Metabolite diffusion in giant muscle fibers of the spiny lobster Panulirus argus
1 Department of Biological Sciences, University of North Carolina at
Wilmington, 601 South College Road, Wilmington, NC 28403-5915, USA
2 Department of Biological Science, Florida State University, Tallahassee,
FL 32306-4370, USA
* Author for correspondence (e-mail: kinseys{at}uncwil.edu)
Accepted 8 August 2002
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Summary |
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Key words: diffusion, giant muscle fibe, nuclear magnetic resonance, spiny lobster, Panulirus argus, crustacea, muscle
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Introduction |
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Creatine phosphate is a substrate for the enzyme creatine kinase (CK),
which catalyzes the reversible transfer of a phosphoryl group from creatine
phosphate to ADP, forming ATP. The time- and orientation-dependence of
diffusion of creatine phosphate in skeletal muscle is of particular interest,
because radial diffusion of creatine phosphate is the principal mechanism for
the transport of ATP equivalents from mitochondria to the myosin ATPases in
the fiber core (Mainwood and Rakusan,
1982; Meyer et al.,
1984
). Several groups have used pulsed-field gradient nuclear
magnetic resonance (PFG-NMR) to measure non-invasively D of creatine
phosphate in muscle cells, and in situ D typically has values that
are 40-50 % of the bulk diffusion coefficient (D0) of
creatine phosphate in water (Moonen et
al., 1990
; van Gelderen et
al., 1994
; Hubley and
Moerland, 1995
; Hubley et al.,
1995
; Kinsey et al.,
1999
; de Graaf et al.,
2000
). The time dependence of radial (D
)
and axial diffusion (D||) of creatine phosphate in
mammalian skeletal muscle was first measured by Moonen et al.
(1990
) and van Gelderen et al.
(1994
). These authors
concluded that the time-dependent decrease in D
was
a consequence of creatine phosphate being restricted within the cylindrical
cell membrane (the sarcolemma), which defines the boundaries of a muscle
fiber. Our own investigations of diffusion in the homogenous red and white
muscles of fish revealed that the decrease in D
with
time was more likely to be a result of subcellular barriers that occur on a
length scale of a few µm (Kinsey et
al., 1999
). Candidate diffusion barriers on this length scale
include the sarcoplasmic reticulum (SR) and mitochondria. Subsequent
measurements by de Graaf et al.
(2000
) on rat skeletal muscle
were interpreted to indicate that radial diffusion of creatine phosphate was
restricted by undefined, cylindrical shaped structures with a diameter of 22
µm, too small to be accounted for by the sarcolemma. Therefore, while
D
of creatine phosphate has been found to have a
consistent pattern of time dependence in skeletal muscle, the barriers that
induce the observed diffusive anisotropy are unresolved.
In the present study, we have used 31P-PFG-NMR to examine the
time dependence of D|| and D
of arginine phosphate (AP), an invertebrate phosphagen analogous to creatine
phosphate, in giant fibers of spiny lobster abdominal muscle. The fibers are
characterized by their extreme size, which can exceed 500 µm in diameter
and 1 cm in length (Jahromi and Atwood,
1971
), as well as a paucity of mitochondria, which in giant
crustacean fibers are almost exclusively localized to the periphery of the
cell (subsarcolemmal mitochondria; Kent
and Govind, 1981
; Tse et al.,
1983
). The large size of the fibers means that aerobic
post-contractile recovery involves diffusion of arginine phosphate over
hundreds of µm, and barriers to diffusion may ultimately limit this
process. Use of giant fibers in the present study minimizes the effect of
restriction within the cylindrical sarcolemma, allowing an examination of
intracellular barriers. In addition, the lack of intermyofibrillar
mitochondria means that the contractile filaments and SR are the only obvious
anisotropic barriers to diffusion. We also used 1H-PFG-NMR to
examine diffusion of protonated metabolites, which differ from AP in their
molecular mass and chemical characteristics. We tested the hypotheses that (i)
all of the metabolites would have similar anisotropic diffusion, (ii) the time
dependence of radial diffusion would be consistent with hindrance by
µm-scale structural barriers such as the SR, and (iii) diffusion
coefficients collected at long diffusion times (i.e. distance-averaged over
intracellular barriers) would be inversely proportional to molecular mass, as
expected in an isotropic solution.
