Cellular distributions of creatine kinase in branchia of
euryhaline tilapia (Oreochromis mossambicus)
Li-Yih
Lin1,2,
Chia-Chang
Chiang2,
Hong-Yi
Gong1,2,
Ching-Yi
Cheng2,3,
Pung-Pung
Hwang1,2,*, and
Ching-Feng
Weng3,*
1 Graduate Institute of Life Sciences, National Defense
Medical Center, Taipei, Taiwan 100; 2 Institute of Zoology,
Academia Sinica, Taipei, Taiwan 115; and 3 Institute of
Biotechnology, National Dong Hwa University, Hualien, Taiwan 974, Republic of China
 |
ABSTRACT |
Although euryhaline
teleosts can adapt to environmental fluctuation of salinity, their
energy source for responding to changes in salinity and osmolarity
remains unclear. This study examines the cellular localization of
creatine kinase (CK) expression in branchia of tilapia
(Oreochromis mossambicus). Western blot analysis of
muscle-type CK (MM form) revealed a high association with salinity changes, but BB and MB forms of CK in the gills of fish adapted to
seawater did not change. With the use of immunocytochemistry, three CK
isoforms (MM, MB, and BB) were localized in mitochondria-rich (MR)
cells and other epithelial cells of tilapia gills. In addition, staining intensity of MM-form CK in MR cells increased after seawater transfer, whereas BB and MB forms did not significantly change. To our
knowledge, this work presents the first evidence of CK expression in MR
cells of tilapia gills, highlighting the potential role of CK in
providing energy for ion transport.
creatine kinase isoform; mitochondria-rich cells; gill
 |
INTRODUCTION |
CREATINE KINASE (CK; EC 2.7.3.2)
catalyzes the reversible transfer of the phosphoryl group from
phosphocreatine to ADP, regenerating ATP. CK participates in an
ubiquitous role to meet the energy demand for homeostasis during
environmental changes. The phosphocreatine/creatine kinase is present
in some excitable tissues, such as Narcine brasiliensis
electric organ (2), and in nonexcitable tissues, such as
Squalus acanthias rectal gland (10),
Gillichthys mirabilis gills (15), and
Oreochromis mossambicus gills (37),
with high and fluctuating energy demand. Plasma CK exhibits the
physiological stress responses in big game fish after capture, perhaps
because of muscle damage and subsequent release of cytosolic soluble CK
in the plasma (36). Total CK activity significantly
declined 20% in the fish (O. mossambicus) brain after
exposure to hypergravity for 7 days (30). Additionally, some evidence has demonstrated seasonal fluctuations of CK in rainbow
trout, Oncorhynchus mykiss (3), variability of
CK isoenzymes in various tissues of trout (22), and
genetic variability in tissue CK among fish species including rainbow
trout and salmon (22, 26). Furthermore, CK provides energy
for ion transport (Na+-K+-ATPase) in the gill
of G. mirabilis when the CK inhibitor iodoacetamide is used
(15). It seems that the phosphocreatine/creatine kinase shuttle in cytosol might coordinate with that in mitochondria to
provide more ATP for the extra energy demand of
Na+-K+-ATPase to pump out excess ions under
hypertonic conditions. The results imply that CK may be a good
candidate for converting the energy after seawater transfer.
Many CK isoforms are identified by their electrophoretic mobility,
tissue and subcellular distribution, and primary sequence (24,
34). Three cytosolic CK isozymes [BB-CK (brain), MM-CK (muscle), and MB-CK (heart, lungs, stomach) (5, 8)] and two mitochondrial forms [sarcomeric MiCK (expressed mainly in heart
and skeletal muscle and probably in some brain cells such as Purkinje
neurons) and ubiquitous MiCK (expressed in many tissues)] are
kinetically very similar but differ in their capacity to associate with
subcellular organelles or protein structures (35, 41). Three types (brain, muscle, and mitochondria) of CK have been demonstrated to exist in teleosts (4, 26). The presence of MM-form, BB-form, and mitochondrial CK has been demonstrated, and
MM-form CK found to predominate, in the gill of G. mirabilis (15). Recently, three different carp (Cyprinus
carpio) muscle CK isoforms have been found, and their expression
may be related to environmental acclimation (31). Multiple
isoforms of CK are present in the muscle of channel catfish,
Ictalurus punctatus (17). Various CK isoforms
retain a phosphocreatine energy shuttle by which ATP generated by
oxidative phosphorylation in mitochondria CK produces creatine
phosphate, which is then transported to the cytosol and used by the
cytosolic CK (MM, MB, or BB) to regenerate ATP at sites of high energy
consumption. After the fish is transferred from a hypotonic
(freshwater) to a hypertonic (seawater) medium, CK, especially the
muscle-form of CK in the tilapia gill, can be affected by salinity,
directly or indirectly (37). Interestingly, the altered
MM-type CK after seawater transfer seems to be a newly synthesized
protein that is involved in osmoregulation. When a euryhaline teleost
goes from seawater to freshwater medium, it tends to lose ions and gain
water, and vice versa (when transferred from freshwater to seawater).
