Effect of Sulfidopeptide Leukotriene Receptor Antagonists on Endotoxin-induced Pulmonary Dysfunction in Awake Sheep1-3
ROBERT F. MILLER, PETER L. LEFFERTS, and JAMES R. SNAPPER4
Introduction Products from both cyclooxygenase and lipoxygenase (5-lipoxygenase) pathways are elevated in sheep lymph and plasma after endotoxemia (1-6), and inhibitors of these pathways attenuate the endotoxin response in awake sheep (4,5, 7). Cyclooxygenaseinhibitors attenuate the early changes in pulmonary hemodynamics, lung mechanics, and lung fluid and solute exchange seen after endotoxin. Studies with thromboxane receptor antagonists suggest that thromboxane is largely responsible for the early changes observed after endotoxemia (7). These inhibitors do not alter the later increases in lung fluid and solute exchange. It is these later alterations that are felt to be analogous to the adult respiratory distress syndrome (ARDS) in humans (8). Additionally, the 5-lipoxygenase product 5-HETE is increased after endotoxin in sheep (5). In the current study, we hypothesized that sulfidopeptide leukotrienes (LTC4 , LTD4 , and LTE4 ) might contribute to the endotoxin response. The sulfidopeptide leukotrienes can reproduce many of the physiologic effects that are seen with endotoxemia, including vascular (9) and bronchial (10, 11) smooth muscle constriction and, in some models and vascular beds, alterations in vascular permeability (12-14). In humans, LTD4 has been shown to be increased in the pulmonary edema fluid from patients with ARDS (15). L-651,392, a 5-lipoxygenase inhibitor, has also been shown to attenuate the early and late alterations caused by endotoxin (6). In the current study, we employed sulfidopeptide leukotriene receptor antagonists to investigate the contribution of sulfidopeptide leukotrienes to endotoxininduced pulmonary dysfunction. The study compares the effects of endotoxin given alone and after pretreatment with two structurally dissimilar sulfidopeptide leukotriene receptor antagonists, L-660,711 (MK-571) and SK&F 104,353.
SUMMARY Westudied the effects of two structurally unrelated sulfldopeptlde leukotrlene recep· tor antagonists on endotoxln·lnduced pUlmonary dysfunction In chronically Instrumented unanesthe· tlzed sheep. The agents employed were L·660,711 (MK·571) (Merck.Frosst, Canada)and SK&F 104,353 (Smith Kline and French, King of Prussia, PA). The efficacy and specificity of the agents were veri· fled In sheep by administering boluses of exogenous leukotrlenes (LTB" LTC" LTD" and LTE,) In doses as great as 100!1gwhile monitoring lung mechanics and vascular pressures. The antagonists blocked the changes In lung mechanics and pulmonary hemodynamics Induced by the sUlfidopep· tide leukotrienes (LTC" LTD" and LTE,) while having no effect on the animals' responses to LTB,. The endotoxin studies were performed by administering endotoxl n alone (Escherichia coli endotox· in 0.75 !1g/kg) or endotoxin after pretreatment with one of the sulfldopeptlde leukotrlene receptor antagonists. In control studies, each animal received a continuous Infusion of one of the receptor antagonists for a duration Identical to that of the endotoxin studies. Neither L-660,711 nor SK&F 104,353 significantly altered the endotoxin·lnduced changes In pUlmonary hemodynamics, lung mechanics, lung fluid and solute exchange, oxygenation, or leukopenia. Peaklung lymph thrombox· ane B,levels were significantly lower In sheep pretreated with L·660,711.When the antagonists were given alone, no effects were seen. Weconclude that (1)sulfldopeptlde leukotrlenes do not measurebly contribute to endotoxln·lnduced pUlmonary dysfunction In chronically Instrumented sheep; (2) sUlfidopeptide leukotrlenes may contribute to thromboxane release after endotoxin. AM REV RESPIR DIS 1992; 148:997-1002
Methods Sheep Preparation Yearling sheep of both sexes weighing 30 to 40 kg were instrumented as previously described (16-18). Under general anesthesia, the efferent duct of the caudal mediastinal lymph node was cannulated with a silastic catheter via a right thoracotomy. Through a second right thoracotomy, the tail of the caudal mediastinal node was resected below the inferior pulmonary ligament, and possible contaminating lymphatics traversing the right hernidiaphragm were interrupted. Silastic balloon-tipped catheters were placed within the pleural space at the time of right thoracotomy. Vascular catheters wereplaced directly into the left atrium and pulmonary artery through a left thoracotomy. Lymphatics traversing the left hemidiaphragm were interrupted through a second left thoracotomy. Through a neck incision, catheters were also placed into the aorta via the carotid artery and into the superior vena cava via the external jugular vein. A tracheostomy was also performed. After surgery,the sheep wereallowed to recover for 7 to 10days prior to experimentation. A no. 10 cuffed tracheostomy tube (Shiley, Irvine, CA) was inserted at the time of experimentation.
