Regulation of branchial Na+/K+-ATPase in common carp Cyprinus carpio L. acclimated to different temperatures
Department of Animal Physiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
* Author for correspondence (e-mail: gertflik{at}sci.kun.nl)
Accepted 31 March 2003
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Summary |
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Key words: temperature acclimation, osmoregulation, Na+/K+-ATPase, cortisol, prolactin, chloride cell, immunohistochemistry, real-time RT-PCR, common carp, Cyprinus carpio
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Introduction |
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Some physiological phenomena in fish that depend critically on temperature
are oxygen consumption (Becker et al.,
1992), immune competence (Le
Morvan-Rocher et al., 1995
), growth and metabolism
(Burel et al., 1996
;
Fine et al., 1996
). Also it
may be anticipated that osmoregulation, involving energised ion pumps in the
gills (and in other osmoregulatory organs such as intestine and kidney), is a
critically temperature-sensitive process. Ambient temperature determines the
hydromineral status of the body fluids of freshwater fish by influencing both
passive and active ion-transport mechanisms. For example, a rise in
temperature will increase passive processes such as loss of ions and gain of
water, thus requiring enhanced osmoregulatory activity. Higher temperatures
will activate enzyme-driven ion transport (defined for example by a
Q10 value or activation energy). Not all components of a
physiological process are equally sensitive to temperature, yet may still
require secondary adjustment to altered key enzyme activities, and thus
eurythermal fish must have developed adequate adaptation strategies to cope
successfully with varying environmental temperatures.
Gills play a crucial role in hydromineral homeostasis of a fish. In
freshwater fish, the function of chloride cells in the branchial epithelium is
uptake of ions (Na+, Cl-, Ca2+) from the
surrounding water (Flik et al.,
1994; Perry,
1997
). The most important and extensively studied enzyme in the
chloride cell is sodium/potassium-activated adenosine triphosphatase
(Na+/K+-ATPase), the enzymatic expression of the sodium
pump, which is under multiple hormonal control
(McCormick, 1995
;
Young et al., 1995
;
Evans, 2002
). Cortisol and
prolactin are considered the most important endocrine factors.
Cortisol is the end product of the
hypothalamicpituitaryinterrenal axis (HPI-axis;
Wendelaar Bonga, 1997) and
stimulates Na+/K+-ATPase activity in many freshwater
teleost fish species, including cichlids
(Dange, 1986
;
Dang et al., 2000a
), salmonids
(Richman and Zaugg, 1987
) and
cyprinids (Abo Hegab and Hanke,
1984
). Cortisol enhances chloride cell numbers
(Richman and Zaugg, 1987
;
McCormick, 1990
) and size
(Madsen, 1990
;
McCormick, 1990
;
Dang et al., 2000a
).
Prolactin, on the other hand, exerts a mainly inhibitory control over
branchial Na+/K+-ATPase activity
(Pickford et al., 1970
;
Madsen and Bern, 1992
), and
reduces chloride cell numbers and activity
(Foskett et al., 1982
).
Moreover, prolactin stimulates Ca2+ uptake
(Flik et al., 1994
) and limits
branchial permeability to water and ions
(Hirano, 1986
;
Wendelaar Bonga et al., 1990
;
Evans, 2002
).
Our previous experiments show that carp acclimated to increasing water
temperature show increased basal plasma cortisol levels
(Arends et al., 1998). In the
present study, we evaluate the hydromineral and endocrine consequences of
acclimation to water at 15, 22 and 29°C. We anticipate that during
temperature acclimation, cortisol and prolactin activities may change to
warrant hydromineral homeostasis via up- or downregulation of
Na+/K+-ATPase activity. Plasma osmolality and ion
composition, gill Na+/K+-ATPase activity and expression
were assessed to establish the end points of the adaptation strategy. The
possible roles of plasma cortisol and pituitary prolactin (the latter
quantified by real-time polymerase chain reaction) in the adaptation process
will be discussed. It appears that the common carp is a species with subtle
mechanisms and unexpected strategies for temperature adaptation.
