Effect of beta -adrenoceptor activation on [Ca2+]i regulation in murine skeletal myotubes

Y. S. Prakash1, H. F. M. van der Heijden2, E. M. Gallant3, and G. C. Sieck1

1 Departments of Anesthesiology and of Physiology and Biophysics, Mayo Clinic and Foundation, Rochester 55905; 3 Department of Veterinary Pathobiology, University of Minnesota, St. Paul, Minnesota 55108; and 2 Department of Pulmonary Diseases, University Hospital Nijmegen, 6500HB Nijmegen, The Netherlands


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study used real-time confocal microscopy to examine the effects of the beta 2-adrenoceptor agonist salbutamol on regulation of intracellular Ca2+ concentration ([Ca2+]i) in myotubes derived from neonatal mouse limb muscles. Immunocytochemical staining for ryanodine receptors and skeletal muscle myosin confirmed the presence of sarcomeres. The myotubes displayed both spontaneous and ACh-induced rapid (<2-ms rise time) [Ca2+]i transients. The [Ca2+]i transients were frequency modulated by both low and high concentrations of salbutamol. Exposure to alpha -bungarotoxin and tetrodotoxin inhibited ACh-induced [Ca2+]i transients and the response to low concentrations of salbutamol but not the response to higher concentrations. Preexposure to caffeine inhibited the subsequent [Ca2+]i response to lower concentrations of salbutamol and significantly blunted the response to higher concentrations. Preexposure to salbutamol diminished the [Ca2+]i response to caffeine. Inhibition of dihydropyridine-sensitive Ca2+ channels with nifedipine or PN-200-110 did not prevent [Ca2+]i elevations induced by higher concentrations of salbutamol. The effects of salbutamol were mimicked by the membrane-permeant analog dibutyryl adenosine 3',5'-cyclic monophosphate. These data indicate that salbutamol effects in skeletal muscle predominantly involve enhanced sarcoplasmic reticulum Ca2+ release.

adenosine 3',5'-cyclic monophosphate; ryanodine receptor; sarcoplasmic reticulum; skeletal muscle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION OF beta -adrenoceptors by agonists such as isoproterenol and salbutamol (a beta 2-adrenoceptor agonist) increases force production in skeletal muscle fibers (3-7, 13, 22). Previous studies on terbutaline effects on isolated rat limb muscle fibers have suggested that the positive inotropic effect of beta -adrenoceptor activation involves enhanced excitation-contraction (EC) coupling, but not via alterations in action potential profiles and Na+-K+ pump activity (4, 5, 7). These results suggest an elevation in intracellular Ca2+ concentration ([Ca2+]i) at the level of the sarcoplasmic reticulum (SR). beta -Adrenoceptor activation may lead to enhanced SR Ca2+ release or decreased Ca2+ reuptake via the SR Ca2+-ATPase.

The effects of beta -adrenoceptor stimulation on [Ca2+]i regulation in skeletal muscle may be mediated through one or more beta -adrenoceptor isoforms and may or may not involve cAMP. Previous studies in skeletal muscles from different species including humans have indicated a predominance of beta 2-adrenoceptors compared with other subtypes (8, 15). Other studies in intact skeletal muscle have suggested a cAMP-dependent mechanism for the action of beta -adrenoceptors (4-6). There is also biochemical evidence that cAMP facilitates SR Ca2+ release and enhances the activity of ryanodine receptor (RyR) channels (21). Furthermore, cAMP accumulation in skeletal muscle upon beta -adrenoceptor stimulation also appears to predominantly involve beta 2-adrenoceptors (25). Accordingly, the purpose of the present study was to investigate the effects of salbutamol, a specific beta 2-adrenoceptor agonist, on [Ca2+]i regulation in dissociated murine skeletal myotubes. Real-time confocal microscopy of [Ca2+]i transients in single myotubes was used to assess the mechanisms by which salbutamol elevates [Ca2+]i.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All procedures used in this study were approved by the Institutional Animal Care and Use Committees of the University of Minnesota and the Mayo Clinic and were in strict accordance with the American Physiological Society Animal Care Guidelines.

Myotube Cultures

Isolation of mouse myoblast cultures was based on a previously described procedure (10-12). Hindlimb muscles of neonatal (1-3 days) mice were enzymatically digested with trypsin and mechanically dissociated. The tissue was then differentially centrifuged and resuspended in DMEM containing 10% FCS. The cells were plated on laminin-coated coverslips at 2 × 105 cells/plate. The plating medium used was 83% DMEM with 4.5 g/l glucose, 15% FCS, 100 U/ml penicillin, and 10 µg/ml streptomycin. After 24 h in culture, the plates were rinsed in Ca2+- and Mg2+-free rodent Ringer solution (in mM: 155 NaCl, 5 KCl, and 10 HEPES; pH 7.4) and the medium was changed to DMEM with 4.5 g/l glucose and 10% FCS. When the cultures were nearly confluent, cell fusion was induced by changing the medium to a mouse maintenance medium (88% DMEM with 4.5 g/l glucose, 10% horse serum, 100 U/ml penicillin, and 10 µg/ml streptomycin). Experiments were performed on myotubes within 8 days after fusion.

