Manuscript Number:
Title: Immunolocalization of Chloride Transporters to Gill Epithelia of Euryhaline Teleosts with Opposite Salinity-induced Na+/K+-ATPase Responses
Article Type: Original research
Keywords: Na+/K+-ATPase; Na+/K+/2Cl- cotransporter; cystic fibrosis transmembrane conductance regulator; anion exchanger 1; chloride channel 3; salinity; gill; teleost.
Corresponding Author: Dr. Tsung-Han Lee, Ph.D.
Corresponding Author's Institution: National Chung Hsing University First Author: Cheng-Hao Tang, Ph.D.
Order of Authors: Cheng-Hao Tang, Ph.D.; I-Da Shen; Yu-Hui Chiu; Tsung-Han Lee, Ph.D.
Abstract: Opposite patterns of branchial Na+/K+-ATPase (NKA) responses were found in euryhaline milkfish (Chanos chanos) and pufferfish (Tetraodon nigroviridis) upon salinity challenge. Because the electrochemical gradient established by NKA is thought to be the driving force for transcellular Cl- transport in fish gills, the aim of this study was to explore whether the differential patterns of NKA responses found in milkfish and pufferfish would lead to distinct distribution of Cl- transporters in their gill epithelial cells indicating different Cl- transport mechanisms. In this study,
immunolocalization of various Cl- transport proteins, including Na+/K+/2Cl- cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 1 (AE1), and chloride channel 3 (ClC-3), were double-stained with NKA, the basolateral marker of branchial mitochondrion-rich cells (MRCs), to reveal the localization of these transporter proteins in gill MRC of FW- or SW-acclimated milkfish and pufferfish. Confocal microscopic observations showed that the localization of these transport proteins in the gill MRCs of the two studied species were similar. However, the number of gill NKA immunoreactive (IR) cells in milkfish and pufferfish exhibited to vary with environmental salinities. An increase in the number of NKA-IR cells should lead to elevation of NKA activity in FW milkfish and SW pufferfish. Taken together, the opposite branchial NKA responses observed in milkfish and pufferfish upon salinity challenge could be attributed to alterations in the number of NKA-IR cells. Furthermore, the localization of these Cl- transporters in gill MRCs of the two studied species was identical. It might depict these two studied species possess similar Cl- transport mechanisms in gills.
Submitted to the Fish Physiology and Biochemistry 2010 Sep. 22nd
Immunolocalization of Chloride Transporters to Gill Epithelia of
Euryhaline Teleosts with Opposite Salinity-induced Na
+/K
+-ATPase
Responses
Cheng-Hao Tang1• I-Da Shen1• Yu-Hui Chiu1•Tsung-Han Lee1,2,*
1. Department of Life Sciences, National Chung-Hsing University, Taichung, 402 Taiwan
2. Department of Biological Science and Technology, China Medical University, Taichung, 404 Taiwan
*Correspondence: Tsung-Han Lee, Ph.D. Department of Life Sciences
National Chung-Hsing University
250 Kuo-Kuang Road, Taichung, 402, Taiwan Tel: +886-4-2285-6141
Fax: +886-4-2287-4740
E-mail: [email protected]
Running title: CHLORIDE TRANSPORTERS IN GILLS OF EURYHALINE TELEOSTS
Abstract
Opposite patterns of branchial Na+/K+-ATPase (NKA) responses were found in
euryhaline milkfish (Chanos chanos) and pufferfish (Tetraodon nigroviridis) upon
salinity challenge. Because the electrochemical gradient established by NKA is
thought to be the driving force for transcellular Cl- transport in fish gills, the aim of
this study was to explore whether the differential patterns of NKA responses found in
milkfish and pufferfish would lead to distinct distribution of Cl- transporters in their
gill epithelial cells indicating different Cl- transport mechanisms. In this study,
immunolocalization of various Cl- transport proteins, including Na+/K+/2Cl
-cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR),
anion exchanger 1 (AE1), and chloride channel 3 (ClC-3), were double-stained with
NKA, the basolateral marker of branchial mitochondrion-rich cells (MRCs), to reveal
the localization of these transporter proteins in gill MRC of FW- or SW-acclimated
milkfish and pufferfish. Confocal microscopic observations showed that the
localization of these transport proteins in the gill MRCs of the two studied species
were similar. However, the number of gill NKA immunoreactive (IR) cells in milkfish
and pufferfish exhibited to vary with environmental salinities. An increase in the
number of NKA-IR cells should lead to elevation of NKA activity in FW milkfish and
milkfish and pufferfish upon salinity challenge could be attributed to alterations in the
number of NKA-IR cells. Furthermore, the localization of these Cl- transporters in gill
MRCs of the two studied species was identical. It might depict these two studied
species possess similar Cl- transport mechanisms in gills.
Keywords Na+/K+-ATPase．Na+/K+/2Cl- cotransporter．cystic fibrosis
transmembrane conductance regulator．anion exchanger 1．chloride channel 3;
Introduction
Euryhaline teleosts are able to survive in environments with a broad range of salinities,
and excellent osmoregulatory mechanisms are required. SW teleosts drink water and
actively excrete excess NaCl via the gills to compensate for passively lost water and
gained salt. In contrast, freshwater (FW) teleosts actively absorb NaCl through the
major osmoregulatory organ, which is the gill (Evans et al. 2005).
Gill epithelium is characterized by the presence of three major cell types: (1)
pavement cells (PVCs), (2) mitochondrion-rich cells (MRCs; chloride cells), and (3)
mucous cells (Laurent et al. 1985). More than 90% of the gill surface epithelium and
usually all lamellar surfaces are characterized by PVCs. Although these cells,
especially on the lamellae, were thought to be the site of transepithelial gas transfer,
recent evidence suggested they might have the roles in ion and acid-base regulation
(Evans, 1987; Evans et al. 2005). Branchial MRCs are characterized by the presence
of high levels of mitochondria and an extensive tubular system continuous with the
basolateral membrane in the cytoplasm (Evans et al. 2005; Hwang and Lee, 2007).
These cells have been reported to be the major site for conducting osmoregulation and
ionoregulation (for review, see Marshall, 2002; Hirose et al. 2003; Evans et al. 2005;
NKA is a ubiquitous, membrane-bound enzyme that actively transports Na+ out
of and K+ into animal cells (Post and Jolly, 1957). Immunocytochemical studies
demonstrated that NKA is mainly localized in the basolateral membrane of MRCs
(McCormick, 1995; Dang et al. 2000; Lee et al. 2000; Wilson and Laurent, 2002). In
some euryhaline teleosts, NKA-immunoreactive (IR) cells are not only presented in
gill filaments but also appeared in the lamellar epithelium (reviewed by Hwang and
Lee, 2007). Furthermore, the current model of osmoregulation in gill MRCs illustrates
that basolateral NKA is essential to create the electrochemical gradient to trigger the
secondary ion transport system during salinity challenge (Marshall, 2002; Hirose et al.
