Nitric oxide synthase in the gill of Atlantic salmon: colocalization with and inhibition of Na+,K+-ATPase
1 Department of Biology, University of Bergen, Bergen High Technology
Centre, N-5020 Bergen, Norway
2 Institute of Biology, University of Southern Denmark, Odense University,
Campusvej 55, DK-5230 Odense M, Denmark
3 Department of Pathology, Lund University, Sölvegatan 25, S-221 85
Lund, Sweden
* Author for correspondence (e-mail: Lars.Ebbesson{at}bio.uib.no)
Accepted 10 January 2005
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Summary |
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Key words: nitric oxide, Na+, K+-ATPase, osmoregulation, parr-smolt transformation, development, metamorphosis, Salmar salmar, fish, teleost
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Introduction |
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Evidence for the role of NO in osmoregulation in fish includes localization
of NOS in osmoregulatory organs and effects on ion regulatory proteins and
vascular tonus. NADPHd has been localized in branchial nerves in the cod
(Gibbins et al., 1995) and
neuroendocrine cells in the catfish gill
(Mauceri et al., 1999
). NO
donors and NOS inhibitors have been shown to decrease and increase,
respectively, the short-circuit current across the killifish opercular
membrane (Evans, 2002
).
Recently, several studies have shown that NO affects
Na+,K+-ATPase (NKA) activity in the kidney and other
tissues in higher vertebrates (Liang and
Knox, 1999
). Tipsmark and Madsen
(2003
) showed for the first
time that NO exerts an inhibitory effect on
Na+,K+-ATPase activity in fish kidney and gill tissue.
In addition, NO is well recognized as a vasodilator in mammals, and more
recently in fish (Fritsche et al.,
2000
; Haraldsen et al.,
2002
). NOS has also been located in nerves along blood vessels in
the gill (Gibbins et al.,
1995
), and therefore the role of NO in osmoregulation may also
include the regulation of blood flow through the gill.
As we begin to accumulate evidence of the role of NO in fish, it is
important to relate this information to other model species with different
life histories to further elucidate basic mechanisms. One potentially
important model is found in anadromous salmonids that undergo a pre-adaptation
to ocean life, called parr-smolt transformation or smoltification, which
involves preparatory behavioral, physiological and morphological changes
(Boeuf, 1993;
Dickhoff, 1993
;
Hoar, 1988
). Sequential
hormone surges (thyroid hormones, growth hormone and cortisol) are central to
this transformation and instrumental in the structural and chemical changes in
the gill in preparation for the subsequent transition to a hyper-osmotic
environment (McCormick, 2001
).
Osmoregulatory changes in the gill during smoltification include increases in
chloride cell number and Na+,K+-ATPase activity, and are
regulated by the changes in the endocrine system
(D'Cotta et al., 2000
;
McCormick, 2001
;
Prunet et al., 1994
;
Seidelin et al., 2001
). It is
widely accepted that the increased abundance of gill
Na+,K+-ATPase enzymes is a preparatory development prior
to seawater entry; however, the functional state of this pool of enzymes while
the fish is still in fresh water is uncertain. To learn more about the
possible regulatory role of NO in this system, we investigated the presence of
NO-producing cells in the gill of Atlantic salmon smolts and determined
possible influence of NO on Na+,K+-ATPase activity.
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Materials and methods |
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Sampling procedures and analysis
In May, when the salmon reached peak smolt status (for details, see
Nilsen et al., 2003), six fish
(mean mass 29.5±0.9 g) were deeply anesthetized with tricaine
methanesulphonate (MS222; Sigma, St Louis, MO, USA) and fixed by vascular
perfusion with 4% paraformaldehyde (PF) in 0.1 mol l-1 phosphate
buffer (PB; in mmol l-1: 28 NaH2PO4, 71
Na2HPO4; pH 7.2). The two central gill arches on either
side were removed and postfixed in the same fixative overnight at 4°C.
Post-fixation was followed by a 2 h rinse in PB after which the gill arches
were placed in 25% sucrose overnight. The samples were then embedded in Tissue
Tek (Miles Inc., Eikhart, IN, USA) and stored at -80°C until sectioning.
