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First published online March 28, 2008
Journal of Experimental Biology 211, 1262-1269 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.013474
Cold induced changes of adenosine levels in common eelpout (Zoarces viviparus): a role in modulating cytochrome c oxidase expression
Alfred Wegener Institute for Polar and Marine Research, Marine Animal Physiology, Am Handelshafen 12, 27570 Bremerhaven, Germany
* Author for correspondence (e-mail: Magnus.Lucassen{at}awi.de)
Accepted 14 February 2008
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Summary |
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Key words: hepatocytes, primary culture, adenosine, temperature acclimation, cytochrome c oxidase, citrate synthase, RNase protection assay
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Pörtner, 2002). These
limits exert their effects on the growth rate of individual specimens and the
abundance of a population, thereby shaping the biogeography of a species
(Pörtner and Knust,
2007).
Many temperate zone poikilotherms possess the ability to acclimate to
various temperatures and thereby shift their thermal tolerance windows. The
limits are indicated by an onset of functional hypoxia, which has to be
overcome during acclimation. The basis of thermal windows and acclimation is
determined at the molecular level, including membrane composition, protein
structure and enzyme functioning, with mitochondria having a key role
(Pörtner et al., 2005).
For example, during cold exposure of temperate zone fish, muscle mitochondrial
density and/or oxidative capacity increase to compensate for the decelerating
effects of low temperatures on metabolic rate and capacity
(Egginton and Sidell, 1989;
Guderley, 2004). This effect is
associated with increments in the activities of mitochondrial enzymes such as
citrate synthase (CS) and cytochrome c oxidase (COX), two commonly
used indicators for mitochondrial capacities
(Battersby and Moyes, 1998;
St-Pierre et al., 1998).
However, liver was found to display different patterns of thermal acclimation.
In temperate zone eelpout and cod, liver CS activities were elevated in the
cold, whereas the activities of COX remained largely unaltered
(Lannig et al., 2003;
Lucassen et al., 2003;
Lucassen et al., 2006). This
may indicate a functional adaptation of mitochondria, to adjust to
tissue-specific demands in the cold, such as enhanced lipid synthesis
(Pörtner et al.,
2005).
Adjustments of mitochondrial content and function are not only induced by
cold or warm acclimation, but also by various other physiological challenges.
For example, an increase of capacity accompanies endurance exercise training
or shivering thermogenesis, and a decrease occurs during hypoxia. The primary
effector has not been established for any of these examples, but it is an
attractive hypothesis that bioenergetic disturbances themselves contribute to
these adjustments. Several bioenergetic factors have been discussed to elicit
proliferation (Hood, 2001;
Leary and Moyes, 2000) and
only recently, nitric oxide emerged as a good candidate in mammals
(Nisoli et al., 2004). It
still needs to be established, whether the same factor(s) and pathway(s) are
involved in ectotherms and in response to all physiological challenges.
One potential bioenergetic signal to modulate thermal adjustments is
adenosine. Adenosine is predominantly produced following a breakdown of
cytosolic ATP levels and thus indicates an acute insufficiency of energy
metabolism. It can be released from the cells by specialized nucleoside
transporters and thus affect the whole organism
(Buck, 2004). Adenosine induces
a number of physiological effects that commonly balance energy demand and
supply at low energy availability, and is therefore often described as a
`retaliatory metabolite' (Newby et al.,
1990). These effects include a reduction of protein synthesis rate
(Tinton et al., 1995), a
decrease of oxygen consumption
(Krumschnabel et al., 2000) or
a stimulation of anaerobic glycolysis
(Lutz and Nilson, 1997).
Adenosine is usually examined in the context of hypoxic or anoxic exposure
(Lutz and Kabler, 1997;
Reipschläger et al.,
1997; Renshaw et al.,
2002). However, since thermal acclimation in fact involves
compensation for temperature-induced hypoxia
(Pörtner, 2002) adenosine
may also play a role in temperature adaptation.