Confocal [Ca2+]i Imaging

Isolated myotubes were plated on laminin-coated glass coverslips, incubated in 5 µM fluo 3-AM (Molecular Probes, Eugene, OR) in Hanks' balanced salt solution (HBSS) at room temperature for 45 min, and then placed on an open slide chamber (Warner Instruments, Hamden, CT) mounted on a Nikon Diaphot inverted microscope. The chamber was perfused with HBSS at 1-2 ml/min at room temperature.

Details of the techniques for real-time confocal imaging of [Ca2+]i have been previously described (23). Briefly, fluo 3-loaded cells were visualized using an Odyssey XL real-time confocal system (Noran Instruments, Middleton, WI) attached to the Nikon microscope and equipped with an Ar-Kr laser. The Odyssey confocal system is capable of acquiring images at a rate up to 480 frames/s. In preliminary studies, we determined an appropriate sampling frequency to acquire the dynamic [Ca2+]i responses of myotubes without frequency aliasing (the appearance of higher frequency signals as being of lower frequency due to inadequate data sampling). An Olympus 40× 1.3-numerical aperture oil immersion objective lens was used for imaging with image size set to 640 × 480 pixels (0.06 mm2/pixel). Optical section thickness was set to 1 µm. With regions of interest (ROIs) of 15 × 15 pixels (13.5 mm2), [Ca2+]i measurements were obtained from volumes of 13.5 mm3 (<1% of total cell volume).

Although fluo 3 is not a ratiometric dye, [Ca2+]i levels can be calibrated using empirical techniques (23). This essentially involves the determination of fluorescence intensities at known [Ca2+]i levels. A fixed combination of laser intensity (20% of maximum) and photomultiplier gain (1,700 from a maximum of 4,096) was set a priori to ensure that pixel intensities within ROIs ranged between 25 and 255 gray levels (GL). A set of fluo 3-loaded myotubes were then sequentially exposed to HBSS containing 10 mM A-23187 (Ca2+ ionophore) and Ca2+ levels ranging from 0 nM (buffered with EGTA) to 10 µM. At each extracellular Ca2+ level, the GL changes in cellular fluorescence levels were recorded after the intracellular and extracellular Ca2+ concentrations had equilibrated in the presence of A-23187. The GL data and corresponding Ca2+ concentrations were then converted to a calibration curve that was used in subsequent protocols to convert fluorescence intensity to nanomolar Ca2+.

Characterization of [Ca2+]i Transients in Myotubes

More than 90% of the myotubes on a coverslip displayed spontaneous [Ca2+]i transients and accompanying contractions that varied in frequency even under resting conditions. Such spontaneous [Ca2+]i transients and contractions have been previously reported by other investigators and are thought to be typical of murine myotubes in vitro (9). The presence of these transients and contractions demonstrates that the EC coupling mechanism found in normal skeletal muscle fibers is also present in these myotubes. Because measurement of cell contraction was not a focus of the present study, the cells were exposed to 1 mM 2,3-butanedione monoxime (BDM) to prevent contractions. Previous studies have demonstrated that BDM interferes with the contractile machinery (actin-myosin cross bridges and related proteins) but does not significantly affect [Ca2+]i (2, 16). Addition of BDM also removed the confounding effect of contractions in the measurement of [Ca2+]i transients in localized regions of the myotube.

The [Ca2+]i transients were characterized by placing ROIs along the length of the cell such that the center of each ROI corresponded with a sarcomere. In an initial set of studies, the amplitude and frequency of spontaneous [Ca2+]i transients were examined at different acquisition rates ranging from 30 frames/s (33-ms resolution) to 480 frames/s (~2-ms resolution) to determine an appropriate sampling frequency with which both the amplitude and frequency could be reliably and reproducibly measured. We found that the amplitude and frequency of [Ca2+]i transients measured at 480 frames/s were not significantly different from those measured at 30 frames/s, indicating that both of these parameters could be reliably measured at a slower acquisition rate. However, at an acquisition rate of 30 frames/s (33-ms resolution), the rise and fall times of each [Ca2+]i transient could not be reliably measured, indicating insufficient sampling. Due to limitations in data storage and analysis, it was not possible to record data at 480 frames/s in the main protocols. Therefore, only a few cells were analyzed at 480 frames/s to characterize the rise and fall time of the [Ca2+]i transients. In these cells, the temporal delay in the peak amplitude between adjacent ROIs was used to estimate the extent of synchronization vs. propagation of the [Ca2+]i response along the length of the cell. In other protocols, data were acquired at 30 frames/s, and only amplitude and frequency were recorded.

