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First published online February 15, 2006
Journal of Experimental Biology 209, 891-906 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02054
Critical temperatures in the cephalopod Sepia officinalis investigated using in vivo 31P NMR spectroscopy
Alfred-Wegener-Institute for Marine and Polar Research, Am Handelshafen 12, 27570 Bremerhaven, Germany
* Author for correspondence (e-mail: fmelzner{at}awi-bremerhaven.de)
Accepted 20 December 2005
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Under control conditions (15°C), changes in muscle phospho-L-arginine (PLA) and inorganic phosphate (Pi) levels could be linearly related to frequently occurring, high-pressure mantle contractions with pressure amplitudes (MMPA) of >0.2 kPa. Accordingly, mainly MMPA of >2 kPa affected muscle PLA reserves, indicating that contractions with MMPA of <2 kPa only involve the thin layers of aerobic circular mantle musculature. On average, no more than 20% of muscle PLA was depleted during spontaneous exercise under control conditions.
Subjecting animals to acute thermal change at an average rate of 1 deg.
h–1 led to significant Pi accumulation (equivalent
to PLA breakdown) and decrements in the free energy of ATP hydrolysis
(dG/d
) at both ends of the temperature window, starting at mean
critical temperatures (Tc) of 7.0 and 26.8°C,
respectively. Frequent groups of high-pressure mantle contractions could not
(in the warm) or only partially (in the cold) be related to net PLA breakdown
in mantle muscle, indicating an oxygen limitation of routine metabolism rather
than exercise-related phosphagen use. We hypothesize that it is mainly the
constantly working radial mantle muscles that become progressively devoid of
oxygen. Estimates of very low dG/d values (–44 kJ
mol–1) in this compartment, along with correlated stagnating
ventilation pressures in the warm, support this hypothesis. In conclusion, we
found evidence for an oxygen limitation of thermal tolerance in the cuttlefish
Sepia officinalis, as indicated by a progressive transition of
routine mantle metabolism to an anaerobic mode of energy production.
Key words: anaerobic metabolism, ventilation, exercise, Cephalopoda, Mollusca, mantle muscle, mantle cavity pressure
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;
Pörtner, 2002a) and has
been supported by the studies on several invertebrates [Arenicola
marina (Sommer et al.,
1997), Maja squinado
(Frederich and Pörtner,
2000), Sipunculus nudus
(Zielinski and Pörtner,
1996), Littorina littorea
(Sokolova and Pörtner,
2003), Laternula elliptica
(Peck et al., 2002) and the
fish species Rutilus rutilus
(Cocking, 1959), Pachycara
brachycephalum (Mark et al.,
2002) and Gadus morhua
(Sartoris et al., 2003;
Lannig et al., 2004)].
According to this hypothesis, ectothermic animals subjected to acute
temperature change first experience a loss in aerobic scope and, second,
oxygen deficiency beyond low and high critical temperature thresholds
(Tc), ultimately leading to a transition to an anaerobic
mode of energy production and time-limited survival. It was further proposed
that oxygen limitation mechanisms first set in at high levels of organismal
complexity, namely the integrated function of the major convection systems of
the oxygen delivery apparatus.
Accordingly, previous studies have identified both ventilatory and
circulatory (where present) systems in invertebrates to be limiting for oxygen
transport during acute temperature change
(Zielinski and Pörtner,
1996; Frederich and
Pörtner, 2000), while circulatory insufficiency was suggested
to be the first limiting process in fish species acutely subjected to high and
low temperature extremes (Heath and
Hughes, 1973; Mark et al.,
2002; Lannig et al.,
2004).
The present study, working with the cuttlefish Sepia officinalis,
will focus on an animal model that, although being an invertebrate, is
characterised by several vertebrate-like features. Namely, sophisticated
behaviours, a closed, high-pressure blood convection system with low blood
volume and a highly efficient counter-current gill gas exchange system similar
to that of fish (Wells and Wells,
1982; Wells and Wells,
1991). These features make the cuttlefish an ideal candidate to
test the proposed universal character of thermal tolerance limitation
mechanisms (Pörtner,
2002a).
An obvious first step is the evaluation of whether or not cuttlefish will
display oxygen-limited thermal tolerance upon acute exposure to low and high
temperature extremes, as evidenced by transition to an anaerobic mode of
energy production at rest. Advent of mitochondrial anaerobiosis in highly
aerobic liver tissue determined high Tc in fish
(Pachycara brachycephalum and Zoarces viviparus), while the
onset of anaerobic metabolism in white muscle only occurred shortly before
death (van Dijk et al., 1999).
Choice of tissues therefore seems to be crucial when investigating
Tcs. Continuously working, aerobic organs with a
permanently high oxygen demand are the first to suffer from oxygen deficiency
and functional failure. Accordingly, we decided to monitor cephalopod mantle
muscle energy status.
Cephalopod mantle tissue is permanently active and is involved in
ventilatory work but also in jet-propelled locomotion: as a result, the mantle
muscle evolved as a complex organ that contains thin outer layers of aerobic
circular muscles, which aid during slow swimming contractions, and thick
layers of anaerobic muscle fibres in the central part of the mantle muscle
organ, which produce the high-pressure amplitude contractions during fast
swimming (Bone et al., 1994a;
Bone et al., 1994b;
Bartol, 2001). The inner and
outer layers of circular fibres possess high densities of mitochondria
(Bone et al., 1981;
Mommsen et al., 1981), leading
to enhanced baseline oxygen demand. Approximately 30% of the mantle volume
consists of radial muscle fibres that have a key role in ventilation
(Milligan et al., 1997). By
contracting, these radial muscle fibres decrease mantle muscle organ diameter
to aid in refilling the mantle cavity with fresh seawater during each
ventilation cycle. Funnel collar flap movements in combination with a passive
relaxation of radial fibres aid during exhalation of the respiratory water.
Contractions of circular muscle fibres are not involved in exhalation
(Bone et al., 1994a).
Due to their permanent workload, muscle tissues active in ventilation are
probably a good indicator tissue for the determination of
Tcs. Other organs/tissues that are less vital during
short-term stresses (i.e. digestive system, reproductive system) may even be
temporarily shut down while essential fuels (nutrients, oxygen) are
reallocated towards the organs that are maintained in continuous operation.