The purpose of this study was to examine (1) if acute temperature changes
affect adenosine concentrations in ectothermic marine fish, (2) if adjustments
in mitochondrial function observed in vivo can be induced in isolated
fish hepatocytes with temperature as a single factor, (3) if adenosine
participates in the regulation of mitochondrial adjustments to temperature,
(4) how adenosine action on mitochondrial functions might be mediated. This
study was performed in the common eelpout, Zoarces viviparus, which
has become a model organism to study thermal acclimation and limitation
(Pörtner and Knust,
2007). Here we describe, for the first time, that adenosine is a
suitable effector to modulate the thermally induced cellular acclimation
response.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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salinity and fed twice per week with
small shrimps (Neomysis integer, Crangon crangon). Animals (mass:
13.5±1.2 g, mean ± s.e.m.) were acutely transferred to
4.0±0.5°C and sampled before (t=0) and after 1 and 3 days
of cold incubation. For the preparation of hepatocytes North Sea eelpout,
caught in the German Bight in spring 2004 (mass: 67.3±25.9 g) were
acclimated to either 4.0±0.5°C (representing mean winter habitat
temperature) or 11.0±0.5°C (close to optimum temperature) and a
salinity of 30 for at least 2 months. During the acclimation period
specimens were fed once per week with Crangon crangon.
Determination of adenosine concentrations
Blood was collected from fish anaesthetised with 0.5 g l–1
MS-222 (3-amino-benzoic-methanosulfonate) by opening the caudal vein. Livers
were excised, immediately frozen in liquid nitrogen and animals were killed by
a cut through the spinal cord. Blood was stored on ice for 4 h to allow
coagulation, centrifuged for 10 min at 5000 g and the serum
was transferred into fresh reaction tubes. Preliminary tests confirmed the
adequacy of this sampling procedure, which kept adenosine levels close to or
below detection limits in unstressed animals.
Serum samples were mixed with 0.2 volumes ice-cold TCA (15% trichloracetic acid), sonicated for 1 min at 0°C in a bath sonicator (Branson, Danbury, CT, USA) and centrifuged (4 min at 16 000 g, 0°C) to precipitate proteins. The supernatants were neutralized with 4 volumes of tri-n-octylamine/1,1,2-trichlortrifluorethane mix (1:4), centrifuged for 3 min at 16 000 g, 0°C, and the upper phase was collected. Frozen liver samples were pulverized under liquid nitrogen, suspended in 3.5 volumes ice-cold TCA and processed as described for serum samples, but the pH of the extracts was adjusted to 9.0–9.4 with 2 mol l–1 NaOH.
Adenosine was determined by capillary electrophoresis (Beckman, Fullerton,
CA, USA) using a method modified after Casey et al.
(Casey et al., 1999). Extracts
were supplied with 0.4 mmol l–1 uric acid as an internal
standard and filtered through a 0.2 µm syringe filter. Samples were
separated on a 50 µm diameter uncoated fused silica capillary with a
current of 30 kV at 40°C. Adenosine peaks were identified by migration
time and sample spiking. Adenosine concentrations were calculated from the
area ratio of adenosine:uric acid using a calibration curve created with
concentrations between 0.5–50 µmol l–1
adenosine.
Isolation of hepatocytes
Hepatocytes were isolated following a procedure modified after Mommsen et
al. (Mommsen et al., 1994).
