Effects of BAPTA on force and
Ca2+ transient during
isometric contraction of frog muscle fibers
Y.-B.
Sun,
C.
Caputo, and
K. A. P.
Edman
Department of Pharmacology, University of Lund, S-223 62 Lund,
Sweden
 |
ABSTRACT |
The effects of
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA) on force and intracellular
Ca2+ transient were studied during
isometric twitches and tetanuses in single frog muscle fibers. BAPTA
was added to the bathing solution in its permeant AM form (50 and 100 µM). There was no clear correlation between the changes in force and
the changes in Ca2+ transient.
Thus during twitch stimulation BAPTA did not suppress the
Ca2+ transient until the force had
been reduced to <50% of its control value. At the same time, the
peak myoplasmic free Ca2+
concentration reached during tetanic stimulation was markedly increased, whereas the force was slightly
reduced by BAPTA. The effects of BAPTA were not duplicated by using
another Ca2+ chelator, EGTA,
indicating that BAPTA may act differently as a
Ca2+ chelator. Stiffness
measurements suggest that the decrease in mechanical performance in the
presence of BAPTA is attributable to a reduced number of active cross
bridges. The results could mean that BAPTA, under the conditions used,
inhibits the binding of Ca2+ to
troponin C resulting in a reduced state of activation of the contractile system.
calcium chelator; cross bridges; fiber stiffness; ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid
 |
INTRODUCTION |
THE CHANGES IN MYOPLASMIC free
Ca2+ concentration
([Ca2+]i)
of frog muscle fibers during contraction have been extensively studied over the last two decades using different
Ca2+ indicators. Blinks et
al. (4), using the photoprotein aequorin, reported that
during twitch contractions the
Ca2+ transient already starts to
decay while tension is still rising. Recent studies have demonstrated
that almost the entire rising phase of the isometric twitch occurs
while
[Ca2+]i
is declining (7, 24). It thus appears that the decay phase of the
Ca2+ transient is an important
determinant of the mechanical response during a single twitch (24). The
decline of
[Ca2+]i
is thought to be governed by the sarcoplasmic reticulum (SR) Ca2+ pump and the intracellular
Ca2+-binding proteins, in
particular, parvalbumin (12). The significance of the SR
Ca2+ pump for the sequestration of
Ca2+ is indicated by the fact that
inhibition of the pump by
2,5-di(tert-butyl)-1,4-benzohydroquinone leads to a dramatic slowing of the decline of
[Ca2+]i
after a contraction (17, 27). Although the SR
Ca2+ pump does play a predominant
role in the decline of
[Ca2+]i,
there is evidence that the
Ca2+-binding protein parvalbumin
contributes to Ca2+ sequestration
in frog twitch muscle fibers (15). Parvalbumins seem to act solely as
Ca2+ buffers in the myoplasm: they
do not exhibit enzymatic activity (22) nor do they associate with
intracellular organelles (13).
To obtain further evidence on the importance of intracellular
Ca2+ buffers in the regulation of
muscle activity, the effects of exogenous
Ca2+ chelators on tension response
and Ca2+ transient were studied in
frog muscle fibers. The high-affinity Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (25) has been used in several previous studies to reduce
the
[Ca2+]i
in isolated muscle fibers (1, 16). For this purpose BAPTA has been
injected into the fiber lumen in relatively high (millimolar) concentrations. After such treatment, the intracellular
Ca2+ transient after stimulation
is drastically reduced (16). In the present experiments, intact single
muscle fibers from Rana temporaria
were exposed to BAPTA in its AM form using a relatively low (50 or 100 µM) concentration in the extracellular fluid. BAPTA-AM diffuses into
the fiber and is hydrolyzed by cellular esterases and trapped inside
the fibers (26). Under the conditions used in the present study, BAPTA
exhibits marked effects on the excitation-contraction coupling by a
mechanism that is apparently different from its Ca2+-buffering action. BAPTA is
found to almost completely abolish the twitch response while only
slightly depressing the Ca2+
transient. At the same time, the peak
[Ca2+]i
reached during tetanic stimulation is markedly increased. These results
could not be foreseen on the basis of previous observations (16). To
distinguish these unexpected effects of BAPTA from the
Ca2+-buffering actions, another
Ca2+ chelator, EGTA, was also
tested in this study.
 |
METHODS |
Preparation and mounting.
