Seawater acclimation causes independent alterations in Na+/K+- and H+-ATPase activity in isolated mitochondria-rich cell subtypes of the rainbow trout gill
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6E 4W1
Author for correspondence (e-mail:
greg.goss{at}ualberta.ca)
Accepted 17 December 2003
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Summary |
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Key words: mitochondria-rich cells, MR cells, sodium, transport, Na+/K+-ATPase, H+-ATPase, rainbow trout, Oncorhynchus mykiss, gill, seawater adaptation
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Introduction |
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In freshwater, the current model for ion exchange links
Cl uptake to HCO 3 secretion
via an anion exchanger. Meanwhile, Na+ uptake is thought
to occur via an Na+ channel linked electrochemically to a
coupled V-type H+-ATPase on the apical membrane
(Lin and Randall, 1991;
Sullivan et al., 1995
;
Wilson et al., 2002
). This
model is based on the requirement to move both Na+ and
H+ against their electrochemical gradients in low ionic strength
media (Avella and Bornancin,
1989
). However, in a higher ionic strength environment such as
seawater, the Na+ concentration gradient is favourable for linked
electroneutral exchange. The mechanism by which the removal of protons is
believed to occur is via an electroneutral sodium/proton exchange
(NHE) system (Claiborne et al.,
1999
; Wilson et al.,
2000
), reducing the ATP requirement for acidbase
regulation. NHE isoforms have been identified by molecular and immunological
methods in the gills of a marine species (Myoxocephalus
octodecimspinosus) and the euryhaline killifish (Fundulus
heteroclitus) (Claiborne et al.,
1999
). Seawater acclimation results in many changes in gill
function including increases in the activity of
Na+/K+-ATPase in MR gill cells
(Mancera and McCormick, 2000
),
concomitant reductions in H+-ATPase
(Lin and Randall, 1993
) and
the appearance of accessory cells on the gill epithelium
(Laurent and Dunel, 1980
).
Recently, it has been proposed that there are separate subtypes of the MR
cells that perform different ionoregulatory functions
(Galvez et al., 2002). Pisam
and co-workers (Pisam et al.,
1987
,
1990
), using morphological
characteristics such as staining, shape, apical surface and location in the
gill, have identified two different MR cells on the gill epithelium of
freshwater teleost fish, which they termed
and ß MR cells. Pisam
et al. (1987
) described two MR
types (
- and ß-cells) in the gills of freshwater teleost fish, but
only one MR cell subtype in the gills of seawater teleost fish (consisting of
the
MR cells). More recently, Wong and Chan
(1999
) used flow cytometry to
track the change in MR gill cell populations during seawater transfer using
relative cell size, granularity and autofluorescence as the defining
characteristics. They demonstrated that in freshwater Japanese eels
(Anguilla japonica) there are two distinct populations of cells that
undergo a transition into morphologically separate seawater cell subtypes.
However, this study did not further assess the function of these presumed MR
gill cell populations. Nonetheless, these studies suggest the existence of
different MR cell subtypes during seawater acclimation.
There has been limited progress made in characterizing the role of MR gill
cell populations in ion and acidbase regulation, partly due to the lack
of any suitable techniques for differentiating between MR cell populations in
live cells. In other epithelia, such as the mammalian cortical collecting duct
(Turnheim, 1991), the frog
skin (Smith, 1971
) and the
turtle urinary bladder (Rich et al.,
2002
), peanut lectin agglutinin (PNA) is known to differentially
bind to MR cell populations based on differences in glycoproteins on the
apical (mucosal) surfaces of these cells. All the aforementioned systems are
tight epithelia that have low rates of passive ion loss across the epithelium
against large electrochemical gradients, and the major cell type (called
`principal cells' in the mammalian collecting duct and `granular cells' in the
frog skin and turtle bladder) has similar features to the fish gill pavement
cells (PVCs). It has been reported that the MR intercalated collecting duct
cells could be differentiated based on the binding of PNA
(Satlin et al., 1992
). Only
one type of intercalated collecting duct cell (ß-type) was found to bind
the PNA. Recently, our lab has demonstrated that a sub-population of MR gill
cells from freshwater rainbow trout bind PNA
(Goss et al., 2001
).
Furthermore, we have developed a method for separation and isolation of
functionally different MR cell subtypes in freshwater rainbow trout based on
differential binding to PNA (Galvez et al.,
2002
). We have also shown that phenamil-sensitive Na+
transport is linked only to the PNA cells
(Reid et al., 2003
). Phenamil
sensitivity suggests the presence of an epithelial sodium channel (ENaC family
of Na+ channels) on the PNA cells only and
establishes a role for this MR cell subtype in Na+ transport.