The physiological response maintains a stable internal milieu, ionic
regulation, and water balance. The organs involved in osmoregulation in
teleosts include the gill, gut, and kidney: the gill is the most
prominent organ. Moreover, the gill filaments contain pavement cells,
mitochondria-rich (MR) cells, mucous cells, and undifferentiated cells.
The MR cells are thought to participate importantly in the ionic
regulation of teleostean fishes. This study attempts to elucidate the
different forms of CK in the specific cells of gills and to investigate the incremental MM-CK association with the increase of bronchial sodium
pump of tilapia after seawater transfer. The results reveal the
existence of MM-, MB-, and BB-forms of CK in the MR cells of the
tilapia gill. There is a coordinated upregulation of
Na+-K+-ATPase and MM-CK in the MR cells of
tilapia gills following transition from freshwater to seawater.
Furthermore, three CK isoforms are also expressed in the pavement and
other epithelial cells of tilapia gills, which showed no increase in CK
level during seawater adaptation.
 |
MATERIALS AND METHODS |
Animals.
Tilapia (O. mossambicus) were originally obtained from the
Tainan Fish Culture Station of the Taiwan Fisheries Research Institute. Euryhaline tilapia can live in both seawater (SW) and freshwater (FW).
All fish were maintained in a FW recirculating tank at 25-28°C in a photoperiodic environment (12:12-h light-dark) at the Institute of
Zoology, Academia Sinica (Taipei, Taiwan). SW was prepared by adding
artificial sea salt to FW. The sampled fish were ~5-7 cm long
and had a body mass of 2.5-4.0 g. Tilapia were directly transferred from FW to 25 parts per thousand (ppt) SW for various periods after being captured in a nylon net. During the period of the experiment (2 h or 2 wk, SW adaptation), fish were reared in a
25-ppt SW tank without feeding. Fish were anesthetized with ice and
killed immediately. The gills were removed and weighed. The tissue was
then homogenized in a homogenization solution (100 mM imidazole-HCl
buffer, pH 7.0, 5 mM Na2EDTA, 200 mM sucrose, and 0.1%
sodium deoxycholate) with a motorized Teflon pestle that rotated at 600 rpm for 20 strokes on ice. After centrifugation (12,000 rpm, 30 min at
4°C), the supernatant was kept at
70°C until the assay was
performed. The total protein content was determined using a protein
assay kit (Bio-Rad, Hercules, CA) and calculated by using bovine serum
albumin (BSA; Sigma, St. Louis, MO) as a standard.
Western blotting.
The homogenate of a gill (total protein 100 µg) was mixed with an
equal volume of 2× electrophoresis sample buffer that contained 250 mM
Tris-base, 2 mM Na2EDTA, 2% SDS, and 5% dithiothreitol. The proteins were separated by electrophoresis on a 4-12%
gradient polyacrylamide slab gel (NuPAGE, Novex, CA) and
electrophoretically transferred to polyvinylidene difluoride (Amersham
Life Science, Amersham, UK). The blots were incubated overnight in 3%
NET buffer (0.25% gelatin, 50 mM NaCl, 50 mM Tris · HCl, 5 mM
EDTA, pH 7.5, and 0.05% Tween 20) and washed three times in PBST
buffer (0.01 M phosphate, 0.09% NaCl, pH 7.5, and 0.05% Tween 20).
Filters were incubated for 1 h with primary antibody: rabbit
anti-human MM-CK polyclonal antibody (Biogenesis) at 1:2,000 dilution
and mouse anti-human MB-CK monoclonal antibody (Biogenesis) and rabbit anti-human BB-CK (Biogenesis) at 1:1,000 dilution. Mouse muscle or
brain tissues were applied as a positive control to check molecular weight and immunoreactions. After being washed three times with PBST
buffer, immunoreactive proteins were visualized with an enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL) according to the
manufacturer's instructions. The differences between the band
intensity of FW and SW were compared using densitometry (Personal Densitometry SI; Molecular Dynamics, Sunnyvale, CA).
In situ fluorescence staining of gill filaments.
For whole mounted branchial staining, tilapia gills were excised and
immediately fixed with 4% paraformaldehyde for 2-3 min at 4°C.
The fixed gills were then washed three times with PBS (0.1 M phosphate
buffer plus 0.09% NaCl) for 5 min each time. The tissues were then
stained with 1 mg/ml concanavalin A conjugated with Texas red (Con A;
Molecular Probes, Eugene, OR) for 30 min at room temperature (RT).
