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-subunit in gills of the teleost
Oreochromis
mossambicus
Department of Zoology, National Chung-Hsing University, Taichung 402, and Institute of Zoology, Academia Sinica, Taipei 115, Taiwan, Republic of China
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Three
isoform-specific antibodies, 6F against the
1-isoform of the
avian sodium pump, HERED against the rat
2-isoform, and Ax2 against the
rat 3-isoform, were used to
detect the expression of
Na+-K+-ATPase
-subunits in gills of a teleost, the tilapia
(Oreochromis mossambicus). Tilapia gill tissue
showed positive reactions to antibodies specific for
1- and
3-isoforms. The results of
immunoblots were converted to numerical values (relative intensities)
by image analysis for comparisons. Relative amounts of
1-like isoform alone and
consequently the ratio of
1-like to
3-like isoforms were higher in
gills of seawater-adapted tilapia than in those of freshwater-adapted
ones, indicating that the two isoforms respond differently to
environmental salinities. In the subsequent immunocytochemical experiments, gill mitochondria-rich cells were demonstrated to immunoreact with antibodies specific for
1- and
3-isoforms. 1-like and
3-like isoforms of gill
Na+-K+-ATPase
are suggested to be involved in the ion- and osmoregulation mechanisms
in tilapia. Moreover, differential expressions of two isoforms may be
associated with different functions, secretion and uptake of ions and
acid-base regulation, in gills of seawater- and freshwater-adapted
tilapia.
chloride cell; sodium pump; ion regulation
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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A UNIVERSAL membrane-bound enzyme,
Na+-K+-ATPase,
actively transports
Na+ out of
and K+ into animal cells. It is
not only crucial for maintaining intracellular homeostasis but also for
providing a driving force for Na+
transport in a variety of osmoregulatory epithelia, including the salt
gland of the invertebrate brine shrimp; elasmobranchial rectal glands;
teleostean gills; reptilian and avian nasal salt glands; and mammalian
kidney tubules, bladder, and intestine. Na+-K+-ATPase
is composed of two noncovalently linked polypeptides, a catalytic
-subunit with a molecular mass of ~110 kDa, and a smaller
glycosylated 
-subunit with a molecular mass of ~55 kDa (27).
Sweadner (26) first showed that two electrophoretically separable forms
of the -subunit exist in mammals. Subsequently, experiments of
molecular cloning and sequencing of cDNA encoding -subunits revealed
the existence of three major -subunit isoforms, designated
1,
2, and
3, in the rat and chicken (30).
Tissue-specific and developmental expression of the -subunit
isoforms was demonstrated in birds and mammals and was suggested to
extend to all vertebrate classes, including teleosts (24, 30).
Tissue-specific expression of different isoforms was suggested to be
associated with various physiological functions. The
1-subunit functions primarily
in a housekeeping capacity to maintain osmotic balance and cell volume regulation, whereas the other -subunits fulfill more specialized requirements for cation transport necessary for differentiated cell-specific functions (12, 21).
Euryhaline teleosts inhabit environments ranging from freshwater to seawater of high salinity. Through effective mechanisms of osmoregulation, teleosts are able to retain an osmotic and ionic constancy in the internal milieus and survive in hypertonic seawater or hypotonic freshwater. Gills are the most important extrarenal organs responsible for osmoregulation in fish. Mitochondria-rich cells (MR cells, i.e., chloride cells) are the main site for active transport of ions in branchial epithelium. MR cells are suggested to have multiple functions: ion secretion in seawater-adapted fish and ion uptake and acid-base regulation in freshwater-adapted fish (5, 8, 10, 15, 16, 23), although the role of MR cells in ion uptake and acid-base regulation is still controversial (23). The biochemical mechanisms for maintenance of constant levels of ions in body fluids depend on the activity of Na+-K+-ATPase, and the activities of gill Na+-K+-ATPase in euryhaline teleosts are affected by environmental salinities and ion concentrations (4, 11, 18, 19). Moreover, biochemical and histochemical studies reveal that most of the Na+-K+-ATPases were found in MR cells (7, 13, 19).