Selection of Myotubes for [Ca2+]i Measurements

At confluence, the coverslip was typically dense with different cell types, including myotubes and fibroblasts. To ensure that [Ca2+]i measurements were obtained only from myotubes, we selected cells that were at least 250 µm in length and 25 µm in width, had apparent sarcomeric structural pattern (see Fig. 1), and did not display any visible processes. There were also a number of seemingly contracted myotubes that were spherical in appearance; these cells were excluded from analysis. At least three cells were selected from each coverslip. Typically, cells from one coverslip were used for only one experimental protocol.

After [Ca2+]i measurements, the cells were fixed in situ using 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After fixation, the cells were permeabilized with 0.5% Triton-X 100 for 20 min and stained with a rabbit polyclonal antibody to RyR (Calbiochem) or with a mouse monoclonal antibody to skeletal muscle myosin (Sigma). The antibody staining was visualized using a indocarbocyanine-conjugated secondary antibody and viewed on a Bio-Rad 500/600 confocal microscope, using previously described techniques (24). Using this approach, we confirmed that the [Ca2+]i measurements were indeed obtained only from myotubes.

Experimental Protocols

Effect of tetrodotoxin (TTX) on spontaneous [Ca2+]i transients. To determine whether the [Ca2+]i transients were accompanied by action potentials, cells were coloaded with fluo 3 and the voltage-sensitive dye RH-414 (5 µM; Molecular Probes) and then exposed to 0.5% TTX in HBSS to block voltage-dependent Na+ channels. The different excitation/emission characteristics of the two indicators allowed the distinction between [Ca2+]i transients and changes in membrane potential. According to the manufacturer's specifications, RH-414 has a sensitivity of ~10% change in fluorescence for a 100-mV change in membrane potential. Due to the relatively low sensitivity of this dye, no attempt was made to convert fluorescence changes to millivolt values. Instead, the dye was used simply to monitor whether changes in membrane potential accompanied the changes in [Ca2+]i.

Effects of caffeine and ryanodine on spontaneous [Ca2+]i transients. To determine whether [Ca2+]i transients involve SR Ca2+ release through RyR channels, cells were exposed to either 10 µM or 1 mM caffeine in HBSS. In a second set of experiments, cells were exposed to 10 µM ryanodine in HBSS.

Effect of ACh stimulation on [Ca2+]i transients. After measurement of [Ca2+]i transients under resting conditions, the cells were successively exposed to a range of ACh concentrations (1 nM, 10 nM, 100 nM, and 1 µM). In a second set of experiments, cells were preexposed to 0.05% alpha -bungarotoxin (BTX) in HBSS to block nicotinic receptors. The cells were then exposed to either 1 nM or 1 µM ACh. In a third set of experiments, cells were preexposed to 10 µM nifedipine or PN-200-110 to inhibit the dihydropyridine-sensitive Ca2+ channels (slow Ca2+ current). The cells were then exposed to 1 nM or 1 µM ACh.

Effect of salbutamol on [Ca2+]i transients. After measurement of [Ca2+]i transients under resting conditions, the cells were successively exposed to a range of salbutamol concentrations (1 nM, 10 nM, 100 nM, and 1 µM; salbutamol obtained from Glaxo-Wellcome).

To determine whether the effect of salbutamol on [Ca2+]i transients is mediated by changes in the generation of action potentials, cells were preexposed to 0.5% TTX and then exposed to either 1 nM or 1 µM salbutamol.

To determine the interactions between salbutamol and dihydropyridine-sensitive Ca2+ channels, cells were preexposed to 10 µM nifedipine or PN-200-110 and then exposed to 1 nM or 1 µM salbutamol.

To determine whether the effects of salbutamol on [Ca2+]i transients were mediated via modulation of SR Ca2+ release, cells were first exposed to 1 mM caffeine and then to 1 nM or 1 µM salbutamol. In a second set of studies, cells were first exposed to 1 nM or 1 µM salbutamol and then to 1 mM caffeine. In a third set of studies, cells were exposed to 10 µM ryanodine and then to 1 nM or 1 µM salbutamol.

Effect of dibutyryl adenosine 3',5'-cyclic monophosphate on [Ca2+]i transients. To determine whether cAMP mimics salbutamol effects on [Ca2+]i transients, cells were exposed to 10 or 100 µM dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), a membrane-permeant cAMP analog, and then exposed to 1 mM caffeine in HBSS.

Statistical Analysis

The effects of various drugs on [Ca2+]i transients were predominantly characterized by changes in the amplitude and frequency of the transients. In some protocols, the rise time and fall time of the [Ca2+]i transients were used to detect differences. Student's t-tests were used to test for statistical significance at a P < 0.05 level. All values are reported as means ± SE. The numbers of myotubes are given with the results for each experimental protocol. A total of 241 myotubes were examined in the present study.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of [Ca2+]i Transients in Myotubes

As mentioned previously, >90% of the myotubes on a coverslip displayed spontaneous [Ca2+]i transients (Fig. 1) and accompanying contractions that were reversibly inhibited by BDM. However, in all the protocols reported here, BDM was used to block contraction.


