Abstract
We investigated the effects, and the mechanism of the effects, of isoproterenol on diaphragmatic contractility and fatigue in septic peritonitis in vitro. Ninety-six rats were divided into two groups of 48. One group (CLP group) was treated with cecal ligation and perforation (CLP) and the other (sham group) was treated with laparotomy. The left hemidiaphragm was removed at 16 h after the operation. We assessed the diaphragmatic contractility by twitch characteristics and force-frequency curves in vitro. Diaphragm fatigue was induced by rhythmically stimulating strips to contract at 60/ min (20 Hz, 0.33-s trains, 1 train/s) over a 4-min period. Force-frequency curves were determined before and after fatigue. Isoproterenol (10− 9, 10− 8, and 10− 7 M), a β -adrenoceptor agonist, was cumulatively administered to the organ bath. Isoproterenol significantly increased diaphragmatic contractility. There were no significant changes in diaphragmatic contractility in the sham group. Isoproterenol (10− 7 M) significantly accelerated diaphragmatic recovery of fatigue and increased cAMP levels both in the sham group and the CLP group. Propranolol (10− 7 M), a general β -adrenoceptor blocker, completely abolished the positive inotropic effect of isoproterenol (10− 7 M) and increased cAMP levels in the CLP group. Dibutyryl cAMP (10− 3 M), a derivative of cyclic AMP, mimicked the effects of isoproterenol in the CLP group. These results suggest that isoproterenol increases diaphragmatic contractility and accelerates diaphragmatic recovery of fatigue in septic peritonitis by activating the adenylate cyclase system. Fujimura N, Sumita S, Narimatsu E, Nakayama Y, Shitinohe Y, Namiki A. Effects of isoproterenol on diaphragmatic contractility in septic peritonitis.
Diaphragmatic dysfunction may contribute to the development of respiratory failure during sepsis (1-3). Diaphragm muscle impairment and/or diaphragm muscle fatigue are thought to be the cause of diaphragmatic dysfunction occurring during sepsis (1). In recent years, much attention has been paid to the usefulness of pharmacologic agents in the treatment of respiratory muscle dysfunction (4-6). However, there are few reports on the treatment of sepsis-induced diaphragm muscle dysfunction.
Recent studies have suggested that beta-agonists increase fresh and fatigued diaphragmatic contractility in vivo and in vitro (4-6). The force-potentiating effect is considered to be due to activation of β-adrenoreceptors (7, 8), and β-adrenoreceptors have been found in the diaphragm (9). The β-adrenoceptor binds to a stimulatory G-protein, and this activates adenylate cyclase, resulting in the formation of cyclic adenosine monophosphate (cAMP) (10). Previous reports suggested that cAMP is the second messenger responsible for the effects of the beta-agonist on skeletal muscle contractility (6, 8).
In our preliminary study (11), we found that isoproterenol and forskoline increased twitch tension in the septic rat diaphragm. Therefore, we hypothesized that isoproterenol has a positive inotropic effect on diaphragmatic contractility during sepsis and that this effect is mediated by an increased cAMP concentration in the diaphragm muscle.
Thus, the present study was designed to (1) estimate the effect of isoproterenol on diaphragmatic contractility and fatigue during sepsis measured in vitro; (2) measure the cAMP concentration in the diaphragm muscle; (3) evaluate the effect of dibutyryl cyclic AMP (db-cAMP), a derivative of cyclic AMP that is more membrane-permeable and less susceptible to intracellular hydrolysis than is cAMP, on diaphragmatic contractility and fatigue during sepsis measured in vitro.
This study was approved by the Committee on Animal Research of Sapporo Medical University. All experiments were conducted on 8- to 10-wk-old male Wistar rats (weighing 250 to 300 g).
