Regulation of branchial V-H+-ATPase, Na+/K+-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias)
Dept of Biological Sciences, University of Alberta, Edmonton, Alberta T5G 2E9, Canada and Bamfield Marine Research Centre, Bamfield, BC V0R 1B0, Canada
* Author for correspondence (e-mail: martint{at}ualberta.ca)
Accepted 11 November 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: dogfish, Squalus acanthias, gill, acid-base regulation, H+-ATPase, Na+/K+-ATPase, NHE, acid infusion, base infusion, alkalosis, acidosis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For branchial acid secretion in marine elasmobranchs two mechanisms have
been proposed. The first involves extrusion of protons in electroneutral
exchange for environmental sodium (i.e. via
Na+/H+ exchangers; NHEs) and the second is via
secretion of protons by a vacuolar-type proton ATPase (H+-ATPase).
At first sight, the former has the advantage of being energetically less
expensive, because it would take advantage of the inward directed sodium
gradient to drive proton secretion. The proposed apical transporters are
members of the NHE family, homologous to the mammalian NHE2 and NHE3. These
transporters would be localized in the same cells as the enzyme
sodium-potassium ATPase (Na+/K+-ATPase) (see
Claiborne et al., 2002). The
second hypothesis is based on the description of H+-ATPase in the
gills of Squalus acanthias by Wilson et al.
(1997
). However, the
relationship between this transporter and acid secretion are based only on the
subapical localization, and an analogy to the
-secreting cells of the
mammalian collecting duct, the frog skin and the turtle urinary bladder
(Brown and Breton, 1996
;
Kirschner, 2004
). Recently,
the H+-ATPase has also been proposed to be involved in base
secretion in a euryhaline elasmobranch, the Atlantic stingray Dasyatis
sabina (Piermarini and Evans,
2001
). These authors found strong cytoplasmic H+-ATPase
staining in cells that were not labeled for
Na+/K+-ATPase. They proposed that H+-ATPase
stored in vesicles could be recruited to the basolateral membrane under
alkalotic stress, but this hypothesis was not investigated further.
Demonstration of cellular remodeling and basolateral localization following
alkalotic stress would support their hypothesis that these cells are involved
in base secretion, in an analogous way to the ß-type intercalated cells
in the mammalian collecting duct and turtle urinary bladder. However, the
Atlantic stingray is a euryhaline myliobatiform, and this model might differ
from the one present in exclusively marine elasmobranchs.
The objective of this study was to examine the involvement of H+-ATPase, Na+/K+-ATPase and NHE2 in the branchial acid-base regulatory mechanism of the dogfish, Squalus acanthias. In order to exacerbate the signals, we infused the fish with acid and base solutions for 24 h. Our results support the role of NHE2 in acid secretion and indicate that increases in H+-ATPase abundance and activity, as well as a change in its subcellular localization, are required for upregulation of base secretion.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies and reagents
Rabbit anti-NHE2 antibody was kindly provided by Dr Mark Donowitz (National
Institutes of Health, Bethesda, Maryland, MD, USA). This antibody was designed
against 87 amino acids of the C-terminal region of mammalian NHE2, so the
protein we detected should be regarded, throughout the text, as a NHE2-like
protein (NHE2-lp). This antibody has been successfully used to detect NHE2-lp
in elasmobranchs (Edwards et al.,
2002). Rabbit anti-Na+/K+-ATPase antibody
was raised against a synthetic peptide corresponding to a part of a highly
conserved region of the
-subunit
(Katoh et al., 2000
;
Katoh and Kaneko, 2003
).
Rabbit anti-H+-ATPase was raised against a synthetic peptide based
on the highly conserved and hydrophilic region in the A-subunit
(Katoh et al., 2003
). A donkey
anti-rabbit fluorescent secondary antibody (Li-Cor Inc., Lincoln, NE, USA) was
used for western analysis. We were unable to run pre-absorption controls
because the corresponding proteins were not available. However, in the western
analyses we always found very distinct bands of about the expected molecular
masses for all the antibodies tested. Together with the absence of signal in
nitrocellulose membranes incubated with blocking buffer without the primary
antibody, it suggests that the antibodies used have an acceptable
specificity.