After being washed three times, the tissues were further fixed and
permeabilized with 70% ethanol for 10 min at
20°C. After being
washed with PBS for 10 min, the Con A-stained gill filaments were
incubated with 10% normal goat serum (NGS) for 30 min to block
nonspecific binding. The tissues were then incubated for 2 h with
mouse anti-chicken Na+-K+-ATPase
-subunit
monoclonal antibody (Developmental Studies Hybridoma Bank), rabbit
anti-human MM-CK polyclonal antibody (Biogenesis), rabbit anti-human
BB-CK polyclonal antibody (Biogenesis), or mouse anti-human MB-CK
monoclonal antibody (Biogenesis) at 1:100 dilution (3% BSA in PBS) at
RT. The stained gill filaments were washed three times with PBS for 5 min each time. The stained gill filaments were then incubated with
FITC-conjugated secondary antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA; 1:200 dilution, 3% BSA in PBS) for 60 min at RT.
Fluorescence staining in frozen sections.
Excised gills from anesthetized tilapia were fixed and permeabilized as
previously described. After being washed with PBS, the gills were
immersed in 30% sucrose for 2 h and then embedded in a cryomatrix
embedding medium (Shandon) at
20°C. Frozen cross sections (15 µm)
were then cut using a cryostat (Bright) and attached to slides coated
with poly-L-lysine (Sigma). To stain creatine kinase, the
fixed gill sections were rinsed with PBS, blocked with 10% NGS, and
then incubated at RT for 2 h with polyclonal antibodies against
MM- or BB-CK or with monoclonal antibodies against MB-CK diluted at
1:100 with PBS that contained 3% BSA. After being washed with PBS, the
sections were further incubated with goat anti-mouse IgG or goat
anti-rabbit IgG conjugated with FITC (Jackson ImmunoResearch
Laboratories; 1:200 dilution) at RT for 1 h. The sections was
double-stained by an additional incubation with monoclonal
5 antiserum or polyclonal antibody TG3 antiserum against
Na+-K+-ATPase (13) diluted at
1:100 for 2 h, followed by incubation with goat anti-mouse or
anti-rabbit IgG conjugated with Texas red (Jackson ImmunoResearch
Laboratories; 1:200 dilution) at RT for 1 h. After washing,
specimens were observed and their positive images were acquisitioned by
using a Leica TCS-NT confocal laser scanning microscope (Leica
Lasertechnik, Heidelberg, Germany) equipped with ×10/0.3, ×20/0.4,
×40/1.2 oil, and ×100/1.35 oil lenses and appropriate filter sets for
simultaneous monitoring of FITC and Texas red. Branchial sections from
FW or SW tilapia were attached side by side on the same slide to
compare staining intensities. Therefore, the sections from various
groups could be stained under the same conditions to reduce artificial
staining bias.
Isolation of epithelial cells.
Fish were quickly anesthetized on ice and killed by transection of the
spinal cord. The gill filaments were separated from gill arcs and
chopped into small segments in PBS. Gill filaments from four tilapia
(2.5-4.0 g) were pooled together. The chopped filaments were
gently agitated in PBS for 30 min to remove blood cells. The filament
segments were incubated in trypsin solution (0.1% trypsin in PBS) for
1 h at RT. After incubation, cells were isolated from digested
tissues by gently and repeatedly passing the tissue suspension through
a wide-bore pipette. The suspension was then passed through a nylon
mesh (mesh size 100 µm) to remove larger tissue fragments. The
resulting cell suspension was centrifuged at low speed (200 rpm, 10 min, 4°C), and the isolation solution was replaced with 4%
paraformaldehyde for fixation over 10 min. The suspension of fixed cell
was washed with PBS and centrifuged again. This cell suspension was
then dropped on poly-L-lysine-coated slides and stored at
20°C for further staining. Isolated cells from FW or SW tilapia
were dropped next to each other on the same slide to compare staining
intensities. Accordingly, the isolated cells treated differently could
be stained under the same conditions to reduce artificial staining
bias. The staining procedure used was the same described previously for
the staining of frozen sections.
Acquiring and analyzing images.
The period of time over which the image was captured, the
photomultiplier tube gain, and the scanning rules of confocal
microscope were optimized before each experiment and maintained
throughout each experiment to standardize the intensity of fluorescence
among experiments. Images were taken with ×40/1.2 oil lenses and
quantified with MetaMorph software (Universal Imaging, Philadelphia,
PA). With respect to the stained bronchial sections, the fluorescence intensities of the sodium pump and CK double-labeled regions of the FW
gill section were measured and compared with those of the SW gill
section on the same slide. The staining intensities of sodium pump and
CK were determined separately from the averaged pixel depth of the
double-labeled regions. The average calculated intensity of one
branchial section referred to a single sample. Ten samples of FW or SW
gill sections were measured and compared. In the cell isolation
experiment, CK and sodium pump double-labeled cells were identified as
MR cells (38), and other cells that expressed only CK were
identified as non-MR cells. MM-CK and sodium pump staining intensities
obtained from 500 non-MR cells and 300 MR cells were measured in both
FW and SW specimens.
