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1 Department of Zoology, University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom; and 2 Laboratoire d'Ecophysiologie et Ecotoxicologie des Systèmes Aquatiques, Unité Mixte de Recherche 5805-OASU, Université Bordeaux 1 and Centre National de la Recherche Scientifique, Arcachon 33120, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Many water-breathing animals have a strategy that consists of maintaining low blood PO2 values in a large range of water oxygenation level (4-40 kPa). This study examines the postprandial changes in O2 consumption, arterial blood PO2, and tissue protein synthesis in the shore crab Carcinus maenas in normoxic, O2-depleted, and O2-enriched waters to study the effects of this strategy on the O2 consumption and peptide bond formation after feeding. In normoxic water (21 kPa), the arterial PO2 was 1.1 kPa before feeding and 1.2 kPa 24 h later. In water with a PO2 of 3 kPa (arterial PO2 0.6 kPa), postprandial stimulation of protein synthesis and O2 consumption were blocked. The blockade was partial at a water PO2 of 4 kPa (arterial PO2 0.8 kPa). An increase in environmental PO2 (60 kPa, arterial PO2 10 kPa) resulted in an increase in protein synthesis compared with normoxic rates. It is concluded that the arterial PO2 spontaneously set in normoxic Carcinus limits the rates of protein synthesis. The rationale for such a strategy is discussed.
crustaceans; oxygen consumption; blood oxygen; hypoxia
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IN AIR-BREATHING HOMEOTHERMS the PO2 in the arterial blood is regulated at 10-14 kPa in resting conditions at sea level (Ref. 6; for reference, PO2 in air at sea level is 20-21 kPa). At the cellular level the most frequently measured values are in the 1- to 3-kPa range in the tissues (43). In numerous water breathers and other poikilotherms, the arterial PO2 is also set at 1-3 kPa (reviewed in Ref. 27). It has been suggested that these low PO2 values are part of a strategy of cell oxygenation termed "the low tissue O2 strategy," which evolved with the origin of aerobic metabolism (27).
In water-breathing animals, there is a strategy of gas-exchange regulation that consists of maintaining PO2 in the arterial blood in an astonishingly low and narrow range, about 5-10 times lower than in homeotherms. Previous studies in crustaceans reported that O2, working in the low PO2 range, acts 1) in a neuromodulator-like manner on the neural networks that generate the masticating and filtering movements of the foregut (5, 31) and 2) limits the oxidative metabolism of locomotor muscles (12).
This paper aims to determine whether the low tissue O2 strategy could affect protein metabolism in a water breather. Around 60 O2-dependent reactions exhibit values of the Michaelis-Menten constant for O2 (Km,O2) that would be rate limited at likely tissue O2 levels (except for cytochrome-c oxidase; Ref. 43). Besides these reactions there may also be a limitation on the rate of energy supply for other processes. If these conditions are present in vivo in water breathers, we would expect that 1) an increase in tissue energy demand would result in an increase in blood PO2 to drive an increase in tissue oxygenation and/or an increase in blood flow; 2) a reduction in environmental PO2 resulting in a decline in blood PO2 would result in a significant reduction in energy-demanding processes, with possibly some anaerobic metabolism taking place; and 3) an increase in environmental PO2 resulting in an increase in blood PO2 would result in an increase in the rates of energy-demanding processes.
We have used the green shore crab Carcinus maenas to test these hypotheses. We have manipulated in vivo protein synthesis, one of the most energetically expensive cellular processes (35), to test the effects of the low arterial PO2 strategy. The minimum theoretical cost for the formation of each peptide bond in a protein is assumed to be five ATPs (46 mmol ATP/g protein synthesized; Ref. 39). Using this theoretical cost in humans, it has been estimated that as much as 15-20% of the basal metabolic rate is due to protein turnover (44). The theoretical cost of protein synthesis has been compared with directly determined costs in a variety of water-breathing animals and in isolated fish cells; protein synthesis costs have been found to be rate dependent and close to, or to exceed, theoretical costs (reviewed in Refs. 19, 41). Overall, protein synthesis may account for up to 40% of the O2 consumption in a variety of aquatic species.
C. maenas is among those water breathers that live chronically with a near-hypoxic arterial PO2 (1-1.5 kPa). The postprandial doubling in O2 consumption 24 h after a meal is not generated through an increase in blood flow rate (25). In crustaceans, the microcirculation in the hepatopancreas (9), a major site of protein synthesis (18), is so well developed that these low arterial PO2s can be considered as quite close to the extracellular PO2. This tissue therefore represents an excellent opportunity to determine the O2 sensitivity of protein synthesis in vivo.
The aims of this study were to examine the postprandial changes in O2 consumption, arterial blood PO2, and tissue protein synthesis in the crab C. maenas in normoxia, hypoxia, and hyperoxia and to investigate how the low physiological oxygenation status in situ may influence in vivo protein synthesis.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and general conditions.
