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
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Abstract
Introduction
Methods
Results
Discussion
References

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(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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.

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.

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.

                              
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Table 1.   Effects of BAPTA and EGTA on force and intracellular Ca2+ transient during isometric twitch and tetanus

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.

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.

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
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(2):C375-C381
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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