Lung Mechanics Chronically instrumented awake sheep were studied whilestanding in a speciallyconstructed pressure-compensated integrated-flow whole-body plethysmograph (8, 18). The tracheostomy tu be was connected to an external valve via flexible noncollapsible tubing. A loose-fitting sling was placed under the sheep to prevent the sheep from lying down while in the plethysmograph. A constant bias flow
(Received in original form May 25, 1989 and in revised form May 26, 1992) 1 From the Center for Lung Research, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee. , Supported by Grant HL-27274 and SCOR in Pulmonary Vascular Diseases Grant HL-19l53 from the National Heart, Lung, and Blood Institl~te. 3 Correspondence and requests for reprints should be addressed to James R. Snapper, M.D., B-1308 Medical Center North, Vanderbilt University, Nashville, TN 37232. . , Established Investigator of the Amen~an Hea,rt Association supported with funds contributed in part by the Middle Tennessee Chapter of the American Heart Association.
997
998
of humidified air was used to reduce the effective dead space of the tubing. Volume (V) was measured by pressure-compensating the integrated signal from the plethysmographic pressure transducer. Flow (V) was obtained by electrically differentiating the volume signal. Airway opening pressure (Pao) was measured in the trachea by a multiple side-hole catheter positioned 0.5 em beyond the distal end of the tracheostomy tube. Pleural pressure (Ppl) was obtained from the pleural balloons. Transpulmonary pressure (Ptp) was the pressure difference between Ppl and Pao. All pressure signals from pressure transducers, catheters, and silastic pleural balloons were tuned to eliminate phasic distortion to 20 Hz. The sheep's lungs were inflated to 40 em H 20 Pao before each set of lung mechanics measurements. Simultaneous V/V and v/Ptp curves were recorded during spontaneous respiration on a dual-beam storage oscilloscope (Tektronix, Beaverton, OR) and photographed for calculation of dynamic lung compliance (Cdyn) and resistance to airflow across the lung (RL). Cdyn was calculated as V divided by Ptp at points of zero flow and expressed in liters per cm H 20 at BTPS. RL was calculated by use of the method of von Neergaard and Wirz (19) by dividing Ptp by V at mid-tidal volume and was expressed as em H 20/L/s at BTPS. FRC was measured by the modified Boyle's Law method of DuBois and coworkers (20). The airway was manually obstructed at end-expiration. While the sheep attempted to breathe against the obstruction, a graph of the change in plethysmographic volume against the change in Pao was traced on the oscilloscope and photographed for calculation of FRC. Lung Lymph Lung lymph was collected continuously throughout each study. Lymph flow (Q1) was determined for 15-min intervals. The lymph was pooled every 30 min in tubes containing small amounts of heparin and indomethacin. Whole blood was collected every 30 min for measurement of plasma proteins, arterial blood gas determinations, leukocyte counts', and thromboxane B2 concentrations. Total protein concentrations in lung lymph and blood plasma were measured by a modified biuret method (21) with an automated system (Autoanalyzer II; TechniconInstruments, Tarrytown, NY). Lymph protein clearance (Clp) was calculated by multiplying lymph flow by the lymph-to-plasma protein ratio (LIP). Blood Fulmonary arterial pressure (Ppa, em H 20), left atrial pressure (PLA, cm H 20), and aortic blood pressure (Psa, mm Hg) were continuously monitored (Model 1208Cpressuretransducers; Hewlett-Packard, Palo Alto, CA). Arterial blood samples were collected every 30 min anaerobically. Pao2 , Pac02 and pH were measured with a pH/blood gas analyzer (Model 158; Corning Medical, Medfield, MA). The alveolar-to-arterial O2 difference
MILLER, LEFFERTS, AND SNAPPER
(AAaPo2 ) on room air was calculated using the alveolar gas equation, assuming a fixed respiratory exchange ratio of 0.8. Wholeblood leukocyte (WBC) counts weredone on an automated system (Coulter counter model ZBI; Coulter Electronics, Hialeah, FL). Thromboxane B2 analyses were performed by enzyme-linked immunoassay as described by Pradelles and coworkers (22)and Wescottand colleagues (23). Protocol Pharmaceutical testing. The efficacy and specificity of L-660,711 and SK&F 104,353 were tested prior to their use in the sheependotoxin studies. Both compounds weretested by observing their ability to inhibit the changes in lung mechanics and hemodynamics induced by exogenously administered leukotrienes. Exogenous leukotrienes werethen administered as boluses into the pulmonary artery. Several