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Fig. 1.   Mature murine skeletal myotubes expressed a sarcomeric structure that was visualized by fixing myotubes in paraformaldehyde and staining for skeletal myosin using a polyclonal antibody. A: antibody staining was visualized using a fluorescently tagged secondary antibody and confocal microscopy. B: mature myotubes displayed spontaneous intracellular Ca2+ concentration ([Ca2+]i) transients that were visualized in fluo 3-loaded cells using real-time confocal microscopy. Box in A indicates a representative region of interest from which [Ca2+]i measurements were made. Similar [Ca2+]i transients (and accompanying contractions) have been reported by other investigators (9) and suggest that excitation-contraction coupling of normal skeletal muscle fibers is present in myotubes in vitro.

In cells that were sampled at 480 frames/s, the rise time was found to range between 4 and 10 ms and the fall time ranged between 14 and 28 ms (Table 1; n = 72). At an acquisition rate of 480 frames/s, the synchrony of [Ca2+]i transients across two to five ROIs within a cell was evaluated by measuring the time difference between peaks of the [Ca2+]i transients (n = 12 cells). At a 2-ms resolution, no difference in the incidence of [Ca2+]i transients was detected across ROIs, indicating that the elevation of [Ca2+]i throughout the myotube was synchronous at the maximum temporal resolution.

                              
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Table 1.   Characteristics of [Ca2+]i transients in murine skeletal myotubes

The amplitude of the spontaneous [Ca2+]i transients was fairly constant within an ROI (<10% coefficient of variation), but there was more variability across ROIs and cells, with values ranging between 75 and 450 nM (Table 1; n = 248). The frequency of spontaneous [Ca2+]i transients was also invariant within a cell (<10% coefficient of variation), but across cells frequencies ranged between 0.5 and 6.7 s-1 (Table 1; n = 248).

Effect of TTX on spontaneous [Ca2+]i transients. The synchrony of [Ca2+]i transients across different ROIs within a cell suggested that the transients were driven by action potentials. In support of that suggestion, in a set of myotubes that were coloaded with fluo 3 and RH-414 (n = 8), the [Ca2+]i transients were accompanied by changes in membrane potential. At a 2-ms resolution, a time delay of 4 ms was detected between the peak of the RH-414 signal and the peak of the [Ca2+]i transient. However, due to hardware limitations of the confocal system, it was not possible to further characterize the temporal relationship between membrane potential changes in the [Ca2+]i transients. Nonetheless, it was observed that exposure to 0.5% TTX irreversibly inhibited spontaneous [Ca2+]i transients (Fig. 2), confirming that they were action potential-driven events.


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Fig. 2.   Effect of tetrodotoxin (TTX; bar) on [Ca2+]i transients. Exposure to TTX inhibited [Ca2+]i transients, confirming that they were action potential-driven events. HBSS, Hanks' balanced salt solution.

Effect of caffeine and ryanodine on spontaneous [Ca2+]i transients. Exposure to 10 µM caffeine resulted in a significant increase in the frequency (163 ± 36% of preexposure values; n = 14; P < 0.05) as well as in the amplitude (119 ± 20% of preexposure values; P < 0.05) of [Ca2+]i transients. Exposure to 1 mM caffeine produced a large transient [Ca2+]i elevation (amplitude 244 ± 39% of spontaneous transients before exposure; n = 15; P < 0.05) and inhibited further repetitive transients (Fig. 3). Washout of caffeine resulted in a slow recovery of spontaneous transients over a 20- to 25-min period.


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Fig. 3.   Effects of caffeine on spontaneous [Ca2+]i transients. Exposure to high caffeine concentrations produced a large [Ca2+]i elevation and inhibited further transients. Washout of caffeine reinitiated transients after ~10 min.

In a second set of experiments, exposure to 10 µM ryanodine produced a slow inhibition (~2 min) of ongoing [Ca2+]i transients with accompanying reductions in frequency as well as amplitude (n = 8).

Effect of ACh stimulation on [Ca2+]i transients. Exposure to ACh resulted in a dose-dependent modulation in the frequency, but not the amplitude, of [Ca2+]i transients (Fig. 4A and Table 2; n = 8).


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Fig. 4.   Effect of ACh on [Ca2+]i transients. A: ACh exposure resulted in a dose-dependent increase in frequency but not in amplitude of transients. Ellipses represent 1-min gaps. B: exposure to alpha -bungarotoxin (BTX) inhibited spontaneous [Ca2+]i transients and prevented subsequent [Ca2+]i response to ACh. C: inhibition of Ca2+ influx current using nifedipine (or PN-200-110, not shown) also inhibited [Ca2+]i transients and prevented response to ACh.


                              
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Table 2.   Effects of ACh and salbutamol on [Ca2+]i transients in murine myotubes

In a second set of experiments, exposure to 0.05% BTX inhibited ongoing spontaneous [Ca2+]i transients (Fig. 4B; n = 14). Subsequent exposure to either 1 nM (n = 8) or 1 µM (n = 6) ACh did not elicit any [Ca2+]i elevation.