We divided the rats into two groups, each containing 48 rats. In the first group (CLP rats), intra-abdominal sepsis was produced using the cecal ligation and perforation (CLP) technique described previously (12). Briefly, under isoflurane and oxygen anesthesia, we performed a laparotomy through a midline abdominal incision. We used volatile anesthetics to anesthetized rats since volatile anesthetics have little effect on diaphragmatic contractility (13). Then we ligated the cecum just below the ileocecal valve with 3-0 silk ligature, so that intestinal continuity was maintained. Using an 18-gauge needle, the cecum was perforated in two locations, 1 cm apart, on the antimesentric surface of the cecum, and the cecum was gently compressed until feces were extruded. The bowel was then returned to the abdomen and the incision was closed with proline sutures for the muscles and 3-0 silk for the skin. Afterward, we observed the rats in a recovery cage for 2 h. Antibiotics were not administered to any of the rats. All rats were resuscitated with saline solution (5 ml/100 g body weight) injected subcutaneously in the back at the time of operation. The rats were deprived of food but had free access to water after the operation. The left hemidiaphragm was removed at 16 h after the operation. The control group (sham group) underwent laparotomy, and the cecum was manipulated but not ligated or punctured. The left hemidiaphragm was also removed at 16 h after the operation and was tested for contractility.
The rats were killed under deep anesthesia of isoflurane. Strips of approximately 10 mm in width, not containing the phrenic nerves, were dissected from the medial aspect of the left hemidiaphragm of each rat. All strips were removed together with the associated ribs and central tendon. The isolated strips were placed in an organ bath containing oxygenated Krebs–Ringer's solution (27° C). The strips were mounted vertically in a tissue chamber, with the central tendon superiorly positioned and attached to a Grass FT-10 force transducer (Grass Instruments, Quincy, MA), which was connected to a micropositioner, and was positioned between two platinum plates. The strips were stimulated with supramaximal currents (1.2 to 1.3 times the current required to elicit maximal tension) delivered via platinum field electrodes. The current was supplied by an amplifier driven by a Grass S48 stimulator. In these experiments, d-tubocurarine (Sigma Chemical Co., St. Louis, MO) at a concentration of 15 μM was added to the Krebs–Ringer's solution to eliminate the activation of intramuscular nerve branches.
The strips were allowed to equilibrate in the organ bath for 20 min. The optimal force-length (L0) relationship was then determined by adjusting the micropositioner between intermittent stimulations of the muscle strips. All stimulations during the study were performed at L0. Muscle contractile characteristics were then assessed according to twitch characteristics and the force-frequency relationship.
Peak twitch tension, contraction time (time to peak tension), and half-relaxation time (time required for tension to decay from maximum to half-maximum) were calculated from single twitches (0.1 Hz stimulation) that were recorded at a high speed (100 mm/s).
Force-frequency relationships were determined by stimulating the diaphragm strips tetanically at frequencies from 10 to 100 Hz over 10-Hz increments. An interval of 10 s was used between stimuli, and the pulses were 0.2 ms in duration with a train duration of 400 ms. Force-frequency relationships were determined 20 min after determination of L0.
The strips were fatigued over a 4-min period (20 Hz, 330 ms train and 670 ms rest, 1 train/s). Muscle fatigability was then assessed by examining the rate of fall of tension over a 4-min contraction.
The diaphragmatic strips were homogenized in 10 volumes of Hank's balanced salt solution (without calcium and magnesium) containing 5 mM EDTA with a homogenizer. The homogenates were centrifuged at 1,000 × g for 10 min at 4° C. The extraction of cAMP from the supernatant was carried out by the solid-phase extraction method using Amprep SAX columns (Amersham, Buckinghamshire, UK). The concentrations of cAMP were measured by the Biotrak cAMP enzyme immunoassay system (Amersham). The resulting pellets were solubilized and used to measure protein concentrations by the Lowry method (14).
Experiment 1. Effect of isoproterenol on diaphragmatic contractility and fatigue. Twenty-four rats were used to measure diaphragmatic contractility (n = 12) and the effect of fatigue (n = 12) after the operation.
After the equilibrating period, 10−9, 10−8, and 10−7 M doses of the beta adrenoceptor agonist isoproterenol (Sigma Chemical) were cumulatively administered to the organ bath. Diaphragmatic contractility was assessed for each dose.
