Energy budget of hepatocytes from Antarctic fish (Pachycara brachycephalum and Lepidonotothen kempi) as a function of ambient CO2: pH-dependent limitations of cellular protein biosynthesis?
Alfred-Wegener-Institut für Polar- und Meeresforschung, Ökophysiologie und Ökotoxikologie, Postfach 120161, D-27515 Bremerhaven, Germany
* Author for correspondence (e-mail: hpoertner{at}awi-bremerhaven.de)
Accepted 21 July 2003
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Summary |
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Key words: hypercapnia, CO2, Antarctic fish, Pachycara brachycephalum, Lepidonotothen kempi, oxygen consumption, metabolic rate, respiratory acidosis, protein synthesis, hepatocyte
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Introduction |
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The currently rising concentrations of anthropogenic CO2 in
atmosphere and surface waters (Haugan and
Drange, 1996), and anticipated scenarios of industrial
CO2 disposal in the deep sea, suggest that CO2 will
again become a more general stress factor in aquatic environments, with as yet
unknown effects in species that were not previously affected. Plans to dispose
of anthropogenic CO2 in the deep sea (an idea originally forwarded
by Marchetti, 1977
,
1979
) will expose animals
living in the aphotic zone to elevated CO2 levels, with the highest
concentrations at the site of disposal
(Auerbach et al., 1996
).
The most notable animal groups that will be threatened by increasing ocean
PCO2 levels include cold-water deep-sea fishes,
which at present live in an environment that is characterized by great
temporal and horizontal spatial stability of physical and chemical conditions
(Childress, 1995). Therefore,
it is strongly suggested that deep sea fauna will be sensitive to any change
that occurs suddenly and is well beyond the range of conditions under which
this fauna has evolved (Hädrich,
1996
). General questions arise as to which physiological
mechanisms define the limits of tolerance to elevated CO2 levels,
and whether the physiologically `old' mechanisms of adaptation displayed by
organisms from unstable, hypoxic environments are still in use and effective
in extant fauna from stable deep sea and pelagic waters.
Apart from investigations of the mechanisms of acid-base regulation, little
is known about the influence of hypercapnia on key physiological processes
that define the long-term survival and productivity of a species. Relevant
data have mostly been collected for invertebrates that dwell in environments
that are regularly prone to combined hypercapnia and hypoxia exposures. These
animals can decrease their energy demand far below the standard metabolic
rate. The phenomenon of hypercapnia-induced metabolic depression has been
extensively investigated in the sipunculid Sipunculus nudus, a marine
worm from intertidal and subtidal sandy sediments. Under conditions of severe
respiratory acidosis, the oxygen consumption of whole animals is reduced
(Pörtner et al., 1998)
and linked to a decrease in the metabolic rate of isolated muscle tissue of up
to 45% (Reipschläger and
Pörtner, 1996
; Langenbuch
and Pörtner, 2002
). These reductions in cellular energy
turnover are partly realized by diminished costs of net H+
excretion and slower regulation of intracellular pH (pHi;
Pörtner et al., 2000
).
Concomitant shifts in cellular N metabolism indicate that a change in the use
of amino acid substrates as well as a decrease of protein biosynthesis rates
occurs under acidosis (Langenbuch and
Pörtner, 2002
). Furthermore, a drop in neuronal and motor
activity, caused by the accumulation of the neurotransmitter adenosine,
supports metabolic depression during hypercapnia and particularly during
exposure to combined anoxia and hypercapnia
(Reipschläger et al.,
1997
). The latter data clearly show that the problem of
CO2 tolerance is not only defined at the cellular level. Limiting
factors or processes can become effective at all levels of organisation, from
specific cellular metabolic pathways to the functional integration of
different tissue types into the whole organism under the control of the
central nervous system. Comparable data do not exist at this level of detail
for any other animal phyla, including the vertebrates.
