Anesth Analg 2003;96:1679-1782
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
Olprinone for the Treatment, but Not Prevention, of Fatigue-Induced Changes in Guinea-Pig Diaphragmatic Contractility
Aki Uemura, MD*,
Yoshitaka Fujii, MD*,
Hidenori Toyooka, MD*,
Setsuko Suzuki ,
Kohei Sawada, PhD, and
Hideyuki Adachi, PhD
*Department of Anaesthesiology, University of Tsukuba Institute of Clinical Medicine; and
Tsukuba Research Laboratories, Eisai Co, Ltd, Tsukuba City, Ibaraki, Japan
Address correspondence and reprint requests to Yoshitaka Fujii, Department of Anaesthesiology, University of Tsukuba Institute of Clinical Med, 2-1-1, Amakubo, Tsukuba City, Ibaraki 305-8576, Japan. Address e-mail to yfujii{at}md.tsukuba.ac.jp
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Abstract
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Olprinone, a phosphodiesterase III inhibitor, improves the contractility in fatigued diaphragm in vivo, but no data are available for the treatment and prevention of fatigue-induced changes in vitro. We therefore examined the efficacy of Olprinone for the treatment and prevention of fatigue-induced changes in guinea-pig diaphragmatic contractility. The guinea-pig diaphragm strips were randomly allocated according to dose of Olprinone (0, 10-6, 10-5, and 10-4 M) (n = 7 each) and were stimulated directly in an organ bath. Diaphragmatic contractility was measured by assessing twitch tension and force at 20-Hz and 100-Hz stimulation. Diaphragmatic fatigue was induced by generating rhythmic, repetitive contractions produced by 20-Hz stimulation for 5 min. In the first experiment, after the fatigue-producing period, Olprinone was administered to the organ bath for 5 min. In the second experiment, Olprinone was pretreated for 5 min, and then diaphragmatic fatigue was produced. In Experiment 1, after a fatigue-producing period, tetanic force to each stimulus decreased from baseline values (P < 0.05). Olprinone 10-5–10-4 M caused an increase in force at both stimuli from fatigued values (P < 0.05). In Experiment 2, no change in tetanic force was observed by pretreatment with Olprinone (0–10-4 M). After producing fatigue, tetanic force to each stimulus decreased from baseline values (P < 0.05). These results suggest that Olprinone 10-5–10-4 M improves the fatigue-induced changes in guinea-pig diaphragmatic contractility and that pretreatment with Olprinone does not prevent diaphragmatic fatigability.
IMPLICATIONS: Olprinone is effective for the treatment, but not prevention, of fatigue-induced changes in guinea-pig diaphragmatic contractility.
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Introduction
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The diaphragm is the most important inspiratory muscle in the respiratory pump. In normal subjects and patients with chronic obstructive lung disease, respiratory muscle (diaphragm) fatigue may play a major role in precipitating respiratory failure (1,2). A number of studies have shown that methylxanthines, ß2-agonists, digoxin, dopamine, and dobutamine enhance contractility in a fatigued diaphragm (3–7). In addition to these pharmacological approaches, we have evaluated the efficacy of phosphodiesterase (PDE) III inhibitors, including amrinone, milrinone, and Olprinone, at clinical doses used for the improvement of contractility in a fatigued canine diaphragm (8–10) and have suggested that Olprinone is most effective (10). The precise mechanism by which Olprinone improves contractility in fatigued diaphragm is not known. However, Olprinone increases diaphragmatic contractility by influencing calcium transport across the cell membrane (10). There have been no reports to investigate the efficacy of Olprinone for the treatment and prevention of fatigue in diaphragm in vitro. This study was designed to estimate the effect of Olprinone on the fatigue-induced changes in guinea-pig diaphragmatic contractility and to evaluate the effect of pretreatment with Olprinone on diaphragmatic fatigability in an isolated guinea-pig diaphragm.
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Methods
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Our animal research committee approved the protocol, and care of the animals was in agreement with guidelines for ethical animal research in the University of Tsukuba.
