Influence of salinity on the localization of Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and CFTR anion channel in chloride cells of the Hawaiian goby (Stenogobius hawaiiensis)
1 USGS, Leetown Science Center, Conte Anadromous Fish Research Center,
Turners Falls, MA 01370, USA
2 Department of Biology, University of Massachusetts, Amherst, MA 01003,
USA
3 Fish Endocrinology Laboratory, Department of Zoology/Zoophysiology,
Göteborg University, Box 463, S405 30 Göteborg, Sweden
4 Marine Biology Program, Florida International University, 3000 NE 151st
St, North Miami, FL 33181, USA
* Author for correspondence (e-mail: stephen_mccormick{at}usgs.gov)
Accepted 9 September 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Na+/K+-ATPase, Na+/K+/2Cl- cotransporter, NKCC, CFTR, teleost fish, chloride secretion, salinity, Stenogobius hawaiiensis, goby
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To date, no single study has examined the influence of salinity on all three of the major transporters thought to be involved in chloride secretion. Based on the current model of chloride secretion in fish, we hypothesize that chloride cells should contain high levels of Na+/K+-ATPase, NKCC and CFTR and that all three are potentially upregulated and their localization altered by exposure to increased salinity. In the present study, we have examined the influence of salinity on chloride cells and the immunolocalization of the Na+/K+-ATPase, NKCC and CFTR in the gills of the euryhaline Hawaiian goby (Stenogobius hawaiiensis).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
After 10 days at the acclimation salinity, fish were anesthetized with 200 mg l-1 MS-222, length and weight were recorded and the gill tissue was removed. Fish weighed between 0.4 g and 2.5 g, and mass did not differ significantly among groups. The branchial bone was trimmed away from two gill arches, and the primary filaments were placed in SEI buffer (150 mmol l-1 sucrose, 10 mmol l-1 Na2EDTA, 50 mmol l-1 imidazole, pH 7.3) and frozen on dry ice for analysis of Na+/K+-ATPase activity. Two gill arches were placed in 4% buffered formalin for two days and then this solution was replaced with 70% ethanol.
Antibodies
Rabbit polyclonal antisera directed against 17 amino acids from a highly
conserved region of the -subunit of salmon
Na+/K+-ATPase (Ura
et al., 1996
) and diluted 1:500 was used for immunocytochemical
detection of Na+/K+-ATPase. A mouse monoclonal antibody
(T4) directed against the 310 amino acids at the carboxyl terminus of the
human colonic Na+/K+/2Cl- cotransporter
(NKCC1; obtained from the Developmental Studies Hybridoma Bank developed under
the auspices of the NICHD and maintained by the University of Iowa, Department
of Biological Sciences, Iowa City, IA, USA) was used at a concentration of
0.25 µg ml-1. This antibody has been shown to be specifically
immunoreactive with NKCC (both secretory and absorptive forms) from many
vertebrates, including teleost fish (Lytle
et al., 1992
; Pelis et al.,
2001
). A mouse monoclonal antibody (24:1; R&D Systems, Boston,
MA, USA) against 104 amino acids at the carboxyl terminus of the human CFTR
was used at 0.4 µg ml-1. The carboxyl terminus of CFTR is highly
conserved among vertebrates, and this antibody has previously been shown to be
specifically immunoreactive with CFTR from several vertebrates, including
teleost fish. Alexa-Fluor 488 goat anti-mouse and Alexa Fluor 546 goat
anti-rabbit (Molecular Probes, Eugene, OR, USA) were used as secondary
antibodies. Antibody control experiments (primary antibodies without secondary
antibody, and secondary antibody without primary antibody) showed no specific
staining and low background. Double staining of the same sections was
performed for Na+/K+-ATPase and NKCC, and
Na+/K+-ATPase and CFTR.
