beta 2-Adrenoceptor agonists reduce the decline of rat diaphragm twitch force during severe hypoxia

H. F. M. van der Heijden, L. M. A. Heunks, H. Folgering, C. L. A. van Herwaarden, and P. N. R. Dekhuijzen

Department of Pulmonary Diseases, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands


    ABSTRACT
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

The aim of the present study was to investigate the in vitro effects of the short-acting beta 2-adrenoceptor agonist salbutamol and the long-acting beta 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 beta -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 beta 2-adrenoceptor agonist-related processes.

contractile properties; respiratory muscles; salmeterol; salbutamol


    INTRODUCTION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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. beta 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 beta 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 beta 2-adrenoceptor agonists on diaphragm contractile properties during hypoxia have not been investigated. Whether the new long-acting beta 2-adrenoceptor agonists like salmeterol have similar inotropic effects on in vitro diaphragm contractility is not known.

Because beta 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 beta 2-adrenoceptor agonists can be blocked by propranolol (a nonselective beta -antagonist).

The purpose of the present study, therefore, was to investigate the effects of short- and long-acting beta 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 beta 2-adrenoceptor-related processes, we investigated whether propranolol blocked the effects of salbutamol and salmeterol during hypoxia.


    METHODS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Study design. The effects of treatment with beta 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-alpha '-[[[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 beta 2-adrenoceptor-related processes, propranolol (a nonselective beta -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.


    RESULTS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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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Table 1.   Tissue bath Krebs solution pH, PCO2, and PO2

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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Table 2.   Diaphragm Pt, Po, and Pt-to-Po ratio at 30-min intervals during hyperoxia and hypoxia in vitro

Effects of beta 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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Fig. 1.   Diaphragm twitch force (Pt) during hyperoxia (open symbols) and hypoxia (closed symbols). Circles, control group; triangles, salbutamol treatment; squares, salmeterol treatment (all concentrations 1 µM). For clarity, means ± SE are indicated separately at start and end of curves only. Control diaphragm Pt was significantly lower during hypoxia compared with hyperoxic control value (# P < 0.01). Because an overall repeated-measures ANOVA analysis resulted in multiple significant interactions between the different factors (hypoxia, treatment, and repeated measurements of force), statistical analysis was performed within hyperoxic and hypoxic groups subsequently. No significant treatment effects were found within hyperoxic group. However, during hypoxia, both salbutamol and salmeterol significantly increased Pt curve compared with hypoxic control value (* P < 0.01 by repeated-measures ANOVA).

Salmeterol did not significantly alter repetitive Pt generation during hyperoxia (Fig. 1). However, Po was significantly reduced by salmeterol treatment, both expressed as specific force and as a percentage of initial Po (P < 0.05; Table 2). This resulted in a significantly increased Pt-to-Po ratio (P < 0.05; Table 2). Significant interactions were found between salmeterol treatment and time by repeated-measures analysis of Pt (P < 0.001). This interaction was also found for Po and the Pt-to-Po ratio (P < 0.001). Post hoc analysis did not show a significant difference in Pt, but Po was decreased after 60 and 90 min (Table 2). As a result, the Pt-to-Po ratio was increased (Table 2).

Effects of beta 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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Fig. 2.   Diaphragm Pt during hypoxia. , Control group; black-triangle, salbutamol treatment; , salmeterol treatment; open circle , propranolol treatment (1 µM); triangle , salbutamol plus propranolol treatment; , salmeterol plus propranolol treatment (all concentrations 1 µM). For clarity, means ± SE are indicated separately at start and end of curves only. Both salbutamol and salmeterol significantly increased Pt curves compared with control values during hypoxia (* P < 0.01 by repeated-measures ANOVA). In salbutamol plus propranolol- and salmeterol plus propranolol-treated groups, Pt curves were significantly decreased compared with control values (# P < 0.05 by repeated-measures ANOVA). Propranolol treatment did not significantly alter Pt curve during hypoxia compared with hypoxic control value (P = 0.341).

During hypoxia, salmeterol treatment increased Pt compared with that in time-matched control strips (P < 0.01; Fig. 2). A significant interaction with time was found (P < 0.001), and post hoc analysis showed that significant increases in Pt were present from ~30 min onward. When expressed as a percentage of initial Pt, similar effects were found (Fig 1, Table 2). However, in contrast to salbutamol treatment, the effect of salmeterol was sustained throughout the experiment. Po was not altered by salmeterol during hypoxia, and no interactions were found with time (Table 2). As a result, the Pt-to-Po ratio was increased (P < 0.001; Table 2). A significant interaction between salmeterol treatment and time was present (P < 0.001), but the Pt-to-Po ratio was significantly increased at all time points (Table 2).

Effects of propranolol during hypoxia. Propranolol itself had no effect on Pt (Fig. 2), Po, or the Pt-to-Po ratio (Table 2). However, when expressed as a percentage of initial Pt, propranolol significantly reduced percent Pt (P < 0.05). In contrast, coadministration of propranolol completely blocked the inotropic effects of both salbutamol and salmeterol on Pt during severe hypoxia (Fig. 2). In fact, coadministration of propranolol with salbutamol or salmeterol significantly decreased Pt (in N/cm2) compared with that in control strips (P < 0.05; Fig. 2). Po and the Pt-to-Po ratio were not affected by the coadministration of propranolol (Table 2).


    DISCUSSION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

The present study shows that both short-acting and long-acting beta 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 beta -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 beta 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 beta 2-adrenoceptor agonist treatment is not known.

Effects of beta 2-adrenoceptor agonist treatment. The present study shows that treatment with short- and long-acting beta 2-adrenoceptor agonists reduced the hypoxia-induced decline in diaphragm Pt. The exact mechanism by which beta 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 beta 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).