The purpose of the present study was twofold. The first objective was to
examine changes in MR cell subtype populations (PNA+ and
PNA) during seawater acclimation to compare with the
morphological data obtained by Pisam et al.
(1987) and Wong and Chan
(1999
). The second objective
was to use our ability to isolate sub-populations of MR cells to independently
examine changes in Na+/K+- and H+-ATPase
activity in each of the MR cell subtypes during seawater acclimation.
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Materials and methods |
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Fish used in the seawater transfer experiments were removed from the
holding facility and transferred to a recirculating system containing
10 (
30% full-strength) seawater. The water temperature and
photoperiod were unchanged. Seawater was made by diluting Instant Ocean brand
salts (Aquarium Systems Inc., Mentor, OH, USA) in dechlorinated Edmonton tap
water. Fish were acclimated to 10
for at least one week before
transfer to a second system containing full-strength seawater (30
) for
at least an additional week before sampling. Salinity of seawater was checked
weekly and adjusted as necessary. Experimental animals were randomly selected
from holding tanks and killed by overdose (1 g l1 MS-222;
Syndel, Vancouver, BC, Canada) followed by cephalic blow.
Materials
Streptavidin-conjugated Alexa fluor 594 and Mitotracker Green-FM were
obtained from Molecular Probes (Eugene, OR, USA). The magnetic cell separation
(MACS) system and streptavidin-conjugated microbeads used for MACS were
purchased from Miltynei Biotech (Auburn, CA, USA). All other reagents used for
experimentation were obtained from Sigma Chemicals, St Louis, MO, USA.
Gill digestion
Gill arches were removed from fish, rinsed in dechlorinated tap water and
lightly blotted to remove excess water. Gill filaments were removed and placed
into ice-cold Cortland's saline (143 mmol l1 NaCl, 5 mmol
l1 KCl, 1.5 mmol l1 CaCl2, 1
mmol l1 MgSO4, 5 mmol l1
NaHCO3, 3 mmol l NaH2 PO4 5 mmol
l1 glucose, pH 7.8). Filaments were digested in 0.2 mg
ml1 collagenase (type 1A) in Cortland's saline for 20 min at
18°C with continuous agitation at 300 revs min1.
Digested filaments were then scraped with glass slides and filtered through
254 µm (gravity) and 96 µm (vacuum) nylon mesh to remove large debris.
The filtrate was then resuspended in Ca2+-free phosphate-buffered
saline (PBS; 137 mmol l1 NaCl, 2.7 mmol l1
KCl, 4.3 mmol l1 Na2HPO4, 1.4 mmol
l1 NaH2PO4 and adjusted to pH 7.8) for
centrifugation at 1500 g for 5 min at 4°C. Dispersed cells
were washed twice with at least 10x volume PBS, pelleted by
centrifugation and resuspended in 1 ml red cell lysis buffer (154 mmol
l1 ammonium chloride) for exactly 60 s to lyse the
erythrocytes. After 1 min, the cells were rapidly diluted by the addition of
45 ml PBS and then centrifuged for 5 min (1500 g). The
resultant cell pellet was washed twice with 40 ml PBS and was finally
resuspended in 2 ml PBS. The cells were then layered on a discontinuous
Percoll density gradient (1.03, 1.05 and 1.09 g ml1 Percoll)
and centrifuged at 2000 g for 45 min. The MR cells from the
1.051.09 g ml1 Percoll interface were collected, spun
down and either used immediately in experiments or further separated into the
PNA and PNA+ cell fractions as outlined
previously by Galvez et al.
(2002).
Fluorescence microscopy
The percentage of MR cells binding PNA was determined by labelling cells
with a biotin-conjugated PNA (40 µg ml1, 20 min), washing
the cells in PBS, followed by a double labelling with streptavidin-conjugated
Alexa fluor 594 (20 µg ml1, 15 min) and Mitotracker
Green-FM (40 µg ml1, 15 min). Cells were washed in PBS to
remove unbound dyes and placed on glass slides for differential interference
contrast (DIC) microscopy (Nikon Eclipse TE300) and fluorescence imaging
(TE-FM epi-fluorescence attachment). Fluorescence microscopy was performed
using epi-illumination via a Xenon arc lamp (Lambda LS; Sutter
Instruments, Novato, CA, USA). Plan-Fluor objectives at either 40x or
100x (oil immersion) were used and images were digitally captured on a
12-bit CCD camera (Cooke SensiCam, Kelheim, Germany). Mitotracker Green-FM and
Alexa fluor 594 were excited at 495±5 nm and 560±2 nm,
respectively, and emission was measured using 540±25 nm and
630±30 nm filters, respectively. Images were binned at 2x2 to
increase the sensitivity of fluorescence capture, and final images were
adjusted using Adobe Photoshop 6 for contrast and brightness only.