In order to identify and characterise the key processes determining the
CO2 tolerance of vertebrates, and deep sea fishes, especially, it
is crucial to choose an appropriate model organism. Due to extreme
difficulties in the collection and handling of deep sea animals, we decided
instead to study a representative of the Antarctic benthic fauna that displays
physiological characteristics very similar to those in the deep sea, namely
hypometabolism and life in the permanent cold. Analysis of the influence of
elevated PCO2 on the cellular physiology of the
Antarctic eelpout Pachycara brachycephalum was the main focus of the
present study. This member of the cosmopolitan familiy Zoarcidae is endemic to
the Southern Ocean, lives at aphotic depths as far down as 1800 m and displays
a sluggish benthic lifestyle, which contributes to an exceptionally low
metabolic rate that is typical of zoarcids
(Wells, 1987;
Anderson, 1994
;
van Dijk et al., 1999
). To
investigate whether effects seen in this species are representative of
cold-water fish, the analyses were repeated with specimens of
Lepidonotothen kempi. This benthopelagic species probably has a
circum-Antarctic distribution and lives at depths between 100 and 900 m
(Gon and Heemstra, 1990
).
As a first step, we examined whether cellular effects similar to those seen in isolated tissue of the marine invertebrate S. nudus are elicited by experimental hypercapnia in cold-water fish. To this end, correlated changes in pH, PCO2 and aerobic metabolic rate were investigated in isolated hepatocytes from P. brachycephalum and L. kempi. We also determined whether and to what extent a shift in the partitioning of cellular energy occurs under conditions of raised CO2 concentration, including effects on the essential and energetically costly process of protein biosynthesis. Cycloheximide, a specific inhibitor of eucaryotic protein synthesis by inactivating peptidyl transferase activity of the ribosomal 60S subunit, was used to determine what proportion of changes in overall oxygen consumption of the cell were due to protein synthesis.
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Materials and methods |
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Preparation and incubation of isolated hepatocytes
Experiments were carried out onboard RV `Polarstern'. Fish were
anaesthesized with a lethal dose of MS222 (0.5 g l-1) in seawater.
To date no effect of the substance on standard metabolic rate or protein
synthesis in fish has been reported (Hove
and Moss, 1997). However, rainbow trout immersed for periods
longer than 15 min had significantly decreased hepatic glycogen
concentrations, probably mediated by catecholamine release
(Palace et al., 1990
; Perrier
and Bernier, 1998). To avoid this effect we minimized exposure times to MS222
to around 5 min.
The liver was excised and placed on ice in medium 1 (Hepes 10 mmol l-1, NaCl 240 mmol l-1, KCl 5 mmol l-1, KH2PO4 0.4 mmol l-1, Na2HPO4 0.3 mmol l-1, NaHCO3 4 mmol l-1, glucose 5.6 mmol l-1, pH 7.40). Using a small disposable syringe, medium 1 was injected into the hepatic vascular system and the liver blanched immediately due to the clearance of blood from the organ. The perfusate was discarded. Subsequently, the liver was perfused for 20-30 min by repeated injection of 3 ml of ice-cold medium 2 (medium 1 containing 750 U ml-1 Sigma collagenase type IV and 1% bovine serum albumin). Afterwards the liver tissue was finely minced with scissors and incubated for another 45 min on ice in medium 2. Once every 10 min, the tissue was gently torn apart with a plastic Pasteur pipette. After digestion, the resultant cell slurry was poured through a 150 µm nylon mesh to remove large fragments. Remaining blood cells were removed by centrifugation (2-4 min, 80 g, 2°C) and washing of the cellular pellet with medium 3 (medium 1 with 1% BSA). The supernatant and the layer of red cells on top of the hepatocytes were removed using a Pasteur pipette. Isolated hepatocytes were resuspended in 2-3 ml of medium 4 (medium 1 containing 1% BSA, 2 ml 100 ml-1 Life Technologies amino acid mix and 2 mmol l-1 MgSO4). Cell density was determined in a Fuchs-Rosenthal counting chamber and adjusted to 3.5x107 cells ml-1 (L. kempi) or 1.8x107 cells ml-1 (P. brachycephalum), respectively. Cell viability was assessed by examining for the exclusion of Trypan Blue. On average, more than 96% of the L. kempi cells and 93% of the P. brachycephalum cells excluded the dye. In all analyses cells were kept in medium 4 on ice for 1 h before further experimentation.