Common shore crabs, C. maenas, were collected locally from
Arcachon Bay in France. They were transported and kept for at least 3 wk in the Marine Biological Station to allow acclimatization (30). The seawater had a constant salinity of
30-32
, and pH and temperature were 8.0-8.3 and 15°C,
respectively. All the crabs were in the size range 39-67 g and
were in the intermoult stage. During the acclimatization period the
crabs were fed on mussels, Mytilus edulis (41.0 ± 7.5% protein dry wt) twice a week.
Experimental conditions.
The experimental tanks were isolated from vibrations with antivibrating
benches. Salinity was 32, and temperature was 15.0 ± 0.5°C.
The PCO2 was set at 0.10 ± 0.01 kPa and
the pH at 7.80 ± 0.50 depending on the water titration alkalinity
by using a pH-PCO2-stat (7).
Shadow areas and a patch of dim light were provided to induce resting
behavior. Different oxygenation levels were obtained by bubbling a
N2-O2-CO2 gas mixture via a
laboratory-constructed O2-stat connected to a calibrated
electrode (E5046 Radiometer) and Radiometer oxygen meter (PHM72 Mk2).
With this system the PO2 in the water was
maintained in a very narrow range (±0.5 kPa of the nominal value). In
hypoxic water conditions, the crabs could not reach the water surface
and aerate the water in their branchial chambers. During the 5- to
7-day period before the beginning of the experiment, the crabs were
starved to reduce the variation in their physiological condition,
stimulate their appetite, and synchronize all individuals before
feeding. Animals were fed with mussel flesh (2.8 ± 0.1% of the
animal's fresh weight) in normoxic water (water
PO2 = 20-21 kPa) before any change in
water PO2. O2 consumption, the rate
of protein synthesis, arterial blood PO2, and
lactate concentrations were determined before and after these changes.
O2 consumption measurements.
Experiments were performed on 42 crabs weighing 53 ± 1 g.
The O2 consumption of individual animals
(µmol · min
1 · kg1)
was measured in an open-flow respirometer (vol 160 ml) maintained at
15°C. The technique used was similar to that described in Ref. 29. Its main characteristics were 1) the
respirometer was equipped with a pH-PCO2-stat,
2) a laboratory-made automatic device (continuously monitored PO2 in the exit water and adjusted
the water flow through the respirometer to clamp
PO2 inspired by the animals at either 21, 4, or
3 kPa), and 3) a rotor to ensure a homogeneous composition in the respirometer. It should be noted that the water
PO2 was measured automatically with electrodes
manipulated by remote control to ensure nonstressful conditions. Each
crab was initially placed in an open tank (20 l, renewal rate 0.5 l/min) filled with water that had been equilibrated to a
PO2 of 21 kPa and a
PCO2 of 0.1 kPa. The crabs were allowed to
settle for 24 h before recordings were made, and then they were
transferred within 2-3 min to the respirometer (at time
t0) where the PO2 was 21 kPa.
Reference preprandial O2 consumption measurements were made
the next morning from 9:00 to 10:00 AM. Animals were allowed access to
food at t0 + 24 h for 30 min (from
10:00 until 10:30 AM). For feeding and to avoid contamination in the
respirometer, the animals were temporarily transferred to a feeding
chamber for 30 min (vol 400 ml, same water composition as that in the
respirometer). They were then transferred back to the respirometer
within approximately 2-3 min, and O2 consumption
measurements were performed 2, 4, 6, 24, and 48 h later.
Blood analysis.
Experiments were carried out on 175 Carcinus (weighing
58 ± 1 g). At least 3 days before the beginning of the
experiments, animals kept in normoxic conditions were prepared for
arterial blood sampling by drilling a hole in the carapace above the
heart; a thin layer of cuticle was left in place and a piece of rubber was glued over it. All animals were fed with mussels, one mussel per
crab (fresh weight 2 g) in normoxic water (salinity = 35; water PO2 = 20-21 kPa;
water PCO2 = 0.1 kPa) and gently
transferred 30 min later to test tanks with water at
PO2 of 21, 4, or 3 kPa. Individuals
(n = 6 per water oxygenation level) were sampled only once. Arterial blood samples (150 µl) were collected by gently removing crabs from the water and puncturing the heart through the
rubber membrane with capillary glass tubes equipped with a needle.
Samples were obtained within the first minute of emersion. This
sampling technique was critically assessed in Ref. 30. After sampling, blood was stored on ice to prevent clotting and to slow
down metabolic reactions. Arterial PO2 was
determined within 2-3 min on 100-µl samples with an E5046
Radiometer polarographic electrode thermostated at 15°C. The
electrode was calibrated with a zero PO2
solution (S4150 Radiometer) and seawater-equilibrated with a precision
gas mixture (O2 fraction = 4%). As shown in Ref. 30,
Fig. 2, this calibration procedure, which improves analysis quality in the low range, does not preclude the measurement of high
blood PO2 values. L-Lactate
concentrations (mmol/l) were determined spectrophotometrically on the
50 µl remaining blood using an enzymatic method (Boehringer kit 139 084) following the precautions described in Ref. 14.