2003; Hwang and Lee, 2007). Moreover, most euryhaline teleosts exhibit acclimated
changes in NKA activity following salinity changes (Marshall, 2002), and two
opposite patterns of NKA responses have been reported: (1) higher NKA in
hyperosmotic media, and (2) higher NKA in hyposmotic media (Hwang and Lee,
2007).
According to the current model of the Cl- transport mechanisms in MRCs, the
driving force for Cl- secretion is the electrochemical gradient established by NKA in
the basolateral membrane, and various Cl- transporting proteins are localized in either
the apical or basolateral membrane to deliver Cl- (Hirose et al. 2003; Evans et al.
Na+/ K+ /2Cl- cotransporter (NKCC), and cystic fibrosis transmembrane conductance
regulator Cl- channel (CFTR) (Hirose et al. 2003; Evans et al. 2005). Meanwhile, the
current model of transepithelial Cl- movements in FW fish gills is controversial.
Wilson et al. (2000a) postulated that AE1 expressed in the apical surface of gill
epithelial cells might play a role in Cl- uptake in FW tilapia and coho salmon
(Oncorhynchus kisutch; Wilson et al. 2002). In addition, the basolateral exit step for
Cl- absorption is less studied. The candidates for the basolateral Cl- channel including
the members of the chloride channel (CLC) family that are highly expressed in the
osmoregulatory organs (Hirose et al. 2003). Miyazaki et al. (1999) demonstrated that
transcripts of ClC-3, a member of the CLC family, are highly expressed in the
osmoregulatory organs of Mozambique tilapia (Oreochromis mossambicus).
Moreover, basolaterally located ClC-3 was demonstrated in the spotted green
pufferfish (Tetraodon nigroviridis) (Tang et al., 2010) and Mozambique tilapia (Tang
and Lee, 2010). Therefore, ClC-3 was a target candidate to study the basolateral
chloride channel in the present study.
Milkfish (Chanos chanos) is a marine inhabitant widely distributes throughout
the tropical and subtropical Indo-Pacific Ocean (Bagrinao, 1994). It occurs naturally
and is commercially cultured in fresh, brackish, and oceanic waters as well as in
peripheral FW inhabitant whose native range covers the rivers and estuaries of the
Southeast Asia (Rainboth, 1996). Both species are able to tolerate a direct transfer
from FW to SW or vice versa in experimental manipulation (Lin et al. 2003; 2004;
2006). Compared with SW conditions, milkfish have higher gill NKA responses in FW (described as the “higher NKA in hyposmotic media” response) (Lin et al. 2003; 2006). However, the pattern of branchial NKA responses in pufferfish is opposite to that of milkfish (described as the “higher NKA in hyperosmotic media” response) (Lin et al. 2004). Therefore, the aim of this study was to investigate whether the
different/opposite NKA responses would trigger distinct Cl- transport mechanisms
indicated by differential localization of Cl- transporters in gill MRCs of euryhaline
teleosts. Furthermore, changes in the distribution and number of the immunoreactive
cells in gills were also compared to discuss the acclimation strategies of the two
Materials and methods
Fish and experimental conditions
Juvenile milkfish (Chanos chanos) with 15.7±5.2 cm total length and 19.1± 5.9 g
body weight were obtained from a fish farm in Chia-Yi, Taiwan. Spotted green
pufferfish (Tetraodon nigroviridis) with 6.7±1.9 cm in total length and 7.4±2.6 g body
weight were purchased from a local aquarium.
All fish used in this study were kept in seawater (SW; 35‰) at 28±1°C with a
daily 12 h photoperiod for at least 4 weeks. SW was prepared from local tap water by
the addition of proper amounts of synthetic sea salt (Instant Ocean, Aquarium
Systems, Mentor, OH, USA). The SW-acclimated milkfish and pufferfish were then
transferred to either SW([Na+], 582.86 mM; [K+], 10.74 mM; [Ca2+], 15.75 mM;
[Mg2+], 32.92 mM; [Cl-], 520.84 mM) or fresh water (FW; [Na+], 2.60 mM; [K+],
0.04 mM; [Ca2+], 0.58 mM; [Mg2+], 0.16 mM; [Cl-], 0.18 mM) for more than 4 weeks
before experiments. The water was continuously circulated through fabric-floss filters.
Fish were fed a daily diet of commercial fodder (for milkfish) or dry shrimp (for
pufferfish). The rate of diet mass per body mass was about 1/25.
Antisera/antibodies
transporters (i.e., Na+/K+/2Cl– cotransporter, NKCC; cystic fibrosis transmembrane
conductance regulator, CFTR; chloride channel 3, ClC-3) to fish was previously
demonstrated by immunoblotting (Marshall et al. 2002; Katoh and Kaneko, 2003;
Hiroi and McCormick, 2007; Tang and Lee, 2007). The antisera/antibodies used in the
present study are listed below. (1) Ab-TG3 is a rabbit polyclonal antiserum that
recognizes the NKA α-subunit and was kindly provided by Prof. Pung-Pung Hwang
(Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan). This
antiserum was raised against a 565-amino-acid portion of the tilapia NKA α-subunit (Hwang et al. 1998) and recognizes all α-isoforms. A 1:100 dilution was used for immunofluorescent detection of NKA. (2) α5 is a mouse monoclonal antibody against
the avian NKA α-subunit (Takeyasu et al. 1988). This antibody was purchased from
the Developmental Studies Hybridoma Bank (DSHB, Iowa City, IA, USA). A 1:200
dilution was used for immunofluorescent detection of NKA. This antibody has been
widely used to detect gill NKA of many fish species including milkfish (Lin et al.