Examples for such selective energy allocation towards certain organs and
metabolic depression within other organs/tissues during acute environmental
stressors are manifold in the animal kingdom [e.g. the mammalian diving
response (Hochachka, 2000)
associated with cellular metabolic depression (e.g.
Buck et al., 1993)]. If,
during thermal stress, anaerobic metabolism is needed to fuel ventilatory
muscle contractions, obviously time-limited survival has set in and critical
temperatures are being reached.
Using in vivo 31P NMR spectroscopy, we had a technique
available to continuously monitor concentrations of mantle muscle
intracellular high-energy phosphate compounds and intracellular pH
(pHi) in unrestrained animals. Net utilization of the phosphagen
(phospho-L-arginine, PLA) to fuel muscle contractions is a sign of
the start of anaerobiosis in molluscs. We could simultaneously monitor in
vivo performance of the very muscle fibres observed with the NMR setup by
recording mantle cavity pressure oscillations. Such pressure oscillations in
the mantle cavity are a consequence of rhythmic action of the mantle muscle
organ's ventilatory and locomotory muscles
(Bone et al., 1994a;
Bone et al., 1994b).
The complexity of mantle muscle structure and the complexity of various ventilatory and locomotory functions may be confounding factors in the analysis. It was thus necessary to first learn more about the various mantle cavity pressure patterns present and their influence on muscle tissue energy status under control conditions, to later use the acquired knowledge to distinguish between (putative) effects of high-pressure mantle contractions related to spontaneous exercise and those of ventilatory pressure cycles on tissue energy status at thermal extremes.
We hypothesized that at both high and low temperatures, mantle muscle metabolism would need to switch to anaerobic metabolism during resting conditions to sustain ventilatory activity. This report therefore concentrates on muscle energy status and the effects of ventilatory activity on mantle metabolism. It will, at the same time, address the possibly interfering effects of spontaneous, exercise-related high-pressure circular muscle contractions on muscle energy status. In a companion study (F.M., C.B. and H.-O.P., manuscript submitted for publication), we analyse how the pressure patterns generated during resting ventilation relate to oxygen extraction from the ventilatory stream, metabolic rate and the costs of ventilatory movements.
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, and
water pH between 8.0 and 8.2. All animals were raised in the same 3
m3 volume tank. Five animals (104.2±7.4 g wet mass, mean
± s.d.) were used for experimentation.
Experimental protocol
Experimental animals were starved for 24 h and then transferred to the
experimental set-up. Surgery was conducted on the first day, followed by an
overnight acclimatization period within the experimental chamber (control 1).
In vivo 31P NMR spectra showed that anaesthesia during
surgery resulted in a transient accumulation of inorganic phosphate
(Pi), which could be fully reversed within 4–6 h of recovery
under control conditions. On the second day, animals were cooled from control
temperature (15°C) to a lower critical temperature, then warmed to and
kept at control temperature overnight (control 2), after which they were
finally warmed until an upper critical temperature was reached on the third
day. Temperature was changed in a stepwise procedure at an average rate of 1
deg. h–1. Specifically, a 3°C temperature change was
accomplished during the first hour of a 3 h period, while temperature was kept
constant for in vivo 31P NMR spectroscopy and mantle
cavity pressure measurements during the two subsequent hours. Assay
temperatures were 14°C/11°C/8°C on the second day, and
17°C/20°C/23°C/26°C on the third experimental day.
Temperatures were changed further at a rate of 1 deg. h–1 if
critical temperatures were not reached within the outlined temperature window.
As the accumulation of Pi due to phosphagen utilization indicates
limited energy production by aerobic metabolism, the appearance of significant
Pi peaks in in vivo 31P NMR spectra and their
persistence over an extended time period (>60 min) defined critical
temperatures.
In vivo 31P NMR spectroscopy and mantle pressure oscillations
To implant a catheter for ventilatory monitoring, animals were
anaesthetized with a 0.4 mol l–1 MgCl2 solution
that was mixed 1:1 with seawater (Messenger, 1985) at 15°C for 3–3.5
min, then placed (ventral side up) on a wet leather cloth to prevent skin
injuries. During surgery, animals were perfused with aerated seawater (with
0.04 mol l–1 MgCl2) through the funnel aperture. A
PE cannula, required to record postbranchial pressure, was connected to a
23-gauge hypodermic needle, led through the entire mantle cavity and then fed
through the posterior ventro-lateral section of the mantle muscle. Cannulae
(Portex PE tubing, i.d. 0.58 mm, o.d. 0.96 mm, flared at the opening) were
held in place by two 4 mm-diameter plastic washers on the inside and outside,
embracing the mantle muscle in a sandwich-like fashion. PE tubes were
connected to MLT-0699 disposable pressure transducers, signals were amplified
with a ML-110 bridge amplifier and were further fed into a PowerLab/8SP data
acquisition system (AD Instruments GmbH, Spechbach, Germany). Pressure
transducers were calibrated daily.
Following surgery, animals were placed in a Perspex perfusion chamber
analogous to the one used by Mark et al.
(Mark et al., 2002) for
eelpout (Fig. 1A). Plastic
sliders within the chamber could be adjusted to the animals' dimensions and
used to restrict the space available for roaming activity. The chamber was
connected to a closed recirculation seawater system and placed within the
magnet as described by Bock et al. (Bock et
al., 2002). Water quality was maintained with a protein skimmer
(Aqua Care, Herten, Germany) and a nitrification filter (Eheim Professional 2;
Eheim, Deizisau, Germany). Water quality was monitored daily, and parameters
were kept within the limits indicated above.
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10–20% by tissues from the organ sac and coelomic fluid.
As Sepia officinalis mantle muscle tissue is characterized by high
phosphagen (PLA) concentration [34 µmol g–1 wet mass
(Storey and Storey, 1979)], it
is quite likely that the in vivo 31P NMR spectra almost
exclusively represent the adenylate pool and pHi of the mantle
musculature.