Protein synthesis measurements. Six starved crabs were transferred to experimental tanks (control group) and exposed in normoxia (at time t0) where water PO2 was 21 kPa. The next morning (t0 + 24h), they were injected with radiolabel, returned to water, and killed 60 min later. Samples of hepatopancreas, heart, claw, and leg muscles were taken for analyses (see below). The same morning previously starved crabs kept in normoxia were given a single meal of Mytilus edulis (t0 + 24 h) equivalent to 2.8 ± 0.1% of the animal's fresh weight. The animals were transferred after 30 min to experimental tanks in normoxia (water PO2 = 21 kPa, n = 21), hypoxia (water PO2 = 4 kPa, n = 23; or PO2 = 3 kPa, n = 24), or hyperoxia (water PO2 = 60 kPa, n = 6). In each water oxygenation condition the animals were injected with a radiolabel at 2, 5, 24, and 48 h postfeeding except in hyperoxia where they were only injected at 24 h. As in reference condition, samples of hepatopancreas, heart, claw, and leg muscles were taken 60 min after injection.
Rates of protein synthesis in tissues were determined after the injection of a single "flooding dose" of [3H]phenylalanine (21). The injection solution consisted of 135 mM phenylalanine and L-[2,6-3H]phenylalanine (Amersham) at 100 µCi/ml (3.7 MBq/ml) at a dose of 1.0 ml · 100 g1 · live wt1. The
injection was made into the blood sinus at the base of the third
walking leg. No O2 consumption measurements were made on these animals because of the elevation of the O2
consumption that occurs through handling (21).
After injection the animals were returned to the normoxic, hypoxic, or
hyperoxic conditions. After a mean incubation time of 1 h
(21), the animals were killed by destroying the brain. Two
hundred-milligram samples of hepatopancreas, heart, claw, and leg
muscles were taken, individually wrapped in plastic bags, immediately
frozen in liquid nitrogen, and stored at 
80°C for further analysis.
Dissections took place in within 5 min (from killing the animal to the
removal of the final tissue sample). The whole body was also frozen.
Tissue samples were homogenized while still frozen in 2% perchloric
acid (PCA), thus separating the "free" intracellular (PCA soluble)
and "protein-bound" (insoluble) phenylalanine fractions, and the
precipitate was treated as described in Ref. 8. The frozen
whole bodies were broken up and homogenized in 200 ml 0.2 M PCA. The
resulting homogenate was thoroughly mixed and a subsample weighed into
a tarred centrifuge tube for further analysis as described above. The
phenylalanine-specific radioactivity of the free pool, the free-pool
phenylalanine concentration, and protein-bound phenylalanine specific
radioactivity were determined as described by Refs. 20 and
42.
The fractional rate of protein synthesis ks as a
percentage of the total protein mass of each tissue or whole body per
day was calculated as
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1 · nmol
phenylalanine1), Sa is the free-pool
phenylalanine specific activity at time t
(disintegrations · min1 · nmol
phenylalanine1), t is the time between
injection and killing of the animal (in min), and 1,440 is the number
of minutes in a day. The ks was calculated for
each whole body and for each tissue. Protein determinations were
treated as described in Ref. 21. The RNA content and the RNA activity (kRNA, the amount of protein in g
being synthesized per g of RNA per day) were calculated as described in
Ref. 21.
Validation of the methodology. To check if hypoxia, through a putative change of blood flow rate, could alter the tissue distribution of radiolabel in the organism, another twelve crabs were randomly selected, transferred to the experimental tanks, and exposed to hypoxia (3 kPa). After an incorporation period of 30, 60, or 90 min, they were killed and samples were taken to measure the phenylalanine specific radioactivity of the tissue free pool and the incorporation of tritiated phenylalanine into tissue proteins.
Statistics. Data are expressed as individual values in histograms of frequency distribution and/or as means ± SE. Paired t-tests were used to test significant changes in O2 consumption and blood O2 status before and after feeding. Otherwise, differences between normoxic and hypoxic conditions were evaluated using the Mann-Whitney U test or Student's t-test. ANOVA (followed where applicable by Tukey's multiple comparison test) was used to compare tissue-specific stabilized free pool specific activities, protein synthesis rates, RNA to protein ratio, and translational efficiencies calculated after different times for each tissue. Linear regression analysis was used to establish protein labeling during the time course. P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Postprandial O2 consumption and supply in normoxia.