2003, 2006) and pufferfish (Lin et al. 2004). (3) T4 is a mouse monoclonal antibody
against the Na+/K+/2Cl– cotransporter (NKCC) and is directed against the 310 amino
acids at the carboxyl terminus of the human colon NKCC. This antibody was
purchased from the DSHB (Iowa City, IA, USA). The concentration of 0.25 g·ml–1
This antibody has been shown to be specifically immunoreactive with NKCC (both
secretory and absorptive forms) from many vertebrates, including teleost fish (Pelis et
al., 2001; Marshall et al., 2002; McCormick et al., 2003; Wu et al., 2003; Hiroi and
McCormick, 2007). (4) A mouse monoclonal antibody of cystic fibrosis
transmembrane conductance regulator (CFTR) (R&D Systems, Boston, MA, USA)
that is directed against 104 amino acids at the carboxyl terminus of the human CFTR
was used at a concentration of 0.4 g·ml–1 (1:500 in dilution). The carboxyl terminus of
CFTR is highly conserved among vertebrates. This antibody has previously been
shown to be specifically immunoreactive with CFTR of teleost fish (Marshall et al.
2002; Katoh and Kaneko, 2003; McCormick et al. 2003; Wilson et al. 2004; Hiroi et
al. 2005). (5) A rabbit polyclonal antiserum of anion exchanger 1 (AE1) against 300
amino acids of AE1 of tilapia was kindly provided by Prof. Pung-Pung Hwang
(Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan). The
similarity between the antigen of AE1 antiserum and the putative AE1 protein in
pufferfish (accession number: CAF92111) is 74%. This antiserum was used at a
dilution of 1:100 for immunofluorescent detection of AE1. (6) Anti-ClCn3 was a
rabbit polyclonal antibody against chloride channel 3 (ClC-3) (Alomone Labs,
Jerusalem, Israel) directed against the highly conserved residues 592-661 (70 amino
ClC-3-like protein in gills of pufferfish (Tang and Lee, 2007; Tang et al. 2010) and
tilapia (Tang and Lee, 2010). A 1:100 dilution was used in this study. (7) Alexa Fluor
488-conjugated goat anti-mouse and Alexa-Fluor 546-conjugated goat anti-rabbit
antibodies (Molecular Probes, Eugene, OR, USA) were used as secondary antibodies
and diluted 1:50 and 1:500, respectively, to detect primary antisera/antibodies from
mouse or rabbit. Preliminary experiments of negative controls (cryosections stained
with only primary or secondary antibodies) demonstrated that either non-specific
staining or over-staining of the background was not found (data not shown).
Fixation and cryosections of gills
First, gill arches of the left and right sides were excised and fixed immediately in a
mixture of methanol and DMSO (4:1 v/v) at -20°C for 3 h (Lin et al., 2003; 2004;
2006). Fixed samples were then washed with phosphate buffered saline (PBS; 137.00
mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, 1.76 mM KH2PO4, pH=7.4). The arch
and one row of the filaments of each gill sample were removed. The remaining
filaments were perfused with 30% sucrose in PBS for 1 h at room temperature. The
tissue was then mounted in O.C.T. (optimal cutting temperature) compound
(Tissue-Tek, Sakura, Torrance, CA, USA) for cryosection. Longitudinal sections of
Walldorf, Germany) at -25°C. The sections were placed on 0.01% poly-L-lysine
(Sigma, St. Louis, MO, USA) coated slides and kept in slide boxes at -20°C before
staining.
Immunofluorescent double staining
Cryosections were rinsed with PBS three times for 3 min then incubated in 5% bovine
serum albumin (Sigma, St. Louis, MO., USA) and 2% Tween 20 (Merck, Hohenbrunn,
Germany) in PBS for 0.5 h at room temperature. The cryosections were then washed
three times with PBS and incubated with primary polyclonal antibodies (Ab-TG3,
AE1, or ClCn3) diluted in PBS for 1 h at room temperature. After incubation, the
cryosections were washed several times with PBS, exposed to secondary antibody
(Alexa Flour 546 goat anti-rabbit antibody) at room temperature for 2 h, and washed
several times with PBS. After the first staining, the cryosections were subsequently incubated with primary monoclonal antibodies (α5, T4, or CFTR) diluted in PBS and incubated overnight at 4°C. After incubation, the cryosections were washed several
times with PBS, exposed to secondary antibody (Alexa Fluor 488 goat anti-mouse
antibody) at room temperature for 2 h, then washed several times with PBS. Then,
cryosections were mounted with ClearmountTM mounting solution (Zymed, South San
fluorescent microscope (Olympus BX50, Tokyo, Japan). Micrographs were taken
within 3 h after staining by confocal laser scanning microscopy or the fluorescent
microscope to recognize immunolocalization for subsequent immunoreactive cell
counting and morphometric analyses.
Confocal laser scanning microscopy
To determine and compare the localization of ion transporters between milkfish and
pufferfish, double immunofluorescent stained cryosections of gills were examined
with a Zeiss LSM 510 inverted laser scanning microscope (Hamburg, Germany)
equipped with an argon laser (488 and 543 nm) for excitation. The
immunofluorescent images of NKA, NKCC, CFTR, AE1, and ClC-3 were obtained
with the Alexa Fluor 488/546 filter set (BP505-530 for 488 and LP 560 for 546)
controlled by the Zeiss LSM image software. With the filter set, the emission
wavelengths of Alexa Flour 488- and Alexa Flour 546-conjugated antibodies were
separated and transmitted to different photomultipliers. The micrographs taken from
each photomultiplier were subsequently merged for simultaneous visualization of the
labels of different colors. At least three individuals of SW or FW groups (n=3) were
examined to confirm the results of immunolocalization for different Cl- transporters of
Quantification of NKA- and NKCC-Immunoreactive (IR) cells in gills of the milkfish
Preliminary observation revealed that all NKA-IR cells were NKCC-IR in
SW-acclimated milkfish. However, only a portion of NKA-IR cells expressed NKCC
in FW-acclimated milkfish. Hence, NKA- and NKCC-IR cells in FW milkfish gills
were quantified to clarify differential localizations of these two transporters. After
double staining of longitudinal cryosections with NKA and NKCC primary antibodies,
the immunoreactive cells of the cryosections of gills were observed and quantified
directly using a fluorescent microscope (Olympus BX50). NKA- and NKCC-IR cells
in the filaments (F) and lamellae (L) were counted separately on every 100 μm of gill
filament of the cryosections. Lamellar regions of the gills were defined as the parts
projecting from filaments. The interlamellar regions of filaments, including the
regions between bases of lamellae, were considered as filament areas. Three regions
on each filament and five filaments of each individual were randomly selected for
quantification. Results were expressed as the numbers of immunoreactive cells (i) per
100 μm of filaments and (ii) per 100 μm of lamellae. Mean numbers of NKA- or
NKCC-IR cells were obtained from eight individuals (n=8) of either the SW or FW
group.
