Journal of Applied Physiology
Vol. 82, No. 1, pp. 324-328, January 1997
GAS EXCHANGE, MECHANICS, AND AIRWAYS

Eglin-c prevents monocrotaline-induced ventilatory dysfunction

Y. L. Lai^1,2 and K.-R. Zhou²

¹ Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China; and ² Division of Pharmacology and Experimental Therapeutics, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Lai, Y. L., and K.-R. Zhou. Eglin-c prevents monocrotaline-induced ventilatory dysfunction. J. Appl. Physiol. 82(1): 324-328, 1997.The present study was carried out to investigate the relationship between elastase and monocrotaline (MCT)-induced ventilatory dysfunction in rats. To accomplish this, we used an elastase inhibitor eglin-c to suppress the activity of endogenous elastase. Thirty-five young Sprague-Dawley rats were randomly divided into six groups: control, MCT, eglin-c (1), eglin-c (2), eglin-c (1)+MCT, and eglin-c (2)+MCT. Rats in the control group received no treatment. Each MCT rat received a single subcutaneous injection of MCT (60 mg/kg) 1 wk before the functional test. Each eglin-c (1) rat was intratracheally instilled with eglin-c (9 mg/rat) twice in 1 wk. Each eglin-c (2) rat was intratracheally instilled with eglin-c (9 mg/rat) five times in 1 wk. Both eglin-c+MCT groups were treated with the combination of eglin-c (1) or eglin-c (2) and MCT. In the MCT group, there were significant decreases in dynamic respiratory compliance, maximal expiratory flow rate at 50% total lung capacity, and the slopes of the maximal expiratory flow-%total lung capacity curve and the maximal expiratory flow-static recoil pressure curve. However, in the eglin-c (1)+MCT and eglin-c (2)+MCT groups, all of the above-mentioned MCT-induced changes were prevented. All ventilatory values of the eglin-c (1) and eglin-c (2) groups were not significantly different from those of the control group. These results demonstrate that eglin-c treatment prevents MCT-induced ventilatory dysfunction and suggest that endogenous elastase may play an important role in MCT-induced inflammation-mediated ventilatory abnormality.

elastase inhibitor; tissue injury; inflammation; airway constriction; airway remodeling

INTRODUCTION

IT HAS BEEN DEMONSTRATED that monocrotaline (MCT) treatment increases lung inflammatory cells, which can release numerous mediators and elastase (20). Released elastase may cause destruction of the lung (9). Elastase-induced tissue damage can augment tissue repair (5), including increased growth in both connective tissue (10) and smooth muscle (16). The increases in connective tissue and smooth muscle are closely related to the development of pulmonary hypertension (24). In addition to alterations in pulmonary vasculature, MCT also causes ventilatory dysfunction, including an increase in lung resistance and decreases in lung compliance and carbon dioxide diffusing capacity (15). The ventilatory dysfunction appears 1-2 wk before the detection of pulmonary hypertension. Furthermore, MCT-induced changes in airways (increase in lung resistance) and in the lung (decrease in lung compliance) can be acutely reversed by a bronchodilator terbutaline (27).

It has been demonstrated that eglin-c, an inhibitor for serine elastase and cathepsin G (21), suppressed endogenous elastase generated in the lungs of rats (14). In addition, Todorovich-Hunter et al. (24) showed that increased serine elastolytic activity associated with lung injury may mediate MCT-induced pulmonary vascular changes. Because ventilatory dysfunction precedes pulmonary hypertension in MCT-treated rats (15), we hypothesized that eglin-c can attenuate or prevent MCT-induced ventilatory abnormalities. To test this hypothesis, eglin-c was intratracheally instilled to inhibit endogenous elastase in the lungs of rats and to determine whether eglin-c prevents MCT-induced ventilatory dysfunction.

MATERIALS AND METHODS

Thirty-five young Sprague-Dawley rats weighing 232 ± 4 g were randomly divided into six groups: 1) control (n = 6), 2) MCT (n = 6), 3) eglin-c (1) (n = 6), 4) eglin-c (2) (n = 5), 5) eglin-c (1)+MCT (n = 6), and 6) eglin-c (2)+MCT (n = 6). Rats in the control group received no treatment. Each animal in the MCT group received a single subcutaneous injection of MCT (Trans-World Chemicals) in a dosage of 60 mg/kg 1 wk before the functional test. MCT and its vehicle were prepared according to the method of Hilliker et al. (8). Eglin-c (Ciba-Geigy) was dissolved in saline, and the volume of intratracheal instillation was 0.15 ml/rat. Each eglin-c (1) rat was intratracheally instilled with eglin-c (9 mg/rat) twice in 1 wk. Each eglin-c (2) rat was intratracheally instilled with eglin-c (9 mg/rat) five times in 1 wk. For the eglin-c (1)+MCT group, rats were intratracheally instilled with eglin-c (9 mg/rat) twice in 1 wk and also received a single subcutaneous MCT treatment (60 mg/kg); the first intratracheal instillation with eglin-c was given 1 day before MCT treatment. Each eglin-c (2)+MCT rat was similarly treated as the eglin-c (1)+MCT rat, except eglin-c was intratracheally instilled five times in 1 wk.

