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Arachidonic acid metabolites do not mediate toluene diisocyanate-induced airway hyperresponsiveness in Guinea pigs
PROSTAGLANDINS
ARACHIDONIC ACID METABOLITES DO NOT MEDIATE TOLUENE DIISOCYANATEINDUCED AIRWAY HYPERRESPONSIVENESS IN GUINEA PIGS’
Terry Gordon2-3, James E. Thompson2, and Dean Sheppard Cardiovascular Research Institute, Department of Medicine, Northern California Occupational Health Center, University of California, San Francisco, San Francisco, CA 94110
ABSTRACT Arachidonic acid metabolites have previously been demonstrated to mediate the airway hyperresponsiveness observed in guinea pigs and dogs after exposure to ozone. Guinea pigs were treated with indomethacin (a cyclooxygenase inhibitor), U-60,257 (pirfprost, a 5 lipoxygenase inhibitor), or BW755c (a lipoxygenase and cyclooxygenase inhibitor) and exposed to air or 3 ppm TDI. Airway responsiveness to acetylcholine aerosol was examined 2 h after exposure. In control animals, the provocative concentration of acetylcholine which caused a 200% increase in pulmonary resistance over baseline (PC2oo) was significantly less (~~0.05) after exposure to TDI (8.6 + 2.0 mg/ml, geometric mean + geometric SE, n=lO) than after exposure to air (23.9 + 2.5 mg/ml, n=14). The airway responsiveness to acetylcholine in animals treated with indomethacin or pirfprost and exposed to TDI was not different from that of control animals exposed to TDI. Treatment with BW755c enhanced the airway hyperresponsiveness observed in animals exposed to TDI without altering the PC2oo of animals exposed to air. The PC200 of animals treated with BW755c and exposed to TDI (2.3 + 0.8 mg/ml, n=8) was significantly lower than the PC200 of control animals exposed to TDI (PC 0.025). These results suggest that products of arachidonic acid metabolism are not responsible for TDI-induced airway hyperresponsiveness in guinea pigs. BW755c. however, appears to potentiate the TDI-induced airway hyperresponsiveness to acetylcholine by an as yet unidentified mechanism. INTRODUCTION Acute airway injury and inflammation are commonly associated with an acute increase in the Arachidonic acid metabolites are known to play important roles in acute inflammation. A number of these metabolites have direct effects on airway smooth muscle. Thus, several studies have examined the contributions of arachidonic acid metabolites to the acute increases in airway responsiveness that follow exposure to various inflammatory stimuli (4-6). In dogs, the increase in airway responsiveness caused by exposure to ozone (6) platelet activating factor (7). or inhaled antigen (8) can be attenuated by specific inhibitors of thromboxane synthesis, in viva responsiveness of airway smooth muscle to contractile stimuli (l-3).
‘This research was supported in part by a grant from the California Medical Research and Education Fund of the American Lung Association of California and by USPHS Grants No. HL35222 and HL-33259. 2Supported by the USPHS Pulmonary Faculty Training Grant No. HL-07185. 3Reprint requests to Terry Gordon, Chest Service, room 5K1, San Francisco General Hospital, 1001 Potrero Ave, San Francisco, CA, 94110.