When all these results were taken into account, the hypothesis that
gills may express multiple isoforms of
Na+-K+-ATPase
to perform various functions was proposed (9). Previous biochemical
studies provide some clues for this hypothesis. Gill Na+-K+-ATPase
from freshwater- and seawater-adapted fish differed in enzyme kinetics
(6, 22). On the other hand, in recent studies different genes encoding
Na+-K+-ATPase
-subunits have been cloned and sequenced, and the expression of
these genes in the gills and kidneys was reported in several teleosts
(2, 3, 14, 25). Some other studies also examined the expression of the
protein of
Na+-K+-ATPase
-subunit in fish gills by Western blotting (9, 35). These results,
however, could not confirm the possibility of the presence of more than
one type of
Na+-K+-ATPase
-subunit in gills of fish as suggested previously (9), because only
the probe derived from one type of isoform was used in the same species
studied. A similar situation may occur in the case of Western blots,
which were performed with antibodies that were not isoform specific (9,
35).
Obviously, there are still no direct and convincing data to demonstrate
the presence of multiple isoforms of
Na+-K+-ATPase
in gills of freshwater- and/or seawater-adapted teleosts. In
the present study, several antibodies were used to demonstrate tissue-specific distribution of various
Na+-K+-ATPase
-subunit isoforms in the teleost tilapia
(Oreochromis mossambicus). The amount of
different isoforms expressed in the gills of tilapia adapted to
different environments was compared, and the localization of different
isoforms in gill MR cells was also investigated.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals
Tilapia, O. mossambicus, one of the most popular model species for research of fish osmoregulation (5, 8, 11, 13, 15, 16, 19), was used for the present study. Male or female tilapia of 6-12 g body weight were obtained from laboratory stocks. The fish were reared separately in aerated local tap water and 33% seawater at 26-29°C with a daily 12:12-h photoperiod. The water was continuously circulated through fabric-floss filters and partially refreshed every 3 days. Fish were fed with a daily diet of commercial pellets.Preparation of Tissue Homogenates and Gill Epithelial Cells
Gills, kidneys, heart, and brain were excised and blotted dry. Gills from each individual fish yielded one sample, whereas other organs (kidneys, heart, and brain) were pooled from two or three fish to make one sample with enough protein for immnuoblottings. Gill epithelia were immediately scraped off the underlying cartilage with a scalpel, and other organs were cut into small pieces. All subsequent operations were carried out in ice. Tissues were suspended in homogenization solution (100 mM imidazole-HCl buffer, pH 7.6; 5 mM Na2EDTA; 200 mM sucrose; and 0.1% sodium deoxycholate). Homogenization was performed in a glass Potter-Elvehjem homogenizer with a motorized Teflon pestle at 600 rpm for 20 strokes. The homogenate was then filtered through nylon gauze of 106-µm-square rectangular mesh. The residue was suspended in homogenization solution and again filtered through gauze. The filtrates were subjected to gel electrophoresis and immunoblotting.To enrich epithelial cells (33), gill tissues were cut into small pieces in a dissociated buffer (2 mM Na2EDTA and 1% Percoll in PBS) and stirred slowly at 4°C for 30 min. Dissociated gill cells were filtered through nylon gauze (described above) and then gently layered on a 20% Percoll discontinuous gradient. After centrifugation with the use of swing rotors at 1,000 g and 4°C for 10 min, the white layer of epithelial cells was separated from the red layer of blood cells. Isolated gill epithelial cells were washed with PBS and centrifuged at 1,000 g and 4°C for 5 min; the pellet was finally subjected to homogenization as described above. Protein concentrations of the homogenates (whole tissues or isolated epithelial cells) were determined with the reagents of Bio-Rad Protein Assay Kit using bovine serum albumin as a standard.
Na+-K+-ATPase Antibodies
Three primary antibodies against the catalytic subunit of Na+-K+-ATPase were used in the present study: 1) mouse monoclonal antibody 6F raised against the1-isoform of the avian sodium
pump (31), 2) rabbit polyclonal
antibody raised against the HERED sequence of the rat
Na+-K+-ATPase
2-isoform (24), and
3) rabbit polyclonal antibody Ax2 raised against the rat axolemma (predominantly
3-isoform of sodium pump) (29).
Monoclonal antibody 6F was kindly provided by Dr. Douglas M. Fambrough
(Dept. of Biology, Johns Hopkins Univ., Baltimore, MD), the polyclonal
antibody HERED was provided by Dr. Thomas A. Pressley (Dept. of
Physiology, Texas Tech Univ. Health Science Center, Lubbock, TX), and
the polyclonal antibody Ax2 was provided by Dr. Kathleen J. Sweadner
(Neurosurgical Research Unit, Massachusetts General Hospital, Boston,
MA).