Unless otherwise mentioned, the reagents used in this study were purchased from Sigma (St Louis, MO, USA).
Surgery and acid-base infusions
A total of 16 animals (2.28±0.31 kg) were removed from the housing
tank and cannulated for this study. We used fish of equivalent sizes to
standardize infusion rates. Fish were caught by hand, anesthetized with MS-222
(1:10000), and transferred to an operating table, where the gills were
irrigated with aerated seawater containing MS-222. Two cannulae (PE-50,
Clay-Adams, Parsippany, NJ, USA) were fitted into the caudal vein and artery.
The incision was sutured with stitches and a small volume of a heparanized (50
i.u. ml-1 Na+-heparin) 500 mmol l-1 NaCl
solution was injected before blocking the end of the tubing with a pin. The
animals were transferred to experimental boxes (36 l) with aerated flowing
seawater. After a 24 h recovery period, the venous cannula was connected to a
Gilson miniplus peristaltic pump (Middleton, WI, USA) and the experimental
solution was infused at a rate of 4.04±0.83 ml h-1
kg-1. The arterial cannula allowed us to obtain blood samples
during the course of the experiment.
In order to induce acidosis or alkalosis in the blood, fish were infused
with either 125 mmol l-1 HCl or 250 mmol l-1
NaHCO3 to achieve nominal H+ and
HCO3- infusion rates of 500 and 1000 µmol
kg-1 h-1. The actual acid and base loads were
495±79 and 981±235 µmol kg-1 h-1,
respectively. These concentrations were selected after previous trial
experiments and reference to other published work
(Gilmour et al., 2001;
Wood et al., 1995
). Infusion
of acid at a nominal rate of 1000 µmol. kg-1 h-1 in
early experiments proved to be fatal after
6 h, possibly as a result of
haemolysis.
To minimize osmotic disturbances, the osmolarity of the infusion solutions was adjusted to 1000 mOsm with the addition of NaCl. Animals infused with 500 mmol l-1 NaCl served as control. Table 1 shows the base, acid and NaCl load in each of the treatments. In addition, four other animals were subjected to surgery, but no cannula was inserted into the caudal vein or artery. These fish represented the sham-operated group, and they were otherwise treated exactly the same as the experimental fish.
|
Blood samples
Arterial blood samples (300 µl) were taken at times 0, 1, 3, 6, 12 and
24 h. After the blood extraction, an equal volume of heparanized 500 mmol
l-1 NaCl solution was injected into the fish to minimize changes in
blood volume and prevent clotting. Blood samples were used for haematocrit
analysis (50 µl) and pH determination (
80 µl). The rest of the
sample was centrifuged at 12 000 g and plasma osmolarity and
total CO2 were measured immediately. The remaining plasma was
preserved at -80°C for later sodium and chloride concentrations
assays.
Analytical procedures on plasma samples
Osmolarity was measured with a micro osmometer (Precision Systems Inc.,
Natick, MA, USA). A pH-sensitive electrode (Radiometer, Copenhagen, Denmark)
was used to measure blood pH. The total CO2 content was determined
in a Cameron chamber equipped with a CO2 electrode (Radiometer,
Copenhagen). Na+ concentration was read by flame spectrometry
(Perkin-Elmer model 3300, Norwalk, CT, USA). Cl- concentration was
measured by the mercuric thiocyanate method
(Zall et al., 1956).
Terminal sampling
After 0 (sham) or 24 h of infusion, fish were killed by injection of 3 ml
of a saturated KCl solution. Samples of gill were excised and snap-frozen in
liquid nitrogen for later western blot and ATPase analyses. Other gill samples
were immersed in fixative for immunohistochemistry and electron microscopy
(see below).
Immunohistochemistry
Gill samples for immunohistochemistry were fixed in 3% paraformaldehyde,
0.1 mol l-1 cacodylate buffer (pH 7.4) for 6 h at 4°C and
dehydrated in a graded ethanol series. After embedding in paraffin, 4 µm
sections were cut from gill filaments. Sections from the trailing and leading
edges, as well as from the middle portion of the filament, were placed in
glycerol-albumin (Mayer's fixative)-coated slides (1 section per slide).