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suggesting that these increases in airway responsiveness are mediated by the cyclooxygenase product thromboxane. In guinea pigs, however, the increase in airway responsiveness caused by ozone was not inhibited by indomethacin (9), suggesting that cyclooxygenase products such as thromboxane are not involved in that species. In related experiments, ozone-induced airway responsiveness in guinea pigs was attenuated by various inhibitors or antagonists of metabolites of the 5-lipoxygenase pathway of arachidonic acid metabolism, including piriprost, BW755c, and FPL-55712 (9,lO). We have been studying the acute increase in airway responsiveness in guinea pigs caused by exposure to toluene diisocyanate (TDI), a precursor of polyurethane that is a well known cause of occupational asthma. To determine the role of arachidonic acid metabolites in TDI-induced airway hyperresponsiveness, we examined the effects of three different inhibitors of arachidonic metabolism (indomethacin - a cyclooxygenase inhibitor, piriprost - a 5-lipoxygenase inhibitor, and BW755c - a mixed cyclooxygenase and lipoxygenase inhibitor) on airway responsiveness to inhaled acetylcholine in guinea pigs exposed to air or 3 ppm TDI. METHODS Male Hartley outbred guinea pigs (virus free, Charles River Breeding Laboratories, Wilmington, MA) weighing between 236 and 592 g were fed standard guinea pig chow (Purina Lab Chow, St. Louis, MO) and water ad /&i&m and housed in hanging steel mesh cages. Treatments Treatment of animals with various inhibitors of arachidonic acid metabolism was initiated prior to a 1h exposure to air or 3 ppm TDI. One group of animals received intraperttoneal (ip) injections of 10 mglkg indomethacin (lyophilized preparation dissolved in saline, a gift of Dr. Morton Rosenberg, Merck, Sharp, and Dohme, West Point, PA) twice per day for 2 days prior to exposure and 5 mg/kg at 30 min before and 30 min after exposure to air (n=6) or TDI (n=6). Another group of animals was treated with intra-arterial injections of 20 mg/kg BW755c (a gift of Dr. Salvador Moncada, Wellcome Research Laboratories, Beckenham, England) 15 min before and 15 min after exposure to air (n=5) or TDI (n=8). An additional group of animals was treated with intravenous injections of U60,257 (piriprost, a gift of Dr. Michael Bach, Upjohn Co., Kalamazoo, Ml) in 5 doses of 10 mglkg each at 30 min intervals beginning 5 min before exposure to air (n=5) or TDI (n=8) and ending approximately 30 min before the acetylcholine aerosol challenges. For intravascular drug administration, PElO or PE50 lntramedic tubing (dead space of 50 to 100 ul, Becton, Dickinson and Co., Parsippany, NJ) was surgically implanted into the jugular vein or carotid artery, respectively. Following each injection of drug, the catheter dead space was cleared with heparinized saline. Drug injections were given in a volume of 1 ml/kg. Airway responsiveness to acetylcholine was also determined in untreated control animals exposed to air (n=9) or TDI (n=6). To examine the possible effect of surgery and the placement of indwelling catheters, intravenous catheters were inserted into anesthetized animals prior to exposure to air (n=5) or TDI (n=4). Saline was given in 5 injections of 1 ml/kg in a manner identical to that used for the administration of piriprost. Animals were exposed to TDI in a 22 liter dynamic exposure chamber constructed of acrylic plastic and lined with a Teflon overlay. The TDI vapor was generated by passing dry filtered air through the head space of a flask containing TDI (80% 2,4 TDl/20% 2.6 TDI, Aldrich Chemical, Milwaukee, WI). The flask was maintained at 40°C in a water bath. The TDI vapor was diluted with filtered air in a glass mixing chamber before it entered the exposure chamber. The exposure and generation systems were enclosed in a fume hood and all tubing in contact with the gas mixture was made of glass or Teflon. TDI concentrations were monitored at IeaSt twice
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during the 1 h exposure by a modified Marcali method (11, 12). Animals were exposed to filtered air in an identical chamber under similar flow and exposure conditions. Animals treated with drugs or saline via indwelling intravenous or intraartenal catheters were placed in restrainers and exposed head only to air or TDI. All other animals were exposed unrestrained via whole body exposure in the air or TDI chambers. Following exposure, animals were anesthetized with intraperitoneal injections of chloralose (Sigma). Additional injections were given as necessary. A tracheal cannula was inserted after additional local lidocaine anesthesia and a 23 gauge polyethylene catheter was introduced into the right carotid artery for monitoring arterial pH and blood gases (the tracheal cannula set-up was changed part way through the study and is reflected by a lower baseline RL in animals treated with saline, piriprost, or BW755c than in untreated animals or animals treated with indomethacin). A water-filled polyethylene catheter (PE 90) with one side hole was inserted into the esophagus to measure esophageal pressure as an approximation of intrapleural pressure. The animal was then placed in a whole body plethysmograph and ventilated with a small animal respirator (model 683, Harvard Apparatus, South Natick, MA) at