To measure the respiration of hepatocytes at different extracellular pH
values (pHe) and PCO2, cells were collected
from 130-150 µl samples of the cell suspension and centrifuged for 4 min
(80 g, 2°C). Pellets were resuspended in medium 1 adjusted
to the respective pH (pHe=7.90, 7.20 or 6.50) and
PCO2 (normocapnia: 100% air,
PCO2 =0.03 kPa; hypercapnia: 99% air/1%
CO2, PCO2 =1.01 kPa) and incubated
for 50 min on ice in Eppendorf tubes filled to the top. Oxygen consumption
measurements were conducted in low amino acid medium 1 (in contrast to the
storage medium 4 of the cellular stock solution) to achieve conditions similar
to those presumed for the whole animal during standard metabolism. Conditions
of low pH or hypercapnic stress have been shown to suppress feeding activity
in a variety of organisms (e.g. Bamber,
1987,
1990
) and hepatocytes were
prepared from non-fed fish.
Media used for incubation were bubbled continously using a 2M303/a-F
Wösthoff (Germany) gas mixing pump. The pH of the medium was titrated to
the specific value required using HCl and NaOH, after equilibrating the
solutions with the respective gas mixture. Values were maintained constant
during further bubbling. A pHe of 7.90 was chosen as a control value, as found
in blood plasma from different Antarctic fishes
(Kunzmann, 1991). Acidotic pH
values as low as 6.50 mimic elevated environmental
PCO2 values and were intended to fall slightly
below the range of values reached naturally in fish plasma, e.g. under
conditions of severe metabolic acidosis
(Heisler, 1986a
). To determine
the extent of inhibition of oxygen consumption by cycloheximide, cells were
treated as described above, but the incubation medium contained 35.5 µmol
l-1 cycloheximide (from a stock solution in dimethylsulphoxide) and
cells were incubated for 60 min on ice before oxygen consumption was
determined.
Measurements of cellular oxygen consumption
Oxygen consumption of isolated hepatocytes was measured using a
micro-optode system (PreSens, Neuburg, Germany). The sensor consists of a
fiber optic cable supplied with a standard glass fiber plug to connect to the
optode array. The tip of the sensor is coated with a ruthenium-II-luminophor
complex immobilized in a polymer matrix. Light is emitted from a blue-light
diode and the resulting fluorescence signal is detected and enhanced by a
photomultiplier. Oxygen acts as a dynamic fluorescence quencher of the
luminophor. The intensity, lifetime and modulation of the phase angle of the
fluorescence signal are influenced by the number of oxygen molecules present
and can be measured to calculate oxygen saturation in the medium
(Klimant et al., 1995).
All experiments were performed at 2°C. The cell suspension was constantly stirred in glass microvials (0.25 ml, Supelco, Bellefonte, USA) in a cooling waterbath to ensure a uniform sample temperature. Micro-optodes were implanted in small plastic syringes that could be attached to the top of the glass vials using specially designed adapter lids, thereby providing a gas-tight measuring chamber. A two-point calibration of the system was performed using saturated ascorbic acid solution for 0% and air-bubbled medium for 100% air saturation.
After incubation, cells were recollected by centrifugation and pellets were resuspended in 150 µl of fresh medium of the desired pH and PCO2 (with or without cycloheximide). Using a small Hamilton syringe, the cell suspension into the glass vial and the exact volume and cell number of the solution was noted. After closing the system and stabilising the phase angle signal, measurements were continued for at least 20 min.
Cellular oxygen consumption rates were determined under control conditions (pH 7.90, normocapnia) and compared to the rates under increasing acidotic stress (pH 7.20 and pH 6.50). All experiments were also performed at normocapnic and hypercapnic PCO2 and at the respective pH. The inhibiting effect of cycloheximide was quantified at pH 7.90 and pH 6.50 under both normocapnic and hypercapnic conditions.
Intracellular acid-base variables
The intracellular pH of P. brachycephalum isolated hepatocytes was
measured after incubation using the homogenate technique
(Pörtner et al., 1990).