For each cell culture two fish from the same acclimation temperature were
prepared simultaneously to obtain a sufficient number of cells. Animals were
anaesthetised with 0.5 g l–1 MS-222 and killed by a cut
through the spinal cord. The liver of the first fish was carefully excised and
weighted, transferred immediately to ice-cold solution 1 (magnesium-free
Hank's medium, containing 240 mmol l–1 NaCl, 10 mmol
l–1 Hepes, 5.5 mmol l–1 glucose, 5.4 mmol
l–1 KCl, 4.2 mmol l–1 NaHCO3, 0.4
mmol l–1 KH2PO4, 0.3 mmol
l–1 Na2HPO4, pH 7.4) and perfused
through the vena hepatica with solution 1 to remove blood cells. The second
liver was prepared accordingly. Subsequently both livers were transferred to
one vial and each organ was perfused two times with 2 ml g–1
fresh mass ice-cold collagenase solution [solution 1 + 1% bovine serum albumin
(BSA) + 750 i.u. ml–1 collagenase type IV]. Between
perfusions livers were gently massaged for about 10 min. After finely chopping
the tissue the homogenate was gently shaken on ice for 60 min. Finally, the
suspension was filtered through 250 µm mesh-size gauze, and hepatocytes
were collected by centrifugation (4 min at 70 g, 0°C) and
washed by repeated centrifugation (2 min at 70 g, 0°C) in
solution 1 containing 1% BSA to remove collagenase, lipids and erythrocytes.
Cells were resuspended in culture medium (Leibovitz L-15 medium + 103 mmol
l–1 NaCl + 10 mmol l–1 Hepes + 1% BSA + 5
mmol l–1 glucose + 1% penicillin–streptomycin; pH 7.8
at 4°C) and shaken on ice until being dispersed for primary cell culture.
Cell density and viability was determined in a Fuchs-Rosenthal haemocytometer
dish by Trypan Blue exclusion.
Cell culture and incubation conditions
Cells were incubated in polystyrene 6-well plates. To each well 2 ml of
culture medium was added and plates were pre-cooled to 4.0°C. The
hepatocyte suspension was equally portioned between wells to a concentration
of 2x106 viable cells per well. Culture dishes were briefly
shaken by hand to spread cells on the bottom. Cells were incubated under air
atmosphere at 4.0°C or 11.0°C (±0.1°C), and 5 µmol
glucose were added per 106 cells per day. In parallel to the
control group, cells were exposed to adenosine at both acclimation
temperatures using the same basic culture conditions. Cells were supplied with
100 nmol ml–1 adenosine directly after dispersion and once
every 24 h thereafter. To investigate the potential role of adenosine
receptors, one group of cells was incubated for 30 min with 100 nmol
ml–1 8-phenyltheophylline (8-PT), a selective adenosine
A1-receptor antagonist, always prior to addition of adenosine, and
in another group adenosine was replaced with 100 nmol ml–1
5'-(N-ethylcarboxamido)adenosine (NECA), a non-selective
adenosine receptor agonist. Owing to limited cell numbers, incubations with
8-PT or NECA were only performed with cells from warm-acclimated fish at
11°C.
For sampling of hepatocytes, culture dishes were transferred onto ice without shaking and 1 ml of culture medium was removed. Cells were resuspended in the remaining culture medium and precipitated by centrifugation (2 min at 1000 g, 0°C). After residual medium was carefully removed, cells were immediately frozen in liquid nitrogen. Samples were collected after 48 and 72 h of incubation. Since no differences were seen between the two time points (multi-factorial ANOVA) values were pooled and treated as replicate samples.
In an additional incubation series the viability of hepatocytes during cell culture was determined by Trypan Blue exclusion. Therefore, cells incubated at 4 or 11°C were harvested after 48 and 72 h by resuspension in their culture medium. Three sub-samples were taken for each well and mixed with 1:6 volumes of 0.4% Trypan Blue dye (Sigma, Steinheim, Germany). Numbers of cells that excluded or took up the dye were counted in a Fuchs-Rosenthal haemocytometer dish.
Enzyme activity
Cells were homogenized in 150 µl ice-cold buffer (20 mmol
l–1 Tris–HCl, 1 mmol l–1 EDTA, 0.1%
Tween 20, pH 7.4) by shaking on a Vortex Genie2 (Scientific Industries, New
York, NY, USA) for 2 min at the highest level. COX and CS activities were
determined at 20°C in a thermostatted spectrophotometer (Beckman,
Fullerton, CA, USA) according to Lucassen et al.