To investigate the effects of isoproterenol on diaphragmatic fatigue, a 10−7 M dose of isoproterenol was administered to the organ bath. Force-frequency relationships were determined 20 min before and 30 s after fatigue trials and then again 15 min into the period of recovery after cessation of the fatiguing stimulation paradigm.
A pilot study showed that, at the concentrations used, isoproterenol had no effects on the pH of the oxygenated Krebs–Ringer's solution. Furthermore, this study showed that a maximal increase in twitch tension was reached within 15 min after the addition of isoproterenol and lasted for 20 min. Therefore, a period of 20 min was allowed for thermoequilibration and diffusion of isoproterenol at each dose.
Experiment 2. Effect of propranolol on the positive inotropic effect of isoproterenol on diaphragmatic contractility. Twenty-four rats were used to measure diaphragmatic contractility (n = 12) and the effect of fatigue (n = 12) after the operation.
To determine the mechanism by which isoproterenol acts on the septic rat diaphragm, a 10−7 M dose of the beta blocker propranolol (Sigma Chemical) was administered to the organ bath prior to the administration of a 10−7 M dose of isoproterenol. Diaphragmatic contractility and the effect of fatigue were assessed. A period of 20 min was allowed for thermoequilibration and diffusion of propranolol and isoproterenol.
Experiment 3. Effect of dibutyryl cAMP on diaphragmatic contractility and fatigue. Twenty-four rats were used to measure diaphragmatic contractility (n = 12) and the effect of fatigue (n = 12) after the operation.
A 10−3 M dose of dibutyryl cAMP (Sigma Chemical) was administered to the organ bath. Diaphragmatic contractility was then assessed.
To investigate the effects of dibutyryl cAMP on diaphragmatic fatigue, a 10−3 M dose of dibutyryl cAMP was administered to the organ bath. Force-frequency relationships were determined 20 min before and 30 s after fatigue trials and then again 15 min into the period of recovery after cessation of the fatiguing stimulation paradigm.
A period of 50 min was allowed for thermoequilibration and diffusion of dibutyryl cAMP.
Experiment 4. The diaphragmatic cAMP concentration. Twenty-four rats were used in the fourth experiment.
To determine the mechanism by which isoproterenol acts on the septic rat diaphragm, diaphragmatic strips were incubated in Krebs– Ringer's solution containing a 10−7 M dose of isoproterenol, in Krebs– Ringer's solution containing a 10−7 M dose of propranolol plus isoproterenol, or in Krebs–Ringer's solution alone. Diaphragmatic strips were frozen quickly in liquid nitrogen and stored at −80° C until the assay.
In these experiments, six animals each from the CLP group and the sham group were tested.
The cross-sectional area was calculated by dividing the muscle mass by the length in centimeters, and the density of muscle was assumed to be 1.056 mg/cm3. Tension was calculated as force per unit of cross-sectional area (kg/cm2). Statistics were calculated using a software program (Macintosh StatView J 4.02; Abacus Concepts, Berkeley, CA). Comparisons of force-frequency curves among the groups were made by repeated-measures analysis of variance (ANOVA). Comparisons of the diaphragm muscle wet/dry ratio, twitch characteristics, and peak tetanic tension among the groups were made with a one-way analysis of variance (ANOVA) combined with Scheffe's procedure used for post hoc comparison of data sets. A p value less than 0.05 was considered significant. Data are presented as mean ± SEM.
There were no significant differences between the weights of rats in the following groups: sham group (270.3 ± 5.2 g) and CLP group (276.9 ± 5.6 g). The weight, length, and cross-sectional area of diaphragm preparations were similar in the two groups. There were no significant differences between the dry/ wet weights ratio of diaphragm muscles between two groups: sham group (0.258 ± 0.004 g) and CLP group (0.259 ± 0.003 g).