To determine the percentage of total MR cells made up of PNA+ or PNA cells in the gills of freshwater and seawater-acclimated trout, random view fields were selected on the slide and a DIC image captured for total cell counts. Serial images of each field were captured with fluorescence microscopy. Mitotracker Green-FM fluorescence was used to indicate the MR cells in the field, and Alexa fluor 594 fluorescence was used to determine the percentage of MR cells that were PNA+. At least five random fields were captured and used for each fish. Cell sizes were measured using the calibrated measuring tool located in Slidebook v.3.1.2 (Intelligent Imaging Innovations; Denver, CO, USA) with the DIC images captured from the seawater adaptation experiments.
ATPase assay
An ATPase assay based on a method developed by McCormick
(1993) was adapted to
determine both the ouabain (Na+/K+-ATPase inhibitor)-
and bafilomycin (V-type H+-ATPase inhibitor)-sensitive ATPase
activities. Gill cells were counted using a haemocytometer and used as either
total MR cells or separated into PNA and PNA+
fractions and stored in SEI buffer (250 mmol l1 sucrose, 10
mmol l1 Na2EDTA, 50 mmol l1
imidazole and adjusted to pH 7.3) at 80°C until assays were
performed. Cells were thawed and homogenized with the addition of 0.5% sodium
deoxycholic acid on ice and immediately centrifuged at 5000 g
for 30 s to remove insoluble material. Homogenate (10 µl) from each sample
was added to nine wells in a 96-well plate. This provided three treatments for
each sample [control, ouabain (500 µmol l1) and ouabain
(500 µmol l1) + bafilomycin (50 nmol
l1)] with triplicate measurements of each treatment.
Preliminary experiments demonstrated that H+-ATPase activity in
total MR cells was maximally inhibited between 10 nmol l1
and 100 nmol l1. Therefore, 50 nmol l1 was
chosen as the appropriate dose for our assay. To each well was added 150 µl
of assay mixture [50 mmol l1 imidazole buffer, 2 mmol
l1 phosphoenol pyruvate (PEP), 0.16 mmol
l1 NADH, 0.5 mmol l1 ATP, 3.3 U
ml1 lactate dehydrogenase (LDH), 3.6 U ml1
phosphokinase (PK)], with appropriate drug treatment added, and 50 µl of
salt solution (50 mmol l1 imidazole, 189 mmol
l1 NaCl, 10.5 mmol l1 MgCl2, 42
mmol l1 KCl). The microplate was read at a wavelength of 340
nm in akinetic microplate reader (ThermoMAX; Molecular Devices, Sunnyvale, CA,
USA) at 15 s intervals for 20 min. The average rate for each treatment was
taken from the stable slope and calculated from a standard curve generated
just prior to the assay. Na+/K+-ATPase activity was
obtained by subtracting the ouabain-treated ATPase activity from control
ATPase activity (see McCormick,
1993
). We also modified the assay to assess H+-ATPase
activity by calculating the difference in ATPase activity between the ouabain-
and the oaubain + bafilomycin-treated samples.
Statistical analysis
All statistics were performed with SPSS version 10, using analysis of
variance (ANOVA) followed by multiple comparison tests using Tukey's HSD. In
all cases, the level of significance was set at P<0.05.
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Results |
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Similarly, reductions in bafilomycin-sensitive H+-ATPase activity occur in the same total isolated 1.031.09 cell fraction. When the total gill cell population was examined, bafilomycin-sensitive H+-ATPase activity declined 79% from 0.32±0.07 nmol ADP min1 106 cells1 in freshwater fish to 0.067±0.02 nmol ADP min1 106 cells1 in full-strength seawater-acclimated fish (Fig. 2).
|
Fig. 3AC shows
differential interference contrast (DIC;
Fig. 3A) and fluorescence
microscopic images (Fig. 3B,C)
from the same field of view for total MR cells (Percoll density isolated from
1.051.09 interface) from freshwater rainbow trout. The fluorescence
images permit the identification of cells as MR cells using Mitotracker
(Fig. 3B) and further
distinguish those MR cells as either binding PNA or not
(Fig. 3C). Greater than 95% of
the cells in the fields of view (Fig.
