+-2Cl cotransporter
abundance increased in parallel during both seawater acclimation and
smolting. These data indicate a direct role of the
Na+-K+-2Cl cotransporter in salt
secretion by gill chloride cells of teleost fish.
Salmo salar; chloride cells; teleost; osmoregulation; Na+-K+-ATPase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE
NA+-k+-2cl cotransporter is
an integral membrane protein found in numerous epithelia where it
functions in cell volume regulation and ion transport (4, 10, 23,
25, 42). The Na+-K+-2Cl
cotransporter is widely distributed among many different species of
vertebrates, including the rat, duck, rabbit, dog, cow, and human
(23). Among fish, the
Na+-K+-2Cl cotransporter has been
shown to be present within the intestinal epithelium of the winter
flounder, Pseudopleuronectes americanus (9, 33, 34,
39), and the rectal gland of the spiny dogfish, Squalus
acanthias (22).
Pharmacological studies using p-sulfamoylbenzoic acid
derivatives ("loop diuretics"; e.g., furosemide, bumetanide, and
benzmetanide) indicate that the
Na+-K+-2Cl cotransporter is
involved in salt secretion by marine teleosts. The short-circuit
current of the opercular membrane is a direct measure of chloride
secretion and is inhibited by loop diuretics in seawater-acclimated
killifish, Fundulus heteroclitus (6, 16).
Furthermore, the transepithelial potential of the flounder gill is
reduced after exposure to furosemide (2).
Chloride cells have been shown to be the site of chloride secretion in
the gills of seawater-acclimated teleosts (8). Current models of chloride cell function in euryhaline teleosts indicate that
these cells have unique functions and compositions of transporters depending on the salinity of the surrounding environment (see review,
Ref. 7). The studies cited above indicate that a loop diuretic-sensitive cotransporter is an integral part of the
salt-secreting mechanism of the chloride cell. In the current model of
the seawater chloride cell, basolaterally located
Na+-K+-ATPase creates a sodium gradient that is
used by a basolateral Na+-K+-2Cl
cotransporter to transport sodium, potassium, and two chloride ions
into the cell. Once inside the cell, chloride ions leave down their
electrochemical gradient through an apical Cl channel,
whereas sodium is transported back into the basal lamina via
Na+-K+-ATPase. The buildup of sodium in the
basal lamina allows it to exit into the external media on a
paracellular pathway.
Of these three major transporters involved in salt secretion, only
Na+-K+-ATPase has been definitively localized
to chloride cells (13, 17, 26, 41). Gill
Na+-K+-ATPase activity has been found to
increase during the seawater acclimation of teleosts (5,
14), including Atlantic salmon, Salmo salar
(38). Although salt secretion through chloride cells in
teleosts likely involves the
Na+-K+-2Cl cotransporter, direct
evidence for the presence and localization of this protein in chloride
cells is conspicuously lacking. In addition, there is no information on
the regulation of this protein by environmental salinity or
developmental events.
The study of anadromous fish provides the opportunity to probe the effects of environmental salinity and the development of seawater tolerance on chloride cell-associated proteins. Anadromy is a life history strategy that includes a freshwater and marine phase. This strategy is exemplified by Atlantic salmon, which hatch and remain in freshwater for several years as parr before seaward migration. The time preceding and during migration is a critical developmental period called "smolting." Smolting includes a number of behavioral, morphological, and physiological changes that are preparatory for seawater entry (12). These changes are incomplete in juveniles before the period of downstream migration (presmolts) and are reversed in salmon that do not successfully reach the ocean (postsmolts).
Physiological changes occurring in the gill, kidney, gut, and bladder are responsible for increased salinity tolerance among smolts before seaward migration (30). Chloride cell size and density have been found to increase after the acclimation of Atlantic salmon to seawater and during smolting (18). Numerous studies have found increased gill Na+-K+-ATPase activity during smolting and seawater acclimation (21, 31, 37). These data suggest a direct relationship between increased gill Na+-K+-ATPase activity and increased hypoosmoregulatory ability of smolts.
If the Na+-K+-2Cl cotransporter
is present within the gills of Atlantic salmon and is important for
seawater tolerance, then it is likely that this protein would also be
upregulated during seawater acclimation and smolting. The current study
was designed to determine whether the
Na+-K+-2Cl cotransporter is
present in gill chloride cells of Atlantic salmon. SDS-PAGE and Western
blotting were used to characterize and quantify the gill
Na+-K+-2Cl cotransporter.
