Changes in metabolic rate and N excretion in the marine invertebrate Sipunculus nudus under conditions of environmental hypercapnia : identifying effective acidbase variables
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 31 January 2002
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Summary |
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Key words: Sipunculus nudus, hypercapnia, acidbase variable, intracellular pH, amino acid catabolism
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Introduction |
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One of the most important adaptations during extended periods of combined
hypoxia and hypercapnia seems to be the reduction of the animals' aerobic
energy demand. A number of studies conducted on brine shrimp embryos
(Artemia franciscana) and on land snails (Otala lactea) have
shown that hypercapnia alone is suitable to elicit a depression of metabolic
rate (Barnhart and McMahon,
1988; Barnhart,
1989
; Rees and Hand,
1990
). For brine shrimp embryos the effect of increased
PCO2 is interpreted as mainly mediated by an
acidotic shift in intracellular pH (pHi). Pörtner et al.
(1998
) reported that exposure
to environmental hypercapnia (1% CO2) causes respiratory acidosis
in both intra- and extracellular compartments of the intertidal worm
Sipunculus nudus. While pHi was completely restored within 48 h,
extracellular pH (pHe) remained only partially compensated. In vitro
experiments demonstrated that in Sipunculus nudus isolated body wall
musculature a reduction in pHe rather than a moderate fall in pHi causes a
depression of aerobic energy turnover
(Reipschläger and Pörtner,
1996
).
Pörtner et al. (2000)
demonstrated that a decrease in the rate of acidbase regulation during
extracellular acidosis contributed to metabolic depression. In addition,
further mechanisms are probably involved in the reduction of energy turnover.
At the whole-organism level, metabolic depression is supported by the
downregulation of neuronal and motoric activity through hypercapnia-induced
accumulation of the neurotransmitter adenosine
(Nilsson and Lutz, 1992
;
Reipschläger et al.,
1997
). An extreme reduction of energy demand, as in the brine
shrimp embryos, also includes a depression of cellular protein synthesis
(Hofmann and Hand, 1994
;
Van Breukelen et al.,
2000
).
The question arises whether protein or amino acid metabolism are also
affected during moderate metabolic depression, as observed in Sipunculus
nudus, and which variables might be relevant for triggering such a
change. Earlier work showed a decrease in O/N ratios and an increase in net
acid excretion, indicating enhanced protein or amino acid catabolism during
the period of hypercapnia (Pörtner et
al., 1998), which was possibly related to a shift in the use of
substrate or a decrease in protein synthesis rate. The role of intra-
versus extracellular acidbase status in this context is not
clear.
Therefore, the aim of the present study was to determine the specific
influence of intra- and extracellular pH, PCO2
and [HCO3-] on ammonia excretion of isolated body wall
musculature of Sipunculus nudus compared to changes in tissue
metabolic rate. The use of isolated muscle tissue as a non-perfused viable
preparation allowed us to control extracellular acidbase status and
thereby clamp the steady-state intracellular acidbase variables. Tissue
oxygen consumption, ammonia excretion rates and O/N ratios were analysed under
both hypercapnic (PCO2=1.01 kPa) and
normocapnic (PCO2=0.03 kPa) conditions and
different pHe levels. As outlined previously, correlated changes in metabolic
variables and acidbase status and the comparison of changes under
hypercapnic and normocapnic conditions enable the identification of the
effective acidbase variable that elicits the metabolic shift
(Reipschläger and Pörtner,
1996). Focusing on the specific effects of gaseous CO2,
analyses were always performed under aerobic conditions.
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Materials and methods |
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Experimental procedure
The experimental approach basically followed the rationale of
Reipschläger and Pörtner
(1996) and Pörtner et al.
(2000
). For the preparation of
isolated body wall musculature, individuals were killed by `decapitating' them
behind the base of the introvert retractor muscles. The animals were opened
dorsally and all inner organs including the ventral nerve cord were removed.
The body wall musculature was cut transversally to obtain three more or less
equal-sized pieces. Each piece was used for one experiment. The tissue was
fixed with a fine needle and thread onto a plastic frame to ensure full
equilibration with the ambient medium.
Each tissue preparation was first subjected to 15h of normocapnic control
conditions at pH 7.90. For each incubation the three tissue pieces of one
animal were placed together in a closed recirculating system containing a
volume of 81 of 34 artificial sea water (455 mmol l-1 NaCl,
10 mmol l-1 KCl, 24 mmol l-1
MgCl2.6H2O, 10 mmol l-1
CaCl2.6H2O, 28 mmol l-1
MgSO4.7H2O) with 0.1 g l-1 streptomycin,
105 i.u. l-1 penicillin and 20 mmol l-1 Hepes
at a temperature of 15±0.5 °C. The medium was equilibrated and
bubbled continuously with a hypoxic gas mixture of 40% air, 60% nitrogen
supplied by a gas mixing pump (2M303/a-F, Wösthoff, Germany). Moderate
hypoxia (PO2=8.47 kPa) was chosen since
normoxic PO2 levels can be damaging to this
sediment-dwelling animal.