Statistics.
Values are given as means ± SD. The level of significance is
P < 0.05 in a two-tailed test. Student's
t-test was used to compare the staining intensities of FW
sections or cells with those of SW sections or cells.
 |
RESULTS |
After tilapia were transferred to SW within 2 h and SW
adapted, Western blot showed that MM-form CK in the gill of tilapia was
highly associated with salinity change (Fig.
1). However, no significant elevations in
the prevalence of BB and MB forms were observed in 25-ppt SW after
transfer and SW adaptation (Fig. 1).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Western blot of 3 creatine kinase (CK) isoforms (MM-,
MB-, and BB-CK) in freshwater (FW) tilapia, tilapia that were
transferred to seawater (SW) for 2 h, and SW-adapted tilapia. Each
lane contained 100 µg of protein.
|
|
Con A-conjugated Texas red used to stain the gills yielded positive
staining, confirming that Con A was located at the apical surface of MR
cells in whole mount gill filaments. Con A staining was used to
identify MR cells and to analyze distribution of sodium pumps and CK
isoforms in MR cells and other cell types in the gill filaments.
Thereafter, the Con A-positive gill filaments were stained with mouse
anti-chicken Na+-K+-ATPase
-subunit
monoclonal antibody, with polyclonal antibodies against MM- or BB-form
CK or with monoclonal antibodies against MB-form CK. The images
revealed that Na+-K+-ATPase or MB-CK was
labeled concomitantly with Con A in the MR cells of the FW tilapia gill
(Fig. 2, a and b).
Results demonstrate that three CK antibodies labeled on the gill
filaments exhibited a similar pattern; that is, MR cells and other
epithelial cells are labeled with three CK isoforms (Fig.
2b; images of MM- and BB-CK not shown).

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 2.
In situ fluorescence staining of gill filaments:
a, double labeling of Con A (red) and sodium pump (green);
b, double labeling of Con A (red) and MB-CK (green) in
FW-adapted tilapia gills. Magnification, ×400).
|
|
Frozen sections were then used for
Na+-K+-ATPase and CK isoform double staining to
further establish the expression pattern of the three CK isoforms in
the various cell types of the branchial epithelium. The confocal images
of FW branchial sections verified the expression of three CK isoforms
in MR cells, which extensively expressed
Na+-K+-ATPase on their tubular systems (Figs.
3-6).
Moreover, CK was also expressed in other epithelial cells, including
pavement cells and undifferentiated cells, and in the connective tissue
beneath the epithelial layer. The three different CK isoforms expressed similar patterns in FW branchial frozen sections (Figs. 4-6). They were localized not only in the cytoplasm of epithelial cells but also
in the cell membrane, thus outlining the boundaries of the epithelial
cells. Branchial sections from FW- and SW-acclimated tilapia were
stained simultaneously, and the relative fluorescence intensities of CK
and sodium pump in MR cells (sodium pump-stained regions) were
measured. Staining intensities of MM-CK were significantly higher
in SW frozen sections (Fig.
7b) than in FW frozen sections (Fig. 7a and Table 1) but
showed no significant difference in the other two isoforms (MB- and
BB-CK) (Fig. 7 and Table 1). The expression of sodium pump was
also found to be stronger in SW MR cells than in FW MR cells (Table 1).

View larger version (74K):
[in this window]
[in a new window]
|
Fig. 3.
a, Double labeling of sodium pump (red) and
MM-CK (green) in branchial sections of FW tilapia; b, signal
from MM-CK staining is isolated by double labeling. Original
magnification, ×400. Arrowheads, mitochondria-rich (MR)
cells.
|
|

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 4.
Fluorescence staining of frozen sections: a,
double labeling of sodium pump (red) and MM-CK (green) in branchial
frozen sections of FW tilapia; b, signal from MM-CK staining
is isolated by double labeling. Original magnification, ×1,000.
Arrowheads, MR cells.
|
|

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 5.
Fluorescence staining of frozen sections: a,
double labeling of sodium pump (red) and MB-CK (green) in branchial
frozen sections of FW tilapia; b, signal from MB-CK staining
is isolated by double labeling. Original magnification, ×1,000.
Arrowheads, MR cells.
|
|

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 6.
Fluorescence staining of frozen sections: a,
double labeling of sodium pump (red) and BB-CK (green) in branchial
frozen sections of FW tilapia; b, signal from BB-CK staining
is isolated by double labeling. Magnification, ×1,000. Arrowheads, MR
cells.
|
|

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 7.