Immunocytochemistry was used to localize the
Na+-K+-2Cl cotransporter to cells
in the gill and to determine changes in immunoreactive cell number,
size, shape, and staining intensity. Changes in the quantity and
location of this protein during seawater acclimation and smolting were
determined using the T4 monoclonal antibody (23).
The results of these experiments provide direct evidence that the
Na+-K+-2Cl cotransporter is
present within gill chloride cells of teleost fish. The upregulation of
the Na+-K+-2Cl cotransporter
during seawater acclimation and smolting provides strong support for a
crucial role of this protein in the mechanism of ion secretion.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and experimental protocols.
To monitor the Na+-K+-2Cl
cotransporter in the gill during seawater acclimation, Atlantic salmon
parr were acclimated to a 1,100-liter flow-through circular tank with
temperatures held constant at 10 ± 0.5°C for 2 wk. On the basis
of their size (<12 cm fork length), these fish were not expected to
smolt. On January 31, 2000, one-half of the fish (n = 16) were moved into an identical tank in a closed recirculating system
maintained at 10 ± 0.5°C and 15 parts per thousand (ppt)
salinity. On February 14 and 28, the salinity was increased to 25 and
30 ppt, respectively. Freshwater controls (n = 16) and
seawater-acclimated fish (n = 16) were sampled on March
7, 2000. All fish were maintained on simulated natural photoperiod and
fed commercial Atlantic salmon diet (Zeigler Brothers) daily throughout
the study period.
cotransporter in the gill before, during, and after smolting, juvenile
Atlantic salmon were transferred in January 2000 into a 1,100-liter
flow-through circular tank with fresh water supplied from the
Connecticut River. These fish were expected to smolt based on their
size (>14 cm fork length). Fish were maintained under simulated
natural photoperiod and within 0.5°C of ambient river temperatures
and fed commercial salmon diet daily throughout the experimental
period. Juvenile fish were sampled on February 16, May 9, and June 29 (n = 8). Atlantic salmon parr (<12 cm fork length)
maintained under identical conditions were sampled on May 9 (n = 16) for reference. Seawater tolerance of juvenile
Atlantic salmon was determined by measuring their ability to regulate
plasma ions in 24-h seawater challenge tests. Twenty-four-hour seawater
challenges were conducted in a 1,100-liter closed recirculating
seawater system maintained at 35 ppt with temperatures ±1.0°C of
ambient river temperatures. Seawater challenges were performed on March
14 (n = 7) and May 7 (n = 7). Plasma
ion levels (Na+ and Cl) were measured using
an electrolyte analyzer (AVL Scientific).
At the time of sampling, all fish were anesthetized with 100 mg/l
MS-222 (pH 7.0, 12 mM NaHCO3), weighed to the nearest
0.1 g, and fork lengths were recorded. To measure gill
Na+-K+-ATPase activity, gill biopsies (5 or 6 gill filaments) were placed in 100 µl of ice-cold SEI (in mM: 250 sucrose, 10 Na2EDTA, and 50 imidazole) and stored at
80°C. A single gill arch from each fish was removed, plunged into
fixative (80% absolute methanol-20% dimethyl sulfoxide at 20°C)
and stored at 20°C for later use in immunocytochemistry. The
remaining gill tissue was removed, snap-frozen on dry ice, and stored
at 80°C to be later used for gel electrophoresis. For
seawater-challenged juvenile Atlantic salmon, blood was drawn from the
caudal blood vessels into a 1-ml ammonium heparinized syringe and
centrifuged at 8,000 g for 5 min at 4°C. Plasma was
separated and stored at 80°C.
Antibodies.
T4 monoclonal antibodies developed against the carboxy terminus of the
human colonic Na+-K+-2Cl
cotransporter were used as the primary antibody for the
Na+-K+-2Cl cotransport protein in
the Atlantic salmon gill. The T4 antibody, developed by Dr. Christian
Lytle and Dr. Bliss Forbush III, 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. This antibody was used at a concentration of 600 µg/ml for
Western blots and 300 µg/ml for immunocytochemistry.
-subunit (generously provided by Dr. Kazuhiro Ura, Ref.
41). This antibody was used at a dilution of 1:500 for immunocytochemistry.