After being starved for 5 days and after feeding, there was a peak in
O2 consumption (from 14.8 ± 0.7 up to 36.0 ± 3.3 µmol · min1 · kg1)
at 2 h after feeding (Fig.
1A). This was followed by a
plateau from 6 to 24 h (O2 consumption = 26.6 ± 1.9 µmol · min1 · kg1
at 6 h; 26.9 ± 1.9 µmol · min1 · kg1
at 24 h). The O2 consumption returned to the reference
value at 48 h after feeding. The reference arterial
PO2 in our resting and settled condition was
only 1.1 ± 0.1 kPa despite an inspired PO2 of 20-21 kPa (Fig. 1B).
Four hours after feeding, the arterial PO2
significantly increased to 1.6 ± 0.2 kPa (P < 0.05, Mann-Whitney test) from the reference value. Two hours later it
decreased to 1.2 ± 0.1 kPa and remained at this value until
24 h after the meal. The low arterial PO2s
were not accompanied by any rise in blood lactate concentration, which
demonstrated the absence of any noticeable anaerobic metabolism
occurring during the specific dynamic action (SDA) (Fig.
1C).
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Postprandial tissue protein synthesis rates in normoxia.
The changes in fractional rates of protein synthesis after feeding are
shown in Fig. 2. In all tissues the
maximal rates were reached within 5 h after feeding
(P < 0.05). The fractional rates of protein synthesis
were highest in the hepatopancreas where they doubled (ANOVA
P < 0.05) from 5.4 ± 0.3%/day before feeding to
10.9 ± 0.3 and 11.6 ± 2.2%/day at 5 and 24 h after
feeding, respectively (not significantly different between 5 and
24 h, ANOVA, P > 0.05). Rates of protein
synthesis increased significantly (ANOVA, P < 0.05) by
threefold in the heart. Smaller increases occurred in the leg and claw
muscle.
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Postprandial O2 consumption and supply in
O2-limited conditions.
The effect of exposing animals previously fed in normoxia to water with
a PO2 of 4 kPa (Fig.
3A) was the abolition of the initial transient peak of O2 consumption. Instead the
O2 consumption rose to a maximum value at 2 h after
food intake and remained at around this value for 24 h
(O2 consumption = 26.1 ± 0.9 and 24.2 ± 1.9 µmol · min1 · kg1
at 2 and 24 h after feeding, respectively; significantly different from the preprandial values, Fig. 3A; not different from the
postprandial normoxic values at 4, 6, and 24 h). At a
PO2 of 3 kPa (Fig. 3A), the
situation was completely different. The postprandial increase was
reduced to 18.8 ± 1.2 µmol · min1 · kg1
2 h after feeding (significantly different from the preprandial value). This value did not statistically change until 24 h after feeding when it increased to 19.3 ± 1.2 µmol · min1 · kg1.
Overall these results represent SDA responses at 6 h of 11.8 µmol · min1 · kg1
at 21 kPa, 13.0 µmol · min1 · kg1
at 4 kPa, and 3.3 µmol · min1 · kg1
at 3 kPa. In all situations the O2 consumption returned to
the prefeeding level 48 h after feeding.
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Postprandial tissue protein synthesis rates in O2-limited conditions: validation of the methodology. It was possible that hypoxia could have induced changes in blood flow rate that could have interfered with the distribution of the injected phenylalanine throughout the animal, resulting in poor flooding of the tissues and problems with the interpretation of the protein synthesis values. To eliminate this possibility, the time course of flooding of the free pools and rate of incorporation of radiolabel into proteins were determined at a water PO2 of 3 kPa in starved animals.
The mean free-pool phenylalanine radioactivities in the heart, claw, leg, and the hepatopancreas (Table 1) were elevated within 30 min of injection and did not change significantly at each time interval (ANOVA, P > 0.05). The phenylalanine-specific radioactivity of the solution injected was 1,856.3 ± 53.03 dpm/nmol phenylalanine (n = 4). In all tissues the values were significantly lower than the specific radioactivity of the injection solution (ANOVA, P < 0.05). Hypoxia (3 kPa) at 60 min after the injection had no significant effect on free pool specific activity, irrespective of tissue (Table 1). One hour after the flooding dose injection, in 3 kPa the free phenylalanine levels in the claw tissue were ninefold above the normal values of 200-700 nmol phenylalanine/g fresh wt (8), demonstrating a large increase in the phenylalanine free pool due to the flooding dose of the amino acid. There were significant linear correlations between protein-bound phenylalanine radioactivity (Sb) and time under hypoxia at 3 kPa in starved animals (Table 1). From the intercept of the regression line, it is possible to estimate how soon after injection the radiolabel began to be incorporated into body protein. The intercepts did not differ significantly from zero, confirming that protein labeling had begun immediately after the injection. The commonly accepted criteria for the successful measurement of protein synthesis by the flooding dose method have therefore been met in crabs living in water with a PO2 of 3 kPa. This also implies that the circulation and diffusional processes were not impaired even in the extremely hypoxic conditions we studied.