Cryosections (7 μm) of gills of FW- or SW-acclimated pufferfish were cut parallel to
the long axis of the filament and mounted onto slides coated with poly-L-lysine. To
quantify the distribution of NKA-IR cells, cryosections were immunohistochemically stained with the monoclonal antibody to the NKA α-subunit (α5) followed by a commercial kit (PicTure™, Zymed, South San Francisco, CA, USA) for visualization of the immunoreaction. The immunostained cryosections were then counterstained
with hematoxylin (Merck, Hohenbrunn, Germany) and observed under a microscope
(Olympus BX50).
Preliminary observation revealed that in either SW- or FW-acclimated pufferfish,
NKA-IR cells were mainly distributed in gill filaments. Hence, the numbers of
NKA-IR cells in the filaments were counted. The filament regions can be defined as
the basal part of the lamella extending 5 μm up from the filament plus the width of the
filament itself. For each individual, 15 areas on the filaments were randomly selected.
Length of the filament was also measured to normalize cell counts to a fixed length
(100 μm). Results are expressed as the numbers of immunoreactive cells per 100 μm
of the filaments. NKA-IR cells were obtained from eight pufferfish (n=8) of either the
SW or FW group.
Values are expressed as the means ± S.E.M. For milkfish, data were compared using a
one-way analysis of variance (ANOVA). Post hoc comparisons were made using a Tukey‟s test with the differences considered to be statistically significant at P<0.05. For pufferfish, unpaired Student‟s t-test (P<0.05 was set as the level of significance) was used for analysis.
Results
Immunolocalization of Cl- Transporters
Milkfish
Localization of Na+/K+-ATPase (NKA) and Na+/K+/2Cl- cotransporter (NKCC) or
cystic fibrosis transmembrane conductance regulator (CFTR) on cryosections was
determined by using monoclonal antibodies against NKCC (T4) or CFTR with rabbit
polyclonal antiserum (Ab-TG3) to the α-subunit of NKA for double staining.
Immunolocalization of NKA in the frozen longitudinal sections from the gills of
seawater (SW) milkfish was mainly observed in the filaments (Figs. 1b, h). In
freshwater (FW) milkfish, however, both filaments and lamellae were positively
immunostained with the NKA antiserum (Figs. 1e, k). The amount of fluorescence in
the negative controls was used to define the background staining (data not shown). No
other type of cells in the gills could be immunostained with signals higher than
background. The immunolocalization of NKCC in SW milkfish gills (Fig. 1a) was
identical to that of NKA (Fig. 1b), with basolateral localization. In FW milkfish, only
a portion of NKA-immunoreactive (IR) cells expressed basolateral NKCC in both gill
filaments and lamellae (Fig. 1f). CFTR was detected in the apical regions of the
NKA-IR cells of SW-acclimated milkfish (Figs. 1g, i), and no immunoreactivity for
Distribution of NKA and anion exchanger 1 (AE1) or chloride channel 3 (ClC-3)
on longitudinal sections was examined by using a polyclonal antiserum to AE1 or a
polyclonal antibody (ClCn3) to ClC-3 together with a monoclonal antibody to the α-subunit of NKA (α5) for double staining. The immunolocalization of NKA detected by the monoclonal antibody (α5) was the same as the result using polyclonal
antiserum (Ab-TG3) and revealed the basolateral localization of NKA in filaments of
SW fish (Figs. 2g, h) and in both filaments and lamellae of FW individuals (Figs. 2e,
k). The results showed that AE1 colocalized with NKA in the basolateral membrane
of SW (Fig. 2c) and FW milkfish (Fig. 2f). Furthermore, ClC-3 was also basolaterally
colocalized with NKA in the gills of SW- (Figs. 2g, h) and FW-acclimated (Figs. 2j, k)
milkfish (Figs. 2i, l).
Pufferfish
The immunoreactions for NKCC (Figs. 3a, d) and NKA were colocalized in the
basolateral membrane of NKA-immunoreactive (NKA-IR) cells in the gill filaments
(Figs. 3c, f). The Cl- secretion channel, CFTR, (Fig. 3g) was localized to the apical
membrane of NKA-IR cells in the gill filaments of SW pufferfish (Fig. 3i).
Immunoreactivity of branchial CFTR, however, was undetectable in FW individuals
(Fig. 3j, l).
colocalized with NKA (Figs. 4b, e) in the gill filaments (longitudinal sections; Figs.
4c, f). Moreover, the immunoreactions of ClC-3 (Figs. 4g, h) and NKA (Figs. 4h, k)
in SW or FW pufferfish gills were also colocalized (Figs. 4i, l).
Quantification of the Number of Immunoreactive Cells
Fig. 5 illustrates that the numbers of NKA- and NKCC-IR cells in filaments of
SW milkfish were significantly higher than those in lamellae. Compared to those of
SW milkfish, however, the numbers of NKA- and NKCC-IR cells in the lamellae of
FW milkfish were evidently increased. The number of NKCC-IR cells but not
NKA-IR cells was significantly higher in the filaments of SW milkfish than in those
of FW milkfish. In addition, the number of NKCC-IR cells in both filaments and
lamellae were significantly lower than NKA-IR cells in FW milkfish.
In pufferfish, NKA-IR cells were mainly presented in the gill filaments in either
SW or FW individuals (Figs. 6A, B). Meanwhile, the number of NKA-IR cells in the
gill filaments of SW individuals (19.133 ± 0.32) was significantly higher than that of
Discussion
Localizations of Chloride Transporters in the Gills of SW-acclimated Teleosts
Branchial mitochondrion-rich cells (MRCs) of fish are the major site responsible for
ion secretion and absorption in hyperosmotic seawater (SW) and hyposmotic fresh
water (FW), respectively (Hirose et al. 2003; Evans et al. 2005; Hwang and Lee,
2007). Identifying the localization of ion transport proteins in apical or basolateral
membranes of MRCs is essential for studying the potential functions in ionoregulation
(Pelis et al. 2001; Wilson et al. 2000a; 2000b; 2002; McCormick et al. 2003; Hiroi et
al. 2005). A working model for the mechanisms of Cl- secretion by the gill MRCs of
SW teleosts is that basolateral NKA provides the driving force in the form of an
electrochemical gradient for the transport of Cl- from plasma into the cell via
Na+/K+/2Cl- cotransporter (NKCC) in the basolateral membrane. Then, Cl- is secreted
via apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels.