After 15 h of incubation, the control rate of oxygen consumption was determined for two of the three tissue segments in artificial sea water medium at pHe 7.90. Water samples were taken before and after the measurement for determination of the control rate of ammonia excretion. The third piece of tissue was freeze-clamped and stored in liquid nitrogen for further analyses of control levels of intracellular acidbase variables.
Subsequently, both remaining tissues were subjected to a second incubation
period of 45 h under normocapnic (40% air, 60% nitrogen;
PCO2 0.03 kPa) or hypercapnic (40% air, 59%
nitrogen, 1% CO2; PCO2 1.01 kPa)
conditions in media at one of various pH values. Normocapnic incubations were
carried out at pH 7.90, 7.20 or 6.70 and hypercapnic incubations at pH 7.90,
7.55, 7.20 or 6.70. Values of 7.2 and 7.55 were chosen because they represent
the plasma pHe of acute, uncompensated hypercapnia (1% CO2) and the
steady-state pHe reached after compensation of hypercapnic acidosis,
respectively (Pörtner et al.,
1998). The lowest medium pH of 6.7 mimics the higher environmental
PCO2 conditions that prevent full compensation
of tissue pHi in vivo. 1% CO2 was used as a high
PCO2 level experienced by the animals in their
natural environment. Normocapnic as well as hypercapnic solutions were
equilibrated with the corresponding gas mixture for several hours and the pH
was then adjusted by the addition of solid NaHCO3. Media
bicarbonate levels used to set the respective pHe covered a broad range of
concentrations reaching from 0.7 mmol l-1 to 27.0 mmol
l-1. They are referred to as extracellular bicarbonate
concentrations in the following text. The appropriate amount was calculated
from the HendersonHasselbach equation using a value of
pK''' determined according to Heisler
(1986
). Medium pH was checked
at the beginning and at the end of the experiment to make sure that variations
remained within ±0.03 pH units of the initial value.
After 45 h of incubation in the different media, rates of oxygen consumption and ammonia excretion were determined again. Water samples were removed before and after the period of PO2 recording and finally tissues were freeze-clamped. Water and tissue samples were stored under liquid nitrogen for further analyses.
Analyses
Oxygen consumption rates were determined by closed-system respirometry in
40 ml respiration chambers equipped with polarographic oxygen sensors
(Eschweiler, Germany). The chambers were filled with media identical to those
that had been used for tissue equilibration. Oxygen consumption was recorded
for 2-3 h. Tissue oxygen consumption rates were calculated after correction
for the electrode drift and for the minor oxygen consumption of the medium
caused by bacterial growth (approximately 10% of experimental value).
Measurements in chambers without tissue demonstrated that bacterial growth was
effectively inhibited by the added antibiotics.
For the quantification of ammonia concentrations, water samples were taken
from the respiration chambers before and after oxygen measurements and
analysed enzymatically according to Bergmeyer
(1984). Tissue excretion rates
were calculated from the difference in ammonia concentrations between water
samples. To determine tissue ammonia concentrations body wall musculature was
ground under liquid nitrogen and extracted in ice-cold perchloric acid, as
described by Beis and Newsholme
(1975
). Extracts were pH
neutralized with 5 mol l-1 KOH and solid
K2CO3/KHCO3 (1:6 w/w).
O/N ratios were calculated from the amount of atomic oxygen consumed by the tissue and the amount of ammonia-N excreted during the same period, reflecting the contribution of protein or amino acid metabolism, respectively, to overall metabolic rate.
Intracellular acidbase variables were analysed using the homogenate
technique (Pörtner et al.,
1990). Tissue samples were ground under liquid nitrogen and the
frozen powder (80-120 mg) was added to 0.2 ml of a solution containing KF (160
mmol l-1) and nitrilotriacetic acid (1 mmol l-1) in a
0.6 ml Eppendorf tube. The tube was filled to the top with the same solution,
closed and mixed on a vortex mixer. After brief centrifugation (15 s, 20 000
g, 4°C), the supernatant was used for pHi measurement.