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Tissue protein synthesis rates.
The influence of exposing the crabs to 4 and 3 kPa, forcing the
arterial PO2 down to 0.7 or 0.5 kPa,
respectively, compared with the normoxic control is presented in Fig.
4. Protein synthesis rates were tissue
specific. At a water PO2 of 4 kPa and arterial blood PO2 of 0.7-0.8 kPa (Fig.
3B), the time course of changes in the rates of protein
synthesis in claw and leg muscle appeared simply delayed as they did
not differ from the normoxic values from 10 to 48 h in fed animals
(Fig. 4). In the hepatopancreas the situation was completely different.
There was 1) a transient and significant decrease occurring
from 2 to 5 h and 2) no increase in protein synthesis
from 24 to 48 h compared with the unfed value. In the heart there
was an intermediate status as the rates of protein synthesis reached
the normoxic reference value for fed animals at 10 h and then was
limited at 24 and 48 h (no difference with the reference unfed
value).
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Tissue RNA to protein concentrations and RNA translational
efficiencies.
The ranking of the tissues in terms of RNA to protein ratios was as
previously described (21). There was no significant change
in the concentration of RNA with hypoxia in the tissues either
expressed in relation to wet weight or protein. Therefore, the amount
of protein synthesized per unit RNA (kRNA)
showed the same response as protein synthesis rates in hepatopancreas,
heart, leg, and claw in normoxia and hypoxia (3 and 4 kPa) (Table
2).
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Relationship between blood PO2 and
tissue-specific protein synthesis rate at 24 h.
To gain more insight into the relationship between the ability
to synthesize proteins and the blood oxygenation status, we studied the
correlation between the arterial PO2 and the
rate of protein synthesis at 24 h after feeding. At that time
there should be no more food in the foregut (32), and
Figs. 2-4 suggest a plateau in the parameters studied. There were
no statistically significant relationships between arterial blood
PO2 and fractional rates of protein synthesis
in the claw, leg, and heart muscles, but there was a clear relationship
between blood PO2 and fractional rates of
protein synthesis in the hepatopancreas (Fig.
5). Therefore we performed an experiment
during which we transferred fed Carcinus into hyperoxic
water where PO2 was so high (60 kPa) that it
exceeded the animals' regulation capacity (28). The
result of this experiment where the mean arterial
PO2 increased to 10.9 ± 1.8 kPa (measured 24 h after the transfer, n = 20) is presented in
Fig. 6. For clarity, data were combined
with previous results from Fig. 5. In all tissues the distribution of
the experimental points followed a single exponential equation of the
type y = y0 + a(1 bx) with a good
resolution power (0.906 < R2 < 0.999), which shows that the arterial PO2
explained 90-99% of the total variability. Three different
situations were observed. First, in the claw muscle and the heart, the
improved O2 conditions in hyperoxia allowed a large
increase in protein synthesis (+189 and +280%, respectively,
P < 0.05). Second, in the hepatopancreas the change
was much smaller as the ks was 13.4 ± 1.1%/day at an arterial PO2 of 1.2 ± 0.1 kPa and 16.7 ± 1.3%/day at arterial PO2
of 10.9 ± 1.8 kPa, which represents a 20% increase. Third, in
the leg muscle, the arterial PO2 change did not
lead to any ks improvement. The calculated
arterial PO2s at which 50 and 99% of the
reaction were occurring in vivo (P50 and P99,
respectively) were 1.1 and 5.9 kPa in the claw muscle, 1.3 and 5.9 kPa
in the heart, 0.8 and 2.2 kPa in the hepatopancreas, and 0.6 and 0.9 kPa in the leg muscle.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study we present evidence that in normoxic water, i.e., in unlimited O2 supply conditions, a crustacean exhibiting the low blood oxygenation strategy can adjust its arterial PO2 to a very low level despite the increased O2 demand associated with protein synthesis and the possibility to maximize protein synthesis efficiency in hepatopancreas, heart, and claw muscles with slightly higher PO2s. A comparison of various rates of protein synthesis determined in steady state 24 h after feeding at various experimentally manipulated arterial PO2 (including the use of superoxygenated blood) suggested that in reference normoxic conditions the spontaneously set arterial PO2 is limiting the rate of protein synthesis in the claw and heart muscles as well as in the hepatopancreas but not in the leg muscle. In the hepatopancreas, which is the major site of protein synthesis, the rate of synthesis was only 80% of its maximum value although a PO2 change from 1.2 to 2.2 kPa would be enough to achieve 100% of the maximum rate. Consequently, the present findings suggest that the low blood oxygenation strategy reported in water breathers can contribute very significantly to the energy budget, which raises the question why these animals maintain such low oxygenation levels in their internal milieu.
