Free
Meeting Abstracts  |   September 1995
Effect of Aminophylline on High-energy Phosphate Metabolism and Fatigue in the Diaphragm
Author Notes
  • (Ide) Postdoctoral Fellow, Department of Environmental Health Sciences.
  • (Nichols, Eleff) Associate Professor, Department of Anesthesiology/Critical Care Medicine.
  • (Buck) Assistant Professor, Department of Surgery.
  • (Shungu) Postdoctoral Fellow, Department of Radiology.
  • (Robotham, Traystman) Professor, Department of Anesthesiology/Critical Care Medicine.
  • (Fitzgerald) Professor, Department of Environmental Health Sciences.
  • Received from the Departments of Anesthesiology/Critical Care Medicine, Surgery, and Radiology, Division of Pediatric Intensive Care, The Johns Hopkins Medical Institutions, Baltimore, Maryland. Submitted for publication August 18, 1994. Accepted for publication May 2, 1995. Presented in part at the meeting of the American Thoracic Society, San Francisco, California, May 1993.
  • Address reprint requests to Dr. Nichols: Division of Pediatric Intensive Care, Children's Medical Surgical Center 7110, The Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, Maryland 21287-3711.
Article Information
Meeting Abstracts   |   September 1995
Effect of Aminophylline on High-energy Phosphate Metabolism and Fatigue in the Diaphragm
Anesthesiology 9 1995, Vol.83, 557-567.. doi:
Anesthesiology 9 1995, Vol.83, 557-567.. doi:
Methods: Bilateral phrenic stimulation was used to pace the diaphragm in pentobarbital-anesthetized piglets (6-10 weeks old; n = 44). Esophageal and abdominal pressures were measured to calculate transdiaphragmatic pressure (Pdi) (Pdi = abdominal pressure - esophageal pressure) as an index of force output. Activation was determined by the amplitude of the compound action potential of the diaphragmatic electromyogram. Aerobic metabolism was assessed with a31Phosphorus MRS surface coil on the right hemidiaphragm with the animal in a 4.7-T magnet. The animals were divided into four groups based on aminophylline loading dose: saline, aminophylline 10 mg/kg (A10), aminophylline 20 mg/kg (A20), and aminophylline 40 mg/kg (A40). After aminophylline loading the diaphragm was paced for 25 min followed by a 10-min recovery.
Results: Aminophylline concentrations were 12.2 plus/minus 0.7, 21.9 plus/minus 2.4, and 44.9 plus/minus 3.6 mg/l in the A10, A20, and A40 groups, respectively. Compound action potential amplitude decreased in all groups by 30% after 25 min of pacing. Conversely, Pdi remained at 100 plus/minus 3% of the initial value after 5 min of pacing in the A40 group but decreased to 75 plus/minus 3% in the saline group. Pdi recovered completely (103 plus/minus 17%) in the A40 group but remained depressed (72 plus/minus 6%) in the saline group. Pdi values were intermediate in the A10 and A20 groups. MRS data revealed inadequate energy supply/demand ratio in the saline group such that the ratio of inorganic phosphate to phosphocreatine (Pi/PCr) increased to 1.01 plus/minus 0.09 after 5 min of pacing. Pi/PCr remained unchanged in the A40 group and was intermediate in the A10 and A20 groups. beta-Adenosine triphosphate and intracellular pH did not differ among groups or as a function of pacing. Diaphragmatic blood flow increased from a resting value of 35-60 to 300-410 ml *symbol* min sup -1 *symbol* 100 g sup -1 during pacing in all groups and was not affected by aminophylline dose.
Conclusions: Aminophylline, in a dose-dependent fashion, delays the onset of fatigue and improves recovery from fatigue. Delayed fatigue is associated with improved aerobic metabolism as reflected in a low Pi/PCr ratio.
Key words: Energy metabolism: adenosine triphosphate; inorganic phosphate; intracellular pH; phosphocreatine. Ions: calcium. Measurement techniques: magnetic resonance spectroscopy. Muscle: diaphragm. Pharmacology: aminophylline.
DIAPHRAGMATIC fatigue may contribute to chronic respiratory failure, difficulty in separation from mechanical ventilation, and apnea in newborn infants. [1,2] Given the morbidity, mortality, and cost of therapy for these conditions, drug therapy to improve diaphragm contractility is an important goal. Of the drugs, which have been investigated including beta-adrenergic agonists, [3] cardiac glycosides, [4] and phosphodiesterase inhibitors, aminophylline (a nonspecific phosphodiesterase inhibitor) is the most widely studied example. [5,6] Despite this extensive study, there remains uncertainty about the effectiveness and mechanism of action of aminophylline in treating diaphragmatic fatigue.
Previously, we have investigated the relation between high-energy phosphate metabolism and neural activation in the fatiguing diaphragm using31Phosphorus magnetic resonance spectroscopy (MRS) and the compound action potential (CAP) amplitude of the diaphragmatic electromyogram (EMG). [7] Although inadequate energy supply or decreased activation of the diaphragm has been proposed as a mechanism for diaphragmatic fatigue, we have shown that these mechanisms are not mutually exclusive in the piglet diaphragm activated by phrenic nerve pacing. Phosphocreatine (PCr) consumption and inorganic phosphate (Pi) production leading to a Pi/PCr ratio [nearly equal] 1 and indicative of inadequate energy supply/demand ratio occur with the onset of fatigue. [7] The Pi/PCr ratio does not return toward control levels until activation and force output of the diaphragm have decreased with prolonged diaphragmatic fatigue.
The purpose of the current study was to investigate the mechanism of action of aminophylline on diaphragmatic fatigue and to attempt to validate the relation between inadequate energy metabolism and diaphragmatic fatigue. We hypothesized that aminophylline (1) reduces fatigue of the diaphragm, (2) improves high-energy phosphate metabolism, and (3) exhibits dose-dependent effects.
Materials and Methods
Animal Preparation
Our preparation has been described previously (Figure 1). [7] In brief, piglets (n = 44; 6-10 weeks old) weighing 10-16 kg were anesthetized with pentobarbital intravenously (20-mg/kg loading dose followed by 10-15-mg *symbol* kg sup -1 *symbol* h sup -1 continuous infusion). The animals' lungs were ventilated by tracheostomy with a Harvard animal ventilator to maintain normocapnia and arterial Oxygen2tension > 100 mmHg.
Figure 1. Animal preparation. The animal's lungs are ventilated by tracheostomy. Transdiaphragmatic pressure is measured with esophageal and abdominal balloon-tipped catheters. Diaphragm is paced with bilateral phrenic nerve stimulation in the chest. Nuclear magnetic resonance (NMR) spectroscopy surface coil is adjacent to right abdominal diaphragm. Left ventricular and aortic catheters are used are used for microsphere injection and withdrawal, respectively. a = artery. (Reproduced with permission from Nichols et al. [7])
Figure 1. Animal preparation. The animal's lungs are ventilated by tracheostomy. Transdiaphragmatic pressure is measured with esophageal and abdominal balloon-tipped catheters. Diaphragm is paced with bilateral phrenic nerve stimulation in the chest. Nuclear magnetic resonance (NMR) spectroscopy surface coil is adjacent to right abdominal diaphragm. Left ventricular and aortic catheters are used are used for microsphere injection and withdrawal, respectively. a = artery. (Reproduced with permission from Nichols et al. [7])
Figure 1. Animal preparation. The animal's lungs are ventilated by tracheostomy. Transdiaphragmatic pressure is measured with esophageal and abdominal balloon-tipped catheters. Diaphragm is paced with bilateral phrenic nerve stimulation in the chest. Nuclear magnetic resonance (NMR) spectroscopy surface coil is adjacent to right abdominal diaphragm. Left ventricular and aortic catheters are used are used for microsphere injection and withdrawal, respectively. a = artery. (Reproduced with permission from Nichols et al. [7])
×
Venous catheters were placed in the left and right internal jugular veins for maintenance fluid, continuous pentobarbital and aminophylline administration. Arterial catheters were placed in the left carotid artery for blood sampling, in the left ventricle via the right femoral artery for microsphere injection, and in the abdominal aorta via the left femoral artery for microsphere withdrawal.
