Intracellular calcium translocation during the contractionrelaxation cycle in scorpionfish swimbladder muscle
1 Department of Physiology, School of Medicine, Teikyo University, 2-11-1,
Kaga, Itabashi-ku, Tokyo 173-8605, Japan
2 Department of Physiology, Juntendo Medical College of Nursing, 2-2,
Takasu, Urayasu City, Chiba 279-0023, Japan
* Author for correspondence at present address: Department of Biological Sciences, School of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka-City, Kanagawa 259-1293, Japan (e-mail: sugi{at}med.teikyo-u.ac.jp)
Accepted 3 December 2003
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Summary |
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Key words: scorpionfish, Sebastiscus marmoratus, swimbladder muscle, intracellular Ca translocation, contractionrelaxation cycle, electron probe X-ray microanalysis, sarcoplasmic reticulum, Ca2+ release, Ca2+ uptake
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Introduction |
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In striated muscles of teleosts, both the SR and the myofibrils are
extremely well aligned in the transverse direction, so that the well-developed
SR components are invariably located at the same regions in each sarcomere
(Fawcett and Revel, 1961). The
present work was undertaken to give clear information about intracellular Ca
translocation during the contractionrelaxation cycle in vertebrate
striated muscle, using the swimbladder muscle (SBM) of a scorpionfish
Sebastiscus marmoratics. The extremely well aligned SR components in
the SBM fibres provide an opportunity to determine the calcium distribution in
the SR components during the contractionrelaxation cycle by
quantitative X-ray microanalysis of SBM fibre cryosections. It is shown that,
in SBM fibres, calcium released from the TC is taken up by the LT and the FC
of the SR, thus supporting the observations of Winegrad
(1965
,
1968
) in frog skeletal
muscle.
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Materials and methods |
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Quick-freezing
The method used for quick-freezing the SBM fibres was essentially the same
as in our previous study (Suzuki et al.,
1993). Briefly, the preparation was mounted horizontally in an
experimental chamber filled with the experimental solution
(1720°C); one end of the preparation was clamped, while the other
end was connected to a strain gauge (UL-2, Shinko, Tokyo, Japan) to record
isometric force. The middle portion of the fibres was placed in a narrow space
between two gold plates (Balzer, Liechtenstein), closely facing each other.
The fibres were made taut by stretching them to
1.2 times the slack
length (sarcomere length,
2.5 µm).
SBM fibres were quick-frozen at rest, during isometric tetanus, and at 0.1,
1, 3 and 5 s after onset of relaxation. The fibres were made to contract
isometrically by applying supramaximal AC current (100 Hz) through the
connections at both fibre ends, and recording the isometric forces on a chart
recorder (Graphtec, Tokyo, Japan). Before freezing, the experimental solution
in the chamber was drained, and the fibres were quick-frozen by jetting liquid
propane (190°C) from two nozzles to the gold plates and the fibres.
The frozen fibres were removed from the experimental chamber together with the
two gold plates, and put into liquid N2 to be stored until
cryosections were cut.
Cryosections
Longitudinal cryosections (200 nm thick) were cut from the frozen SBM
fibres at 110°C on a cryoultramicrotome (LKB Nova; LKB Produkter,
Bromma, Sweden), and then carefully sandwiched between two cooled Ni-grids
with thin carbon films. In the cryochamber kept at 125°C, the
Ni-grid sandwich was transferred to a freeze-drying aluminium container,
equipped with a thermoelectric device to monitor temperature during freeze
drying. The mass of the container was enough to maintain temperatures below
95°C for 24 h in a vacuum chamber. The freeze-drying container was
then brought out of the cryochamber, further cooled to 196°C with
liquid N2, and then put into the vacuum chamber of a freeze-drying
machine (FD-2A; Eiko Engineering, Tokyo, Japan) to be freeze-dried at
106 torr (1.3x104 Pa) and at
95°C for 24 h. After the freeze-drying procedure, the Ni-grid
sandwich was split apart, and the cryosections were lightly evaporated in
vacuo with carbon for X-ray microanalysis.
