Inhaled olprinone improves contractility of fatigued canine diaphragm

A. Uemura, Y. Fujii* and H. Toyooka

Department of Anaesthesiology, University of Tsukuba Institute of Clinical Medicine, 2-1-1, Amakubo, Tsukuba City, Ibaraki 305-8576, Japan *Corresponding author

Accepted for publication: October 29, 2001


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Diaphragmatic fatigue is implicated as a cause of respiratory failure. This study was undertaken to evaluate the effects of inhaled olprinone, a newly developed phosphodiesterase III inhibitor, on the contractility of fatigued diaphragm in dogs.

Methods. Diaphragmatic fatigue was induced by intermittent supramaximal bilateral electrophrenic stimulation at a frequency of 20 Hz stimulation applied for 30 min. When fatigue was established, group I (n=8) received inhaled vehicle; group II (n=8) received inhaled olprinone 1 mg; group III (n=8) received inhaled olprinone 2 mg. Diaphragmatic contractility was assessed by transdiaphragmatic pressure (Pdi, cm H2O).

Results. In the presence of fatigue, in each group, Pdi at low-frequency (20 Hz) stimulation decreased from baseline values (P<0.05), whereas Pdi at high-frequency (100 Hz) stimulation did not change. In groups II and III, during olprinone administration, Pdi at both stimuli increased from fatigued values (20 Hz stimulation: group II (mean (SD)) 10.8 (1.0) to 12.5 (1.3), group III 10.9 (1.7) to 15.0 (3.0); 100 Hz stimulation: group II 20.1 (1.9) to 22.6 (1.3), group III 20.6 (2.0) to 24.5 (2.0), P<0.05). The increase in Pdi was larger in group III than in group II (P<0.05).

Conclusions. Inhaled olprinone produces a dose-dependent improvement in contractility of fatigued canine diaphragm.

Br J Anaesth 2002; 88: 408–11

Keywords: ventilation, diaphragm, fatigue; pharmacology, phosphodiesterase III inhibitor, olprinone; dog


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Phosphodiesterase (PDE) III inhibitors have been evaluated for therapeutic potential in the treatment of congestive heart failure.1 2 In addition to these pharmacological properties, we have shown that amrinone, milrinone, and olprinone administered intravenously (i.v.) improve contractility of fatigued diaphragm that is implicated as a cause of respiratory failure, and that olprinone is most efficacious against diaphragmatic fatigue.35 Recently, Myou and colleagues6 have demonstrated that inhaled olprinone exhibits bronchodilater activity in asthmatic patients. However, there have been no reports investigating the effects of inhaled olprinone on contractility of fatigued diaphragm. The purpose of the present study was to examine the efficacy of inhaled olprinone for the improvement of diaphragm muscle function in dogs.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The procedure was approved by our animal research committee, and the care of animals was in agreement with guideline for ethical animal research. Twenty-four healthy mongrel dogs weighing 10–15 kg were anaesthetized with pentobarbitone (25 mg kg–1 loading dose plus 2 mg kg–1 h–1 maintenance dose) i.v. to abolish spontaneous movement. Muscle relaxants were not used. Animals were placed in the supine position, their tracheas were intubated with a cuffed tracheal tube, and the lungs were ventilated mechanically with a mixture of oxygen and air (FIO2=0.4) to maintain PaO213.3 kPa, PaCO2=4.7–5.3 kPa, and pHa=7.35–7.45. The right femoral artery was cannulated to monitor arterial pressure and to obtain blood gas samples for blood gas analysis. Arterial blood gas tensions were measured every 30 min. The right femoral vein was cannulated to administer fluids (Ringer’s lactate solution 10 ml–1 kg–1 h–1), pentobarbitone and bicarbonate to maintain plasma HCO3 concentration within normal range. Rectal temperature was monitored continuously and maintained at 37 (1)°C.

