Seasonality of energetic functioning and production of reactive oxygen species by lugworm (Arenicola marina) mitochondria exposed to acute temperature changes
1 Alfred Wegener Institute for Polar and Marine Research, Columbusstrasse
27568 Bremerhaven, Germany
2 International University Bremen, Campus Ring 1, 28759 Bremen,
Germany
* Author for correspondence (e-mail: dabele{at}awi-bremerhaven.de)
Accepted 22 April 2004
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Summary |
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Key words: lugworm, Arenicola marina, mitochondria, ROS, proton leak, metabolic regulation, temperature
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Introduction |
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As important ROS producers, mitochondria are prone to become immediate
targets for ROS-induced molecular damage. This leads to disturbance of
mitochondrial energetic functioning in a primary assault
(Yan et al., 1997;
Brand, 2000
), whereas slowly
accumulating damage of the mitochondrial DNA causes mitochondrial degeneration
and enhances the process of cellular ageing
(Sastre et al., 2000
;
St-Pierre et al., 2002
).
Mitochondrial ROS production depends on the magnitude of membrane potential
(
) in isolated mitochondria
(Korshunov et al., 1997
;
Brand, 2000
), and not so much
on the rate of electron transport. The
-threshold value for
significant ROS production is just above state 3
level
(Korshunov et al., 1997
) and,
indeed, most investigations do not detect substantial ROS production under
phosphorylating state 3 conditions. Mild uncoupling of the proton gradient
through futile cycling of protons through the inner mitochondrial membrane
dissipates
and reduces proton motive force (Skulachev,
1996
,
1998
;
Korshunov et al., 1997
;
Brand, 2000
), thereby
preventing overflow of electrons from mitochondrial complexes I and III. High
proton motive force slows respiratory electron transport and leads to an
increased reduction of complex III ubiquinone (QH), which then leaks electrons
into the matrix and presumably also to the intermembrane space
(St-Pierre et al., 2002
).
Here, the electrons react with molecular oxygen to form superoxide and
H2O2 (for a review, see
Brand, 2000
). Accordingly, the
maximal proportion of proton leakage through the inner mitochondrial membrane
determines the range over which an animal can shift mitochondrial
, in order to optimise respiratory efficiency, while avoiding
deleterious ROS production during transient ADP exhaustion
(Korshunov et al., 1997
). An
adjustable proton leak rate could contribute towards controlling the low
intracellular PO2, especially in water
breathing animals (see also Massabuau,
2003
), as it increases oxygen consumption also under resting
conditions (Brand, 2000
). This,
and the limitation of ubisemiquinone (QH·) accumulation, gave rise to
the idea of an antioxidant function of `mild uncoupling' of
under
physiological conditions (Skulachev,
1996
,
1998
;
Brand, 2000
).
In the present study we isolated mitochondria from lugworms of an intertidal population during summer (July) and winter (February), and measured mitochondrial energetics, membrane potential and ROS production. The aim was to investigate the functional changes caused by seasonal temperature acclimatisation and the higher energy demand during the reproductive cycle. Specifically, the following questions were addressed. (i) How does seasonal acclimatisation affect mitochondrial function, ROS production and mitochondrial density in summer compared to winter animals? (ii) What is the interdependence between phosphorylation efficiency, membrane potential and ROS production in lugworms? (iii) Is ROS formation related to the energetic state of the mitochondria (state 3 vs state 4 respiration)? (iv) Is ROS production controlled under thermal stress (warming and cooling) in mitochondria during summer and winter by increasing the leak, or would the animals have to respond by increasing cellular antioxidant stress defence?
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Materials and methods |
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For the experiments on mitochondrial physiology, animals were kept at constant temperatures of 1°C (winter animals) and 10°C (summer animals) for 27 weeks. One group of late winter animals was acclimated to 10°C for a period of 2130 weeks, to follow the time course of mitochondrial density changes. In addition, mitochondrial counts were done in residual individuals from the winter animal group, kept at 1°C, after finishing the physiological experiments at 14, 18 and 22 weeks after collection. The numbers of mitochondria per cell in summer animals maintained at 10°C were counted immediately after collection and again after 4 weeks.
Isolation of mitochondria
After removing head and tail of the worm, the body wall tissue was opened,
the intestine removed and the remaining tissue rinsed with seawater and
blotted dry. Tissues from 24 animals were pooled per isolation,
yielding a total of 3.54 g fresh mass. Part of the pooled tissue was
frozen in liquid nitrogen and stored at 80°C for subsequent
enzymatic measurements.