Gel Electrophoresis and Immunoblotting
Proteins within the homogenates were fractionated by electrophoresis on SDS containing 10% polyacrylamide gels (20 or 100 µg of protein/lane), except that the homogenates were heated at 37°C for 5 min rather than at higher temperatures. Rat brain microsomes (UBI, Lake Placid, NY) were used as a positive control for immunoblotting. The separated proteins were then transferred to PVDF membrane (Immobilon Transfer Membranes; Millipore, Bedford, MA) by electroblotting. Protein bands on the gel were visualized by Coomassie brilliant blue staining. The presence of transferred proteins on the blots was confirmed by staining with Amido black. After preincubation for 2 h in PBST buffer [137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.2% (vol/vol) Tween 20, pH 7.4] containing 2% (wt/vol) nonfat dried milk to minimize nonspecific binding, the blots were incubated for 1 h with primary antibody diluted in PBST (200-fold dilution), washed in PBST, and reacted for 30 min with alkaline phosphatase-conjugated secondary antibodies (goat anti-mouse IgG, Pierce, Rockford, IL; goat anti-rabbit IgG, Sigma, St. Louis, MO; both diluted 1:1,000). Blots were developed after incubation with 0.015% nitro-blue tetrazolium, 0.007% bromochloroindolyl phosphate in a reaction buffer containing 100 mM Tris, 100 mM NaCl, and 5 mM MgCl2, pH 9.5. Immunoblots were scanned and imported as joint photographic experts group files into a commercial software package (Image-Pro Plus, Version 1.2, Silver Spring, MD). Stained bands in the finished images were analyzed and converted to numerical values to show the relative intensities of the immunoreactive bands.Four repeated immunoblottings of tilapia organs (Fig. 1) were conducted using different samples. For immunoblottings of tilapia gills (Fig. 2), four individuals from each group (seawater or freshwater) were used.
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Immunocytochemistry and Confocal Microscopy
Concanavalin A and Na+-K+-ATPase-subunits. Because Concanavalin A (ConA) is located
at the apical surface of gill MR cells, whole mount preparations of
gill filaments were made for the double labeling. Gills were excised
and immediately fixed in 4% paraformaldehyde-1% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 5 min at 4°C. The briefly fixed
gill filaments were first stained with 1% ConA (conjugated with Texas
red, Sigma) for 20 min. After being permeablized with 75% ethanol for
3 min and being incubated with 10% normal goat serum (Jackson, West
Grove, PA) in PBS for 30 min, the gill filaments were stained with
1-specific monoclonal antibody
(6F, diluted 1:50) or
3-specific polyclonal antibody (Ax2, diluted 1:50) for 1 h at room temperature. Then goat anti-mouse IgG (or goat anti-rabbit IgG) conjugated with fluorescein
isothiocyanate (FITC; Jackson, diluted 1:100) was added for another 1 h. The antibodies were diluted in PBS that contained 0.05% Tween 20 and 3% bovine serum albumin (Sigma). Between each step, three 10-min washes in PBS were performed.
Stained gill filaments were observed by a Bio-Rad MRC 600 confocal
laser scanning microscope that was attached to a Nikon microscope and
equipped with an argon laser (488 and 514 nm) for excitation. The
Na+-K+-ATPase
1 (or
3) images (FITC) were taken
under an A1 (BP 525-555 nm) filter set (Fig.
3A),
whereas ConA images (Texas red) were obtained with the use of an A2 (LP
600 nm) filter set (Fig. 3B).
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Na+-K+-ATPase
1 and
3.
Gills were excised and immediately fixed in 4% paraformaldehyde-1%
glutaraldehyde in 0.1 M PB (pH 7.4) for 1 h at 4°C. After
rinsing briefly with PB, 10-µm-thick frozen cross sections of the
gill filaments were made with a cryostat (Bright, Cambridgeshire, UK).
The subsequent procedures for double staining were similar to those
above, except that the two primary antibodies
(1 and
3) were simultaneously added
for 1-h staining and then two secondary antibodies, goat anti-mouse IgG
conjugated with FITC (for 1)
and goat anti-rabbit IgG conjugated with tetramethylrhodamine isothiocyanate (TRITC; for
3), were also simultaneously
added for another 1 h. Control sections were processed in parallel
without primary antibody or substitution of primary antibody with
nonimmune normal rabbit or mouse serum.