Single muscle fibers were dissected from the anterior
tibialis muscle of R. temporaria. The
frogs were killed by decapitation followed by destruction of the spinal
cord. After dissection, the fibers were mounted horizontally in a
thermostatically controlled Perspex chamber between a force transducer
(AE801, Aksjeselskapet Mikroelektronikk, Horten, Norway) and a
stainless steel hook fixed to the bottom of the experimental chamber.
Clips of aluminum foil were attached to the tendons, and the side parts
of the clips were tightly folded around the hooks on the force
transducer and the opposite attachment site. The setting of the clips
was carefully adjusted to minimize any lateral, vertical, or twisting
movements of the fiber during contractile activity.
The Ringer solution had a composition (in mM) of 115.5 NaCl, 2.0 KCl,
1.8 CaCl2, and 2.0 Na2HPO4 + NaH2PO4
(pH 7.0). BAPTA-AM or EGTA-AM (Molecular Probes, Eugene, OR) was
dissolved in pure DMSO to a concentration of 25 mM. The stock solution
of BAPTA-AM or EGTA-AM was then mixed with Ringer solution to provide a
final concentration of either 50 or 100 µM. Higher concentrations of these two agents in Ringer solution were not performed due to their
limited solubility.
The temperature of the bathing fluid varied between 10 and 12°C in
the different experiments but was constant to within ±0.2°C during any given experiment.
The sarcomere length of the resting fiber was set to 2.2 µm by direct
microscopy at ×400 magnification. The fibers were stimulated by
passing rectangular current pulses (0.2-ms duration) between two
platinum plate electrodes placed symmetrically on either side of the
fiber. The stimulus strength was 15-20% above the threshold. During the experiment, the fiber was stimulated to produce a twitch and
a tetanus at regular 3-min intervals until constant responses were
obtained. This initial control period lasted for at least 45 min.
Measurement of fiber stiffness.
A full description of the technique used for measuring fiber stiffness
has been given by Edman and Lou (9), and it is only outlined here. For
this measurement, the fiber was mounted between a force transducer
(resonant frequency, ~19 kHz) and an electromagnetic puller. A 4-kHz
length oscillation with a peak-to-peak amplitude of ~1.5
nm/half-sarcomere was applied by the puller to one end of the fiber
throughout the stimulation period starting ~3 s before the
stimulation began. The stiffness was measured as the change in force
that resulted from the length oscillation and could be read out on line
as a separate signal as described in detail before (9). The force
signal was recorded without the superimposed force oscillations by
using a notch filter that produced maximum damping at 4.0 kHz.
Estimation of the intracellular
Ca2+ transients.
The intracellular
Ca2+ transients were monitored by
using the Ca2+-sensitive
fluorescent indicator, fluo 3 (21). The loading procedure has been
described previously (5). In brief, the fiber was immersed in Ringer
solution containing ~20-40 µM fluo 3-AM (Molecular Probes) for
~45 min at room temperature. The fiber was thereafter immersed in
ordinary Ringer solution for at least 20 min before experimentation.
The muscle chamber was mounted on the stage of a Zeiss inverted
microscope (Axiovert 35) equipped with an epifluorescence attachment.
The light source was a 100-W Hg lamp driven by a stabilized power
supply. The set of filters used for fluo 3 was (excitation, dichroic,
and barrier, respectively) 450-490, 510, and 520 nm. A shutter was
used to illuminate the fiber only during recording of the light signal.
The light signal was collected from an area with a diameter of ~1 mm
that was kept constant during the experiment.
The
[Ca2+]i
was calculated from the fluo 3 signal by taking account of the on
(k+) and off
(k
) rate
constants for the Ca2+-fluo 3 complex following the procedure described by Caputo et al. (5).
The numerical values of
k
and
k+ for fluo 3 in
the myoplasm have recently been estimated to be 33.5 s
1 and 13.1 µM
1 · s
1,
respectively, at 16°C (14). Accounting for the temperature dependence of the
k
and
k+ (20), the
following values of
k
and
k+ were derived
and used in the present study (10-12°C):
k
= 32.0 s
1 and
k+ = 12.0 µM
1 · s
1.
The
[Ca2+]i
at rest was taken to be 0.07 µM (for references see Ref.
5).
The change in fluorescence above the resting level was expressed as a
fraction of Fmax
Frest, where
Fmax and
Frest denote, respectively, the
maximum and resting fluorescence signals.
Fmax
Frest was determined at the end of
the experiment by exposing the fiber to a solution containing 0.1 mg/ml
saponin and 95 mM CaCl2 as described previously (5).