Mitochondria were prepared after Sommer and Pörtner
(2002). Between 2.3 and 2.5 g
of fresh tissue were minced in 35 ml isolation buffer [550 mmol l-1
glycine, 250 mmol l-1 sucrose, 40 mmol l-1 Tris/HCl, 4
mmol l-1 EDTA; 1% (w:v) bovine serum albumin (BSA); 1 µg
ml-1 aprotinin, pH 7.5 at 20°C] using scissors. The tissue was
transferred to a teflon/glass homogeniser (type: Potter Elvejhem; Sartorius
BBI Systems, Melsungen, Germany) and homogenised with 57 passes. After
centrifugation for 8 min at 1300 g and 0°C, the
supernatant was stored on ice and the pellet resuspended and homogenised a
second time. Following a second centrifugation, supernatants were combined and
centrifuged for 15 min at 10 000 g and 0°C to sediment
mitochondria. The resulting pellet was resuspended in 2 ml assay medium (600
mmol l-1 glycine, 160 mmol l-1 KCl, 5 mmol
l-1 K2HPO4, 20 mmol l-1 Na-Hepes,
4 mmol l-1 EDTA, 3 mmol l-1
MgCl26·H2O, 1 µg ml-1 aprotinin, 1%
(w:v) BSA, pH 7.5 at 20°C) and kept on ice. Portions of this isolate and
of the assay medium were frozen for protein determination according to a
Biuret method, modified after Kresze
(1988
), using 5% (w:v)
deoxycholate to resolve membrane proteins.
Measurements of mitochondrial respiration and coupling
The measurements of mitochondrial respiration were carried out in a
respiration chamber using Clarke-type oxygen electrodes (Eschweiler, Kiel,
Germany). Measurements were performed at habitat temperature: 10°C for
summer animals and 1°C for winter animals, respectively. Both types of
mitochondria were also measured at the other temperature, thus representing
cold exposure (1°C) for summer animal mitochondria, and heating to
10°C for winter animal mitochondria, to test the mitochondrial reaction to
acute changes of temperature.
Recording was done using an Eschweiler M 200 oxymeter (Kiel, Germany)
connected to a Linseis (Selb, Germany) two-channel chart recorder. For each
measurement, the chambers were filled with 768 µl of
O2-saturated assay medium, 20 µl of a 50% BSA solution, 5 µl
of the myokinase inhibitor
P1,P5-adenosine-5'-pentaphosphate
(Ap5A) in water (5 µmol l-1), 2 µl of complex I
inhibitor rotenone (10 µmol l-1), 3.3 mmol l-1 of
respiratory substrate sodium succinate and 200 µl of mitochondrial
suspension. Rates of oxygen consumption were measured at constant temperature,
while continuously stirring the mitochondrial suspension at 300 revs
min-1. After approximately 5 min, ADP was added to a final
concentration of 150 µmol l-1 to initiate state 3 respiration
under saturating conditions. State 4 respiration was recorded after all ADP
had been consumed (Chance and Williams,
1955). After addition of 2 µg ml-1 of the ATPase
inhibitor oligomycin, state 4+ respiration was recorded, which comprises the
oxygen consumption by the proton leak through the inner mitochondrial
membrane, plus the amount of oxygen molecules converted to ROS per unit time
(Brand et al., 1994a
;
Heise et al., 2003
).
The oxygen solubility (ßO2) of the assay medium
at experimental temperatures was calculated after Johnston et al.
(1994). Oxygen consumption
measurements were corrected for electrode drift at 100%
PO2 and at 0%
PO2. The respiratory control ratio (RCR) was
calculated by dividing state 3 by state 4 respiration, according to Estabrook
(1967
), or by state 4+
respiration, following Pörtner et al.
(1999
; RCROl). ADP/O ratios
were calculated by dividing the amount of ADP added by the amount of molecular
oxygen consumed during state 3 respiration
(Chance and Williams, 1955
;
Estabrook, 1967
).