In a third set of experiments, exposure to either 10 µM nifedipine (n = 9) or PN-200-110 (n = 12) also inhibited ongoing [Ca2+]i transients (Fig. 4C). Subsequent exposure to either 1 nM (n = 12) or 1 µM (n = 9) ACh did not elicit any [Ca2+]i elevation.

Effect of salbutamol on [Ca2+]i transients. Exposure to salbutamol resulted in a dose-dependent increase in the frequency as well as in the amplitude of ongoing [Ca2+]i transients (Fig. 5A and Table 2; n = 31). Exposure to 1 µM salbutamol resulted in a large [Ca2+]i transient and prevented subsequent [Ca2+]i transients.


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Fig. 5.   Effect of salbutamol (SLB) on [Ca2+]i transients. A: salbutamol exposure resulted in a dose-dependent increase in frequency as well as in amplitude of transients. Exposure to high salbutamol concentrations resulted in a large transient, akin to response to caffeine. Ellipses represent 1-min gaps. B: even in presence of TTX, salbutamol produced a large [Ca2+]i transient, indicating that effects of salbutamol are not mediated via an effect on action potential. C: as with TTX, preexposure to nifedipine did not prevent subsequent [Ca2+]i response to salbutamol.

Preexposure to 0.5% TTX inhibited spontaneous [Ca2+]i transients and prevented a subsequent [Ca2+]i response to 1 nM salbutamol (n = 8). However, even in the presence of TTX, 1 µM salbutamol produced a large [Ca2+]i transient (Fig. 5B).

As with TTX, preexposure to 10 µM nifedipine (n = 7) or PN-200-110 (n = 8) prevented a subsequent [Ca2+]i response to 1 nM salbutamol but did not prevent the large [Ca2+]i transient in response to 1 µM salbutamol (Fig. 5C).

Exposure to 1 mM caffeine produced a large [Ca2+]i transient (944 ± 38 nM). In the continued presence of caffeine, there was no [Ca2+]i response to 1 nM salbutamol (n = 9) and an extremely small response to 1 µM salbutamol (n = 10) (Fig. 6A), compared with the response with salbutamol alone (15 ± 4% of salbutamol response; 6 ± 3% of caffeine response). In a second set of studies, exposure to 1 nM salbutamol did not have a significant impact on the subsequent [Ca2+]i response to 1 mM caffeine (n = 13). However, exposure to 1 µM salbutamol significantly blunted the subsequent [Ca2+]i response to caffeine (n = 15; Fig. 6B). In a third set of studies, preexposure to 10 µM ryanodine inhibited the subsequent [Ca2+]i response to both 1 nM (n = 7) and 1 µM (n = 9) salbutamol (Fig. 6C).


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Fig. 6.   Interaction between ryanodine receptor (RyR) channels and salbutamol. A: exposure to caffeine produced a large [Ca2+]i transient. In continued presence of caffeine, subsequent [Ca2+]i response to salbutamol was considerably blunted. B: conversely, preexposure to salbutamol significantly blunted subsequent [Ca2+]i response to caffeine. C: preexposure to a blocking concentration of ryanodine inhibited subsequent [Ca2+]i response to salbutamol, indicating that salbutamol induces sarcoplasmic reticulum Ca2+ release through RyR channels.

Effect of DBcAMP. Exposure to 10 µM DBcAMP increased the frequency and amplitude of spontaneous [Ca2+]i transients (n = 16; Fig. 7). Exposure to 100 µM DBcAMP produced a large [Ca2+]i elevation and blunted the subsequent response to caffeine.


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Fig. 7.   Effect of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). Exposure to membrane-permeant cAMP analog DBcAMP increased frequency and amplitude of spontaneous [Ca2+]i transients. Exposure to high concentrations of DBcAMP produced a large [Ca2+]i elevation (not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study is a direct demonstration of beta -adrenoceptor-induced SR Ca2+ release via RyR channels in single skeletal muscle cell preparations. Dose-dependent differences in the interactions between different drugs and salbutamol suggest effects on Ca2+ influx at lower concentrations. The fact that the [Ca2+]i response to DBcAMP mimics the response to salbutamol suggests that salbutamol-induced SR Ca2+ release occurs through a cAMP-dependent mechanism. On the other hand, the lack of an effect of TTX or BTX on the [Ca2+]i response to salbutamol indicates that beta -adrenoceptor action on the action potential is not significant.