Eight adjacent, serial, 10 µm-thick cryostat sections were collected on
frozen Superfrost Plus glass slides (Merck, Germany), dried at 60°C for 10
min then stored -80°C in airtight boxes. In situ hybridization,
immunocytochemistry and NADPHd histochemistry were performed on parallel
cryosections. Adjacent sections were used for hybridization with the antisense
and sense probes, for NOS immunocytochemistry, including specificity tests in
which the primary antibody was excluded, or for NADPHd histochemistry. For
comparison with smolts, parr gills were sampled in March as described above
and sections from both stages placed on the same slide for NADPHd
histochemistry.
Immunocytochemistry
The sections were rehydrated in phosphate-buffered saline (PBS; in mmol
l-1: 3.2 NaH2PO4, 7.8
Na2HPO4; pH 7.2) for at least 10 min followed by 30 min
incubation with 3% H2O2 to remove endogenous peroxidase
activity and then rinsed 2x15 min each, in PBS. The sections were then
rinsed in PBS containing 0.25% Triton X-100 (PBS-TX) for 10 min and incubated
in either rabbit anti-rat nNOS diluted 1:1500 (a gift from Prof. Fahrenkrug,
Bispebjerg Hospital Copenhagen, Denmark), rabbit anti-mouse iNOS from Affinity
Bioreagents Inc. (Golden, CO, USA) diluted 1:2000, rabbit anti-mouse eNOS from
Affinity Bioreagents Inc. diluted 1:2000, or
Na+,K+-ATPase -subunit diluted 1:600 (a gift from
Dr S. Adachi, Hokkaido University, Hakodate, Japan) overnight at room
temperature. The secondary antibody, anti-rabbit IgG diluted 1:50 (Dako,
Glostrup, Denmark), was applied for 30 min, rinsed 2x10 min each in
PBS-TX followed by incubation with rabbit PAP complex (Dako, Glostrup,
Denmark) diluted 1:50 for 30 min and rinsed for 10 min in PBS-TX and 10 min in
0.05 mol l-1 Tris-HCl, pH 7.6. The sections were then reacted in 50
ml Tris-HCl with 25 mg diaminobenzidine (Sigma, St Louis, MO, USA) and 250
µl 3% H2O2 for 10 min, followed by dehydration, and
mounted with Permount.
NADPHd histochemistry
For NADPHd histochemistry, sections were rinsed in PBS for 10 min followed
by a rinse in Tris-HCl (0.01 mol l-1, pH 7.6) for 10 min. Sections
were incubated for 90 min at 37°C in Tris-HCl (0.01 mol l-1, pH
7.6) containing 0.125 mmol l-1 Nitroblue Tetrazolium and 1 mmol
l-1 ß-NADPH. Sections were then rinsed for 5 min in Tris-HCl
followed by three rinses for 10 min in PBS. Sections were mounted in Kaisers
glycerol gelatin (Merck, Darmstadt, Germany).
In situ hybridization
Expression of salmon Na+, K+-ATPase -subunit
mRNA (GenBank accession number AJ250809;
Seidelin et al., 2001
) in the
gills was visualized by in situ hybridization with digoxigenin
(DIG)-labeled cRNA probes. The Na+,K+-ATPase-
subunit inserts of approximately 890 base pairs in the pCR-Blunt II-TOPO
vector (Invitrogen, Carlsbad, CA, USA) were transformed, amplified and
isolated from overnight cultures using QIAGEN Maxi Plasmid Kit. The inserts
were digested with the appropriate restriction enzymes (NotI and
SpeI; Promega, Madison, WI, USA). Antisense and sense cRNA probes
were synthesized with T7 RNA polymerase and SP6 RNA polymerase, respectively,
using DIG RNA labeling Kit (Roche Diagnostics, Mannheim, Germany). DIG
incorporation and the concentration of the probes were estimated by spot tests
(Roche Diagnostics).