3A) were found to be MR cells, as demonstrated in
Fig. 3B. The percentage of
PNA+ cells was calculated from the number showing PNA fluorescence
(Fig. 3C). Fig. 3DF shows DIC and
fluorescence images of the same field of view for a 30 acclimated
(seawater) trout. Clearly, the relative number of PNA+ cells in the
field of view has greatly increased from the freshwater situation
(Fig. 3F). Also noted during
the image capture of gill cells from seawater-acclimated rainbow trout is the
30% increase in size of the PNA+ cells relative to the size of
PNA+ cells from freshwater rainbow trout. Quantification of this
size change was performed, and this increase in size concurs with the increase
in size of MR cells during seawater acclimation
(Fig. 4).
|
|
These fluorescence images were used to quantify the relative numbers of the
different MR cell subtypes during transition from freshwater to seawater. In
freshwater, the total MR cell fraction is primarily made up of
PNA cells. However, in either the 10 (not shown) or
the 30
acclimated fish, almost all MR cells in the observed fields are
PNA+ (Fig. 3).
Quantification of these changes was performed by counting the total number of
MR cells and the number of PNA+ MR cells in each corresponding
field, to give the percentage of the MR cells that were PNA+
(minimum five fields per fish). In freshwater, approximately 35% of the MR
cells are PNA+. After seawater acclimation, 7894% of the MR
cells are PNA+ (Fig.
5). Of note, these percentages represent only the relative
distribution of the PNA+ and PNA cells and cannot
distinguish between an increase in one cell type and a decrease in another.
Unfortunately, absolute cell counts are not possible due to variable loss of
total cells throughout the protocol, with each step involving suction and/or
handling of the cells. However, the relative distribution of cells should
remain the same within a single fraction in a single isolation. A potential
complication was that the mobility of PNA cells in the
Percoll gradients might have changed during acclimation of fish to seawater.
However, MR cells were not detected in any of the other Percoll fractions
(data not shown), indicating that seawater-acclimated MR cells are in fact all
migrating to the same Percoll density fraction as found for freshwater MR
cells and that monitoring the change in relative distribution of
PNA+:PNA cells is valid.
|
The total MR cell fraction was separated into PNA and
PNA+ using the MACS system
(Galvez et al., 2002), and
ouabain-sensitive (Na+/K+-ATPase) activity was measured.
The PNA+ fraction showed a decrease in
Na+/K+-ATPase activity, on a per cell basis, upon
transfer of rainbow trout from freshwater to 100% seawater, while the
PNA fraction demonstrated the opposite response.
PNA Na+/K+-ATPase activity (on a per
cell basis) was found to significantly increase during acclimation to
full-strength seawater (Fig.
6).
|
We separated the total MR gill cells into PNA+ and
PNA fractions and assessed bafilomycin-sensitive
H+-ATPase activity. Under freshwater conditions,
H+-ATPase activity in PNA cells was the same as
in PNA+ cells but was substantially (90%) decreased after
acclimation to either 10
or 30
seawater
(Fig. 7). Interestingly, we
found that H+-ATPase in the PNA+ cells of freshwater
trout was also high and a similar trend, with a 73% reduction in activity,
occurred during seawater acclimation.
|
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Discussion |
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Levels of Na+/K+-ATPase have been used extensively as
an index of transport capacity in fish exposed to a variety of conditions
including seawater transfer (Yoshikawa et
al., 1993; Mancera and
McCormick, 2000
). We have already demonstrated that the MR cell
fraction possesses high levels of Na+/K+-ATPase activity
compared with the other fractions in the Percoll density separation
(Galvez et al., 2002
). Activity
and abundance of Na+/K+-ATPase in total gill homogenates
have been demonstrated to increase with seawater acclimation and have been
used numerous times as `indicators' of readiness for migration or transition
into seawater (see review by McCormick,
1995
). Our data are consistent with these previous findings that
Na+/K+-ATPase activity is elevated in the total gill
cell fraction during transition to seawater
(Fig. 1). We demonstrate that
Na+/K+-ATPase activity is approximately fivefold higher
in the gills of seawater-acclimated trout compared with that observed in the
freshwater fish gill. This increase is in agreement with previously published
figures (Epstein et al., 1980
;
Kultz et al., 1992
;
Wilson et al., 2002
). However,
the present paper is the first to separate and independently analyze this
response into distinct MR cell fractions based on binding of PNA to the cell
membrane. We found that the two cell fractions respond differently to seawater
acclimation. While the Na+/K+-ATPase activity of
PNA+ cell types is significantly greater than that of the
PNA cells under freshwater conditions,
Na+/K+-ATPase activity in the PNA
cells of seawater-acclimated fish is increased while that of PNA+
cells is decreased.