Murine Myotube Model

In the present study, we used dissociated murine myotubes to examine the effects of salbutamol on [Ca2+]i regulation. A potential issue with such a preparation is whether the EC coupling normally found in skeletal muscle exists in dissociated myotubes. Compared with intact muscle, in which SR Ca2+ release is the predominant factor in [Ca2+]i regulation, Ca2+ influx plays an additional role in [Ca2+]i regulation of dissociated myotubes. Nonetheless, in this case, the presence of action potentials, vigorous contractions associated with elevations in [Ca2+]i (reversibly blocked by BDM), and a clear sarcomeric pattern justifies the cellular preparation. In this regard, it must be emphasized that in the present study the spontaneous action potentials and [Ca2+]i transients, apparently characteristic of this preparation (9), are simply used as a tool to examine salbutamol effects on [Ca2+]i regulation. By characterizing the mechanisms underlying these transients, different parameters such as frequency and amplitude can be used to distinguish whether salbutamol effects are mediated via alterations in membrane events (action potential, Ca2+ currents) or at the SR.

Sampling Issues Associated With Confocal Imaging

The rapid kinetics of the [Ca2+]i transients in skeletal myotubes justify the use of real-time confocal imaging. In this regard, it is of interest that, although the rise and fall times of the [Ca2+]i transients could not be reliably detected at an acquisition rate of 30 frames/s, neither amplitude nor frequency were different when measured at 30 vs. 480 frames/s. Furthermore, there was no significant time-dependent variation in the measured amplitudes or frequencies within an ROI. Therefore, it appears unlikely that the measurements of amplitude and frequency obtained at <480 frames/s were confounded by sampling problems. On the other hand, the relatively minimal resolution with which rise and fall times could be measured even at 480 frames/s prevented a detailed characterization of the actual kinetics of the [Ca2+]i transients in skeletal myotubes and an evaluation of the effects of ACh and/or salbutamol on these parameters.

Effect of ACh on [Ca2+]i Transients

Exposure to ACh resulted in a dose-dependent modulation of the frequency of [Ca2+]i transients but did not significantly affect the amplitude. These data suggest that the effect of ACh was to enhance the activation of the Ca2+ regulatory system but not significantly alter the amount of subsequent Ca2+ released by the SR. Not surprisingly, the inhibition of ACh effects by BTX are consistent with a nicotinic receptor-mediated mechanism of ACh action. Furthermore, the blockade of ACh effects by nifedipine also indicates that the effects of ACh in murine myotubes are likely mediated via dihydropyridine-sensitive Ca2+ channels. However, the relevance of this latter finding to intact skeletal fibers may be only partial, since Ca2+ regulation in murine myotubes also involves Ca2+ influx through these channels, a component absent in intact skeletal muscle.

Effect of Salbutamol on [Ca2+]i Transients

In the present study, we observed that inhibition of [Ca2+]i transients with TTX did not significantly affect the subsequent [Ca2+]i response to high concentrations of salbutamol, suggesting that the site of action for beta -adrenoceptor agonists lies beyond the action potential. Further characterization of the interactions of salbutamol and the action potential was not possible in this study due to severe hardware and software limitations of the imaging system and the lack of calibration for the membrane-sensitive dye. Regardless, an effect of salbutamol on skeletal muscle action potential may not be significant, as some previous studies have determined using electrophysiological techniques to measure membrane potential changes following acute exposure to sympathomimetic amines (1, 22). A study by Cairns and Dulhunty (4) used ouabain to block the Na+-K+ pump and found that force potentiation in hindlimb muscles by terbutaline was unaffected. Furthermore, Cairns and Dulhunty (4) also found that both terbutaline and DBcAMP had no influence on K+ contractures, suggesting that the site of beta -adrenoceptor action is beyond the action potential-generating mechanisms. However, other studies have noted beta -adrenoceptor agonist-induced changes in muscle action potential in both innervated and denervated fast- and slow-twitch muscles (20). The discrepancy in various reports may be due to the variety of beta -adrenoceptor agonists used.

Our results using both nifedipine and PN-200-110 demonstrate that salbutamol-induced elevation of [Ca2+]i, at least at higher concentrations, is not mediated by alterations in the sarcolemmal Ca2+ influx current. These results are in contrast to some previous reports in which beta -adrenoceptor activation or application of cAMP protein kinase resulted in phosphorylation of dihydropyridine-sensitive Ca2+ channels and an enhancement of slow Ca2+ current (19). However, it must be noted that both nifedipine and PN-200-110 inhibited the effect of 1 nM salbutamol, suggesting that at lower concentrations salbutamol may indeed have an enhancing effect on the slow Ca2+ current. In this regard, interpretation of our results may be limited by the fact that direct measurements of Ca2+ channel activity were not performed. It is also possible that salbutamol increases Ca2+ influx through a nifedipine-insensitive pathway (1); however, such pathways have not been reported in mammalian skeletal muscle (4, 18).

The interactive effects of caffeine and salbutamol on [Ca2+]i regulation and the inhibition of salbutamol-induced elevation of [Ca2+]i by high concentrations of ryanodine confirm that salbutamol increases [Ca2+]i by enhancing SR Ca2+ release through RyR channels. The fact that the interaction between salbutamol and caffeine was more pronounced at higher concentrations of salbutamol suggests that at these concentrations increased SR Ca2+ release may be the predominant effect of salbutamol. On the other hand, the lack of an interaction at lower salbutamol concentrations suggests that increased Ca2+ influx may also play a role. From the present data, it is not possible to determine whether these dose-dependent effects are exclusive.