After a right thoracotomy, helical stainless steel stimulating electrodes were positioned around each phrenic nerve and then connected to a nerve stimulator (S8, Grass Medical Instruments, Quincy, MA) for phrenic nerve pacing. A midline laparotomy was performed to place an elliptical (5 x 2 cm) MRS surface coil, which had been tuned to both proton (200 MHz) and phosphorus (80.5 MHz) resonance frequencies, on the abdominal side of the right hemidiaphragm. The surface coil was shielded from surrounding intercostal and abdominal wall muscle with wet gauze padding. Air was evacuated from the chest and the incisions were closed in layers.
Esophageal pressure (Pes) was measured with an air-filled balloon-tipped catheter positioned through an esophagotomy in the neck. A similar catheter was inserted into the abdomen by laparotomy to determine abdominal pressure (Pab). A needle-tipped catheter was introduced into the endotracheal tube lumen to measure airway pressure. Diaphragmatic force output was given by the transdiaphragmatic pressure (Pdi), which was taken as Pab - Pes at end-expiration with the airway occluded. Diaphragmatic fatigue was defined as a decrease in Pdi by > 20% compared with the initial value during pacing. Constant transpulmonary pressure (taken as airway pressure - Pes) was used as an index of constant lung volume during the experiment.
Blood Analyses
Arterial blood samples for pH, CO2tension, and Oxygen2tension were measured with a blood gas analyzer (ABL3, Radiometer America, Cleveland, OH). Hemoglobin, Oxygen2saturation, and Oxygen2content were analyzed with a co-oximeter (IL 282 CO-oximeter, Instrumentation Laboratory, Lexington, MA). Plasma aminophylline concentrations were analyzed by fluorescent polarization immunoassay technique (TDX system, Abbott Laboratories, Dallas, TX) with a sensitivity of 0.4 mg/l and < 1% cross reactivity.
Radiolabeled Microspheres
Approximately 1.3 x 106microspheres (15 plus/minus 0.5 micro meter in diameter) (du Pont-New England Nuclear Products, Boston, MA) were injected over 30 s into the left ventricle for each diaphragmatic blood flow (Qdi) measurement. [8] Withdrawal of the reference blood sample from the abdominal aorta using a withdrawal pump (Harvard Apparatus Co., Millis, MA) began 20 s before the injection and lasted until 5 ml blood had been withdrawn. The withdrawal rate was 2.47 ml/min.
At the conclusion of the experiment the animal was killed with an overdose of Sodium pentobarbital and KCl. The diaphragm was sectioned, weighed, placed in 15-ml Poly Q vials, and counted in a multichannel analyzer (Minaxi model 5330, Packard Instrument Co., Downer's Grove, IL) with a 7.6-cm through-hole NaI crystal. Qdi was calculated as Qdi = CDx (QR/CR) x 100, where CD= counts/g diaphragm; QR= reference blood flow (2.47 ml/min); and CR= total counts in the reference sample.
In Vivo Magnetic Resonance Spectroscopy
MRS data were obtained in 24 animals in a 40-cm-bore, 4.7-T magnet (CSI, General Electric, Freemont, CA). Noise was filtered from the MRS signal by attaching a filter to the stimulating electrodes, which were grounded on the wall of the magnet at the bore aperture. Homogeneity of the magnet field was maximized by adjusting the shim coil current at the start of the experiment. Signal to noise ratio was further improved by gating the signal acquisition to changes in airway pressure, which varied primarily in response to mechanical ventilation. Free induction decays for phosphorus were acquired after 80-W radiofrequency pulses of 85-micro second duration. The interval between successive radiofrequency pulses was determined by airway pressure changes and was usually 3.0 s. A single spectrum was produced by averaging free induction decays over 2 min. Fourier transform of the free induction decay was performed after exponential weighting with a line width of 40 Hz.
Areas of beta-adenosine triphosphate (beta-ATP), PCr, and Pi were computed by a least-squares best-fit technique. To adjust for relaxation effects, the PCr and Pi areas were corrected relative to beta-ATP by factors of 1.3 and 1.1, respectively. The areas of each phosphate peak were expressed as fractions of the total phosphate area to adjust for changes in signal intensity. Based on studies by Chance et al. [9] and Gutierrez et al. [10] we assumed that Pi/PCr greater or equal to 1 reflected a state of inadequate oxidative metabolism in the muscle. Intracellular pH (pHi) was determined by the chemical shift of the Pi. [11] .
Electromyography
Because measurement of CAP amplitude interferes with the MRS signal, a separate group of 20 animals was used to evaluate CAP of the diaphragmatic EMG during pacing with and without aminophylline. The same surgical procedures and protocol were used for this group except for placement of a bipolar stainless steel electrode in the abdominal side of the left hemidiaphragm to measure CAP. A reference electrode was placed in the adjacent rectus abdominis muscle. A copper plate applied to the back of the animal served as the ground electrode.
The CAP electrodes were connected to a signal averager (Quantum 84, Cadwell Laboratories, Kennewick, WA). CAP and Pdi were measured at the onset of pacing and then every 5 min. The CAP amplitude data at each 5 min time point represented the average of 24 individual compound action potentials recorded within an 800-ms interval. Bandpass filters of the signal averager were set at 100 and 10,000 Hz. The CAP amplitude was taken as the distance between the trough of the initial downward deflection and the peak of the subsequent upward deflection.
Protocol
To assess the effects of aminophylline on diaphragmatic force output, activation, and high-energy phosphate metabolism, the animals (n = 44) were divided into four groups: (1) saline control (n = 11), (2) aminophylline 10 mg/kg (A10; n = 11), aminophylline 20 mg/kg (A20; n = 11), and (4) aminophylline 40 mg/kg (A40; n = 11). The aminophylline loading doses of 10, 20, and 40 mg/kg intravenously were followed by continuous infusions of aminophylline of 1, 2, and 4 mg *symbol* kg sup -1 *symbol* h sup -1, respectively. A 60-min recovery was allowed after completion of the surgical procedures. Thereafter, MRS data were acquired every 5 min during four phases of the experiment: (1) control with the diaphragm at rest before drug administration (10 min); (2) diaphragm rest during saline or aminophylline loading (10-20 min); (3) diaphragm pacing and saline or aminophylline continuous infusion (25 min); and (4) recovery (no pacing, 10 min). Diaphragm pacing required stimulation of both phrenic nerves in the chest with supramaximal voltage, stimulation frequency 30 Hz, duty cycle 0.33, and 10 trains/min. These parameters were chosen to mimic a normal respiratory pattern with 10 breaths/min, an inspiratory to expiratory time ratio of 1:2 (duty cycle 0.33), and a phrenic nerve firing rate in the physiologic range (stimulation frequency 30 Hz). [12] The animals' lungs were also mechanically ventilated throughout the experiment to suppress all spontaneous respiratory effort and maintain adequate oxygenation and CO2elimination.