doses within the range of 1 to 100 ug of LTB. and the sulfidopeptide leukotrienes (LTC., LTD., and LTE.) were used while monitoring lung mechanics and PLA, Psa, and Ppa. The leukotrienes were first given alone and then after pretreatment with one of the receptor antagonists. Sheep receiving L-660,711 were given a 1.0 mg/kg bolus followed by a constant infusion of 25 ug/kg/h for the duration of the study. Sheep receiving SK&F 104,353 were given a 5.0 mg/kg bolus followed by a constant infusion of 2.0 mg/kg/h for the duration of the study. Sheep used for pharmaceutical testing werenot used later in the endotoxin studies. Endotoxin studies. A total of 10sheep were used for the endotoxin studies. In six sheep, the effects of endotoxin alone were compared with endotoxin given after pretreatment with L-660,711. In four different sheep, the effects of endotoxin alone were compared with endotoxin given after pretreatment with SK&F 104,353. In each series, the order of experimentation was varied to avoid sequential bias. Sheep receivingendotoxin alone also received a continuous infusion of normal saline, the diluent for the antagonists, to normalize the conditions of the study. Several sheep in each study were also monitored af-
ter receiving the leukotriene receptor antagonists alone. At the end of the 6-h infusion of drug alone, the animals were given 25 ug bolus injections of LTD. into the pulmonary artery, and lung mechanics and pulmonary' hemodynamics were monitored. The sheep were initially placed in the plethysmograph and allowed to stabilize for 1 h prior to experimentation. One hour of baseline vascular pressures, airway mechanics,and lymph and blood collections werethen obtained. In the endotoxin studies, Escherichia coli endotoxin (0.75 ug/kg, prepared by the Westphalmethod from E. coli 055:B5; Difco Laboratories, Detroit, MI) was dissolved in 30 ml of 0.9070 NaCl solution and infused intravenously over 15 min. Sheep receiving a leukotriene receptor antagonist had an additional hour of monitoring after beginning the receptor antagonist infusion. In these sheep, endotoxin was then given as above. Sheep receiving L-660,711 were given a 1.0 mg/kg bolus followed by a constant infusion of 0.25 ug/kg/h for the duration of the study. Sheep receiving SK&F 104,353 were given a 5.0 mg/kg bolus followed by a constant infusion of 2.0 mg/kg/h for the duration of the study. Throughout the study, vascular pressures were continuously monitored and recorded. Cdyn, RL, FRC, and Ql flow were measured every 15 min. Statistics Data are presented as means ± SEM. Twoway analysis of variance with Duncan's multiple range test was used to compare baseline measurements to time points after endotoxin administration. Paired t tests were performed to compare matched time points from the same sheep. Significancewas taken at p < 0.05 (data obtained from sheep exposed to endotoxin alone or endotoxin after pretreatment with L-660,711 are represented in figures 2-4). Similar studies using SK&F 104,353 are presented in table 1 (24, 25).
Results
Pharmaceutical Testing Both L-660,711 and SK&F 104,353 effec-
TABLE 1 EFFECT OF SK&F 104,353 ON ENDOTOXIN-INDUCED PULMONARY DYSFUNCTION IN SHEEP' Baseline Parameter Ppa, em H,O QL, ml/15 min Cdyn, % baseline RL, %' baseline FRC, % baseline WBC, eellslmm' x 1,000 TxB 2 , ng/ml Ll.AaPo" mm Hg
E
E + SK&F
22 ± 2 1.5 ± 0.3 100 100 100 7.7 ± 1.3 0.5 ± 0.13 18 ± 4
20 ± 3 1.2 ± 0.3 100 100 100 6.3 ± 1.8 0.3 ± 0.5 15 ± 4
5 Hours
Peak E + SK&F
E
78 8.0 70 323 80 2.1 26.2 29
± ± ± ± ± ± ± ±
7 1.1 12 52 2 0.8 14 4
62 6.6 37 284 86 3.1 13.6 35
± ± ± ± ± ± ± ±
12 2.1 13 65 6 2.0 12 4
E + SK&F
E
26 3.7 76 156 105 5.4 2.2 29
± ± ± ± ± ± ± ±
3 1.5 8 61 8 1.0 1.5 4
30 ± 4.7 ± 60 ± 203 ± 102 ± 5.0 ± 3.8 ± 35 ±
3 1.8 15 65 6 3.1 2 5
Definitionof abbreviations: E = endotoxin; Ppa = pulmonary artery pressure; OL - lung lymph flow; Cdyn _ dynamic compliance; RL = pulmonary resistance; dAaPo, - change in alveolar-arterial 0, difference. • The data represent means ± SE; n = 4. None of the data points in the endotoxin alone experiments was significantly different from the time-matched points in the studies in which animals were given both endotoxin and SK&F 104,353.
SULFIDOPEPTIDE LEUKOTRIENE RECEPTOR ANTAGONISTS AND ENDOTOXEMIA
50 Fig. 1. Effect of L-660,711 and SK&F 104,353 on the pulmonaryarterial pressure(Ppa)responseto LTD•. Datafrom LTD. aloneareshownas closedcircles. Data obtained after L-660,711 pretreatment are shown as open circles. Data obtained after SK&F 104,353 pretreatment are shownas open triangles.The doses of L-660,711 and SK&F 104,353 were identicalto those used for the endotoxin studies.