Sections were deparaffinized in toluene, hydrated in a decreasing ethanol
series, washed in double distilled water (ddH2O), and then exposed
to 0.6% H2O2 for 30 min to devitalize endogenous
peroxidase activity. After blocking with 2% normal goat serum (NGS) for 30
min, the sections were incubated overnight at 4°C with the respective
antibody, which was diluted in 2% NGS, 0.1% bovine serum albumin, 0.02% limpet
haemocyanin, 0.01% NaN3 in 10 mmol l-1 PBS, pH 7.4. The
anti-Na+/K+-ATPase antibody was diluted 1:4000, and the
antibody against the A-subunit of the H+-ATPase was diluted 1:1000.
In order to look for colocalization of the transporters, consecutive sections
were incubated with different antibodies. The next steps were performed at
room temperature, using the Vectastain ABC kit (Vector Laboratories, CA, USA)
as follows. Sections were incubated with a biotinylated goat anti-rabbit
secondary antibody for 30 min and then incubated with a horseradish
peroxidase-labeled streptavidin solution for 1 h. Sections were rinsed in
ddH2O for 6 min and then in phosphate-buffered saline (PBS) for 2
min inbetween incubations. Bound antibodies was visualized by soaking the
sections in a solution containing 20% w/v 3,3'-diaminobenzidine
tetrahydrochloride (DAB) and 0.005% H2O2 in 50 mmol
l-1 Tris-buffered saline, pH 7.6. DAB reacts with the horseradish
peroxidase, producing a brown coloration. As controls, gill sections from
every fish were incubated without any primary antibody. These sections never
showed specific staining, regardless of the treatment and location in the gill
(trailing or leading edge of the filament). The qualitative description of the
amount of positively labeled cells is based on three pairs of sections from
the leading edge, three pairs of sections from the approximate middle of the
filament, and three pair of sections from the trailing edge. Each pair of
sections contained one section labeled for Na+/K+-ATPase
and the other for H+-ATPase. A minimum of two filaments per fish,
and three fish per treatment were analyzed.
Transmission electron microscopy
Gill samples for transmission electron microscopy (TEM) were fixed in 1.5%
glutaraldehyde, 3% paraformaldehyde, 0.1 mol l-1 cacodylate buffer
(pH 7.4) for 6 h at 4°C, immersed in 50% ethanol for 2 h and stored in 70%
ethanol at 4°C. Samples were rehydrated after arrival to Edmonton,
post-fixed in 2% OsO4 for 2 h, dehydrated in an ethanol graded
series, transferred to propylene oxide and embedded in Epon resin. Ultrathin
sections (90 nm) were cut with an automatic microtome (Reichert Ultracut
E), mounted in copper grids, counter stained with 1% uranyl acetate (1 h) and
0.02% lead citrate (1 min), and observed using a TEM (Philips model 201).