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Materials and methods |
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Plasma parameters
Fish were anaesthetised in 0.1% (v/v) 2-phenoxyethanol (Sigma, St Louis,
USA). Immediately thereafter (always within 2 min), 1 ml blood was taken from
the caudal vessels, using a 1 ml syringe that contained 20 µl of 2% (w/v)
Na2EDTA (Sigma) as anti-coagulant. Blood was transferred to
ice-cold Eppendorf tubes containing 1 trypsin-inhibiting unit (t.i.u.) of
aprotinin (Sigma). After 5 min centrifugation (1000 g,
4°C), plasma was separated from blood cells and stored at -20°C until
analysis.
Plasma cortisol was measured by radioimmunoassay (RIA) as described by
Arends et al. (1998). Plasma
osmolality was measured on a freezing-point depression osmometer (Gonotec,
Berlin, Germany). Plasma Na+ and K+ concentrations were
determined by flame photometry (Radiometer Copenhagen FLM3 flame photometer);
Cl- was measured spectrophotometrically by the formation of
ferrothiocyanate (O'Brien,
1962
). Ca2+ was measured spectrophotometrically with a
commercial kit (Sigma). The free Ca2+ fraction was determined after
filtering heparinised blood plasma over a 10 kDa membrane.
Na+/K+-ATPase
The specific, Na+- and K+-dependent,
ouabain-sensitive ATPase activity was measured in crude gill homogenates
containing saponin, to obtain optimal substrate availability, as described by
Flik et al. (1983).
Homogenates (final protein content 1 mg ml-1) were divided into 12
fractions and triplicate 10 µl samples were incubated for either 15 min at
37°C, 20 min at 29°C, 30 min at 22°C or 45 min at 15°C. The
specific activity was calculated by subtracting the K+-dependent,
ouabain-sensitive ATPase activity from the total ATPase activity. ATP
hydrolysis was assessed by the amount of inorganic phosphate formed
min-1 mg-1 protein under each incubation condition.
Sample protein content was assayed using a commercial protein kit (BioRad,
Hercules, CA, USA).
Histological preparation of the gills was carried out according to standard
protocols (e.g. Dang et al.,
2000b). Briefly, sections were collected from a comparable area in
the trailing edge of the filament where the chloride cells reside. Care was
taken to orient the filaments in a standardised way to obtain cross-sections
through the secondary lamellae perpendicular to the axis of the filament.
After dewaxing, blocking of endogenous peroxidase with 2% (v/v)
H2O2 and blocking of non-specific sites with 10% (v/v)
normal goat serum, the slides were incubated overnight with a monoclonal
antibody against chicken Na+/K+-ATPase (IgG
5,
Developmental Studies Hybridoma Bank, Department of Biological Sciences,
University of Iowa, USA) at a final dilution of 1:500 (v/v). Goat anti-mouse
(Nordic Immunology, Tilburg, The Netherlands) was used as secondary antibody
at 1:150 (v/v) dilution. The slides were subsequently incubated with 1:150
(v/v) diluted mouse peroxidase anti-peroxidase (M-PAP; Nordic). Staining was
performed in 0.025% (w/v) 3,3'-diaminobenzidine (DAB) and 0.0005% (v/v)
H2O2.
Cortisol administration
To determine the effect of exogenous cortisol on
Na+/K+-ATPase activity, a miniosmotic pump (Model 1007D,
Alzet, California, USA) was implanted into the peritoneal cavity of the
anaesthetised fish. This allowed for a stable elevation of plasma cortisol
levels for 1 week without repetitive handling. This approach was used instead
of cortisol injection, which evokes stress responses due to repetitive
handling, or feeding, which yields high individual variation. The minipumps
were filled with cortisol (hydrocortisone; Sigma) at 6 mg kg-1 body
mass using 30% (w/v) 2-hydroxypropyl-ß-cyclodextrin (Sigma) as vehicle.
Control animals received vehicle only. After 1 week, the fish were killed,
blood sampled, and gills were taken and analysed as described above.
Prolactin expression
Relative expression of prolactin was assessed using real-time quantitative
polymerase chain reaction (PCR). Pituitary glands, rapidly removed after
anaesthesia, were brought into 250 l Trizol reagent (Gibco BRL, Gaithersburg,
USA), immediately followed by total RNA extraction according to the
manufacturer's instructions. To ensure complete removal of traces of genomic
DNA, a sample equivalent to 1 µg of total RNA was incubated with 1 unit
DNase I (amplification grade; Gibco BRL) for 15 min at room temperature. To
inactivate DNase, 1 µl of 25 mmol l-1 EDTA was added and the
sample was incubated for 10 min at 65°C to simultanuously linearise RNA.