Change in
also caused pulmonary arterial vasoconstriction as previously reported (26). This effect was not inhibited by either L-660,711 or SK&F 104,353 pretreatment.
40
Ppa
30
(em H2O)
20
999
Endotoxin Studies Vascular pressure measurements. The Ppa responses to endotoxin were similar 10 to those of previous studies (2, 5, 8, 16-18).Endotoxin caused rapid increases 0 Ppa followed by a slow decline over in 3 10 30 100 4 h to a level of 5 to 10 em H 20 above LTD4 (~g) baseline (figure 2). The Ppa response to endotoxin was nearly identical in animals receiving endotoxin alone and in those pretreated with either L-660,711 or SK&F tively blocked the lung mechanics creased to 29070 of baseline, and Ppa in- 104,353. No significant alterations in the changes and pulmonary arterial vaso- creased from 15to 55 em H 20 . L-660,711 systemic arterial or left atrial pressure repressor effects of the sulfidopeptide leu- completely blocked the changes in lung sponses were seen with L-660,711 and kotrienes. LTD4 was the most potent pul- mechanics and hemodynamics caused by SK&F 104,353. monary arterial pressor of the sul- LTD4 (as great as 100!J,g). SK&F 104,353 Lung Lymph. Endotoxin alone profidopeptide leukotrienes in sheep inhibited the pressor response complete- duced a significant and sustained increase followed by LTC4 • LTE4 had no effect. ly for lower doses of LTD4 (10 to 30 ug), in lung Ql (figure 2). The changes in or, A 3- to 30-!J,g bolus of LTD 4 caused Attenuated pressor responses were seen, LIP, and Clp observed after endotoxin marked changes in lung mechanics and however, in the higher doses (30 to 100 werenot affected by pretreating the sheep pulmonary hemodynamics. Cdyn de- ug boluses) (figure 1). Boluses of LTB4 with L-660,711 or SK&F 104,353. Lung mechanics. Endotoxin alone and after pretreatment with L-660,711 or SK&F 104,353 produced significant alterations in Cdyn, RL, and FRC as pre80 (AI IBI viously described for endotoxin alone (5, 8 8, 18). The effects of endotoxin on lung 60 mechanics were not significantly differ6 ent between the animals treated with en~ C ::; r:~ 40 dotoxin alone and those pretreated with o~ "::; '""- 4 either antagonist (figure 3). ~ ~ Circulating white bloodcells. Circulat20 2 ing WBCs fell significantly 1 h after endotoxin and remained significantly low0 0 Endotoxin er than baseline for the 5-h period monii 5 tored after endotoxin administration 3 0 2 4 0 2 3 4 5 (figure 2). The fall in WBCs was attenuTIME (Hours Alter Endotoxin) TIME (Hours After Endotoxin) ated in animals pretreated with L-660,711, but it did not reach statistical significance when compared with the endotoxin group. SK&F 104,353 pretreatment did 60 lei 12 (01 not affect endotoxin-induced leukopenia. .t1AaPo2 difference. Endotoxin caused 10 a rapid and prolonged increase in 0 0 40 8 9 ~aP02' Pretreatment with either the w 0 U e, co E 6 L-660,711 or SK&F 104,353 did not sig~ < ~ ~ nificantly affect this measurement (fig~ ... 20 ure 2). "~ 4 Thromboxane B2 • Lymph lXB2 con2 centrations peaked 1 h after endotoxin 0 0 EndOIOlin administration and gradually apI ~ proached baseline 5 h later (figure 4). 5 3 4 0 0 1 2 3 5 2 4 L-660,711 and SK&F 104,353attenuated TIME (Hours Alter ElldoloxinJ TIME (Hours Afler Endotoxin) peak lymph lXB 2 concentrations and delayed the occurrence of the peak conFig. 2. Effectof L-660,711 on endotoxin-inducedchanges in (A) pulmonary arterial pressure(Ppa);(B) lung lymph flow(OL); (e) alveolar-to-arterial 0, pressuredifference(aAaPO,); and (D)circulatingwhiteblood cell count (WBC). centration from 1.0 to 1.5 h. The differDatafrom the sheep studied with endotoxin alone are shown as closed circles, and those from the same sheep ences in peak lymph lXB2concentrations pretreatedwith L-660,711 are shown as open circles. The data are means ± standard errors; n = 6. None of were statistically significant (p < 0.05) the data points in the endotoxin alone experiments was significantly different from the time-matched points in with L-660,711. the animals pretreated with L-660,711.
.
L
-
£ndOIOtr:ift
-
.. .