The fact that the [Ca2+]i responses to DBcAMP mimic those of salbutamol suggests a cAMP-dependent mechanism and is consistent with previous studies in intact skeletal muscle (4-6). There is already good biochemical evidence that cAMP facilitates SR Ca2+ release and enhances the activity of RyR channels. For example, Meissner (21) demonstrated that cAMP enhances the rate of Ca2+-induced Ca2+ release. Other studies have demonstrated cAMP-dependent protein kinase phosphorylation of RyR channels and accompanying increases in channel activity (14, 26, 28). Accordingly, the inhibition of salbutamol effects by caffeine and ryanodine suggests a common point of action. Of course, it is possible that the weak phosphodiesterase activity of caffeine itself may cause a small elevation in cAMP levels (17). However, the fact that preexposure to salbutamol also diminished the subsequent [Ca2+]i response to caffeine rules out this confounding effect.

From the results of the present study, it is not possible to determine whether salbutamol affects SR Ca2+ reuptake. Studies in cardiac muscle have demonstrated cAMP-dependent phosphorylation of phospholamban, thus increasing Ca2+ loading into the SR for subsequent release (27). Phospholamban is expressed only in slow-twitch skeletal muscle fibers (27). Of course, a limitation of myotube cultures is that it is not easily possible to determine "fiber type," and there is currently no information on whether these myotubes express phospholamban. Nonetheless, in pilot studies, we observed that the fall time of [Ca2+]i transients was decreased by ~20% during salbutamol exposure, suggesting that SR Ca2+ reuptake may have been affected. Further characterization of this issue awaits improvements in the temporal resolution of the imaging system.

In conclusion, the present study demonstrates that acute salbutamol treatment increases [Ca2+]i in skeletal myotubes predominantly by increasing SR Ca2+ release through RyR channels. In comparison, it appears that salbutamol may not significantly affect the action potential or the slow Ca2+ influx current in skeletal myotubes, at least at higher concentrations.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the contributions of John Snover in preparation of the murine myotubes.


    FOOTNOTES

This study was supported by National Institutes of Health Grants HL-37680 (to G. C. Sieck), HL-34817 (to G. C. Sieck), and AR-41270 (to E. M. Gallant) and by grants to H. F. M. van der Heijden from Glaxo-Wellcome (The Netherlands), the Van Walree Foundation, and the Royal Netherlands Academy of Arts and Sciences. Y. S. Prakash was supported by a fellowship from Abbott Laboratories.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. C. Sieck, Div. of Anesthesia Research, Mayo Clinic, Rochester, MN 55905 (E-mail: sieck.gary{at}mayo.edu).

Received 3 September 1998; accepted in final form 19 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arreola, J., J. Calvo, M. C. Garcia, and J. A. Sanchez. Modulation of calcium channels of twitch skeletal muscle fibres of the frog by adrenaline and cyclic adenosine monophosphate. J. Physiol. (Lond.) 393: 307-330, 1987[Abstract].

2.   Backx, P. H., W. D. Gao, M. D. Azan-Backx, and E. Marban. Mechanism of force inhibition by 2,3-butanedione monoxime in rat cardiac muscle: roles of [Ca2+]i and cross-bridge kinetics. J. Physiol. (Lond.) 476: 487-500, 1994[Abstract].

3.   Bowman, W. C., and C. Raper. The effects of adrenaline and other drugs affecting carbohydrate metabolism on contractions of the rat diaphragm. Br. J. Pharmacol. 23: 184-200, 1964.

4.   Cairns, S. P., and A. F. Dulhunty. Beta-adrenergic potentiation of E-C coupling increases force in rat skeletal muscle. Muscle Nerve 16: 1317-1325, 1993[Medline].

5.   Cairns, S. P., and A. F. Dulhunty. Beta-adrenoceptor activation shows high-frequency fatigue in skeletal muscle fibers of the rat. Am. J. Physiol. 266 (Cell Physiol. 35): C1204-C1209, 1994[Abstract/Free Full Text].

6.   Cairns, S. P., and A. F. Dulhunty. The effects of beta-adrenoceptor activation on contraction in isolated fast- and slow-twitch skeletal muscle fibres of the rat. Br. J. Pharmacol. 110: 1133-1141, 1993[Abstract].

7.   Cairns, S. P., H. Westerblad, and D. G. Allen. Changes of tension and [Ca2+]i during beta-adrenoceptor activation of single, intact fibres from mouse skeletal muscle. Pflügers Arch. 425: 150-155, 1993[Medline].

8.   Elfellah, M. S., R. Dalling, I. M. Kantola, and J. L. Reid. Beta-adrenoceptors and human skeletal muscle characterisation of receptor subtype and the effect of age. Br. J. Clin. Pharmacol. 27: 31-38, 1989[Medline].