Prior to in situ hybridization, sections were rehydrated for 10 min in potassium phosphate-buffered saline (KPBS; in mmol l-1: 137 NaCl, 1.4 KH2PO4, 2.7 KCl, 4.3 Na2HPO4; pH 7.3), post-fixed in 4% PF in KPBS for 10 min and permeabilized with proteinase K (5 µg ml-1) in 50 mmol l-1 Tris-HCl (pH 7.5) for 5 min. After a 5 min rinse in Tris-HCl, sections were post-fixed in 4% PF and rinsed for 10 min in KPBS before being treated for 10 min with 0.1 mol l-1 triethanolamine, pH 8.0 and then for 10 min with 0.25% acetic anhydride in triethanolamine, followed by three 5 min rinses in KPBS. Then, after 2 h prehybridization at room temperature (RT) in hybridization buffer (10% dextran sulphate, 50% formamide, 5x SSC, 5x Denhardt's solution, 500 µg ml-1 salmon sperm DNA (Sigma) and 250 µg ml-1 tRNA (Promega) the buffer was removed, and 300-800 ng ml-1 probe in fresh hybridization buffer were applied to the slides. Hybridization was performed for 16 h at 67°C followed by post-hybridization rinses in 5x SSC for 30 min at RT, 30% formamide in 5x SSC for 15 min at 67°C, 0.2x SSC for 2x15 min at 67°C and 0.2x SSC at RT. After 5 min in wash solution (0.1 mol l-1 Tris-HCl, 0.15 mol l-1 NaCl, pH 7.5) the sections were incubated for 1 h in 1% blocking solution (Roche Diagnostics) and then incubated overnight at RT with an alkaline phosphatase-conjugated sheep anti-DIG goat antibody (1:2000; Roche Diagnostics). Sections were then rinsed 3x5 min with wash solution at RT, followed by 5 min in detection solution (0.1 mol l-1 Tris-HCl, 0.1 mol l-1 NaCl, 50 mmol l-1 MgCl2, pH 9.5) at RT, before being incubated with reaction solution (3.4 µl Nitroblue Tetrazolium, 3.5 µl 5-Bromo-4-chloro-3-indoylphosphate and 0.24 mg ml-1 levamisole in detection solution), in darkness at RT. The phosphatase reaction was terminated after 1-3 h with stop solution (10 mmol l-1 Tris-HCl, 1 mmol l-1 EDTA, 0.9% NaCl, pH 8.0) and sections mounted in 50% glycerol (in stop solution).
Colocalization of Na+,K+-ATPase -subunit mRNA and nNOS
The distribution of NADPHd-positive labeling, nNOS, eNOS and
Na+,K+-ATPase immunoreactivity, and the
Na+,K+-ATPase -subunit mRNA on adjacent sections
indicated similar localization of NOS and NKA in chloride cells. The
co-localization within the same cells was confirmed via double labeling for
simultaneous visualization of nNOS and NKA immunolabeled structures and NKA
-subunit mRNA. This was achieved by first processing sections for
in situ hybridization of the NKA
-subunit mRNA as described in
the previous section, but after the stop solution, the sections were processed
for nNOS immunocytochemistry as described above. The sections where then
mounted in 50% glycerol in stop solution.
In vitro Na+,K+-ATPase activity
To test the effects of NO on gill Na+,K+-ATPase
activity in Atlantic salmon, an in vitro study was performed by
administration of NO donors to isolated gill samples and recordings of changes
in rubidium ion uptake. The fish used in this in vitro study were
Atlantic salmon smolts (20-40 g), acclimated to seawater (SW; 35 p.p.t.) at
the Odense University Campus (15°C, 12 h:12 h light:dark artificial
photoperiod) for at least 2 months. They were fed a maintenance diet of
commercial trout pellets (2% body mass every second day).