Immunocytochemistry
Fixed gill tissue was rinsed in 10 mmol l-1 phosphate-buffered
saline (PBS), placed in PBS with 30% (w/v) sucrose for one hour and then
frozen in embedding medium. 7 µm sections were cut in a cryostat at
-24°C, parallel to the long axis of primary filaments and perpendicular to
the attachment of secondary lamellae. The tissue was placed on
poly-L-lysine-coated slides, dried, rinsed with PBS and then
incubated in 2% normal goat serum in PBS for 0.5 h at room temperature. Slides
were exposed to primary antibody in antibody dilution buffer (0.01%
NaN3, 0.1% bovine serum albumin, 2% normal goat serum and 0.02%
keyhole limpet hemocyanin in PBS) and incubated overnight at 4°C. After
incubation, the slides were rinsed several times with PBS, exposed to
secondary antibody at room temperature for 2 h and then rinsed several times
with PBS. The tissue was covered by a cover slip and examined with a Nikon
inverted fluorescent microscope with a mercury lamp. Images were taken within
4 h of completion of staining for subsequent counting and morphometric
analysis.
From each fish, immunoreactive chloride cells on the primary filament and secondary lamellae (tallied separately) were counted from sagittal sections of gill filament (300 µm of primary filament/sagittal section) and expressed per millimeter of primary filament. As in most teleost fish, there was an increasing number of chloride cells from leading to trailing edge, so only sections in the middle of the filament were used to quantify cell number. Mean numbers of chloride cells for each group were obtained using the means calculated from each fish. Cell or staining area (µm2 cell-1), staining intensity (mean gray scale/pixel) and shape factor were also obtained from immunoreactive chloride cells using MetaMorph 4.1.2 (Universal Imaging Corporation, West Chester, PA, USA). A single threshold level for each image and antibody was used to quantify immunoreactive regions. Background staining intensity was obtained by averaging intensity in at least two noncellular regions of each image and subtracting that from each staining intensity value obtained from that image. In order to determine whether the shape of chloride cells and their immunoreactive regions was affected by salinity, shape factor was measured. Shape factor is defined as 4xA/p2 (where A and p are the area and perimeter of the immunopositive regions, respectively), with values close to 1 indicating a circular shape and 0 an elongate shape. Since the NKCC and Na/K-ATPase were measuring the same region of the cell, only the shape factor for Na/K-ATPase is presented. At least 50 immunoreactive chloride cells from several different tissue sections were analyzed from at least five fish from each salinity; more than 1000 chloride cells were examined overall.
Measurement of gill Na+/K+-ATPase
Within one month of sampling, gill Na+/K+-ATPase
activity was measured according to the microassay protocol of McCormick
(1993). Gill filaments were
homogenized in SEI buffer containing 0.1% sodium deoxycholate. Following
centrifugation (3000 g for 0.5 min) to remove large debris,
Na+/K+-ATPase activity was determined by linking ATP
hydrolysis to the oxidation of nicotinamide adenine dinucleotide (NADH),
measured at 340 nm for 10 min at 25°C in the presence or absence of 0.5
mmol l-1 ouabain. Protein content in the gill homogenate was
measured using a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL,
USA). Specific activities were expressed as µmol ADP mg-1
protein h-1.
Statistics
One-way analysis of variance (ANOVA) followed by
StudentNewmanKeuls post-hoc test was used to test for
the effect of salinity on cell number and gill
Na+/K+-ATPase activity. As multiple chloride cells were
measured from each individual, measures of cell area, brightness and shape
factor were analyzed with ANOVA with `individual' nested (contained) within
`salinity'. P<0.05 was used to reject the null hypothesis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Large columnar cells on the primary filament and somewhat smaller cells on
the secondary lamellae were positively stained for
Na+/K+-ATPase (Fig.