Pdi was determined at 5-min intervals in all animals. Pi, PCr, ATP, and pHi were measured in 24 animals: 6 animals in each of the saline, A10, A20, and A40 groups. Simultaneous Qdi determination with the microsphere technique was obtained in 20 animals (5 in each of the four groups). Because there were only six isotopes available, microsphere injection took place at control; during saline or aminophylline loading; at 1, 5, and 25 min of pacing; and during recovery. Diaphragmatic activation as measured by CAP amplitude was determined every 5 min during pacing in 20 animals (n = 5 in each of the four groups).
Statistical Analysis
The data were analyzed by analysis of variance to determine differences between groups and within a single group over time. Critical differences between the mean values were assessed with Scheffe's F test or the paired t test with Bonferroni's correction. P < 0.05 was considered significant. Results are expressed as mean plus/minus SE.
Results
Arterial blood gases and pHa (Table 1), transpulmonary pressure, hemoglobin concentration, and mean arterial pressure did not vary as a function of pacing or aminophylline dose. After the aminophylline loading dose, heart rate increased in the A40 group compared with the saline group (P < 0.05, Table 2).
Table 1. Arterial Blood Gas Data with the Diaphragm at Rest, during Pacing (Pace 0, 10, and 20 min), and Recovery in the Saline, Aminophylline 10 mg/kg (A10), Aminophylline 20 mg/kg (A20), and Aminophylline 40 mg/kg (A40) Groups
Image not available
Table 1. Arterial Blood Gas Data with the Diaphragm at Rest, during Pacing (Pace 0, 10, and 20 min), and Recovery in the Saline, Aminophylline 10 mg/kg (A10), Aminophylline 20 mg/kg (A20), and Aminophylline 40 mg/kg (A40) Groups
×
Table 2. Hemodynamic Data with the Diaphragm at Rest, during Pacing (Pace 0, 10, and 20 min), and Recovery in the Saline, Aminophylline 10 mg/kg (A10), Aminophylline 20 mg/kg (A20), and Aminophylline 40 mg/kg (A40) Groups
Image not available
Table 2. Hemodynamic Data with the Diaphragm at Rest, during Pacing (Pace 0, 10, and 20 min), and Recovery in the Saline, Aminophylline 10 mg/kg (A10), Aminophylline 20 mg/kg (A20), and Aminophylline 40 mg/kg (A40) Groups
×
Plasma aminophylline concentrations varied in a dose-dependent manner (Figure 2). At the onset of pacing, the plasma aminophylline concentration was 12.2 plus/minus 0.7 mg/l in the A10 group, 21.9 plus/minus 2.4 mg/l in the A20 group (P < 0.05 compared with A10 group), and 44.9 plus/minus 3.6 mg/l in the A40 group (P < 0.05 compared with A10 and A20 groups). Aminophylline concentrations decreased gradually over time in all groups despite a continuous infusion of aminophylline.
Figure 2. Plasma aminophylline concentrations ([A]) after administration of aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1 (A10), 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1 (A20), and 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1 (A40).
Figure 2. Plasma aminophylline concentrations ([A]) after administration of aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1 (A10), 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1 (A20), and 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1 (A40).
Figure 2. Plasma aminophylline concentrations ([A]) after administration of aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1 (A10), 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1 (A20), and 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1 (A40).
×
(Figure 3and Figure 4) demonstrate the effects of aminophylline on diaphragmatic force output and activation. The original tracings of Pab, Pes, and CAP show preservation of Pab and Pes (and hence Pdi) despite decreasing CAP amplitude when the animal is treated with aminophylline 40 mg/kg (Figure 3(A)) in contrast to the decrease in Pab, Pes, and CAP amplitude during pacing in a saline control animal (Figure 3(B)). Mean data confirm that aminophylline did not affect CAP amplitude during pacing (Figure 4(A)). CAP amplitude was reduced in all groups by 30% after 25 min of pacing. Conversely, aminophylline had a dose-dependent effect on diaphragmatic force output (Figure 4(B)). Pdi was significantly greater in the A40 group than in the saline group during pacing (P < 0.01) such that fatigue (> 20% decrease in Pdi) was prevented in the A40 animals for the first 20 min of pacing, whereas fatigue occurred in the saline group within the first 5 min of pacing. When the diaphragm was allowed to rest for 10 min after pacing, Pdi returned to baseline in the A40 group (103 plus/minus 16%), whereas Pdi remained depressed in the saline group (72 plus/minus 6%) (P < 0.05 compared with the initial value). The A10 and A20 groups exhibited Pdi levels intermediate between these two extremes at each time point consistent with a dose-dependent effect of aminophylline.
Figure 3. Original tracings of compound action potential (CAP), abdominal pressure (Pab), and esophageal pressure (Pes) during diaphragm pacing with aminophylline 40 mg/kg (A40) (A) compared with saline control (SAL) (B). Note decrease in CAP amplitude in both animals, but Pab and Pes (and hence Pdi) are nearly preserved in the animal given aminophylline but decrease rapidly and persistently throughout pacing in the control animal. (The offset of the CAP baselines does not imply differences in resting potential.) REC = recovery.
Figure 3. Original tracings of compound action potential (CAP), abdominal pressure (Pab), and esophageal pressure (Pes) during diaphragm pacing with aminophylline 40 mg/kg (A40) (A) compared with saline control (SAL) (B). Note decrease in CAP amplitude in both animals, but Pab and Pes (and hence Pdi) are nearly preserved in the animal given aminophylline but decrease rapidly and persistently throughout pacing in the control animal. (The offset of the CAP baselines does not imply differences in resting potential.) REC = recovery.
Figure 3. Original tracings of compound action potential (CAP), abdominal pressure (Pab), and esophageal pressure (Pes) during diaphragm pacing with aminophylline 40 mg/kg (A40) (A) compared with saline control (SAL) (B). Note decrease in CAP amplitude in both animals, but Pab and Pes (and hence Pdi) are nearly preserved in the animal given aminophylline but decrease rapidly and persistently throughout pacing in the control animal. (The offset of the CAP baselines does not imply differences in resting potential.) REC = recovery.