Thereafter, the RNA was reverse transcribed (RT) with 300 ng random primers
(Gibco BRL), 0.5 mmol l-1 dNTPs, 10 units RNase Inhibitor (Gibco
BRL), 10 mmol l-1 dithiothreitol and 200 units
SuperscriptTM RT (Gibco BRL) for 50 min at 37°C. For
quantitative PCR analysis, 5 µl of 50x diluted RT-mix was used as
template in 25 µl amplification mixture, containing 12.5 µl SYBR Green
Master Mix (Applied Biosystems Benelux, Nieuwerkerk aan den IJssel, The
Netherlands) and 3.75 µl of each primer (final concentration 600 nmol
l-1). The primer sets used in the PCR were, for prolactin (165 bp;
303 bp in the case of genomic DNA): forward 5'-CAT CAA TGG TGT CGG TCT
GA-3', reverse 5'-TGA AGA GAG GAA GTG TGG CA-3', and for
ß-actin (154 bp; 255 bp in case of genomic DNA): forward 5'-GCC CCC
AGC ACA ATG AAA A-3', reverse 5'-GGT GGA CGA TGG ATG GTC-3'.
After an initial step at 95°C for 10 min, a real-time quantitative PCR of
40 cycles was performed (GeneAmp 5700, Applied Biosystems), each cycle
consisting of 15 s denaturation at 95°C and 1 min annealing and extension
at 60°C. Cycle threshold (CT) values were determined
and expression of prolactin was calculated as a percentage of ß-actin
expression.
Statistical analyses
In all experiments, differences among groups were assessed by the
non-parametric MannWhitney U-test. Linear regression for
correlation data was based on the least squares method. Significance was
accepted at P<0.05. All values are expressed as means ±
standard error of the mean (S.E.M.).
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Results |
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Na+/K+-ATPase activity assayed under conditions of Vmax (i.e. at 37°C) was twofold higher in gill homogenates from the cold-acclimated group compared to those of the 22°C- and 29°C-adapted group (Fig. 1; N=8, P<0.01). However, Na+/K+-ATPase activity measured at the acclimation temperature of the fish (Vapparent), revealed that the homogenates from the 29°C-acclimated group had the highest apparent Na+/K+-ATPase activity (N=8; P<0.05). Na+/K+-ATPase activity did not differ significantly between the sham-treated group and the untreated group at 22°C, but implantation for 7 days of with a miniosmotic pump filled with cortisol resulted in a significant increase in Na+/K+-ATPase activity (N=8, P<0.05).
|
The Na+/K+-ATPase activity of gill homogenates from carp acclimated to 15, 22 or 29°C at different in vitro assay temperatures is shown in Fig. 2. Discontinuity in the Arrhenius plots of enzyme activity were evident in homogenates from the 15°C-acclimated fish at 29°C and the 22°C-acclimated fish at 22°C. No break in the plot of the 29°C fish was observed over the range of the temperatures tested. Calculated Q10-values for Na+/K+-ATPase activity were approximately 1.7 for the 15 and 22°C-acclimated fish at the higher temperatures and for the 29°C fish at all temperatures tested. At temperatures below the breakpoint, Q10-values were significantly higher and increased up to 2.3.
|
Light microscopical analysis showed that the gill filaments of 15°C-acclimated fish were thicker and the lamellae slightly shorter and thicker than those of 29°C-acclimated fish (Fig. 3). Na+/K+-ATPase immunopositive cells (chloride cells, CCs) were located only in the interlamellar spaces in all fish analysed. CCs in 15°C-acclimated fish were more abundant (105±12 CCs mm-1 versus 62±8 CCs mm-1; N=8, P<0.05), appeared larger and contained visibly more immunoreactive Na+/K+-ATPase than those of 29°C-acclimated fish. The gill structure of 22°C-acclimated fish was very similar to that of the 29°C-acclimated fish, with respect to number and structure of immunoreactive cells (not shown).