For Western blotting, a peroxidase-labeled goat anti-mouse IgG, heavy
and light chain (H+L) was used as the secondary antibody (2 µg/ml; Kirkegaard & Perry Laboratories, Gaithersburg, MD). For
immunocytochemistry, secondary antibodies included fluorescein-labeled goat anti-rabbit IgG (H+L) and Cy3-labeled goat anti-mouse IgG, H+L
(2.5 µg/ml and 2 µg/ml, respectively; Kirkegaard & Perry
Laboratories) for the localization of
Na+-K+-ATPase and the
Na+-K+-2Cl cotransporter, respectively.
Gill Na+-K+-ATPase activity. Gill Na+-K+-ATPase activity was measured by the method of McCormick (27). Gill tissue taken from biopsies was thawed immediately before assay and homogenized in 125 µl of 0.1% sodium deoxycholate SEI buffer for 10-15 s. The resulting homogenate was centrifuged at 5,000 g for 30 s, and the supernatant was retained and assayed for Na+-K+-ATPase activity. Each sample of gill homogenate was plated in quadruplicates of 10 µl. Fifty microliters of salt solution (in mM: 50 imidazole, 189 NaCl, 10.5 MgCl2 × 6 H2O, and 42 KCl) and 150 µl of assay mixture (50 mM imidazole, 2 mM phosphoenolpyruvate, 0.16 mM nicotinamide adenine dinucleotide, 0.5 mM adenosine triphosphate, 3.3 U/ml lactic dehydrogenase, and 3.6 U/ml pyruvate kinase) were added to each well. Each sample was measured four times, twice with ouabain (0.5 mM) and twice without. The kinetic assay was read at a wavelength of 340 nm with a run time of 10 min and intervals of 10 s. The difference between the kinetic reading with and without ouabain is measured as ouabain-sensitive Na+-K+-ATPase activity. Protein concentration of the gill homogenate was determined using the bicinchoninic acid method (BCA Protein Kit, Pierce, Rockford, IL). Na+-K+-ATPase activity is expressed as micromoles ADP per milligram protein per hour.
SDS-PAGE and Western blotting.
SDS-PAGE and Western blotting were used to quantify the amounts of
Na+-K+-2Cl cotransporter present
in the gill. Frozen gill tissue was thawed, rinsed in ice-cold PBS (in
mM: 137 NaCl, 2.7 KCl, 4.3 Na2HPO4, 1.4 KH2PO4 adjusted to pH 7.3), and blotted on a
paper towel. Gill epithelia were cut away from the arches and placed in
10 vol of ice-cold homogenization buffer (2 mM EDTA and 30% sucrose wt/vol in PBS) along with the following protease inhibitors: 0.2 mM
[4-(2-aminoethyl)benzenesulfonylfluoride HCl], 100 µM
N-tosyl phenylalanine chloromethyl ketone, 1 µM
pepstatin A, 10 µM chymostatin, 10 µM leupeptin, and 50 µM
o-phenanthroline. Gill tissue for Atlantic salmon parr were
grouped in pairs to obtain enough protein for electrophoresis. Gill
tissue was homogenized at low speed using a tissue homogenizer (Tekmar;
SDT-182EN fitted with a saw tooth generator) and centrifuged at 5,000 g for 10 min at 4°C. The resulting supernatant was
centrifuged at 20,000 g for 10 min, and the pellet was
removed to discard mitochondria and cellular debris. The supernatant was centrifuged at 48,000 g for 2 h at 4°C. The final
pellet was resuspended in homogenization buffer, and total protein was
determined using the BCA protein assay. Enrichment of the basolateral
membrane, determined by measuring Na+-K+-ATPase
activity, was 15-fold higher than the crude homogenate.
-mercaptoethanol wt/vol, 0.05% bromophenyl blue wt/vol in deionized water adjusted to
pH 6.8) and heated to 60°C for 15 min. Membranes were loaded on 7%
SDS polyacrylamide gels at 50 µg of protein per lane. Gels were run
overnight followed by transfer to immobilon P [polyvinylidene fluoride
(PVDF)] transfer membranes (Millipore, Bedford, MA). PVDF
membranes were immersed for 1.5 h in blocking buffer (7.5% nonfat
dry milk and 0.1% Tween 20 wt/vol in PBS) at room temperature and were
incubated overnight at 4°C in T4 primary antibody diluted in blocking
buffer. PVDF membranes were washed five times in blocking buffer
followed by a 2-h incubation at room temperature in peroxidase-labeled secondary antibody in blocking buffer. PVDF membranes were washed four
times in blocking buffer, once in 0.1% Tween 20 (wt/vol) in PBS, and
once in deionized water. Immunoreactivity was visualized using DAB
buffer [1.38 mM 3,3'-diaminobenzidine tetrahydrochloride (DAB), 0.0084 mM CoCl2, and 0.001% H2O2 wt/vol
in PBS].