Determination of reactive oxygen species (ROS) formation in mitochondrial isolates
Mitochondrial ROS production was determined fluorimetrically by recording
the reaction of the indicator dye homovanillic acid (HVA; Sigma) with hydrogen
peroxidase (H2O2) catalyzed by horseradish peroxidase
(López-Torres et al.,
2002). Briefly 3.6 mg HVA was diluted in 2 ml distilled water to
give a 9.8 mmol l-1 solution. A fluorometer LS 50B (Perkin &
Elmer, Boston, MA, USA; excitation: 312 nm, 2.5 nm slit width; emission: 420
nm, 3.5 nm slit width) equipped with a water-jacketed quartz cuvette
thermostatted to the relevant measuring temperature was used for the ROS
assays. The measurement was carried out using 200 µl mitochondrial isolate
and alongside the respiratory measurements, but omitting BSA. 5 µl of a
6000 U ml-1 superoxide dismutase solution (Sigma) was added, to
convert superoxide anions to H2O2, and 10 µl of horse
radish peroxidase (215 U ml-1, Merck, Darmstadt, Germany) to
catalyze HVA oxidation by H2O2. The assay mixture was
gently stirred throughout the measurement. State 2 was induced with sodium
succinate (3.3 mmol l-1) and state 3 with ADP (150 µmol
l-1). State 2 oxidation was always higher than in state 3 (see
Fig. 1). State 3 terminated
when HVA oxidation, i.e. the fluorescence slope, started to rise again,
indicating exhaustion of ADP and the beginning of state 4 respiration. The
state 4 slope was recorded for a couple of minutes, before adding oligomycin
to induce state 4+. Finally, for calibration of the assay, 440 pmol
H2O2 were added and the immediate increase in
fluorescence recorded.
|
Previous testing of the HVA assay showed that the probe is not sensitive to H2O2 alone, or to oxidation by superoxide anions prior to SOD conversion. The H2O2 induced slope was entirely abolished by catalase.
Measurement of membrane potential
The mitochondrial membrane potential () was measured according
to Brand (1995
), using an
electrode sensitive to the hydrophobic cation triphenylmethylphosphonium
(TPMP+). Four times 2 µl of 0.125 mmol l-1
TPMP+ were added for calibration in a glass cuvette containing 768
µl respiration buffer (0.6 mol l-1 glycine, 0.16 mmol
l-1 KCl, 20 mmol l-1 Na-Hepes (pH 7.5 at 20°C), 4
mmol l-1 EDTA, 5 mmol l-1 K2HPO4,
3 mmol l-1 MgCl2.6.H2O and 1 µg
ml-1 aprotinin), 20 µl of a 50% (w:v) BSA solution, 5 µl of
the myokinase inhibitor
P1,P5-di-adenosine-5'-pentaphosphate
(Ap5A) in water (5 µmol l-1) and 2 µl of complex I
inhibitor rotenone (10 µmol l-1 in ethanol). When the trace was
steady, 200 µl of the mitochondrial suspension were added to give a volume
of 1 ml. After the addition of 3.3 mmol l-1 sodium succinate,
mitochondria were allowed to accumulate TPMP+ and the
extramitochondrial TPMP+ concentration reached a new stable value.
The membrane potential in state 3 was measured in the presence of 150 µmol
l-1 ADP and state 4+ was induced by the addition of 2 µg
ml-1 oligomycin. 1 µl nigericin (80 ng ml-1) was
added to bring the pH gradient (z
pH) to zero. At the end of the
run, the uncoupler FCCP was added to fully dissipate
, so that all
TPMP+ was released by the mitochondria and the external
concentration re-established. Measurements were done in triplicate. The
membrane potential (mV) was calculated using the following equation:
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Measurements of citrate synthase and antioxidant enzyme activities
To analyse the activity of the mitochondrial marker enzyme citrate synthase
(CS; EC 4.1.3.7) approximately 100 mg of frozen tissue was homogenized in
liquid nitrogen and diluted 1:7 (w:v) with 75 mmol l-1 Tris-HCl
buffer that contained 1 mmol l-1 EDTA at pH 7.6 at 20°C.
Samples were homogenized with an Ultraturrax T8 homogenizer (IKA Labortechnik,
Staufen, Germany) and sonicated for 5 min at 0°C using ultrasound. After
10 min centrifugation at 12 000 g at 0°C, CS activity was
determined in the supernatant according to the protocol of Sidell et al.
(1987) at 412 nm at 20°C
and the respective habitat temperatures (1°C, winter animals; 10°C,
summer animals). The assay system contained 75 mmol l-1 Tris-HCl
buffer [pH 8.0 at 20°C), 0.25 mmol l-1 DTNB
((5,5'-dithiobis(2-nitrobenzoic acid) = Ellmann's reagent], 0.6 mmol
l-1 acetylCoA and 130 ml of supernatant (diluted with
H2O). The assay was started by addition of 40 µl 20 mmol
l-1 oxaloacetate and the subsequent absorbance increase recorded.