Western blot analysis
Frozen gill samples were weighed, immersed in liquid nitrogen and
pulverized in a porcelain grinder. The resultant powder was resuspended in
1:10 w/v of ice-cold homogenization buffer (250 mmol l-1 sucrose, 1
mmol l-1 EDTA, 30 mmol l-1 Tris, 100 mg ml-1
PMSF and 2 mg ml-1 pepstatin, pH 7.4) and sonicated on ice for 20
s. Debris was removed by two low speed centrifugations (3000 g
for 5 min, 4°C) and gill membranes were pelleted by a final high speed
centrifugation (20 800 g for 30 min, 4°C). The resulting
pellets were resuspended in homogenization buffer and a sample was saved for
protein determination analysis (BCA protein assay reagent kit, Pierce, IL,
USA), which was performed in triplicate. The remaining sample was combined
with 2x Laemmli buffer (Laemmli,
1970) for western analysis. 30 µg (for NHE2) or 50 µg (for
H+-ATPase) of total protein were separated in a 7.5% polyacrylamide
mini-gel (45 min at 180 V) and transferred to a nitrocellulose (NC) membrane
using a semi-dry transfer cell (Bio-Rad Laboratories, Inc., USA). Following
blocking (5% chicken ovoalbumin in 0.5 mol l-1 Tris-buffered saline
(TBS) with 0.1% Triton X-100, pH 8.0, overnight at 4°C), the NC membranes
were incubated with primary antibodies against either the A-subunit of the
H+-ATPase or NHE2 (1:2500 in blocking buffer) with gentle rocking
at 4°C overnight. After four washes with TBS-Triton X-100 (0.2%), the NC
membrane was blocked briefly for 15 min and incubated with the fluorescent
secondary antibody (4°C overnight). Bands were visualized and quantified
using the Odyssey infra-red imaging system and software (Li-Cor Inc.), which
allows direct linear quantification of western blots. After quantification, NC
membranes probed against H+-ATPase were soaked in stripping buffer
(50 mmol l-1 Tris, pH 8.0, 1% SDS, 0.7% ß-mercaptoethanol) for
30 min at 60°C to remove the previously used antibodies. After stripping,
NC membranes were washed three times with TBS-Triton X-100 (0.2%) (20 min
each), blocked for 15 min, and incubated with the primary antibody against
Na+/K+-ATPase following the protocol described above. To
correct for differences in loading, protein concentration in each lane was
quantified after staining with Coomassie Brilliant Blue. Hence, the amount of
H+-ATPase, Na+/K+-ATPase and NHE2 in each
sample was given by the ratio of antibody/Coomassie Blue fluorescence. Values
are presented relative to the samples from sham-operated fish in each gel.
Membranes incubated with blocking buffer without the primary antibody served
as controls. These membranes did not show any labeling.
ATPase assays
Gill membranes were obtained as described above, with the difference that
homogenization was performed in ice-cold SEID buffer (200 mmol l-1
sucrose, 20 mmol l-1 Na2EDTA, 40 mmol l-1
imidazole, 0.5% Na+-deoxycholic acid;
McCormick, 1993). 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
l-1) and ouabain (500 µmol l-1) + bafilomicyn (50
nmol l-1), with triplicate measurements of each treatment.
Na+/K+-ATPase activity was obtained by subtracting the
ouabain-treated ATPase activity from control ATPase activity (see
McCormick, 1993
).
H+-ATPase activity was assessed by calculating the difference in
ATPase activity between the ouabain- and the ouabain + bafilomycin-treated, as
described by Hawkings et al.
(2004
). Protein concentration
in each well was determined (Pierce, IL, USA) after evaporation of the assay
solution (60°C overnight).
Statistics
All data are given as means ± S.E.M. Differences between
groups were tested using one way analysis of variance (one-way ANOVA) or
repeated-measures (RM)-ANOVA when appropriate. When RM-ANOVA was used,
differences at each sampling time were tested using one-way ANOVA followed by
Dunnet's post test, using the sham-operated or the NaCl-infused fish
as the control treatment. In all cases, the fiducial level of significance was
set at P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Na+/K+-ATPase
Na+/K+-ATPase abundance in gill cell membranes was
significantly increased after acid infusion to 315±88% of the
sham-operated fish (Fig. 2A,B).
However, Na+/K+-ATPase activity did not vary
significantly among the treatments (Fig.
2C), a discrepancy that might be explained by the low number of
samples analyzed (N=4). In all the treatments the number of
immunolabeled cells decreased from the trailing to the leading edge region of
the gill filaments, with no detectable signal in the latter. In the gill
filaments from sham-operated fish and NaCl-infused fish,
Na+/K+-ATPase immunolabeling was mostly found in cells
of the interlamellar region, but some sections also showed extensive labeling
on the lamella. In AIF and BIF, comparatively more
Na+/K+-ATPase-positive cells were present higher on the
lamella (Fig. 3), but it was
not possible to tell if the differences were real owing to high variability in
the control fish. The Na+/K+-ATPase immunostaining was
restricted to the basolateral region of the cells in all the treatments, as
shown in the higher magnification micrographs in
Fig. 4.
|
|
|
H+-ATPase
H+-ATPase abundance in the membrane fraction of gills from BIF
was threefold higher than in gills from the rest of the treatments, as
estimated from western blots (Fig.