|
Fig. 4A shows plasma cortisol values of fish acclimated for 8 weeks to 15, 22 and 29°C, and 22°C-acclimated carp containing an implanted miniosmotic pump filled with cortisol or vehicle (sham-treated). Basal cortisol levels correlated positively with the acclimation temperature within the range of temperatures tested and were, within the range, best-fitted to the equation: plasma cortisol (nmol l-1)=3.86T51 (r2=0.99, P<0.01), where T=temperature. Cortisol-treated fish had 2.4-fold higher circulating cortisol levels than vehicle-treated fish (N=8, P<0.01).
|
Pituitary prolactin expression relative to the housekeeping gene ß-actin, quantified by real-time RT-PCR, was one third as high in the 29°C-acclimated group as in the other two groups (Fig. 4B; N=8, P<0.01).
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Discussion |
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Hydromineral status
Acclimation temperature affects the hydromineral status of common carp,
reflected by different plasma Na+, K+ and Cl-
profiles and total plasma osmolality. We interpret the observation that plasma
Ca2+ levels are perfectly regulated to indicate successful
adaptation of the fish. Changes in plasma Na+, Cl- and
K+ as a result of temperature acclimation have been established
already in many fish species (Burton,
1986), but at variance with our data, a constant plasma ion
composition was reported for carp acclimated to temperatures between 2 and
30°C (Houston et al.,
1970
; Houston and Smeda,
1979
). We observed consistently that plasma osmolality as well as
Na+ and Cl- levels (the major determining ions for
osmolality) are elevated at both 15°C and 29°C when compared to the
values seen in fish at 22°C. We have no clearcut explanation for our
observations, but suggest that the altered endocrine status of the fish (see
below) could at least form the basis for such differences. The adaptive
response and resetting could represent an energetically more profitable
condition for the fish as a result of multiple altered processes in the gills,
as well as in other osmoregulatory organs (e.g. intestine and kidney).
Na+/K+-ATPase activity
When maximum velocity (i.e. under optimal conditions for substrate
concentrations and accessibility as well as temperature, viz.
37°C, yielding an estimate of the total amount of enzyme) was assessed, a
very large rise in Na+/K+-ATPase activity was observed
in the gills from the 15°C-acclimated fish. When we performed
immunohistochemistry on the gills, more and larger chloride cells, containing
more Na+/K+-ATPase immunoreactivity, were observed in
the 15°C-acclimated fish. The immunohistochemical picture is thus in
agreement with the maximum velocity determination of
Na+/K+-ATPase activity. However, note that when the
enzyme activity was assayed at the acclimation temperature of the fish, the
reverse effect was seen: the lowest activity was at 15 and 22°C, and the
apparent highest activity at 29°C. Thus it is important to assay the
enzyme at the acclimation temperature of the fish in order to provide a
physiological interpretation. Furthermore, an intact cell may contain a latent
pool of membrane vesicles carrying the enzyme that would be released and
assayed when a homogenate is made, so biochemical analysis would tend to
overestimate enzyme activity.
It has been demonstrated for many membrane-bound enzymes that a break in an
Arrhenius plot is often caused by a change of membrane fluidity. Membrane
fluidity is largely determined by the lipid composition. Fish acclimated to
lower ambient temperatures respond by lowering the membrane melting point
(Wodtke, 1981;
Hazel, 1984
). As the breaks in
the Arrhenius plot in this study appear to move rightwards with increasing
acclimation temperature, we speculate that the breaks are related to protein
thermo(in)stability or differences in isozyme activities rather than membrane
fluidity. Indeed, different Na+/K+-ATPase isozymes have
been identified in fish gills (Cutler et al.,
1995
,
2000
).
It would seem that carp acclimated to 15°C increase chloride cell numbers and Na+/K+-ATPase expression to compensate for the lower reaction rate that occurs at this temperature. Slight overcompensation might explain the enhanced plasma Na+ and Cl- levels seen in these fish.
Cortisol and prolactin
It was predicted that the altered hydromineral status of carp upon
acclimation to different temperatures results from changes in their endocrine
status, since the ion pumps in the gills are under strict hormonal control.