Unfortunately, sample numbers of L. kempi and thus the amount of
hepatic tissue obtained was far too low to permit parallel analyses of pHi in
this species. Pellets of 45-55 mg cellular fresh mass were resuspended in a
300 µl Eppendorf tube in a solution containing KF (160 mmol l-1)
and nitrilotriacetic acid (1 mmol l-1). The tubes were filled to
the top with approximately 250 µl of the solution to avoid air bubbles,
closed and mixed on a vortex mixer. Intact cells were disrupted by freeze
thawing and sonification (2 min, 2°C). After brief centrifugation (30 s,
20 000 g, 2°C), the supernatant was used for pHi
measurements. Apparent intracellular HCO3-
concentrations were calculated using values of pK''' and the
solubility coefficient
CO2 determined according to Heisler
(1986b
). Well-equilibrated
cellular suspensions have markedly reduced diffusion limitations compared to
tissues, so intracellular PCO2 levels were
assumed to equal extracellular PCO2 levels for
the calculation of bicarbonate concentrations.
Statistics
For each treatment (normocapnia and hypercapnia), oxygen consumption rates
under control and experimental conditions were compared using two-factorial
analyses of variance (ANOVA) or analyses of covariance (ANCOVA). When a
significant influence of a single variable was indicated by ANOVA/ANCOVA, the
different experimental treatments were compared using the Tukey Test. For the
effects of cycloheximide on oxygen consumption rates, values are expressed as
a percentage of the respective control value, to facilitate visual comparisons
between different experimental conditions. Original data were analysed by
Student's t-test for paired samples. Significant changes of
normalized data were determined using the Mann-Whitney rank sum test. In all
cases, P<0.05 was accepted as the level of significant difference.
All values are presented as means ± standard deviation
(S.D.) for preparations of hepatocytes from 4-6 individuals. Two
replicate samples were measured per preparation for each treatment
(combination of pHe and PCO2).
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Results |
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Dependence of oxygen consumption rates on experimental pH values
Rates of oxygen consumption by isolated hepatocytes from both L.
kempi and P. brachycephalum
(Fig. 2A,B) were significantly
influenced by decreasing medium pH (ANOVA; F=23.390,
P<0.001 for P. brachycephalum; F=63.119,
P<0.001 for L. kempi). The parallel lowering in pHi, seen
in P. brachycephalum hepatocytes, correlated in a linear and
significant way (F=37.038, P<0.001) with decreasing rates
of oxygen consumption (see Fig.
3). Under normocapnia and at a low pHe of 7.20
(pHi=7.17±0.07), the rate of oxygen consumption by hepatocytes from
Antarctic eelpout decreased to approximately 85% of the rate measured under
control conditions (pHe 7.90, PCO2 =0.03 kPa)
and fell even further to 66% at pHe 6.50 (pHi=6.98±0.07). The results
obtained for hepatocytes from L. kempi were similar. Oxygen
consumption rates decreased by approx. 17% at pHe 7.20 and 33% at pHe 6.50.
The decline in aerobic metabolic rates was similar during hypercapnic
incubation of hepatocytes from both species. On average, the rates were
approx. 23% below control levels at pHe 7.20 and 37% below at pHe 6.50.
Comparison of normocapnic and hypercapnic data clearly demonstrated that for
identical pHe values there was a significant difference in oxygen consumption
under the two PCO2 treatments in both fishes
(ANOVA; F=11.025, P=0.002 for P. brachycephalum;
F=12.220, P<0.001 for L. kempi). In contrast,
the influence of pHi on oxygen consumption of hepatocytes from P.
brachycephalum displayed no significant dependence on
PCO2 treatment, despite a similar mean
difference between normocapnic and hypercapnic oxygen consumption rates at a
specific pHi value (Fig. 3,
ANCOVA; F=0.002, P=0.964). However, the pHi determinations
showed relatively high standard deviations, possibly due to methodological
reasons, so differences between normocapnic and hypercapnic data might have
been seen with larger (but unavailable) sample numbers.
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Changes in other acid-base variables, PCO2 or extra-/intracellar [HCO3-], were not consistently related to oxygen consumption under any of the experimental conditions analysed. As seen in Fig. 4, intracellular and especially extracellular bicarbonate concentrations differed considerably between both PCO2 treatments at the same pHe, but nevertheless the changes in oxygen consumption rates were similar.