Total CO2 (CCO2) was analysed using
a gas chromatograph (Hach Carle, USA) and apparent
HCO3-concentrations were calculated as
[HCO3-]=CCO2-
CO2xPCO2
using values of pK''' and solubility coefficient
CO2 determined according to Heisler
(1986
).
Statistics
For each treatment (hypercapnia or normocapnia), oxygen consumption rates,
ammonia excretion rates and the resulting O/N ratios under control and
experimental conditions were compared using two-factorial analysis of variance
(ANOVA) or analysis of covariance (ANCOVA). Oxygen consumption and ammonia
excretion rates are expressed as a percentage of the respective control values
at pHe 7.90 and PCO2 0.03 kPa in order to
faciliate comparisons between different experimental conditions. When a
significant influence of a single variable was indicated by ANOVA/ANCOVA, the
different treatments were compared using the StudentNewmanKeuls
post-hoc test. In all cases, P<0.05 was accepted to
indicate a significant difference. All values are calculated as means ±
S.D., N=5-6.
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Results |
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As shown previously, oxygen consumption rates of Sipunculus nudus isolated body wall musculature (Fig. 2A,B) were significantly influenced by decreasing pHe (ANCOVA; F1/37=284.938, P<0.0001) and correlated with the subsequent lowering of pHi (ANCOVA; F1/36=344.170, P<0.0001). Under normocapnia and at a low pHe of 7.20 (pHi=7.06±0.04), oxygen consumption was depressed to approximately 75% of the control rate and fell further to 60% at pHe 6.70 (pHi=6.91±0.03). At pHe 7.20 (pHi=7.18±0.01), hypercapnic tissues exhibited a similar reduction of 25% below the control oxygen consumption rate, but an even greater depression to approximately 54% of control metabolism was observed at the lowest experimental pHe of 6.70 (pHi=7.00±0.03). Comparison of normocapnic and hypercapnic treatments clearly demonstrated that hypercapnic samples consumed the same amount of oxygen as normocapnic ones at different pHi values but at the same pHe values (ANCOVA; F1/37=1.001, P=0.323). For identical pHi values a significant difference in oxygen consumption between the two PCO2 treatments results (ANCOVA; F1/36=26.679, P<0.0001).
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Changes in other acidbase variables, PCO2 or extracellular [HCO3-], were not consistently related to oxygen consumption and ammonia excretion (not shown) under all the experimental conditions analysed. As seen in Fig. 3, medium HCO3- concentrations differed considerably between both PCO2 treatments at the same pHe, but nevertheless oxygen consumption rates were identical.
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Dependence of ammonia excretion rates on experimental pH values
Ammonia excretion rates are expressed as a percentage fraction of the
respective control values determined at pHe 7.90 and
PCO2 0.03 kPa. Even under control conditions,
values at the end of the experimental period never reached 100% of the initial
level, indicating that tissue samples showed a continuous decrease in
metabolic rate possibly owing to progressive relaxation from long-term tonic
contractions and the associated energetic costs
(Fig. 4).
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Fig. 4A depicts the relationship between ammonia excretion rate and pHe. In the high pHe range the rate of ammonia excretion of both normocapnic and hypercapnic tissues remained constant at approximately 90% of the respective control values. Only below a threshold value of pHe 7.20, were rates significantly reduced by approximately 10-15% (ANOVA; F2/26=27.16, P<0.0001) independent of ambient PCO2 (ANOVA; F1/26=0.40, P=0.535). Note that the rate of ammonia excretion in both treatments was still at control levels at a low pHe of 7.20, when a depression of aerobic metabolic rate by 25% had already occurred (see Fig. 2).
The correlation of ammonia excretion with pHi is similar (see Fig. 4B), except that the threshold value for the significant reduction in ammonia excretion (ANCOVA; F1/34=19.37, P<0.0001) was found at a higher pHi in hypercapnic (reduction below pHi=7.15) compared to normocapnic tissue samples (reduction below pHi=7.05). A significant difference between normo- and hypercapnic treatments was evident only for the relationship of ammonia excretion rate and pHi (ANCOVA; F1/34=4.33, P=0.0045), whereas the change of ammonia excretion rate with pHe was the same in normocapnic and hypercapnic treatments.
To make sure that the depression of the rates of ammonia excretion under conditions of low pHe was not caused by an increased accumulation of the protonated form NH4+ in acidotic cells, samples of body wall musculature were analysed for ammonium content. The results showed that tissue ammonia concentration even decreased significantly from 1.10±0.15 µmol NH4+ g-1 fresh mass at pHe 7.90 to 0.63±0.08 µmol NH4+ g-1 fresh mass at pHe 6.70, indicating that ammonia was not trapped intracellularly at low values of pHi.