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Materials and methods |
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NMR procedures
Muscle fibers were prepared as described in Kinsey and Ellington
(1996). Lobsters were placed
on ice for 20 min prior to dissection. The carapace on the dorsal surface of
the abdomen was removed, and bundles of 2-3 giant fibers were isolated from
the deep extensor abdominal muscle. The muscle fibers were tied at either end
with 6-0 surgical silk and suspended in the center of a 1.9 mm i.d. glass
capillary superfusion chamber housed in a NMR probe. The glass capillary
chamber was connected at either end to superfusion lines that were fed through
the bottom of the probe to a pair of peristaltic pumps. The fibers were
continuously superfused at a flow rate of 10 ml min-1 with lobster
saline solution (457 mmol l-1 NaCl, 15 mmol l-1 KCl, 1.8
mmol l-1 MgCl2 and 2.5 mmol l-1
MgSO4, buffered with 10 mmol l-1 Hepes and 10 mmol
l-1 MES, pH 7.5). The temperature of the superfused tissue was
maintained at 20°C using a refrigerated recirculating water bath. The NMR
probe had a horizontal, five-turn solenoidal radiofrequency coil (2.6 mm i.d.)
tunable to 1H and 31P, which surrounded the superfusion
chamber.
Experiments were performed on a Bruker 600 MHz DMX wide-bore spectrometer
with micro-imaging gradient coils (960 mT m-1 maximum gradient
strength) located at the National High Magnetic Field Laboratory in
Tallahassee, FL, USA. Data were acquired and processed using a Silicon
Graphics Indigo workstation and Bruker X-Win NMR software. The probe was
oriented so that the long axis of the superfusion chamber, and hence the long
axis of the muscle fibers, was oriented parallel to the y-axis
imaging gradient. Therefore, both the x and the z gradients
was oriented perpendicular to the muscle fibers. This careful alignment of the
probe and the homogeneous orientation of the giant fibers within the probe
ensured that our measurements of axial diffusion (along the y-axis)
and radial diffusion (along the x- or z-axis) contained
essentially no contamination from fibers oriented at angles off-axis. To
measure D of phosphorylated compounds, 31P-spectra were
acquired at 242 MHz using a bipolar gradient pulse-stimulated echo sequence,
with a longitudinal eddy current delay (BPP-LED;
Cotts et al., 1989;
Wu et al., 1995
;
Gibbs, 1997
) as previously
applied to fish muscle fibers (Kinsey et
al., 1999
). This sequence minimizes both eddy-current-induced
artifacts and the background gradients generated from susceptibility contrast
in heterogenous samples (Fordham et al.,
1996
). To measure D of protonated compounds,
1H-spectra were collected at 600 MHz using the BPP-LED pulse
sequence as above modified to include the CHESS (CHEmical
Shift Selective) water suppression sequences
(Moonen et al., 1990
) inserted
during the relaxation delay and during the mixing time
(Fig. 1). The CHESS sequences
are as described in Kinsey and Ellington
(1996
). In an isotropic
solution, the NMR peak amplitude, A, is described by:
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|
To measure D, a series of spectra were collected with different
values for G. In all experiments, was 2 ms,
was
250µs, the recycle time was 2s, the eddy current delay was 20 ms and
spectra were collected in either 256 or 512 scans. Gradient strengths ranged
from 100 to 960 mT m-1, and the current pulses through the gradient
coils were pre-emphasized so that the magnetic field gradient produced was
square, as shown in Fig. 1A. At
least four data points with different values of G were collected for
each diffusion measurement. Diffusion was measured at seven diffusion times,
which ranged from 20 to 300 ms. The diffusion time was adjusted by changing
values, while all other time variables were constant. The direction in
which diffusion was measured was alternated for each diffusion time (e.g.