1). Their size, shape and location indicated that these were
mitochondrion-rich chloride cells. No other cell types in the gill were
stained above background levels, and antibody controls (lacking primary
antibody) showed only background staining. Na+/K+-ATPase
immunoreactivity was detectable throughout the chloride cell except for the
nucleus and the most apical region of the cell (Figs
1,
2), consistent with a
basolateral/tubular distribution. This pattern of staining was not altered by
salinity. Fish in all salinities had
Na+/K+-ATPase-positive chloride cells on both the
primary filament and secondary lamellae, with most of the cells located on the
primary filament (Figs 1,
3). The number of
Na+/K+-ATPase immunoreactive chloride cells on the
primary filament increased with increasing salinity and was 42% greater in
30 seawater than in freshwater. Chloride cells on the primary filament
were larger than those on the secondary lamellae, and their size increased
slightly but significantly in response to increased salinity (1327%;
Fig. 3). Chloride cells on the
secondary lamellae were more flattened (less round with a lower shape factor;
Table 1) than those on the
primary filament. Shape of chloride cells on the primary filament was not
altered by salinity, whereas chloride cells on the secondary lamellae were
less round (lower shape factor) in 20
and 30
than in
freshwater. The brightness of Na+/K+-ATPase
immunoreactivity was similar in chloride cells in the primary filament and
secondary lamellae and decreased slightly (7.09.4%) in response to
salinity.
|
|
|
The pattern of immunoreactivity for NKCC was nearly identical to that of Na+/K+-ATPase (Fig. 1). All cells that stained for Na+/K+-ATPase also stained for NKCC, and the distribution of NKCC staining within the chloride cells (staining throughout the cell except for the nucleus and most apical region) was the same as for Na+/K+-ATPase immunoreactivity. NKCC staining intensity was slightly higher in chloride cells on the primary filament compared with the secondary lamellae (Table 1). NKCC staining was not significantly affected by salinity in chloride cells on the primary filament but increased with increasing salinity in chloride cells on the secondary lamellae.
The pattern of CFTR staining differed substantially from that of
Na+/K+-ATPase and NKCC. CFTR staining was only present
in cells that were Na+/K+-ATPase positive (i.e. chloride
cells; Figs 2,
4). The distribution of
staining was apical and slightly subapical with a relatively small area of
overlap with Na+/K+-ATPase immunoreactivity. In
freshwater, CFTR immunoreactivity was not very bright and spread over a broad
apical surface. In 20 and 30
salinity, CFTR was more often in
a narrower apical region of the cell and extended deeper into the cell. In
seawater, the brightest staining was usually in the most apical region of the
chloride cell with a less bright subapical region
(Fig. 2). In many chloride
cells of seawater-exposed fish, there was a clear discontinuous, punctate
distribution of CFTR at the apical surface
(Fig. 2),although this
distribution was rarely seen in cells of freshwater fish. In some chloride
cells of seawater-exposed fish, there was a central area of the apical region
that was not stained, indicating that the apical crypt had been
cross-sectioned in these cells and that staining within the non-cellular
portion of the apical crypt was absent.
|
The number of CFTR-positive cells was much greater in the 20 and
the 30
groups than in the freshwater group (4- and 5-fold more,
respectively; Fig. 3). The
relative proportions of chloride cells with CFTR-positive staining were 19%,
62% and 67% in freshwater, 20
and 30
, respectively. The area
of the chloride cells staining positively for CFTR was about one-eighth of
that stained by Na+/K+-ATPase (610
µm2 versus 5080 µm2). In chloride
cells on the primary filament, the CFTR positive area was 62% and 53% greater
in fish in 20
and 30
seawater, respectively, relative to
gobies in freshwater. The mean CFTR staining intensity also increased
significantly after seawater exposure in chloride cells on both the primary
filament and the secondary lamellae.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The intracellular distribution of both Na+/K+-ATPase
and NKCC immunoreactivity in Hawaiian goby gills was identical, staining
throughout the cell except for the nucleus and the most apical region of the
chloride cell. Previous studies using electron microscopy have demonstrated
that Na+/K+-ATPase is present on both the basolateral
membrane and the extensive tubular system that extends through most of the
cell but that is not present in the most apical region of the cell
(Karnaky et al., 1976;
Wilson et al., 2000
). Thus,
the presence of high levels of both Na+/K+-ATPase and
NKCC found within chloride cells in the present study is likely to represent a
basolateral/tubular distribution. Marshall et al.