The phrenic nerves were exposed bilaterally at the neck, and stimulating electrodes were placed around them. Transdiaphragmatic pressure (Pdi) was measured using two thin-walled latex balloons: one positioned in the stomach, the other in the middle third of the oesphagus. Balloons were connected to a differential pressure transducer and an amplifier. Supramaximal electrical stimuli (10–15 V) of 0.1 ms duration were applied for 2 s at low-frequency (20 Hz) and high-frequency (100 Hz) stimulation with an electrical stimulator. The isometric contractility of the diaphragm was evaluated by measuring maximal Pdi after airway occlusion at FRC. Transpulmonary pressure (Ptp), the difference between airway and oesophageal pressures, was maintained by maintaining same lung volume before each phrenic stimulation. End-expiratory diaphragmatic geometry and muscle fibre length during contraction were kept constant by placing a close-fitting plaster cast around the abdomen and lower one-third of the rib cage. Electrical activity of crural (Edi-cru) and costal (Edi-cost) parts of the diaphragm was recorded by two pairs of fishhook electrodes placed through a midline laparotomy: electrodes were positioned into the anterior portion of crural part near the central tendon and the anterior portion of costal part (away from the zone of apposition) in the left hemidiaphragm. Each pair was placed in parallel fibres 5–6 mm apart. The abdomen was then sutured in layers. The signal was rectified and integrated with a leaky integrator with a time constant of 0.1 s, and was regarded as the integrated diaphragmatic electrical activity (Edi-cru, Edi-cost).

Twenty-four dogs were randomly divided into three groups of eight each. After measuring the pre-fatigued (baseline) values of Pdi, Edi-cru, Edi-cost, and haemodynamic variables, in each group, diaphragmatic fatigue was induced by intermittent supramaximal bilateral electrophrenic stimulation applied for 30 min at a frequency of 20 Hz, an entire cycle of 4 s, and a duty cycle of 0.5 (i.e. low-frequency fatigue).7 Olprinone 1 mg (group II), olprinone 2 mg (group III), or vehicle (group I) was inhaled from DeVilbiss 646 nebulizer (DeVilbiss Co., Somerset, PA, USA) operated by compressed air at 5 litre–1 min–1. The nebulizer output was 0.14 ml min–1. The dose of olprinone chosen in this experiment was based on the study of Myou and colleagues.6 Thirty minutes after the start of inhalation, in each group, Pdi, Edi-cru, Edi-cost, heart rate, and mean arterial pressure (MAP) were measured.

Values are mean (SD). Statistical analysis was performed by ANOVA for repeated measurements with Bonferroni’s adjustment for multiple comparison and Student’s t-test, where appropriate. A P value of <0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
No differences in haemodynamic variables and Pdi in the pre-fatigued (baseline) period were observed among the three groups. With an inhalation of olprinone in group III, increases in heart rate (P<0.05) and decreases in MAP (P<0.05) were observed. In groups I and II, there were no haemodynamic changes throughout the experiment (Table 1).


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Table 1 Changes in haemodynamics, Pdi (cm H2O), and %Edi values are mean (SD). HR=heart rate, MAP=mean arterial pressure, Pdi=transdiaphragmatic pressure, Edi-cru=integrated electrical activity of the crural part of diaphragm, Edi-cost=integrated electrical activity of the costal part of diaphragm. *P<0.05 vs baseline; {dagger}P<0.05 vs fatigued; {ddagger}P<0.05 vs group I; +P<0.05 vs group II
 