Electron probe X-ray microanalysis
Electron probe X-ray microanalysis was performed using an analytical
electron microscope (JEM 2000FXS, JEOL, Tokyo, Japan) equipped with an energy
dispersive X-ray microanalyzer (TN5450; Tracor Northern Inc., Middleton,
Wisconsin, USA). Initially, the cold trap attached to the electron microscope
column was cooled with liquid N2 to avoid specimen contamination.
The freeze-dried cryosections on the Ni-grid were mounted on a Be-stage of the
cryotransfer holder (EH-CTH 10; JEOL), and put into the electron microscope,
the cryosections being cooled at 130°C by a supply of liquid
N2. The transmission system of the analytical electron microscope
was operated at an accelerating voltage of 80 kV. For X-ray microanalysis
(spot analysis), the electron beam was focused on a fixed area (diameter, 0.16
µm) under a magnification of 25 000x with a sample current of
1.35 nA. X-ray emissions from the cryosection were collected for a period
of 200 s. To examine whether or not mass-loss and/or contamination in the
cryosection occurred during the collection of X-ray emissions, the change of
spectral peak integral during the course of analysis was monitored using a
software program (Mass-loss monitoring mode) of the TN5450 computer system,
which detected any significant mass-loss and contamination that occurred
during the analysis.
The elemental concentrations were calculated from X-ray spectra in mmol
kg1 dry mass, based on the Hall's quantitative equation
(Hall, 1971;
Shuman et al., 1976
). X-ray
emissions of up to
10.24 KeV were collected, and the intensity of X-ray
continuum was represented by an integral X-ray count in a region of
4.55.5 KeV where no spectral peak appeared. For each X-ray spectrum, a
reference spectrum was obtained from the area including only carbon supporting
film. To calculate elemental concentrations, the intensity of X-ray continuum
from the cryosection was always corrected by subtracting the artificial X-ray
continuum caused by carbon film, Ni-grid and other factors (e.g. contamination
of Si) from the X-ray spectrum.
To determine the intensity of each spectral peak with reasonable accuracy,
elemental spectral peaks were subjected to a multiple least-squares fit to
corresponding elemental standard spectral peaks (Na, Mg, Al, Si, P, S, Cl, K,
Ca and Ni), obtained by analyzing pure chemicals, and stored in the TN5450
computer system. In the case of overlapped spectral peaks such as
K-Kß and Ca-K emissions, the first and the
second derivatives of the spectral peaks were further subjected to a multiple
least-squares fit to the derivatives of the standard spectral peaks.
Conventional electron microscopy
SBM fibres in resting muscle were fixed with a 2.5% glutaraldehyde solution
containing 2 mmol l1 CaCl2 (adjusted to pH 7.2
with 0.1 mol l1 cacodylate buffer), and postfixed with a 2%
osmium tetroxide (OsO4) solution in the same buffer
(Suzuki et al., 2003). The
preparations were then dehydrated through a graded ethanol series, cleared
with propylene oxide, and embedded in Epon 812. Ultrathin sections cut on a
Porter Blum MT-2 ultramicrotome (DuPont Instrument-Sorval, Wilmington,
Delaware, USA) were stained with uranyl acetate and lead citrate, and examined
using a JEOL JEM 2000FXS transmission electron microscope.
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Results |
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Intracellular Ca distribution in resting SBM fibres
A typical example of longitudinal cryosections of the SBM fibres is shown
in Fig. 2. Although striation
patterns are clearly visible, it was not possible to observe the SR components
in SBM fibre cryosections. We therefore performed spot analysis to measure Ca
concentrations in the SR components, assuming that the cryosections contain
the SR components as well as the myofibrils.
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This assumption was proved valid for the following reasons: (1) in resting fibres, the Ca concentration was always highest around the AI boundary, where the TC of the SR were located (see Fig. 4 and Table 2), (2) spot analysis on 20 consecutive regions along the AI boundary in the transverse direction revealed that regions of high [Ca] (>20 mmol kg1 dry mass) were not continuous but separated from each other by regions of low [Ca] (Fig. 3), reflecting the separation of the SR by the myofibrils (Fig. 1A).