(2002
) recently found that
NKCC immunoreactivity occurs throughout the chloride cell in
seawater-acclimated killifish but has a more restricted and asymmetric
basolateral distribution in freshwater-acclimated killifish. As in the present
study, Pelis et al. (2001
)
found that NKCC immunoreactivity occurred throughout the chloride cell in both
freshwater- and seawater-acclimated Atlantic salmon and that the number of
cells staining and the amount of NKCC present in the gill was lower in
freshwater. For the few species examined to date, seawater teleosts seem to
have a consistent basolateral/tubular distribution and high levels of NKCC and
Na+/K+-ATPase present in chloride cells.
CFTR was present at detectable levels only in chloride cells and had a
clear and consistent apical distribution. In freshwater, this distribution was
spread over a relatively broad area of the apical surface. Previous studies
have shown that chloride cells in most freshwater teleosts have a broad apical
surface (Perry, 1997), and the
observed CFTR immunoreactivity in freshwater may reflect the presence of CFTR
over just such a broad apical membrane. In seawater, the orientation of CFTR
immunoreactivity was more often perpendicular to the main axis of the cell,
indicative of staining along a deep apical pit that is characteristic of
seawater chloride cells. There was also a more intense, often punctate
staining in the most apical region, with a less intense subapical region. The
punctate distribution of CFTR may be a function of the structure of the apical
pit. The apical pit is formed primarily by the chloride cell but is
interdigitated by the neighboring accessory cell
(Shiraishi et al., 1997
). This
results in a discontinuity of the chloride cell at the apical surface, and the
presence of CFTR only in chloride cells would explain the punctate
distribution that we observed.
Not all Na+/K+-ATPase-positive cells had positive
CFTR staining, and this may have been due, in part, to the greater area of the
Na+/K+-ATPase-rich cell body relative to the more narrow
apical region, making it inherently less likely that the apical region would
be sectioned. However, the large number of cells sampled in this study makes
it very unlikely that the treatment differences observed were the result of
this limitation caused by sectioning. It seems likely that the observed
differences in the relative number of CFTR-positive chloride cells relates
directly to the greater chloride secretory activity occurring in 20
and 30
salinity. The lower number of chloride cells with apical
staining for CFTR in freshwater may also relate to limits of detectability;
there may in fact be low staining present in the apical region of freshwater
chloride cells that was not detected using the present methods. It is
nonetheless clear that the intensity of staining for CFTR and the size of the
CFTR-positive apical region were greatly increased by exposure to seawater.
During seawater acclimation of killifish, Marshall et al.
(2002
) observed a
`redistribution' of CFTR from the subapical to the apical region of chloride
cells. In the rectal gland of spiny dogfish (Squalus acanthias), peak
CFTR immunofluorescence moved from the subapical to the apical region in
response to vasoactive intestinal peptide, which also caused increased
chloride secretion (Lehrich et al.,
1998
). Ernst et al.
(1994
) found more CFTR
immunoreactivity in the apical region of the salt gland of ducks (Anas
platyrhynchos) after salt loading. These results suggest that an
increased amount or redistribution of CFTR to the apical surface is associated
with increased chloride secretory activity and is consistent with the present
results. Because the size, shape and intensity of staining all differed among
the freshwater- and seawater-acclimated groups, it was not possible to
determine whether a `redistribution' of CFTR had occurred or whether the
increase in CFTR immunofluorescence was simply due to the presence of more
CFTR. More detailed studies will be necessary to examine the intracellular
distribution and de novo synthesis of CFTR in this and other teleosts
during seawater acclimation.