Staining of isoforms MM-CK (a and b),
BB-CK (c and d), and MB-CK (e and
f) in branchial sections of FW (a,
c, e) and SW-adapted (b,
d, f) tilapia. Magnification,
×400. All images were double labeled for CK (green) and sodium pump
(red). Scale bars, 40 µm. Arrowheads, MR cells.
|
|
Furthermore, the expression of MM-CK in isolated epithelial cells from
FW individuals was compared with that in such cells from SW
individuals. Figure 8 depicts the double
staining of MM-CK and sodium pump in isolated epithelial cells from FW
or SW tilapia. The results revealed that MM-CK was expressed in
MR cells of SW and FW, and the expression in MR cells of SW was more
intense than in MR cells of FW (Fig. 9).
However, other sodium pump-negative cells (non-MR cells) did not
significantly differ between FW and SW.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 8.
Double labeling of MM-CK (green) and sodium pump (red) in
epithelial cell isolated from FW (a) or SW-adapted
(b) tilapia. Magnification, ×400. Scale bars, 40 µm.
Arrowheads, MR cells.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 9.
Relative intensities of MM-CK staining in isolated
mitochondria-rich cells (MRC) and non-MRC from FW or SW-adapted
tilapia. *Significantly different (P < 0.05).
Student's t-test was applied to compare the difference
between FW and SW groups.
|
|
 |
DISCUSSION |
This work is the first evidence of CK expressions localized in MR
cells of tilapia gills. MM-form CK in MR cells is increased after SW
transfer, whereas BB and MB forms did not show a significant change.
Three forms of CK (MM, MB and BB) are also present in other epithelial
cells, including pavement cells and undifferentiated cells of gill
filaments. The elevation of MM-CK expression in MR cells of SW tilapia
suggests that MM-form CK provides an energy source to regulate ion
transport during hyperosmoregulation. Under normal conditions, the
activity of CK is already many times higher than that of the
Na+-K+-ATPase in the gills of euryhaline
teleost G. mirabilis (15). The CK system
(likely MM-CK) is directly functionally coupled with
Na+-K+-ATPase, which allows ATP generated by
membrane-bound CK to be directly channeled to the ATPase without
equilibration with the cytosolic bulk phase in G. mirabilis
gills. CK activity is ~80 times higher than that of
Na+-K+-ATPase in tilapia gills
(37). The results obtained elsewhere (37) and
in the present study appear to support the hypothesis that a
coordinated upregulation of Na+-K+-ATPase and
MM-CK occurs in tilapia MR cells. Furthermore, Kultz and Somero
(15) also demonstrated the existence of all the other CK
isoforms, including mitochondria CK, in G. mirabilis gills, suggesting the existence of a complete phosphocreatine shuttle. Recently, mitochondria CK of tilapia gills have been cloned, and the
expressions of RNA are consistent with changes in salinity (C. F. Weng, C. J. Wu, M. J. Lo, and J. L. Wu, unpublished
data). These results suggest that the phosphocreatine shuttle
could be an important system in energetic cross talk between the sites of ATP production and those of its utilization in MR cells, as previously suggested (15). The fact that MM-CK is
upregulated during transition from FW to SW also reinforces the
plasticity of this system. After SW transfer or SW adaptation, the
number and size of MR cells and the activity and amount of
Na+-K+-ATPase in tilapia gills are increased
(14, 38). In the context of this shuttle, which acts as a
spatial rather than a temporal energy buffer, an increase in the number
of Na+-K+-ATPase units will be accompanied by
an almost stoichiometric increase in the expression of MM-CK. This
coordinated upregulation of MM-CK preserves the function of the CK
shuttle as a network for transferring intracellular energy and feedback
signals among the sites of ATP production and utilization. However, the
mechanisms by which an increase in MM-CK activity promotes the
acclimation of SW must be elucidated further.
Previous studies reported that fish experience two phases after
transfer to SW. The first is a crisis phase, in which fish critically
respond to the removal of water from the gill and the gut epithelia
(dehydration), and the second phase is stabilization, during which the
fish remains alive or dies, according to the extent of dehydration
(1, 12, 14). Tilapia died within 4 h after being
directly transferred to SW (35 ppt); however, they survived after
transfer from FW to 25-ppt SW (14, 37). Fish must expend
much energy to react to and compensate for a salinity challenge,
particularly in the gill, intestine, kidney, and brain. Most previous
studies have focused on physiological responses, including those
involving hormones (growth hormone, prolactin, cortisol),
plasma ions (Na+, Cl
, K+) and
osmolarity, glucose and oxygen consumption rate (19), and
water-drinking activity (16) during SW transfer. The
exposure of common carp to salt stress reduces their intake of food and depletes both muscle and liver glycogen stores to meet the requirement for extra energy (6). Few reports have addressed the
energy source of the osmoregulation in fish that faced a change in
salinity. The energy source for the responding organ remains an
interesting issue in understanding osmoregulation after fish are
transferred to SW. Normal demands placed on the
Na+-K+-ATPase during the generation of
electrical currents require large and rapid changes in activity of
CK in the electric organ of N. brasiliensis
(2). By using the CK inhibitor iodoacetamide and measuring the CK activity, recent studies have demonstrated that CK is an energy source for ion transporter
(Na+-K+-ATPase) in the gills of
G. mirabilis and tilapia (O. mossambicus) after
transfer from FW to SW (15, 37). This study is consistent with previous studies and further demonstrates the existence of three
forms of CK (MM, MB, and BB) in MR cells and other epithelial cells in
tilapia gills.