O/N ratios
Fig. 5 shows the dependence
of O/N ratios on pHe (Fig. 5A)
and pHi (Fig. 5B). During
hypercapnia, the ratios initially remained constant with decreasing pHe,
whereas during normocapnia the O/N ratios fell progressively, starting at high
pHe. A significant depression in both normocapnic and hypercapnic tissues
(ANOVA; F2/25=21.63, P<0.0001) to
approximately 70-75 % of the respective control value was, however, observed
at a low pHe value of 6.70. For the dependence of O/N ratios on pHi a
threshold value occurred somewhere between pHi 7.20 and 7.30, below which O/N
ratios of both CO2 treatments started to decrease with falling pHi
(ANCOVA; F1/33=36.19, P<0.001).
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The influence of intracellular as well as extracellular pH was found to be independent of ambient PCO2 because no significant difference in O/N ratios was found between normocapnic and hypercapnic tissues at the same pHe (ANOVA; F1/25=0.55, P=0.465) or pHi (ANCOVA; F1/33=1.45, P=0.237), respectively. Both pHe and pHi seemed to have a significant influence on the pattern of change in O/N ratios, although pHe will influence cellular processes by modifying pHi (see Fig. 1A). For that reason we checked the correlation of pHe and O/N ratios after taking into account the dependence of pHi on pHe. O/N ratios determined at different pHe and the respective pHi values were normalized for the mean of all pHi data (pHimean=6.97) as if O/N ratios had been measured at constant pHi, and thus were solely dependent on pHe. In a subsequent analysis of the relationship between pHe and standardized O/N data, we were no longer able to find a significant influence of pHe (ANOVA; F2/24=0.792, P=0.465).
Another aspect of interest is the relationship between intracellular [HCO3-] and O/N ratios (Fig. 6). Bicarbonate is involved since amino acid degradation results in the net production of bicarbonate and ammonium ions. Fig. 6 shows clearly that there is no overall correlation of intracellular [HCO3-] with O/N ratios, especially during normocapnia. Starting from high pHi values and a low value of 1.33±0.14 mmol l-1 intracellular [HCO3-], normocapnic intracellular [HCO3-] increased significantly with a reduction in O/N ratio. At O/N values below 3.5 a parallel decrease in intracellular [HCO3-] occurred under normo- and hypercapnic conditions.
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Discussion |
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One has to keep in mind that ammonia excretion rates cannot be seen
independently of the respective rates of oxygen consumption and energy
turnover, which are reduced during metabolic depression and, subsequently,
less protein or amino acids should be catabolized. Only at the lowest pHe
tested (6.70), however, were ammonia excretion rates significantly depressed
by approximately 10-15 % in both normocapnic and hypercapnic tissue samples.
At the same time, oxygen consumption had fallen by 40-45 %. O/N ratios reflect
the proportion of amino acids being used to fuel aerobic energy metabolism and
were found to be approximately 4.0-4.5 in Sipunculus nudus isolated
body wall tissue under control conditions. These values show that amino acid
catabolism covers by far the largest fraction, if not all, of the energy
demand in this invertebrate tissue (Cowey
and Corner, 1963; Snow and
Williams, 1971
). O/N ratios reached even lower values during
acidosis. At pHe 6.70 the reduction in ammonia excretion did not prevent a
further decrease in O/N ratios to values of approximately 3.0, since oxygen
consumption rates fell to a greater extent compared to ammonia excretion.
The lowest O/N ratios expected from catabolism of a mix of amino acids in
proportion to their presence in the proteins are around 7.0, and indicate the
sole use of protein (Cowey and Corner,
1963). An O/N ratio lower than this value is expected for the
predominant oxidation of glycine (approximately 1.0). Oxidative metabolism of
alanine or glutamic acid leads to O/N values of 7.0 and 3.0, respectively
(Lehninger, 1975
;
Mayzaud and Conover, 1988
).
Therefore, a decreasing O/N ratio during acidosis suggests a shift in
metabolic substrate. The values found do not suggest elevated overall amino
acid catabolism but rather a shift in the selection of the different amino
acids. A larger participation of asparagine (O/N=3.3), glutamic acid
(O/N=3.0), glutamine (O/N=1.5), glycine (O/N=1.0) or histidine (O/N=3.0) in
metabolic amino acid degradation would result in lower than control O/N
ratios. All of these are non-essential amino acids that could easily be
channelled into the Krebs cycle via
-ketoglutarate, pyruvate
or oxaloacetate. At the same time an enhanced oxidative decarboxylation of
dicarboxylic acids (aspartic and glutamic acid) would yield a higher net
production of HCO3-, supporting the compensation of pHi
under acidotic conditions. However, the question of whether protein synthesis
and turnover rates were lowered under these conditions remains open.