Inhibition of protein biosynthesis by cycloheximide
Depression of cellular oxygen consumption after specific inhibition of
protein biosynthesis by cycloheximide is an indication of the contribution of
protein anabolism to overall metabolic rate. In preliminary titration
experiments the effect of the cycloheximide concentration on the rate of
oxygen consumption was established (data not shown). We incubated cells with
35.5 µmol l-1 (Lefebvre et
al., 1993), 0.7 mmol l-1 and 1 mmol l-1
(Wieser and Krumschnabel,
2001
) cycloheximide. The measured reduction of oxygen consumption
rate did not increase with increased inhibitor concentrations above the value
obtained at 35.5 µmol l-1 cycloheximide. Therefore, we decided
to work with the lowest tested inhibitor concentration to minimize side
effects due to unspecific inhibition of cellular processes.
In normocapnic compared to hypercapnic hepatocytes, there were no detectable significant differences in the response of cellular oxygen consumption rates to cycloheximide under control (pHe 7.90) or acidotic conditions (pHe 6.50) (P=0.862 and P=0.664, respectively, for P. brachycephalum; P=0.716 and P=0.879, respectively, for L. kempi). Our experiments clearly showed that under control (pHe 7.90, PCO2 0.03 kPa) and hypercapnic conditions (pHe 7.90, PCO2 1.01 kPa) in L. kempi and P. brachycephalum liver cells, approx. 20% of the aerobic energy metabolism is due to cellular protein synthesis (Fig. 5). The decline in oxygen consumption to 83.1% (L. kempi) and 77.2% (P. brachycephalum) of the respective control values, measured after incubation of hepatocytes with 35.5 µmol l-1 cycloheximide, was significant in both cases (P<0.001 for L. kempi and P. brachycephalum). Under conditions of severe acidosis (pHe 6.50, pHi 6.98±0.07 in P. brachycephalum) cycloheximide-induced inhibition of cellular oxygen consumption was only 3-5%, i.e. no significant change from the respective control value in L. kempi (P=0.139). The decrease was also small, but was significant, in P. brachycephalum (P=0.016). In both species and under normocapnic and hypercapnic conditions the effect of cycloheximide on oxygen consumption at pHe 7.90 was significant at pHe 7.90 and 6.50 (L. kempi: P=0.008 for normocapnia, P=0.032 for hypercapnia; P. brachycephalum: P<0.001 for normocapnia, P<0.001 for hypercapnia). Provided that there is no pH-dependent shift in the costs of peptide synthesis, these data suggest that protein biosynthesis was downregulated by approx. 80% at pHe 6.50 in both species, regardless of the level of PCO2.
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Discussion |
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Similar to previous findings in muscle tissue of a CO2 tolerant
marine invertebrate, S. nudus, rate of oxygen consumption by
hepatocytes from both Antarctic fish species mirror a drastic reduction in
energy demand, depending on pH, but without any influence by intra- or
extracellular levels of PCO2 and
[HCO3-] (see Fig.
4). In P. brachycephalum hepatocytes, intracellular pH
changed in parallel with pHe. As shown in
Fig. 3, both CO2
treatments yielded some, but not significant, difference in energy turnover at
identical pHi values, whereas for the same pHe values, the differences in
oxygen consumption rates were significant
(Fig. 2). It cannot currently
be excluded that both intra- and extracellular pH have a specific influence on
cellular oxygen demand. At pHe 6.50 oxygen consumption fell by up to 34% in
hepatocytes from both Antarctic fish species, signalling a severe reduction in
hepatic cellular energy turnover during acidosis. This clearly agrees with
previous findings of metabolic depression by respiratory acidosis in other
animal models, including the marine invertebrate S. nudus
(Langenbuch and Pörtner,
2002) or brine shrimp embryos (Artemia franciscana;
Hand and Gnaiger, 1988
).
Similar to Artemia embryos, where hypercapnia and the subsequent
decrease in pHi serve as a signal for the massive downregulation in metabolic
rate, the decrease in hepatic pHi observed in P. brachycephalum
elicited metabolic depression, which may temporarily contribute to protect the
functional integrity of hepatocytes. In support of this conclusion, Sakaida
and Coworkers (1992
) found
reduced mortality of rat hepatocytes after treatment with cyanide (chemical
anoxia) when cells were cultured at pH 6.00 rather than at control pH.