×
Figure 4. (A) Percent compound action potential (CAP) amplitude during diaphragm pacing. Note decreases in percent CAP amplitude are not affected by aminophylline dose. (B) Percent transdiaphragmatic pressure (Pdi) during diaphragm pacing. Note rapid and significant decrease in percent Pdi during saline administration in contrast to preservation of percent Pdi during the first 5 min of pacing and only modest fatigue after 25 min, which recovers completely in the A40 group. Intermediate reductions in percent Pdi occur in the A10 and A20 groups. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 4. (A) Percent compound action potential (CAP) amplitude during diaphragm pacing. Note decreases in percent CAP amplitude are not affected by aminophylline dose. (B) Percent transdiaphragmatic pressure (Pdi) during diaphragm pacing. Note rapid and significant decrease in percent Pdi during saline administration in contrast to preservation of percent Pdi during the first 5 min of pacing and only modest fatigue after 25 min, which recovers completely in the A40 group. Intermediate reductions in percent Pdi occur in the A10 and A20 groups. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 4. (A) Percent compound action potential (CAP) amplitude during diaphragm pacing. Note decreases in percent CAP amplitude are not affected by aminophylline dose. (B) Percent transdiaphragmatic pressure (Pdi) during diaphragm pacing. Note rapid and significant decrease in percent Pdi during saline administration in contrast to preservation of percent Pdi during the first 5 min of pacing and only modest fatigue after 25 min, which recovers completely in the A40 group. Intermediate reductions in percent Pdi occur in the A10 and A20 groups. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
×
(Figure 5(A)) displays the effects of aminophylline on Pi/PCr during pacing. The normal resting Pi/PCr ratio of 0.2-0.3 did not change during saline or aminophylline infusion. Immediately after the onset of pacing, Pi/PCr increased to 1.01 plus/minus 0.09 in the saline group (P < 0.05 compared with rest and with A40 group during pacing). Subsequently, Pi/PCr decreased progressively to 0.61 plus/minus 0.06 by the end of pacing and returned to a resting control value of 0.30 plus/minus 0.03 after a 10-min recovery. In contrast, Pi/PCr did not differ significantly from the resting levels throughout pacing with aminophylline 40 mg/kg. In the A10 and A20 groups, Pi/PCr was intermediate between the saline and A40 levels during pacing such that Pi/PCr increased to 0.56 plus/minus 0.12 (A10) and 0.49 plus/minus 0.14 (A20) immediately after the onset of pacing (P < 0.05 compared with resting value and with saline value at the onset of pacing). Pi/PCr plateaued at these levels for the next 15 min of pacing in contrast to the sharp increase and subsequent decrease in Pi/PCr in the saline group.
Figure 5. Magnetic resonance spectroscopic data in the diaphragm from 24 animals during saline (S) or aminophylline administration. (A) Inorganic phosphate/phosphocreatine ratio (Pi/PCr) with and without aminophylline administration during rest, pacing, and recovery. Pi/PCr approaches 1 immediately after the onset of pacing in saline-treated animals. Conversely, A40 treatment prevents the increase in Pi/PCr. A10 and A20 treatments produce intermediate increases in Pi/PCr. (B) Adenosine triphosphate (ATP) concentration does not change as a function of pacing or aminophylline dose. (C) intracellular pH (pHi) does not vary during pacing as a function of aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 5. Magnetic resonance spectroscopic data in the diaphragm from 24 animals during saline (S) or aminophylline administration. (A) Inorganic phosphate/phosphocreatine ratio (Pi/PCr) with and without aminophylline administration during rest, pacing, and recovery. Pi/PCr approaches 1 immediately after the onset of pacing in saline-treated animals. Conversely, A40 treatment prevents the increase in Pi/PCr. A10 and A20 treatments produce intermediate increases in Pi/PCr. (B) Adenosine triphosphate (ATP) concentration does not change as a function of pacing or aminophylline dose. (C) intracellular pH (pHi) does not vary during pacing as a function of aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 5. Magnetic resonance spectroscopic data in the diaphragm from 24 animals during saline (S) or aminophylline administration. (A) Inorganic phosphate/phosphocreatine ratio (Pi/PCr) with and without aminophylline administration during rest, pacing, and recovery. Pi/PCr approaches 1 immediately after the onset of pacing in saline-treated animals. Conversely, A40 treatment prevents the increase in Pi/PCr. A10 and A20 treatments produce intermediate increases in Pi/PCr. (B) Adenosine triphosphate (ATP) concentration does not change as a function of pacing or aminophylline dose. (C) intracellular pH (pHi) does not vary during pacing as a function of aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
×
(Figure 5(B)) shows beta-ATP in the presence and absence of aminophylline during rest and pacing. The resting beta-ATP area referenced to the total phosphate area was approximately 0.2 in all groups. Neither pacing nor aminophylline infusion affected beta-ATP subsequently. Similarly, pHi was approximately 7.2 in all groups and remained constant during pacing regardless of aminophylline dose (Figure 5(C)). These changes are evident in Figure 6, which depicts original MRS scans during saline (Figure 6(A)) and aminophylline 40 mg/kg (Figure 6(B)) administration.
Figure 6. (A) Original31Phosphorus nuclear magnetic resonance (NMR) spectroscopic spectra from a saline (S)-treated animal. Note the increase in inorganic phosphate (Pi) and the decrease in phosphocreatine (PCr) peak area during pacing. ATP = adenosine triphosphate. (B) Similar original spectra from an animal treated with aminophylline 40 mg/kg (A40). Note minimal change in Pi and PCr peaks during pacing compared with the resting state. ATP presents as 3 individual peaks (gamma, alpha, and beta). Because quantitative changes are the same for the individual peaks, only beta-ATP peak area is analyzed. Amn = aminophylline.
Figure 6. (A) Original31Phosphorus nuclear magnetic resonance (NMR) spectroscopic spectra from a saline (S)-treated animal. Note the increase in inorganic phosphate (Pi) and the decrease in phosphocreatine (PCr) peak area during pacing. ATP = adenosine triphosphate. (B) Similar original spectra from an animal treated with aminophylline 40 mg/kg (A40). Note minimal change in Pi and PCr peaks during pacing compared with the resting state. ATP presents as 3 individual peaks (gamma, alpha, and beta). Because quantitative changes are the same for the individual peaks, only beta-ATP peak area is analyzed. Amn = aminophylline.
Figure 6. (A) Original31Phosphorus nuclear magnetic resonance (NMR) spectroscopic spectra from a saline (S)-treated animal. Note the increase in inorganic phosphate (Pi) and the decrease in phosphocreatine (PCr) peak area during pacing. ATP = adenosine triphosphate. (B) Similar original spectra from an animal treated with aminophylline 40 mg/kg (A40). Note minimal change in Pi and PCr peaks during pacing compared with the resting state. ATP presents as 3 individual peaks (gamma, alpha, and beta). Because quantitative changes are the same for the individual peaks, only beta-ATP peak area is analyzed. Amn = aminophylline.
×
Qdi is affected by pacing but not by aminophylline (Figure 7). Once pacing commenced, Qdi increased to 300-410 ml *symbol* min sup -1 *symbol* 100 g sup -1 at 1 and 5 min and then decreased to 200-250 ml *symbol* min sup -1 *symbol* 100 g sup -1 in all groups by the end of pacing.
Figure 7. Diaphragmatic blood flow (Qdi) determined at six time points: control (C), saline or aminophylline infusion (S/A), diaphragm pacing at 1, 5, or 25 min (P-1, P-5, P-25), and recovery (REC) in the four groups. Qdi increases during diaphragm pacing but does not differ depending on aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 7. Diaphragmatic blood flow (Qdi) determined at six time points: control (C), saline or aminophylline infusion (S/A), diaphragm pacing at 1, 5, or 25 min (P-1, P-5, P-25), and recovery (REC) in the four groups. Qdi increases during diaphragm pacing but does not differ depending on aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 7. Diaphragmatic blood flow (Qdi) determined at six time points: control (C), saline or aminophylline infusion (S/A), diaphragm pacing at 1, 5, or 25 min (P-1, P-5, P-25), and recovery (REC) in the four groups. Qdi increases during diaphragm pacing but does not differ depending on aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
×
Discussion
The major findings of this study are that (1) aminophylline delays the onset of diaphragmatic fatigue despite a reduction in diaphragmatic activation during pacing; (2) aminophylline limits the increase in Pi/PCr that is otherwise observed during fatigue; (3) the effects of aminophylline on Pdi and Pi/PCr cannot be explained by differences in Qdi; and (4) the effects of aminophylline are dose-dependent.