The slide was then observed with a Bio-Rad MRC 1000 confocal
laser scanning microscope. The microscope was attached to a Zeiss microscope, and a krypton-argon laser (488 nm and 568 nm) was equipped
for excitation. The
Na+-K+-ATPase
1 images (FITC) were taken
under a 515/32 filter set, whereas
Na+-K+-ATPase
3 images (TRITC) were obtained
with the use of a 580/32 filter set. Figures
4 and 5
demonstrate that the mutual interference of the two filter sets was
excluded, because the 1 image
(FITC) could not be observed under the 580/32 filter set (Fig.
4B) and the
3 image (TRITC) could not be
observed under the 515/32 filter set (Fig.
5B).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Immunoblotting of
Na+-K+-ATPase
1 and
3
-subunits among vertebrates (9), we were able to detect the
distribution of three rat or chicken isoform-specific antibodies
of
Na+-K+-ATPase
-subunit in different tissues, e.g., brain, kidneys, heart, and
gills of freshwater-adapted tilapia (Fig. 1), and the results revealed
a tissue-specific distribution of the three isoforms. We detected a
major immunoreactive band of the
1-specific antibody with a
relative molecular mass of ~100 kDa in the homogenates from kidneys
and gills. We failed to detect any major bands of the
2-specific antibody HERED. We
detected a major protein band of the
3-specific antibody Ax2 in
brain, heart, and gills, but not in kidneys, of freshwater-adapted
tilapia.
To reconfirm the expression of
1-like and
3-like isoforms of
Na+-K+-ATPase
in tilapia gills, enriched gill epithelial cells, instead of whole gill
tissues, were used in the subsequent immunoblotting. The results
demonstrate the coexistence of the protein of
1-like and
3-like subunits of
Na+-K+-ATPase
in gills of both freshwater- and seawater-adapted tilapia (Fig. 2).
Moreover, the two isoforms appeared to respond differently to
environmental salinities. The results of Western blots (Fig. 2) based
on image analysis (see MATERIALS AND
METHODS) indicate that the amount of
Na+-K+-ATPase
1-like subunit in the seawater
group was significantly higher than that in the freshwater group, but
no significant difference in the amount of
3-like subunit was found
between the two groups (Table 1).
Consequently, the ratio of
1-like to
3-like subunits in the seawater
group was also higher than that in the freshwater group (Table 1).
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Double Staining of
Na+-K+-ATPase
1 and
3 in MR Cells
1-antibody. All cells that were
stained with 1-specific antibody (Fig. 3A) were also labeled
with ConA (Fig. 3B). Similar results
were obtained in the case of double staining of ConA and 3-specific antibody (data not
shown). These results confirm that Na+-K+-ATPase
1-like and
3-like subunits are stained
exclusively in MR cells.
Figure 6 shows the confocal images of a
frozen cross section of gill filament that was doubly stained with both
antibodies specific for
Na+-K+-ATPase
1 and
3. The staining patterns of
these two isoforms of enzyme are not identical in the same gill
sections (compare Fig. 6A with
6B). Some
Na+-K+-ATPase
3-positive MR cells were weakly
stained with the 1-specific antibody (Fig. 6B), whereas several
Na+-K+-ATPase
1-positive MR cells were weakly
or negatively stained with the
3-specific antibody (Fig.
6A). However, it should be noted
there were a few MR cells strongly stained with both these two isoforms
of the enzyme (Fig. 6, A and
B).
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Negative control experiments, in which normal goat serum was used instead of the isoform-specific antibodies, have been conducted (data not shown) to confirm the above positive results.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present study demonstrates for the first time that two isoforms,
1- and
3-like, of
Na+-K+-ATPase
are expressed in gill MR cells of the euryhaline teleost tilapia
(O.
mossambicus) and that the
expressions of the two isoforms in the gills vary depending on
environmental salinities to which the fish are adapted. These results
suggest that these two isoforms of
Na+-K+-ATPase
-subunit are involved in the mechanisms of ion regulation in
freshwater- and seawater-adapted tilapia.
Tissue-specific and developmental expression of the -subunit
isoforms has been reported in birds and mammals and was also suggested
to extend to all vertebrate classes, including teleosts (24, 27).