The peak amplitude of the fluo 3 signal recorded during the isometric
twitch under control conditions in the same experiments, expressed as a
fraction of Fmax
Frest, was determined in each
case, and the mean value (±SE) of the ratio was 0.37 ± 0.02 (n = 14). The latter value was used for calibrations in all experiments presented in this study.
Recording and analysis of data.
The optical signals, the output of the force
transducer, and the stimulation signals were fed into a data
acquisition and analysis system (Asystant+, Asyst Software
Technologies, Rochester, NY). The data were collected at a sample rate
of 1 kHz and were stored on diskettes for later analysis.
All statistics are given as means ± SE. Student's paired
t-test was used for determination of
statistical significance. P < 0.05 (two-tailed) was considered statistically significant.
 |
RESULTS |
Effects of BAPTA on force response and
Ca2+ transient.
Figure 1 shows
the effect of BAPTA on the isometric twitch and tetanus in fibers that
were first exposed to 50 µM BAPTA-AM for 40 min and thereafter
reimmersed in the standard Ringer solution for the rest of the
experiment. The twitch force can be seen to be more effectively
suppressed by BAPTA than was the maximum tetanic response. As
illustrated in Fig. 1, the peak twitch tension decreased steadily as
the fiber was immersed in the BAPTA-AM solution, indicating that BAPTA
gradually accumulated inside the muscle fiber. The time for the peak
twitch force to decline to 50% of its control value in the presence of
50 µM BAPTA-AM was 23.6 ± 2.1 min
(n = 5). The results suggest that the
hydrolysis of BAPTA-AM inside the fiber was relatively slow, since the
peak twitch force continued to decrease after withdrawal of BAPTA-AM
from the bathing medium (Fig. 1).

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Fig. 1.
Changes in peak twitch force (squares) and maximum
tetanic force (circles) of frog single muscle fibers in presence of 50 µM BAPTA-AM (filled symbols) and after withdrawal of BAPTA from
bathing solution (open symbols). Means ± SE of 5 experiments.
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Figure 2 illustrates the effects of BAPTA
on isometric force and intracellular
Ca2+ transient during twitch and
tetanus. It can be seen that, at the time when the twitch force had
been reduced to ~50% of its control value in the presence of BAPTA
(Fig. 2Ab), the peak amplitude of
the corresponding Ca2+ transient
was virtually unaffected (Fig.
2Bb). The
Ca2+ transient during the twitch
still remained only slightly depressed (Fig.
2Bc) when the twitch force had
declined to 10% of the control (Fig.
2Ac). By further exposure to BAPTA,
the twitch response was almost completely abolished
(Fig. 2Ad). At this
stage, the Ca2+ transient after a
single stimulus was markedly depressed (Fig. 2Bd).

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Fig. 2.
Effects of BAPTA on force
(A and
C) and
Ca2+ transient
(B and
D) in a single muscle fiber during
isometric twitch (A and
B) and tetanus
(C and
D). BAPTA was loaded into fiber by
exposing fiber to 100 µM BAPTA-AM (see text).
a: Control.
b and
c: Twitch force reduced to ~50 and
10% of its control value, respectively.
d: Twitch force virtually abolished by
BAPTA. Traces for b,
c, and
d were obtained 22, 35, and 85 min,
respectively, after exposure of fiber to BAPTA-AM. Note that
[Ca2+]i
recorded during tetanic stimulation rises above control level in
presence of BAPTA. Sarcomere length, 2.2 µm; temperature,
10.4°C.
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The possibility that the decrease in isometric force during BAPTA
treatment was associated with an equivalent decline in fiber stiffness
was explored. To this end, force and stiffness were recorded
simultaneously throughout the contraction period as described in
METHODS. A series of recordings was
first performed to establish the relationship between force and
stiffness at different tension levels during tetanic stimulation in
ordinary Ringer solution. This was achieved by imposing a release step
during the tetanus plateau so as to produce a drop in tension to zero
load followed by redevelopment of force at the new length. The
amplitude of the release step was large enough to slacken the fiber, to
ensure that all attached cross bridges were dissociated before the
tension started to rise. Data from such measurements, plotted in Fig. 3, outline the relationship between force
and stiffness in the fully activated fiber in control Ringer solution.