Digital photographs were taken immediately after incubation with DAB.
Band staining intensity was measured from the digital photographs using
Image calc (C. H. A. van de Lest, Dutch Asthma Foundation). To obtain staining intensity, an entire lane on a Western
blot is selected, and Image calc scans the selected part of the image
from top to bottom, averaging the 8-bit gray scale values that are
located on each horizontal line. The average 8-bit gray scale values on
each horizontal line are then summed to obtain cumulative 8-bit gray
scale values for each particular band. To standardize for differences
in background intensity between Western blots, the background 8-bit
gray scale value was subtracted from each horizontal line average 8-bit
gray scale value. Na+-K+-2Cl
cotransporter abundance, as measured by staining intensity, is recorded
as cumulative 8-bit gray scale.
Enzymatic deglycosylation. Enzymatic deglycosylation was performed on gill tissue from four smolts to determine the degree to which the protein isolated on Western blots was glycosylated. After isolation, plasma membranes were brought up in deglycosylation buffer (2 mM EDTA and 1% SDS wt/vol in PBS). Protein content was determined through the BCA method as described above. Enzymatic deglycosylation was accomplished by adding N-glycosidase-F (0.02 U/5 µg protein) to the samples and incubating at 20°C for 18 h. After deglycosylation, plasma membrane samples were electrophoresed on 7% polyacrylamide gels, blotted, and stained using the procedures mentioned previously.
Immunocytochemistry.
Immunocytochemical procedures were modified from Ginns et al.
(10). After fixation (80% absolute methanol-20% dimethyl
sulfoxide) at 20°C, tissue was placed on ice and allowed to warm
before being placed in Ringer at 4°C. The tissue was then
equilibrated in cryoprotectant (30% sucrose wt/vol in Ringer) before
being embedded in Histo Prep Embedding Media (Fisher Scientific).
Tissue was frozen (25°C), cryosectioned at 10 µm, placed on warm
poly-L-lysine-subbed slides, and washed twice with
high-salt Ringer (360 mM NaCl and 1% BSA wt/vol in Ringer). Sections
were washed three times in glycine wash (50 mM glycine and 1% BSA
wt/vol in Ringer) and incubated overnight in primary antibodies at
4°C (T4 and anti-Na+-K+-ATPase -subunit
diluted in 0.1% NaN2 and 1% BSA wt/vol in Ringer). Both
primary antibodies were used together during colocalization studies.
After incubation with primary antibodies, tissue was washed five times
in high-salt Ringer, incubated for 2 h at 4°C in secondary
antibodies, and washed twice in Ringer before viewing. All washes were
10 min in length. Controls omitting the primary antibodies were
performed and yielded no immunoreactivity. For colocalization studies,
controls consisted of omitting one of each of the secondary antibodies,
and no cross-reactivity was detected.
cotransporter
immunoreactive chloride cells on the primary filament and secondary
lamellae were tallied separately. From each fish, immunoreactive
chloride cells were counted from 10 sagittal sections of gill filament
and expressed per millimeter of primary filament. Mean numbers of
primary and secondary immunoreactive chloride cells for each group were
obtained using the means calculated from each fish. Due to the uneven
distribution of chloride cells in the gill, an entire piece of gill
arch was sectioned, and images were randomly selected from 10 of the
resulting sagittal sections. Cell area (µm2/cell), shape
factor, and staining intensity were also obtained from primary and
secondary immunoreactive chloride cells. Shape factor is defined as
4
A/P2 (with A and P being area and perimeter,
respectively), with values close to one indicating a round shape and
less than one a more elongated shape. Immunoreactive cell staining
intensity is measured as average 8-bit gray scale. To standardize for
differences in background staining intensity among images, background
8-bit gray scale was subtracted from the average 8-bit gray scale
values from each immunoreactive cell. Fifty primary and fifty secondary immunoreactive chloride cells were analyzed from a minimum of five
sagittal sections of gill filament from each fish. Fewer immunoreactive
chloride cells on the secondary lamellae were analyzed from
seawater-acclimated parr because fewer cells were present on the
secondary lamellae. Means for each group were calculated using the
means from individual fish. Cell number, size, shape factor, and
staining intensities were obtained using MetaMorph 4.1.2 (Universal
Imaging 1992-2000).