CS activity was calculated using the molar extinction coefficient
=13.61
ml µmol-1 cm-1 of the DTNB-SH-CoA complex formed.
Catalase (CAT; EC 1.11.1.6) was extracted into 50 mmol l-1
potassium phosphate buffer (pH 7.0 at 20°C, 1:11, w:v) and measured
according to Aebi (1985
).
Superoxide dismutase (SOD; E.C. 1.15.1.1) activity in crude homogenates was
measured according to Livingstone et al.
(1992
). The test uses the
xanthine oxidase/xanthine system to generate superoxide anions at a rate that
reduces cytochrome c with an absorbance slope of exactly 0.02
absorbance units min-1. One unit of SOD activity in the sample
reduces cytochrome c reduction by 50%.
Cell isolation and mitochondrial fluorescence staining
For cell isolations, a 50 mg piece of freshly sampled body wall tissue was
rinsed thoroughly with filtered seawater and finely chopped on ice. To remove
blood, the tissue pieces were gently washed with ice cold filtered seawater.
Two digestive enzymes, 757 U hyaluronidase (Merck) and 10145 U trypsin (Sigma)
were added, and allowed to stand overnight at 8°C in the dark. In the
morning, digestion was continued for approx. 1 h at room temperature under
constant shaking. Cells were filtered through gauze and left to settle at the
bottom of the vial. 100 µl of 50% BSA (w:v) were added to quench the
digestive enzyme activity. 15 µl of Mito-tracker Green solution (Molecular
Probes, Leiden, The Netherlands; 50 µg dissolved in 740 µl
dimethylsulfoxide) were added to the cell suspension to stain the mitochondria
during 20 min of gentle shaking at room temperature.
Stained cells were kept on ice and mitochondrial counts performed with a
confocal microscope (Leica IRBE; Bensheim, Germany) using the setup described
in Abele et al. (2002). Cells
from 35 animals were studied during one sampling and at least 10 cells
were counted per animal. Mitochondria in cells from winter animals were
counted during four samplings and from summer animals during two sampling
events. Additionally, every second week, counts were performed in cells from
winter animals acclimated to 10°C under experimental conditions to monitor
the changes on long term exposure to higher temperatures.
Statistics
Data were tested for normality of distribution (KolmogorovSmirnov
test) and homogeneity of variance (Levene test). Significant changes
(P<0.05) in normally distributed data were evaluated by analysis
of variance (ANOVA) and a NewmanKeuls post hoc test was
employed to determine significant differences between data pairs. A Statistica
version for Windows was employed for the statistics.
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Results |
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Respiratory coupling rate (RCR; state 3/state 4 respiration) was lower in summer animals at habitat temperature (10°C: 4.79±0.46, N=5) than in winter animals at both temperatures (10°C: 6.53±0.71, N=11; 1°C: 6.63±1.72, N=8) (P< 0.05). On cooling from 10° to 1°C, RCRs in summer animal mitochondria increased significantly to 6.32±1.32 (P<0.01, N=6). Addition of oligomycin decreased state 4 respiration by about 25% in each measurement, augmenting RCROl, especially in summer animals at 1°C to 12.66±3.11 (N=5), whereas in winter animals RCROl was 8.78±0.97 (N=10) at 10°C and 9.76±1.81 (N=8) at 1°C. Summer animals at 10°C had an RCROl of 6.99±0.78 (N=6).
ADP/O ratios with sodium succinate were between 1.50 and 1.58 and did not differ among groups, with one exception: warming of winter animal mitochondria to 10°C resulted in lower ADP/O ratios (1.43±0.05, N=9, P<0.01) compared to summer animals at 10°C (1.58±0.09, N=7).
ROS (i.e. H2O2) production relative to mitochondrial
oxygen uptake (%-H2O2/O2) could be calculated
(Table 1), because both data
sets were measured in the same isolate and were related to mitochondrial
protein. Calculations are based on the stoichiometric approximation of 2 moles
of oxygen being univalently reduced to 2 mol superoxide, to give 1 mol
H2O2 in the reaction:
2O2·- + 2H+
1H2O2 + 1O2. State 3
%-H2O2/O2 was around 0.25% and lower in both
seasonal groups at both temperatures, compared to the oxygen consumption due
to ROS formation under non-phosphorylating state 4 conditions. In states 4 and
oligomycin-induced 4+, summer animal mitochondria cooled to 1°C
(N=5) reduced a maximal proportion of 23.07±8.14% of consumed
oxygen to ROS. Although the absolute level of H2O2
formation per mg protein increased at high temperature
(Fig. 3), warming generally
reduced the proportion of O2 uptake leading to
H2O2 production in both seasonal groups
(Table 1) and in all
states.