In vivo 31P NMR spectra [sweep width, 5000 Hz; flip
angle, 45° (pulse shape bp 32; pulse length 200 µs); repetition time
(TR), 1 s; scans, 256; duration, 3 min 40 s] were acquired every 21.3 min to
measure pHi, and changes in pHi were represented by the
position of the Pi signal relative to the position of the PLA
signal. pHi was calculated using the PLA vs Pi
shift equation obtained by Doumen and Ellington
(Doumen and Ellington, 1992),
using a pKa value determined by Pörtner
(Pörtner, 1990) for an
ionic strength of I=0.16. pKa values were adjusted
according to temperature (Kost,
1990). 31P NMR spectra were processed automatically
using TopSpin V1.0 software (BrukerBioSpin MRI GmbH, Ettlingen, Germany) and a
macro (written by R.-M. Wittig, AWI) to finally yield integrals of all major
peaks within the spectrum (Bock et al.,
2001), as these correlate with the amount of substance within the
detection volume (= sensitive volume) of the 31P NMR coil
(Fig. 1B). Flow-weighted images
to examine blood flow in major blood vessels were also generated directly
before and after the collection of 31P NMR spectra but will be
treated separately. Concentrations of metabolites (ATP, PLA, Pi)
were expressed as percentages of the total 31P signal intensities.
This was found necessary, as animals were free to move to some extent in the
chamber both vertically and horizontally (for a maximum of 5 mm in either
direction, to assure unrestrained ventilatory movements), thus altering
overall in vivo 31P NMR signal intensities:
 | (1) |
-ATP]+[ß-ATP]+[
-ATP]+[Pi] is the sum of
the five major 31P NMR peak integrals that constituted >98% of
the overall 31P signal (see Fig
5A). As a precondition for such an approach, it is necessary that
no major phosphate export from the mantle muscle takes place. Finke et al.
could demonstrate that the sum of adenylates and inorganic phosphate (ATP,
ADP, AMP, PLA, Pi) in squid mantle muscle (Lolliguncula
brevis) was similar in control and exercised animals, as was the sum of
all arginine-containing metabolites [PLA, octopine (Oct),
L-arginine (Arg)] (Finke et
al., 1996). Storey and Storey also found the sum of
arginine-containing metabolites to be relatively stable in cuttlefish mantle
muscle following hypoxia and exhaustive exercise (decreases of less than 10%,
minor releases of Oct in the bloodstream), but they did not determine
inorganic phosphate concentrations (Storey
and Storey, 1979). Still, both studies suggest that anaerobic
metabolites mostly remain in the mantle muscle organ in cephalopods, thus
giving validity to our approach. For better visualisation, percentages of
concentrations were transformed into molar quantities, assigning [PLA] control
values found in cuttlefish mantle muscle
(Storey and Storey, 1979)
([PLA]=33.6 µmol g–1 wet mass) to [PLA] controls in our
study (see Table 1) and
evaluating the other metabolite concentrations accordingly. Free energy change
of ATP hydrolysis (dG/d, kJ mol–1) was estimated from
NMR visible metabolites as described by Pörtner et al.
(Pörtner et al., 1996),
with apparent equilibrium constants of arginine kinase and myokinase adjusted
to changing temperatures. The identity of enthalpies of the arginine and
creatine kinase reactions was confirmed by the analysis of the
temperature-dependent equilibrium constant of arginine kinase under cellular
conditions simulated according to Pörtner et al.
(Pörtner, 1990;
Pörtner et al., 1990).
This analysis yielded a standard apparent enthalpy value (
H at pH 7.0
and 1 mmol l–1 free Mg2+) for
KappAK of –11.87 kJ mol–1 (H.-O.P.,
unpublished data), which is close to the one (–11.93 kJ
mol–1) determined by Teague and Dobson for creatine kinase
(Teague and Dobson, 1992).
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Concentrations of Arg and Oct were estimated using published values
(Storey and Storey, 1979) and
assuming that a decrease in 1 µmol g–1 wet mass [PLA]
results in a concomitant 0.67 µmol g–1 wet mass increase
in [Arg] and a 0.33 µmol g–1 wet mass increase in [Oct]
(Storey and Storey, 1979) (as
witnessed during moderate and severe hypoxia and during exercise).
Intracellular free [Mg2+] was estimated from 31P NMR
spectra as described by Doumen and Ellington
(Doumen and Ellington,
1992).
Mantle cavity pressure analysis
Pressure oscillations in the cephalopod mantle cavity are generated to
create both a ventilatory water stream past the gills (mainly by concerted
action of the collar flap muscles of the funnel apparatus and radial mantle
muscles) and a jet stream to elicit swimming and escape movements (mainly by
action of circular and radial mantle muscles). While the former is associated
with low-pressure amplitudes, the latter can cause amplitudes of up to 25 kPa
(Wells and Wells, 1991).
According to Bone et al., slow swimming in cuttlefish starts with pressure
amplitudes of 0.1–1 kPa, while maximum ventilation pressure amplitudes
(at rest) are 0.15 kPa (Bone et al., 1994). For an analysis of the interfering
influence of spontaneous activity on temperature-dependent muscle energetics,
we set a pressure threshold of 0.2 kPa as the starting point for
non-ventilation-related mantle pressure generation. All such pressure
oscillations will be termed `swimming jets' (SJ) hereafter. For all five
animals, frequencies of SJs of >0.2 kPa amplitude were analysed at all
temperatures. In addition, mean mantle pressure amplitudes of these SJs
(MMPASJ) were recorded (and grouped into 12 amplitude classes, i.e.
0.2–1 kPa, 1–2 kPa, [...], 11–12 kPa).
3523 randomly chosen SJs from all temperatures and animals were used to
develop a relationship of SJ duration vs measurement temperature to
calculate the fraction of experimental time spent with non-ventilation
pressure generation. Also, the relationship of swimming jet amplitude
(MMPASJ) vs swimming jet mean pressure (MMPSJ =
mean pressure of SJ peaks) was determined to convert MMPASJ to
MMPSJ at all temperatures. Routine ventilation pressures
(MMProut) were analysed in similar ways. Thus, we could calculate
total mean mantle pressure:
 | (2) |
t1 is the time interval (min
h–1) spent with swimming jets, and
t2 is the time interval (min h–1)
spent with routine ventilation.
Spontaneous exercise impacts on mantle metabolism
Mantle contractions associated with high-amplitude pressure pulses were
present at all temperatures. The major challenge in the present study was thus
to distinguish between the effects of spontaneous exercise and the effects of
routine ventilatory mantle muscle activity (related to ventilation only) on
muscle metabolic status. An extensive base of control 31P NMR
spectra at 15°C was available to investigate activity patterns and
patterns of metabolite change in the mantle organ.