(Lucassen et al., 2003). Prior
to measurement of CS activity, homogenates were sonicated in a bath sonicator
(Branson, Danbury, CT, USA) for 5 min at 0°C.
RNA isolation and construction of probes
Total RNA from hepatocytes was isolated using the RNeasy kit (Qiagen,
Hilden, Germany) according to the manufacturer's protocol for animal cells.
RNA was quantified in a spectrophotometer (Eppendorf, Hamburg, Germany) with
A260:A280 ratios always >1.8. Additionally, the
integrity of RNA was verified by formaldehyde agarose gel electrophoresis
(Sambrook et al., 1989).
For sequencing of the Z. viviparus COX4 gene, mRNA was isolated
from total RNA with the Oligotex kit (Qiagen, Hilden, Germany). RT–PCR
was performed according to the method of Lucassen et al.
(Lucassen et al., 2003) with
gene-specific primers (Table 1)
designed homologous to published sequences, using the MacVector 7.0 program
package (Accelrys, Oxford, UK). The PCR reaction containing 1.5 mmol
l–1 MgCl2 was performed with 32 cycles of 45 s
denaturation at 94°C, 2 min annealing at 57±6°C and 1 min
elongation at 72°C. Separation, cloning and analyses of PCR fragments was
performed as described by Mark et al.
(Mark et al., 2006).
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The 5' and 3' termini of COX4 mRNA were identified with the
RLM–RACE kit (Ambion, Austin, TX, USA) following the manufacturer's
manual. Gene-specific backward primers for 5'-RACE and forward primers
for 3'-RACE were designed according to the partial sequence determined
for COX4 of Z. viviparus. Forward primers for amplification of the
5' terminus and backward primers for the 3' terminus,
corresponding to the adapter sequence, were provided with the kit. Cloning and
analysis of positive clones was performed as for RT–PCR fragments
(Mark et al., 2006).
For construction of the COX4-specific probe, primers for a 183 bp fragment
were designed appropriate to the coding sequence and used for RT–PCR as
described above. A 255 bp COX2 fragment was isolated from an existing 507 bp
fragment [(Lucassen et al.,
2003); accession no.: AY227660] by PCR with the primers given in
Table 1. Existing fragments
were applied for the construction of CS (369 bp; accession no.: AY382597) and
β-actin (215 bp; accession no.: AY227657) probes
(Lucassen et al., 2003;
Mark et al., 2006).
Quantification of RNA
Gene-specific RNA transcripts of CS and the two COX subunits were
quantified with a ribonuclease protection assay (RPA) using the RPA III kit
(Ambion, Austin, TX, USA), with β-actin as an internal standard to
correct for loading differences. The construction of templates from the PCR
clones described above and the in vitro synthesis of
[
-32P]UTP-labelled antisense probes was performed according
to the method of Lucassen et al. (Lucassen
et al., 2003). To obtain equal intensities for protected
fragments, specific radioactivity was applied as follows: 1000 Ci mmol
l–1 for CS and COX4 probes, and 45 Ci mmol
l–1 for COX2 and β-actin probes.
The RPA was adjusted to low RNA amounts (2 µg) following the
manufacturer's instructions. Accordingly, the amount of each radiolabelled
probe was lowered to 20 000 cts min–1 per sample, and
hepatocyte RNA was supplied with an equal quantity of yeast RNA, to improve
the formation of pellets. Sample RNA was simultaneously hybridized to all
antisense probes at 42°C. RNAse treatment was performed with an RNaseA/T1
dilution of 1:100. RNA:RNA hybrids were precipitated and separated by
denaturing PAGE (Lucassen et al.,
2003). Radioactivity was detected and quantified in a phosphor
storage imaging system (Fuji, Tokyo, Japan) using the AIDA software package
(raytest, Straubenhardt, Germany).