D
was measured at 20 ms followed by measurement of
D|| at 20 ms). Several determinations of D
could be made for each fiber preparation.
Analysis
The time dependence of intracellular diffusion coefficients is influenced
by fixed structures in the cytoplasm, and the highly organized structure of
muscle would be expected to have a different effect on
D and D||. ATP equivalents
must diffuse from subsarcolemmal mitochondria to the fiber core, so barriers
to radial diffusion directly impact cellular energy transport. Several
dominant structural barriers are likely to affect radial diffusion, such as:
(i) the nm-scale myofilament lattice, which consists primarily of the
filamentous contractile proteins actin and myosin, (ii) µm-scale
subcellular membranes, which in lobster abdominal muscle principally
constitute the SR, and (iii) the 1x102µm-scale sarcolemmal
membrane, which is the cylindrical membrane that delineates individual muscle
cells. Two of these barriers, the myofilament lattice and the sarcolemmal
membrane, both have known dimensions and their effect on radial diffusion can
be predicted.
We have previously used a volume-averaging approach
(Carbonell and Whitaker, 1984)
to model the time dependence of D
in the myofilament
lattice, and details of this procedure are available in Kinsey et al.
(1999
). The effect of
restriction within the cylindrical sarcolemma on the time dependence of
diffusion has been approximated by Gibbs
(1997
), by interpolating
between the theoretical long- and short-time asymptotic behavior of
D
to yield:
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Results |
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Those peaks that were examined always had linear decreases in signal
amplitude as G was increased at all of the diffusion times that were
analyzed. Fig. 3 illustrates
this linearity as well as the characteristic difference in attenuation slopes
associated with axial and radial diffusion. The time dependence of
D|| and D is presented in
Fig. 4. The unrestricted, bulk
diffusion coefficient D0 of a metabolite in water would be
expected to be higher than D in the crowded environment inside a
muscle fiber. D0 of arginine phosphate has been previously
determined to be 4.05x10-6 cm2 s-1
(Ellington and Kinsey, 1998
),
which is considerably higher than the D values for arginine phosphate
in lobster muscle (Fig. 4A).
The most obvious pattern in both the 31P- and 1H-NMR
derived data was the orientation-dependence of diffusion within lobster muscle
fibers, where D
was lower than
D|| at all diffusion times for all of the metabolites
(Fig. 4). From 20 to 100 ms, a
substantial reduction in D
was apparent for arginine
phosphate and for betaine. This pattern was not resolved for the two small
1H resonances arising from arginine/arginine phosphate and
-CH/-CH2 groups since we were unable to measure D at the
shortest diffusion times. In contrast to the pattern for radial diffusion,
D|| was only slightly time-dependent for all of the
compounds and demonstrated no rapid decrease at short diffusion times. The
D values in both orientations were fairly stable after about 100 ms,
as would be expected for diffusion through a porous medium.
|
|
Since radial diffusion of arginine phosphate from subsarcolemmal mitochondria to the fiber interior is important in aerobic post-contraction recovery in lobster fibers, it is of interest to examine the effect of intracellular barriers on the net movement of arginine phosphate. Fig. 5 shows the extent to which the radial root-mean-square (RMS) displacement of arginine phosphate in lobster muscle deviates from that of arginine phosphate in a non-restricted environment. At the maximal diffusion time of 300 ms measured in this study, arginine phosphate diffused a distance of only 7 µm, whereas in an unrestricted environment, it would diffuse twice that distance.
|
The principal cause of the diffusive anisotropy can be inferred from the
model data presented in Fig. 6,
which predicts the effects of the myofilament lattice and the sarcolemmal
membrane on the normalized radial diffusion coefficient of arginine phosphate
(D/D0). The results of this
analysis would be nearly identical for the other metabolites. The predicted
effect of the nm-scale myofilament lattice leads to a very rapid reduction in
D
/D0 (<0.2 ms), after which a
steady-state diffusion coefficient is reached
(Fig. 6A). In contrast,
restriction of metabolites within the 300 µm diameter sarcolemma of lobster
fibers only minimally impacts radial diffusion
(Fig. 6B). In fact,
D
is only about 5% less than D0
at a diffusion time of 300 ms (Fig.