Besides the identification of potential changes in N metabolism, another
aim of the study was to analyse the regulatory role of individual
acidbase variables for the different physiological processes. With
respect to oxygen consumption and ammonia excretion rates, the statistical
evaluation clearly showed that both processes are influenced by pHe. Neither
PCO2 nor extracellular
[HCO3-1] exert a regulatory influence. In line with
previous studies (Reipschläger and
Pörtner, 1996;
Pörtner et al., 2000
),
the pHe-dependent regulation of oxygen consumption rates seems to be partly
mediated by an inhbition of net proton transport across the cell membrane
during acidosis. The resulting decrease in the overall rate of acidbase
regulation and a shift to more energy-efficient transport (see below) lowers
the cost of this cellular process and contributes to an energy-saving strategy
during metabolic depression.
With respect to the effect of acidbase status on O/N ratios, no clear picture emerged from the statistical evaluation of the present data, for both pHe and pHi showed a significant influence. However, when the correlation of intra- and extracellular pH was considered from the calculation of pHi-normalized O/N data, no specific effect of extracellular pH on O/N ratios could be observed. Therefore, changes in N metabolism seem to be determined predominantly by intracellular pH, which in itself is influenced by pHe.
A decrease in pHi may also not directly affect N metabolism, which led us
to investigate any potential effect of intracellular bicarbonate. If we
exclude a further influence of pHe-inhibited ion exchange on metabolic rate in
the low range of pHe and pHi, changes in intracellular
[HCO3-], ammonia excretion and O/N ratios are
consistently correlated. The transient but significant accumulation of
intracellular HCO3- with a decrease in pHe from 7.90 to
7.20 (see Fig. 6, normocapnic
data) cannot be explained by a shift of pHi regulation from the
Na+/H+ antiporter to Na+-dependent
Cl-/HCO3- exchange. Although investigations
by Pörtner et al. (2000)
indicate a predominant use of this more ATP-efficient transport system under
conditions of acidosis, the exchange rates of all membrane proteins
participating in pHi homeostasis were downregulated below control rates in
acidotic tissue, leaving no room for any accumulation of bicarbonate. It seems
very likely that the changes in intracellular [HCO3-],
clearly visible in the normocapnic data of
Fig. 6, have to be viewed in
close connection with N metabolism. In hypercapnic tissue samples, such
findings are hidden by high ambient HCO3- concentrations
at high pHe, which lead to a strong passive influx of
HCO3- and elevated pHi levels (see
Fig. 1). On the one hand,
bicarbonate, resulting from the decarboxylation of the amino acid
-carboxylic group, represents an end product of protein degradation; on
the other hand, it plays an important role in the buffering of intracellular
pH. An augmented breakdown of dicarboxylic amino acids (e.g. glutamic acid
and, after deamination, asparagine or glutamine), thought to be favoured under
conditions of acidosis, may explain the higher intracellular
[HCO3-] in normocapnic tissue at pHe 7.20. As the medium
pH was further reduced to pHe 6.70, overall amino acid catabolism fell owing
to decreased energy demand, causing the normocapnic intracellular
[HCO3-] to fall below the respective control value (see
Fig. 6). A full compensation of
pHi, as reported by Pörtner et al.
(1998
) for the whole animal
after 48 h of incubation at 1 % CO2, would have been supported by
metabolic bicarbonate production. The mechanism that triggers the
disproportional use of dicarboxylic amino acids and their amines remains
unclear; however, it probably involves the decrease in pHi. This leaves the
accumulation of intracellular bicarbonate as a dependent process which does
not exert a regulatory role.
In summary, the present data provide evidence for an influence of intracellular acidbase variables on N metabolism, specifically on the selection of amino acids used by catabolism. This effect is correlated with a decrease in pHi. The shift in N metabolism, leading to a decrease in O/N ratios despite lowered ammonia excretion rates, is evident under conditions of extreme acidosis. This indicates that under pronounced metabolic depression catabolism prefers low O/N amino acids like asparagine, glutamine or their dicarboxylic acids. These changes cause a release of bicarbonate and, thus, support the regulation of pHi.
It remains to be investigated whether, during long-term exposure to high levels of PCO2, this metabolic shift may damage the cellular protein pool and also influence growth and reproduction. As a working hypothesis to be examined in further studies we propose a downregulation of protein biosynthesis at low levels of pHi. Free amino acids originating from ongoing protein degradation would not be incorporated in the cellular protein pool, but rather would be diverted into catabolic energy metabolism.
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