Fourteen male guinea pigs weighing 400–500 g were used in this study. Animals were anesthetized with ether and then killed by decapitation. Immediately, the entire diaphragm was removed and placed in chilled Krebs-Ringer’s solution, the composition of which was (in mM): NaCl 118.4, KCl 11.0, NaHCO3 25.0, CaCl2 2.5, MgSO4 1.3, KH2PO4 1.2, and glucose 11.0, equilibrated with a mixture of 95% O2 and 5% CO2. Four diaphragm strips, approximately 10 x 5 mm, were dissected from the left hemidiaphragm by making incisions parallel to the muscle fibers; each strip included portions of the central tendon and rib on its distal ends for use as points of attachment. The isolated strips were placed in an organ bath containing Krebs-Ringer’s solution and bubbled continuously with 95% O2 and 5% CO2 mixed gas. The organ bath was maintained at 37°C by using a thermostatically controlled circulating pump. The strips were mounted vertically in separate 10-mL water-jacketed tissue chambers. The origin of each muscle was secured by a steel hook in the bath, and the central tendon of each strip was tied to a silk thread attached to an isometric force transducer that was connected to a micropositioner. The strips were stimulated with supramaximal currents (1.5 times the current required to elicit maximal tension) delivered via platinum field electrodes (20 x 15 mm; 5 mm apart). The current was generated by an electrical stimulator. Force transducer output was amplified by using an amplifier and recorded by using a digital multipen recorder. The strips were allowed to equilibrate in the organ bath for 60 min. The optimal force-length (L0) relationship was then determined by adjusting the micropositioner between intermittent stimulations of the strips. All stimulations during the experiment were performed at L0. Muscle contractile properties were then assessed from the twitch tension (0.2-ms duration; supramaximal current) and force-frequency relationship. This relationship was determined by stimulating the diaphragm strips tetanically at low-frequency (20-Hz) and high-frequency (100-Hz) stimulation. An interval of 15 s was used between stimuli, and pulses were 0.2 ms in duration with train duration of 200 ms.
Seven guinea pigs were used to assess the efficacy of Olprinone for the treatment of fatigue-induced changes in diaphragmatic contractility. Twenty-eight diaphragm strips were randomly allocated according to dose of Olprinone (0 (no drug control), 10-6, 10-5, and 10-4 M) (n = 7 each) and stimulated directly in the organ bath. After equilibration, diaphragmatic fatigue was induced by rhythmic, repetitive contractions produced by trains of 20-Hz stimulation (200-ms train duration; 0.5 duty cycle; 60 trains/min) over a period of 5 min. After the fatigue-induced stimulation, Olprinone was directly added to the organ bath by using pipette and was kept for 5 min. Diaphragmatic contractility was measured by assessing twitch tension and force at 20-Hz and 100-Hz stimulation.
Twenty-eight diaphragm strips obtained from another seven guinea pigs were used to assess the efficacy of Olprinone for the prevention of fatigue-induced changes in diaphragmatic contractility. These strips were pretreated with Olprinone (0, 10-6, 10-5, and 10-4 M) (n = 7 each) for 5 min, and then diaphragmatic fatigue was induced by rhythmic, repetitive contractions produced by trains of 20-Hz stimulation (200-ms train duration; 0.5 duty cycle; 60 trains/min) over a period of 5 min. Twitch and force-frequency relationship were measured to evaluate diaphragmatic contractility.
Data are mean ± SE. Statistical analysis was performed by one-way analysis of variance (ANOVA) for repeated measurements with Bonferroni adjustment for multiple comparisons and Student’s t-test, where appropriate. P < 0.05 was considered significant.
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Results
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The L0 and weights of the strips were similar between the two experiments and averaged 10.5 ± 0.2 mm and 18.0 ± 0.5 mg, respectively. Twitch tension decreased from baseline values (P < 0.05) after producing fatigue and increased from fatigued values (P < 0.05) during the Olprinone 10-5–10-4 M administration. In a fatigued period, force at 20-Hz and 100-Hz stimulation decreased from baseline values (P < 0.05). Olprinone 10-5–10-4 M caused an increase in force at both frequencies of stimulation from fatigued values (P < 0.05) (Table 1).