Because anti-NKCC (T4) and anti-CFTR (R&D Systems, Boston, MA, USA)
antibodies have been used to detect NKCC and CFTR in several teleost species and
the specificity has been confirmed (Wilson et al. 2000b; Pelis et al. 2001; Marshall et
al. 2002; Katoh and Kaneko, 2003; Wu et al. 2003; Hiroi et al. 2005; Hiroi and
McCormick, 2007; Tang and Lee, 2007), these two antibodies were used to detect the
localization of NKCC and CFTR was basolateral and apical, respectively, in branchial
MRCs of SW milkfish and pufferfish. The results show that the Cl- secretion
mechanism is identical in these two studied euryhaline species despite they have
opposite NKA expression patterns upon salinity challenge. In addition, NKCC
includes two isoforms, NKCC1 and NKCC2. Because of the prominent expression of
NKCC1 on basolateral confinement and it has been confirmed to operate in
chloride-secreting function, NKCC1 is considered to be the secretory isoform.
However, NKCC2 is a kidney-specific isoform and function to regulate in Cl
-absorption (Lytle et al. 1995). Therefore, the basolaterally localized gill NKCC in
milkfish and pufferfish should be NKCC1.
Localizations of Chloride Transporters in Gills of FW-acclimated Teleosts
Cl- uptake by FW fish gill is generally considered to be via Cl-/HCO3- anion
exchanger (AE) (Evans et al. 2005). AE1 is a member of the SLC4 gene family, and
is the most abundant transmembrane protein of the human erythrocytes (Davis et al.
2002). Sullivan et al. (1996) verified that AE1 mRNA was typically observed by
MRCs by in situ hybridization. Furthermore, Wilson et al. (2000a; 2002) used a
polyclonal antibody raised against rainbow trout AE1 to verify that branchial AE1
was expressed in the apical surface of MRCs in tilapia and coho salmon. The AE1
reveal a single immunoreactive band in the gills of pufferfish (Tang and Lee, 2007).
However, the localization of branchial AE1 in FW- and SW-acclimated two studied
species differs from previous findings in tilapia and coho salmon (Wilson et al. 2000a;
2002). In zebrafish (Danio rerio), mRNA of the AE1 paralog was localized to a
specific group of skin and gill cells, whereas AE1 mRNA expression was not affected
by low-Cl- artificial FW (Hwang, 2009). Similar to zebrafish, branchial AE1 in
milkfish and pufferfish might not be involved in Cl- absorption. The non-erythrocyte
AE1 protein is expressed in the basolateral membrane of alpha-intercalated cells in
mammalian kidney (Alper et al. 1989) and has a role in acid secretion. Accordingly,
AE1 might have a role in acid-base regulation or modulation of intracellular [Cl-]
homeostasis in fish gills. Meanwhile, the basolateral localization of gill AE1 implied
that another AE protein may be presented in the apical membrane that is responsible
for Cl- uptake. Piermarini et al. (2002) illustrated that pendrin was expressed in the
apical membrane of specialized ionocytes in the gill of the Atlantic stingray (Dasyatis
sabina) and might be involved in Cl- uptake. Recent studies further verified that
SLC26 anion transporters mediated branchial chloride uptake in adult zebrafish (Perry
et al. 2009). The members of the SLC26 family are, therefore, proposed to be the
candidates for performing the function of Cl- absorption in the apical membrane of
Cl- uptake mechanisms are accomplished through apical AE and the basolateral
chloride channel of branchial MRCs. However, the basolateral chloride channel of
MRCs is less studied. The CLC family has multiple roles in biological membranes,
and ClC-3 is a member that is ubiquitously expressed in diverse tissues of mammals
(Jentsch et al. 2002; 2005). In hyposmotic medium, ClC-3 represents a major
molecular entity responsible for native volume sensing of outwardly rectifying anion
channels in various cell types (Kawasaki et al. 1994; Duan et al. 1997; 2001; Hermoso
et al. 2002; Wang et al. 2000; 2003; Vessey et al. 2004). In fish, ClC-3 was cloned
from cDNA libraries of the euryhaline tilapia (Oreochromis mossambicus), and it was
found that OmClC-3 mRNA was broadly expressed in different tissues in FW- and
SW-acclimated tilapia (Miyazaki et al. 1999). Using the whole-mount staining
method, our previous study demonstrated that ClC-3-like protein and NKA were
co-localized in the basolateral membrane of gill MRCs of pufferfish (Tang et al.
2010). The present study simultaneously examined the localization of gill ClC-3-like
protein in two euryhaline species by double-immunostaining of cryosections and
found that ClC-3-like protein were basolaterally localized in gill NKA-IR cells in both
two studied species. It was thus suggested that ClC-3-like protein might have a role in
basolateral Cl- uptake in the gill MRCs of euryhaline teleosts. To our knowledge, this
teleosts (i.e., milkfish).
NKCC-Immunoreactive (IR) Cells in Gills of Euryhaline Teleosts
The most widely accepted model of Cl- regulation in FW MRCs includes NKA,
Na+/Cl- cotransporter (NCC) and AE proteins, but not NKCC (Hirose et al. 2003;
Evans et al. 2005; Hwang and Lee, 2007; Evans, 2010). In this study, however,
immunological detection revealed that basolateral NKCC exists in gill MRCs of
FW-acclimated milkfish and pufferfish. The basolaterally localized NKCC was also
reported in gills of FW-acclimated Atlantic salmon (Salmo salar) (Pelis et al. 2001;
Hiroi and McCormick, 2007), Hawaiian goby (Stenogobius hawaiiensis) (McCormick
et al. 2003), lake trout (Salvelinus namaycush) and brook trout (Salvelinus fontinalis)
(Hiroi and McCormick, 2007). Pelis et al. (2001) suggested that branchial NKCC
presented in FW MRCs might conduct the function of cell volume regulation.
Immunoreactivity against NKCC was found in some but not all NKA-IR cells (MRCs)
in the gills of Atlantic salmon parr (Pelis et al. 2001) and FW milkfish, and the
number of gill NKCC-IR cells evidently decreased when compared with Atlantic
salmon smolt (Pelis et al. 2001) and SW milkfish, implying that NKCC has a less
critical role in ion absorption in these two species. In contrast to milkfish,
immunoreactivity against NKCC was found in all MRCs of SW- and FW-acclimated
antibody to detect the protein expression of NKCC in gill membrane fraction and
revealed that the NKCC protein was detectable only in SW pufferfish (Tang and Lee,
2007). Therefore, we presumed that gill NKCC might exist mainly in the cytoplasm
rather than the plasma membrane of the MRCs in FW pufferfish.