Cortisol and prolactin are the two classical
Na+/K+-ATPase regulators in fish, having opposite
effects. Cortisol is a stimulator of Na+/K+-ATPase
activity, and is often therefore referred to as a `seawater-adapting hormone',
as branchial Na+ secretion in seawater requires enhanced
Na+/K+-ATPase activity
(Epstein et al., 1980;
McCormick, 1995
). Prolactin,
on the other hand, is referred to as the `freshwater-adapting hormone',
inhibiting Na+/K+-ATPase activity and ensuring low
water- and ion-permeability of the gills
(Hirano, 1986
;
Wendelaar Bonga et al., 1990
;
Bern and Madsen, 1992
). A
critical role for cortisol, not just in seawater fish but also in freshwater
fish, cannot be denied as both cortisol and prolactin appear to be necessary
to maintain ionic homeostasis in hypophysectomised freshwater catfish
(Parwez and Goswami,
1985
).
Our data confirm the results of Arends et al.
(1998) and Van den Burg et al.
(2003
), who showed that rising
temperatures induce increased basal plasma cortisol levels. Unexpectedly, we
found no relationship between basal plasma cortisol levels and branchial
Na+/K+-ATPase abundance. Yet, after 1 week of
exogenously administered cortisol, Na+/K+-ATPase
activity was clearly enhanced, which is in agreement with earlier studies
(Abo Hegab and Hanke, 1984
;
De Boeck et al., 2001
). It
would seem that circulating cortisol levels must exceed a certain threshold
level to act as a stimulator of branchial Na+/K+-ATPase
activity. Indeed, it has been shown in freshwater rainbow trout that chloride
cells contain at least two cortisol receptors, a mineralocorticoid receptor
(MR) having a high affinity and a glucocorticoid receptor (GR) with a lower
affinity (Ducouret et al.,
1995
; Colombe et al.,
2000
; Sloman et al.,
2001
). Assuming a similar situation in carp, it follows that the
enhanced Na+/K+-ATPase activity after exogenously
administered cortisol results from a GR-mediated effect of cortisol. An
attractive hypothesis proposed by Sloman et al.
(2001
), based on GR and
MR-pharmacological studies, is that the more sensitive MR is upregulated in
situations when chloride cell proliferation is required, for example in
ion-deficient water. A similar activation of a silent MR would explain our
observations in the 15°C-acclimated carp, while the circulating basal
cortisol levels are too low for a GR-mediated effect. The differences in basal
cortisol levels observed here may thus reflect more so the metabolic status of
the fish at different temperatures.
Pituitary prolactin expression was downregulated in the 29°C-acclimated
group. Assuming that 8 weeks of acclimation results in a steady resetting of
the protein expression machinery in a cell, we speculate that differences in
mRNA levels reflect equivalent differences in protein production and
secretion, implying that 29°C-acclimated carp have lower plasma prolactin
levels (which we cannot determine directly by radioimmunoassay) than the 15
and 22°C-acclimated fish. This is in agreement with plasma measurements in
rainbow trout Oncorhynchus mykiss
(Rand-Weaver et al., 1995) and
newt Cynops pyrrhogaster
(Takahashi et al., 2001
) kept
at different ambient temperatures. Since prolactin limits membrane
permeability to water and ions (Hirano,
1986
; Wendelaar Bonga et al.,
1990
; Evans,
2002
), our observation that 29°C-acclimated fish have a higher
apparent Na+/K+-ATPase activity is in accordance with an
enhanced branchial permeability as a result of downregulated prolactin
expression, leaving plasma mineral composition within a physiological
range.
Concluding remarks
Taken together, the hydromineral status of carp appears to be reset when
the fish is acclimated to different temperatures. To cope with different
temperatures, the eurythermal carp exploits various strategies resulting in
subtle yet significant readjustments of hydromineral balance. Clearly,
branchial Na+/K+-ATPase activity is differentially
regulated. We interpret our data to indicate that carp, at higher ambient
temperatures, rely on a temperature-enhanced branchial enzymatic pump
activity. A higher apparent Na+/K+-ATPase activity,
combined with enhanced branchial permeability, ensures ionic homeostasis. At a
lower ambient temperature, by contrast, branchial
Na+/K+-ATPase expression is upregulated to counteract
the temperature-inhibited activity of the sodium pump, perhaps via a
mineralocorticoid receptor. Understanding the role of cortisol in temperature
adaptation requires further study of the regulation of cortisol receptors in
the gills.
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Acknowledgments |
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