6B). It is expected that the rate of decay of
D
/D0 with time will be
considerably less in the large fibers of lobsters than in cells of `normal'
dimensions. In Fig. 6C the
combined effect of these two types of structural features has been removed
from the arginine phosphate D
values, and compared
to the bulk diffusion coefficient for arginine phosphate
(4.05x10-6 cm2s-1). Here, the residual
time dependence of AP D
values presumably is caused
by structures other than the myofibrillar lattice or the sarcolemmal
membrane.
|
Fig. 7 shows the
relationship between intracellular D values and the reciprocal of the
square root of molecular mass. This relationship should be linear in an
isotropic solution, as well as in a porous medium at long diffusion times
(i.e. when D reaches a steady state). We examined
D|| values at a diffusion time of 100ms. This selection
was made, not only because the D values have largely stabilized at
this point, but also because measurements of D|| at a
diffusion time of 100ms are available for lactate and alanine in lobster
abdominal muscle fibers (Kinsey and
Ellington, 1996), and these data can be included in the analysis.
In addition, we were able to estimate D|| of ATP by
averaging values from three diffusion times collected from a muscle
preparation that yielded an adequate signal-to-noise ratio and linear
attenuation plots. We arrived at a steady-state value of
D|| (100-300 ms diffusion time) of 1.13±0.23
cm2s-1 (N=3), which is consistent with previous
measurements of ATP diffusion in skeletal muscle
(de Graaf et al., 2000
). The
longtime behavior of D|| can be seen to be linearly
related to the square root of molecular mass
(Fig. 7).
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Discussion |
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Several previous studies have focused on diffusion of the phosphagen,
creatine phosphate, in skeletal muscle, because of its role in cellular energy
transport (Meyer et al., 1984;
Walliman et al., 1992
). The
invertebrate phosphagen, arginine phosphate, fulfils the same role in
crustacean muscle and is responsible for the vast majority of high-energy
phosphate flux (Ellington and Kinsey,
1998
). We will therefore focus on arginine phosphate in the
present discussion. An important feature of our data is that the time
dependence of arginine phosphate diffusion was nearly identical to that
previously observed for creatine phosphate diffusion in white muscle fibers
from cold-acclimated goldfish Carasius auratus
(Kinsey et al., 1999
). In this
previous work, as in the present study, we used small tissue samples and
carefully oriented the fibers axially along the y-imaging gradient in
order to measure D|| or D
. In
both fish white muscle and P. argus muscle,
D|| of the phosphagen (creatine phosphate or arginine
phosphate) was essentially independent of time over the range of diffusion
times measured (Fig. 4A).
However, we know that D|| must decrease in a
time-dependent fashion prior to our earliest measurements to account for the
fact that D||<D0. Similarly, the
time course over which phosphagen D
was reduced was
the same in fish white muscle and P. argus muscle. In both types of
muscle, D
reached a fairly constant value at a
diffusion time of 75-100ms (Fig.