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Thus, the distribution of Ca in the SR components of resting fibres was determined by spot analysis at the centre of five consecutive regions along the half sarcomeres, i.e. the Z region, containing the FC; I and A1 regions, containing the TC; A2 region, containing the LT; and H region, containing the FC (Figs 1B, 2). As can be seen in Fig. 4A, the [Ca] in the I and A1 regions (42.7±5.0 and 43.6±5.4 mmol kg1 dry mass, means ± S.E.M., N=36, respectively), were much higher than those in the Z, A2 and H regions (23.7±2.1, 13.3±2.1 and 9.9±1.7 mmol kg1 dry mass, respectively, N=36) (P<0.001).
Table 1 shows concentrations of several elements including Ca in the five consecutive regions of resting fibres. Except for Ca, the concentrations of all the elements were nearly constant in all the five regions. The high K and low Na concentrations indicate that the fibres had been frozen without any serious damage to the surface membrane. The concentrations of elements other than Ca did not change significantly whether the fibres were frozen during contraction or at various times after the onset of relaxation.
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Intracellular Ca distribution in contracting SBM fibres
The SBM fibres were quick-frozen at various stages of the
contractionrelaxation cycle, and the Ca concentrations in the five
regions along the half sarcomere at these various stages are summarized in
Table 2. In fibres frozen at
the peak of the mechanical response to electrical stimulation, the [Ca] in the
A1 region (19.8±2.7 mmol kg1 dry mass, N=25)
was significantly lower than that in resting fibres (P<0.01),
while the [Ca] in the H region (16.5±2.3 mmol kg1 dry
mass, N=25) was significantly higher than that in resting fibres
(P<0.05) (Fig. 4B,
Table 2). [Ca] in the Z, I and
A2 regions did not change significantly from the corresponding values in
resting fibres, though the mean [Ca] in the I region decreased to 31.5 mmol
kg1 dry mass, and that in the A2 region increased to 16.1
mmol kg1 dry mass.
These results are consistent with the release of Ca from the TC into the myoplasm, if the increase in [Ca] in the Z, A2 and H regions is interpreted as being due to the increase in the myoplasmic [Ca2+] in these regions. The insignificant changes in [Ca] in the Z and I regions may be a consequence of the distance between the TC and FC being too short in these regions.
Intracellular Ca distribution after onset of relaxation
SBM fibres were frozen at various times after onset of relaxation. At 0.1 s
after onset of relaxation, the isometric force fell to 50% of the maximum
value. In fibres quick-frozen at this stage, [Ca] in the A1, A2 and H regions
were significantly higher than the corresponding values in contracting fibres
(P<0.05 for the A1 and A2 regions and P<0.01 for the H
region; Table 2).
The isometric force fell to zero at 1 s after onset of relaxation. In
fibres quick-frozen at this stage, the [Ca] in the H region was significantly
lower than the value in the previous stage (P<0.05), while the
[Ca] in the other regions did not change significantly
(Fig. 4D; see also
Table 2). In fibres frozen at 3
and 5 s after onset of relaxation (or at 2 s and
4 s after
completion of relaxation), [Ca] in the five regions gradually returned to the
values in resting fibres (Fig.
4E,F and Table 2);
thus, [Ca] in all the five regions at 5 s after onset of relaxation did not
differ significantly from the corresponding values in resting fibres.
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Discussion |
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The [Ca] in the TC obtained in the present study (40 mmol
kg1 dry mass) is much smaller than the value reported for
the TC in resting frog muscle fibres (117 mmol kg1 dry mass;
Somlyo et al., 1981
). In frog
skeletal muscle fibres, however, the SR volume is less than 5% of the whole
fibre volume (Peachey, 1965
;
Mobley and Eisenberg, 1975
),
whereas the SR volume in SBM fibres is
25% of the whole fibre volume
(Suzuki et at., 2003
). Taking
this into consideration, the total amount of Ca in the TC lumen in the SBM
fibres may be rather larger than that in frog fibres.
Release of Ca from the TC into the myoplasm
In SBM fibres quick-frozen at the peak of mechanical response to electrical
stimulation, the [Ca] in the A1 region was significantly smaller than the
corresponding value in resting fibres (P<0.01), while the [Ca] in
the H region was significantly higher (P<0.05)
(Fig. 4B, Table 2). The mean [Ca] in the
A2 region was also higher than that in resting fibres, though the difference
was not significant. These results can be taken to reflect Ca release from the
TC into the myoplasm, if the increased [Ca] is assumed to result from
increased myoplasmic [Ca2+] around the SR.