Increased salinity resulted in many more chloride cells showing positive
CFTR staining, and the area and intensity of staining also increased. Based on
a 5-fold increase in cell number, a 53% increase in cell area (which roughly
translates to a doubling of the cell volume) and a 29% increase in staining
intensity, we can roughly calculate that a 13-fold increase in CFTR occurred
in the primary gill filament after seawater acclimation. The calculated
increase in CFTR on the secondary lamellae was 4.4-fold, and combining the two
yields an overall estimated 10-fold increase in CFTR for the entire gill. This
is likely to be a minimal estimate for change, as the staining intensity is
only semiquantitative and could substantially underestimate the change in
amount of CFTR. It is clear from these results, however, that CFTR is
upregulated during seawater acclimation, and this provides strong
circumstantial evidence that CFTR is involved in chloride secretion carried
out by chloride cells. Gill CFTR mRNA levels increase within 8 h of seawater
exposure in killifish (Marshall et al.,
1999). Two forms of CFTR mRNA have been found in Atlantic salmon,
both of which increase after seawater exposure; CFTRII increases transiently,
and CFTRI shows a more sustained increase
(Singer et al., 2002
). It will
be of interest to examine the time course of changes in response to salinity
to determine how quickly changes in CFTR message and protein can occur in the
Hawaiian goby and other teleosts and how it relates to temporal changes in
gill morphology and ion fluxes.
Based on differences in the number, size and staining intensity of
Na+/K+-ATPase immunoreactivity in freshwater- and
seawater-acclimated fish (similar to the calculations above for CFTR), we can
calculate that there was a 46% increase in the amount of
Na+/K+-ATPase in gill tissue following seawater
acclimation. Gill Na+/K+-ATPase activity of Hawaiian
gobies increased by 24% following seawater acclimation, although there was no
statistically significant difference between freshwater fish and those exposed
to 30 salinity. We consider the measurement of activity to be more
quantitative than this rough approximation based on immunoreactivity, but the
two values are nonetheless similar. In many teleost species, gill
Na+/K+-ATPase increases several fold following seawater
acclimation (McCormick, 1995
).
This is not universally true, however, and there are many species in which
Na+/K+-ATPase does not change or is lower following
seawater acclimation, often in species of marine ancestry. The relatively
moderate change in number of chloride cells and the lack of significant change
in gill Na+/K+-ATPase activity in response to seawater
is consistent with the marine ancestry of amphidromous gobies in which
occurrence in freshwater is considered to be a derived trait
(Chubb et al., 1998
).
The Na+/K+/2Cl- cotransporter occurs in
two major isoforms: a secretory isoform (NKCC1) and an absorptive isoform
(NKCC2). The antibody used in the present study recognizes both of these
isoforms in a wide variety of vertebrates. It is likely that the NKCC
immunoreactivity in chloride cells of the Hawaiian goby is the secretory
isoform. In most tissues, the secretory form has been found only on the
basolateral membrane and tubular systems of epithelial cells, whereas the
absorptive form is found only on the apical membrane. The only exception to
this general rule is the choroid plexus, where both NKCC1 and
Na+/K+-ATPase are found on the apical membrane
(Haas and Forbush, 2000). The
present finding of NKCC immunoreactivity throughout the chloride cells
indicates a basolateral/tubular distribution and, by analogy, suggests that
this is the secretory isoform. This is also suggested by the known bumetanide
sensitivity of chloride secretion carried out by the chloride cell
(Degnan et al., 1977
). The
greater number of NKCC-immunoreactive cells in seawater suggests that this
transporter has increased in quantity following seawater acclimation, similar
to results with other teleosts (Flik et
al., 1997
; Pelis et al.,
2001
).