Aminophylline Effects on Force Output and Activation
Our data suggest a direct effect of aminophylline on diaphragmatic muscle with improved diaphragmatic force output and delayed onset of fatigue during pacing despite decreasing CAP amplitude (Figure 4). This result is consistent with previous findings in animals [13] and humans [14] in which aminophylline had no effect on diaphragmatic EMG but increased Pdi. Our findings are not consistent with reports that suggest aminophylline acts primarily through increased activation either at the brainstem or neuromuscular junction. The data supporting a central stimulatory effect of aminophylline come primarily from neonates in whom aminophylline is used to treat apnea of prematurity. These studies have documented a reduction in the frequency of central apnea, but have not measured diaphragmatic EMG. [15] Hence, it is possible that the central effects of aminophylline lie in the regular initiation of discharges from the respiratory center rather than in an increase in amplitude of those discharges. Similarly, aminophylline has been reported to antagonize neuromuscular blockade with pancuronium, raising the possibility that aminophylline may act by increasing cyclic adenosine monophosphate at the motor endplate, which in turn increases acetylcholine release. [16] This mechanism has been called into question by the finding that neuromuscular blockade produced by vecuronium is not antagonized by aminophylline. [17] Furthermore, the observed improvement in strength after aminophylline administration during neuromuscular blockade with pancuronium also may be explained by increased myoplasmic Calcium2+ concentration resulting from the aminophylline (which would leave diaphragmatic EMG unchanged) rather than increased neurotransmitter release (which should increase diaphragmatic EMG). Thus our data and others', [18] in which diaphragmatic activation was measured directly, show that the primary action of aminophylline is within the muscle rather than along the activation pathway with the exception of stabilizing respiratory center discharge in patients with central apnea.
Although others' work [18] supports our result of improved diaphragmatic force output with aminophylline, some investigators have noted no effect [6] or even decreased force output. [19] These differing results reflect the complexity of the interaction between aminophylline and respiratory muscle, including factors such as aminophylline dose, developmental stage, muscle fiber type composition, and type of diaphragmatic contractions (paced vs. spontaneous breathing). The particular importance of a large aminophylline dose is confirmed by our data.
Aminophylline Effects on High-energy Phosphate Metabolism
In a dose-dependent manner, aminophylline prevents the increase in Pi/PCr ratio while preserving force output of the diaphragm during pacing. Others [20] have shown that contractile failure of cardiac and limb muscle as a result of inadequate energy supply relative to demand is accompanied by an increase in Pi/PCr to greater or equal to 1. We have demonstrated previously the same phenomenon in the diaphragm. [7] Marsh et al. [21] used MRS to demonstrate that theophylline administration improves forearm contractile force in association with improved aerobic metabolism. The current report extends these observations on the link between energy metabolism and force output by demonstrating that force output can be improved with administration of aminophylline, which also prevents the increase in Pi/PCr in the diaphragm.
Intracellular pH was approximately 7.2 in all groups during diaphragm pacing and was not affected by the onset of fatigue or by the administration of aminophylline (Figure 5(C)). We have shown previously that diaphragmatic fatigue induced by phrenic nerve pacing is not associated with significant changes in pHi. [7] Others have also noted that changes in pHi cannot account for muscle fatigue, [22] possibly because lactate production during muscle stimulation is balanced by H+ consumption during PCr hydrolysis. [23] .
The few studies on the effects of aminophylline on pHi have provided conflicting results. Connett [24] noted an association between intracellular alkalosis and Calcium2+ release in frog sartorius muscle during aminophylline administration. Conversely, Shee et al. [25] found no effect of aminophylline on pHi in the resting state and a decrease in pHi during stimulated rat diaphragm contractions in the presence of aminophylline. These differing results may reflect different muscle fiber types or difference between in vitro, superfused muscle versus in vivo, perfused muscle. When adult subjects voluntarily increase forearm contraction to the point of exhaustion, intracellular acidosis is noted and is ameliorated by theophylline administration. [21] Intracellular acidosis may be more likely during progressively increasing voluntary contractions to the point of maximum output as opposed to the constant stimulation frequency producing submaximal paced contractions used in this investigation. Alternatively, differences in fiber type composition may play a role. Regardless of the reported effects of aminophylline on pHi, these studies agree with our finding that the improvement in force output in the presence of aminophylline is not mediated by an effect on pHi.
ATP was unaffected by pacing or aminophylline in our study. This observation is consistent with our previous data [7] and that of others. [26] The constancy of ATP during fatiguing contractions reflects the activity of the creatine kinase reaction to buffer changes in ATP at the expense of PCr hydrolysis and Pi production. Extreme hypoxia or prolonged maximal tetanic contractions may overwhelm the creatine kinase buffer and lead to ATP depletion. [27] In general, studies in humans showing ATP depletion during fatigue have used maximal voluntary contraction to exhaustion. [28] Our experimental protocol used constant stimulation intensity to produce submaximal contractions of the diaphragm, and the extreme conditions described above were not used. Finally, it is possible that changes ATP concentration in subcellular compartments are obscured because the 31P MRS measurement of ATP provides the average ATP in the volume of tissue sampled.
We used supramaximal phrenic nerve stimulation to activate the diaphragm to control the stimulus intensity. However, this method leads to recruitment of all diaphragm fibers simultaneously rather than the incremental recruitment observed during spontaneous breathing. If fatigue and the increase in Pi/PCr develop because of early activation of fatigable, glycolytic type IIb fibers in the diaphragm (which might not have been activated during spontaneous breathing), then it is possible that aminophylline improves force output and aerobic metabolism by preferentially affecting type IIb fibers. This possibility could not be tested in our model because the MRS surface coil samples a volume of tissue that is approximately the size of the diameter of the coil and that contains all fiber types.
Aminophylline Effects on Diaphragmatic Blood Flow
Qdi increased during pacing, but the increases in Qdi were not affected by aminophylline dose (Figure 7). Thus the improved force output and aerobic energy metabolism in the presence of the large dose of aminophylline cannot be explained by increased Qdi. Our series confirms the results of a previous study on Qdi in the piglet by Mayock et al. [29] The pattern of Qdi changes during muscle contraction may depend on developmental stage, because studies in adult humans and animals have shown increased blood flow during aminophylline administration.
Limitations of the Study
The study design sought to assess the metabolic and mechanical effects of various doses of aminophylline on the diaphragm in vivo. This design limited the ability to control heart rate in the various groups because high dose aminophylline produces tachycardia. However, we do not believe the tachycardia in the A20 and A40 groups affected the results because there was no difference in Qdi between the groups.
Aminophylline has been shown to recruit expiratory muscles in the spontaneously breathing dog. [18] It is unknown whether this effect is also seen during diaphragm pacing. However, it raises the possibility that expiratory muscle recruitment could have led to changes in diaphragm length during pacing that in turn may have affected force output and metabolism. Although we cannot exclude small changes in length, the protocol was designed to minimize this possibility by obtaining measurements during end-expiration with the airway occluded. Constant transpulmonary pressure during the experiment suggests that there were no major changes in lung volume.
Relation between Force Output and Aerobic Metabolism
We speculate that the known effects of aminophylline on intracellular Calcium2+ (Cai2+) metabolism may explain the relation between force output and metabolism. Aminophylline increases Cai2+ concentration through sarcolemmal Cai2+ channels and by increased Cai2+ release from the sarcoplasmic reticulum. [30] Increased Cai2+ leads to increased crossbridge formation and force output. Aminophylline may also improve relaxation of the diaphragm by enhancing Calcium2+ sequestration into sarcoplasmic reticulum after a contraction. [31] The improved relaxation leads a more optimal diaphragm length and hence increased force output during the next contraction. Conversely, delayed relaxation may reduce contractile force, as has been shown in cardiac muscle, in which relaxation defects have been associated with heart failure, [32] myocardial ischemia, [33] and "stunned" myocardium. [34] .