Tissue preparations and assay procedure was done according to Tipsmark and
Madsen (2001) with only minor
modifications. Briefly, gill arches were excised of the cartilage and rinsed
in salmon Ringer's solution equilibrated with 99% O2/1%
CO2 (in mmol l-1: 140 NaCl, 15 NaHCO3, 3.5
KCl, 1.5 CaCl2, 1.0 NaH2PO4, 0.8
MgSO4, 5.0 D-glucose and 5.0 N-2 Hepes; pH 7.8)
and then cut into small samples (5-10 mg). The samples were equilibrated for
30-60 min in Ringer's prior to experimentation. To measure Rb+
uptake, the Ringer's was replaced with Rb+-Ringer's solution
containing 3.5 mmol l-1 RbCl instead of KCl. The gill samples were
incubated in Rb+-Ringer's for 10 min. Ouabain-sensitive
Rb+ uptake (in situ NKA activity) was calculated as the
difference between total uptake and uptake in samples both pre-incubated (10
min in Ringer's) and incubated (in Rb+-Ringer's) with 1 mmol
l-1 ouabain. After incubation the samples were washed in
Tris-sucrose buffer (4x15 min at 0°C; mmol l-1: 10 Tris,
260 sucrose, pH 7.8), blotted onto filter paper and weighed. Ions were
extracted overnight at 4°C in 5% trichloroacetic acid (TCA). Potassium
chloride (20 mmol l-1) was added and Rb+ was determined
in the extracts by atomic absorption spectrophotometry at 780.8 nm (Perkin
Elmer 2380 Mountain View, CA, USA). Rubidium uptake was expressed as nmol mg
wet weight-1 h-1. The effects of SNP (1 mmol
l-1; Sigma) and PAPA-NONOate (NOC-15; 0.5 mmol l-1;
Sigma) on NKA activity were measured as ouabain-sensitive Rb+
uptake in gill tissue from SW-acclimated Atlantic salmon. The samples were
pre-incubated 10 min with the NO donors before the assay.
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Results |
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Using the antisense probe for in situ hybridization, expression of
the Na+,K+-ATPase -subunit mRNA was found in
cells on the primary filament and lamellae
(Fig. 2A) and the sense NKA
mRNA probe revealed no staining (Fig.
2B). The distribution of the NKA
-subunit mRNA is
consistent with the distribution of the NKA
-subunit protein, by
immunocytochemistry (Fig. 1D).
Moreover, the NKA mRNA and protein were found to be colocalized within the
same cells by double labeling (Fig.
2C,E). Double labeling with the NKA mRNA and nNOS revealed
localization of these enzymes within the same cells
(Fig. 2D,F).
|
NADPHd histochemical staining increased in intensity and distribution from parr (Fig. 3A,C) to smolt (Fig. 3B,D) in particular cells on the secondary lamellae.
|
In vitro Na+,K+-ATPase activity
The in vitro effects of both NO donors, SNP (1 mmol
l-1) and NOC-15 (0.5 mmol-1), revealed a significant 30%
inhibition of Na+,K+-ATPase activity (measured as
ouabain-sensitive Rb+ uptake) in gill tissue from SW-acclimated
Atlantic salmon (Table 1).
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Discussion |
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Previously, the localization of NOS and/or NADPHd in the gills of fish has
only been reported in the nitrergic nerve fibers coursing along the efferent
branchial artery in the puffer fish Takifugu niphobles
(Funakoshi et al., 1999).
Also, NADPHd activity has been localized to parasympathetic postganglionic
neurons in branchial nerves nIX and nX in the cod Gadus morhua
(Gibbins et al., 1995
).
Judging by the location and shape of the present NO-producing cells, they
appear identical to chloride cells. To investigate this further, gill sections
were probed for the presence of NKA
-subunit mRNA and protein, which
are particularly abundant in chloride cells in the gill of Atlantic salmon
smolts (Karnaky et al., 1976
;
Pelis et al., 2001
;
Wilson and Laurent, 2002
). The
protein and messenger encoding for the NKA
-subunit were coexpressed
primarily in cells at the base of the lamellae and interlamellar space. In
addition,
-subunit protein was found sporadically in cells along the
lamellae, where
-subunit mRNA was not heavily expressed. These cells
may represent FW-type ionocytes that are about to retreat as the salmon smolt
enters SW. FW-type chloride cells in lamellar positions are known to disappear
when salmonids are moved into SW (Seidelin
et al., 2000
; Uchida et al.,
1996
). Thus, there is a reduced mRNA expression in these cells
even though they maintain the abundance of active protein. Direct evidence for
colocalization of the enzyme with nNOS was obtained from double labeling
experiments, combining nNOS immunocytochemistry with
Na+,K+-ATPase in situ hybridization. These
experiments clearly showed that putative SW-type chloride cells are
functionally equipped with both ion-transport
(Na+,K+-ATPase) and NO-production (NOS) capacity. In
this study, FW Atlantic salmon at the peak of smolt development were used for