4A). The nearly identical patterns of D
of creatine phosphate and arginine phosphate in fish white muscle and P.
argus muscle, respectively, suggest that the barriers that restrict
diffusion are the same in both tissues. White muscle fibers from goldfish that
have been acclimated to cold have an extensive SR and relatively few core
(intermyofibrillar) mitochondria (Tyler
and Sidell, 1984
). Giant glycolytic fibers from crustaceans also
have an extensive SR, and are virtually devoid of intermyofibrillar
mitochondria (S. T. Kinsey, unpublished results; Jahromi and Atwood, 1969;
Tse et al., 1983
). Therefore,
with respect to diffusion barriers, the principal difference between the two
types of muscle fibers is the fiber size. White muscle fibers from goldfish
are approximately 100 µm in diameter
(Kinsey et al., 1999
), while
P. argus fibers used in the present study were approximately 300
µm in diameter. The fact that the time dependence of
D
was identical in these two fibers of dramatically
different sizes indicates that restriction by the sarcolemmal membrane cannot
account for the observed reduction in D
.
Other studies have found a time-dependent decrease in D of
creatine phosphate in mammalian skeletal muscle that is similar to that
observed in the present paper, but the interpretations have been quite
different (Moonen et al.,
1990; van Gelderen et al.,
1994
; de Graaf, 2000). In rat quadriceps muscle in vivo,
apparent limits to creatine phosphate displacement at long diffusion times
indicated a compartment with an axial dimension of 44 µm, which the authors
suggested corresponded to the length of a muscle fiber
(Moonen et al., 1990
). These
diffusion measurements were made on muscles that were oriented at an angle
relative to the diffusion-weighting gradients, and D was measured in
only one direction. Subsequently, van Gelderen et al.
(1994
) examined the time
dependence of diffusion in rabbit skeletal muscle in vivo along the
x, y and z axes of the laboratory frame (the muscle fibers
were not oriented with respect to these axes). The orientation- and
time-dependent values of D were used to calculate the trace of the
diffusion coefficient (DTr), assuming restriction within a
cylindrical compartment. DTr is invariant to orientation,
which is sometimes important in in vivo experiments where orientation
of fibers may not be controllable or when fiber orientation is not uniform.
From the time-dependent pattern of DTr, van Gelderen et
al. (1994
) estimated that the
cell diameter of rabbit skeletal muscle was 17 µm. De Graaf et al.
(2000
) recently conducted a
similar analysis of the time dependence of DTr, also
fitted to a cylindrical model, in rat skeletal muscle in vivo. These
authors estimated that cylindrical compartments with diameters of 16 and 22
µm restricted the motion of ATP and creatine phosphate, respectively.
However, de Graaf et al. (2000
)
recognized that these dimensions were considerably smaller than the dimensions
of rat skeletal muscle cells, and they concluded that yet-to-be-defined,
subcellular, cylindrical barriers restrict ATP and creatine phosphate
diffusion in skeletal muscle.
In contrast to the conclusions drawn from studies of diffusion in mammalian
skeletal muscle, we believe that the principal barrier that induces the
anisotropy observed in PFG-NMR measurements is the reticulated SR membrane.
Several lines of evidence support this conclusion. First, our modeling results
clearly indicate that the time-dependent reduction in diffusive flux through
the nm-scale thick and thin filament lattice is too rapid to be detected by
NMR methods (Fig. 6A). This
intuitively satisfying result is very robust to moderate variation in the
estimate of myofilament porosity or filament dimensions. Second, restriction
within a cylinder the size of most skeletal muscle fibers has only a slight
effect on the observed time dependence of D
(Fig. 6B)
(Gibbs, 1997
;
Kinsey et al., 1999
). This was
partly what motivated us to select crustacean giant muscle fibers for this
study, since at physiologically relevant diffusion times the effect of the
sarcolemma on D
is virtually undetectable. This
leaves us with intracellular barriers with length scales intermediate between
the myofilament lattice, which has a nm-length scale, and the sarcolemma,
which has a 1x102 µm-length scale. In lobster giant muscle
fibers, the only obvious barrier is the SR membrane, and the associated
sarcolemmal invaginations, or clefts, that are peculiar to crustaceans
(Peachey, 1967
;
Selverston, 1967
). The SR is a
reticulated membrane envelope that wraps around each myofibril or a bundle of
2-3 myofibrils. Each myofibril is
1 µm in diameter, so the cylindrical
partial membrane of the SR would serve as a strong barrier to radial
diffusion, but would not be expected to hinder axial diffusion. The
sarcolemmal clefts in crustaceans are partial membranes that project radially
into the fiber core with a linear spacing of approximately 50 µm
(Peachey, 1967
;
Selverston, 1967
). These
membranous structures would therefore be expected to have some effect on
radial, but not axial, diffusion in crustacean muscle. Whereas the SR will
constitute a similar barrier to radial diffusion in crustacean, fish and
mammalian skeletal muscle, the sarcolemmal clefts are specific to crustaceans.