Statistical analyses
Statistical significance was tested at the P
0.05 level.
Outliers were identified on the 95% significance level with Nalimov's test
(Noack, 1980). Adenosine
concentrations were analysed using one-factor ANOVA and the post-hoc
Student–Newman–Keuls test. Differences in freshly isolated
hepatocytes were analysed using a t-test. The impact of acclimation
temperature in primary cell culture was observed with multi-factorial ANOVA.
The effects of different treatments during cell culture in each acclimation
group were analysed in a pairwise manner using repeated measures ANOVA and the
post-hoc Student–Newman–Keuls test. Linear regressions
and squared correlation coefficients were calculated using SigmaStat 3.0. Data
are given as means ± s.e.m. (N=4–10).
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RESULTS |
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Effects of temperature and adenosine on enzyme activities
The effect of temperature acclimation in vivo on mitochondrial
enzyme activities was determined in freshly isolated hepatocytes of Z.
viviparus acclimated at 4 and 11°C. Citrate synthase (CS) activity
per 106 cells was significantly higher, by 44±10%, in cells
from cold-acclimated compared with those from warm-acclimated animals
(Fig. 3A), whereas activities
of cytochrome c oxidase (COX) remained unaffected by whole animal
acclimation (Fig. 3D). During
cell culture, the original acclimation temperature in vivo influenced
the response of both enzymes to incubation temperature. Warm incubation of
hepatocytes from 4°C-acclimated eelpout caused a significant decrease of
CS and COX activities, by 26±4% and 30±4%, respectively,
compared with their cold-incubated counterparts
(Fig. 3B,E). By contrast,
enzyme activities in cells isolated from warm-acclimated fish were not altered
by cold or warm incubation (Fig.
3C,F). Adenosine treatment of isolated hepatocytes had no impact
on CS, but significantly affected COX activities. In cells from
cold-acclimated eelpout, adenosine treatment resulted in a reduction of COX
activities by 16±8% at 4°C and by 18±8% at 11°C compared
with their respective control cells (Fig.
3E). In cells from warm-acclimated Z. viviparus COX
activities remained more or less unaffected by the 4°C incubation
temperature, whereas adenosine treatment during warm incubation significantly
reduced COX activities, by 25±12% compared to the untreated control at
11°C (Fig. 3F).
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The expression of COX mRNA was measured by use of the mitochondrial encoded COX2 and the nuclear encoded COX4 subunits, both displaying a similar pattern. In accordance with unchanged enzyme activities, long-term temperature acclimation of Z. viviparus (in vivo) resulted in equal mRNA levels for both subunits in freshly isolated hepatocytes (Fig. 4D,G). However, temperature significantly affected the response of hepatocytes to different incubation conditions. In cells from cold-acclimated Z. viviparus, warm incubation without adenosine induced a significant reduction in COX2 and COX4 mRNA levels, by 34±6% and 37±6%, respectively (Fig. 4E,H). By contrast, adenosine treatment, which had caused a drop in COX activities, significantly increased the expression of COX2, by 27±7% at 4°C and 95±7% at 11°C, and of COX4 by 81±14% at 11°C. The increase in COX2 and COX4 expression was less pronounced in hepatocytes from warm-acclimated eelpout, but followed the same pattern. COX4 mRNA levels were maintained at significantly, by 34±10%, higher levels in cold-than in warm-incubated cells under control conditions, and were increased to levels found in 4°C controls, when treated with adenosine at 11°C (Fig. 4I). For COX2 expression, no effect occurred as a result of incubation temperature, but adenosine treatment at 11°C resulted in mRNA levels 66±20% higher than in control cells (Fig. 4F).