Isoproterenol (10−7 M) did not induce contracture both the sham group and the CLP group. The effects of isoproterenol on diaphragmatic twitch characteristics are shown in Table 1. The peak twitch tension in the CLP group was significantly lower than that in the sham group (p < 0.01). The contraction time in the CLP group was significantly decreased compared with that in the sham group (p < 0.01). The half relaxation time in the CLP group was significantly longer than that in the sham group (p < 0.05).
| Groups | Twitch Characteristics | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ISP (M) | CONT | 10−9 | 10−8 | 10−7 | ||||||
| Sham | TT, kg/cm2 | 0.48 ± 0.03 | 0.48 ± 0.02 | 0.48 ± 0.03 | 0.49 ± 0.03 | |||||
| CT, ms | 41.8 ± 0.7 | 46.3 ± 1.5§ | 46.5 ± 0.5‖ | 46.7 ± 0.7‖ | ||||||
| 1/2RT, ms | 29.5 ± 1.2 | 30.7 ± 0.3 | 30.2 ± 0.2 | 31.0 ± 0.4 | ||||||
| CLP | TT, kg/cm2 | 0.17 ± 0.01‡ | 0.19 ± 0.01 | 0.23 ± 0.01‖ | 0.24 ± 0.01‖ | |||||
| CT, ms | 32.1 ± 1.4‡ | 33.1 ± 1.4 | 37.6 ± 1.8‖ | 39.7 ± 1.6‖ | ||||||
| 1/2RT, ms | 46.3 ± 6.2† | 40.3 ± 4.9§ | 33.7 ± 2.8§ | 32.2 ± 3.3‖ | ||||||
The average increase in twitch tension induced by each dose in the sham group was as follows : 10−9 M, −0.9 ± 1.8%; 10−8 M, 0.5 ± 3.0%; 10−7 M, 2.8 ± 2.9%. There were no significant changes in peak twitch tension and half-relaxation time in the sham group in any dose. Isoproterenol significantly prolonged contraction time in the sham group (10−9 M, p < 0.05; 10−8 and 10−7 M, p < 0.01). The average increase in twitch tension induced by each dose in the CLP group was as follows: 10−9 M, 18.5 ± 11.9%; 10−8 M, 44.6 ± 18.8%; 10−7 M, 51.3 ± 23.1%. Isoproterenol significantly increased peak twitch tension in the CLP group in a dose-dependent manner (10−8 and 10−7 M, p < 0.01). Isoproterenol significantly prolonged contraction time in the CLP group (10−8 and 10−7 M, p < 0.01), whereas it significantly decreased half-relaxation time (10−9 and 10−8 M, p < 0.05; 10−7 M, p < 0.01).
The effects of isoproterenol on diaphragmatic force-frequency curves are shown in Figure 1. Force-frequency curves for the CLP group are significantly lower than those for the sham group (p < 0.01). The peak tetanus tension was 1.81 ± 0.06 kg/ cm2 in the sham group and 1.14 ± 0.11 kg/cm2 in the CLP group. Peak tetanus tension in the CLP group was significantly lower than that in the sham group (p < 0.01).
Fig. 1. Diaphragmatic force-frequency curves after cumulative administration of isoproterenol in the sham and CLP groups. Isoproterenol caused a significant upward shift in the force-frequency curves for the CLP group (10−8 and 10−7 M, p < 0.01).
[More] [Minimize]Isoproterenol did not induce any significant changes in the force-frequency curves for the sham group. The peak tetanus tension induced by each dose in the sham group was as follows: control, 1.81 ± 0.06 kg/cm2; 10−9 M, 1.83 ± 0.06 kg/cm2; 10−8 M, 1.83 ± 0.07 kg/cm2; 10−7 M, 1.84 ± 0.07 kg/cm2. The average increase in peak tetanus tension induced by each dose in the sham group was as follows: 10−9 M, 1.1 ± 0.8%; 10−8 M, 0.9 ± 1.7%; 10−7 M, 1.4 ± 1.6%. Isoproterenol did not induce any significant changes in peak tetanus tension in the sham group.