Na+/K+-ATPase (NKA) Immunoreactive Cells in Gills of Euryhaline Teleosts
NKA is highly expressed in the gill epithelia and the distribution of
NKA-immunoreactive (IR) cells in the gill of euryhaline teleosts could be classified
into three categories: (1) NKA-IR cells appeared only in the filamental epithelia of
both FW- and SW-acclimated fish, (2) in addition to filaments, NKA-IR cells
occurred abundantly in the lamellar epithelia of the FW-acclimated individuals, and (3)
NKA-IR cells were found in both gill filaments and lamellae of either FW- or
SW-acclimated fish (reviewed in Hwang and Lee, 2007). The distribution of NKA-IR
cells in milkfish and pufferfish could be classified into categories (2) and (3),
respectively. Although MRCs in lamellae were proposed to be the site of ion uptake
in hyposmotic conditions while those in the filaments were proposed to be for salt
secretion in hyperosmotic conditions in several studies (Uchida et al. 1996; Sasai et al.
1998; Hirai et al. 1999). Lin and Sung (2003) supported the hypothesis of Laurent and
Perry (1990) and Dang et al. (2000) that the functions of filament and lamellar MRCs
-transporters in NKA-IR cells in both filaments and lamellae of FW-acclimated
milkfish provided more evidence that MRCs in filaments and lamellae might have
similar functions.
Milkfish is a “higher NKA in hyposmotic media ” species, and the number of NKA-IR cells was significantly higher in FW milkfish due to a significant increase of
NKA-IR cells in lamellae. Hence, the lamellar MRCs are likely crucial for the
elevation of gill NKA responses in milkfish challenged by hypotonic conditions.
Whereas pufferfish is a “higher NKA in hyperosmotic media” species, and the
distribution pattern of branchial NKA-IR cell was different from that of milkfish. In
contrast to milkfish, the number of NKA-IR cells in SW pufferfish was significantly
higher than that of the FW individuals. This pattern is similar to the observations
made in Hawaiian goby, in which higher NKA-IR cell numbers and NKA activity
were reported in gills of the SW-acclimated group compared to the FW group
(McCormick et al. 2003). Therefore, an increase in the number of gill NKA-IR cells
in SW pufferfish might lead to higher NKA activity (Lin et al. 2004) as well as the
relative protein abundance of NKA, NKCC, and CFTR (Tang and Lee, 2007) in this
group of euryhaline teleosts.
Conclusion
distinct Cl- transport mechanisms in the gills of euryhaline teleosts. However, our
results showed that the Cl- regulatory mechanisms were similar between the two
studied species. This similarity might reflect that those Cl- transporter proteins are
important for euryhaline teleosts acclimated to various salinities, although their
primary nature habitats are different. Moreover, the opposite salinity-induced NKA
responses of the two studied species may result from the increase in NKA-IR cells in
lamellae of FW milkfish and filaments of SW pufferfish, respectively. The results of
this study depict a more detailed Cl- transport model than was previously described in
the FW gill MRCs, and the mechanisms of Na+ absorption will be investigated in our
future studies.
Acknowledgements
The rabbit polyclonal antiserum to tilapia AE1 was kindly provided by Prof.
Pung-Pung Hwang (Institute of Cellular and Organismic Biology, Academia Sinica,
Taipei, Taiwan). The monoclonal antibodies T4 and α5 were obtained from the
Developmental Studies Hybridoma Bank developed under the auspices of the NICHD
and maintained by the Department of Biological Sciences, University of Iowa, Iowa
City, IA. This study was supported by a grant to T.H.L. from the National Science
Council of Taiwan (NSC 93-2313-B-005-007).
References
Alper SL, Natale J, Gluck S, Lodish HF, Brown D (1989) Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase. Proc Natl Acad Sci 86:5429-5433.
Bagrinao T (1994) Systematics, distribution, genetics and life history of milkfish, Chanos chanos. Environ Biol Fish 39:23-41.
Crear D (1980) Observations on the reproductive state of milkfish populations (Chanos chanos) from hypersaline ponds on Christmas Island (Pacific Ocean). Proc World Maricul Soc 11:548-566.
Dang, ZC, Balm PH, Flik G, Wendelaar Bonga SE, Lock RAC (2000) Cortisol increases Na+/K+-ATPase density in plasma membranes of gill chloride cells in the freshwater tilapia Oreochromis mossambicus. J Exp Biol 203:2349-2355. Davis EM, Musch MW, Goldstein L (2002) Transfection of an inducible trout anion
exchanger (AE1) into HEK-EcR cells. J Exp Zool 293:46-57. Duan D, Winter C, Cowley S, Hume JR, Horowitz B (1997) Molecular
identification of a volume-regulated chloride channel. Nature 390:417-421.
Duan D, Zhong J, Hermoso M, Satterwhite CM, Rossow CF, Hatton WJ, Yamboliev I, Horowitz B, Hume JR (2001) Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody. J Physiol 531:437-444.
Evans DH (1987) The fish gill: site of action and model for toxic effects of environmental pollutants. Environ Health Perspect 71:47-58.
Evans DH (2010) A brief history of the study of fish osmoregulation: the central role of the Mt. Desert Island Biological Laboratory. Frontiers Physiol 1:13.1-13.10. Evans DH, Piermarini PM, Choe K (2005) The multifunctional fish gill: dominant site
nitrogenous waste. Physiol Rev 85:97-177.
Hermoso M, Satterwhite CM, Andrade YN, Hidalgo J, Wilson SM, Horowitz B, Hume JR (2002) ClC-3 is a fundamental molecular component of
volume-sensitive outwardly rectifying Cl– channels and volume regulation in HeLa cells and Xenopus laevis oocytes. J Biol Chem 277:40066-40074.
Hirai N, Tagawa M, Kaneko T, Seikai T, Tanaka M (1999) Distributional changes in branchial chloride cells during freshwater adaptation in Japanese sea bass
Lateolabrax japonicus. Zool Sci 16:43-49.
Hiroi J, McCormick S D (2007) Variation in salinity tolerance, gill Na+/K+- ATPase, Na+/K+/2Cl– cotransporter and mitochondria-rich cell distribution in three salmonids Salvelinus namaycush, Salvelinus fontinalis and Salmo salar. J Exp Biol 210:1015-1024.
Hiroi J, McCormick SD, Ohtani-Kaneko R, Kaneko T (2005) Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na+/K+-ATPase, Na+/K+/2Cl- cotransporter and CFTR anion channel. J Exp Biol 208:2023-2036.