The decrease in Na+/K+-ATPase activity during
seawater adaptation in both PNA+ and PNA cells
seems contradictory given that total gill Na+/K+-ATPase
activity is increased. We believe that there are at least two possible
explanations for these changes. The first is that the noted decrease in
Na+/K+-ATPase activity is a result of an overall
decrease to half of the value on an individual cell basis. However, to have
this occur and still be congruent with increases in total
Na+/K+-ATPase activity on a whole gill basis, there must
be a large increase in the total number of PNA+ MR cells in
seawater fish, thereby increasing total Na+/K+-ATPase
activity. When examining sections of gills by light microscopy, although there
has been a substantial (up to fivefold in some studies) increase in the total
number of chloride cells in seawater-adapted fish
(Borgatti et al., 1992;
Shikano and Fujio, 1998
) this
cannot entirely account for the noted drop in the average cell
Na+/K+-ATPase activities found in the present study. A
second possible explanation for the noted decrease in
Na+/K+-ATPase activity in PNA+ MR cells
during seawater adaptation is methodological. It is possible that accessory
cells that are expressed only in seawater may also be PNA+ and also
migrate to the same fraction as the MR PNA+ chloride cells in the
Percoll density gradient. If accessory cells are found in the PNA+
fraction then our technique for isolating PNA+ cells in seawater
fish would result in a mixed population of MR chloride cells and accessory
cells, making interpretation of the results difficult. Accessory cells are
known to possess lower Na+/K+-ATPase activity compared
with MR cells, as demonstrated by ouabain-binding studies
(McCormick, 1990
), but can be
considered relatively mitochondria-rich, at least when compared with
surrounding PVCs (Laurent and Dunel,
1980
). In our assay, the accessory cells would appear as MR, as
the assay is only a relative comparison to the PVC fraction. By combining the
two cell types into one population, we would predict a decrease in
Na+/K+-ATPase activity depending on the relative number
of accessory cells in each preparation. We cannot differentiate between
PNA+ MR cells and accessory cells once cells are dispersed.
Accessory cells are usually identified under the electron microscope by their
close association with `MR chloride cells' in the base of the lamellae. The
lack of specific gill localization of the accessory cells does not allow for
distinct identification. This is a potential problem with isolation and
characterization of PNA+ cells in seawater-adapted fish and makes
final interpretation of these data difficult. We must find a specific marker
to identify one of the cell types in seawater fish to resolve these
differences.
The current model for ion exchange in the cells of freshwater fish links
Na+ uptake to a rheogenic H+-ATPase. This
H+-ATPase drives the uptake of Na+ through an apical
epithelial Na+ channel (probably a phenamil-sensitive ENaC type
Na+ channel, although direct molecular evidence is lacking) by
creating a negative potential inside the cell (see review by
Perry, 1997). We have
previously shown that there is an acid-inducible, phenamil-sensitive
Na+ uptake attributed to the PNA cells in
freshwater trout gills (Reid et al.,
2003
). Furthermore, western blot analysis shows that the
expression of H+-ATPase is elevated in the PNA
cells, relative to that of PNA+ cells, and increased during
hypercapnic acidosis but not during infusion of bicarbonate. Of note, however,
was the relatively high expression of H+-ATPase in the
PNA+ cells under freshwater control conditions similar to that
found earlier in our lab (Galvez et al.,
2002
).
In seawater, the requirement for H+-ATPase-driven Na+
uptake is reduced or eliminated due to a favourable Na+ gradient
into the fish. It has been shown that Na+ movement in a seawater
situation is more likely through an energetically favourable sodium/proton
exchange mechanism (NHE; Claiborne et al.,
1999). Reductions in H+-ATPase immunoreactivity
(Lin et al., 1994
;
Piermarini and Evans, 2001
)
and biochemical activity (Lin and Randall,
1993
; Wilson et al.,
2002
) have been previously demonstrated during seawater
acclimation in fish. These results are comparable to those seen in the present
study (Fig. 2), with total MR
cell H+-ATPase activity decreased during seawater acclimation to
about one-third of the activity of that of the freshwater fish.