The increase in Cai2+ may also improve aerobic energy metabolism within muscle. [35] Once myoplasmic Calcium2+ concentrations have increased, mitochondrial Calcium2+ uptake is mediated by an electrophoretic uniporter mechanism that is stimulated by increasing myoplasmic Calcium2+ concentration. [36] Conversely, Calcium2+ efflux from mitochondria by the Sodium sup +/Calcium2+ exchanger is inhibited by increased extramitochondrial Calcium2+ concentrations. [37] The resultant increase in intramitochondrial Calcium2+ concentration activates three intramitochondrial dehydrogenases: pyruvate dehydrogenase, [38] isocitrate dehydrogenase, [39] and alpha-ketoglutarate dehydrogenase. [40] The increase in activity of these Calcium2+ sensitive enzymes leads to an increase in turnover of the Krebs cycle, an increase in NADH production, and ultimately an increase in overall oxidative phosphorylation of adenosine diphosphate to ATP. Thus changes in intracellular Calcium2+ concentration balance the consumption of ATP by muscle work with ATP production by oxidative phosphorylation.
A variety of compounds, including epinephrine, phenylephrine, and caffeine, stimulate intramitochondrial dehydrogenases and oxidative metabolism. [41-43] This stimulatory effect is blocked by ruthenium red, an inhibitor of Calcium2+ uptake into mitochondria. [43] The effects of aminophylline on Calcium2+ -sensitive intramitochondrial dehydrogenase activity have not, to our knowledge, been investigated directly. However, our data are consistent with the interpretation that aminophylline raises intracellular Calcium2+ such that the increase in myosin adenosine triphosphatase activity during diaphragmatic work is balanced by increased aerobic metabolism and oxidative phosphorylation, which in turn is reflected in the low Pi/PCr ratio during pacing. The finding that another methylxanthine, caffeine, affects aerobic metabolism in a similar dose-dependent fashion further supports this interpretation. [43] Because Qdi is unchanged with aminophylline, it is likely that the effects of aminophylline on aerobic metabolism are associated with increased Oxygen2extraction and consumption.
In summary, we have demonstrated that aminophylline improves aerobic metabolism and delays the onset of fatigue in the diaphragm in a dose-dependent manner. We speculate that increased Cai2+ concentration resulting from aminophylline provides the link between aerobic metabolism and force output. Even if sufficient improvement in diaphragmatic force output may not be uniformly achievable with nontoxic aminophylline doses, a clearer understanding of the relation between energy metabolism and force output may permit the development of other drugs to increase diaphragmatic force by preserving aerobic metabolism.
REFERENCES
Lopes JM, Muller NL, Bryan MH, Bryan AC: Synergistic behavior of inspiratory muscles after diaphragmatic fatigue in the newborn. J Appl Physiol 51:547-551, 1981.
Cohen CA, Zagelbaum G, Gross D, Roussos C, Macklem PT: Clinical manifestations of inspiratory muscle fatigue. Am J Med 73:308-316, 1982.
Ebata T, Fujii Y, Toyooka H: Dobutamine increases diaphragmatic contractility in dogs. Can J Anaesth 39:375-380, 1992.
Aubier M, Murciano D, Viires N, Lebargy F, Curran Y, Seta JP, Pariente R: Effects of digoxin on diaphragmatic strength generation in patients with chronic obstructive pulmonary disease during acute respiratory failure. Am Rev Respir Dis 135:544-548, 1987.
Levy RD, Nava S, Gibbons L, Bellemare F: Aminophylline and human diaphragm strength in vivo. J Appl Physiol 68:2591-2596, 1990.
Polaner DM, Kimball WR, Fratacci MD, Wain JC, Torres A, Kacmarek RM, Zapol WM: Effects of aminophylline on regional diaphragmatic shortening after thoracotomy in the awake lamb. ANESTHESIOLOGY 77:93-100, 1992.
Nichols DG, Buck JR, Eleff SM, Shungu DC, Robotham JL, Koehler RC, Traystman RJ: Diaphragmatic fatigue assessed by 31P-magnetic resonance spectroscopy in vivo. Am J Physiol 264:C1111-C1118, 1993.
Nichols DG, Howell S, Massik J, Koehler RC, Gleason CA, Buck JR, Fitzgerald RS, Traystman RJ, Robotham JL: Relationship of diaphragmatic contractility to diaphragmatic blood flow in newborn lambs. J Appl Physiol 66:120-127, 1989.
Chance B, Leigh JS Jr, Clark BJ, Maris J, Kent J, Nioka S, Smith D: Control of oxidative metabolism and oxygen delivery in human skeletal muscle: A steady-state analysis of the work/energy cost transfer function. Proc Natl Acad Sci U S A 82:8384-8388, 1985.
Gutierrez G, Pohil RJ, Andry JM, Strong R, Narayana P: Bio-energetics of rabbit skeletal muscle during hypoxemia and ischemia. J Appl Physiol 65:608-616, 1988.
Moon RB, Richards JH: Determination of intracellular pH by 31P magnetic resonance. J Biol Chem 248:7276-7278, 1973.
Iscoe S, Dankoff J, Migicovsky R, Polosa C: Recruitment and discharge frequency of phrenic motoneurones during inspiration. Respir Physiol 26:113-128, 1976.
Sigrist S, Thomas D, Howell S, Roussos C: The effect of aminophylline on inspiratory muscle contractility. Am Rev Respir Dis 126:46-50, 1982.
Okubo S, Konno K, Ishizaki T, Kubo M, Suganuma T, Takizawa T: Effect of theophylline on respiratory neuromuscular drive. Eur J Clin Pharmacol 33:85-88, 1987.
Barrington KJ, Finer NN: A randomized, controlled trial of aminophylline in ventilatory weaning of premature infants. Crit Care Med 21:846-850, 1993.
Dretchen KL, Morgenroth VH, Standaert FG, Walts LF: Azathioprine: Effects on neuromuscular transmission. ANESTHESIOLOGY 45:604-609, 1976.
Daller JA, Erstad B, Rosado L, Otto C, Putnam CW: Aminophylline antagonizes the neuromuscular blockade of pancuronium but not vecuronium. Crit Care Med 19:983-985, 1991.
Decramer M, Deschepper K, Jiang TX, Derom E: Effects of aminophylline on respiratory muscle interaction. Am Rev Respir Dis 144:797-802, 1991.
Esau SA: Slowing of relaxation in the fatiguing hamster diaphragm is enhanced by theophylline. J Appl Physiol 65:1307-1313, 1988.
Gutierrez G, Pohil RJ, Andry JM, Strong R, Narayana P: Bio-energetics of rabbit skeletal muscle during hypoxemia and ischemia. J Appl Physiol 65:608-616, 1988.
Marsh GD, McFadden RG, Nicholson RL, Leasa DJ, Thompson RT: Theophylline delays skeletal muscle fatigue during progressive exercise. Am Rev Respir Dis 147:876-879, 1993.
Idstrom JP, Subramanian VH, Chance B, Schersten T, Bylund-Fellenius AC: Oxygen dependence of energy metabolism in contracting and recovering rat skeletal muscle. Am J Physiol 248:H40-H48, 1985.
Sahlin K, Ren JM: Relationship of contraction capacity to metabolic changes. J Appl Physiol 67:648-654, 1989.