Figure 3 also includes measurements of force and stiffness performed at
the peak of the isometric twitch and on the plateau of the isometric tetanus before the introduction of BAPTA and at different times after
addition of BAPTA-AM to the Ringer fluid. The results clearly demonstrate that the force-to-stiffness relationship was essentially the same in the presence and absence of BAPTA. The force-to-stiffness ratio, and therefore the force output per cross bridge, was thus unaffected by BAPTA under the conditions used. This supports the view
that the decrease in contractile force recorded in the presence of
BAPTA-AM may be accounted for by fewer attached cross bridges.

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Fig. 3.
Relationship between fiber stiffness and isometric
force measured in a single muscle fiber before and after exposure to 50 µM BAPTA-AM. Open squares are measurements at different tension
levels during redevelopment of force after a release step during
tetanus plateau in control Ringer solution. Triangles and circles are
measurements performed at peak of isometric twitch and on plateau of
isometric tetanus, respectively, before (open symbols) or after (solid
symbols) introduction of BAPTA. Note that decrease in active force
after exposure to BAPTA is associated with a decrease in fiber
stiffness and that same force-stiffness relationship holds true as in
control Ringer solution.
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The depressant effect of BAPTA on twitch force was, to a great extent,
overcome during tetanic stimulation in that repetitive stimuli led to a
continuous rise of force (Fig. 2C).
Even at a stage when the single twitch response had nearly disappeared, the tetanic tension climbed to ~80% of the maximum force recorded under control conditions (Fig.
2Cd). It is noteworthy that the Ca2+ transient, being markedly
depressed at the onset of the stimulation volley, finally reached a
higher level at the end of the tetanus period than before treatment
with BAPTA (Fig.
2Dd). Results similar to those shown
in Fig. 2 were obtained in altogether eight experiments and are
summarized in Table 1.
The above results seem to indicate that BAPTA does not simply act by
chelating Ca2+ in the myofibrillar
space. It was therefore of interest to investigate whether the effects
of BAPTA were reproducible by another frequently used
Ca2+ chelator, EGTA.
Effects of EGTA on force response and
Ca2+ transient.
EGTA was loaded into the fibers by immersing the fiber
in Ringer solution containing 100 µM EGTA-AM using the same procedure as employed for BAPTA-AM (see
METHODS). Figure
4 shows the effects of EGTA on isometric
force and intracellular Ca2+
transient during twitch and tetanus. The effects of EGTA on the isometric twitch and tetanus were similar to those produced by BAPTA.
However, a considerably longer time was required for EGTA to depress
the twitch force. The time for the peak twitch tension to decline to
50% of its control value in the presence of 100 µM EGTA-AM was 44.3 ± 11.7 min (n = 4), i.e., nearly
twice as long as required with 50 µM BAPTA-AM (see earlier).

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Fig. 4.
Effects of EGTA on force
(A and
C) and
Ca2+ transient
(B and
D) in a single muscle fiber during
isometric twitch (A and
B) and tetanus
(C and
D). EGTA was loaded into fiber by
exposing fiber to 100 µM EGTA-AM (see text).
a: Control.
b and
c: Twitch force reduced to ~50 and
10% of its control value, respectively.
d: Twitch force almost abolished by
EGTA. Traces for b,
c, and
d were obtained 30, 75, and 110 min,
respectively, after exposure of fiber to EGTA-AM. Note that
[Ca2+]i
recorded during tetanic stimulation is steadily reduced below control
value in presence of EGTA. Sarcomere length, 2.2 µm; temperature,
10.7°C.
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Similar to the situation with BAPTA, the peak amplitude of the
Ca2+ transient was depressed
proportionally less than was the twitch force in the presence of EGTA.
The effects of the two agents on the time course of the
Ca2+ transient were, on the other
hand, markedly different. This is illustrated in Fig.
5, which shows comparable records of the
isometric twitch force and the
Ca2+ transient under control
conditions and after depression of the twitch to ~10% of the control
value by BAPTA and EGTA. In spite of a moderate decrease in amplitude
of the Ca2+ transient in the
presence of BAPTA, the decay phase of
[Ca2+]i
coincided well with that recorded in the control (Fig.
5A). This is attributable to a
slowing of the decay phase of the
Ca2+ transient that was induced by
BAPTA. The rate of decay of the Ca2+ transient was determined by
fitting a single-exponential function to the first 50 ms of the decay
phase, as described previously (24). The rate
constant of
[Ca2+]i
decay during twitch contraction was reduced by BAPTA with high statistical significance (P < 0.001) at a time when the twitch force had been depressed to 50 and
10% of its control value, respectively (Table 1). EGTA was likewise
found to reduce the amplitude of the
Ca2+ transient but, contrary to
the situation with BAPTA, the rate of decay of the transient was not
significantly changed (Table 1). As a consequence, the duration of the
Ca2+ transient was substantially
abbreviated by EGTA (Fig. 5B).