The serum levels of CK showed significant variation in the presence of
environmental stressors (acute handling and transport stress) in
channel catfish (7). Plasma CK exhibited physiological stress responses in big game fish (36). Brain CK activity
responded to hypergravity (30) or hypertonic conditions
(37) in tilapia. The rising CK levels were associated with
pathology in Atlantic salmon (Salmo salar) (9,
25) and in sea bass (Dicentrarchus labrax)
(20). Marine fish, red seabream (Pagrus major),
and Pacific mackerel (Scomber japonicus) have less
thermostable muscle CK than carp (C. carpio)
(21). The expression of three different muscle CK isoforms
in carp may be related to thermoacclimation (31).
Recently, we determined that CK activity and content were elevated
within 2 h after transfer from FW to SW, and only MM-form CK was
affected by changes in salinity (37). In the present study, the MR cells of SW-adapted tilapia expressed MM-form CK more
strongly than did than FW individuals, confirming that the elevations
of MM-form CK are associated with changes in salinity. Furthermore, two
different muscle-type isoforms (CKM1 and CKM2) were cloned in our
laboratory. The expression of the two isoforms shows opposite
responses, and CKM1 expresses an increasing pattern, suggesting that
different CKMs are influenced by an acute salinity challenge (11).
In the past decade, some enzymes
[Na+-K+-ATPase (38),
H+-ATPase and Na+/NH
-ATPase
(41), carbonic anhydrase (29)], ion
exchangers and cotransporters
[Cl
/HCO
and
Na+/H+ (39),
Na+/NH
(40),
Na+-K+-2Cl
(23)],
ion channels [Na+ channel (39),
K+ channel (32), CFTR-like Cl
channel (40)], and receptors [prolactin receptor
(38), glucocorticoid receptor (33),
angiotensin II receptor (18)] were found to be localized
in MR cells of fish gills. This study reports the first evidence of the
localization of CK expression in MR cells of tilapia gills. The
function of BB-CK protein in neurons may involve ATP regeneration to
maintain ATP availability to, for example, the
Na+-K+-ATPase. Accordingly, in vivo studies
have suggested a direct coupling of CK-ATPase (2). An
increase in CKB activity and brain energy metabolism constitutes a
mechanism for coupling energy production (ATP) and energy storage
(creatine phosphate) to meet rapidly increasing cellular energy demands
(35). Muscle-type CK is functionally coupled to
Na+-K+-ATPase activity, providing ATP for the
ATPase reaction (27, 28). Taking all these facts into
account, the increase in CK is associated with increased
Na+-K+-ATPase activity and more MR cells in
fish gills after SW transfer. The CK localized in MR cells may couple
with the Na+-K+-ATPase to maintain ion balance
under hypertonic conditions.
 |
ACKNOWLEDGEMENTS |
We thank the National Science Council of the Republic of China for
financially supporting this research under Contract No. NSC
90-2313-B-259-002.
 |
FOOTNOTES |
*
P.-P. Hwang and C.-F. Weng contributed equally to this
work. Address for reprint requests and other correspondence: C.-F. Weng, Institute of Biotechnology, National Dong Hwa Univ., Hualien, Taiwan 974, ROC (E-mail:cfweng{at}mail.ndhu.edu.tw).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 25, 2002;10.1152/ajpcell.00087.2002
Received 28 February 2002; accepted in final form 9 September 2002.
 |
REFERENCES |
1.
Bath, RN,
and
Eddy FB.
Transport of nitrite across fish gills.
J Exp Zool
214:
119-121,
1979[ISI].
2.
Blum, H,
Balschi JA,
and
Johnson RG.
Coupled in vivo activity of creatine phosphokinase and the membrane-bound Na+, K+-ATPase in the resting and stimulated electric organ of the electric fish Narcine brasiliensis.
J Biol Chem
266:
10254-10259,
1991[Abstract/Free Full Text].
3.
Bucher, F.
Organ patterns and natural fluctuations of blood enzymes of rainbow trout, Salmo-gairdneri Rich.
Comp Biochem Physiol B
96:
795-800,
1990[ISI].
4.
Coppes, ZL,
Vecchi SD,
Ferreira E,
and
Hirschhorn M.
Multilocus isozyme systems in fish.
Comp Biochem Physiol B
96:
11-13,
1990.
5.
Dawson, DM,
Eppenberger HM,
and
Kaplan NO.
