Department of Pulmonary Diseases, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands
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ABSTRACT |
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The aim of the present study was to investigate
the in vitro effects of the short-acting
2-adrenoceptor
agonist salbutamol and the long-acting
2-adrenoceptor agonist
salmeterol on hypoxia-induced rat diaphragm force reduction. In vitro
diaphragm twitch force (Pt) and
maximal tetanic force (Po) of
isolated diaphragm muscle strips were measured for 90 min during
hyperoxia (tissue bath PO2 83.8 ± 0.9 kPa and PCO2 3.9 ± 0.1 kPa) or severe hypoxia (PO2 7.1 ± 0.3 kPa and PCO2 3.9 ± 0.1 kPa) in the presence and absence of 1 µM salbutamol or 1 µM
salmeterol. During hyperoxia, salbutamol and salmeterol did not
significantly alter the time-related decreases in
Pt and
Po (to ~50% of initial values).
Salbutamol had no effects on Po or the
Pt-to-Po
ratio. Salmeterol treatment significantly reduced Po and increased the
Pt-to-Po
ratio during hyperoxia (P < 0.05 compared with control value). Hypoxia resulted in a severe decrease in
Pt (to ~30% of initial value)
and Po after 90 min. Both
salbutamol and salmeterol significantly reduced the decline in
Pt during hypoxia
(P < 0.05). The reduction in
Po was not prevented. Salbutamol increased Pt rapidly but
transiently. Salmeterol had a delayed onset of effect and a longer
duration of action. Addition of 1 µM propranolol (a nonselective
-adrenoceptor antagonist) did not alter
Pt,
Po, or the
Pt-to-Po
ratio during hypoxia but completely blocked the inotropic effects of
salbutamol and salmeterol, indicating that these effects are dependent
on
2-adrenoceptor
agonist-related processes.
contractile properties; respiratory muscles; salmeterol; salbutamol
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INTRODUCTION |
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DYSFUNCTION OF THE RESPIRATORY MUSCLES frequently occurs in patients with severe chronic obstructive pulmonary disease (COPD) (20). Alterations like hypoxia, hypercapnia, and electrolyte disorders are important factors that may impede respiratory muscle function (2, 9, 16, 26, 27, 29). In patients with severe COPD, chronic hypoxia may be present, often in combination with hypercapnia. Furthermore, during exacerbations of their disease, acute-on-chronic respiratory failure may occur. Hypoxia significantly affects respiratory muscle function; it reduces in situ diaphragm contractility (2), in vitro diaphragm twitch force (26), and in vitro isotonic and isometric diaphragm fatigue resistance (24). Also, in healthy subjects, fatigue resistance (measured by inspiratory resistive breathing) is reduced by hypoxia (13).
Therefore, pharmacological interventions that (partly) reverse these
deleterious effects of hypoxia on respiratory muscle function are of
clinical importance.
2-Adrenoceptor agonists are of
special interest because these drugs are already widely used in the
treatment of bronchoconstriction and exacerbations of asthma or COPD.
Furthermore, previous studies (1, 32) have shown that
2-adrenoceptor agonists like
salbutamol and terbutaline can improve normal rat diaphragm contractile
properties under optimal in vitro conditions. These inotropic effects
are increased by foreshortening (31) and are also present during
fatigue (8, 19, 30) and in vivo metabolic acidosis (12). The effects of
2-adrenoceptor agonists on
diaphragm contractile properties during hypoxia have not been
investigated. Whether the new long-acting
2-adrenoceptor agonists like
salmeterol have similar inotropic effects on in vitro diaphragm
contractility is not known.
Because 2-adrenoceptor agonists
like salbutamol have an inotropic effect on in vitro (1, 31, 32) and in
vivo (8, 12, 19, 30) diaphragm contractile properties, we hypothesized that the observed decrease of twitch force induced by hypoxia (26) is
reversed by these drugs. In addition, we hypothesized that salmeterol
will have a sustained effect during hypoxia in comparison to salbutamol
and that the effects of these
2-adrenoceptor agonists can be
blocked by propranolol (a nonselective
-antagonist).