5A,B). The H+-ATPase specific activity was also
significantly higher in the BIF (Fig.
5C). Similarly to Na+/K+-ATPase, the
H+-ATPase immunoreactive signal decreased from the trailing to the
leading edge region, being absent in the latter. This was the case for all the
treatments. In sham-operated and NaCl-infused fish, H+-ATPase
immunostaining was concentrated in the interlamellar region of the gills and
at the base of the lamella. Qualitatively, there appear to be more
H+-ATPase-positive cells in gill sections from AIF and BIF,
especially on the lamella (Fig.
6). Control and AIF, H+-ATPase-positive cells showed a
diffuse signal throughout the cytoplasm, slightly stronger in the apical
region (Fig. 7A,B). This
sub-cellular localization is similar to that previously described by Wilson et
al. (1997). Remarkably, the
H+-ATPase immunoreactivity in gills from BIF was distinctly
confined to the basolateral region of the cells
(Fig. 7C).
|
|
|
Colocalization of Na+/K+-ATPase and H+-ATPase
Piermarini and Evans (2001)
reported that in the gills of Atlantic stingray, the cells rich in
Na+/K+-ATPase did not show positive labeling for
H+-ATPase, and vice versa. To address this possibility in
the dogfish, we incubated consecutive 4 µm sections with antibodies against
either Na+/K+-ATPase or H+-ATPase. We then
identified cells in both sections and looked at the localization of the two
transporters. We found only a minority of cells that were positive for both
Na+/K+-ATPase and H+-ATPase
(Fig. 8). No obvious
differences could be seen among the treatments.
|
NHE2
To determine the involvement of a NHE2-like protein in the branchial
acid-base regulatory mechanism of the dogfish, we performed quantitative
immunoblottings on gill membrane samples of the fish from the various
treatments. All samples had a specific 80 kDa band, but the acid-infused
sample was the only one with a significantly different relative intensity
(213±5% of the sham-operated fish;
Fig. 9). We attempted to
perform colocalization studies for Na+/K+-ATPase,
H+-ATPase and NHE2, but, unfortunately, the anti NHE2-antibody did
not work for immunohistochemistry, despite trying some antigen retrievals
techniques (pre treatment with 1% SDS, 10 mmol l-1 citric acid
buffer at 97°C).
|
Transmission electron microscopy
We found mitochondria-rich (MR) cells in the filament and lamella of fish
gills from all the treatments. The MR cell fine structure was similar to that
previously reported by Laurent
(1984) and Wilson et al.
(2002
), among others. MR cells
had an ovoid appearance, with large numbers of mitochondria, long microvilli,
abundant subapical vesicles and numerous basolateral membrane infoldings
(Fig. 10). No qualitative
differences in the ultrastructure of MR cells were found in either AIF or
BIF.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blood acid-base status during infusions
The rate of acid infusion was of 495±79 µmol kg-1
h-1. As expected, blood pH showed a tendency to drop, reaching
statistical significance after 6 h of infusion, 0.45 pH units lower than
at the beginning of the experiment. However, by t=12 h blood pH had
recovered to control values, where it remained until the end of the infusions.
Moreover, we also found a transient, although not statistically significant,
increase in blood total CO2 at t=12 h. Put together, this
suggests an augmentation in the capacity for acid excretion and
HCO3- reabsorption after 6 h of continuous acid-loading
due to increased expression of proteins involved in these mechanisms.
Nevertheless, we cannot rule out a contribution of intracellular buffering and
net H+ transfer into this compartment as a potential mechanism for
regulation of extracellular pH independently of branchial ionic transfers.
This is a question that will form the basis for further experimentation.
HCO3- was infused at an even higher rate, roughly
twice as much as H+ (981±235 µmol kg-1
h-1; the rationale for the mismatch is explained in the Materials
and methods) producing immediate increases in blood pH and in plasma total
CO2. By t=6 and t=24 h these parameters
equilibrated at 8.0 pH units and 10 mmol l-1 CO2,
suggesting that a new steady state status was reached by increased by
HCO3- secretion and/or H+ reabsorption.