We calculated an approximate estimate of the amount of Ca released from the
TC to the myoplasm from the difference in mean [Ca] of the I and A1 regions in
resting and contracting fibres (43.2-26.7=17.5 mmol kg1 dry
mass). Assuming that the TC volume is 60% of the SR volume
(Suzuki et al., 2003
), the
dilution rate of released Ca is
0.15. Consequently, the myoplasmic
concentration of the released Ca is
2.6 mmol kg1 dry
mass (17.5x0.15). If the water content of the SBM fibres is assumed to
be
70% (Somlyo et al.,
1981
), the 2.6 mmol kg1 dry mass is equivalent
to 1.11 mmol l1 fibre H2O, a value nearly equal
to that reported for frog muscle fibres (
1 mmol l1
fibre H2O; Somlyo et al.,
1981
).
Uptake of Ca by the SR components
In the fibres frozen at 0.1 s after onset of relaxation, the [Ca] in the Z,
A1, A2 and H regions were significantly higher than the corresponding values
in resting fibres (P<0.05 for the Z, A1 and A2 regions and
P<0.01 for the H region) (Fig.
4C, Table 2). These
results are consistent with all the SR components starting to take up the
myoplasmic Ca2+ at the early phase of relaxation. At 1 s after
onset of relaxation, the [Ca] in the H region became significantly smaller
than the value at the previous stage of relaxation (P<0.05)
(Fig. 4D,
Table 2). The mean [Ca] was
also smaller in the Z and A2 regions, and larger in the I and A1 regions
(Fig. 4D,
Table 2). At 3 s after onset of
relaxation, the [Ca] in the A2 and the H regions decreased significantly
compared to the values at the previous stage (P<0.05 and 0.01,
respectively) (Fig. 4E,
Table 2). The mean [Ca] was
also decreased in the Z region, but increased in the I and A1 regions
(Fig. 4E, Table 2). These results seem to
indicate that the Ca, already taken up by the LT and FC in the previous stage,
is moving to the TC.
At 5 s after onset of relaxation, the [Ca] in the A1 region became significantly higher than the value at the previous stage (P<0.05), and the mean [Ca] in the I region was also higher than the value at the previous stage. Thus, the [Ca] in both the I and A1 regions returned to levels much higher than those in all the other regions (P<0.001) (Fig. 4F, Table 2), indicating that the Ca distribution along the SR components returned to that seen in resting fibres within 5 s after the onset of relaxation.
Mechanism of the Ca translocation in the SR components during relaxation
In the present study, we have clearly answered the question of whether the
myoplasmic Ca2+ is taken up by the LT (Winegrad,
1965,
1968
) or by the TC
(Somlyo et al., 1981
) during
relaxation of vertebrate striated muscle fibres. The answer we have obtained
is intermediate between the above two cases; after onset of relaxation in the
SBM fibres, the myoplasmic Ca2+ is taken up not only by the LC and
FC but also by the TC (Fig. 4,
Table 2), which is consistent
with the uniform distribution of Ca pump proteins all over the SR membrane
except for the triadic junctional region
(Jorgensen et al., 1979
;
Saito et al., 1984
). As can be
seen in Fig. 4CF and
Table 2, the increases in [Ca]
in the Z, A1, A2 and H regions, reflecting Ca uptake by all the SR components,
are most prominent in the early phase of relaxation (at 0.1 s after its
onset). The reason why Somlyo et al.
(1981
) did not observe Ca
uptake by the LC and FC might be partly because they did not measure [Ca]
during relaxation, and partly because the LT and FC are much less developed in
frog muscle fibres than in SBM fibres.
Muscle fibres of cold-blooded animals are known to contain parvalbumin, a
Ca-binding protein buffering the myoplasmic [Ca2+]
(Gillis, 1985). If parvalbumin
is present in SBM fibres, it may increase the rate of relaxation to a certain
extent. However, possible Ca2+-binding to parvalbumin may not
qualitatively affect the present results, since parvalbumin distributes
uniformly in the myoplasm.