To recall the origin of this strategy, we already suggested that these low PO2s might reflect the level of O2 in the environment when animals first evolved, about 2 billion years ago, in the Proterozoic age (27). An analysis of the literature also clearly shows that there are many reasons to maintain tissue PO2 in a low range protected against O2 excess and O2 toxicity, i.e., production of reactive oxygen species (ROS) since the origin of life. That ROS damage cells is indeed a major hypothesis with respect to cell alteration and aging (13). Superoxide dismutase, a specialized enzyme aimed at protecting cells against O2 toxicity, is thought to have appeared very early in evolution, but without doubt, preventing high PO2 is also the simplest and must efficient tool to limit ROS production. To summarize briefly how water breathers adjust low arterial PO2s, it has been shown in crayfish and in sheatfish (27) that in a large range of water oxygenation level (4-5 to 40 kPa), ventilation is inversely related to the water PO2 and adjusted so that the level of oxygenation in the branchial cavities tends to remain more or less constant. This is based on the facts that 1) there are carotid body-like O2 chemoreceptors located in the branchial cavities, 2) there is no strong argument in favor of a central O2 chemosensitivity, and 3) PO2 in the fluids leaving the branchial cavities, arterialized blood and expired water, stay within a narrow range, independent of changes in water PO2. Note, in addition, that the existence of this strategy was also reported in molluscs (27).
The P50 we determined was in the range of 1 kPa. To compare
this with published in vitro Km values for
O2-utilizing enzymes (expressed as O2
concentrations, Ref. 43), we made the following assumptions: 1) the partial pressure of a gas is close to
its activity, 2) intracellular PO2
where protein synthesis occurs is equal at best to the arterial
PO2, and 3) the O2
solubility in Carcinus blood is 10.5 µmol · l1 · kPa1.
Based on these assumptions, EC50 values (effective
concentration at which 50% of the maximum rate occurs in µmol/l)
were at 15°C, 11 µmol/l in the claw muscle, 14 µmol/l in the
heart, 8 µmol/l in the hepatopancreas, and 6 µmol/l in the leg
muscle. Interestingly, all these estimates are in the range found in
mammals (43).
Respiratory changes in normoxic and hypoxic conditions. The near doubling of the O2 consumption during the SDA at 24 h fits well with the normal results in most water-breathing animals (21, 22, 25). This study showed that postprandial O2 consumption was abruptly reduced by exposing animals to water at a PO2 of 3 kPa (arterial PO2 = 0.5-0.6 kPa) but not to water PO2 of 4 kPa (arterial PO2 of 0.6-0.8 kPa). The large rise in blood lactate concentrations occurring at the lowest PO2 demonstrated an increased requirement for anaerobic metabolism and is characteristic of the arterial PO2 at the anaerobic threshold (Fig. 3).
Five hours after feeding there is a transient peak in normoxia in arterial PO2 (Fig. 1B). Previous work on lobster Homarus gammarus has shown that in unfed lobster the arterial PO2 is 1-2 kPa and doubles within 1 h after feeding before returning to control values 24 h later (5). Feeding accelerates both pyloric and gastric network activity in H. gammarus for 24-48 h. Using in vitro and in vivo studies with the crustacean stomatogastric nervous system that drives rhythmic activity of the muscles producing foregut movements, it has been demonstrated that specific PO2 change influences pyloric activity in a neuromodulatory-like manner at the stomatogastric ganglion (STG; 5, 31). We suggest that a similar mechanism exists in C. maenas. The transient arterial PO2 peak in normoxia (see Fig. 1) could modulate the facility of the Carcinus STG rather than reflecting the energy demand of protein synthesis. The associated O2 consumption peak could thus be due to masticatory and filtering activity but could also be associated with a transient loading and unloading of O2 stores in each body compartment during this unsteady period.Protein synthesis after a single meal in normoxic and hypoxic conditions. In normoxia there was a 30% rise in the fractional rates of protein synthesis by 2 h postfeeding (Fig. 2) with a maximum reached at 5 h and then a return to prefeeding values within 48 h in the hepatopancreas. A rapid response of protein synthesis to refeeding (significantly elevated above the rate in the fasted fish by 3 h) in various tissues of rainbow trout has been demonstrated (33). In the present study, protein synthesis rates were elevated 5 h after a single meal with respect to the prefeeding values. The current work agrees with that of Ref. 21 in C. maenas, which found that the hepatopancreas, gill, heart, proventiculus, leg muscle, and claw muscle protein synthesis rates increased 3 h after feeding in normoxic conditions; this increase occurred between 2 and 5 h after the meal in the present study. The ranking of the tissues in terms of their protein synthesis rates was similar to that previously reported from crustacean studies (21) and is in agreement, so far as the tissues