Connett RJ: Association of increased pHi with calcium ion release in skeletal muscle. Am J Physiol 234:C110-C114, 1978.
Shee CD, Wright AM, Cameron IR: The effect of aminophylline on function and intracellular pH of the rat diaphragm. Eur Respir J 3:991-996, 1990.
Miller RG, Boska MD, Moussavi RS, Carson PJ, Weiner MW: 31P nuclear magnetic resonance studies of high energy phosphates and pH in human muscle fatigue: Comparison of aerobic and anaerobic exercise. J Clin Invest 81:1190-1196, 1988.
Kushmerick MJ, Meyer RA: Chemical changes in rat leg muscle by phosphorus nuclear magnetic resonance. Am J Physiol 248:C542-C549, 1985.
Hirvonen J, Nummela A, Rusko H, Rehunen S, Harkonen M: Fatigue and changes of ATP, creatine phosphate, and lactate during the 400-m sprint. Canadian Journal of Sports Sciences 17:141-144, 1992.
Mayock DE, Twiggs GA, Standaert TA, Watchko JF, Woodrum DE: The effect of aminophylline on diaphragm blood flow in the piglet. Pediatr Res 26:196-199, 1989.
Aubier M, Murciano D, Viires N, Lecocguic Y, Pariente R: Diaphragmatic contractility enhanced by aminophylline: Role of extracellular calcium. J Appl Physiol 54:460-464, 1983.
Sham JS, Jones LR, Morad M: Phospholamban mediates the beta-adrenergic-enhanced Calcium sup 2+ uptake in mammalian ventricular myocytes. Am J Physiol 261:H1344-H1349, 1991.
Grossman W: Diastolic dysfunction in congestive heart failure. N Engl J Med 325:1557-1564, 1991.
Aroesty JM, McKay RG, Heller GV, Royal HD, Als AV, Grossman W: Simultaneous assessment of left ventricular systolic and diastolic dysfunction during pacing-induced ischemia. Circulation 71:889-900, 1985.
Charlat ML, O'Neill PG, Hartley CJ, Roberts R, Bolli R: Prolonged abnormalities of left ventricular diastolic wall thinning in the 'stunned' myocardium in conscious dogs: Time course and relation to systolic function. J Am Coll Cardiol 13:185-194, 1989.
McCormack JG, Halestrap AP, Denton RM: Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol Rev 70:391-425, 1990.
Denton RM, McCormack JG: Calcium sup 2+ transport by mammalian mitochondria and its role in hormone action. Am J Physiol 249:E543-E554, 1985.
Hayat LH, Crompton M: Evidence for the existence of regulatory sites for Calcium sup 2+ on the Sodium sup +/Calcium sup 2+ carrier of cardiac mitochondria. Biochem J 202:509-518, 1982.
Denton RM, Randle PJ, Martin BR: Stimulation by calcium ions of pyruvate dehydrogenase phosphate phosphatase. Biochem J 128:161-163, 1972.
Denton RM, Richards DA, Chin JG: Calcium ions and the regulation of NAD sup + -linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues. Biochem J 176:899-906, 1978.
McCormack JG, Denton RM: The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem J 180:533-544, 1979.
McCormack JG, England PJ: Ruthenium red inhibits the activation of pyruvate dehydrogenase caused by positive inotropic agents in the perfused rat heart. Biochem J 214:581-585, 1983.
Oviasu OA, Whitton PD: Hormonal control of pyruvate dehydrogenase activity in rat liver. Biochem J 224:181-186, 1984.
Kunz WS, Kuznetsov AV, Gellerich FN: Mitochondrial oxidative phosphorylation in saponin-skinned human muscle fibers is stimulated by caffeine. FEBS Lett 323:188-190, 1993.
Figure 1. Animal preparation. The animal's lungs are ventilated by tracheostomy. Transdiaphragmatic pressure is measured with esophageal and abdominal balloon-tipped catheters. Diaphragm is paced with bilateral phrenic nerve stimulation in the chest. Nuclear magnetic resonance (NMR) spectroscopy surface coil is adjacent to right abdominal diaphragm. Left ventricular and aortic catheters are used are used for microsphere injection and withdrawal, respectively. a = artery. (Reproduced with permission from Nichols et al. [7])
Figure 1. Animal preparation. The animal's lungs are ventilated by tracheostomy. Transdiaphragmatic pressure is measured with esophageal and abdominal balloon-tipped catheters. Diaphragm is paced with bilateral phrenic nerve stimulation in the chest. Nuclear magnetic resonance (NMR) spectroscopy surface coil is adjacent to right abdominal diaphragm. Left ventricular and aortic catheters are used are used for microsphere injection and withdrawal, respectively. a = artery. (Reproduced with permission from Nichols et al. [7])
Figure 1. Animal preparation. The animal's lungs are ventilated by tracheostomy. Transdiaphragmatic pressure is measured with esophageal and abdominal balloon-tipped catheters. Diaphragm is paced with bilateral phrenic nerve stimulation in the chest. Nuclear magnetic resonance (NMR) spectroscopy surface coil is adjacent to right abdominal diaphragm. Left ventricular and aortic catheters are used are used for microsphere injection and withdrawal, respectively. a = artery. (Reproduced with permission from Nichols et al. [7])
×
Figure 2. Plasma aminophylline concentrations ([A]) after administration of aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1 (A10), 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1 (A20), and 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1 (A40).
Figure 2. Plasma aminophylline concentrations ([A]) after administration of aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1 (A10), 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1 (A20), and 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1 (A40).
Figure 2. Plasma aminophylline concentrations ([A]) after administration of aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1 (A10), 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1 (A20), and 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1 (A40).
×
Figure 3. Original tracings of compound action potential (CAP), abdominal pressure (Pab), and esophageal pressure (Pes) during diaphragm pacing with aminophylline 40 mg/kg (A40) (A) compared with saline control (SAL) (B). Note decrease in CAP amplitude in both animals, but Pab and Pes (and hence Pdi) are nearly preserved in the animal given aminophylline but decrease rapidly and persistently throughout pacing in the control animal. (The offset of the CAP baselines does not imply differences in resting potential.) REC = recovery.
Figure 3. Original tracings of compound action potential (CAP), abdominal pressure (Pab), and esophageal pressure (Pes) during diaphragm pacing with aminophylline 40 mg/kg (A40) (A) compared with saline control (SAL) (B). Note decrease in CAP amplitude in both animals, but Pab and Pes (and hence Pdi) are nearly preserved in the animal given aminophylline but decrease rapidly and persistently throughout pacing in the control animal. (The offset of the CAP baselines does not imply differences in resting potential.) REC = recovery.
Figure 3. Original tracings of compound action potential (CAP), abdominal pressure (Pab), and esophageal pressure (Pes) during diaphragm pacing with aminophylline 40 mg/kg (A40) (A) compared with saline control (SAL) (B). Note decrease in CAP amplitude in both animals, but Pab and Pes (and hence Pdi) are nearly preserved in the animal given aminophylline but decrease rapidly and persistently throughout pacing in the control animal. (The offset of the CAP baselines does not imply differences in resting potential.) REC = recovery.