Functional testings. For physiological determination of the lung function, the rat was anesthetized with an intraperitoneal injection of ethyl carbamate (1.0-1.5 g/kg). After the surgical insertion of a tracheal cannula, each rat was placed supine inside a 2.8-liter whole body plethysmograph. The rat was then paralyzed with an intravenous injection of gallamine triethiodide (10 mg/kg) and artificially ventilated with a model 680 Harvard Rodent Respirator. According to our previous study (28), the parameters for the artificial ventilation were a respiratory rate of 60 breaths/min and tidal volume (VT) of 6-8 ml/kg. Airway opening pressure (Pao) and VT were measured during artificial ventilation, and both parameters were recorded on a Grass polygraph. Dynamic respiratory compliance (Crs) was calculated as the ratio of the VT-to-Pao difference between the end of expiration and the end of inspiration. Functional residual capacity (FRC) was determined with the use of the neon dilution method (13). To examine alterations in elastic properties of the lung, the pressure-volume (PV) maneuver for the total respiratory system was performed according to a previous method (14). To obtain the maximal expiratory flow-volume (MEFV) curve, the animal was inflated to total lung capacity (TLC; lung volume at Pao = 30 cmH₂O) three times; the third inflation was via a solenoid valve. At the peak volume during the third inflation, the inflation valve was shut off and immediately another solenoid valve for deflation was automatically turned on. The deflation valve was connected to a 20-liter container that had a pressure of 40 cmH₂O (subatmospheric). The negative pressure of 40 cmH₂O produced maximal expiratory flow (_max) (13). MEFV curve was recorded and stored on a cathode-ray storage oscilloscope (model V-134, Hitachi). All reported lung volumes are at standard body termperature and pressure.

To explore the effect of recoil pressure on _max, the maximal expiratory flow-static recoil (MFSR) curve (17) was derived from both MEFV and PV curves. Slopes of both MEFV and MFSR curves were obtained with a linear regression program. Values used for the slope fittings include 1) _max between 20 and 80% TLC from the MEFV curves and 2) _max between 0 and 6 cmH₂O Pao from the MFSR curves. The range of correlation coefficients for the MEFV slopes was 0.84-0.99, and that for the MFSR slopes was 0.96-1.00. The _max rate at a specific lung volume and the slope of the MFSR curve were used as indexes of the alteration in airway dimension.

Statistical analysis. Data are means ± SE. Analysis of variance was used to establish the statistical significance of differences among groups. After analysis of variance, Dunnett's test was used to differentiate differences between experimental and control groups and Scheffe's test was employed to analyze differences between any two experimental groups. Differences were regarded as significant at P < 0.05.

RESULTS

Body weight, TLC, FRC, _max at 50% TLC, and Crs for all six groups are shown in Table 1. Compared with the control group, body weight, TLC, FRC, _max at 50% TLC, and Crs were significantly reduced in the MCT group. However, these respiratory parameters (except for body weight) were not significantly changed in the eglin-c (1)+MCT and eglin-c (2)+MCT groups. In both the eglin-c (1) and eglin-c (2) groups, all parameters were very close to those of the control group with the exception of body weight in the eglin-c (2) group. The reasons for the weight loss in the eglin-c and eglin-c+MCT groups are not clear but might be related to the handling of animals.

Table 1. Body weight and respiratory parameters in six groups of rats Group n BW, g TLC, ml FRC, ml  max 50, ml/s Crs, ml/cmH2O Control 6 260 ± 4  12.15 ± 0.60  2.08 ± 0.15  69 ± 2  0.31 ± 0.01  MCT 6 219 ± 5a 8.73 ± 0.64a 1.51 ± 0.08a 43 ± 3e 0.21 ± 0.01e Eglin-c (1) 6 249 ± 7  12.01 ± 0.44d 1.99 ± 0.08  70 ± 2  0.34 ± 0.02  Eglin-c (2) 5 228 ± 9b 11.18 ± 0.53  1.94 ± 0.10  68 ± 2  0.29 ± 0.01  Eglin-c (1) + MCT 6 225 ± 10a 11.60 ± 0.46d 2.03 ± 0.11d 71 ± 2  0.31 ± 0.01  Eglin-c (2) + MCT 6 208 ± 8a,c 11.23 ± 0.40  1.84 ± 0.08  69 ± 3  0.29 ± 0.01 Values are means ± SE; n, no. of rats. BW, body weight; TLC, total lung capacity; FRC, functional residual capacity; max 50, maximal expiratory flow at 50% of TLC; Crs, dynamic respiratory compliance; MCT, monocrotaline treated; eglin-c (1), rats intratracheally instilled with eglin-c (9 mg/rat) twice weekly; eglin-c (2), rats intratracheally instilled with eglin-c (9 mg/rat) 5 times weekly. a Significantly different at P < 0.01 compared with control group values. Significantly different at P < 0.05 compared with values from: b control group; c eglin-c1 group; d MCT group; e all other groups.