Hirose S, Kaneko T, Naito N, Takei Y (2003) Molecular biology of major components of chloride cells. Comp Biochem Physiol B 136:593-620. Hwang PP (2009) Ion uptake and acid secretion in zebrafish (Danio rerio). J
Exp Biol 212: 1745-1752.
Hwang PP and Lee TH (2007) New insights into fish ion regulation and mitochondrion-rich cells. Comp Biochem Physiol A 148:479-497.
Hwang PP, Fang MJ, Tsai JC, Huang CJ, Chen ST (1998) Expression of mRNA and protein of Na+-K+-ATPase α subunit in gills of tilapia (Oreochromis
Jentsch TJ, Stein V, Weinreich F, Zdebik AA (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82:503-568.
Jentsch T J, Poet M, Fuhrmann JC, Zdebik AA (2005) Physiological functions of CLC Cl- channels gleaned from human genetic disease and mouse models. Annu Rev Physiol 67:779-807.
Katoh F, 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 206:113-4123.
Kawasaki M, Uchida S, Monkawa T, Miyawaki A, Mikoshiba K, Marumo F, Sasaki S (1994) Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells. Neuron 12:597-604.
Laurent P, Hobe H, Dunel-Erb S (1985) The role of environmental sodium chloride relative to calcium in gill morphology of freshwater salmonid fish. Cell Tissue Res 240:675-692.
Laurent P, Perry S (1990) The effects of cortisol on gill chloride cell morphology and ionic uptake in the freshwater trout, Salmo gairdneri. Cell Tissue Res
259:429-442.
Lee TH, Hwang PP, Shieh YE, Lin CH (2000) The relationship between „deep-hole‟ mitochondria-rich cells and salinity adaptation in the euryhaline teleost,
Oreochromis mossambicus. Fish Physiol Biochem 23:133-140.
Lin CH, Tsai RS, Lee TH (2004) Expression and distribution of Na, K-ATPase in gill and kidney of the green spotted pufferfish, Tetraodon nigroviridis, in response to salinity challenge. Comp Biochem Physiol A 138:287-295.
air-breathing fishes. Physiol Biochem Zool 76:215-228.
Lin YM, Chen CN, Lee TH (2003) The expression of gill Na, K-ATPase in milkfish, Chanos chanos, acclimated to seawater, brackish water, and fresh water. Comp Biochem Physiol A 135:489-497.
Lin YM, Chen CN, Yoshinaga T, Tsai SC, Shen ID, Lee TH (2006) Short-term effects of hyposmotic shock on Na+/K+-ATPase expression in gills of the euryhaline milkfish, Chanos chanos. Comp Biochem Physiol A 143:406-415. Lytle C, Xu J, Biemesderfer D, Forbush BIII (1995) Distribution and diversity of
Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol Cell Physiol 269, 1496-1505.
Marshall WS (2002) Na+, Cl−, Ca2+ and Zn2+ transport by fish gills: retrospective review and prospective synthesis. J Exp Zool 293:264-283.
Marshall WS, Lynch EM, Cozzi RRF (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 SD (1995) Hormonal control of gill Na+,K+-ATPase and chloride cell function. In: Wood CM, Shuttleworth TJ, editors. Cellular and Molecular Approaches to Fish Ionic Regulation. New York: Academic Press. p 285-315. McCormick SD, Sundell K, Bjornsson BT, Brown CL, Hiroi J (2003) 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). J Exp Biol 206:4575-4583.
Miyazaki H, Uchida S, Takei Y, Hirano T, Marumo F, Sasaki S (1999) Molecular cloning of CLC chloride channels in Oreochromis mossambicus and their functional complementation of yeast CLC gene mutant. Biochem Biophys Res
Commun 255:175-181.
Pelis RM, Zydlewski J, McCormick SD (2001) Gill Na+-K+-2Cl- cotransporter abundance and location in Atlantic salmon: effects of seawater and smolting. Am J Physiol 280:R1844-R1852.
Perry SF, Gilmour KM (2006) Acid–base balance and CO2 excretion in fish:
unanswered questions and emerging models. Respir Physiol Neurobiol 154:199-215.
Perry SF, Vulesevic B, Grosell M, Bayaa M (2009) Evidence that SLC26 anion transporters mediate branchial chloride uptake in adult zebrafish (Danio rerio). Am J Physiol 297:R988-R997.
Piermarini PM, Verlander JW, Royaux IE, Evans DH (2002) Pendrin
immunoreactivity in the gill epithelium of a euryhaline elasmobranch. Am J Physiol 283:R983-R992.
Post RL, Jolly PC (1957) The linkage of sodium, potassium, and ammonium active transport across the human erythrocyte membrane. Biochim Biophys Acta 25:118-128.
Rainboth WJ (1996) Fishes of the Cambodian Mekong. In: FAO Species Identification Field Guide for Fishery Purposes. Rome: FAO. p 265.
Sullivan GV, Fryer JN, Perry SF (1996) Localization of mRNA for the proton pump (H+-ATPase) and Cl-/HCO3-exchanger in the rainbow trout gill. Can J Zool 74:2095-2103.
Takeyasu K, Tamkum MM, Renaud KJ, Fambrough DM (1988) Ouabain-sensitive (Na++K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the (α-subunit of an avian sodium pump). J Biol Chem 263:4347-4354. Tang CH, Lee TH (2007) The effect of environmental salinity on the protein
transmembrane conductance regulator, anion exchanger 1, and chloride channel 3 in gills of a euryhaline teleost, Tetraodon nigroviridis. Comp Biochem Physiol A 147:521-528.
Tang CH, Lee TH (2010) Ion-deficient environment induces the expression of basolateral chloride channel, ClC-3-like protein, in gill mitochondrion-rich cells for chloride uptake of the tilapia, Oreochromis mossambicus. Physiol Biochem Zool (in press).
Tang CH, Hwang LY, Lee TH (2010) Chloride channel ClC-3 in gills of the euryhaline teleost, Tetraodon nigroviridis: expression, localization and the possible role of chloride absorption. J Exp Biol 213:683-693.
Tresguerres M, Katoh F, Orr E, Parks SK, Goss GG (2006) Chloride Uptake and Base Secretion in Freshwater Fish: A Transepithelial Ion-Transport Metabolon? Physiol Biochem Zool 79:981-996.
Uchida K, Kaneko T, Yamauchi K, Hirano T (1996) Morphometrical analysis of chloride cell activity in the gill filaments and lamellae and changes in
Na+,K+-ATPase activity during seawater adaptation in chum salmon fry. J Exp Zool 276:193-200.