Although Na+/K+-ATPase, NKCC and CFTR were all
present in the gill chloride cells of seawater-acclimated Hawaiian goby at
high concentration, there were also detectable levels of all three
transporters, especially Na+/K+-ATPase and NKCC, in
freshwater chloride cells. There are two likely explanations for this that are
not mutually exclusive. First, these elevated levels in freshwater may be
present to allow for the euryhalinity that is apparently characteristic of
this species. By maintaining relatively high levels of
Na+/K+-ATPase, NKCC and, to a lesser extent, CFTR, the
animal has a greater capacity for moving into seawater at any time than it
otherwise would; i.e. the existing transporters could be immediately activated
rather than requiring de novo synthesis. Other teleosts that make a
limited number of seasonal migrations into seawater over their lifetime, such
as anadromous salmonids, can time the appearance of these transport proteins
to coincide with seawater entry (see Pelis
et al., 2001). Further studies are required to determine how the
euryhalinity of this species changes during ontogeny and whether this is
accompanied by changes in these ion transporters. Another explanation is that
these transporters are involved in ion uptake. This seems quite likely in the
case of Na+/K+-ATPase, which is involved in moving
Na+ from the interior of the chloride cell into the blood, as well
as providing ionic and electrical gradients used by other transporters
involved in ion uptake (Marshall,
2002
). To date, there is no direct evidence that NKCC is involved
in ion uptake by the fish gill. Wilson et al.
(2000
) hypothesized that
Na+/K+-ATPase and/or NKCC may be involved in ammonia
excretion by the mudskipper gill through substitution of
NH4+ for K+.
Exposure to low ion concentrations (i.e. deionized freshwater) results in a
proliferation of cells on the secondary lamellae in several teleosts
(Avella et al., 1987;
Perry, 1997
). In some species,
such as American shad (Alosa sapidissima), the number of chloride
cells on the secondary lamellae is high in freshwater fish but greatly reduced
in seawater fish (Zydlewski and McCormick,
2001
). Pisam et al.
(1987
) has described two
morphologically distinct chloride cells, with the alpha-chloride cell more
often in association with the circulation of the secondary lamellae. These
results have led to speculation that chloride cells on the secondary lamellae
are involved in ion uptake, whereas those on the primary filament are involved
in salt secretion. In the Hawaiian goby, there were still a large number of
chloride cells on the secondary lamellae after seawater exposure, and they had
similar immunoreactivity of Na+/K+-ATPase, NKCC and CFTR
to that of chloride cells on the primary filaments. Other than the more
flattened appearance of chloride cells on the secondary lamellae, their
morphological appearance and localization of transporters did not differ. Our
results therefore do not provide evidence for differential function of
chloride cells on the primary and secondary lamellae in the Hawaiian goby. It
thus appears that among teleosts there are species-specific differences in
chloride cell morphology and localization following changes in environmental
ion concentrations. For instance, in the striped bass (Morone
saxatilis), there are virtually no chloride cells on the secondary
lamellae in either freshwater or seawater
(Madsen et al., 1994
).
Varsamos et al. (2002
) have
recently found that the number of chloride cells on the secondary lamellae is
higher in both freshwater and concentrated seawater (70
) compared with
35
seawater. Findings such as these suggest that chloride cells on the
secondary lamellae may relate more to increased demand for salt regulation
(either uptake or secretion) than just ion uptake. Our results indicate that
Na+/K+-ATPase, NKCC and CFTR are found specifically in
chloride cells of the Hawaiian goby. Na+/K+-ATPase and
NKCC have a basolateral/tubular distribution, whereas CFTR is present in the
apical pit region, often in a discontinuous, punctate arrangement. The number
of Na+/K+-ATPase- and NKCC-immunoreactive chloride cells
and their size increase slightly after seawater adaptation. The number of
cells with CFTR-immunoreactive apical regions increases dramatically after
seawater exposure, as does the area and brightness of CFTR immunoreactivity.
These results verify previously proposed models for the function and
localization of these transport proteins in chloride cells of teleost fish and
indicate that they are all involved in chloride secretion by gill chloride
cells.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avella, M., Masoni, A., Bornancin, M. and Mayer-Gostan, N. (1987). Gill morphology and sodium influx in the rainbow trout (Salmo gairdneri) acclimated to artificial freshwater environments. J. Exp. Zool. 241,159 -169.
Chubb, A. L., Zink, R. M. and Fitzsimons, J. M.