Creatine kinase: evidence for a dimeric structure.
Biochem Biophys Res Commun
21:
346-353,
1965[ISI][Medline].
6.
De Boeck, G,
Vlaeminck A,
Van der Linden A,
and
Blust R.
The energy metabolism of common carp (Cyprinus carpio) when exposed to salt stress: an increase in energy expenditure or effects of starvation?
Physiol Biochem Zool
73:
102-111,
2000[ISI][Medline].
7.
Ellsaesser, CF,
and
Clem LW.
Blood serum chemistry measurements of normal and acutely stressed channel catfish.
Comp Biochem Physiol A
88:
589-594,
1987[ISI][Medline].
8.
Eppenberger, HM,
Dawson DM,
and
Kaplan NO.
The comparative enzymology of creatine kinases. I. Isolation and characterization from chicken and rabbit tissues.
J Biol Chem
242:
204-209,
1967[Abstract/Free Full Text].
9.
Ferguson, HW,
Rice DA,
and
Lynas JK.
Clinical pathology of myodegeneration (pancreas disease) in Atlantic salmon (Salmo salar).
Vet Rec
119:
297-299,
1986[ISI][Medline].
10.
Friedman, DL,
and
Robert R.
Purification and localization of brain-type creatine kinase in sodium chloride transporting epithelia of the spiny dogfish, Squalus acanthias.
J Biol Chem
267:
4270-4276,
1992[Abstract/Free Full Text].
12.
Hwang, PP.
Tolerance and ultrastructural response of branchial chloride cells to salinity changes in the euryhaline teleost Oreochromis mossambicus.
Mar Biol (Berl)
94:
643-649,
1987.
13.
Hwang, PP,
Fang MJ,
Tsai JC,
Huang CJ,
and
Chen ST.
Expression of mRNA and protein of Na-K-ATPase
subunit in gills of tilapia (Oreochromis mossambicus).
Fish Physiol Biochem
18:
363-373,
1998[ISI].
14.
Hwang, PP,
Sun CM,
and
Wu SM.
Changes of plasma osmolality, chloride concentration and gill Na+,K+-ATPase activity in tilapia (Oreochromis mossambicus) during seawater acclimation.
Mar Biol (Berl)
100:
295-299,
1989.
15.
Kultz, D,
and
Somero GN.
Ion transport in gill of the euryhaline fish Gillichthys mirabilis is facilitated by a phosphocreatine circuit.
Am J Physiol Regul Integr Comp Physiol
268:
R1003-R1012,
1995[Abstract/Free Full Text].
16.
Lin, LY,
Weng CF,
and
Hwang PP.
Effects of cortisol on water drinking in SW transferred tilapia (Oreochromis mossambicus) larvae.
Physiol Biochem Zool
73:
283-289,
2000[ISI][Medline].
17.
Liu, Z,
Kim S,
Kucuktas H,
and
Karsi A.
Multiple isoforms and an unusual cathodic isoform of creatine kinase from channel catfish (Ictalurus punctatus).
Gene
275:
207-215,
2001[ISI][Medline].
18.
Marsigliante, S,
Acierno R,
Maffia M,
Muscella A,
Vinson GP,
and
Storelli C.
Immunolocalisation of angiotensin II receptors in icefish (Chionodraco hamatus) tissues.
J Endocrinol
154:
193-200,
1997[Abstract].
19.
McCormick, SD.
Hormonal control of gill Na+,K+-ATPase and chloride cell function.
In: Fish Physiology, edited by Wood CM,
and Shuttleworth TJ.. New York: Academic, 1995, vol. 14, p. 285-315.
20.
Meassager, JL,
Stephan G,
Quentel C,
and
Baudin LF.
Effects of dietary oxidized fish oil and antioxidant deficiency on histopathology, haematology, tissue and plasma biochemistry, Dicentrarchus labrax.
Aquat Living Resour
5:
205-214,
1992.
21.
Nakagawa, T,
and
Nagayama F.
Enzymatic properties of fish muscle creatine kinase.
Comp Biochem Physiol B
98:
349-354,
1991[ISI].
22.
Paaver, TK.
Electrophoretic variability of proteins and genetic features of breed groups and stocks of rainbow trout, Salmo gairdneri, reared in the USSR.
Voprosy Ikhtiologii
28:
595-603,
1988.
23.
Pelis, RM,
Zydlewski J,
and
McCormick SD.
Gill Na+-K+-2Cl
cotransporter abundance and location in Atlantic salmon: effects of seawater and smolting.
Am J Physiol Regul Integr Comp Physiol
280:
R1844-R1852,
2001[Abstract/Free Full Text].
24.
Perryman, MB,
Kerner SA,
Bohlmeyer TJ,
and
Roberts R.
Isolation and sequence analysis of a full-length cDNA for human M creatine kinase.