Pdi values at different stages and changes of Edi-cru and Edi-cost (%Edi-cru and %Edi-cost, respectively) from baseline values are shown in Table 1. Three groups were comparable with regard to Pdi to each stimulus during baseline condition. In each group, after producing fatigue, Pdi at low-frequency (20 Hz) stimulation decreased from baseline values (P<0.05), whereas Pdi at high-frequency (100 Hz) stimulation did not change. In groups II and III, Pdi at both stimulation frequencies increased from fatigued values (P<0.05) during olprinone inhalation. The increase in Pdi to each stimulus was larger in group III than in group II (P<0.05). In group I, the speed of recovery from fatigue was slower at 20 Hz stimulation than at 100 Hz stimulation. No changes in %Edi-cru and %Edi-cost were observed throughout the study in any group.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The pressure generated by the diaphragm (Pdi) after a given electrical stimulus depends on its length and geometry.8 A major determinant of length and geometry of the diaphragm is lung volume. In this study, lung volume was strictly controlled, because the airway was occluded at end-expiratory lung volume during the measurements and the end-expiratory Ptp was maintained constant before each stimulus. The deformation of thoracoabdominal structures was also avoided by placing a cast around the abdomen and lower one-third of the rib cage. Therefore, changes in Pdi observed in this experiment can be regarded as the result of changes in diaphragmatic contractility.

Hypoxaemia, hypercapnia, and metabolic acidosis decrease contractility of the diaphragm.9 10 The PaO2, PaCO2, pHa, and HCO3 concentration were controlled within normal ranges in this study. Therefore, these factors that could have affected diaphragmatic contractility were excluded. In addition, it has been reported that pentobarbitone, at the dose used in this experiment, does not affect diaphragmatic contractility.11 This was also in accordance with our results in group I showing no change in Pdi.

Low-frequency fatigue is of particular clinical importance because the spontaneous, natural rate of phrenic nerve discharge is mainly in the low-frequency ranges (i.e. 5–30 Hz).12 Therefore, the effect of inhaled olprinone on contractility in fatigued diaphragm induced by 20 Hz stimulation (i.e. low-frequency fatigue) was examined. Following a fatigue-producing period, in each group, Pdi at 20 Hz stimulation decreased from baseline values (P<0.05), whereas Pdi at 100 Hz stmulation did not change. The results of group I, in which Pdi was observed without inhalation of olprinone in fatigued diaphragm, showed that the speed of recovery from fatigue was relatively slower at 20 Hz stimulation than at 100 Hz stimulation, and also showed that Edi to each stimulus did not change. This was in agreement with previous studies by us.35

PDE III inhibitors—including amrinone, milrinone, and olprinone—administered i.v. at doses used clinically have been shown previously to increase contractility of fatigued diaphragm.35 Amrinone (10 µg kg–1 min–1) increases Pdi by 55% at 20 Hz stimulation and by 9% at 100 Hz stimulation; milrinone (0.5 µg kg–1 min–1) increases Pdi by 69% and by 23% at each stimulus; olprinone (0.3 µg kg–1 min–1) increases Pdi by 85% and by 35% at each stimulus.35 Thus, PDE III inhibitors affect Pdi at 20 Hz stimulation more when compared with Pdi at 100 Hz stimulation. We have no explanation for this difference, but olprinone is more effective than amrinone or milrinone for the augmentation of contractility in fatigued diaphragm. In the present study, Pdi at 20 and 100 Hz stimulation increased from fatigued values (P<0.05) when olprinone was administered by inhalation in groups II and III. The increase in Pdi at both stimuli was also larger in group III than in group II (P<0.05). These observations suggest that inhaled olprinone, in addition to i.v. olprinone, improves contractility of fatigued diaphragm, in a dose-related manner.

Diaphragmatic blood flow is an important determinant factor in diaphragm muscle function12 and is also maintained relatively constant within a certain limit of perfusion pressure.13 When MAP increases to >9.3 kPa, diaphragmatic blood flow is not reduced.14 In this study, however, MAP <9.3 kPa was not observed with an inhlation of olprinone in groups II and III. Therefore, the decrease in MAP during olprinone inhalation would not affect diaphragmatic contractility. Additionally, cardiovascular effects were not evident in group II (no changes in heart rate and MAP) during olprinone inhalation while the effects on Pdi at both stimuli were significant. This suggests that the action of inhaled olprinone on heart rate and MAP are not important for its effects on contractility in the fatigued diaphragm.