the analyses. One of the most prominent characteristics of this developmental
stage of the salmon is its readiness for hypo-osmoregulation
(Björnsson et al., 1989
;
Seidelin et al., 2001
). While
still residing in FW at the smolt stage, very high levels of the
Na+,K+-ATPase enzyme localized in typical SW-type
chloride cells have been reported in numerous studies
(Pelis et al., 2001
;
Pisam et al., 1988
;
Ura et al., 1997
). If these
fish are transferred to SW, the osmotic disturbance is minimal compared to the
parr stage, with low levels of gill Na+,K+-ATPase,
suggesting that chloride cell development and high levels of
Na+,K+-ATPase are part of the development of
hypo-osmoregulatory ability as a pre-adaptation to seawater entry. In order to
maintain hydro-mineral balance while still in fresh water during the final
stages of smoltification, it is, however, important to prevent excessive ion
loss. Therefore, in situ inhibition of the
Na+,K+-ATPase might be anticipated. The presence of NO
donors in vitro does indeed significantly inhibit
Na+,K+-ATPase by 30%, providing preliminary evidence
that this may indeed be one of the important functions of NOS activity in the
salmon smolt gill, i.e. to reduce hydrolytic
Na+,K+-ATPase activity, thereby deactivating ion
excretion until SW is encountered. This part of the study was done using gills
from SW-acclimated smolts, which have roughly the same total hydrolytic
capacity as measured in gills of FW smolts. The increase in NADPHd staining
from parr to smolt shown here further support this role. The increase in
staining in smolts may represent stimulated NOS gene expression in existing
chloride cells or be related to the general increase in chloride cell
abundance associated with smoltification. With regard to the stimulus for
increased NOS expression we speculate that thyroid hormones may have some role
during smoltification. Thyroid hormones stimulate NOS gene expression in
mammals (Ueta et al., 1995
)
and are characteristically high during the mid to later stages of
smoltification. Taken together, our data suggest an important role of NO in
inhibiting the peak Na+,K+-ATPase activity while the
smolt is still in FW, thus preventing excessive ion loss. Furthermore, these
results are in agreement with the notion that NO plays an important role as an
inhibitor of development once a certain competence is reached
(Bishop and Brandhorst,
2003
).
Within the last decade, NO has proved to influence ion regulation in
several tissues. In mammals, NO has been shown to have inhibitory effects on
Na+,K+-ATPase activity in kidney
(Liang and Knox, 1999), liver
(Muriel and Sandoval, 2000
)
and brain (Sato et al., 1997
).
Stimulatory effects have also been shown in the trachea
(de Oliveira Elias et al.,
1999
) and heart (Gupta et al.,
1994
), as well as opposing influences depending on the kidney
segment (Ortiz and Garvin,
2002
). Inhibition of Na+,K+-ATPase activity
by NO was recently found in the brown trout (Salmo trutta L.) kidney
and gill (Tipsmark and Madsen,
2003
), and there is support that the action is mediated by local
production of cGMP. Juxta-localization of NOS and
Na+,K+-ATPase has recently been reported using
immunocytochemistry in the killifish opercular membrane
(Evans, 2002
), which differs
from our findings showing them to be colocalized. The physiological
significance of this observation in killerfish was supported by data showing
the inhibition by NO of short-circuit current (Isc) across
the killifish opercular skin (Evans,
2002
).
In addition to putative chloride cells, NADPHd staining was also localized
to structures in the secondary lamellae. These appeared as occasional bands or
punctuate structures, similar to the extracellular arrangement of collagen
strands found in association with branchial pillar cells
(Hughes and Weibel, 1972;
Olson, 2002
). This staining
may be an artifact, as immunocytochemistry did not reveal similar structures.
However, if real, the presence of NADPHd staining in this location of the gill
may suggest a role of NO in regulating vascular resistance within the gill.
Pillar cells may be contractile and respond to paracrine control
(Olson, 2002
), and NO is well
recognized as a vasodilator in mammals, and more recently in fish
(Fritsche et al., 2000
;
Haraldsen et al., 2002
).
In conclusion, this study suggests a role of NO in regulation of branchial ion transport. Colocalization of NOS activity and the essential ion pump, Na+,K+-ATPase in chloride cells of FW salmon smolts suggests an important physiological inhibition by NO of salt extrusion at a critical developmental stage.
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Acknowledgments |
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References |
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