Isoproterenol did, however, increase force generation and cause an upward shift in the force-frequency curves for the CLP group (10−8 and 10−7 M, p < 0.01). The peak tetanus tension induced by each dose in the CLP group was as follows: control, 1.05 ± 0.03 kg/cm2; 10−9 M, 1.12 ± 0.03 kg/cm2; 10−8 M, 1.24 ± 0.04 kg/cm2; 10−7 M, 1.21 ± 0.04 kg/cm2. The average increase in peak tetanus tension induced by each dose in the CLP group was as follows: 10−9 M, 7.0 ± 2.0%; 10−8 M, 18.3 ± 5.0%; 10−7 M, 16.0 ± 5.5%. Isoproterenol significantly increased peak tetanus tension in the CLP group (10−8 and 10−7 M, p < 0.01).
The peak twitch tension induced by each dose in the CLP group was as follows: control, 182 ± 16 g/cm2; plus propranolol (10−7 M), 144 ± 16 g/cm2; plus propranolol (10−7 M) and isoproterenol (10−7 M), 144 ± 15 g/cm2. The effects of propranolol on diaphragmatic force-frequency curves on CLP rats are shown in Figure 2. The peak tetanic tension induced by each dose in the CLP group was as follows: control, 1.27 ± 0.14 kg/cm2; plus propranolol (10−7 M), 1.17 ± 0.13 kg/cm2; plus propranolol (10−7 M) and isoproterenol (10−7 M), 1.18 ± 0.14 kg/cm2. There were no significant change in peak twitch tension, force-frequency curves, and peak tetanic tension among the three treatments. The positive inotropic effect of isoproterenol on diaphragmatic contractility in CLP rats was completely abolished after a 20-min exposure to propranolol (10−7 M). These results indicated that force potentiation of isoproterenol requires β-receptor activation.
Fig. 2. Diaphragmatic force-frequency curves after administration of propranolol followed by isoproterenol in the CLP group. There were no significant changes in force-frequency curves.
[More] [Minimize]The cAMP levels in the diaphragm muscle after isoproterenol and propranolol plus isoproterenol are shown in Figure 3. There were significant differences in the cAMP concentration in the diaphragm muscle between the sham group and the CLP group (p < 0.05). Isoproterenol significantly increased cAMP levels in the diaphragm muscle of both the sham (p < 0.05) and the CLP groups (p < 0.01). The effects of isoproterenol on cAMP production were completely abolished after a 20-min exposure to propranolol (10−7 M) both in the sham and the CLP groups.
Fig. 3. Changes in diaphragmatic cAMP concentration after administration of isoproterenol and propranolol followed by isoproterenol in the sham and CLP groups. #p < 0.05 compared with the sham group. *p < 0.05 compared with control values. **p < 0.01 compared with control values.
[More] [Minimize]The effects of dibutyryl cAMP on diaphragmatic twitch characteristics are shown in Table 2. The average increase in twitch tension in the sham group was −4.5 ± 2.9%. Dibutyryl cAMP did not induce any significant changes in peak twitch tension, contraction time and half-relaxation time in the sham group.
| Groups | Twitch Characteristics | |||||
|---|---|---|---|---|---|---|
| db-cAMP (M) | CONT | 10−3 | ||||
| Sham | TT, kg/cm2 | 0.44 ± 0.04 | 0.43 ± 0.04 | |||
| CT, ms | 34.5 ± 1.1 | 32.4 ± 1.2 | ||||
| 1/2RT, ms | 24.8 ± 1.1 | 27.7 ± 0.3 | ||||
| CLP | TT, kg/cm2 | 0.17 ± 0.02‡ | 0.23 ± 0.02§ | |||
| CT, ms | 25.8 ± 1.6‡ | 35.9 ± 1.4‖ | ||||
| 1/2RT, ms | 34.0 ± 3.4† | 29.5 ± 3.4§ | ||||
The average increase in twitch tension in the CLP group was 38.8 ± 8.2%. Dibutyryl cAMP significantly increased peak twitch tension in the CLP group (p < 0.05) and significantly prolonged contraction time in the CLP group (p < 0.01). Dibutyryl cAMP also significantly decreased half-relaxation time in the CLP group (p < 0.05).