The mean MEFV curves in the six groups of rats are illustrated in Fig. 1. _max values at 20, 40, 60, and 80% TLC from the MCT group were significantly reduced compared with values from the control group. However, these decreases in _max were prevented by eglin-c [including the treatments of eglin-c (1)+MCT and eglin-c (2)+MCT].

The PV curves of the total respiratory system in the six groups of rats are shown in Fig. 2. Compared with the control group, the PV curve of the MCT group was significantly shifted to the right and downward, whereas the shift was markedly attenuated or abolished in the eglin-c (1)+MCT and eglin-c (2)+MCT groups.

The MFSR curve of the MCT group also indicated a significant rightward and downward shift. However, this shift was prevented by both eglin-c (1)+MCT and eglin-c (2)+MCT treatments (Fig. 3).

The slopes of the MEFV curve and the MFSR curve are listed in Table 2. Compared with the control group and all other groups, the slopes of the MEFV and _max-pressure curves were significantly decreased, especially in the _max-pressure curve in the MCT group.

Table 2. Slopes of max-volume and max-pressure curves Group n  max-Volume Slope, ml · s1 · %TLC1  max-Pressure Slope, ml · s1 · cmH2O 1 Control 6 1.43 ± 0.08  11.46 ± 0.57  MCT 6 1.01 ± 0.08* 5.75 ± 0.65* Eglin-c (1) 6 1.42 ± 0.02  11.65 ± 0.63  Eglin-c (2) 5 1.42 ± 0.02  11.38 ± 0.85  Eglin-c (1) + MCT 6 1.40 ± 0.04  11.96 ± 0.64  Eglin-c (2) + MCT 6 1.45 ± 0.04  12.86 ± 0.57 Values are means ± SE; n, no. of rats. max-volume slope, slope of maximal expiratory flow-% TLC curve; max-pressure slope, slope of maximal expiratory flow-static recoil pressure curve. * Significantly different compared with values from all other groups, P < 0.05.

DISCUSSION

We showed that MCT caused decreases in TLC, FRC, _max at 50% TLC, and the slope of the MFSR curve, indicating MCT-induced ventilatory dysfunction. These MCT-induced alterations in ventilatory features were significantly attenuated by eglin-c administration. The physiological significance of our results are discussed below.

MCT-induced inflammation. After MCT, abnormalities in the lung parenchyma include interstitial edema and elastolysis (26) and an increased number of inflammatory cells such as mast cells (12), leukocytes (22), and platelets (10). The release and/or production of several inflammatory mediators were increased after MCT treatment. Stenmark et al. (22) found that MCT administration increases the level of arachidonic acid metabolites in the lungs. Leukotriene C₄ increased 1-wk after treatment with MCT, whereas thromboxane and leukotriene D₄ increased 3 wk after MCT treatment. After treatment with MCT, the level of platelet-activating factor increased in some rats at 1-3 wk (19). In addition, the administration of platelet-activating factor antagonist WEB 2086 attenuated vascular leakage and right ventricular hypertrophy caused by MCT. Kanai et al. (11) found increased plasma serotonin at 12 h to 3 days after MCT treatment. They also found that serotonin antagonist attenuated pulmonary hypertension, right ventricular hypertrophy, and medial thickening of pulmonary arteries induced by MCT. Furthermore, the precursor of proinflammatory kinins, kininogen, and its mRNA increased in the lungs at 10 and 20 days after MCT treatment (1).