are comparable, with the results from fish (11, 37). Tissues such as hepatopancreas and gills have higher rates of synthesis than muscle and heart, which in turn have higher rates than skeletal muscle. The liver is an important organ in the metabolism and utilization of nutrients from the diet and makes a significant contribution to protein metabolism in the whole body due to its high rate of protein turnover (11). The reduction in fractional protein synthesis rate during hypoxia was most marked in the hepatopancreas. The hepatopancreas responds to hypoxia by a downregulation of protein synthesis when the arterial PO2 is 0.5 kPa. This downregulation can be assumed to be an energy-saving process in a tissue, which is a major contributor to meal-stimulated protein synthesis (at least after a period of starvation) (34). Survival of vertebrates under anoxic or hypoxic conditions is achieved by a drastic reduction in ATP-consuming process, coupled with an increased ATP production via anaerobic pathways, allowing maintenance of cellular ATP concentrations and cell function (17). In contrast, in anoxia- and hypoxia-intolerant animals, cellular ATP concentrations rapidly decrease during anoxia, leading to loss of ion gradients, a rise in intracellular Ca2+ concentrations, and multiple cellular damaging processes, leading to death (17). Therefore as part of the general downregulation of metabolism, there is a decrease in protein synthesis in anoxia/hypoxia-tolerant animals on exposure to these conditions, since protein synthesis is an energetically expensive process accounting for a large proportion of cellular energy consumption (3). Mammalian tissues have been found also to reduce rates of protein synthesis in conditions of hypoxia (reviewed in Ref. 20). In isolated hepatocytes of rainbow trout, protein synthesis started to fall below an extracellular PO2 of 2 kPa (36). During anoxia, protein synthesis rates of isolated liver cells of the turtle Chrysemys picta bellii were reduced by 92% (reviewed in Ref. 20). Crucian carp living in complete anoxia reduce liver protein synthesis rates by 90% or more compared with normoxic values; however, rates of protein synthesis in the brain are maintained in anoxia at normoxic rates (42). The decrease in protein synthesis rates during hypoxia (compared with the normoxic values) in this study suggested that invertebrates as well as fish downregulate protein synthesis in a tissue-specific manner when exposed to hypoxic conditions.
It seems unlikely that food processing in the gut makes a major contribution to the SDA because work in plaice showed no detectable increase in O2 consumption after an indigestible meal of kaolin (4). In contrast, the synthesis of macromolecules, especially proteins, can be very costly in energetic terms and so could be a substantial contributor to postprandial metabolism. A significant proportion of the SDA should be accounted for by an elevation of protein synthesis (23). Protein synthesis is an important determinant of O2 consumption after feeding in cod (26). On the basis of protein synthesis costs derived from the literature (2), it may contribute as much as 44% of the observed postprandial rise in O2 consumption. An interesting observation in the present study was that at arterial PO2 of 0.7 kPa (inspired PO2 of 4 kPa), O2 consumption had doubled from 6 to 24 h postfeeding while the rate of protein synthesis was limited to the reference prefeeding level in normoxia. This shows that at this arterial PO2, the major O2-consuming process (which is the mitochondria pool) was still consuming O2 at its normal rate. The ability to supply ATP at the rate required by the energy-demanding process as well as the O2-sensitive key step that impaired the protein synthesis remains to be studied.RNA concentrations and translational efficiency. Overall, in this study there was no reduction in tissue RNA-to-protein ratios in hypoxia. Studies on mammals have also shown no reduction in tissue RNA-to-protein ratios in hypoxia-exposed rats (38). It would be expected, perhaps, that there would be a downregulation of RNA synthesis under hypoxia concurrent with the reduction in protein synthesis as an energy-saving strategy. However, Smith et al. (42) showed that there was little agreement between RNA and protein synthesis rates within individual tissues in anoxia, suggesting that RNA synthesis may comprise a fixed cost of protein synthesis that probably cannot be reduced under O2-limited conditions (36). A number of studies in endo- and ectotherms have shown a linear relationship between RNA concentration, expressed as RNA to protein ratio, and fractional rates of protein synthesis (21, 33). In the present study there was a linear correlation between protein synthesis rates and RNA to protein ratio between tissues. The rates of protein synthesis relative to the RNA concentration were radically elevated after a meal in normoxia. Changes in RNA activity (kRNA) after a meal have also been found in fish (33) and rat muscle (15). In the rat, hypoxia has been shown to reduce kRNA by 20%-35% in several tissues but not in skeletal muscle (38).