-
Erws'otoltln
1000 {AI
MILLER, LEFFERTS, AND SNAPPER
50
100 75
40
m'" Cd" IX
50
CI>
c:
>
ROBERT F. MILLER, PETER L. LEFFERTS, and JAMES R. SNAPPER4
Introduction Products from both cyclooxygenase and lipoxygenase (5-lipoxygenase) pathways are elevated in sheep lymph and plasma after endotoxemia (1-6), and inhibitors of these pathways attenuate the endotoxin response in awake sheep (4,5, 7). Cyclooxygenaseinhibitors attenuate the early changes in pulmonary hemodynamics, lung mechanics, and lung fluid and solute exchange seen after endotoxin. Studies with thromboxane receptor antagonists suggest that thromboxane is largely responsible for the early changes observed after endotoxemia (7). These inhibitors do not alter the later increases in lung fluid and solute exchange. It is these later alterations that are felt to be analogous to the adult respiratory distress syndrome (ARDS) in humans (8). Additionally, the 5-lipoxygenase product 5-HETE is increased after endotoxin in sheep (5). In the current study, we hypothesized that sulfidopeptide leukotrienes (LTC4 , LTD4 , and LTE4 ) might contribute to the endotoxin response. The sulfidopeptide leukotrienes can reproduce many of the physiologic effects that are seen with endotoxemia, including vascular (9) and bronchial (10, 11) smooth muscle constriction and, in some models and vascular beds, alterations in vascular permeability (12-14). In humans, LTD4 has been shown to be increased in the pulmonary edema fluid from patients with ARDS (15). L-651,392, a 5-lipoxygenase inhibitor, has also been shown to attenuate the early and late alterations caused by endotoxin (6). In the current study, we employed sulfidopeptide leukotriene receptor antagonists to investigate the contribution of sulfidopeptide leukotrienes to endotoxininduced pulmonary dysfunction. The study compares the effects of endotoxin given alone and after pretreatment with two structurally dissimilar sulfidopeptide leukotriene receptor antagonists, L-660,711 (MK-571) and SK&F 104,353.
SUMMARY Westudied the effects of two structurally unrelated sulfldopeptlde leukotrlene recep· tor antagonists on endotoxln·lnduced pUlmonary dysfunction In chronically Instrumented unanesthe· tlzed sheep. The agents employed were L·660,711 (MK·571) (Merck.Frosst, Canada)and SK&F 104,353 (Smith Kline and French, King of Prussia, PA). The efficacy and specificity of the agents were veri· fled In sheep by administering boluses of exogenous leukotrlenes (LTB" LTC" LTD" and LTE,) In doses as great as 100!1gwhile monitoring lung mechanics and vascular pressures. The antagonists blocked the changes In lung mechanics and pulmonary hemodynamics Induced by the sUlfidopep· tide leukotrienes (LTC" LTD" and LTE,) while having no effect on the animals' responses to LTB,. The endotoxin studies were performed by administering endotoxl n alone (Escherichia coli endotox· in 0.75 !1g/kg) or endotoxin after pretreatment with one of the sulfldopeptlde leukotrlene receptor antagonists. In control studies, each animal received a continuous Infusion of one of the receptor antagonists for a duration Identical to that of the endotoxin studies. Neither L-660,711 nor SK&F 104,353 significantly altered the endotoxin·lnduced changes In pUlmonary hemodynamics, lung mechanics, lung fluid and solute exchange, oxygenation, or leukopenia. Peaklung lymph thrombox· ane B,levels were significantly lower In sheep pretreated with L·660,711.When the antagonists were given alone, no effects were seen. Weconclude that (1)sulfldopeptlde leukotrlenes do not measurebly contribute to endotoxln·lnduced pUlmonary dysfunction In chronically Instrumented sheep; (2) sUlfidopeptide leukotrlenes may contribute to thromboxane release after endotoxin. AM REV RESPIR DIS 1992; 148:997-1002
Methods Sheep Preparation Yearling sheep of both sexes weighing 30 to 40 kg were instrumented as previously described (16-18). Under general anesthesia, the efferent duct of the caudal mediastinal lymph node was cannulated with a silastic catheter via a right thoracotomy. Through a second right thoracotomy, the tail of the caudal mediastinal node was resected below the inferior pulmonary ligament, and possible contaminating lymphatics traversing the right hernidiaphragm were interrupted. Silastic balloon-tipped catheters were placed within the pleural space at the time of right thoracotomy. Vascular catheters wereplaced directly into the left atrium and pulmonary artery through a left thoracotomy. Lymphatics traversing the left hemidiaphragm were interrupted through a second left thoracotomy. Through a neck incision, catheters were also placed into the aorta via the carotid artery and into the superior vena cava via the external jugular vein. A tracheostomy was also performed. After surgery,the sheep wereallowed to recover for 7 to 10days prior to experimentation. A no. 10 cuffed tracheostomy tube (Shiley, Irvine, CA) was inserted at the time of experimentation.