The purpose of the present study, therefore, was to investigate the
effects of short- and long-acting
2-adrenoceptor agonists (salbutamol and salmeterol, respectively) on rat diaphragm contractile properties during severe hypoxia in vitro and to compare these effects
with hyperoxic conditions. To ascertain that these effects are mediated
via
2-adrenoceptor-related
processes, we investigated whether propranolol blocked the effects of
salbutamol and salmeterol during hypoxia.
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METHODS |
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Study design. The effects of treatment
with 2-adrenoceptor agonists on
in vitro rat diaphragm contractile properties were studied either
during standard optimal in vitro conditions (hyperoxia) or during
severe hypoxia. In both conditions, the effects of salbutamol or
salmeterol treatment were studied and compared with those of the
untreated control group.
General procedures. Adult male outbred Wistar rats aged 16-18 wk and with a mean weight of 376 ± 5 (SE) g were used. The animals were housed under standard conditions and were fed ad libitum.
The rats were anesthetized with pentobarbital sodium (70 mg/kg ip; Narcovet, Opharma, Arnhem, The Netherlands). A tracheotomy was performed, and a polyethylene cannula was inserted. The animals were mechanically ventilated with 100% oxygen. The diaphragm and adherent lower ribs were quickly excised after a combined laparotomy and thoracotomy, and they were immediately submersed in cooled, oxygenated Krebs solution at pH ~7.4. This Krebs solution consisted of 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM KH2PO4, 24 mM NaHCO3, 7 mM glucose, and 25 µM D-tubocurarine (Sigma, Bornem, Belgium).
From the central costal region of the right hemidiaphragm, two rectangular strips were dissected parallel to the long axis of the muscle fibers. Silk sutures were tied firmly to both ends. The strips were suspended in two tissue baths containing Krebs solution, maintained at 37°C, and perfused with a 95% O2-5% CO2 mixture. The central tendon end was connected to an isometric force transducer (Sensotec model 31/1437-10, Columbus, OH) mounted on a micrometer. Two large platinum stimulating electrodes were placed parallel to the bundles. Stimuli were applied with a pulse duration of 0.2 ms and a train duration of 400 ms and were delivered by a stimulator (ID-electronics, University of Nijmegen, Nijmegen, The Netherlands) activated by a personal computer. To ensure supramaximal stimulation, the strips were stimulated at ~20% above the voltage at which maximal forces were obtained (~6 V through the stimulation electrodes). Data acquisition and storage of the amplified signal were performed via a Dash-16 interface on a personal computer (Twist-trigger software, ID-electronics). Both strips were placed at their optimal length, defined as the length at which peak twitch force (Pt) was obtained. After a 15-min thermoequilibration period (i.e., before the start of treatments), maximal Pt and maximal tetanic force (Po) were determined under normal (hyperoxic) conditions.
Treatment groups. The diaphragm strips
were randomly allocated to the treatment groups, and different
treatments were given to each of the two muscle strips obtained from
one animal. Immediately after the thermoequilibration period, perfusion
of the tissue baths was either maintained with the 95%
O2-5%
CO2 gas mixture (hyperoxia) or was
switched to a gas mixture containing 3%
O2-5% CO2-92%
N2 (hypoxia). Subsequently,
treatments were started by adding the drug or an equal volume of saline
to the Krebs solution in the tissue baths. A concentration of 1 µM
was studied for both salbutamol (Glaxo Wellcome, Zeist, The
Netherlands) and salmeterol {(±)-4-hydroxy-'-[[[6-(4-phenylbutoxy)hexyl]amino]methyl]-1,3-benzenedimethanol; Glaxo Wellcome Research and Development, Stevenage, UK}. Before the start of the experiments, salmeterol was dissolved in glacial acetic acid and diluted to stock concentrations with phosphate buffer
(pH 7.0). Salbutamol solutions were made from 0.5 mg/ml vials of
salbutamol (Ventolin).
Subsequently, maximal Pt was measured at 2-min intervals for 90 min and maximal Po was measured every 30 min. The Pt-to-Po ratio was calculated every 30 min. The pH of the Krebs solution was monitored at regular intervals and was not affected throughout the experiment nor was it different between treatment groups. At the end of this protocol, pH, PO2, and PCO2 of the Krebs solution in the tissue baths were measured in both the hyperoxic and hypoxic conditions (Corning 178 pH/blood gas analyzer, Medfield, MA). Finally, the length of each diaphragm strip was measured with a micrometer (Mitutoyo model 560-128, Veenendaal, The Netherlands), and the strips were weighed. Cross-sectional area was calculated by dividing diaphragm strip weight (in g) by strip length (in cm) times specific density (1.056). Forces are expressed per cross-sectional area (in N/cm2).