First, consecutive in vivo 31P NMR spectra were
analysed for changes in [PLA] and [ATP] parallel to changes in
[Pi]:
 | (3) |
[Met] is the concentration change of a given metabolite,
[Met]n+1 is the concentration of the given metabolite obtained with
spectrum n+1, and [Met]n is the concentration of
metabolite obtained with spectrum n. Changes in pHi were
calculated in a similar fashion. A total of 282 intervals from all five
animals was used for such comparisons. This was done to elaborate patterns of
correlated concentration changes between the respective metabolites and
pHi and to investigate the degree to which phosphagen resources are
commonly used under control conditions.
As a second step, an attempt was made to correlate metabolic changes
observed within the mantle muscle with non-ventilatory muscle contractions (of
pressure amplitudes >0.2 kPa). It is known from previous work that such
mantle muscle contractions are fuelled in part by phosphagen breakdown, as
aerobic metabolism cannot provide sufficient amounts of ATP to match demand at
very high ATP fluxes (e.g. Pörtner et
al., 1993; Finke et al.,
1996). For this, in vivo 31P NMR spectra were
scanned for relative increases in [Pi] (which is equivalent to a
net phosphagen breakdown). A total of 30 intervals from all five animals was
randomly selected, and all associated mantle contractions within each interval
were analysed. To find a (putative) causal relationship between spontaneous
exercise and metabolite changes in the mantle organ, it was necessary to
identify those suitable time intervals and pressure amplitudes that actually
have an effect on muscle phosphagen stores. In an exercise study on squid
(Illex illecebrosus), it could be demonstrated that mantle phosphagen
levels returned close to control levels within 10 min after fatiguing
exercise, where PLA had decreased by –22.5 µmol g–1
wet mass from an initial concentration of >30 µmol g–1
wet mass (Pörtner et al.,
1993). Thus, in our case, a major spontaneous exercise event,
occurring 20 min prior to 31P NMR spectrum acquisition, might not
be reflected in the latter due to a putative rapid recovery phase of PLA
stores.
Consequently, intervals (21 min 20 s between the acquisition of two 31P NMR spectra plus the acquisition time of the second spectrum; thus, a total of 25 min) were divided into 11 two-minute segments and one 3 min segment (s1–s12; see Fig. 1c). Both the frequency and amplitude of SJs greater than 0.2 kPa were determined for each segment. Pressure amplitudes were grouped into classes with the following class means: 0.6 kPa (=0.2–1 kPa), 1.5 kPa (=1–2 kPa) [...] up to 11.5 kPa (=11–12 kPa) and a jet index (JI, in kPa segment–1) obtained for each segment by adding the products of class amplitude means and jet numbers within the respective amplitude classes. For example, five jets between 2 and 3 kPa and three jets between 5 and 6 kPa within one segment yield a JI of (5x2.5)+(3x5.5)=29 kPa. 12 variables were created by adding JIs from segments within intervals `a' to `l' (see Fig. 1C). Furthermore, each of these variables was split up by varying the pressure threshold used for JI calculation (only jets >0.2 kPa or >1 kPa or >2 kPa, [>...], or >11 kPa used for calculations). Thus, for our example above, a JI calculated from jets >3 kPa would result in 3x5.5=16.5 kPa. This variation in duration of the interval and in the pressure amplitudes taken for JI calculation created a total of 12x12=144 different variables that could be tested in an iterative linear regression analysis to explain a maximum of the variability observed in [Pi] changes as obtained by successive in vivo 31P NMR spectra. In a similar fashion, maximum jet density (JD) of Sjs >0.2, >1 [>...], >11 kPa within segments of intervals a to l (Fig. 1C) was calculated (again, 144 possible variables) as a second factor that might influence [Pi] changes in mantle muscle tissue. For example, JD was 15 for jets >1 kPa if we considered interval d (see Fig. 1C), and the density of jets >1 kPa (in jets 2 min–1) in segments s9, s10, s11 and s12 were 9, 12, 15 and 3.
Statistics
Simple linear, exponential and sigmoidal regression analyses were performed
using SigmaPlot 8.0 (SSPS Inc., Point Richmond, CA, USA). Multiple linear
regression analysis was also performed using Statistica (Statsoft, Tulsa, OK,
USA), as were all other statistics. Comparisons between values grouped
according to temperature or animal were conducted using one-factorial analysis
of variance (ANOVA) and subsequent post-hoc testing with
Student–Newman–Keuls. t-tests were used to compare SJ
frequencies obtained during the day with those obtained at night.
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57 min of each control hour.
The remaining time was filled with spontaneous high-pressure mantle
contractions of >0.2 kPa. Occurrence of such SJs was observed in all
animals, with pressure amplitudes distributed as shown in
Fig. 2A. It appeared that
roughly 73% of all non-routine ventilation pressure cycles of >0.2 kPa were
characterized by an amplitude lower than 2 kPa. Only 0.2% of all pressure
cycles of >0.2 kPa showed pressure amplitudes of 11–12 kPa.
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In vivo 31P NMR spectroscopy
Concentrations of in vivo 31P NMR visible metabolites
and pHi were found to be as variable as mantle pressure recordings.
Fig. 3 gives an example: data
are shown from 14 subsequent control 31P NMR spectra taken from
animal 4 between 23:22 h and 04:47 h during the second control phase of the
experiment (at constant 15°C). While muscle ATP concentrations remained
constant over the whole period, [PLA] decreased from 33.4 to 23.8 µmol
g–1 wet mass ([PLA]=–29%) between 50 and 100
min. This was mirrored by a concomitant increase in [Pi] from 0.8
to 11.6 µmol g–1 wet mass. Recovery of the phosphagen pool
in combination with a decrease in [Pi] started at t=100
min and lasted for 150–200 min. pHi values followed a similar
pattern when compared with [PLA] but were delayed by 50 min. During the
first 25 min of phosphagen utilization, pHi increased from 7.43 to
7.49. Thereafter, pHi values decreased continuously to a minimum
value of 7.32 at t=150 min. A value close to control was reached
again after 325 min. The observed Pi accumulation of 11.6 µmol
g–1 wet mass was the highest observed in any of the five
animals under control conditions (Table
1).