Lung Mechanics Chronically instrumented awake sheep were studied whilestanding in a speciallyconstructed pressure-compensated integrated-flow whole-body plethysmograph (8, 18). The tracheostomy tu be was connected to an external valve via flexible noncollapsible tubing. A loose-fitting sling was placed under the sheep to prevent the sheep from lying down while in the plethysmograph. A constant bias flow
(Received in original form May 25, 1989 and in revised form May 26, 1992) 1 From the Center for Lung Research, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee. , Supported by Grant HL-27274 and SCOR in Pulmonary Vascular Diseases Grant HL-19l53 from the National Heart, Lung, and Blood Institl~te. 3 Correspondence and requests for reprints should be addressed to James R. Snapper, M.D., B-1308 Medical Center North, Vanderbilt University, Nashville, TN 37232. . , Established Investigator of the Amen~an Hea,rt Association supported with funds contributed in part by the Middle Tennessee Chapter of the American Heart Association.
997
998
of humidified air was used to reduce the effective dead space of the tubing. Volume (V) was measured by pressure-compensating the integrated signal from the plethysmographic pressure transducer. Flow (V) was obtained by electrically differentiating the volume signal. Airway opening pressure (Pao) was measured in the trachea by a multiple side-hole catheter positioned 0.5 em beyond the distal end of the tracheostomy tube. Pleural pressure (Ppl) was obtained from the pleural balloons. Transpulmonary pressure (Ptp) was the pressure difference between Ppl and Pao. All pressure signals from pressure transducers, catheters, and silastic pleural balloons were tuned to eliminate phasic distortion to 20 Hz. The sheep's lungs were inflated to 40 em H 20 Pao before each set of lung mechanics measurements. Simultaneous V/V and v/Ptp curves were recorded during spontaneous respiration on a dual-beam storage oscilloscope (Tektronix, Beaverton, OR) and photographed for calculation of dynamic lung compliance (Cdyn) and resistance to airflow across the lung (RL). Cdyn was calculated as V divided by Ptp at points of zero flow and expressed in liters per cm H 20 at BTPS. RL was calculated by use of the method of von Neergaard and Wirz (19) by dividing Ptp by V at mid-tidal volume and was expressed as em H 20/L/s at BTPS. FRC was measured by the modified Boyle's Law method of DuBois and coworkers (20). The airway was manually obstructed at end-expiration. While the sheep attempted to breathe against the obstruction, a graph of the change in plethysmographic volume against the change in Pao was traced on the oscilloscope and photographed for calculation of FRC. Lung Lymph Lung lymph was collected continuously throughout each study. Lymph flow (Q1) was determined for 15-min intervals. The lymph was pooled every 30 min in tubes containing small amounts of heparin and indomethacin. Whole blood was collected every 30 min for measurement of plasma proteins, arterial blood gas determinations, leukocyte counts', and thromboxane B2 concentrations. Total protein concentrations in lung lymph and blood plasma were measured by a modified biuret method (21) with an automated system (Autoanalyzer II; TechniconInstruments, Tarrytown, NY). Lymph protein clearance (Clp) was calculated by multiplying lymph flow by the lymph-to-plasma protein ratio (LIP). Blood Fulmonary arterial pressure (Ppa, em H 20), left atrial pressure (PLA, cm H 20), and aortic blood pressure (Psa, mm Hg) were continuously monitored (Model 1208Cpressuretransducers; Hewlett-Packard, Palo Alto, CA). Arterial blood samples were collected every 30 min anaerobically. Pao2 , Pac02 and pH were measured with a pH/blood gas analyzer (Model 158; Corning Medical, Medfield, MA). The alveolar-to-arterial O2 difference
MILLER, LEFFERTS, AND SNAPPER
(AAaPo2 ) on room air was calculated using the alveolar gas equation, assuming a fixed respiratory exchange ratio of 0.8. Wholeblood leukocyte (WBC) counts weredone on an automated system (Coulter counter model ZBI; Coulter Electronics, Hialeah, FL). Thromboxane B2 analyses were performed by enzyme-linked immunoassay as described by Pradelles and coworkers (22)and Wescottand colleagues (23). Protocol Pharmaceutical testing. The efficacy and specificity of L-660,711 and SK&F 104,353 were tested prior to their use in the sheependotoxin studies. Both compounds weretested by observing their ability to inhibit the changes in lung mechanics and hemodynamics induced by exogenously administered leukotrienes. Exogenous leukotrienes werethen administered as boluses into the pulmonary artery. Several