However, direct proof of teleostean -subunit expression has not been
available until now. With recent advances in molecular cloning, the
genes of
Na+-K+-ATPase
-subunits have been isolated from several teleosts, including white
sucker (Catostomus
commersoni; -subunit (26), European eel (Anguilla
anguilla;
1 subunit) (2), Atlantic salmon
(Salmo salar; partial sequence of
-subunit) (3), brown trout (Salmo trutta; partial sequence of
-subunit), and other species (14). The mRNA of - or
1-subunit has been found to
increase after acclimation of freshwater fish to seawater (2, 14, 17) or during smoltification (3). So far, two or more genes of Na+-K+-ATPase
-subunits have never been found in the same fish species studied. On
the other hand, Ura et al. (35) raised a polyclonal antibody against a
synthetic oligopeptide that was based on the sequences of
Na+-K+-ATPase
-subunits from different vertebrates and invertebrates, and Hwang et
al. (9) established a polyclonal antibody that was raised against the
fusion protein derived from a partial
Na+-K+-ATPase
1 cDNA of tilapia
(O.
mossambicus). Neither antibody showed isoform-specific specificity. Because of the high identities of
cDNA sequences of different isoforms, Northern blots using one isoform
as the probe cannot discriminate between the mRNAs of two isoforms of
similar size. A similar possibility may occur in the case of Western
blots with antibodies that are general for all isoforms.
With the use of three isoform-specific antibodies, the present study
provides direct evidence for the presence of multiple isoforms of
Na+-K+-ATPase
-subunit in different organs of a teleost.
In accordance with Takeyasu et al. (30) and Pressley (24), sequences of
NH2-terminal and of 11 amino acids
(487-497) in the central portion of the
Na+-K+-ATPase
-subunit are markedly different between isoforms. Cutler et al. (2)
reported that
Na+-K+-ATPase
1 of European eel shared 8 of
11 amino acids (487-497) with the consensus
1 sequence compared with 3 of
11 or 1 of 11 with the 2 or
3 sequences, respectively; and
the NH2-terminal region of eel
1 is also more similar to that
of 1 than to those of the other
isoforms. The amino acid 384-504 of
Na+-K+-ATPase
1 of tilapia was found to show
83.5-84.3, 73.3, and 76.0-76.9% homologies with those of rat
and chicken 1,
2, and
3, respectively (9). In
accordance with Hwang et al. (9), the
Na+-K+-ATPase
-subunit of white sucker (C. commersoni) and electric ray
(Torpedo
californica) shows the highest
homology with 1 and 3 of rat and chicken,
respectively. On the other hand, mouse monoclonal antibody 6F, which
was raised against the
1-isoform of the avian sodium
pump, has been demonstrated to be
1 specific and to show broader
species specificity (1). The rabbit polyclonal antibody, which was
raised against the HERED sequence of the rat Na+-K+-ATPase
2-isoform, shows species
specificity for mammals only (24), suggesting that the possibility for
the presence of 2 in fish still
cannot be excluded. Rabbit polyclonal antibody Ax2 raised against rat
axolemma (predominately 3 of
the sodium pump) has been found to have good reactivity with
3 but poor reactivity with
2 (20). On the basis of this
information, the present results indicate that at least two isoforms of
Na+-K+-ATPase
-subunit, 1-like and
3-like, are expressed in
tilapia tissues, including gills. However, we must note that the
relative expression of these isoforms in tissues of tilapia appears to be different from what has been reported in mammals. Indeed, Cutler et
al. (2) also found that the mRNA expression pattern of
1 in various organs of the
European eel was different from that of mammals. With regard to this
discrepancy, two possibilities are proposed.
1) The functions, and thus the
relative expression, of these isoforms in fish may be different from
those in mammals. 2) Because the
antibodies used in the present study are heterogeneous for tilapia,
there may be some problem of crossreaction. The abundance of isoforms
in some organs of tilapia may be too low to be detected by these
heterogeneous antibodies. It is necessary to raise antibodies specific
to fish isoforms to answer the above questions.