The decrease in Po found in the salmeterol-treated hyperoxic and salbutamol-treated hypoxic groups may indicate a progressive depletion of the SR Ca2+ pool. Also, the stronger decline in Pt in the salbutamol-treated group toward the end of the experiment may be a result of such a Ca2+ depletion, which would be in agreement with the proposed mechanism of action. However, during hypoxia, no such effect of salbutamol or salmeterol was found on Pt or Po. An alternative explanation for the different effect of salbutamol and salmeterol on Po during hyperoxia may be found in differences in the pharmacological activity or non-beta 2-adrenoceptor-related processes (18) or in the switching of G protein coupling (7). However, because these effects are not consistently found throughout the present study, it is likely that other processes are also involved in this decrease in Po. Because these effects were not evaluated in this study, we do not have a clear explanation for these findings.

The effect of hypoxia on SR Ca2+ release or on other mechanisms that may influence the inotropic effects of beta 2-adrenoceptor agonists in hypoxic skeletal muscles is not known. One study has found a small effect of salbutamol treatment on diaphragm Pt in dogs during metabolic acidosis (12), but no studies were performed under hypoxic conditions. Prolonged hypoxia has been shown to reduce the expression of beta 1-adrenoceptors in cardiac muscle and of beta 2-adrenoceptors in pulmonary and systemic arteries (25). In the lung, hypoxia increased the density and binding affinity of beta 2-adrenoceptors (3). Furthermore, the in vitro regulation of the beta 2-adrenoceptor agonist gene in hamster smooth muscle cells was found to be critically dependent on pH during hypoxia (22). Whether such changes are present within the time frame of the present study and what functional consequences such changes may have is not clear.

Coadministration of propranolol plus salbutamol or salmeterol during hypoxia reduced Pt. This effect was also found for propranolol alone when Pt is expressed as a percentage of initial Pt. This is in agreement with an earlier report (17) in which a selective beta 2-adrenoceptor antagonist reduced force generation in gastrocnemius muscle preparations. It has been suggested that this could be the result of a blockade of endogenous catecholamines (17). However, it is unlikely that such an effect was of importance in the present in vitro experiment. Alternatively, these findings may indicate changes in oxidative enzyme activity (14).

Methodological considerations. In the present study, alteration of tissue bath oxygenation and addition of beta 2-adrenoceptor agonists and/or antagonists were performed simultaneously. These changes do not immediately exert their effect, and all parameters are likely to have different equilibration times. We did not include separate time frames for either hypoxia (diffusion time) or the onset of action of salbutamol or salmeterol in the diaphragm strips. The use of different time frames would certainly have affected force production because at 37°C, a time-related decrease of in vitro force production is present. In such an experimental design, the use of separate time-matched control groups would have been obligatory.

Not surprisingly, the present study shows that salbutamol has a more rapid onset of action and a larger effect on Pt compared with those with salmeterol. This can be explained by differences in lipophilicity and a lower efficacy (partial agonist) of salmeterol compared with salbutamol (11). Furthermore, diaphragm Pt was decreased further in both hypoxia and hyperoxia when salmeterol started to have an effect. This may have reduced the inotropic effect of salmeterol. However, the duration of action of salmeterol was longer compared with that of salbutamol, which is in agreement with its pharmacological properties (11).

In the present study, very high concentrations (1 µM) of salbutamol and salmeterol were used. At these high concentrations, salmeterol may have non-beta 2-adrenoceptor properties due to its high lipophilicity (18). However, the experiments in which propranolol was added to salbutamol or salmeterol treatment show that these non-beta 2-adrenoceptor-related processes did not play a role in the inotropic effects of salmeterol on diaphragm Pt during hypoxia. In earlier studies (31, 32), salbutamol was shown to have significant inotropic effects at the clinically relevant concentration of 0.05 µM. Preliminary studies with salmeterol showed that lower concentrations had similar but less pronounced effects during hypoxia. To reach maximal effects and to simplify comparison between the two drugs, we chose the high concentration of 1 µM for both compounds.

Clinical relevance. In patients with severe COPD, chronic hypoxia can frequently be found, often in combination with hypercapnia. Furthermore, during exacerbations of their disease, acute hypoxia or acute-on-chronic hypoxia may be present. This is of particular interest because hypoxia may reduce diaphragm contractile properties in situ (2) and in vitro (26) and may reduce fatigue resistance in vitro (24) and in humans (13). Dysfunction of the respiratory muscles frequently occurs in patients with severe COPD (20). Metabolic changes like hypoxia, hypercapnia, and electrolyte disorders are important factors that may impede (the already compromised) respiratory muscle function (2, 9, 16, 26, 27, 29). In this clinical situation, beta 2-adrenoceptor agonists are often used for bronchodilation. Previous studies have shown that beta 2-adrenoceptor agonists like salbutamol and terbutaline can improve diaphragm contractile properties under optimal in vitro conditions (1, 32) and after fatigue (8, 19, 30). The present study shows that the decrease in diaphragm contractility under hypoxic conditions can be partially prevented by the addition of beta 2-adrenoceptor agonists in vitro. This might be of importance in the treatment of incipient or manifest respiratory muscle fatigue in COPD patients, but clinical studies are recommended to evaluate the effects of beta 2-adrenoceptor agonists in these situations.


    ACKNOWLEDGEMENTS

Salmeterol base was kindly donated by Glaxo Wellcome Research and Development (Stevenage, UK). We thank W. Janssen for expert biotechnical assistance.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

1.   Aubier, M., N. Viires, D. Murciano, G. Medrano, Y. Lecocguic, and R. Pariente. Effects and mechanism of action of terbutaline on diaphragmatic contractility and fatigue. J. Appl. Physiol. 56: 922-929, 1984[Abstract/Free Full Text].

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