Effects of adenosine receptor antagonists and agonists
A potential role for adenosine–receptor interactions in eliciting
adenosine effects was tested by the addition of 8-PT, an antagonist for
adenosine A1 receptors, and
5'-(N-ethylcarboxamido)adenosine (NECA), a potent non-selective
agonist for adenosine receptors (Ralevic
and Burnstock, 1998). 8-PT- and NECA-treated samples were compared
to the analogous control and adenosine-treated groups of the respective
preparations.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Therefore, adenosine might act as a hormone-like effector
in response to bioenergetic challenges such as temperature changes. However,
although there is no literature available concerning the effect of
temperature, elevated adenosine levels have frequently been reported following
anoxic or hypoxic conditions. In the brain of epaulette shark, adenosine
levels increased during acute anoxia, whereas the levels of phosphorylated
adenylates (ATP+ADP+AMP) remained virtually unchanged
(Renshaw et al., 2002). Since
acute temperature changes result in functional hypoxia
(Pörtner, 2001;
Pörtner, 2002), similar
effects may occur during thermal response. Sartoris et al.
(Sartoris et al., 2003) found
unchanged ratios of phosphorylated adenylates during thermal acclimation of
common eelpout. In the present study, adenosine concentrations were found to
increase significantly in blood serum and liver during 24 h after a
temperature shift from optimum (11°C) to a typically experienced low
temperature (4°C). Increased levels were still visible after 3 days of
incubation (Fig. 1). Changes in
adenosine concentrations are typically small but significant and therefore
elicited by only minor reductions in ATP levels. These observations suggest
that adenosine might be a suitable signal to support metabolic adaptation in
response to such bioenergetic disturbances.
Adenosine concentrations observed in eelpout after cold exposure were
similar to those previously reported for the heart of short-horned sculpin
during acute anoxia (MacCormack and
Driedzic, 2004). Hypoxic conditions usually cause an increase in
adenosine levels for minutes or hours
(Renshaw et al., 2002;
Lutz and Kabler, 1997). By
contrast, cold-induced adenosine accumulation in Z. viviparus
persisted for at least 3 days (Fig.
1), possibly as long as the hypoxic challenge remained
uncompensated for. After 3 days of cold exposure liver adenosine
concentrations remained high, whereas serum adenosine had already
significantly decreased (Fig.
1). In our previous study on eelpout, cold compensation of energy
metabolism in liver, monitored by mitochondrial enzymes, was shown to become
visible after 4 days (Lucassen et al.,
2003). In line with thermally induced hypoxia and functional
insufficiency an extended exposure to temperature change may result in a
prolonged elevation of adenosine levels until the seasonal acclimation process
is well underway. This would allow adenosine to contribute to the cold
acclimation process.
Impact of temperature and adenosine on mitochondrial enzymes
To test for a potential role of adenosine as a systemic effector in thermal
adaptation, we investigated its influence on the acclimation response of
isolated hepatocytes from cold- versus warm-acclimated fish.
Therefore, we monitored the activity and expression of the mitochondrial key
enzymes citrate synthase (CS) and cytochrome c oxidase (COX) in isolated
hepatocytes. Elevated activities of the mitochondrial matrix enzyme CS in
conjunction with conserved capacities of membrane bound COX typify the cold
acclimation response of eelpout liver
(Lucassen et al., 2003). By
contrast, both enzymes typically increase in parallel in red and white muscle
(Lannig et al., 2003;
Lucassen et al., 2006). In
line with earlier observations, cold acclimation of Z. viviparus in
vivo had no impact on COX activities
(Fig. 3D), but resulted in an
increase of CS activities (Fig.
3A). The mRNA levels of CS
(Fig. 4A) and of two COX
subunits, the mitochondrial-encoded subunit COX2
(Fig. 4D) and the
nuclear-encoded subunit COX4 (Fig.
4G), were not affected by in vivo temperature
acclimation. The acclimation profile of freshly isolated hepatocytes is thus
in line with the situation in liver of thermally acclimated specimens
(Lucassen et al., 2003).