Vessey JP, Shi C, Jollimore CA, Stevens KT, Coca-Prados M, Barnes S, Kelly ME (2004) Hyposmotic activation of ICl, swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane. Biochem. Cell Biol 82:708-718.
Wang GX, Hatton WJ, Wang GL, Zhong J, Yamboliev I, Duan D, Hume JR (2003) Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells. Am J Physiol 285:H1453-H1463.
currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition. J Physiol 524:63-75.
Wilson JM, Laurent P (2002) Fish gill morphology: inside out. J Exp Zool 293:192-213.
Wilson JM, Laurent P, Tufts BL, Benos DJ, Donowitz M, Vogl AW, Randall DJ (2000a) NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization. J Exp Biol 203:2279-2296.
Wilson JM, Randall DJ, Donowitz M, Vogl AW, Ip AKY (2000b) Immunolocalization of ion-transport proteins to branchial epithelium
mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri). J Exp Biol 203:2297-2310.
Wilson JM, Whiteley NM, Randall DJ (2002) Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation. Physiol Biochem Zool 75:237-249.
Wilson JM, Antunes JC, Bouça PD, Coimbra J (2004) Osmoregulatory plasticity of the glass eel of Anguilla anguilla: freshwater entry and changes in branchial ion-transport protein expression. Can J Fish Aquat Sci 61:432-442.
Wu YC, Lin LY, Lee TH (2003) Na+, K+, 2Cl--cotransporter: a novel marker for identifying freshwater- and seawater-type mitochondria-rich cells in gills of euryhaline tilapia, Oreochromis mossambicus. Zool Stud 42:186-192.
Figure legends
Fig. 1. The immunolocalization of Na+/K+-ATPase (NKA), Na+/K+/2Cl- cotransporter
(NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR) in frozen
longitudinal sections of seawater (SW) and freshwater (FW) milkfish gills. The
cryosections were double stained with anti-NKCC (green; a and d) or anti-CFTR
(green; g and j) and anti-NKA (red; b, e, h and k). The merged (c and f) images of
double-stained gill sections show that NKCC is colocalized with NKA in the
basolateral membrane of NKA-immunoreactive cells (mitochondrion-rich cells;
MRCs) in FW and SW milkfish. Moreover, i and l show that CFTR is present in the
apical membrane of MRCs in SW-acclimated milkfish rather than FW-acclimated
individuals. F, filament; L, lamella.
Fig. 2. The immunolocalization of Na+/K+-ATPase (NKA), anion exchanger 1 (AE1),
and chloride channel 3 (ClC-3) in frozen longitudinal sections of seawater (SW) and
freshwater (FW) milkfish gills. The cryosections were double stained with anti-AE1
(red; a and d) or anti-ClC-3 (red; g and j) and anti-NKA (green; b, e, h and k). The
and l) are colocalized with NKA in the basolateral membrane of
NKA-immunoreactive cells (mitochondrion-rich cells; MRCs) in FW- and
SW-acclimated milkfish. F, filament; L, lamella.
Fig. 3. The immunolocalization of Na+/K+-ATPase (NKA), Na+/K+/2Cl- cotransporter
(NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR) in frozen
longitudinal sections of seawater (SW) and freshwater (FW) pufferfish gills. The
cryosections were double stained with anti-NKCC (green; a and d) or anti-CFTR
(green; g and j) and anti-NKA (red; b, e, h and k). The merged (c and f) images of
double-stained gill sections show that NKCC is colocalized with NKA in the
basolateral membrane of NKA-immunoreactive cells (mitochondrion-rich cells;
MRCs) in FW and SW pufferfish. Moreover, i and l show that CFTR is present in the
apical membrane of MRCs in SW-acclimated pufferfish but not FW-acclimated fish. F,
filament; L, lamella.
Fig. 4. The immunolocalization of Na+/K+-ATPase (NKA), anion exchanger 1 (AE1),
and chloride channel 3 (ClC-3) in frozen longitudinal sections of seawater (SW) and
freshwater (FW) pufferfish gills. The cryosections were double stained with anti-AE1
(red; a and d) or anti-ClC-3 (red; g and j) and anti-NKA (green; b, e, h and k). The
merged images of double-stained gill sections show that AE1 (c and f) and ClC-3 (i
NKA-immunoreactive cells (mitochondrion-rich cells; MRCs) in FW and SW
pufferfish. F, filament; L, lamella.
Fig. 5. The number of Na+/K+-ATPase (NKA)-immunoreactive (IR) cells and
Na+/K+/2Cl– cotransporter (NKCC)-IR cells in frozen longitudinal sections of gills of
milkfish acclimated to seawater (SW) or fresh water (FW). Different letters indicate
significant differences between cell numbers in filaments and lamellae or SW and FW
acclimation. The asterisk indicates that the number of NKCC-IR cells in filaments or
lamellae is significantly lower than that of NKA-IR cells in FW milkfish (n=8, one
way ANOVA followed by Tukey‟s comparison, P<0.05). Values are represented as
means ± S.E.M.
Fig. 6. Na+/K+-ATPase (NKA) immunostaining in longitudinal sections of gills of (A)
seawater (SW)- and (B) freshwater (FW)-acclimated pufferfish. (C) Quantification of
NKA-immunoreactive (IR) cells in gills of SW and FW fish (n=8). The asterisk
indicates that the number of NKA-IR cells in filaments of SW pufferfish is
significantly higher than that of FW individuals (Student‟s t-test; P＜0.05). Values were represented as means ± S.E.M. F, filaments; L, lamellae; arrows, NKA-
immunoreactive cells.
Fig. 7. The proposed model of distribution of Na+/K+-ATPase (NKA) and chloride
SW-acclimated fish, basolateral Na+/K+-ATPase (NKA) provides the driving force in
the form of a transmembrane Na+ gradient for transport of Cl- into the cell via the
Na+/K+/2Cl- cotransporter (NKCC) and the anion exchanger 1 (AE1). Cl- leaves the
cell to external media via cystic fibrosis transmembrane conductance regulator (CFTR)
anion channels in the apical membrane, and feedback enters the blood through Cl
-channels (ClC-3) in the basolateral membrane. (B) The MRCs of FW milkfish and
pufferfish utilize basolateral NKA to provide the driving force in the form of a
transmembrane Na+ gradient for the uptake of Cl- into the cell via an unknown Cl
-transporter. Then, the Cl- exits the cells to the blood through a basolateral ClC-3
Fig. 7
(A)
(B)