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[PLA]
changed linearly with [Pi], with a negative slope close to
one (Fig. 4B), while ATP
concentrations remained stable at 7–9 µmol g–1
wet mass (ANOVA, F7,272=1.83, P<0.07;
Fig. 4D). pHi
changed linearly with increasing [Pi] between less than
–3 and 3 µmol g–1 wet mass
(Fig. 4A) but deviated
significantly from this relationship once [Pi] accumulated above 3 µmol
g–1 wet mass. pHi fell back to 7.44 (control pH
was 7.45; see Table 1) at those
higher inorganic phosphate accumulations. From 282 intervals analysed for
Fig. 4, only nine intervals
(3%) were characterized by such a high increase in [Pi].
Correspondingly, intracellular [H+] changed linearly in the
respective range of [Pi] values
(Fig. 4C). Increases in
[Pi] of >3 µmol g–1 wet mass did not result
in a further proton buffering but rather led to a net increase in
[H+] by 3 nmol l–1.
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It seemed obvious that fluctuations observed in the concentrations of
high-energy phosphates and in pHi should be related to the
frequency of high-amplitude swimming jets. In a first iterative step of
univariate linear regression analysis, 36 different jet index (JI) variables
were identified that could explain significant fractions of variability in
[Pi]. These differed in the length of the time interval as
well as in the pressure threshold chosen for JI calculation. Twelve of these
are shown in Table 2. Intervals
of 13–15 min for JI calculation (corresponding to intervals f and g in
Fig. 1C) resulted in
regressions with the highest r2. Omitting mantle pressure
cycles below 2 kPa also resulted in better regressions, while JIs exclusively
calculated from SJs greater than 3 kPa failed to explain a similar amount of
variability in [Pi]
(Table 2). The best single
variable identified was a JI constructed from mantle pressure cycles greater
than 2 kPa during the last 15 min of each 25 min interval:
 | (4) |
[Pi] in µmol g–1 wet mass and JI
in kPa were calculated from SJs >2kPa within interval g (see
Fig. 1C). This regression could
explain 84% of [Pi] variability. Accordingly, 20 jets of an
amplitude between 2 and 3 kPa (JI=50) would result in a [Pi]
of roughly 2 µmol g–1 wet mass, which is equivalent to a
6% decline in mantle muscle phosphagen reserves. Inclusion of a second
variable, jet density (JD), into the model significantly enhanced the fraction
of explainable [Pi] variability to 89%:
 | (5) |
[Pi] in µmol g–1 wet mass and JI
calculated from SJs >2 kPa within interval g, and JD calculated from jets
>5 kPa within interval g (see Fig.
1C).
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As high spontaneous SJs could be related to the accumulation of inorganic phosphate observed in mantle muscle, true control values for [ATP], [PLA] and pHi were calculated only from spectra with [Pi]<1.5 µmol g–1 wet mass, as this was the maximum [Pi] found during prolonged routine ventilation sequences [Table 1; only sporadic (<10) jets of pressure amplitudes of <1 kPa for at least 30 min]. Mean pHi values were comparable between animals, except for animal 3, which showed a significantly higher mean muscle pHi. Variability in [H+i] was low under control conditions, with a relative standard deviation (CV) of about ±15% at a mean concentration of 36.1 nmol l–1. ATP concentrations were comparable between animals 2–5, while animal 1 had a significantly lower muscle [ATP] than animals 2, 4 and 5. Although there were significant differences found in [PLA] between animals, it has to be considered that all mean concentrations were found within a range of –1.6 to +2.8% of the mean value of 33.6 µmol g–1 wet mass. The ratio of [PLA] over [ATP] proved to be relatively stable between animals (4.0–4.6), with ratios being comparable from animals 2–5 and only animal 1 being characterised by a significantly higher ratio (Table 1).
Acute temperature change
Mantle pressure
Bouts of spontaneous mantle muscle activity (SJs >0.2 kPa) could be
observed at all temperatures (Fig.
2B), with no significant differences between temperatures
(F6,28=1.5, P<0.22). A trend towards a higher
frequency of spontaneous swimming jets is evident with rising temperature
between 8 and 23°C. Examination of SJs at all temperatures yielded a
linear regression (r2=0.89, N=3523 SJs) for the
duration of individual SJs in relation to temperature:
 | (6) |
 | (7) |
1 and 3% of the total experimental time spent performing
high-pressure swimming jets. With a mean SJ amplitude of 1.7 kPa (see above),
the impact of relatively few high pressure cycles constituted a significant
fraction of total mantle pressure (MMPtot); 20–36% of
MMPtot were produced by high-pressure SJs
(Fig. 2C) in the investigated
temperature range. Owing to the high variability in SJ frequency
(Fig. 2B), these differences
were not significant, although a trend towards reduced pressure generation by
SJs is evident between 23 and 26°C.
In vivo 31P NMR spectroscopy
In vivo 31P NMR spectra
(Fig. 5A) revealed that despite
the changes in metabolic rate and ventilatory power output observed over the
entire temperature range (F.M., C.B. and H.-O.P., manuscript submitted for
publication), muscle [ATP] remained constant (ANOVA;
F6,28=0.13, P<0.99) at around 8 µmol
g–1 wet mass (Fig.
5B). The situation was different for the other metabolites.
Although we could not detect significant differences in muscle [PLA] (ANOVA;
F4,20=1.48, P<0.25) and [Pi]
(ANOVA; F4,20=1.68, P<0.20;
Fig. 5B,C) between 11 and
23°C, there was a trend towards decreasing [PLA] values between 17 and
23°C, which was mirrored by increasing [Pi] values in this
respective interval. Intracellular pH decreased with rising temperature in a
linear fashion (r2=0.87, F1,5=33.4,
P<0.007):
 | (8) |
Therefore, grouping animals into pre-Pi accumulation (group A,
means of in vivo 31P NMR spectra 60 min prior to
Pi accumulation) and Pi accumulation [group B, means of
in vivo 31P NMR spectra during Pi accumulation
(60 min duration), with the start of accumulation defined as at least two
successive spectra with a [Pi] of >1.5 µmol
g–1 wet mass] enabled us to improve the resolution of
metabolic patterns at both ends of the temperature window
(Table 3). In the cold, phase B
mean temperature was 7°C. From phase A to B, [PLA] had decreased to 30.9
µmol g–1 wet mass while [ATP] remained constant.
pHi was also comparable between phases A and B. At the warm end of
the temperature spectrum, the picture was similar. At a mean temperature of
26.8°C, [PLA] decreased to 30.5 µmol g–1 wet mass,
while [ATP] and pHi did not change from phase A to B. Looking at the last
31P NMR spectra taken at each temperature (Bextreme in
Table 3) illustrates that at
high temperatures, pHi is significantly decreased compared with
phase A. A trend towards decreased pHi values is also visible at
the low-temperature Bextreme, although it is not (yet) significant.