Data analysis.
For salinity acclimation, data were analyzed using the Student's
t-test or Mann-Whitney U test when appropriate.
The Mann-Whitney U test was also used to compare plasma ion
levels (Na+ and Cl) in seawater-challenged
juvenile Atlantic salmon in March and May. Analyses of the effects of
smolting were conducted using a one-way analysis of variance or the
Kruskal-Wallis test on ranks when the assumption of normality was not
attained. Multiple comparisons were made using Tukey's honestly
significant difference test. To establish the relationship
between gill Na+-K+-2Cl
cotransporter abundance and Na+-K+-ATPase
activity, a simple linear regression was performed. All statistical
analyses were deemed significant at the = 0.05 level and
conducted using Sigma Stat 2.0 (Jandel).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Characterization and localization of the gill
Na+-K+-2Cl
cotransporter.
Western blots of Atlantic salmon gill tissue membrane preparations
revealed three broadly stained bands with molecular masses centered at
285, 160, and 120 kDa when probed with the T4 anti- Na+-K+-2Cl cotransporter antibody
(Fig. 1). On enzymatic deglycosylation, all three bands shifted downward with final masses of 230, 127, and 93 kDa. When staining intensity was low on Western blots, only the upper
band was visible.
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cotransporter was
localized primarily to large, columnar cells on the primary gill
filament of seawater-acclimated Atlantic salmon parr, and
Na+-K+-ATPase was immunolocalized to these same
cells (Fig. 2A). On the basis
of size, location, and colocalization with
Na+-K+-ATPase,
Na+-K+-2Cl
cotransporter immunoreactivity occurred in chloride cells. In freshwater-acclimated parr, the
Na+-K+-2Cl cotransporter was also
localized to chloride cells along with Na+-K+-ATPase. There were, however, chloride
cells only immunoreactive for Na+-K+-ATPase
present on the primary filament and secondary lamellae. No detectable
staining occurred in the other major cell types of the gill epithelia
(pavement, pillar, and mucous cells) or in red blood cells or
cartilage. Immunoreactive staining appeared to be distributed evenly
throughout chloride cells, except for the absence of staining in
nuclei.
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Salinity acclimation.
Gill Na+-K+-2Cl cotransporter
abundance, as measured by cumulative 8-bit gray scale from all three
bands on Western blots, increased 2.5-fold in parr acclimated to
seawater (Fig. 3A). The same
molecular mass bands (285, 160, and 120 kDa) were observed in gill
tissue from freshwater- and seawater-acclimated salmon (Fig. 1). Gill Na+-K+-ATPase activity increased 2.3-fold in
seawater-acclimated parr (Fig. 3A).
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cotransporter
immunoreactive chloride cells on the primary filament increased
3.6-fold in number, whereas cells on the secondary lamellae decreased
by a factor of 2.5 (Fig. 3B). The size of immunoreactive
chloride cells on the primary filament and secondary lamellae increased
approximately twofold after seawater transfer (Table
1). There were no differences in the
shape, as measured by shape factors, or staining intensities of primary
and secondary immunoreactive chloride cells among freshwater- and
seawater-acclimated parr.
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Smolting.
Seawater-challenged juvenile Atlantic salmon in May had significantly
lower (n = 7, P < 0.05, Mann-Whitney
U test) plasma Na+ (180.6 ± 3.4 mM) and
Cl (160.9 ± 3.7 mM) levels than fish sampled in
March (Na+, 194.6 ± 3.8 mM; Cl,
178.5 ± 4.8 mM), indicating higher salinity tolerance among fish
in May. In May, high levels of gill
Na+,K+-ATPase activity (12.1 ± 0.42 µmol ADP · mg
protein1 · h1), the appearance of
silvering and darkened fin margins, and higher salinity tolerance
confirmed that the fish were smolts.
cotransporter
abundance, as measured by cumulative 8-bit gray scale from all three
bands on Western blots, was low in presmolts (February), increased
3.3-fold in smolts (May), and declined 85.8% in postsmolts by June
(Fig. 4A). Gill
Na+-K+-2Cl cotransporter
abundance of parr sampled in May was 77.5% less than in smolts.