The consecutive response of isolated hepatocytes to temperature clearly
depended on the previous acclimation temperature of the cells in
vivo. Warm incubation of cells from cold-acclimated fish induced a muscle
type reduction of the activities of both mitochondrial enzymes
(Fig. 3B,E) and a decrease of
mRNA expression of both COX subunits (Fig.
4E,H). In line with an earlier study on catfish hepatocytes
(Koban, 1986), this suggests
that fish liver cells without any systemic input may display basic adjustment
to warming.
By contrast, cold incubation of hepatocytes from warm-acclimated eelpout
left enzyme activities unchanged (Fig.
3C,F) and solely increased the mRNA levels of COX4
(Fig. 4I). These findings are
in line with the concept that a cold-induced mismatch of energy demand and
supply becomes effective at a high organisational level, the intact animal, as
a consequence of limitations in oxygen supply
(Pörtner, 2001;
Pörtner, 2002). The
decrease in the metabolic rate of isolated cells in the cold parallels the
reduction of energy demand and occurs at ample oxygen supply. This
experimental situation alleviates the pressure to acclimate, an observation,
which indicates that cold acclimation in vivo occurs in response to
systemic signal(s).
Adenosine probably participates as a modulator of thermal acclimation. When applied to isolated hepatocytes, the metabolite had no effect on CS, but distinctly affected COX activities. Although in vivo acclimation of Z. viviparus changed neither the initial activities nor the mRNA expression of COX, the cellular response to adenosine was found to depend on the thermal origin of the cells. Responsiveness to adenosine was enhanced in hepatocytes from cold-acclimated fish, however, the effects were more pronounced at higher incubation temperatures.
The adenosine effect comprised two major components, a decrease of COX
activity (Fig. 3E,F) and a
concomitant increase of COX mRNA expression
(Fig. 4E,F,H,I). The activity
of COX, the terminal oxygen-consuming step of the respiratory chain, is often
used as an approximation for the aerobic capacity of the cells
(Kadenbach et al., 2000).
Thus, it can be assumed that adenosine reduces the capacity of aerobic energy
production. This is in line with observations by Krumschnabel et al.
(Krumschnabel et al., 2000),
who found reduced oxygen consumption rates under the acute effect (10–30
min) of adenosine in trout hepatocytes in parallel to a decrease of protein
synthesis rate. They assumed that the deceleration of oxygen uptake was due to
diminished cellular ATP demand caused by adenosine. Such an effect may be
paralleled by the reduction of COX capacities as observed in the present
study.
The suppressing effect on aerobic capacities is contrasted by the
stimulating effect of adenosine on the expression of COX. Since this effect
was more pronounced in cells incubated at 11°C, adenosine treatment of
isolated hepatocytes abolished the warming induced reduction of COX
expression. As a result, COX mRNA remained at similar levels at both
incubation temperatures (Fig.
4E,H,I), resembling the expression pattern obtained during in
vivo temperature acclimation (Fig.
4D,G). Adenosine treatment thus results in a discrepancy between
the levels of transcription (increased) and the capacity of the enzyme
(decreased). Similarly, loose coordination between message levels and enzyme
functional capacity was found in the time-course of whole animal acclimation
(Lucassen et al., 2003).
Adenosine may thus influence the coordination of transcriptional and
translational activities or cause posttranslational modification of the enzyme
proteins. Besides, the discrepancy might result from incomplete response of
the cell culture within the experimental time. However, while establishing the
cell culture system we found steady state levels for COX activities
established within the first 48 h, which were conserved for the following 3
days (data not shown). Because of restricted survival time of eelpout
hepatocytes in primary culture, a further extension of the incubation period
was not feasible. A more delayed response of the functional levels compared to
the mRNA response seems to be rather unlikely.
The question arises of how adenosine exerts these effects. A
receptor-mediated action was investigated by application of the adenosine
A1 receptor antagonist 8-PT and the non-selective receptor agonist
NECA. Both ligands have been used to block and stimulate adenosine receptors
in other fish species, respectively
(Krumschnabel et al., 2000;
Rosati et al., 1995). However,
8-PT could not prevent the effects of adenosine, and NECA failed to reduce COX
activities in eelpout liver cells (Fig.