The effects of dibutyryl cAMP on diaphragmatic force-frequency curves are shown in Figure 4. Force-frequency curves for the CLP group were significantly lower than those for the sham group (p < 0.01). The peak tetanus tension was 1.21 ± 0.21 kg/cm2 in the sham group and 0.69 ± 0.08 kg/cm2 in the CLP group. Peak tetanus tension in the CLP group was significantly lower than that in the sham group (p < 0.01).
Fig. 4. Diaphragmatic force-frequency curves after administration of dibutyryl cAMP in the sham and CLP groups. Dibutyryl cAMP caused a significant upward shift in the force-frequency curves for the CLP group (p < 0.05).
[More] [Minimize]The peak tetanus tension in the sham group was as follows: control, 1.88 ± 0.09 kg/cm2 and 10−3 M, 1.86 ± 0.09 kg/cm2. The average increase in peak tetanus tension in the sham group was −20.8 ± 0.3%. Dibutyryl cAMP did not induce any significant changes in peak tetanus tension and force-frequency curves in the sham group.
Dibutyryl cAMP increased force generation and caused an upward shift in force frequency curves for the CLP group (p < 0.05). The peak tetanic tension in the CLP group was as follows: control, 1.16 ± 0.10 kg/cm2 and 10−3 M, 1.45 ± 0.10 kg/ cm2. The average increase in peak tetanic tension in the CLP group was 25.5 ± 2.5%. Dibutyryl cAMP significantly increased peak tetanus tension in the CLP group (p < 0.05).
The effects of isoproterenol and dibutyryl cAMP on diaphragmatic fatigability are shown in Table 3. Isoproterenol and dibutyryl cAMP significantly increased diaphragmatic fatigability in the CLP group (p < 0.01).
| Groups | Fatigability (%) | |||||
|---|---|---|---|---|---|---|
| CONT | ISP | db-cAMP | ||||
| Sham | 42.4 ± 1.4 | 58.0 ± 2.1‡ | 60.4 ± 1.5‡ | |||
| CLP | 27.2 ± 2.9† | 67.5 ± 4.9‡ | 54.0 ± 2.8‡ | |||
The effects of isoproterenol and dibutyryl cAMP on diaphragmatic fatigue are shown in Figure 5. Rhythmic electrical stimulation elicited reductions in tension production in all animals. Isoproterenol significantly cause an upward shift in the force-frequency curves 15 min after fatigue for the sham and CLP groups (p < 0.01). Dibutyryl cAMP significantly cause an upward shift in the force-frequency curves 15 min after fatigue for the sham and CLP groups (p < 0.01).
Fig. 5. Diaphragmatic force-frequency curves of prefatigue and 30-s and 15-min postfatigue in the sham and CLP groups. Force is expressed as the percentage of prefatigue maximal tension for a given stimulation frequency. Isoproterenol (10−7 M) and dibutyryl cAMP (10−3 M) caused a significant upward shift in the force-frequency curves after fatigue for the CLP group (p < 0.01).
[More] [Minimize]The effect of isoproterenol (10−7 M) on fatigued diaphragmatic contractility in CLP rats was completely abolished after a 20-min exposure to propranolol (10−7 M) (Figure 6).
Fig. 6. Diaphragmatic force-frequency curves of 15-min postfatigue after administration of isoproterenol and propranolol followed by isoproterenol in the CLP group. The effect of isoproterenol on fatigued diaphragmatic contractility in the CLP rats was completely abolished after exposure to propranolol.
[More] [Minimize]The present study demonstrated that isoproterenol increased diaphragmatic contractility and accelerated diaphragmatic recovery of fatigue in the CLP rats. Isoproterenol increased diaphragmatic cAMP levels. Propranolol completely abolished the positive inotropic effect of isoproterenol and increased cAMP levels in the CLP rats. Dibutyryl cAMP mimicked the effects of isoproterenol, suggesting that activation of the adenylate cyclase system plays a role in the effects of isoproterenol on diaphragmatic contractility and fatigue in CLP rats.