Elastase and inflammation in MCT-induced ventilatory dysfunction. MCT caused decreases in lung volumes, _max at 50% TLC, and the slope of the MFSR curve, indicating MCT-induced ventilatory dysfunction. MCT also caused rightward shifts of both the PV and MEFV curves. If the PV curves in Fig. 2 were plotted as a percentage of observed TLC, they would superimpose on one another. This phenomenon has been demonstrated by Gibson and Pride (6) in the lung with fibrosing alveolitis. They explained that the rightward shift of the PV curve is caused simply by replacement of some units by indistensible fibrous tissue and retention of normal function of the surviving alveoli. We demonstrated previously that a bronchodilator, terbutaline, prevents MCT-induced rightward shifts of both PV and MEFV curves (27). The rightward shift of the PV curve and decrease in lung volume (shrinkage of the lung) could be produced by replacement of some units by constricted airways in this study. This reasoning is based on the fact that MCT causes decreases in _max and the slope of MFSR curve, which indicate airway constriction. Therefore, the MCT-induced changes in PV and MEFV curves may mainly be due to reversible airway constriction. This type of reversible bronchoconstriction has been demonstrated by Colebatch and Mitchell (2) in freshly excised lungs ventilated with warm oxygenated Krebs solution. The addition of a bronchoconstrictor histamine caused a rightward shift in the PV curve. This rightward shift was, however, reversed by ventilating the lungs with a solution containing smooth muscle relaxant isoproterenol (2).

These MCT-induced alterations in ventilatory parameters were significantly attenuated by the administration of an elastase inhibitor, eglin-c. The mechanism for this inhibitory effect of eglin-c is speculative. Because eglin-c inhibits elastase that is mainly released from inflammatory cells, we reason that elastase plays an important role in the ventilatory abnormality through inflammation. It is possible that MCT induces lung tissue injury and inflammation, which is augmented by elastase. Stockley (23) pointed out that elastase can augment inflammation via several ways. First, elastase can stimulate epithelial cells to produce interleukin-8, a potent neutrophil chemoattractant (18) that, in turn, causes more neutrophil recruitment. Second, elastase damages the neutrophil C3bi receptor (25) and immunoglobulins (4). Subsequently, these actions delay phagocytic clearance of organisms. Third, elastase can damage epithelium, reduce ciliary beating, and result in mucus retention and bacterial proliferation. The accumulation of phagocytes in the lungs during inflammation can release oxygen radicals and some mediators (e.g., leukotrienes and thromboxane), thus causing constriction of airway smooth muscle. As mentioned above, we have previously found that terbutaline blocks ventilatory dysfunction 1 wk after MCT treatment. Therefore, the mediator-induced airway constriction should contribute significantly to the ventilatory abnormality. Contrary to the ventilatory dysfunction, eglin-c cannot prevent the decrease in body weight caused by MCT (Table 1). This could be related to the handling of animals during eglin-c treatment.

It is also possible that MCT-induced phagocyte recruitment increases oxygen radical production that, in turn, can damage elastin and other proteins. Proteins that have been damaged are recognized and selectively degraded by proteolytic systems (3). The enhanced protein degradation can initiate proliferation. Faris et al. (5) demonstrated an increase in protein synthesis by the cells 20 h after elastase exposure. This type of surge in protein synthesis after elastase treatment should augment proliferation and remodeling in airways. Several investigators found an increased proliferation in the lungs after MCT administration. Hacker (7) observed that MCT treatment causes an increase in DNA synthesis of the whole lung. An increase in DNA synthesis in 20- to 200-mm pulmonary arteries was detected 7 days after administration of MCT pyrrole (16). Similarly, Kameji et al. (10) found an increase in collagen synthesis in the pulmonary artery of MCT-treated rats. Although more studies are needed to prove the point, it is possible that a decrease in airway dimension due to proliferation of airway smooth muscle may also occur in MCT-treated animals. According to the results of previous studies of Lai and co-workers (15, 27, 28), we analyze the effect of airway proliferation as follows. Airway proliferation and remodeling can contribute significantly to the MCT-induced ventilatory dysfunction under the following three conditions: 1) the rate of proliferation and remodeling in airways is faster than that in pulmonary arteries; 2) the newly proliferated airway smooth muscle is not completely relaxed during resting state; and 3) the newly proliferated airway smooth muscle is also very sensitive to terbutaline. Otherwise, airway proliferation and remodeling should play only a minor role in this type of ventilatory dysfunction. More studies are needed to elucidate this contributing role of airway proliferation and remodeling.

In summary, we observed that eglin-c (an elastase inhibitor) attenuated MCT-induced ventilatory dysfunction. It is speculated that the MCT-induced increase in elastolytic activity plays an important role in the progression in ventilatory abnormality.

ACKNOWLEDGEMENTS

We express deep appreciation to Dr. H. P. Schnebli, Ciba-Geigy Research Department, Basel, Switzerland, for providing eglin-c.

FOOTNOTES

Address for reprint requests: Y. L. Lai, Dept. of Physiology, College of Medicine, National Taiwan Univ., No. 1, Sect. 1, Jen-Ai Rd., Taipei 100, Taiwan.

Received 5 September 1995; accepted in final form 3 September 1996.

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