Energetic cost of protein synthesis. It has been suggested that the postprandial increase in O2 consumption after feeding is a reflection of the energy cost of a surge in protein synthesis, with the total animal's O2 consumption representing the sum of the increases in O2 demand of the individual tissues (18). The contribution that protein synthesis makes to O2 consumption can be calculated from the energy cost of protein synthesis. Increasing rates of protein synthesis resulted in reduced cost (36). When the minimal and experimentally determined costs of protein synthesis were used, this process can account for from 24 to 52% of the total O2 consumption in normoxic conditions in this study. O2 consumption devoted to protein synthesis falls in hypoxia 4 kPa from 19 to 31% and in hypoxia 3 kPa from 15 to 22%. Overall, the percentage of the energy consumption used for protein synthesis in C. maenas is comparable with some of the values obtained for some fish and fish cells (reviewed in Ref. 41). Except for the results from fish cells, calculation of the contribution that protein synthesis makes to whole animal O2 consumption produces values ranging from 20 to 40%: C. maenas, 19-37%; octopus, 35-51%; mytilus, 20%; cod, 20-40%; salmonids, 20-40%; and tilapia, 37% (reviewed in Ref. 41).
Why is the blood oxygenation status limiting protein synthesis after feeding? In fed and normoxic Carcinus, the arterial PO2 was only 1.2 kPa despite the tissue apparent P99 of 5.9 kPa in claw muscle and heart, 2.2 kPa in the hepatopancreas, and 0.9 kPa in the leg muscles (Fig. 6). It is only in the latter tissue that the rate of protein synthesis was not O2 limited in normoxic animals, possibly due to a good match between a relatively low rate of synthesis and a well-developed microcirculation for a proportionally small amount of tissue (16). It remains then to discuss why the blood oxygenation was set at such a low value at the expense of higher rates of protein synthesis. We suggest an analysis based on a strategy in terms of costs and benefits at the whole body level. We list here three hypotheses, which are not mutually exclusive. First, we know that O2 is not only used in mitochondria for ATP production and that changes of local PO2 in crustaceans can play a role for example as a neuromodulator-like substance (31) or in modulating fast muscle activity (12). Moreover, in mammalian tissue where local PO2 is also in the 1- to 3-kPa range, it has been proposed that the production of reactive oxygen species (ROS) can act as signaling molecules enabling cells to regulate electrical activity (1) and O2-dependent gene expression (10). In addition, most of the metabolic reactions in which O2 participates directly could be affected by in situ PO2 fluctuations (24, 43). A narrow PO2 regulation at given apparent set points may thus be crucial. Second, one must differentiate between the notions of O2 flux and quantity of free O2 at the tissue level. In crustaceans, immediately after food ingestion, the cardiac output increases significantly and the blood flow approximately doubles in most arteries supplying the hepatopancreas (32). Present data show that this is performed at low PO2 in Carcinus, which shows that the O2 flux from gills to cells was largely enhanced by a change of circulatory blood flow at low O2 activity. A large body of literature has been devoted to problems of O2 toxicity and ROS production (13). To keep tissue PO2 at low levels can be considered as the first protective step against oxidative stress even if protective enzymes do exist. Finally, it has recently been proposed that the low blood PO2 value regulated during the daytime in crustaceans might contribute to shaping their resting behavior via direct limiting action on the oxidative metabolism of the locomotor muscles themselves (12). Based on the observation that after feeding a crab stays resting in hiding places (40), the limitation problem we describe in this work could also be a consequence of this particular behavior and another example of a hierarchy in the normal physiological repertoire of the crustacean life cycle. As the resting and hiding comportment is associated with a diminished exposure period to predators and consequently an increased probability to survival for the species, it could also be a strong enough drive that dominates the short-term benefit of a 100% transformed diet. In conclusion, postprandial stimulation of protein synthesis and O2 consumption (the SDA response) under hypoxia and hyperoxia were used as a test bed to investigate the effects of the low blood O2 strategy in the green crab C. maenas. PO2 in the arterial blood, O2 consumption, and in vivo protein synthesis rates were measured to define the in vivo P50, that is the PO2 value permitting 50% of the maximum reaction rate. We found that in vivo P50 ranged from 0.6 to 1.3 kPa, which is in the same order of magnitude as the actual arterial PO2 in normoxic Carcinus. The results suggest that even in air-equilibrated water, i.e., in conditions where O2 supply is not limited by any environmental constraint, the blood oxygenation status in resting green crabs can be set at such low values that it limits the rate of protein synthesis in most tissues. This is the first time that such a bioenergetic strategy has been described.| |
ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. R. Smith.
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FOOTNOTES |
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This research was carried out while E. Mente had a grant from the Commission of the European Communities under Contract No. FAIR GT96/1292 and A. Legeay had a grant from the French Ministry of Research and Education.
Address for reprint requests and other correspondence: E. Mente, Dept. of Zoology, Univ. of Aberdeen, Tillydrone Ave., Aberdeen AB24 2TZ, United Kingdom (E-mail: e.mente{at}abdn.ac.uk and massabuau{at}ecotox.u-bordeaux.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpregu.00193.2002
Received 1 April 2002; accepted in final form 2 September 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) and at different times after food
intake (
). * Significantly different from the
prefeeding value.