Biochem Biophys Res Commun
140:
981-989,
1986[ISI][Medline].
25.
Rodger, HD,
Murphy TM,
Drinan EM,
and
Rice DA.
Acute skeletal myopathy in farmed Atlantic salmon, Salmo salar.
Dis Aquat Organ
12:
17-23,
1991[ISI].
26.
Rottiers, DV,
Redell LA,
Booke HE,
and
Amaral S.
Differences in stocks of American shad from the Columbia and Delaware rivers.
Trans Amer Fish Soc
121:
132-136,
1992[ISI].
27.
Saks, VA,
Belikova YO,
and
Kuznetsov AV.
In vivo regulation of mitochondrial respiration in cardiomyocytes: specfic restriction for intracellular diffusion of ATP.
Biochim Biophys Acta
1074:
302-311,
1991[ISI][Medline].
28.
Saks, VA,
Lipina NV,
Sharov VG,
Sminov VN,
Chazov EI,
and
Grosse R.
The localization of the MM isoenzyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited Na+-K+-ATPase.
Biochim Biophys Acta
465:
550-558,
1977[ISI][Medline].
29.
Sender, S,
Bottcher K,
Cetin Y,
and
Gros G.
Carbonic anhydrase in the gills of seawater- and freshwater-acclimated flounders, Platichthys flesus: purification, characterization, and immunohistochemical localization.
J Histochem Cytochem
47:
43-50,
1999[Abstract/Free Full Text].
30.
Slenzka, K,
Appel R,
and
Rahmann H.
Brain creatine kinase activity during ontogeny of the cichlid fish Oreochromis mossambicus and the clawed toad Xenopus laevis, influence of gravity?
Neurochem Int
22:
405-411,
1993[ISI][Medline].
31.
Sun, HW,
Hui CH,
and
Wu JH.
Cloning, characterization, and expression in Escherichia coli of three creatine kinase muscle isoenzyme cDNAs from carp striated muscle.
J Biol Chem
273:
33774-33780,
1998[Abstract/Free Full Text].
32.
Suzuki, Y,
Itakura M,
Kashiwagi M,
Nakamura N,
Matsuki T,
Sakuta H,
Naito N,
Takano K,
Fujita T,
and
Hirose S.
Identification by differential display of a hypertonicity-inducible inward rectifier potassium channel highly expressed in chloride cells.
J Biol Chem
274:
11376-11382,
1999[Abstract/Free Full Text].
33.
Uchida, K,
Kaneko T,
Tagawa M,
and
Hirano T.
Localization of cortisol receptor in branchial chloride cells in chum salmon fry.
Gen Comp Endocrinol
109:
175-185,
1998[ISI][Medline].
34.
Villarreal, LG,
Ma TS,
Kerner SA,
Roberts R,
and
Perryman MB.
Human creatine kinase: isolation and sequence analysis of cDNA clones for the B subunit, development of subunit specific probes and determination of gene copy number.
Biochem Biophys Res Commun
144:
1116-1127,
1987[ISI][Medline].
35.
Wallimann, T,
and
Hemmer W.
Creatine kinase in non-muscle tissues and cells.
Mol Cell Biochem
133-134::
193-220,
1994[ISI].
36.
Wells, RM,
McIntyre RH,
Morgan AK,
and
Davie PS.
Physiological stress responses in big gamefish after capture: observations on plasma chemistry and blood factors.
Comp Biochem Physiol A
84:
565-571,
1986[ISI][Medline].
37.
Weng, CF,
Chiang CC,
Gong HY,
Chen Mark HC,
Lin Cliff JF,
Cheng CY,
Hwang PP,
and
Wu JL.
Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus).
Physiol Biochem Zool
75:
29-36,
2002[ISI][Medline].
38.
Weng, CF,
Lee TH,
and
Hwang PP.
Immuno-localization of prolactin receptor in the mitochondria-rich cells of the euryhaline tilapia, Oreochromis mossambicus gill.
FEBS Lett
405:
91-94,
1997[ISI][Medline].
39.
Wilson, JM,
Laurent P,
Tufts BL,
Benos DJ,
Donowitz M,
Vogl AW,
and
Randall DJ.
NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization.
J Exp Biol
15:
2279-2296,
2000.
40.
Wilson, JM,
Randall DJ,
Donowitz M,
Vogl AW,
and
Ip AK.
Immunolocalization of ion-transport proteins to branchial epithelium mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri).
J Exp Biol
15:
297-310,
2000.
41.
Wyss, M,
Smeitink J,
Wevers RA,
and
Wallimann T.
Mitochondrial creatine kinase: a key enzyme of aerobic energy metabolism.
Biochim Biophys Acta
1102:
119-166,
1992[ISI][Medline].
Am J Physiol Cell Physiol 284(1):C233-C241
0363-6143/03 $5.00
Copyright © 2003 the American Physiological Society