doses within the range of 1 to 100 ug of LTB. and the sulfidopeptide leukotrienes (LTC., LTD., and LTE.) were used while monitoring lung mechanics and PLA, Psa, and Ppa. The leukotrienes were first given alone and then after pretreatment with one of the receptor antagonists. Sheep receiving L-660,711 were given a 1.0 mg/kg bolus followed by a constant infusion of 25 ug/kg/h for the duration of the study. Sheep receiving SK&F 104,353 were given a 5.0 mg/kg bolus followed by a constant infusion of 2.0 mg/kg/h for the duration of the study. Sheep used for pharmaceutical testing werenot used later in the endotoxin studies. Endotoxin studies. A total of 10sheep were used for the endotoxin studies. In six sheep, the effects of endotoxin alone were compared with endotoxin given after pretreatment with L-660,711. In four different sheep, the effects of endotoxin alone were compared with endotoxin given after pretreatment with SK&F 104,353. In each series, the order of experimentation was varied to avoid sequential bias. Sheep receivingendotoxin alone also received a continuous infusion of normal saline, the diluent for the antagonists, to normalize the conditions of the study. Several sheep in each study were also monitored af-
ter receiving the leukotriene receptor antagonists alone. At the end of the 6-h infusion of drug alone, the animals were given 25 ug bolus injections of LTD. into the pulmonary artery, and lung mechanics and pulmonary' hemodynamics were monitored. The sheep were initially placed in the plethysmograph and allowed to stabilize for 1 h prior to experimentation. One hour of baseline vascular pressures, airway mechanics,and lymph and blood collections werethen obtained. In the endotoxin studies, Escherichia coli endotoxin (0.75 ug/kg, prepared by the Westphalmethod from E. coli 055:B5; Difco Laboratories, Detroit, MI) was dissolved in 30 ml of 0.9070 NaCl solution and infused intravenously over 15 min. Sheep receiving a leukotriene receptor antagonist had an additional hour of monitoring after beginning the receptor antagonist infusion. In these sheep, endotoxin was then given as above. Sheep receiving L-660,711 were given a 1.0 mg/kg bolus followed by a constant infusion of 0.25 ug/kg/h for the duration of the study. Sheep receiving SK&F 104,353 were given a 5.0 mg/kg bolus followed by a constant infusion of 2.0 mg/kg/h for the duration of the study. Throughout the study, vascular pressures were continuously monitored and recorded. Cdyn, RL, FRC, and Ql flow were measured every 15 min. Statistics Data are presented as means ± SEM. Twoway analysis of variance with Duncan's multiple range test was used to compare baseline measurements to time points after endotoxin administration. Paired t tests were performed to compare matched time points from the same sheep. Significancewas taken at p < 0.05 (data obtained from sheep exposed to endotoxin alone or endotoxin after pretreatment with L-660,711 are represented in figures 2-4). Similar studies using SK&F 104,353 are presented in table 1 (24, 25).
Results
Pharmaceutical Testing Both L-660,711 and SK&F 104,353 effec-
TABLE 1 EFFECT OF SK&F 104,353 ON ENDOTOXIN-INDUCED PULMONARY DYSFUNCTION IN SHEEP' Baseline Parameter Ppa, em H,O QL, ml/15 min Cdyn, % baseline RL, %' baseline FRC, % baseline WBC, eellslmm' x 1,000 TxB 2 , ng/ml Ll.AaPo" mm Hg
E
E + SK&F
22 ± 2 1.5 ± 0.3 100 100 100 7.7 ± 1.3 0.5 ± 0.13 18 ± 4
20 ± 3 1.2 ± 0.3 100 100 100 6.3 ± 1.8 0.3 ± 0.5 15 ± 4
5 Hours
Peak E + SK&F
E
78 8.0 70 323 80 2.1 26.2 29
± ± ± ± ± ± ± ±
7 1.1 12 52 2 0.8 14 4
62 6.6 37 284 86 3.1 13.6 35
± ± ± ± ± ± ± ±
12 2.1 13 65 6 2.0 12 4
E + SK&F
E
26 3.7 76 156 105 5.4 2.2 29
± ± ± ± ± ± ± ±
3 1.5 8 61 8 1.0 1.5 4
30 ± 4.7 ± 60 ± 203 ± 102 ± 5.0 ± 3.8 ± 35 ±
3 1.8 15 65 6 3.1 2 5
Definitionof abbreviations: E = endotoxin; Ppa = pulmonary artery pressure; OL - lung lymph flow; Cdyn _ dynamic compliance; RL = pulmonary resistance; dAaPo, - change in alveolar-arterial 0, difference. • The data represent means ± SE; n = 4. None of the data points in the endotoxin alone experiments was significantly different from the time-matched points in the studies in which animals were given both endotoxin and SK&F 104,353.
SULFIDOPEPTIDE LEUKOTRIENE RECEPTOR ANTAGONISTS AND ENDOTOXEMIA
50 Fig. 1. Effect of L-660,711 and SK&F 104,353 on the pulmonaryarterial pressure(Ppa)responseto LTD•. Datafrom LTD. aloneareshownas closedcircles. Data obtained after L-660,711 pretreatment are shown as open circles. Data obtained after SK&F 104,353 pretreatment are shownas open triangles.The doses of L-660,711 and SK&F 104,353 were identicalto those used for the endotoxin studies.