Evidently, similar cellular effects are still present in species that live
permanently in highly oxygenated environments like the Antarctic oceans and
display a sluggish mode of life and low levels of energy turnover compared to
other, warmer water fish. Nevertheless, the question arises as to whether the
observed decrease in cellular oxygen consumption is an integral part of a
regulated whole-body metabolic depression that will extend the time period
that an animal can survive on stored fuel supplies during periods of
environmental stress (Guppy et al.,
1994
), or whether it should be seen as a single rudimentary
mechanism which is `old' on an evolutionary time scale.
Previous analyses of the influence of variable acid-base parameters on
trout hepatocyte metabolism revealed a similar effect of reduced pHe and pHi
on the depression of cellular metabolism (production of CO2 and
glucose from lactate; Walsh et al.,
1988). Moreover, specific effects of CO2 on the
pathways of lactate metabolism have been postulated
(Walsh et al., 1988
), which
indicate that the shifting acid-base equilibria may be associated with
specific changes in metabolic equilibria. When interpreting such data it needs
to be borne in mind that lactate may change cellular energy turnover
(Pinz and Pörtner, 2003
).
Specific CO2 effects are not evident in total energy turnover
analysed by oxygen consumption (this study). Overall, a reduction of aerobic
metabolism in fish hepatocytes is brought about by the synergistic inhibition
by acid-base parameters of both energy-producing (see
Walsh et al., 1988
) and
energy-consuming processes (see below). For comparison, and in contrast to our
study, short-term exposure of goldfish or trout hepatocytes to the low pHe of
6.60 did not cause significant metabolic depression
(Krumschnabel et al.,
2001
).
Inhibition of protein synthesis
Under conditions of environmental stress and thus limited energy supply,
survival is supported by a parallel and coordinated reduction in ATP-producing
and -consuming processes, which includes protein biosynthesis as a major
contributor to cellular ATP turnover. In fish, the central site of secondary
metabolism is liver, which is responsible for the synthesis and secretion of a
variety of essential proteins (e.g. lipoproteins or fibrinogen), and therefore
relies upon a highly active protein synthesis machinery
(Houlihan, 1991) and may be
most severely affected by a non-transient reduction in protein biosynthesis
rates due to permanent disturbances of cellular energy metabolism.
Growing evidence indicates that the costs of protein synthesis may have
been overestimated in many previous studies, due to secondary effects on
cellular energy metabolism by use of overly high cycloheximide concentrations.
In isolated goldfish and trout hepatocytes, oxygen consumption rates followed
an inverse hyperbolic function with rising cycloheximide concentrations,
reaching a maximum effect at not less than 15 mmol l-1. However,
incorporation of labelled amino acids into cellular protein was already fully
blocked by 25 µmol l-1 cycloheximide
(Wieser and Krumschnabel,
2001). To ensure accurate measurements of the percentage
contribution of protein synthesis to metabolic rate we established that the
inhibiting effect was saturated at 35.5 µmol l-1 without further
reduction in the rate of oxygen consumption at higher levels. These findings
show that at this concentration of inhibitor, unspecific inhibition of other
cellular processes could very well have been excluded.
A significant decrease of cellular oxygen consumption rates to approx. 80%
of the control rates was observed upon inhibition by 35.5 µmol
l-1 cycloheximide under control conditions (normocapnia, pHe 7.90)
in hepatocytes from both L. kempi and P. brachycephalum.
These values mirror protein synthesis costs that account for 20% of the
hepatic basal energy metabolism and are well within the range of values given
for other ectotherm hepatocytes in the literature. A study by Land et al.
(1993) on turtle hepatocytes
reported a 36% contribution of protein synthesis to metabolic rate while Fuery
et al. (1998
) determined a
value of 12% in liver slices of Bufo marinus. These differences are
not surprising as both oxygen consumption and rate of protein synthesis in
liver, can vary enormously, depending on nutritional and hormonal status
(Reeds et al., 1985
).