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|
The mitochondrial membrane potential () was generally higher in
summer than in winter animals in all respiratory states. At 10°C the state
4
with nigericin was 179.3±7.3 mV (N=5) in summer
and significantly higher than in winter animals (165.4±5.5 mV,
N=10) (P<0.033).
correlated positively with
respiration rates in states 3 and 4+ (Fig.
4). Likewise, rates of H2O2 production
correlated with the membrane potential (
) in states 4 and 4+ (an
example is shown for state 4+ in Fig.
5). Thus higher H2O2 production was found at
higher
, whereas the dependence of ROS formation on mitochondrial
membrane potential was much steeper in summer than in winter animals.
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Seasonal variability of enzyme activities in A. marina body wall tissue
Table 2 lists the activities
of the antioxidant enzymes superoxide dismutase (SOD) and catalase, as well as
the mitochondrial marker enzyme citrate synthase (CS) in body wall tissue
homogenates assayed at 20°C and habitat temperatures. Q10
values were close to 2 for SOD and CS and around 1.5 for catalase in both
seasonal groups.
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Seasonal variations of mitochondrial densities
Counts of Mito-tracker Green stained mitochondria in body wall cells from
both seasonal groups displayed 1.6 times higher (P<0.01)
mitochondrial densities in recently collected winter (68±7,
N=14) vs (pre-spawning) summer animals (43±14,
N=15, see Table 3). In
line with this finding, the protein content of the mitochondrial isolates was
1.5 times higher in winter (2.6±0.8 mg protein ml-1
mitochondrial isolate) than in summer (1.7±0.4 mg protein
ml-1 mitochondrial isolate). This indicates that an increase in
mitochondrial densities might be a seasonal cold compensation mechanism in
lugworms. Mean cell diameter and volume were slightly (5%) higher in summer
animals, thus contributing somewhat to the lower mitochondrial density. During
prolonged maintenance (4 weeks) of winter animals at 1°C under laboratory
conditions, a decrease of mitochondrial numbers to 48±6
µm-3 of cell volume occurred, which was attributed to the
prolonged maintenance and reduced burrowing activity of lugworms in the
aquaria. Acclimation of winter animals at 10°C for at least 10 and up to
30 weeks did not result in significant changes of mitochondrial densities or
cell size, although the very last value was somewhat lower.
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Discussion |
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We found lower mitochondrial densities in body wall cells from summer
compared with winter animals. However, in vitro rates of
protein-specific mitochondrial oxygen consumption more than 3 times higher
than winter rates can easily overcompensate for the lower mitochondrial
density in summer (Fig. 6, dark
bars). Isolated mitochondria can only serve as a model of the in vivo
situation and the effect may not be as pronounced under cellular conditions
where the ATP/ADP ratios are much closer to state 4 and rarely resemble fully
ADP-saturated state 3 conditions
(Guderley, 1998). However, the
elevated metabolic activity in the pre-spawning animals from July can be
traced down to the elevated oxygen consumption rates of isolated mitochondria.
Mitochondria of post-spawning worms from late summer (September) isolated by
Sommer and Pörtner (2004
)
clearly displayed lower protein-specific respiration rates, in keeping with
the lower oxygen uptake of the whole animal at that time of the year
(Fig. 6; after
Schöttler, 1989
).
Table 4 illustrates that
higher mitochondrial respiration rates go hand in hand with higher proton
motive force in isolated mitochondria from pre-spawning summer animals
compared to mitochondria from winter lugworms. As a consequence, summer
mitochondria produce a higher proportion of ROS, especially in state 4+
(Fig. 5). As sudden warming can
induce oxidative stress in marine ectotherms (Abele et al.,
1998b,
2002
), and environmental
conditions in intertidal environments are more changeable in summer than in
winter, lower mitochondrial density is likely to reduce the risk of damage
from mitochondria-borne ROS in pre-spawning summer animals. Additionally, the
higher leak in isolated mitochondria from summer (10°C, see
Table 4) compared to winter
animals could balance ROS formation by a mild uncoupling of the proton motive
force. These preventive antioxidant mechanisms might be crucial under high
energy requirements, as the animals face reproduction and high temperatures.