In this study, isoproterenol exerted a positive inotropic effect on diaphragmatic contractility and caused an upward shift in the force-frequency curves during the recovery period after fatigue in the CLP rats. The effects of sympathomimetic amines on mammalian skeletal muscle are different in slow-twitch fibers (type I) and fast-twitch fibers (type II) (15, 16). Sympathomimetic amines increase peak twitch tension in type II fibers of mammalian skeletal muscle but decrease peak twitch tension in type I fibers (15). The positive inotropic effect of sympathomimetic amines on type II muscle fibers is much greater in fatigued than in nonfatigued fibers. The negative inotropic effect of sympathomimetic amines on type I muscle fibers is not potentiated after fatigue (15). During sepsis, hyperventilation, increased transpulmonary resistance, and decreased lung compliance secondary to lung disease increase the work of breathing and the energy demand of respiratory muscles (17). Respiratory muscle fatigue occurs easily under such conditions (3). Furthermore, underlying muscle weakness renders the diaphragm muscle more susceptible to fatigue (18). Type II muscle fibers are more easily fatigued than are type I muscle fibers. Isoproterenol might improve fatigued type II muscle fibers in CLP rats.
On the other hand, a positive inotropic effect on diaphragmatic contractility was not observed in the sham rats. The diaphragm is a mixed skeletal muscle containing 40% type I, 30% type IIa, and 30% type IIb fibers (19). The positive inotropic effect of isoproterenol on type II muscle fibers might be diminished by the negative inotropic effect of isoproterenol on type I muscle fibers; consequently, the net effect of isoproterenol on the diaphragm might be zero.
Isoproterenol increased twitch tension in the CLP rats in a dose-dependent manner. However, the corresponding increase in tetanic tension was relatively small. In particular, the increase in tetanic tension during high-frequency stimulation was small. Isoproterenol increased twitch tension of the fatigued canine diaphragm in a dose-dependent manner, but the corresponding increase in tetanic tension was small (4). The explanation for this might be that isoproterenol decreased the half relaxation time, leading to decreased twitch duration, so that less force was developed by a train of twitches of given amplitude (4, 5). The small effect of isoproterenol on tetanic tension might be due to a decreased half relaxation time.
Propranolol completely abolished the positive inotropic effect of isoproterenol on diaphragmatic contractility in the CLP rats, suggesting that the positive inotropic effect of isoproterenol on diaphragmatic contractility in the CLP rats requires β-receptor activation. Isoproterenol binds to membrane-bound β-receptors and exerts its effect via a stimulatory G-protein that activates the adenylate cyclase system, resulting in the formation of cAMP (10). cAMP activates protein kinase, which controls many biochemical events through phosphorylation of target proteins (20). In this study, isoproterenol increased cAMP tissue levels and propranolol abolished the increase in diaphragmatic cAMP levels and the positive inotropic effect of isoproterenol on diaphragmatic contractility in the CLP rats. It has been suggested that cAMP is responsible for the inotropic effect of catecholamines on mammalian skeletal muscle (8, 16, 21). We tested the hypothesis that an increased concentration of myoplasmic cAMP is responsible for the positive inotropic effect of isoproterenol on diaphragmatic contractility in CLP rats using dibutyryl cAMP. We used a high concentration (10−3 M) of dibutyryl cAMP to examine the effects of β-receptor activation on diaphragmatic contractiltiy under in vitro conditions because of diffusion limitation in the case of a whole muscle preparation, a high concentration (10−3 M) of dibutyryl cAMP is required to examine the effects of an increased concentration of myoplasmic cAMP on diaphragmatic contractility (8). Dibutyryl-cAMP mimicked the effect of isoproterenol on diaphragmatic contractility in CLP rats. These results suggest that cAMP is responsible for the positive inotropic effect of isoproterenol on diaphragmatic contractility in CLP rats. In other words, activation of the adenylate cyclase system might be responsible for the positive inotropic effect of isoproterenol on diaphragmatic contractility in CLP rats.