Under conditions of severe acidosis (pHe 6.50, pHi=6.98±0.07) the
fraction of cycloheximide-sensitive oxygen consumption decreased significantly
from 20% (pHe 7.90) to 3-5% in hepatocytes from both species, reflecting, on
average, an 80% reduction in protein synthesis of both L. kempi and
P. brachycephalum liver cells. An even more pronounced reduction was
observed in Artemia franciscana embryos, where protein synthesis was
suppressed to 3% of control values after 4 h of anoxia or aerobic acidosis due
to a global arrest of transcription and translation
(Hofmann and Hand, 1994;
van Breukelen et al., 2000
). A
less extreme response to anoxia was seen in hepatocytes from an
anoxia-tolerant turtle. The downregulated process of protein synthesis
contributed 33% to the global 88% metabolic depression measured under
conditions of anoxia (Land et al.,
1993
; Land and Hochachka,
1994
). Compared to the turtle data, the contribution of protein
synthesis inhibition to the reduction in metabolic rate in hepatocytes of
Antarctic fish was much higher. Almost 60% of the depression in oxygen
consumption under acidosis can be attributed to the decline in protein
synthesis in both species.
Interestingly, the capacity for protein synthesis was maintained in
anoxia-tolerant goldfish hepatocytes even in the metabolically depressed
state. In contrast, the more anoxia-sensitive trout hepatocytes experienced a
massive downregulation of protein synthesis during anoxia (by 50%,
Wieser and Krumschnabel,
2001). Unfortunately, pH effects were not investigated in these
studies, so cause and effect relationships remain elusive. The percentage
changes in protein synthesis of trout hepatocytes and the even larger changes
in liver cells from Antarctic fish may suggest a high sensitivity to
hypercapnia-induced acidosis in the polar fish. Here pH-dependent inhibition
of protein biosynthesis is responsible for the largest fraction of the decline
in cellular energy turnover at low pH. Perturbations in the activity of
Na+/K+-ATPase or mitochondrial proton leak, both other
major contributors to aerobic energy demand of a single cell, were likely to
have been very small. In a short-term scenario, the maintenance of
transmembrane ion gradients may be more important than the synthesis of new
proteins for maintaining the integrity of the hepatocytes. However, it appears
likely that a pronounced limitation of protein synthesis will compromise the
functional integrity of the organ on an extended time scale.
Summary and conclusions
The present study examined the correlated changes in acid-base variables
and in oxygen consumption of hepatocytes from Antarctic fish during
normocapnia and hypercapnia. The aerobic energy metabolism of fish hepatocytes
was pH-dependent, with a significant and large reduction in the fraction of
cellular energy turnover allocated to protein synthesis during extra- and
intracellular acidosis. One has to keep in mind that liver accounts for only
2-3% of total body mass in L. kempi and P. brachycephalum.
Thus, it is difficult to extrapolate the present findings to the whole animal
level. Effects seen in isolated hepatocytes may contribute to whole organism
metabolic depression under conditions of uncompensated extra- and
intracellular acidosis, provided that other tissues, especially the much
larger fraction of muscle tissue, respond in similar ways to the liver.
Accordingly, it remains unclear from the present data if the 33-34% decrease
in hepatic aerobic metabolic rate contributes to whole organism energy
savings. At the cellular level, the mechanisms leading to a marked reduction
in cellular protein synthesis and, thereby, energy turnover may be similar in
marine invertebrates like the CO2-tolerant intertidal worm S.
nudus (Langenbuch and Pörtner,
2002) and in fishes. The relevance of the present cellular
findings for whole organism responses to CO2 in Antarctic fish is
currently under investigation in this laboratory. An estimation of the
adaptational flexibility of Antarctic (and deep sea) fish under conditions of
CO2 stress requires the analysis of whole body metabolic rates as
well as the identification of key physiological processes and mechanisms at
all organizational levels (e.g. energy and protein metabolism in muscle
tissue, cardiorespiratory system, neurotransmitter patterns in the brain).
Additional long-term dose-response studies are required to establish critical
in vivo thresholds for the growth and reproductive success of various
groups of animals. The main objective in all of these studies should be the
development of a comprehensive picture of short-, medium- and, especially,
long-term effects of hypercpania on animal physiology that will enable us to
predict possible long-term consequences for the complex network of marine
ecosystems due to future passive or active (dumping) accumulation of
anthropogenic CO2 in the oceans.
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