(1998). Patterns of MtDNA variation in Hawaiian freshwater
fishes: the phylogeographic consequences of amphidromy. J.
Hered. 89,8
-16.
Cutler, C. P. and Cramb, G. (2002). Two isoforms of the Na+/K+/2Cl- cotransporter are expressed in the European eel. Biochim. Biophys. Acta 1566,92 -103.[Medline]
Degnan, K. J. (1984). Chloride secretion by teleost gill and operculum. In Chloride Transport Coupling in Biological Membranes and Epithelia (ed. G. A. Gerencser), pp.360 -391. Amsterdam: Elsevier Science.
Degnan, K. J., Karnaky, K. J. and Zadunaisky, J. A. (1977). Active chloride transport in the in vitro opercular skin of a teleost (Fundulus heteroclitus), a gill-like epithelium rich in chloride cells. J. Physiol. 271,155 -191.[Medline]
Ernst, S. A., Crawford, K. M., Post, M. A. and Cohn, J. A. (1994). Salt stress increases abundance and glycosylation of CFTR localized at apical surfaces of salt gland secretory cells. Am. J. Physiol. Cell. Physiol. 36,C990 -C1001.
Flik, G., Kaneko, T., Greco, A. M., Li, J. and Fenwick, J. C. (1997). Sodium dependent ion transporters in trout gills. Fish Physiol. Biochem. 17,385 -396.[CrossRef]
Foskett, J. K. and Scheffey, C. (1982). The chloride cell: definitive identification as the salt-secretory cell in teleosts. Science 215,164 -166.[Medline]
Haas, M. and Forbush, B. (2000). The Na-K-Cl cotransporter of secretory epithelia [Review]. Ann. Rev. Physiol. 62,515 -534.[CrossRef][Medline]
Karnaky, K. J., Kinter, L. B., Kinter, W. B. and Stirling, C. E. (1976). Teleost chloride cell II. Autoradiographic localization of gill Na,K-ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments. J. Cell Biol. 70,157 -177.[Abstract]
Keys, A. and Willmer, E. N. (1932). `Chloride secreting cells' in the gills of fishes, with special reference to the common eel. J. Physiol. 76,368 -378.
Lehrich, R. W., Aller, S. G., Webster, P., Marino, C. R. and
Forrest, J. N. (1998). Vasoactive intestinal peptide,
forskolin, and genistein increase apical CFTR trafficking in the rectal gland
of the spiny dogfish, Squalus acanthias acute regulation of
CFTR trafficking in an intact epithelium. J. Clin.
Invest. 101,737
-745.
Lytle, C., Xu, J. C., Biemesderfer, D., Haas, M. and Forbush,
B. (1992). The Na-K-Cl cotransport protein of shark rectal
gland. I. Development of monoclonal antibodies, immunoaffinity purification,
and partial biochemical characterization. J. Biol.
Chem. 267,25428
-25437.
Madsen, S. S., McCormick, S. D., Young, G., Endersen, J. S., Nishioka, R. S. and Bern, H. A. (1994). Physiology of seawater acclimation in the striped bass, Morone saxatilis (Walbaum). Fish Physiol. Biochem. 13, 1-11.
Marshall, W. S. (2002). Na+, Cl-, Ca2+ and Zn2+ transport by fish gills: retrospective review and prospective synthesis [Review]. J. Exp. Zool. 293,264 -283.[CrossRef][Medline]
Marshall, W. S., Bryson, S. E., Midelfart, A. and Hamilton, W. F. (1995). Low-conductance anion channel activated by cAMP in teleost Cl- secreting cells. Am. J. Physiol. Reg. Integr. C. 37,R963 -R969.
Marshall, W. S., Emberley, T. R., Singer, T. D., Bryson, S. E.
and McCormick, S. D. (1999). Time course of salinity
adaptation in a strongly euryhaline estuarine teleost, Fundulus
heteroclitus: a multivariable approach. J. Exp.