The magnitude of the acid-base disturbances induced by our infusions is
severe. However, even greater drops in the blood pH of dogfish have been
reported to occur in situations such as exhaustive exercise
(Richards et al., 2003) and
environmental hypercapnia (Claiborne and
Evans, 1992
). Similarly, unpublished results by C. Wood, M.
Kajimura, T. Mommsen and P. Walsh show that arterial blood pH increases
significantly between 3 h and 9 h after feeding. In their study, arterial pH
peaked at 8.1 compared to control conditions of 7.9 pH units, values very
similar to those obtained in our study.
Ion transporting cell subtypes
The immunostained sections suggest that there are at least two potential
subpopulations of ion-transporting cells in the gills of the dogfish:
H+-ATPase-rich cells and Na+/K+-ATPase-rich
cells, whose roles in acid-base regulation are discussed below. These findings
are in agreement with previous research in the Atlantic stingray by Piermarini
and Evans (2001), who
similarly found independent H+-ATPase- and
Na+/K+-ATPase-rich cells in the gills. In contrast, we
also found a small proportion of cells that labeled positive for both
transporters. However, this result must be treated with caution, since these
cells may either represent a true subpopulation of cells carrying the two
transporters or may represent an artifact of using two antibodies on
consecutive sections. Regardless, the relatively small proportion of
double-labeled cells likely indicates that, if they represent a true subtype,
their role in the branchial acid-base regulation in the dogfish would be of
minor importance compared to either the H+-ATPase- or
Na+/K+-ATPase-rich cells.
Acid secretion
It is generally accepted that the primary gill acid-secretory mechanism in
marine fish involves apical Na+/H+ exchange through
specialized gill cells. This hypothesis is based on the inward directed
Na+ gradient that fish face in seawater, which could drive acid
secretion, and supported by several studies that show the presence of either
NHE2- and/or NHE3-like proteins in the gills of hagfish
(Choe et al., 2002),
squaliform, myliobatiform and rajiform elasmobranchs
(Choe et al., 2002
;
Edwards et al., 2002
) and a
variety of teleosts (Claiborne et al.,
1999
; Choe et al.,
2002
; Wilson et al.,
2000a
). The increase in NHE2 abundance in our acid-infused fish is
evidence for the involvement of this protein in branchial acid secretion in
the dogfish. Unfortunately, we were unable to detect its cellular localization
as immunohistochemical analysis was not possible. However, based on numerous
studies in both mammalian tissue (see
Féraille and Doucet,
2001
) and in other marine fishes
(Claiborne et al., 2002
;
Edwards et al., 2002
;
Wilson et al., 2000a
), we
propose that NHE2 in dogfish is also localized on the apical membrane of
polarized epithelial cells rich in Na+/K+-ATPase.
Supporting this theory, we found an increase in abundance of the
-subunit of the Na+/K+-ATPase after acid
infusion, suggesting that Na+/K+-ATPase is involved in
powering electroneutral apical Na+/H+ by secondary
active transport. In order for this hypothesis to be tested, the generation of
specific antibodies for dogfish NHE2 is imperative.
Another mechanism to increase net branchial H+ secretion could be to use a V-type H+-ATPase located on the apical membrane. While we found an increased number of H+-ATPase-rich cells on the lamella of AIF, H+-ATPase abundance and activity in gill membrane fractions remained unchanged compared to the control fish. This apparent contradiction could be due to the fact that H+-ATPase in AIF and control fish is mainly located on cytoplasmic vesicles. Our assays for activity and quantity involved differential centrifugation that loses the cytoplasmic fraction, which may explain the discrepancy in our current findings. At this point it is worth noting that using H+-ATPases to extrude protons to seawater - a hypernatric and typically alkaline milieu - seems to be an energetically less convenient alternative than electroneutral Na+/H+ exchange. Regardless, we do see an apparent increase in the number of H+-ATPase-positive cells after acid infusion, the reasons for this increase remain obscure. It is thus possible that both mechanisms (NHE2 and H+-ATPase) contribute to acid secretion, as in the proximal tubule and thick ascending limb of the mammalian kidney (reviewed by Gluck and Nelson, 1992).