Twitch tension did not change with an administration of Olprinone (10-6, 10-5, and 10-4 M) and decreased from baseline values after producing fatigue (P < 0.05). No change in force at 20-Hz and 100-Hz stimulation was observed by pretreatment of Olprinone (10-6–10-4 M). After the fatigue-induced period, force at both frequencies of stimulation decreased from baseline values (Table 2).
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Discussion
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Olprinone, a newly developed inhibitor of PDE III, improves cardiac performance not only through its inotropic effects, but also through its vasodilating effects (11,12). Olprinone increases contractility of cardiac muscle by selectively inhibiting PDE III and accumulating cyclic adenosine monophosphate intracellularly, which, in turn, induces the activation of Ca2+ transport from the sacroplasmic reticulum (13). In addition to these pharmacological properties, we have demonstrated that Olprinone improves contractility in a fatigued canine diaphragm (10). To clarify the mechanism responsible for the effect of Olprinone on contractility of fatigued diaphragm in dogs, combined Olprinone and nicardipine, a calcium antagonist that inhibits Ca2+ influx into diaphragm muscle, was administered. Consequently, augmentation of diaphragmatic contractility by Olprinone was abolished by administering nicardipine, suggesting that Olprinone may increase contractility in fatigued diaphragm by influencing Ca2+ transport across the cell membrane (10).
The results of the first experiment showed that Olprinone 10-5–10-4 M caused an increase in twitch tension and force to each stimulus from fatigued values in guinea-pig diaphragm strips (P < 0.05). This was in agreement with our previous study in vivo demonstrating that Olprinone 0.3 µg · kg-1 · min-1 increased diaphragmatic contractility (as assessed by transdiaphragmatic pressure) during fatigued conditions. These results suggest that Olprinone improves contractility in fatigued diaphragm in vitro as well as in vivo. Why the fatigue-induced changes in diaphragmatic contractility in vitro were enhanced during Olprinone administration is not known. The mechanism of action of Olprinone for the improvement of fatigue in diaphragmatic contractility in vitro may be similar to that in vivo. Further studies are required to elucidate the mechanism of its beneficial effect on diaphragm strips.
The dose (10-6–10-4 M) of Olprinone used in this study was based on a previous study on isolated guinea-pig cardiac muscle (11). We found no report establishing the effective dose of Olprinone for improving fatigue-induced changes in guinea-pig diaphragmatic contractility. In the current study, Olprinone 10-5–10-4 M increased twitch tension and force at 20-Hz and 100-Hz stimulation from fatigued values (P < 0.05) and accelerated the recovery from diaphragmatic fatigue. However, there was no difference in twitch tension and force at both frequencies of stimulation between the control (no drug) and Olprinone 10-6 M groups. Therefore, Olprinone 10-5 M was the minimum effective dose for the improvement of fatigue-induced change.
In the second experiment, no change in twitch tension and force at both stimuli was observed by pretreatment with Olprinone (10-6–10-4 M), and force to each stimulus decreased from baseline values. Thus, the efficacy of Olprinone for the prevention of fatigued-changes in diaphragmatic contractility could not be shown. However, this is the first report to evaluate the effect of Olprinone administered before producing fatigue on diaphragmatic fatigability. Consequently, pretreatment with Olprinone does not prevent fatigability in the isolated guinea-pig diaphragm.
Although the action of Olprinone was different in the treatment and prevention of fatigue in the diaphragm, from a therapeutic point of view, of particular importance is the characterization of the effect of Olprinone on fatigue-induced change in diaphragmatic contractility. Diaphragmatic fatigue may contribute to the development of respiratory failure (1,2). An improvement in fatigued diaphragmatic contractility because of PDE III inhibitors, such as Olprinone, could be of great value in patients with chronic obstructive pulmonary disease related to the fatigue of respiratory muscle (diaphragm).
In conclusion, Olprinone 10-5–10-4 M improves the fatigue-induced changes in guinea-pig diaphragmatic contractility, and pretreatment with Olprinone does not prevent diaphragmatic fatigability.
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References
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Accepted for publication February 10, 2003.
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Abstract
Introduction