Nevertheless, absolute ROS production rates in isolated mitochondria from
pre-spawning summer animals were fourfold higher (0.12±0.02 nmol
H2O2 min-1 mg-1 protein) than
those from winter specimens (0.03±0.003 nmol H2O2
min-1 mg-1 protein) and explain the significantly higher
antioxidant activities we measured in Arenicola body wall tissue in
July (Table 2). We conjecture
that in winter, when habitat temperatures are low and less variable and worms
are less active, a smaller mitochondrial membrane leak sustains constant and
high phosphorylation efficiency at minimal risk of causing an increase of the
percentage conversion of oxygen to ROS, due to the low membrane potential.
Given the low metabolic rates of winter animals this is obviously tolerable,
even taking into account the worms' already low antioxidant enzyme activities,
further hampered by the Q10-dependent thermal slow down.
|
Comparatively low proton leak rates in A. marina body wall mitochondria
According to Brand (2000),
between 15 and 35% of state 3 oxygen consumption drive the proton leak in
cells from ectothermal and endothermal vertebrates so far studied. We have
previously reported that marine mud clams also fall into this range
(Abele et al., 2002
;
Heise et al., 2003
). However,
in Arenicola mitochondria the percentage rate of oligomycin-saturated
state 4+ respiration ranged far lower and, at 10°C, amounted for only
1114.5% of state 3 oxygen consumption
(Table 4). Mitochondrial oxygen
consumption makes up only 80% of overall cellular respiration, a surplus of
20% being extra mitochondrially consumed
(Brand et al., 1994a
).
Therefore, only approximately 10% of the cellular oxygen uptake actually
drives the proton leak in lugworms. Similarly low values (<10% of state 3
respiration) were found in mitochondria isolated from the body wall tissue of
a sipunculid worm (Buchner et al.,
2001
). So, what makes these tissues different from others?
The vast majority of data in proton leak research have been obtained in
vertebrate studies. Most vertebrate tissues respond to various environmental
or internal stimuli with high flexibility of muscular and general metabolic
activity. High long-chain polyunsaturated fatty acid content in mammalian
mitochondrial membranes is thought to enable a higher proton leak rate and, in
part, is held responsible for the higher metabolic rates and heat production
in endotherms, compared with ectotherms
(Brand et al., 1994b;
Brookes et al., 1998
). In
contrast, body wall tissue of marine infaunal worms displays low and,
moreover, strictly oxyconforming metabolic rates. Although Arenicola
marina possesses gills, 50% of its overall oxygen uptake is directly
cutaneous (Mangum, 1976
).
PO2 in the burrow environment is around 100
Torr (14 kPa; Mangum, 1976
),
and pumping activity is oxyconforming, limiting metabolic and motoric
flexibility in response to other environmental stimuli.
Fig. 7 describes the
dependence of state 4+ substrate oxidation rates on mitochondrial membrane
potential in both seasonal groups of lugworms and compares values for
Xenopus toad and rainbow trout, taken from Brookes et al.
(1998). At the same membrane
potential, lugworms display lower state 4+ respiration rates than trout,
suggesting that both leak and substrate oxidation rates are reduced in worms,
compared to a comparatively active fish. In winter lugworm mitochondria, the
balance between substrate oxidation and membrane potential is close to the
conditions found in toad, a relatively slow amphibian. A high proton leak
enables a tissue to perform quick adjustments in response to sudden changes of
energy requirements by reducing the leak to power phosphorylation, or by
increasing the leak to prevent overflow of ROS production
(St-Pierre et al., 2000
). Body
wall tissues of marine worms may not need this flexibility to the same extent
as more active vertebrates, since they rely on sustained slow rates of motor
activity for ventilatory purposes. This goes along with high RCRs in A.
marina mitochondria. Under unstressed habitat conditions, the cells of
lugworms are not exposed to sudden oxygen deprivation and subsequent oxygen
flushing. Metabolic adjustments in these tissues are slow and function on a
time scale of hours and upwards. However, the present and earlier
(Sommer and Pörtner,
2004
) experiments indicate that on exposure to acute temperature
stress, when oxygen transport to the tissue ceases (low temperatures), or
cannot further compensate for the high metabolic expenditures (heat stress),
the changes of the proton leak rate, however small, can still confer some
metabolic flexibility and moderate antioxidant function in these slow moving
animals.
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Acknowledgments |
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