1 Departments of Anesthesiology
and of Physiology and Biophysics, The present
study used real-time confocal microscopy to examine the effects of the
adenosine 3',5'-cyclic monophosphate; ryanodine
receptor; sarcoplasmic reticulum; skeletal muscle
ACTIVATION OF The effects of 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
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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
-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-adrenoceptors by agonists such as
isoproterenol and salbutamol (a
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
-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).
-Adrenoceptor activation may lead to enhanced SR
Ca2+ release or decreased
Ca2+ reuptake via the SR
Ca2+-ATPase.
-adrenoceptor stimulation on
[Ca2+]i
regulation in skeletal muscle may be mediated through one or more
-adrenoceptor isoforms and may or may not involve cAMP. Previous
studies in skeletal muscles from different species including humans
have indicated a predominance of
2-adrenoceptors compared with
other subtypes (8, 15). Other studies in intact skeletal muscle have
suggested a cAMP-dependent mechanism for the action of
-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
-adrenoceptor
stimulation also appears to predominantly involve
2-adrenoceptors (25).
Accordingly, the purpose of the present study was to investigate the
effects of salbutamol, a specific
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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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%
-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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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.
|
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.
|
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 s1 (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.
|
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.
|
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).
|
|
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.
|
|
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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DISCUSSION |
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The present study is a direct demonstration of -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
-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 forOur 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 -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.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the contributions of John Snover in preparation of the murine myotubes.
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FOOTNOTES |
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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.
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