Base secretion
The samples from the BIF showed an increase in all three of the variables
related to H+-ATPase tested. Although no statistical analysis was
performed, it was apparent that more H+-ATPase-rich cells were
located on the lamella and on the gill filament of BIF than of control fish,
together with a significantly higher H+-ATPase abundance and
activity in gill membranes. The immunostained sections provided further
information regarding the involvement of H+-ATPase in base
secretion. In contrast with the rest of the treatments, H+-ATPase
was distinctly confined to the basolateral region of gill cells in the BIF. To
compensate for an alkalosis, excess HCO3- would need to
be secreted by the gills and/or H+ retained within the body. We
believe that H+-ATPases are inserted in the basolateral membrane
under alkalotic stress and function to rid the cell of excess H+
generated by hydration of CO2 by intracellular CA, which is present
in dogfish gills (Swenson and Maren,
1987; Wilson et al.,
2000b
). Furthermore, when gill intracellular CA was selectively
blocked in fish made alkalotic by NaHCO3 infusion, there was a
significant reduction of HCO3- secretion
(Swenson and Maren, 1987
),
suggesting an involvement in acid-base regulation via an apical anion
exchange. Current research in our lab is focused on elucidating the identity
of the apical anion exchanger. By analogy to the Atlantic stingray
(Piermarini et al., 2002
), the
most promising lead points to a pendrin-like protein.
In summary, we have established the presence of at least two types of ion-transporting cells, Na+/K+-ATPase- and H+-ATPase-rich cells in the gills of a marine stenohaline elasmobranch. Based on responses to acid and base infusions, we propose that a NHE2-like protein participates in acid secretion, and that basolateral V-H+-ATPases are involved in net base secretion.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brown, D. and Breton, S. (1996).
Mitochondria-rich, proton-secreting epithelial cells. J. Exp.
Biol. 199,2345
-2358.
Choe, K. P., Morrison-Shetlar, A. I., Wall, B. P. and Claiborne, J. B. (2002). Immunological detection of Na+/H+ exchangers in the gills of a hagfish, Myxine glutinosa, an elasmobranch, Raja erinacea, and a teleost, Fundulus heteroclitus. Comp. Biochem. Physiol. 131A,375 -385.
Claiborne, J., Blackston, C., Choe, K., Dawson, D., Harris, S.,
Mackenzie, L. and Morrison-Shetlar, A. (1999). A mechanism
for branchial acid excretion in marine fish: identification of multiple
Na+/H+ antiporter (NHE) isoforms in gills of two
seawater teleosts. J. Exp. Biol.
202,315
-324.
Claiborne, J. B., Edwards, S. L. and Morrison-Shetlar, A. I. (2002). Acid-base regulation in fishes: cellular and molecular mechanisms. J. Exp. Zool. 293,302 -319.[CrossRef][Medline]
Claiborne, J. B. and Evans, D. H. (1992). Acid-base balance and ion transfers in the spiny dogfish (Squalus acanthias) during hypercapnia: a role for ammonia excretion. J. Exp. Zool. 261,9 -17.
Edwards, S. L., Donald, J. A., Toop, T., Donowitz, M. and Tse, C. M. (2002). Immunolocalisation of sodium/proton exchanger-like proteins in the gills of elasmobranchs. Comp. Biochem. Physiol. 131A,257 -265.
Féraille, E. and Doucet, A. (2001).
Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the
kidney: hormonal control. Physiol. Rev.
81,345
-418.
Gilmour, K. M., Perry, S. F., Bernier, N. J., Henry, R. P. and Wood, C. M. (2001). Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion. Physiol. Biochem. Zool. 74,477 -492.[CrossRef][Medline]
Gluck, S., Nelson, R., Lee, B., Wang, Z., Guo, X., Fu, J. and
Zhang, K. (1992). Biochemistry of the renal V-ATPase.
J. Exp. Biol. 172,219
-229.
Hawkings, G. S., Galvez, F. and Goss, G. G.
(2004). Seawater acclimation causes independent alterations in
Na+/K+- and H+-ATPase activity in isolated
mitochondria-rich cell subtypes of the rainbow trout gill. J. Exp.
Biol. 207,905
-912.