Olprinone, administered i.v., increases contractility of cardiac muscle by selectively inhibiting PDE III and accumulating cyclic AMP intracellularly, which, in turn, induces the activation of Ca2+ transport from the sarcoplasmic reticulum.14 In a previous report by us,5 to clarify the mechanism responsible for the effects of olprinone administered i.v. on contractility of fatigued diaphragm, combined olprinone and nicardipine, a calcium antagonist which inhibits Ca2+ influx into diaphragmatic muscle,15 was administered. Augmentation of Pdi by olprinone was abolished by administation nicardipine, suggesting that olprinone may increase contractility of fatigued diaphragm by influencing Ca2+ transport across the cell membrane. Why the fatigued diaphragmatic contractility was enhanced during olprinone inhalation is not known. The mechanisms of action of inhaled olprinone in the improvement of contractility in fatigued diaphragm may be similar to that of i.v. olprinone. However, further studies with inhaled olprinone and nicardipine are required.

In summary, inhaled olprinone produced a dose-dependent improvement, in contractility of fatigued canine diaphragm.


    Acknowledgement
 
The authors are very grateful to Takuo Hoshi MD for his technical assistance.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
1 Lejemtel TH, Keung E, Sonnenblick EH, et al. Amrinone: a new non-glycoside, non-adrenergic cardiotonic agent effective in the treatment of intractable myocardial failure in man. Circulation 1979; 59: 1098–104[Abstract]

2 Karlsberg RP, DeWood MA, DeMaria AN, et al. Comparative efficacy of short-term intravenous infusions of milrinone and dobutamine in acute congestive heart failure following acute myocardial infarction. Clin Cardiol 1996; 19: 21–30[ISI][Medline]

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5 Fujii Y, Takahashi S, Toyooka H. The effect of olprinone compared with milrinone on diaphragmatic muscle function in dogs. Anesth Analg 1999; 89: 781–5[Abstract/Free Full Text]

6 Myou S, Fujimura M, Kamio Y, et al. Bronchodilater effect of inhaled olprinone, a phosphodiesterase 3 inhibitor, in asthmatic patients. Am J Respir Crit Care Med 1999; 160: 817–20[Abstract/Free Full Text]

7 Aubier M, Farkas G, DeTroyer A, et al. Detection of diaphragmatic fatigue in man by phrenic nerve stimulation. J Appl Physiol 1981; 50: 538–44[Abstract/Free Full Text]

8 Grassino A, Goldman MD, Mead J, Sears TA. Mechanics of the human diaphragm during voluntary contraction: statics. J Appl Physiol 1978; 44: 829–39[Abstract/Free Full Text]

9 Esau SA. Hypoxic, hypercapnic acidosis decreases tension and increases fatigue in hamster diaphragmatic muscle in vitro. Am Rev Respir Dis 1989; 139: 1410–7[ISI][Medline]

10 Howell S, Fitzgerald RS, Roussos C. Effect of uncompensated and compensated metabolic acidosis on canine diaphragm. J Appl Physiol 1985; 59: 1376–82[Abstract/Free Full Text]

11 Ide T, Kochi T, Isono S, Mizuguchi T. Effect of sevoflurane on diaphragmatic contractility in dogs. Anesth Analg 1992; 74: 739–46[Abstract]

12 Roussos C, Macklem PT. The respiratory muscles. N Engl J Med 1982; 307: 786–97[ISI][Medline]

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15 Satoh H, Endoh M. Effects of a new cardiotonic agent 1,2-dihydro-6-methyl-2-oxo-5-[imidazol(1,2-a)pyridine-6-yl]-3-pyridine carbonitrile hydrochloride monohydrate (E-1020) on contractile force and cyclic AMP metabolism in canine ventricular muscle. Jpn J Pharmacol 1990; 52: 215–24[ISI][Medline]





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