×
Figure 4. (A) Percent compound action potential (CAP) amplitude during diaphragm pacing. Note decreases in percent CAP amplitude are not affected by aminophylline dose. (B) Percent transdiaphragmatic pressure (Pdi) during diaphragm pacing. Note rapid and significant decrease in percent Pdi during saline administration in contrast to preservation of percent Pdi during the first 5 min of pacing and only modest fatigue after 25 min, which recovers completely in the A40 group. Intermediate reductions in percent Pdi occur in the A10 and A20 groups. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 4. (A) Percent compound action potential (CAP) amplitude during diaphragm pacing. Note decreases in percent CAP amplitude are not affected by aminophylline dose. (B) Percent transdiaphragmatic pressure (Pdi) during diaphragm pacing. Note rapid and significant decrease in percent Pdi during saline administration in contrast to preservation of percent Pdi during the first 5 min of pacing and only modest fatigue after 25 min, which recovers completely in the A40 group. Intermediate reductions in percent Pdi occur in the A10 and A20 groups. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 4. (A) Percent compound action potential (CAP) amplitude during diaphragm pacing. Note decreases in percent CAP amplitude are not affected by aminophylline dose. (B) Percent transdiaphragmatic pressure (Pdi) during diaphragm pacing. Note rapid and significant decrease in percent Pdi during saline administration in contrast to preservation of percent Pdi during the first 5 min of pacing and only modest fatigue after 25 min, which recovers completely in the A40 group. Intermediate reductions in percent Pdi occur in the A10 and A20 groups. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
×
Figure 5. Magnetic resonance spectroscopic data in the diaphragm from 24 animals during saline (S) or aminophylline administration. (A) Inorganic phosphate/phosphocreatine ratio (Pi/PCr) with and without aminophylline administration during rest, pacing, and recovery. Pi/PCr approaches 1 immediately after the onset of pacing in saline-treated animals. Conversely, A40 treatment prevents the increase in Pi/PCr. A10 and A20 treatments produce intermediate increases in Pi/PCr. (B) Adenosine triphosphate (ATP) concentration does not change as a function of pacing or aminophylline dose. (C) intracellular pH (pHi) does not vary during pacing as a function of aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 5. Magnetic resonance spectroscopic data in the diaphragm from 24 animals during saline (S) or aminophylline administration. (A) Inorganic phosphate/phosphocreatine ratio (Pi/PCr) with and without aminophylline administration during rest, pacing, and recovery. Pi/PCr approaches 1 immediately after the onset of pacing in saline-treated animals. Conversely, A40 treatment prevents the increase in Pi/PCr. A10 and A20 treatments produce intermediate increases in Pi/PCr. (B) Adenosine triphosphate (ATP) concentration does not change as a function of pacing or aminophylline dose. (C) intracellular pH (pHi) does not vary during pacing as a function of aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 5. Magnetic resonance spectroscopic data in the diaphragm from 24 animals during saline (S) or aminophylline administration. (A) Inorganic phosphate/phosphocreatine ratio (Pi/PCr) with and without aminophylline administration during rest, pacing, and recovery. Pi/PCr approaches 1 immediately after the onset of pacing in saline-treated animals. Conversely, A40 treatment prevents the increase in Pi/PCr. A10 and A20 treatments produce intermediate increases in Pi/PCr. (B) Adenosine triphosphate (ATP) concentration does not change as a function of pacing or aminophylline dose. (C) intracellular pH (pHi) does not vary during pacing as a function of aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
×
Figure 6. (A) Original31Phosphorus nuclear magnetic resonance (NMR) spectroscopic spectra from a saline (S)-treated animal. Note the increase in inorganic phosphate (Pi) and the decrease in phosphocreatine (PCr) peak area during pacing. ATP = adenosine triphosphate. (B) Similar original spectra from an animal treated with aminophylline 40 mg/kg (A40). Note minimal change in Pi and PCr peaks during pacing compared with the resting state. ATP presents as 3 individual peaks (gamma, alpha, and beta). Because quantitative changes are the same for the individual peaks, only beta-ATP peak area is analyzed. Amn = aminophylline.
Figure 6. (A) Original31Phosphorus nuclear magnetic resonance (NMR) spectroscopic spectra from a saline (S)-treated animal. Note the increase in inorganic phosphate (Pi) and the decrease in phosphocreatine (PCr) peak area during pacing. ATP = adenosine triphosphate. (B) Similar original spectra from an animal treated with aminophylline 40 mg/kg (A40). Note minimal change in Pi and PCr peaks during pacing compared with the resting state. ATP presents as 3 individual peaks (gamma, alpha, and beta). Because quantitative changes are the same for the individual peaks, only beta-ATP peak area is analyzed. Amn = aminophylline.
Figure 6. (A) Original31Phosphorus nuclear magnetic resonance (NMR) spectroscopic spectra from a saline (S)-treated animal. Note the increase in inorganic phosphate (Pi) and the decrease in phosphocreatine (PCr) peak area during pacing. ATP = adenosine triphosphate. (B) Similar original spectra from an animal treated with aminophylline 40 mg/kg (A40). Note minimal change in Pi and PCr peaks during pacing compared with the resting state. ATP presents as 3 individual peaks (gamma, alpha, and beta). Because quantitative changes are the same for the individual peaks, only beta-ATP peak area is analyzed. Amn = aminophylline.
×
Figure 7. Diaphragmatic blood flow (Qdi) determined at six time points: control (C), saline or aminophylline infusion (S/A), diaphragm pacing at 1, 5, or 25 min (P-1, P-5, P-25), and recovery (REC) in the four groups. Qdi increases during diaphragm pacing but does not differ depending on aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 7. Diaphragmatic blood flow (Qdi) determined at six time points: control (C), saline or aminophylline infusion (S/A), diaphragm pacing at 1, 5, or 25 min (P-1, P-5, P-25), and recovery (REC) in the four groups. Qdi increases during diaphragm pacing but does not differ depending on aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
Figure 7. Diaphragmatic blood flow (Qdi) determined at six time points: control (C), saline or aminophylline infusion (S/A), diaphragm pacing at 1, 5, or 25 min (P-1, P-5, P-25), and recovery (REC) in the four groups. Qdi increases during diaphragm pacing but does not differ depending on aminophylline dose. S = saline; A10 = aminophylline 10 mg/kg followed by 1 mg *symbol* kg sup -1 *symbol* h sup -1; A20 = 20 mg/kg followed by 2 mg *symbol* kg sup -1 *symbol* h sup -1; and A40 = 40 mg/kg followed by 4 mg *symbol* kg sup -1 *symbol* h sup -1.
×
Table 1. Arterial Blood Gas Data with the Diaphragm at Rest, during Pacing (Pace 0, 10, and 20 min), and Recovery in the Saline, Aminophylline 10 mg/kg (A10), Aminophylline 20 mg/kg (A20), and Aminophylline 40 mg/kg (A40) Groups
Image not available
Table 1. Arterial Blood Gas Data with the Diaphragm at Rest, during Pacing (Pace 0, 10, and 20 min), and Recovery in the Saline, Aminophylline 10 mg/kg (A10), Aminophylline 20 mg/kg (A20), and Aminophylline 40 mg/kg (A40) Groups
×
Table 2. Hemodynamic Data with the Diaphragm at Rest, during Pacing (Pace 0, 10, and 20 min), and Recovery in the Saline, Aminophylline 10 mg/kg (A10), Aminophylline 20 mg/kg (A20), and Aminophylline 40 mg/kg (A40) Groups
Image not available
Table 2. Hemodynamic Data with the Diaphragm at Rest, during Pacing (Pace 0, 10, and 20 min), and Recovery in the Saline, Aminophylline 10 mg/kg (A10), Aminophylline 20 mg/kg (A20), and Aminophylline 40 mg/kg (A40) Groups
×