9.   Flucher, B. E., and S. B. Andrews. Characterization of spontaneous and action potential-induced calcium transients in developing myotubes in vitro. Cell Motil. Cytoskeleton 25: 143-157, 1993[Medline].

10.   Gallant, E. M., E. M. Balog, and K. G. Beam. Slow calcium current is not reduced in malignant hyperthermic porcine myotubes. Muscle Nerve 19: 450-455, 1996[Medline].

11.   Gallant, E. M., and R. C. Jordan. Porcine malignant hyperthermia: genotype and contractile threshold of immature muscles. Muscle Nerve 19: 68-73, 1996[Medline].

12.   Gallant, E. M., N. S. Taus, T. F. Fletcher, L. R. Lentz, C. F. Louis, and J. R. Mickelson. Perchlorate potentiation of excitation-contraction coupling in mammalian skeletal muscles. Am. J. Physiol. 264 (Cell Physiol. 33): C559-C567, 1993[Abstract/Free Full Text].

13.   Gonzalez-Serratos, H., L. Hill, and R. Valle-Aguilera. Effects of catecholamines and cyclic AMP on excitation-contraction coupling in isolated skeletal muscle fibres of the frog. J. Physiol. (Lond.) 315: 267-282, 1981[Abstract].

14.   Hymel, L., H. Schindler, S. D. Yang, M. Inui, S. Reif, and S. Fleischer. Protein kinase/phosphatase modulation of purified skeletal muscle calcium release channel activity in planar bilayers (Abstract). Biophys. J. 55: 307a, 1989.

15.   Jensen, J., O. Brors, and H. A. Dahl. Different beta-adrenergic receptor density in different rat skeletal muscle fibre types. Pharmacol. Toxicol. 76: 380-385, 1995[Medline].

16.   Kostanas, G., S. M. Holroyd, I. R. Wendt, and C. L. Gibbs. Intracellular Ca2+, force and activation heat in rabbit papillary muscle: effects of 2,3-butanedione monoxime. J. Mol. Cell. Cardiol. 25: 1349-1358, 1993[Medline].

17.   Kramer, G. L., and J. N. Wells. Xanthines and skeletal muscle: lack of relationship between phosphodiesterase inhibition and increased twitch tension in rat diaphragm. Mol. Pharmacol. 17: 73-78, 1980[Abstract].

18.   Lamb, G. D., and T. Walsh. Calcium currents, charge movements and dihydropyridine binding in fast- and slow-twitch muscle of rat and rabbit. J. Physiol. (Lond.) 393: 595-617, 1987[Abstract].

19.   Lu, X., L. Xu, and G. Meissner. Phosphorylation of dihydropyridine receptor II-III loop peptide regulates skeletal muscle calcium release channel function. Evidence for an essential role of the beta-OH group of Ser687. J. Biol. Chem. 270: 18459-18464, 1995[Abstract/Free Full Text].

20.   McArdle, J. J., and J. D'Alonzo. Effects of terbutaline, a beta-adrenergic agonist, on the membrane potentials of innervated and denervated fast- and slow-twitch muscles. Exp. Neurol. 71: 134-143, 1981[Medline].

21.   Meissner, G. Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum. J. Biol. Chem. 259: 2365-2374, 1984[Abstract/Free Full Text].

22.   Oota, I., and T. Nagai. Effects of catecholamines on excitation-contraction coupling frog single twitch fiber. Jpn. J. Physiol. 27: 195-213, 1977[Medline].

23.   Prakash, Y. S., M. S. Kannan, and G. C. Sieck. Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells. Am. J. Physiol. 272 (Cell Physiol. 41): C966-C975, 1997[Abstract/Free Full Text].

24.   Prakash, Y. S., S. M. Miller, M. Huang, and G. C. Sieck. Morphology of diaphragm neuromuscular junctions on different fibre types. J. Neurocytol. 25: 88-100, 1996[Medline].

25.   Roberts, S. J., and R. J. Summers. Cyclic AMP accumulation in rat soleus muscle: stimulation by beta2- but not beta3-adrenoceptors. Eur. J. Pharmacol. 348: 53-60, 1998[Medline].

26.   Seiler, S., A. D. Wegener, D. D. Whang, D. R. Hathaway, and L. R. Jones. High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated and are degraded by Ca2+-activated protease. J. Biol. Chem. 259: 8550-8557, 1984[Abstract/Free Full Text].

27.   Tada, M., M. Kadoma, M. Inui, and J. Fujii. Regulation of Ca2+ pump from cardiac sarcoplasmic reticulum. Methods Enzymol. 157: 107-154, 1988[Medline].

28.   Timerman, A. P., C. C. Chadwick, and S. Fleischer. Phosphorylation states of skeletal muscle ryanodine receptor and smooth muscle inositol 1,4,5-trisphosphate receptor (IP3Rec) (Abstract). Biophys. J. 57: 286a, 1990.


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