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Fig. 5.
Comparative effects of BAPTA
(A) and EGTA
(B) on isometric twitch
(top) and
Ca2+ transient
(bottom) from 2 experiments. Records
from same fibers as in Figs. 2 and 4. Solid lines, controls; dashed
lines, time when isometric twitch had been reduced to ~10% of its
control value. Note that, in presence of BAPTA, time course of decay of
[Ca2+]i
coincides with that of control record despite a moderate decrease in
amplitude. In contrast, duration of
Ca2+ transient is shortened in
presence of EGTA.
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Unlike the finding in the presence of BAPTA, there was no tendency of
the Ca2+ transient to increase
above the control level during tetanic stimulation in the presence of
EGTA (Fig. 4). On the contrary, the
[Ca2+]i
attained during tetanic stimulation was steadily reduced during exposure to EGTA-AM. This difference in relation to BAPTA is striking when Figs. 2D and
4D are compared. The isometric tetanus
is here moderately depressed in both cases. However, although
[Ca2+]i
can be seen to be markedly increased in the presence of
BAPTA, [Ca2+]i
is reduced to merely one-third of its control value in
the presence of EGTA.
 |
DISCUSSION |
The effects of BAPTA, a high-affinity
Ca2+ chelator (25), were studied
on the mechanical response and the
Ca2+ transient in frog single
muscle fibers. The fibers were exposed to BAPTA in its permeant AM form
using relatively low (50 or 100 µM) concentrations in the bathing
medium. The results demonstrate that BAPTA produced a marked depression
of the isometric twitch associated with a moderate reduction of the
tetanic force. These effects would, a priori, be expected to result
from the Ca2+-buffering action of
BAPTA, since it has previously been reported that BAPTA, in millimolar
concentrations, virtually eliminates the
Ca2+ transient in response to
electrical stimulation (16). However, the contractile changes observed
in this study did not show any clear correlation with the
Ca2+ transient in the presence of
BAPTA. Thus during twitch stimulation the peak amplitude of the
Ca2+ transient did not start to
decline until the force had been reduced to <50% of its control
value in the presence of BAPTA. Furthermore, during tetanic stimulation
[Ca2+]i
actually increased above the control level while the
force was moderately reduced by BAPTA. It is furthermore of interest to
note that, due to a lower rate of decline of
[Ca2+]i
in the presence of BAPTA, the decay phase of the
Ca2+ transient coincided well with
that in the control even after the twitch had been depressed to ~10%
of the control value by BAPTA. This is a pertinent finding in view of
our previous observation (24) that the decay phase of the
Ca2+ transient is a far more
important determinant of the mechanical response during an isometric
twitch than is the peak amplitude of the transient. The same conclusion
has recently been reached by Johnson et al. (18). The marked depression
of the isometric twitch by BAPTA observed under these conditions
therefore cannot be attributed to the changes of the
Ca2+ transient. Clearly these
observations cannot be explained by the
Ca2+-buffering action of BAPTA.
The effects of BAPTA on force and
Ca2+ transient were compared with
the changes produced by an alternative
Ca2+ chelator, EGTA, likewise
applied in its permeant AM form. Unlike BAPTA, EGTA depressed the
Ca2+ transient both during the
twitch and during the tetanus. The decrease in twitch amplitude by EGTA
was associated with only a slight reduction of the peak amplitude of
the Ca2+ transient, but EGTA
shortened the duration of the Ca2+
transient, leading to considerably lower
[Ca2+]i
during the decay phase of the Ca2+
transient than in the control (Fig. 5). As pointed out in the foregoing, the latter effect can be presumed to be mainly responsible for the decrease of the twitch response (18, 24). In further contrast
with BAPTA, at a stage when the isometric tetanus was reduced by EGTA
there was a corresponding decrease in
[Ca2+]i
recorded during tetanic stimulation. The contractile changes induced by
EGTA thus seem to be in full accord with the
Ca2+-buffering ability of this
agent. That is, by chelating the
Ca2+, EGTA inhibits the activation
of the contractile machinery, thus reducing the number of cross bridges
formed during muscle contraction. The comparison with EGTA further
strengthens the view that BAPTA exerts some specific effect that is not
mediated by chelation of Ca2+ in
the myofibrillar space.