Free-energy changes of ATP hydrolysis |dG/d
|
decreased by 3.3 kJ mol–1 at the low temperature
Bextreme and by 5.1 kJ mol–1 at the high
Bextreme. Mean values did not drop below 50 kJ
mol–1, if one assumes that phosphagen utilization is
distributed evenly among all muscle fibre types [radial and circular fibres
(r+c) in Table 3] present in
the sensitive volume of the 31P NMR coil.
|
Having established that changes in [PLA] and [Pi] can be caused by SJs
under control conditions, it was necessary to investigate whether changes at
extreme temperatures were also due to locomotory exercise or due to routine
ventilation activity. Regression models were tested to establish a link
between SJs and inorganic phosphate accumulation in the mantle muscle organ at
extreme temperatures (phase B). Testing the same set of variables as for the
control situation (see above), a significant regression model
(r2=0.49, F2,29=14.3,
P<0.001) could be established for the low extreme temperature
situation
 | (9) |
[Pi] in µmol g–1 wet mass, JI
calculated within interval l (Fig.
1C) from jets of >2kPa, and JD calculated in interval l from
jets of >5 kPa. While this significant model could explain roughly half of
the encountered variability in [Pi] at low temperatures, we
could not identify a single significant regression model at high
temperatures.
In a second step, we looked at [Pi] variability during 25 min
intervals with no high-pressure jets of >0.2 kPa present. At both low and
high extreme temperatures (phase B), a mean (± s.d.) accumulation of
[Pi] could be found (high temperature,
[Pi]=1.15±0.3 µmol g–1 wet mass,
N=8 intervals; low temperature,
[Pi]=0.53±0.43 µmol g–1 wet mass,
N=9 intervals), while during (randomly) chosen control intervals with
no high-pressure cycles present, no inorganic phosphate increases could be
found at all ([Pi]=–1.65±1.9 µmol
g–1 wet mass, N=8 intervals). Rather, negative
[Pi] values dominated under control conditions, as periods
without any SJs at all were found predominantly during recovery times from
(spontaneous) exercise.
In summary, we have found significant increases in [Pi] (= phosphagen use) in the mantle muscle organ at both high and low temperature extremes in all five animals investigated. At a control temperature of 15°C, [Pi] variability could almost completely (89%) be explained by the occurrence of high-pressure SJs. At high extreme temperatures, SJs could not be related to the observed increases in [Pi]. Inorganic phosphate accumulation was also observed during intervals without any SJs present, and thus was likely to be caused by elevated levels of routine ventilation alone. At low extreme temperatures, apparently both processes (routine ventilation energy demands and spontaneous exercise energy demands) contributed to [Pi] accumulation.
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Gaede, 1980;
Finke et al., 1996;
Pörtner et al., 1991;
Pörtner et al., 1993;
Pörtner et al., 1996;
Zielinski, 1999; Zielinski et
al., 2000).
Control conditions
During control conditions, we had the unique possibility to study the
effects of facultative mantle muscle exercise on the animals' intracellular
energy status. We could show that animals displayed a higher activity (as
evidenced by the occurrence of SJs with an amplitude of >0.2 kPa) during
1–3% of the total time. Similar levels of activity were recently found
for a tropical cuttlefish in its natural habitat [S. apama was found
to be active (as estimated from elevated mantle pressures) for approximately
3% of the day (Aitken et al.,
2005)].
Control mantle muscle organ adenylate levels obtained in our study, as
witnessed by the ratio of [PLA] over [ATP], are comparable with other studies
that analysed metabolite concentrations invasively. Our ratio of 4.2 (0.4
s.d.) compares well with a value of 3.9 for S. officinalis
(Storey and Storey, 1979), 4.4
for the squid Lolliguncula brevis
(Finke et al., 1996) or 3.5
for the squid Loligo pealei
(Pörtner et al., 1993).
We were able to clearly demonstrate that PLA stores were being utilized during
high-pressure SJs and could establish a quantitative relationship between
exercise levels and decreases in [PLA] or increases in [Pi],
respectively. The best relationship obtained suggests that SJs with a MMPA of
>2 kPa cannot be fuelled entirely by means of aerobic energy production,
similar to findings in the squid L. brevis
(Finke et al., 1996). This
fits the picture that slow-swimming (`cruising') mantle pressure amplitudes in
S. officinalis do not seem to exceed 2 kPa (fig. 1b in
Wells and Wells, 1991). Bone
et al. could demonstrate that during slow swimming at pressure amplitudes
between 0.1 and 1.0 kPa, (aerobic) circular fibres become involved in the
pressure generating process (Bone et al.,
1994a). Thus, it is quite likely that slow swimming at <2 kPa
can be fuelled entirely by metabolism of the thin aerobic muscle layers of the
mantle periphery. More extreme exercise with pressure amplitudes of >2 kPa
progressively involves central anaerobic fibres
(Bone et al., 1994b) that
exploit their phosphagen reserves in order to propel the animal at higher
speeds. Gradual involvement of central (mainly) anaerobic fibres (rather than
an all-or-nothing transition) to support aerobic fibres at an increasing
workload has been recently demonstrated for swimming squid (L.
brevis) (Bartol,
2001).
The fact that we found a linear relationship between jet indices
constructed from mantle pressure amplitudes multiplied with jetting
frequencies and phosphagen use is not surprising, as pressure production
should be a direct function of circular mantle muscle fibre force generation.
Muscle fibre force has been found to depend on the number of crossbridges in
the force generating state per cross-sectional area (e.g.
Wannenburg et al., 1997);
thus, mantle cavity pressure in cephalopods should be directly proportional to
ATP flux rates in working mantle muscle. This corresponds to the results of
Webber and O'Dor, who found correlated changes in mantle pressure integral and
whole-animal rate of oxygen consumption
(
O2) during various levels
of exercise in a squid (I. iIllecebrosus)
(Webber and O'Dor, 1986).