Changes in gill Na+-K+-2Cl
cotransporter abundance paralleled changes observed in gill
Na+-K+-ATPase activity. Linear regression
analysis of Na+-K+-2Cl
cotransporter abundance against Na+-K+-ATPase
activity indicated a significant and positive correlation (n = 32, P < 0.001, r2 = 0.86) between the relative abundance
of both proteins. Gill Na+-K+-ATPase activity
was low in presmolts, increased more than twofold in smolts, and
declined in postsmolts. Gill Na+-K+-ATPase
activity of parr in May was 41% of the activity in smolts.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This is the first published study to identify, characterize, and
localize the Na+-K+-2Cl
cotransporter in the gill of any teleost fish. The T4 antibody has been
shown to recognize both the absorptive and secretory isoforms of the
cotransporter from 23 different cell types among a wide variety of
species (23). This includes rectal gland tissue from one
elasmobranch fish, the spiny dogfish (S. acanthias). With
the use of the T4 antibody and Western blots in this study, Atlantic
salmon gill contained three bands with molecular masses centered at
285, 160, and 120 kDa (Fig. 1). The
Na+-K+-2Cl cotransporter has been
shown to exhibit a wide variety of sizes depending on the species
and/or tissue: 195 kDa in the shark rectal gland (22), 180 kDa in the kidney of winter flounder, P. americanus (39), and 164 kDa in the human colon (23). In
the gills of killifish (F. heteroclitus), the
Na+-K+-2Cl cotransporter is
~190 kDa large, and between 210 and 260 kDa in the gills of rainbow
trout, Onchorynchus mykiss (B. Forbush III, personal
communication). Similar sizes for the upper band isolated on Western
blots in this study and for the
Na+-K+-2Cl cotransporter in trout
(a familial relative) further validate the isolation of the Atlantic
salmon gill Na+-K+-2Cl cotransporter.
Studies characterizing the
Na+-K+-2Cl cotransporter on
Western blots have reported enzymatic deglycosylation of the
protein (22, 23, 32). These studies identify a shift of
20-60 kDa on deglycosylation depending on the tissue source. In
this study, the three bands shifted down to 230, 127, and 93 kDa,
respectively, when deglycosylated. This indicates a similar pattern of
glycosylation for the protein isolated on Western blots in this study
and provides additional evidence that the isolated protein is the
Na+-K+-2Cl cotransporter. The
band at 285 kDa likely represents the glycosylated form, and the band
at 230 kDa represents the core mass of the cotransporter. The lower
molecular mass bands (160 and 120 kDa) likely represent degradation
products. Three bands (180, 110-70, and 50 kDa)
corresponding to the Na+-K+-2Cl
cotransporter in winter flounder intestine have previously been reported (39). The large amount of degradation present on
Western blots may be due to the protein being in different stages of
glycosylation. An alternative explanation, however, is that the
lipophilic nature of the
Na+-K+-2Cl cotransporter may
affect its migration through SDS-PAGE gels.
The Na+-K+-2Cl cotransporter was
localized specifically to chloride cells and was present at low or
nondetectable levels in other cell types in the gill (Fig.
2A). Positively stained cells were identified as chloride
cells on the basis of their colocalization with
Na+-K+- ATPase, size, morphology, and
location within the gill. The current model of seawater chloride cells
includes both the Na+-K+-2Cl
cotransporter and Na+-K+- ATPase on the
basolateral surface of the cell. Except for the nucleus,
Na+-K+-2Cl cotransporter
immunoreactivity occurred throughout chloride cells, and
immunoreactivity was similar to that exhibited by
Na+-K+-ATPase. Previous work using
[3H]ouabain has localized
Na+-K+-ATPase to the tubular system of chloride
cells (17). Although the tubular system spreads throughout
the chloride cell, it is contiguous with the basolateral surface. A
similar even distribution of
Na+-K+-ATPase-specific anthroylouabain staining
in chloride cells of the tilapia (Oreochromis mossambicus)
and the long-jawed mudsucker (Gillichthys mirabilis) has
been described (26). Similar immunocytochemical staining
of the Na+-K+-2Cl cotransporter
and Na+-K+-ATPase in this study suggests that
the cotransporter is also present on the basolateral surface of
chloride cells. Further research at the electron microscopic level will
be necessary to confirm this.