In vertebrate skeletal muscle, the Ca in the TC lumen is largely bound to
the Ca-binding protein calsequestrin, localized at high concentrations in the
TC (Meissner, 1975;
Jorgensen et al., 1979
;
Guo and Campbell, 1995
). In
toadfish SBM, however, a small concentration of calsequestrin is also present
in the LC (Franzini-Armstrong et al.,
1987
). These reports suggest that calsequestrin concentration is
highest in the TC and lowest in the FC. It therefore seems likely that the Ca
taken up by all the SR components during relaxation gradually moves towards
the TC along the calsequestrin concentration gradient. Much more experimental
work is needed to prove the mechanism of intracellular translocation stated
above.
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Acknowledgments |
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References |
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Ebashi, S. and Endo, M. (1968). Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol. 18,123 -183.[CrossRef][Medline]
Fawcett, D. W. and Revel, J. P. (1961). The sarcoplasmic reticulum of a fastacting fish muscle. J. Cell Biol. 10 Suppl,89 -109.
Franzini-Armstrong, C., Kenny, L. J. and Varriana-Marston, E. (1987). The structure of calsequestrin in triads of vertebrate skeletal muscle: A deepetch study. J. Cell Biol. 105,49 -56.[Abstract]
Gillis, J. M. (1985). Relaxation of vertebrate skeletal muscle. A synthesis of the biochemical and physiological approaches. Biochem. Biophys. Acta 811,97 -145.[Medline]
Guo, W. and Campbell, K. P. (1995). Association
of triadin with the ryanodine receptor and calsequestrin in the lumen of the
sarcoplasmic reticulum. J. Biol. Chem.
270,9027
-9030.
Hall, T. A. (1971). The microprobe assay of chemical elements. In Physical Techniques in Biological Research (ed. G. Oster), pp. 157-275. New York: Academic Press.
Jorgensen, A. O., Kalnins, V. and MacLenan, D. H. (1979). Localization of sarcoplasmic reticulum proteins in rat skeletal muscle by immunofluorescence. J. Cell Biol. 80,372 -384.[Abstract]
Meissner, G. (1975). Isolation and characterization of two types of sarcoplasmic reticulum vesicles. Biochim. Biophys. Acta 389, 51-68.[Medline]
Mobley, B. A. and Eisenberg, B. R. (1975). Sizes of components in frog skeletal muscle measured by methods of stereology. J. Gen. Physiol. 66,31 -45.[Abstract]
Peachey, L. D. (1965). The sarcoplasmic
reticulum and transverse tubules of the frog's sartorius. J. Cell
Biol. 25,209
-231.
Saito, A., Seiler, S., Chu, A. and Fleischer, S. (1984). Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J. Cell Biol. 99,875 -885.[Abstract]
Shuman, H., Somlyo, A. V. and Somlyo, A. P. (1976). Quantitative electron probe microanalysis of biological thin sections: methods and validity. Ultramicroscopy 1, 317-339.[Medline]
Somlyo, A. V., Gonzalez-Serratos, H., Shuman, H., McClellan, G. and Somlyo, A. P. (1981). Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron-probe study. J. Cell Biol. 90,577 -594.[Abstract]
Somlyo, A. V., McClellan, G., Gonzalez-Serratos, H. and Somlyo,
A. P. (1985). Electron probe X-ray microanalysis of
post-tetanic Ca2+ and Mg2+ movements across the
sarcoplasmic reticulum in situ. J. Biol. Chem.
260,6801
-6807.
Suzuki, S., Oshimi, Y. and Sugi, H. (1993). Freeze-fracture studies on the cross-bridge angle distribution at various physiological states and the thin filament stiffness in single skinned frog muscle fibers. J. Electron Microsc. 42,107 -116.[Medline]
Suzuki, S., Hino, N. and Sugi, H. (2003).
Ultrastructural organization of the transverse tubule and the sarcoplasmic
reticulum in a fish sound-producing muscle. J. Electron
Microsc. 52,337
-347.
Winegrad, S. (1965). Autoradiographic studies
of intracellular calcium in frog skeletal muscle. J. Gen.
Physiol. 48,455
-479.
Winegrad, S. (1968). Intracellular calcium
movements of frog skeletal muscle during recovery from tetanus. J.
Gen. Physiol. 51,65
-83.