The inclusion of a second variable into the regression model (jet density) significantly enhanced the fraction of explainable variation in inorganic phosphate concentration changes. This implies that high amplitude jets of >5 kPa pose a higher threat to cellular phosphagen reserves when they occur in quick succession, rather than distributed over a longer time interval. This could be due to oxygen depletion or aerobic fibre fatigue at high jet density, resulting in pressure generation exclusively by anaerobic fibres during high jet density, high-pressure time intervals.
It should be emphasized that PLA stores were never used extensively under control conditions (15°C) and during facultative, spontaneous exercise. Mean maximum decreases in [PLA] (= increases in [Pi]; see Table 1) under control conditions amounted to 6.66 µmol g–1 wet mass, which corresponds to roughly 20% of phosphagen reserves, although one animal (replicate 4, Fig. 3) depleted 35% of its phosphagen reserves on one occasion.
Fig. 3 demonstrated that
during initial phosphagen transphosphorylation and Pi accumulation,
pHi can be buffered and remains unchanged. pHi decreased
only during prolonged phosphagen utilization, probably due to glycolysis and
concomitant octopine formation (Storey and
Storey, 1979; Pörtner,
1987; Pörtner,
2002b). Work on in vitro preparations of scallop
(Argopecten irradians) contracting phasic adductor muscles supports
this conclusion (Chih and Ellington,
1985). While, during initial exercise (40 muscle contractions),
proton consumption by phosphagen utilization exceeded proton production by
octopine formation, resulting in a net alkalosis of about 0.09 pH units
([H+]=–16 nmol l–1), further exercise
(40–200 contractions) led to progressively declining pHi
values due to glycolytic proton production outmatching proton consumption by
the phosphagen. Fig. 4 provides
a more quantitative picture and shows clearly that anaerobic metabolism is
seldom employed to the degree that net cellular acidification occurs. Only 3%
of all (randomly chosen) intervals analysed for
Fig. 4 showed a [Pi]
accumulation of >3 µmol g–1 wet mass. Such a degree of
phosphagen utilization goes along with the onset of muscle acidosis,
suggesting that glycolytic proton production outmatches proton buffering by
phosphagen use.
Apparently, cuttlefish avoid intracellular acidification by terminating
exercise in most cases as soon as glycolytic proton production equals
phosphagen proton buffering capacity. Upon removal of inorganic phosphate
during recovery, a glycolytic proton surplus, which cannot be buffered,
results in a slight decrease in pHi
(Fig. 4A,B). Potentially
adverse effects of decreased pHi values on muscle function
(reviewed by Fitts, 1994) are
thus shifted into recovery phases. Absolute changes in intracellular proton
activities are low (in the nmole range;
Fig. 4B), a feature also
observed by Chih and Ellington (Chih and
Ellington, 1985). A recent in vivo 31P NMR
spectroscopy study on forced scallop exercise
(Bailey et al., 2003) confirmed
the metabolic patterns obtained in the older in vitro study on
stimulated adductor muscle preparations.
Acute temperature change
Fig. 5 depicts the changes
in mantle organ metabolite levels with temperature. Despite the dramatic
changes in ventilatory power output over the entire temperature range, [ATP]
is strictly conserved (Fig.
5A), a phenomenon commonly encountered in studies on muscles of
marine ectothermic animals subjected to acute temperature change (i.e.
Mark et al., 2002;
Sartoris et al., 2003;
Zielinski, 1999) and generally
referred to as the `stability paradox'
(Hochachka and Somero, 2002).
[PLA] was also constant between 11 and 23°C.
We could not find any evidence for an alphastat pattern of pHi
regulation (Reeves, 1972; see
Burton, 2002 for a review) in
the investigated temperature range. Typically, changes of around –0.018
pH units deg.–1 are expected to ensure constant levels of
imidazol and protein ionization. For fish species, such a pattern could be
demonstrated in white muscle (Borger et
al., 1998; Van Dijk et al.,
1997; Van Dijk et al.,
1999; Bock et al.,
2001). As for molluscs, an alphastat pattern of pHi
regulation was absent in the stenothermal marine bivalve Limopsis
marionensis (Pörtner et al.,
1999). Despite confounding effects of mantle muscle exercise at
all temperatures, we found a decrease in pHi with temperature by
about –0.006 pH units deg.–1 over the full temperature
range examined. Omitting pHi values at 8 and 26°C, as
phosphagen utilization was observed to start at these temperatures, gives an
even lower rate of change of –0.004 pH units deg.–1.
Between 11 and 17°C [the typical natural temperature range of this
population of cuttlefish in the English Channel
(Boucaud-Camou and Boismery,
1991)], pHi values are nearly identical. The absolute
temperature-dependent changes in pHi are <0.05 units between 11
and 23°C, which is lower than the range of change observed during
facultative exercise at control temperature. Future studies should address the
time dependence of pHi regulation in response to temperature. Our
study focused on short-term temperature effects and may not have allowed
mantle pHi to fully reach new steady-state values after each
thermal challenge. As it stands, the question of temperature-dependent
pHi regulation in cephalopods must remain open.
Patterns of metabolite changes observed at extreme temperatures exactly mirrored those during exercise under control conditions. The start of phosphagen utilization was observed in all five animals at both temperature extremes during phase B. Mean temperatures during this phase were 26.8°C and 7°C.
High Tc
The analysis of mechanisms at the high end of the temperature spectrum
proved to be easier than at the low end. As no relationship between the few
SJs (see Fig. 2B,C) and the use
of the phosphagen could be established and, also, since [Pi]
increases could be found during periods of ventilation at rest, obviously
aerobic metabolic limitation had set in independent of effects of spontaneous
SJ exercise at warm temperatures. Still, the results are difficult to
interpret as mantle muscle is a complex organ that consists of different
muscle fibre types with different functions
(Bone et al., 1981;
Bone et al., 1994a;
Bone et al., 1994b;
Bartol, 2001). Radial muscle
fibres aid in refilling the mantle cavity during ventilation by contracting
and thus enlarging mantle cavity volume. Bone et al. were the first to
demonstrate (Bone et al.,
1994a) that expiration in the cuttlefish under control conditions
(18°C) and at rest (mantle pressure amplitudes of 0.05–0.15 kPa) is
not brought about by contraction of the outer, aerobic layers of circular
fibres but rather by the movements of the collar flaps (muscular funnel
appendages) (see Tompsett,
1939) that expel water rhythmically from the mantle cavity.