In the hyperoxic group, three treatment regimens were studied. Either
salbutamol (1 µM; n = 8 strips),
salmeterol (1 µM; n = 10 strips), or
an equal volume of saline (~50 µl;
n = 9 strips) was added to the tissue
baths. In the hypoxia group, five treatment regimens were investigated:
saline control (n = 24 strips), salbutamol (n = 18 strips),
salmeterol (n = 19 strips),
propranolol (n = 6 strips), salbutamol
plus propranolol (n = 7 strips), and
salmeterol plus propranolol (n = 7 strips). To determine whether the effects of salmeterol and salbutamol
on diaphragm muscle contractile properties during hypoxia were mediated
via 2-adrenoceptor-related
processes, propranolol (a nonselective
-adrenoceptor antagonist;
Sigma) was added to the Krebs solution in the tissue baths
simultaneously with either salmeterol or salbutamol. This was done
immediately after the thermoequilibration period after perfusion was
switched to the hypoxic gas mixture. In these experiments, a
concentration of 1 µM was used for all drugs.
The various combinations of oxygen supply and treatments were investigated in random order; the investigator (H. v. d. Heijden) was blinded with regard to treatment throughout the experiment. This study was approved by the Animal Experiments Committee of the University of Nijmegen, and it was performed in accordance with the Dutch National Guidelines for Animal Care.
Data analysis. Treatment effects were determined with two-way analysis of variance (ANOVA) in a repeated-measures design, with treatment and oxygen state (hypoxia or hyperoxia) as grouping variables. Because significant interactions were found between the three factors, i.e., treatment, the absence or presence of hypoxia, and repeated force measurements, treatment effects were subsequently analyzed in a repeated-measures design within the hypoxic and hyperoxic groups. Post hoc analysis (Student-Newman-Keuls test) was used to compare differences in Po and the Pt-to-Po ratio in independent treatment groups. Results were considered significant at P < 0.05. The SPSS package (version 6.1.3, Chicago, IL) was used for statistical analysis. All data are expressed as means ± SE.
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RESULTS |
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Verification of tissue bath hypoxia.
Perfusion of the tissue baths with the hypoxic gas mixture
significantly reduced the PO2 in the
Krebs solution to ~7 kPa compared with ~84 kPa in the hyperoxic
group (P < 0.001; Table
1). No differences were found in pH or
PCO2 between hypoxia and hyperoxia, and no differences were found in PO2,
PCO2, or pH between treatment groups.
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Diaphragm strip dimensions and specific
forces. Average diaphragm strip length ranged from 21.8 ± 0.5 to 23.0 ± 0.4 mm. Diaphragm muscle strip weight ranged
from 39.7 ± 1.4 to 49.5 ± 3.4 mg. No significant differences
were found between treatment groups for either strip length or strip
weight (one-way ANOVA with subsequent Student-Newman-Keuls post hoc
test). The initial Pt and initial maximal Po are listed in Table
2. These measurements were performed at the
end of the thermoequilibration period, before the start of treatment
and under standard hyperoxic conditions. No differences were found
between any treatment groups in the pretreatment period (one-way
ANOVA).
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Effects of 2-adrenoceptor
agonist treatment during hyperoxia.
Salbutamol treatment did not significantly alter repetitive diaphragm
Pt during hyperoxia. Also,
when expressed as a percentage of the initial
Pt, no significant overall effect
was found (Fig. 1, Table 2). Salbutamol did
not affect Po and the
Pt-to-Po
ratio either during hyperoxia (Table 2). However, significant
interactions were found between salbutamol treatment and time by
repeated-measures analysis of Pt
and
Pt-to-Po
ratio (P < 0.01). This may indicate that salbutamol initially increased
Pt (and
Pt-to-Po
ratio) but that this effect was not sustained throughout the experiment
(Fig. 1).
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Effects of 2-adrenoceptor
agonist treatment during hypoxia.