However, the sarcolemmal clefts are an incomplete membrane with relatively
large linear spacing, and the effect of this potential barrier on radial
diffusion is likely to be minimal.
As described above, de Graaf et al.
(2000) have proposed an
alternative view of the subcellular structures that induce diffusive
anisotropy in skeletal muscle. However, we know of no cylindrical barriers in
skeletal muscle that have a length scale of 16-22 µm as described in their
study (de Graaf et al., 2000
).
We contend that their determination of a length scale of this magnitude is
derived from the fact that these authors fit their diffusion data to a model
of restriction within an impermeable cylinder. Thus, while their method of
determining DTr has great utility when fiber orientation
cannot be controlled, we do not believe it yields relevant length scales of
barriers to diffusion.
Part of the difficulty in assessing the effect of the SR (and sarcolemmal
clefts) is the complex three-dimensional structure of these membranes. For
instance, we know that diffusion through the SR will lead to a time-dependent
reduction in D until a steady-state value is
reached, as is the case for diffusion through the thick and thin filament
lattice (Fig. 6A). However, the
effect of the SR on diffusion will be a function of two variables,
neither of which are adequately described or easily modeled. Both the membrane
porosity and the length scale in the direction of diffusion will have an
impact on the time dependence of D
. Because of this
complication, our conclusions are drawn from the fact that barriers in muscle
fibers from mammals, fish and crustaceans induce the same time-dependent
anisotropy despite the fact that they are dramatically different with respect
to cell size, mitochondrial density, and the presence or absence of
sarcolemmal clefts. All of these tissues have in common a well-developed SR,
however, and the function of this organelle in Ca2+ dynamics
demands that its radial length scale is relatively invariant, regardless of
the type of skeletal muscle.
Another finding of the present study is that when the effect of the thick
and thin filament lattice and restriction within the sarcolemma are
mathematically removed, the intracellular D values at short diffusion
times do not differ substantially from D0
(Fig. 6C). This is in agreement
with previous studies, which indicated that at short diffusion times the
cytoplasm viscosity was not significantly higher than that of water
(Luby-Phelps et al., 1993;
van Gelderen et al., 1994
;
de Graaf et al., 2000
). In
addition, the steady-state diffusion of metabolites in P. argus
muscle had diffusion coefficients that were inversely proportional to their
molecular mass (Fig. 7). This
is consistent with the idea that when diffusion is temporally (and hence
spatially) averaged across intracellular structural barriers, the cytoplasmic
space in lobster abdominal muscle behaves as would be expected for a
well-mixed, bulk solution.
Diffusion analyses such as the present study are useful for probing the
nature of the intracellular environment, but they also raise the following
question: does the diffusive flux of metabolites impose constraints on muscle
function? The abdominal muscles in lobsters, crayfish and shrimp are used
solely for a rapid series of `tail-flips' that propel the animal away from
potential predators at a high velocity. The abdominal muscle bundles are
composed almost exclusively of fast-twitch, glycolytic fibers. These
fast-twitch fibers of lobsters and crabs tend to be the largest, often
exceeding 500 µm in diameter and more than 1 cm in length
(Jahromi and Atwood, 1971;
Tse et al., 1983
). In
addition, nearly all of the mitochondria in these fibers are located
peripherally near the sarcolemmal membrane (subsarcolemmal mitochondria;
Kent and Govind, 1981
;
Tse et al., 1983
). This
arrangement of mitochondria forms a cylinder of aerobic metabolic potential
that has essentially the same dimensions as the muscle fibers themselves.