Our stiffness measurements (Fig. 3) indicate that the decrease in
mechanical performance in the presence of BAPTA is attributable to a
reduced number of active cross bridges with no change in the force
output of the individual bridge. This provides evidence that the state
of activation of the contractile system is reduced in the presence of
BAPTA in spite of the fact that the
[Ca2+]i
is little affected during the twitch and actually rises above the
control level during tetanic stimulation. This opens the possibility that BAPTA impedes the binding of
Ca2+ to the regulatory sites of
troponin, resulting in a reduced state of activation of the contractile
system. This inhibitory effect of BAPTA, which leads to almost complete
suppression of the isometric twitch, may be largely overcome during
tetanic stimulation, as the increase in
[Ca2+]i
during the tetanus will compensate for the reduced affinity of
Ca2+ for the troponin-binding
sites. This is in line with the observation that the tetanic force is
only slightly reduced at a stage when the isometric twitch has been
virtually abolished by BAPTA. So far there is no biochemical data in
the literature to support or oppose the idea that BAPTA interferes with
the binding of Ca2+ to troponin.
There is evidence, however, that BAPTA does interact with several other
Ca2+-binding proteins in the
muscle fiber, e.g., parvalbumin and calmodulin (6).
It has been pointed out previously that the amount of
Ca2+ released from SR is much
greater than indicated by the Ca2+
transient (3). The amplitude of the
Ca2+ transient during a twitch
contraction is thus generally found to be 2-10 µM, whereas the
total Ca2+ released from SR is
estimated to be several hundred micromolar (2, 11). This discrepancy is
explained by the fact that the major portion of the released
Ca2+ is rapidly bound to various
intracellular structures, most significantly to the
Ca2+-specific sites of troponin C,
which are able to bind ~140 µM Ca2+ (8). If BAPTA at a low
intracellular concentration predominantly acts as an inhibitor of
Ca2+ at the troponin-binding
sites, the
[Ca2+]i
would tend to increase while the mechanical response is reduced. However, with increasing concentration of BAPTA in the myoplasm, the
Ca2+ transient will finally be
reduced. This is in line with the observation that, after long exposure
of the fiber to BAPTA-AM, the Ca2+
transient during a single twitch (and during the early phase of a
tetanus) becomes greatly depressed (Fig. 2). Parvalbumin is generally
thought to serve as a temporary sink and storage site for
Ca2+ in the myoplasm before
Ca2+ is finally taken up by the SR
(12). However, the Ca2+-buffering
capacity of parvalbumin may be reduced in the presence of BAPTA (6).
This may contribute to the conspicuous climb of
[Ca2+]i
above the control level during tetanic stimulation at a stage when
BAPTA causes a moderate depression of the tetanic force (Fig. 2).
An alternative explanation of some of the present findings could be
that BAPTA and fluo 3 are nonuniformly distributed within the fiber
volume. For example, if fluo 3 were mainly confined to a compartment
close to the Ca2+-release channel
of the SR, while BAPTA reached its highest concentration around the
myofilaments, the concentration of
Ca2+ in the close vicinity of
troponin might be lower than indicated by the fluo 3 signal. However,
recent studies based on confocal imaging techniques (19, 23) make such
a mechanism appear highly unlikely in that fluo 3 is found to be
distributed quite uniformly within the sarcomeres with only a minor
increase in fluo 3 fluorescence in the Z-line region of the resting
fiber (19). Furthermore, nonuniform distribution of BAPTA and fluo 3 would not explain the observation that the
Ca2+ transient is
increased during tetanic stimulation at a time when the
twitch response is greatly reduced in the presence of BAPTA.
 |
ACKNOWLEDGEMENTS |
We are grateful to Britta Kronborg for excellent technical
assistance.
 |
FOOTNOTES |
The study was supported by grants from the Swedish Medical Research
Council (project no. 14X-184) and the Medical Faculty, Lund University,
Lund, Sweden.
Some of the results were presented in a preliminary form (10).
Present addresses: Y.-B. Sun, The Randall Institute, King's College
London, 26-29 Drury Lane, London WC2B 5RL, UK; C. Caputo, Laboratorio de Biofisica del Musculo, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas, Caracas 1020A, Venezuela.
Address for reprint requests: K. A. P. Edman, Dept. of Pharmacology,
University of Lund, Sölvegatan 10, S-223 62 Lund, Sweden.
Received 11 December 1997; accepted in final form 21 April 1998.
 |
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