Change in
also caused pulmonary arterial vasoconstriction as previously reported (26). This effect was not inhibited by either L-660,711 or SK&F 104,353 pretreatment.
40
Ppa
30
(em H2O)
20
999
Endotoxin Studies Vascular pressure measurements. The Ppa responses to endotoxin were similar 10 to those of previous studies (2, 5, 8, 16-18).Endotoxin caused rapid increases 0 Ppa followed by a slow decline over in 3 10 30 100 4 h to a level of 5 to 10 em H 20 above LTD4 (~g) baseline (figure 2). The Ppa response to endotoxin was nearly identical in animals receiving endotoxin alone and in those pretreated with either L-660,711 or SK&F tively blocked the lung mechanics creased to 29070 of baseline, and Ppa in- 104,353. No significant alterations in the changes and pulmonary arterial vaso- creased from 15to 55 em H 20 . L-660,711 systemic arterial or left atrial pressure repressor effects of the sulfidopeptide leu- completely blocked the changes in lung sponses were seen with L-660,711 and kotrienes. LTD4 was the most potent pul- mechanics and hemodynamics caused by SK&F 104,353. monary arterial pressor of the sul- LTD4 (as great as 100!J,g). SK&F 104,353 Lung Lymph. Endotoxin alone profidopeptide leukotrienes in sheep inhibited the pressor response complete- duced a significant and sustained increase followed by LTC4 • LTE4 had no effect. ly for lower doses of LTD4 (10 to 30 ug), in lung Ql (figure 2). The changes in or, A 3- to 30-!J,g bolus of LTD 4 caused Attenuated pressor responses were seen, LIP, and Clp observed after endotoxin marked changes in lung mechanics and however, in the higher doses (30 to 100 werenot affected by pretreating the sheep pulmonary hemodynamics. Cdyn de- ug boluses) (figure 1). Boluses of LTB4 with L-660,711 or SK&F 104,353. Lung mechanics. Endotoxin alone and after pretreatment with L-660,711 or SK&F 104,353 produced significant alterations in Cdyn, RL, and FRC as pre80 (AI IBI viously described for endotoxin alone (5, 8 8, 18). The effects of endotoxin on lung 60 mechanics were not significantly differ6 ent between the animals treated with en~ C ::; r:~ 40 dotoxin alone and those pretreated with o~ "::; '""- 4 either antagonist (figure 3). ~ ~ Circulating white bloodcells. Circulat20 2 ing WBCs fell significantly 1 h after endotoxin and remained significantly low0 0 Endotoxin er than baseline for the 5-h period monii 5 tored after endotoxin administration 3 0 2 4 0 2 3 4 5 (figure 2). The fall in WBCs was attenuTIME (Hours Alter Endotoxin) TIME (Hours After Endotoxin) ated in animals pretreated with L-660,711, but it did not reach statistical significance when compared with the endotoxin group. SK&F 104,353 pretreatment did 60 lei 12 (01 not affect endotoxin-induced leukopenia. .t1AaPo2 difference. Endotoxin caused 10 a rapid and prolonged increase in 0 0 40 8 9 ~aP02' Pretreatment with either the w 0 U e, co E 6 L-660,711 or SK&F 104,353 did not sig~ < ~ ~ nificantly affect this measurement (fig~ ... 20 ure 2). "~ 4 Thromboxane B2 • Lymph lXB2 con2 centrations peaked 1 h after endotoxin 0 0 EndOIOlin administration and gradually apI ~ proached baseline 5 h later (figure 4). 5 3 4 0 0 1 2 3 5 2 4 L-660,711 and SK&F 104,353attenuated TIME (Hours Alter ElldoloxinJ TIME (Hours Afler Endotoxin) peak lymph lXB 2 concentrations and delayed the occurrence of the peak conFig. 2. Effectof L-660,711 on endotoxin-inducedchanges in (A) pulmonary arterial pressure(Ppa);(B) lung lymph flow(OL); (e) alveolar-to-arterial 0, pressuredifference(aAaPO,); and (D)circulatingwhiteblood cell count (WBC). centration from 1.0 to 1.5 h. The differDatafrom the sheep studied with endotoxin alone are shown as closed circles, and those from the same sheep ences in peak lymph lXB2concentrations pretreatedwith L-660,711 are shown as open circles. The data are means ± standard errors; n = 6. None of were statistically significant (p < 0.05) the data points in the endotoxin alone experiments was significantly different from the time-matched points in with L-660,711. the animals pretreated with L-660,711.
.
L
-
£ndOIOtr:ift
-
.. .
-
Erws'otoltln
1000 {AI
MILLER, LEFFERTS, AND SNAPPER
50
100 75
40
m'" Cd" IX
50
CI>
c:
>