Biol. 202,1535
-1544.
Marshall, W. S., Lynch, E. A. and Cozzi, R. F.
(2002). Redistribution of immunofluorescence of CFTR anion
channel and NKCC cotransporter in chloride cells during adaptation of the
killifish Fundulus heteroclitus to sea water. J. Exp.
Biol. 205,1265
-1273.
McCormick, S. D. (1993). Methods for non-lethal gill biopsy and measurement of Na+,K+-ATPase activity. Can. J. Fish. Aquat. Sci. 50,656 -658.
McCormick, S. D. (1995). Hormonal control of gill Na+,K+-ATPase and chloride cell function. In Fish Physiology, Volume XIV, Ionoregulation: Cellular and Molecular Approaches (ed. C. M. Wood and T. J. Shuttleworth), pp. 285-315. New York: Academic Press.
Morgan, J. D., Sakamoto, T., Grau, E. G. and Iwama, G. K. (1997). Physiological and respiratory responses of the Mozambique tilapia (Oreochromis mossambicus) to salinity acclimation. Comp. Biochem. Physiol. 117,391 -398.[CrossRef]
Pelis, R. M., Zydlewski, J. and McCormick, S. D.
(2001). Gill Na+-K+-2Cl(-)
cotransporter abundance and location in Atlantic salmon: effects of seawater
and smolting. Am. J. Physiol. Reg. Integr. C.
280,R1844
-R1852.
Perry, S. F. (1997). The chloride cell: structure and function in the gills of freshwater fishes [Review]. Annu. Rev. Physiol. 59,325 -347.[CrossRef][Medline]
Pisam, M., Caroff, A. and Rambourg, A. (1987). Two types of chloride cells in the gill epithelium of a freshwater-adapted euryhaline fish: Lebistes reticulatus; their modifications during adaptation to saltwater. Am. J. Anat. 179, 40-50.[Medline]
Shiraishi, K., Kaneko, T., Hasegawa, S. and Hirano, T. (1997). Development of multicellular complexes of chloride cells in the yolk-sac membrane of tilapia (Oeochromis mossambicus) embryos and larvae in seawater. Cell Tissue Res. 288,583 -590.[CrossRef][Medline]
Silva, P., Solomon, R., Spokes, K. and Epstein, F. H. (1977). Ouabain inhibition of gill Na-K-ATPase: relationship to active chloride transport. J. Exp. Zool. 199,419 -426.[Medline]
Singer, T. D., Clements, K. M., Semple, J. W., Schulte, P. M., Bystriansky, J. S., Finstad, B., Fleming, I. A. and McKinley, R. S. (2002). Seawater tolerance and gene expression in two strains of Atlantic salmon smolts. Can. J. Fish. Aquat. Sci. 59,125 -135.[CrossRef]
Ura, K., Soyano, K., Omoto, N., Adachi, S. and Yamauchi, K. (1996). Localization of Na+, K+-ATPase in tissues of rabbit and teleosts using an antiserum directed against a partial sequence of the alpha-subunit. Zool. Sci. 13,219 -227.[Medline]
Varsamos, S., Diaz, J. P., Charmantier, G., Flik, G., Blasco, C. and Connes, R. (2002). Branchial chloride cells in sea bass (Dicentrarchus labrax) adapted to fresh water, seawater, and doubly concentrated seawater. J. Exp. Zool. 293, 12-26.[CrossRef][Medline]
Wilson, J. M., Randall, D. J., Donowitz, M., Vogl, A. W. and Ip,
A. Y. (2000). Immunolocalization of ion-transport proteins to
branchial epithelium mitochondria-rich cells in the mudskipper
(Periophthalmodon schlosseri). J. Exp. Biol.
203,2297
-2310.
Zydlewski, J. and McCormick, S. D. (2001). Developmental and environmental regulation of chloride cells in young American shad, Alosa sapidissima. J. Exp. Zool. 290, 73-87.[CrossRef][Medline]