An important contribution of the present paper is the analysis of
independent changes in H+-ATPase activity in the
PNA MR cell population. When the total MR gill cell fraction
was separated into PNA subtypes, we found that seawater adaptation resulted in
a notable decrease in H+-ATPase activity in both the
PNA and the PNA+ cells. The decreases in
H+-ATPase activity found in PNA cells during
seawater adaptation were predicted based on previous attribution of
Na+ uptake to this particular cell type. In freshwater, the
PNA cell type is responsible for Na+ uptake, as
demonstrated by Reid et al.
(2003), where acid-stimulated,
phenamil-sensitive Na+ uptake could only be found in the
PNA cell fraction and not in the PNA+ fraction.
Theoretically, Na+ uptake from low ionic strength media requires
active H+ extrusion to the water via an apical
H+-ATPase to provide the driving gradient for Na+
uptake. This mechanism for Na+ uptake was first proposed by Avella
and Bornancin (1989
) based on
the frog skin model of Na+ uptake
(Ehrenfeld et al., 1985
; Harvey
and Ehrenfeld, 1986
,
1988
). A similar mechanism has
also been demonstrated for acidbase regulation in the mammalian kidney
(Brown and Breton, 2000
). In
the kidney, two types of MR cells exist, termed
- and ß-(acid- and
base-secreting, respectively) intercalated cells. These cells have similar
morphological and physiological characteristics to the MR cells in the fish
gill. The results of this and previous experiments are consistent with this
model and allow us to suggest that the PNA cell is the
-MR cell in the fish.
Of note, our terminology is inconsistent (opposite) with that of the
- and ß-MR cells coined by Pisam et al.
(1987
). Our terminology
follows functionally from the
-(acid-secreting) and
ß-(base-secreting) intercalated cells in the mammalian kidney and is
based primarily on the fact that the PNA cell types in both
tissues are responsible for Na+ uptake and acid excretion while the
PNA+ cells in the kidney have been demonstrated to be involved in
base secretion (apical Cl/HCO3
exchange). Pisam et al. (1987
)
based their observations on morphological (staining) characteristics that,
unfortunately, do not appear to match the functional equivalents in the
mammalian kidney.
During seawater acclimation, external Na+ rises to a level that
favours inward-directed Na+ movement without the need for an
energetically expensive apical H+-ATPase. Our results, showing a
reduction in PNA (-MR cell) H+-ATPase
during seawater acclimation, are consistent with this model. Other authors
(Lin et al., 1994
;
Piermarini and Evans, 2001
)
have also documented a reduction in H+-ATPase immunoreactivity
during seawater acclimation and also the appearance of immunoreactivity of the
NHE-2 isoform of the Na+/H+ exchanger family in
seawater-adapted fish (Wilson et al.,
2002
).
We were surprised at the relatively high levels of H+-ATPase
activity found in the freshwater PNA+ cells and the concomitant
reductions that occurred during seawater acclimation. The fact that
PNA cells appear to perform Na+
uptake/H+ excretion in freshwater suggests that PNA+
cells are responsible for Cl uptake/base excretion. While
the reduction in H+-ATPase activity during seawater adaptation in
PNA cells may be due to methodological problems, with the
possibility of accessory cell contamination in the seawater PNA+,
the high levels of H+-ATPase activity in freshwater suggest that
the H+-ATPase may play a significant role in Cl
uptake and acidbase regulation in freshwater fish. Chloride uptake from
very dilute freshwater does not have a favourable driving electrochemical
gradient from the water to the fish. Similarly, a strict gradient for
HCO3 from the blood to the water is not
sufficient to provide a driving gradient for HCO3
excretion under normal conditions. The relatively high levels of expression of
H+-ATPase in PNA+ cells under freshwater conditions
suggest that Cl uptake may be linked to the activity of
H+-ATPase, although the mechanism(s) is as yet undetermined.
Immunocytochemical localization of the anion exchanger Pendrin through
Pendrin-like immunoreactivity has been demonstrated for euryhaline stingrays
(Piermarini et al., 2002) but
has not yet been demonstrated for salmonids. Our future research focus will
take advantage of our ability to isolate MR cells into separate populations
and to use this technique to functionally characterize these individual cell
populations and their adaptations during environmental changes. The power of
this approach is demonstrated in the above results, whereby independent
changes in biochemical activity in separate cell types are unmasked by the
ability to separate the MR cells.
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Acknowledgments |
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Footnotes |
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References |
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