The upregulation of the
Na+-K+-2Cl cotransporter during
seawater acclimation and the localization of this transport protein to
chloride cells is further evidence for the role of this protein in ion
secretion by the gill. Seawater acclimation of Atlantic salmon parr
increased gill Na+-K+-2Cl
cotransporter abundance and Na+-K+-ATPase
activity (Fig. 3A). Similarly, the number (Fig.
3B) and size (Table 1) of
Na+-K+-2Cl cotransporter
immunoreactive chloride cells on the primary filament, and the size of
immunoreactive chloride cells on the secondary lamellae increased
significantly after seawater transfer. Although this is the first study
to quantify Na+-K+-2Cl
cotransport protein in the gill, other studies in a large number of
teleosts have demonstrated increases in gill
Na+-K+-ATPase activity after seawater transfer
(see reviews, Refs. 7, 15, 24,
28). Gill Na+-K+-2Cl cotransporter
abundance increased 2.5-fold after seawater acclimation, and gill
Na+,K+-ATPase activity experienced a similar
2.5-fold increase (Fig. 3A). The strong correlation
(P < 0.001, r2 = 0.86)
between the gill Na+-K+-2Cl
cotransporter and Na+-K+-ATPase suggests a
possible mechanistic link between these two transport proteins. A
similar association between the abundance of
Na+-K+-2Cl cotransporter and
Na+-K+-ATPase has been shown in other
ion-transporting tissues (23). Because the cotransport of
sodium and chloride across the basolateral surface of seawater chloride
cells is dependent on the sodium gradient created by
Na+-K+-ATPase, parallel regulation of these
proteins during seawater acclimation is not surprising.
Gill Na+-K+-2Cl cotransporter
abundance increased during the developmental process of smolting,
coinciding with increased gill Na+-K+-ATPase
activity and increased salinity tolerance (Fig. 4A). Other studies examining smolting in salmonids have reported preparatory changes in the osmoregulatory physiology of salmonids in the spring associated with increased salinity tolerance. These preparatory changes
include increased gill Na+-K+-ATPase activity
(21, 38) and chloride cell number and size (18). In this study, smolts had increased numbers of
Na+-K+-2Cl cotransporter
immunoreactive chloride cells on the primary filament (Fig.
4B) and enlarged immunoreactive chloride cells on the
primary filament and secondary lamellae (Table 2). The number of
immunoreactive chloride cells on the secondary lamellae did not change
throughout the developmental period of smolting. The increased levels
of Na+-K+-2Cl cotransporter,
Na+-K+-ATPase, and numbers of chloride cells in
Atlantic salmon gills are likely to be underlying causes of the
increased seawater tolerance observed during smolting.
Na+-K+-2Cl cotransporter
immunoreactive chloride cells on the primary filament of parr were more
round than immunoreactive chloride cells of presmolts, smolts, and
postsmolts (Table 2). It has been observed that -chloride cells,
which are more prevalent in freshwater parr than smolts, are more
rounded than seawater -chloride cells (36). The
staining intensities of
Na+-K+-2Cl cotransporter
immunoreactive chloride cells (primary and secondary) were elevated in
presmolts and smolts, indicating more protein per cross-sectional area.
After smolting, gill Na+-K+-2Cl
cotransporter abundance and
Na+-K+-2Cl cotransporter
immunoreactive chloride cell number (primary filament), size (secondary
lamellae), and staining intensities (primary filament and secondary
lamellae) were reduced to levels that were comparable to those seen in
parr (May). Many physiological changes associated with smolting,
including increased gill Na+-K+-ATPase activity
are lost after smolting, resulting in a loss of hypoosmoregulatory
ability in postsmolts (29).