4). Furthermore, hepatocytes continuously consumed adenosine, a
process stimulated at elevated temperature (data not shown). These
observations suggest diffusive entry, and intracellular action of adenosine.
Assuming a receptor-mediated action in accordance with earlier studies on fish
hepatocytes, adenosine concentrations about one order of magnitude higher than
the observed concentrations have been used in our cell culture system. Higher
adenosine levels might be necessary to compensate possible insensitivity of
the isolated cells, but also facilitate the diffusive entry of the metabolite.
Since the actual concentration in incubated cells was not measurable because
of the small sample size, we cannot exclude higher intracellular adenosine
levels in vitro compared to in vivo, which might have
influenced the response of the cells in vitro.
Although no data exist for an intracellular action in fish, adenosine may
act through different mechanisms in hepatocytes. First, adenosine can be
reconverted to AMP by adenosine kinase and give rise to subsequent ATP
synthesis (Bontemps et al.,
1983). ATP is known as an allosteric inhibitor of COX but also
acts as a noncompetitive inhibitor of CS in fish
(Hochachka and Lewis, 1970),
thus adenosine treatment might result in reduced activities of both enzymes
which has, however, not been observed here. Accumulation of ATP upon thermal
acclimation of the cells appears unlikely. Second, high intracellular
adenosine concentrations prevent the hydrolysis of
S-adenosylhomocystein (SAH), a competitive inhibitor of most
S-adenosylmethionine (SAM)-dependent methyltransferases
(Kloor and Oswald, 2004),
which are involved in the methylation of many molecules, e.g. proteins, DNA
and RNA (Chiang et al., 1996).
In knockout mice deficient for the synthesis of hepatic SAM, the levels of
COX1 and COX2 proteins were found to be only half of those in wild-type mice,
whereas the levels of COX2 mRNA remained unaltered, indicating a translational
downregulation of COX (Santamaria et al.,
2003). Although the underlying mechanisms still need to be
investigated, the inhibition of SAM-dependent methyltransferases by adenosine
may account for the mismatch between the expression and activity levels of
COX, observed in adenosine-treated eelpout hepatocytes. The adenosine-related
increase in the expression of both the nuclear and the mitochondrial encoded
COX subunits is most remarkable and suggests coordinated regulation of nuclear
and mitochondrial genes, thereby substantiating the observed effects. However,
with the data at hand and the sparse literature available, the mechanism of
how adenosine affects COX transcription remains to be elaborated.
Conclusions and perspectives
In summary, the lack of cold acclimation in isolated fish hepatocytes
in vitro and the differences between warm acclimation patterns in
whole animals versus cells isolated from cold-acclimated specimens
indicate the involvement of systemic control in thermal acclimation. The
accumulation of adenosine observed during cold exposure in vivo would
allow for a role for adenosine in thermal acclimation. Our findings suggest
that adenosine specifically modulates mitochondrial functioning. mRNA from
nuclear and mitochondrial encoded COX subunits were found to be increased
under adenosine treatment, resulting in an expression pattern in isolated
hepatocytes similar to the one found during whole animal acclimation. By
contrast, functional levels of COX were decreased in the presence of
adenosine, possibly mediated by the inhibition of methyltransferases. The
detailed mechanisms of action including the stimulating effect of adenosine on
COX transcription await further investigation. Since in vivo thermal
acclimation of liver mitochondria involves the modulation of CS, the lack of
an effect of adenosine on CS activity and expression levels indicate that
further signals remain to be identified.
LIST OF ABBREVIATIONS
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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include samples with adenosine concentration below
detection limit. For these samples the detection limit of the method was used
for calculations. *Significant difference from other time points;
significant difference from control (t=0). Values
are means ± s.e.m. (N=9–10).