By using four site-specific-directed polyclonal antibodies, Pressley
(24) found that both catfish brain and gill show positive reactions to
the LEAVE-directed antibody (general for all
Na+-K+-ATPase
-subunits), whereas only catfish brain was found to positively react
with TED-directed antibody (specific for
Na+-K+-ATPase
3). Pressley (24) indicated the possibility of the presence of multiple isoforms in teleosts and argued that the gill must
express an isoform other than
3. The inconsistency between
Pressley's results and the present data on gills should not merely be
ascribed to the different species studied. Rather, differences in the
antibodies (as described above, TED vs. Ax2) and the experimental
methods used in Pressley's study and the present studies should be
considered. Pressley used the homogenates of gill tissue, whereas in
the present study we used homogenates of enriched gill epithelial
cells. The enrichment procedure apparently enhances the reaction of the
epithelial cells to the
3-specific antibody (Ax2).
It has been well documented that tissue-specific, cell-specific, and
developmental stage-specific distribution of
Na+-K+-ATPase
-isoforms are of physiological significance. Previous studies on the
correlation of isoform distribution and enzyme kinetics in mammals
indicate that 1-,
2-, and
3-isoforms differ in the
affinities to cardiac glycoside,
Na+ and
K+ ions, etc. (12, 27). Recent
studies further demonstrate that not only isoform-specific but also
tissue-specific differences in the expression of
Na+-K+-ATPase
-subunits are related to the apparent affinities for both
Na+ and
K+ ions (21, 32). Jewell and
Lingrel (12) and Munzer et al. (21) have previously hypothesized that
1 represents a
"housekeeping" form of
Na+-K+-ATPase
that is capable of responding to typical physiological demands and
that, in the case of neurons or other excitable tissues, the larger
influxes of Na+, such as those
likely to occur during repeated action potentials, overwhelm the
capacity of housekeeping pumps and require the involvement of other
types of isoforms that can deal with an excess of intracellular Na+. According to the present
results, two isoforms of
Na+-K+-ATPase
-subunits are differentially expressed in the gills of sweater- and
freshwater-adapted tilapia and appear to be of physiological significance. Considering the dramatic differences in
ionic compositions of seawater and freshwater environments, the
intracellular and extracellular conditions in the gill cells of fish
adapted to the two environments may be subtly different. Different
combinations of two or more isoforms with different enzymatic
characteristics, rather than only one type of isoform, may be necessary
to carry out excretion and uptake of various ions. Indeed, previous
studies indicated that branchial
Na+-K+-ATPase
shows differences in the enzyme kinetics and optimal activity conditions between freshwater- and seawater-adapted coho salmon, Oncorhynchus
kisutch (6), or rainbow trout (22).
The present results provide some clues for this inference; however,
further studies are needed to confirm it.
Most tissues express more than one isoform because of the multicellular
composition of these tissues and because different isoforms are
expressed in a cell-specific manner (28). For example, in the central
nervous system of rat, neural cell bodies have been found to contain
predominantly one of three -subunits; however, mixtures of any two
isoforms or all three have also been found. Glia were observed to
express either 1 or
2 or both. In the case of
myelinated tracts, some had predominantly
3 in the axons, others had
1 and
2 (20, 28). In the present
study we found that some gill MR cells express both
1-like and
3-like subunits of
Na+-K+-ATPase,
whereas others predominantly express either
1-like or 3-like. It is not surprising to
find heterogeneity at the level of enzymatic expression in animals
other than mammals. However, this provides some clues for our previous
hypothesis concerning the structures and functions of gill MR cells.
Recent studies report that several types of gill MR cells with
different structures are found in freshwater- and seawater-adapted fish
(8, 10, 13, 15, 34). Moreover, the composition of these different types
of MR cells was found to correlate with the ionic composition in
environments, suggesting that these MR cells are responsible for the
transport of different ions in fish adapted to diverse environments
(15, 34). To test this hypothesis, further studies are needed to
examine whether these types of MR cells show subtle differences in the
expression of 1-like,
3-like, or both subunits of
Na+-K+-ATPase.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. Douglas M. Fambrough, Dr. Thomas A. Pressley, and Dr. Kathleen J. Sweadner for providing antibodies. Special thanks are also given to Guan-Yuh Cho for assistance in confocal microscopy and image processing.
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FOOTNOTES |
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This study was supported by grants to P.-P. Hwang from the National Science Council, Taiwan, Republic of China (NSC-84-2311-B001-057 and NSC-85-2311-B001-067).
Address for reprint requests: P.-P. Hwang, Institute of Zoology, Academia Sinica, Taipei, Taiwan 11529, Republic of China.
Received 21 May 1998; accepted in final form 27 October 1997.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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