Although there is strong evidence for the role of the
Na+-K+-2Cl cotransporter in ion
secretion, the presence of this protein in gill chloride cells of
freshwater-acclimated fish is less easily explained (Fig. 1). Atlantic
salmon parr can tolerate gradual increases in environmental salinity
(3). The presence of the Na+-K+-2Cl cotransporter may
reflect the euryhaline life history of Atlantic salmon and indicate a
moderate level of readiness for seawater entry. The concentration of
the Na+-K+-2Cl cotransport
protein in chloride cells may also serve a physiological function in
freshwater. Freshwater chloride cells have been implicated in ion
uptake, calcium uptake, and acid-base metabolism (see Ref. 35). However, the most widely accepted models for these
functions include Na+-K+-ATPase but not the
Na+-K+-2Cl cotransporter. The
presence of Na+-K+-2Cl
cotransporter immunoreactivity in some but not all chloride cells exhibiting Na+-K+-ATPase immunoreactivity in
freshwater-acclimated parr (Fig. 2A) suggests that the
cotransporter plays a less critical role than Na+-K+-ATPase in ion absorption. Rather than a
direct role, the greater concentration of the
Na+-K+-2Cl cotransporter in
chloride cells in freshwater may reflect the cell's increased demand
for volume regulation as an active ion transport cell.
Chloride cells on the primary filament increase in size and number
after seawater transfer of most teleost fish (see reviews, 19, 20, 28).
An increase in the number (Table 1) and size (Fig. 3B) of
immunoreactive chloride cells on the primary filament was observed in
this study, whereas chloride cells on the secondary lamellae declined
in number. It has been demonstrated that chloride cells on the
secondary lamellae of chum salmon (Oncorhynchus
keta) fry are greatly reduced after seawater transfer
(40), whereas they are totally absent in
seawater-acclimated Atlantic salmon smolts (36). Chloride
cells on the secondary lamellae proliferate during exposure to ion-poor
water (1). These data suggest that chloride cells on the
primary filament are involved in ion secretion, whereas chloride cells
on the secondary lamellae take up ions. Because the current model of
the freshwater chloride cell lacks an
Na+-K+-2Cl cotransporter, if
chloride cells on the secondary lamellae are solely involved in ion
uptake, we would expect to see low levels of the
Na+-K+-2Cl cotransporter in
secondary lamellar chloride cells. This in fact was not the case,
because chloride cells on the primary filament and secondary lamellae
were stained with similar intensity (Tables 1 and 2). Furthermore, in
seawater-acclimated parr, immunoreactive chloride cells on the primary
filament (116.5 ± 5.8 µm2) and secondary lamellae
(102.8 ± 13.7 µm2) were similar in size. The
present study, therefore, does not provide evidence that there are two
functionally distinct chloride cells on the primary filament and
secondary lamellae. It is possible that in Atlantic salmon there is a
single chloride cell type that provides an absorptive or secretory role
(bifunctional) depending on development and the salinity of the
external environment. By tracking in vivo sequential changes in
individual chloride cells it was recently shown that individual
chloride cells in the yolk sac membrane of tilapia (O. mossambicus) embryos and larvae increase in size after freshwater
to seawater transfer (11), suggesting that individual
cells can change from ion uptake to ion secretion, and are therefore bifunctional.
The current study demonstrates that the
Na+-K+-2Cl cotransporter is
present in gill chloride cells of Atlantic salmon and is upregulated
after seawater acclimation and during the developmental process of
smolting. Changes in gill Na+-K+-ATPase were
parallel to changes in the
Na+-K+-2Cl cotransporter,
suggesting a mechanistic link between the proteins in ion secretion by
gill chloride cells. These observations provide the first direct
support for the inclusion of the
Na+-K+-2Cl cotransporter in the
current models of chloride cell ion secretion by the gills of teleost fish.
| |
ACKNOWLEDGEMENTS |
|---|
We thank J. Kunkel for methodological assistance and Dr. K. Ura for generously providing the antibody against Na+-K+-ATPase. G. B. Zydlewski made many helpful comments in review of the manuscript.
| |
FOOTNOTES |
|---|
Present addresses: J. Zydlewski, Abernathy Fish Technology Center, US Fish and Wildlife Service, Longview, WA 98632; R. Pelis, University of Connecticut, BBS#4, Rm. 021, Physiology and Neurobiology, U-4156, 3107 Horsebarn Hill Rd., Storrs, CT 06269-4156.
Address for reprint requests and other correspondence: R. M. Pelis, Univ. of Connecticut, Physiology and Neurobiology, U-4156, BBS #4, Rm. 021, 3107 Horsebarn Hill Rd., Storrs, CT 06269-4156 (E-mail: ryan.pelis{at}.uconn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 October 2000; accepted in final form 24 January 2001.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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; n = 16, means ± SE) in FW- and
SW-acclimated Atlantic salmon parr (A). *Significantly
different, P < 0.05. Student's t-test and
Mann-Whitney U test for
Na+-K+-2Cl