There are important functional implications to the combination of extreme
cellular dimensions and highly localized aerobic machinery. Rapid, aerobically
powered contraction is logically forbidden, since this would require rapid
diffusive flux of ATP-equivalents (i.e. arginine phosphate) over a distance of
hundreds of microns. The displacement of molecules via diffusion,
, scales as the square-root of the diffusion time, t:
. The steady-state value of
D for arginine phosphate in situ is
1x10-6 cm2 s-1
(Fig. 5), meaning that it takes
several minutes for arginine phosphate to diffuse from subsarcolemmal
mitochondria at the fiber periphery, to the sites of ATP demand at the fiber
core of a crustacean giant muscle fiber. In contrast, muscle that contracts
aerobically has relevant diffusion distances that can be traversed in
milliseconds. Since the escape-response in lobsters is induced by a short-term
burst of muscle activity (England and
Baldwin, 1983
), reliance on anaerobic metabolism (phosphagen
hydrolysis and glycolysis) to supply ATP poses no obvious problems. However,
post-contractile recovery ultimately relies on aerobic metabolism, and the
large dimensions of crustacean fibers and barriers to radial diffusive flux
would be expected to severely limit the rates of these processes.
The metabolic restrictions imposed by extreme diffusion distances are
apparent in the pattern of post-exercise recovery in crustacean fast-twitch
muscles, which differs substantially from the mammalian paradigm. An early
phase of recovery is the resynthesis of phosphagen and restoration of ionic
gradients across membranes, which allows a second round of high-force
contractions. In mammalian skeletal muscle, creatine phosphate is rapidly
resynthesized by aerobic processes
(Kushmerick, 1983;
Meyer, 1988
). In crustacean
muscle, however, AP stores are replenished via anaerobic
glyogenolysis (Ellington, 1983
;
Kamp, 1989
), presumably
because the large diffusion distances and barriers to diffusive flux make
aerobic recovery unacceptably slow. The aerobic phase of recovery in
crustaceans that follows includes the resynthesis of glycogen, processing of
lactate and restoration of muscle pH, and these processes occur over a
protracted time course of several hours (Milligan et al., 1990;
Henry et al., 1994
). Whether
the combination of extreme distances and hindered diffusion in giant fibers is
the proximate cause for the slow recovery process is not known. Other
properties, such as metabolic potential and oxygen supply, may ultimately
limit aerobic metabolism. In this scenario, excessive diffusion distances
would not be limiting per se, but would simply not be selected
against. However, it is clear that the large diffusion distances and barriers
to diffusion impose severe limits on the rate of post-contractile recovery,
even if aerobic metabolic potential and oxygen supply were increased. This
argument also raises issues as to the nature of the SR in fast-contracting,
aerobic fibers. Here, a substantial SR is required for rapid
excitationcontraction coupling, but it must be sufficiently permeable
to permit high rates of ATP-equivalent flux from mitochondria to the myosin
ATPase.
In summary, metabolite diffusion in lobster giant muscle fibers is
anisotropic, indicating that the barriers to radial diffusion are more
substantial than those that hinder axial diffusion. The similarity of the time
dependence of D in crustacean muscle to that found
in skeletal muscle from mammals and fish suggests a common diffusive barrier,
despite the fact that these tissues differ dramatically in fiber diameter and
mitochondrial density. The time-dependent reduction in
D
is consistent with barriers on a µm scale, and
the most likely candidate is the SR membrane system. The combination of large
diffusion distances and barriers to diffusive flux appear to severely limit
the capacity for aerobic metabolic processes in crustacean giant muscle
fibers.
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Acknowledgments |
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