Isoproterenol increased cAMP tissue levels both in the CLP and sham rats. However, diaphragmatic contractility in the sham rats was not changed after administration of isoproterenol and dibutyryl-cAMP. The positive inotropic effect of cAMP on type II muscle fibers might be diminished by the negative inotropic effect of cAMP on type I muscle fibers; consequently, the net effect of increases in cAMP levels on the diaphragm in sham rats might be zero.
Isoproterenol was found to increase twitch tension in the CLP rats. Isoproterenol might exert this effect via activation of sarcoplasmic reticulum Ca2+ pumps (21) or an increase in the inward Ca2+ current (16). In other words, the increases in cAMP tissue levels enhanced either Ca2+ release from the sarcoplasmic reticulum or the influx of Ca2+ to the muscle cells. Other possible cAMP-mediated mechanisms, including the alteration of intracellular Ca2+ exchange (22), sodium-potassium transportation (23), and myosin phosphorylation (24), might also be responsible for the positive inotropic effect of isoproterenol on diaphragmatic contractility in CLP rats.
Isoproterenol and dibutyryl-cAMP were found to decrease the half relaxation time in the CLP rats. It has been suggested that the relaxant effect of catecholamines on skeletal muscle fibers is mediated by the cyclic AMP-dependent phosphorylation of phospholamban (25). The cyclic-AMP-dependent phosphorylation of phospholamban results in a marked increase in the rate of Ca2+ transport and plays a role in the acceleration of Ca2+ uptake into the sarcoplasmic reticulum (26, 27). Isoproterenol and dibutyryl-cAMP might accelerate the phosphorylation of phospholamban, leading to an acceleration in SR calcium uptake. The cAMP/ phospholamban system might be responsible for this isoproterenol-induced acceleration of relaxation in CLP rats.
Although diaphragmatic contractility was reduced, cAMP tissue levels increased after CLP. This phenomenon has also been observed during diaphragm muscle fatigue (6, 28). The increase in the cAMP tissue levels after CLP might represent diaphragm muscle fatigue caused by sepsis. Another possible explanation for this increase in the cAMP tissue levels after CLP is that sepsis caused protein degradation (29), and therefore the normalized cAMP value by mg protein might overestimate cAMP tissue levels.
Recent evidence has suggested that oxygen-derived free radicals play an important role in the development of diaphragmatic dysfunction during sepsis (1, 2). β-adrenoceptors are a part of the membrane and oxygen-derived free radicals may damage these membrane constituents. The effects of sepsis or oxygen-derived free radicals on the adenylate cyclase system in diaphragm muscle are not fully understood. Incubation of cardiac membranes with xanthine plus xanthine oxidase increased the affinity and decreased the density of β2-adrenoceptors (30). β-adrenoceptors in the rat heart undergo an externalization from light vesicles to sarcolemma during the early phase of sepsis, whereas they are internalized from the surface membrane to intracellular sites during the late phase of sepsis (31). Proinflammatory cytokines induce a decrease in β-adrenoceptor responsiveness secondary to reduced adenylate cyclase activity (32-34). The cyclic-AMP-dependent phosphorylation of phospholamban was inhibited in the canine cardiac sarcoplasmic reticulum during endotoxic shock (35). Isoproterenol increased diaphragmatic contractility and cAMP tissue levels in the CLP rats; however, diaphragmatic contractility is still depressed compared with that of the sham rats. There might be a diaphragm muscle injury and/or dysfunction in the adenylate cyclase system elicited by oxygen- derived free radicals.
In conclusion, we found that isoproterenol increases diaphragmatic contractility and accelerates diaphragmatic recovery of fatigue in CLP rats. Activation of the adenylate cyclase system might be responsible for the positive inotropic effect of isoproterenol on the CLP rat diaphragm.
Supported by Grants-in-Aid for Scientific Research Nos. 09671579 and 10671435 from the Japanese Ministry of Education, Science, Sports and Culture.
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