Hypoxia impaired diaphragm Pt
generation compared with hyperoxia (Fig. 1). During hypoxia, salbutamol
rapidly increased diaphragm Pt
compared with that in time-matched control strips
(P < 0.001; Fig.
2, Table 2). A similar effect was found
when Pt was expressed as a
percentage of initial Pt (Fig. 1).
A significant interaction was found between salbutamol treatment and
time for repeated Pt measurements,
indicating that the effect varied throughout the experiment
(P < 0.001). Post hoc analysis
showed that Pt was increased from
~15 to ~60-75 min after the start of the protocol. Overall, salbutamol did not alter Po, both
when expressed as specific force (Table 2) and as a percentage of
initial Po. However, a significant interaction with time was found (P < 0.001). After 90 min of hypoxia, Po was significantly lower in
salbutamol-treated diaphragm strips compared with those from control
animals (Table 2). The
Pt-to-Po ratio was increased compared with that in control strips
(P < 0.001; Table 2). Again, a
significant interaction was found between salbutamol treatment and time
(P < 0.001). Post hoc analysis
showed that the
Pt-to-Po
ratio was significantly increased at all time points during the
experiment (Table 2).
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DISCUSSION |
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The present study shows that both short-acting and long-acting
2-adrenoceptor agonists improve
diaphragm muscle Pt generation during severe hypoxia in
vitro. This partially restored the hypoxia-induced decline in
Pt. The significant interactions
found between treatment and time in these repeated force measurements
indicate that the magnitude of these effects varied during the
experiment. Salbutamol had a more rapid onset of action compared with
salmeterol, but salmeterol had a longer duration of action. Toward the
end of the protocol, salbutamol reduced
Po during hypoxia and the decline in Pt also appeared to be
stronger. In contrast, neither salbutamol nor salmeterol affected
Pt during hyperoxia. Salmeterol
decreased Po during hyperoxia,
whereas salbutamol had no effect on
Po. Finally, the inotropic effects
of both salmeterol and salbutamol were blocked by propranolol,
indicating that these processes are dependent on
-adrenoceptor-mediated processes.
Severity of hypoxia. In the present in vitro experiments, Krebs solution PO2 was reduced to ~7 kPa to induce hypoxia compared with ~84 kPa in the hyperoxic group. Because oxygenation of the diaphragm muscle strips in these experiments is dependent solely on diffusion of oxygen into the core region of the strips, this partial pressure can be considered as severe hypoxia. Besides, at in vitro experimental conditions similar to our hyperoxia experiments (PO2 ~84 kPa), significant hypoxia may be present in centrally situated muscle fibers when the critical radius of ~0.6 mm is exceeded at 37°C (23). These authors showed that at temperatures higher than 25°C, Pt and Po in both the soleus and extensor digitorum longus muscles decreased with the duration of in vitro incubation. This was accompanied by a temperature-dependent depletion of glycogen content in the central portions of these muscles (23). However, in contrast to whole muscle preparations, diaphragm strip thickness was well within this critical radius (31, 32). In previous studies of hypoxic effects on diaphragm contractility, similar levels of in vitro PO2 (26) or slightly higher levels of PO2 (~10-20 kPa) were studied in rat (27), hamster (9), and mouse diaphragms (24) and in situ in the dog diaphragm (2). It is difficult to compare those results with the present findings because higher duty cycles and stimulation frequencies will accelerate the decline in force (2, 33). In the hamster diaphragm, hypoxia (~16 kPa) partly reduced the force-frequency curve, and it reduced submaximal tetanic tension (160 ms, 25-Hz stimulation) by ~35% (9). In the mouse diaphragm, isotonic and (to a lesser extent) isometric fatigue resistances were dramatically reduced by hypoxia (~10 kPa) (24). During fatigue, the rat diaphragm relaxation rate was increased (33). The rat diaphragm Pt was reduced to ~10% of initial Pt after 25 min of stimulation at 0.5 Hz and 0.1-ms pulse duration at a PO2 of 4-5 kPa (26). At a PO2 of ~20 kPa, a similar stimulation protocol reduced rat diaphragm Pt by ~60% after 30 min (27). In the present study, a much lower duty cycle was used (with a stimulation frequency of ~0.01 Hz and a pulse duration of 0.2 ms). With this stimulation paradigm, Pt was decreased by ~70% after 90 min at a PO2 of ~7 kPa compared with a reduction of ~50% at a PO2 of ~84 kPa. This again shows that hypoxia has a clear additive effect on the in vitro decline of Pt.