Maximum resting ventilation MMPA recorded in our experimental animals were
lower than 0.15 kPa (F.M., C.B. and H.-O.P., submitted), thus we assume that
during our entire experimental series, radial fibres had been the only
constantly working myofilaments within the sensitive volume of our
31P NMR coil (Fig.
1B). Our companion study revealed that ventilation pressures
stagnate at temperatures beyond 26°C.
Fig. 6A shows ventilation
pressure amplitudes (at rest) at temperatures close to the upper
Tc for two experimental animals (the ones with the highest
and the lowest Tcs), while
Fig. 6B gives correlated
increases in [Pi] (circles). It is quite evident from this figure
that declining ventilation pressures and phosphagen use are tightly coupled
(all other experimental animals showed similar patterns). We thus conclude
that an energetic limitation of radial muscle fibres is responsible for the
observed increases in [Pi] and the correlated decreases in
ventilation pressures once phosphagen usage starts. Radial fibres have a
mitochondrial content as low as that of central `anaerobic' circular fibres
(Bone et al., 1981;
Mommsen et al., 1981) and thus
may be especially sensitive to enduring ventilation exercise at high
intensities. Considering that radial fibres constitute about 30% of total
mantle volume in the cuttlefish (Milligan
et al., 1997), and assuming that solely radial fibres deplete
their phosphagen stores as ventilation pressures increase while, on the other
hand, circular fibre energy status remains constant, it is possible to
estimate metabolite changes for the radial fibre compartment. Based on such
considerations, radial fibre |dG/d | for animals
1 and 5 would drop severely as phosphagen use proceeds
(Fig. 6B). Following such a
rationale, metabolite changes were recalculated for phase B (denoted r in
Table 3) for all five animals.
Thus, mean [PLA] reserves would decrease by 75% from 33 to 8.5 µmol
g–1 wet mass, mirrored by an increase in [Pi] in
the same range and a 25% reduction in [ATP]. pHi would be
significantly decreased to 7.19 and |dG/d | would
drop to below 44 kJ mol–1 in the radial fibre compartment of
the mantle organ. These calculations correspond to similar
|dG/d | values for mantle muscle of three species
of squid following fatiguing exercise, which ranged from 42 to 47 kJ
mol–1 (Pörtner et
al., 1996), while values for two species of exercise-fatigued
eelpout (P. brachycephalum, Z. viviparous)
(Hardewig et al., 1998) white
muscle ranged from 46.6 to 48 kJ mol–1. Possibly, reductions
in the free energy of ATP hydrolysis as calculated for cuttlefish radial
muscle could contribute to muscle fibre fatigue in that vital muscle due to
functional impairments of ATPase functions. Kammermeier et al. found a drop in
contractile performance of perfused rat hearts (38°C) below a
|dG/d | of 48 kJ mol–1
(Kammermeier et al., 1982).
They calculated threshold values of |dG/d |
required for proper function of the various ATPases engaged in muscular work
to range from 45 to 53 kJ mol–1
(Kammermeier, 1987;
Kammermeier, 1993). Only
recently, Jansen et al. found maintenance of [Na+i]
homeostasis prevented by |dG/d | values below 50
kJ mol–1 due to a limitation of the sodium pump
(Na+/K+-ATPase) in perfused rat hearts
(Jansen et al., 2003). Less
information on critical |dG/d | values for muscle
function is available for ectothermic animals: Combs and Ellington calculated
an energy requirement of 41 kJ mol–1 for blue mussel
(Mytilus edulis) sarcolemmal Ca2+–ATPase
(Combs and Ellington, 1995). A
minimum |dG/d | value of 46 kJ
mol–1 for the sodium pump of crayfish abdominal muscle was
calculated by the same authors (Combs and
Ellington, 1997), although they found changes in
[Na+i] homeostasis well above 50 kJ
mol–1 already. They speculated that global
|dG/d | might not reflect the
|dG/d | close to the sodium pump.
|
Also, both elevated intracellular proton and inorganic phosphate
concentrations have frequently been connected with muscular fatigue
independent of |dG/d |
(Allen and Westerblad, 2001;
Fitts 1994). Increased
[Pi] is thought to reduce muscle force by reversing the
force-generating Pi release step by mass action
(Hibberd et al., 1985).
According to Debold et al. (rat muscle fibres), the [Pi] dependency
of muscle fatigue is temperature related, with a more pronounced effect of
[Pi] at low temperatures
(Debold et al., 2004). For
ectothermic (marine) animals such studies have, to our knowledge, not been
undertaken.
Judging from the presented results and literature data, it appears that progressive phosphagen usage is indeed causative of the observed stagnation in ventilatory pressure generation at high temperature extremes, although the exact mechanisms still need to be elucidated.
Low Tc
At low temperatures, we could demonstrate that SJs contributed
significantly to increases in [Pi]. 50% of variability in
[Pi] could be attributed to facultative exercise of circular
anaerobic fibres, while the other half remained unexplained. As neither SJ
frequency nor the fraction of MMPSJ in MMPtot increased
at lower temperatures (similar frequencies/pressures were observed between 8
and 11°C; Fig. 2B,C) and no
Pi accumulation occurred at 11°C
(Fig. 5B), a major loss in
aerobic scope for spontaneous activity had evidently occurred towards low
temperature extremes, resulting in the net use of phosphagen stores for
spontaneous activity. The other 50% of variability that could not be explained
by the linear regression is likely due to a limitation in oxygen flux towards
radial muscles, thus limiting their active role in ventilation. It seems that,
at low temperatures, all muscle fibre types might suffer from phosphagen
breakdown. Accordingly, we calculated |dG/d |
values assuming that mantle phosphagen depletion took place more
homogeneously. In Bextreme, |dG/d |
had dropped from 55 to 51 kJ mol–1. This does not exclude the
existence of heterogeneity or even of putative intracellular
|dG/d | gradients. Hubley et al. calculated
distinct intracellular concentration gradients of the vertebrate phosphagen,
creatine phosphate (PCr), and free energy of ATP hydrolysis in working fish
white muscle, depending on the maximum distance from the nearest mitochondrion
(Hubley et al., 1997). Maximum
intracellular |dG/d | gradients of 7 kJ
mol–1 were calculated. Interestingly, the study suggests that
in fish white muscle such |dG/d | gradients will
be less pronounced in animals acclimated to high temperature