Heisler, N. (1988). Acid-base regulation. In Physiology of Elasmobranch Fishes (ed. T. J. Shuttleworth), pp. 215-252. Berlin: Springer-Verlag.
Katoh, F., Hyodo, S. and Kaneko, T. (2003).
Vacuolar-type proton pump in the basolateral plasma membrane energizes ion
uptake in branchial mitochondria-rich cells of killifish Fundulus
heteroclitus, adapted to a low ion environment. J. Exp.
Biol. 206,793
-803.
Katoh, F. and Kaneko, T. (2003). Short-term
transformation and long-term replacement of branchial chloride cells in
killifish transferred from seawater to freshwater, revealed by
morphofunctional observations and a newly established `time-differential
double fluorescent staining' technique. J. Exp. Biol.
206,4113
-4123.
Katoh, F., Shimizu, A., Uchida, K. and Kaneko, T. (2000). Shift of chloride cell distribution during early life stages in seawater-adapted killifish, Fundulus heteroclitus. Zool. Sci. 17,11 -18.
Kirschner, L. B. (2004). The mechanism of
sodium chloride uptake in hyperregulating aquatic animals. J. Exp.
Biol. 207,1439
-1452.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227,680 -685.[Medline]
Laurent, P. (1984). Gill internal morphology. In Fish Physiology, vol. 10A eds H. W. S. and D. W. Randall), pp. 73-183. New York: Academic Press.
McCormick, S. D. (1993). Methods for non-lethal gill biopsy and measurements of Na+/K+-ATPase activity. Can. J. Fish Aquat. Sci. 50,656 -658.
Piermarini, P. M. and Evans, D. H. (2001).
Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills
of a euryhaline stingray (Dasyatis sabina): effects of salinity and
relation to Na+/K+-ATPase. J. Exp.
Biol. 204,3251
-3259.
Piermarini, P. M., Verlander, J. W., Royaux, I. E. and Evans, D. H. (2002). Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch. Am. J. Physiol. 283,R983 -R992.
Richards, J., Heigenhauser, G. and Wood, C. (2003). Exercise and recovery metabolism in the Pacific spiny dogfish (Squalus acanthias). J. Comp. Physiol. 173B,463 -474.
Shuttleworth, T. J. (1988). Salt and water balance-extrarenal mechanisms. In Physiology of Elasmobranch Fishes (ed. T. J. Shuttleworth), pp.171 -199. Berlin: Springer-Verlag.
Swenson, E. R. and Maren, T. H. (1987). Roles of gill and red cell carbonic anhydrase in elasmobranch HCO3- and CO2 excretion. Am. J. Physiol. 253,R450 -R458.[Medline]
Wilson, J. M., Morgan, J. D., Vogl, A. W. and Randall, D. J. (2002). Branchial mitochondria-rich cells in the dogfish Squalus acanthias. Comp. Biochem. Physiol. 132A,365 -374.
Wilson, J., Randall, D., Donowitz, M., Vogl, A. and Ip, A.
(2000a). Immunolocalization of ion-transport proteins to
branchial epithelium mitochondria-rich cells in the mudskipper
(Periophthalmodon schlosseri). J. Exp. Biol.
203,2297
-2310.
Wilson, J. M., Randall, D. J., Vogl, A. W., Harris, J., Sly, W. S. and Iwama, G. K. (2000b). Branchial carbonic anhydrase is present in the dogfish, Squalus acanthias. Fish Physiol. Biochem. 22,329 -336.[CrossRef]
Wilson, J. M., Randall, D. J., Vogl, A. W. and Iwama, G. K. (1997). Immunolocalization of proton-ATPase in the gills of the elasmobranch, Squalus acanthias. J. Exp. Zool. 278, 78-86.[CrossRef][Medline]
Wood, C. M., Part, P. and Wright, P. A. (1995).
Ammonia and urea metabolism in relation to gill function and acid-base balance
in a marine elasmobranch, the spiny dogfish (Squalus acanthias).
J. Exp. Biol. 198,1545
-1558.
Zall, D. M., Fischer, M. D. and Garner, Q. M. (1956). Photometric determination of chloride in water. Anal. Chem. 28,1665 -1678.