Mechanisms of force decline during hypoxia. Hypoxia affects virtually all physiological processes, and it impairs the energy supply of living cells. In addition, hypoxia is often accompanied by changes in PCO2 and intracellular as well as extracellular pH (9, 26, 27). Hypercapnia reduced the capacity of the unfatigued human diaphragm to generate force during voluntary contractions (16). Both hypoxia and hypercapnia lowered intracellular pH (26, 27), and the combination of hypercapnia, hypoxia, and acidosis had greater detrimental effects than either abnormality alone (9). In the present study, (extracellular) pH and PCO2 were similar in the hypoxic and hyperoxic groups, and only PO2 was modified.
The metabolic and structural mechanisms by which skeletal muscle force
production is reduced during acute hypoxia have recently been reviewed
by Sieck and Johnson (29). These mechanisms include altered membranous
ionic conductance (33) reducing sarcolemmal excitability,
downregulation of mitochondrial enzymes, increased production of
reactive oxygen-derived species (ROS), and effects on muscle blood flow
in vivo (29). Hypoxia may alter membrane conduction of
K+,
Cl, and possibly
Na+ (28, 33), but whether
2-adrenoceptor agonists affect
these processes is disputed (4, 5). A reduced sarcolemmal excitability may contribute to neuromuscular transmission failure, which may predominantly affect type IIb muscle fibers as a result of their high
actomyosin ATPase activity (29). However, in the present in vitro
experiments, diaphragm muscle strips were directly stimulated and
neuromuscular transmission was blocked by the addition of D-tubocurarine to the Krebs
solution. Also, alterations in muscle blood flow in response to acute
hypoxia are unlikely to be involved in these in vitro experiments.
Therefore, downregulation of oxidative enzymes such as succinate
dehydrogenase or cytochrome-c oxidase may be important mechanisms in the reduction of force generation induced by acute hypoxia during the present experiments. This downregulation may be the result of ROS formation and is encountered after repetitive diaphragm stimulation (15, 29). ROS and scavengers of
ROS are increasingly implicated in modulating contractile properties in
skeletal and respiratory muscles. In skeletal muscles, a low level of
ROS is essential for excitation-contraction coupling and is obligatory
for optimal contractile function (21). However, whether ROS formation
is altered by
2-adrenoceptor
agonist treatment is not known.
Effects of 2-adrenoceptor
agonist treatment.
The present study shows that treatment with short- and long-acting
2-adrenoceptor agonists reduced
the hypoxia-induced decline in diaphragm
Pt. The exact mechanism by which
2-adrenoceptor agonists exert
their inotropic effect on respiratory muscles is not fully understood.
These mechanisms may involve excitation-contraction coupling of
skeletal muscle. In earlier experiments, the inotropic effect of
salbutamol was blocked by ryanodine, which indicates that this effect
is most likely mediated by an improvement of sarcoplasmic reticulum
(SR) Ca2+ release (31). This is in
agreement with previous findings reported by Cairns and Dulhunty (4, 5)
and Cairns et al. (6). They further showed that
enhancement of sodium-pump activity, dihydropyridine-sensitive
Ca2+ currents, glycolysis, and
altered action potentials are unlikely to be involved in the mechanisms
of action
2-adrenoceptor
agonists (4, 5). These findings in mammalian skeletal muscle are in
line with earlier experiments conducted with frog skeletal muscle,
showing that adrenaline treatment potentiated
Pt by modulating calcium channels
(10).
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ACKNOWLEDGEMENTS |
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Salmeterol base was kindly donated by Glaxo Wellcome Research and Development (Stevenage, UK). We thank W. Janssen for expert biotechnical assistance.
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FOOTNOTES |
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This study was supported by a grant from Glaxo Wellcome (Zeist, The Netherlands).
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: P. N. R. Dekhuijzen, Dept. of Pulmonary Diseases, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands (E-mail: R.Dekhuijzen{at}long.azn.nl).
Received 26 June 1998; accepted in final form 10 December 1998.
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