a frequency of 90 breaths/minute. Rectal temperature was maintained at approximately 37”- 38°C with a heat lamp. Tidal volume was adjusted to yield an artertal pH of 7.35 to 7.45. Transpulmonary pressure (the difference between airway and esophageal pressure) was measured with a Sanborn 2688 transducer (Hewlett-Packard, Palo Alto, CA). Tidal volume was measured by a differential pressure transducer (MP-45, Validyne, Northridge, CA) connected to the plethysmograph and was electronically differentiated to obtain flow. Pulmonary resistance (RL) was calculated with an analogue computer (model 6, Buxco, Sharon, CT) and recorded continuously. To determine airway responsiveness, a dose-response curve to an inhaled acetylcholine aerosol was constructedat 1 l/2 to 2 h after exposure to air or TDI. The aerosol was generated with a DeVilbiss 646 nebulizer fSomerset. PA) driven bv 8 Vmin of medical arade breathina air and was delivered via the tracheal cannula. The aerosol delivery system was a modificationof that described by Hulbert and co-workers (13) and has been described previously. A largebore 3-way stopcock distal to the cannula was used for rapid switching from the animal respirator to the aerosol delivery train (dead space from the ventilator to the animal was 0.83 ml). Pulmonary function measurements were monitored continuously throughout the experiment and an initial challenge of 5 tidal volume breaths of a saline aerosol was followed by successive doubling doses of acetylcholine aerosol (initial dose was 0.5 mglml) at 2 min intervals until RL increased 500% above the post-saline value. Acetylcholine-induced bronchoconstriction was rapid and RL values were chosen as the peak changes obtained during the 2 min intervals. mistical
Analv&
For comparison of airway responsiveness, the provocative concentration of acetylcholine which caused a 200% increase in RL above the baseline saline value (PC2oo) was calculated for each animal by logllinear interpolation between the doses immediately before and after the 200% increase in RL. A one-way analysis of variance and a Neumann-Keuls test were used to examine group differences in PC20o. Statistical comparfsons were made between treatment groups and their matched controls (i.e., untreated and unrestrained controls versus indomethacin animals and catheterized and restrained controls versus piriprost and BW755c animals. RESULTS As we have reported in previous studies, untreated/saline treated animals exposed to TDI had significantly lower values for PC200 than did untreated/saline treated animals exposed to air
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(8.6 + 2.0 mg/ml, geometric mean + geometric SE after TDI and 23.9 + 2.5 mg/ml after air, peO.005). As expected, treatment with saline had no effect on PC200 in either TDI or airexposed animals (PC200 was 6.3 + 2.9 mglml in TDI-exposed saline-treated animals and 10.6 + 3.1 mg/ml in TDI-exposed untreated animals, 22.3 + 2.3 mg/ml in air-exposed, saline-treated animals and 24.8 + 3.8 mglml in air-exposed untreated animals) (Figure 1). Treatment with
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Fiaure 1: A) The effect of treatment with saline, BW755c or piriprost on the airway responsiveness to acetylcholine aerosol in guinea pigs exposed to air or 3 ppm TDI. The ordinate represents the provocative concentration of acetylcholine (mglml) on a log2 scale. The columns represent the geometric mean concentration of acetylcholine calculated to cause a 200% increase in pulmonary resistance over baseline. The error bars represent the geometric SE. Animals were treated with saline (n=5 for air and n=4 for TDI), BW755c (n=5 for air and n=8 for TDI), or pirfprost (n=5 for air and n=8 for TDI). The asterisks indicate that the mean PC200 was significantly less (~~0.05) for each group of animals exposed to TDI than for the similarly treated group of animals exposed to air. The double asterisk indicates that the mean PC200 was significantly less (pcO.025) in BW755ctreated animals exposed to TDI than in saline-treated or piriprost-treated animals exposed to TDI. B) The effect of treatment with indomethacin on the airway responsiveness to acetylcholine aerosol in guinea pigs exposed to air or 3 ppm TDI. The left hand columns show the values for untreated guinea pigs exposed to air (n=9) or TDI (n=6). The right hand columns show the values for indomethacin-treated guinea pigs exposed to air (n=6) or TDI (n=6). The asterisks indicate that the mean PC200 was significantly less (~~0.05) in untreated or indomethacin-treated animals exposed to TDI than in similarly treated animals exposed to air. indomethacin and with piriprost also had no effect on the PC2cc of animals exposed to either TDI or air (7.0 + 4.3 mg/ml and 22.0 + 2.8 mgml in animals treated with indomethacin and
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exposed to TDI and to air, respectively; 10.2 + 3.5 n-g/ml and 30.3 + 5.5 mg/ml in animals treated with piriprost and exposed to TDI and to air). Thus, PC200 was significantly decreased after TDI exposure in both treatment groups (p
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E&.uuQ: The effect of treatment with BW755c on the airway responsiveness to acetylcholine aerosol in guinea pigs exposed to air or 3 ppm TDI. The ordinate represents the provocative concentration of acetylcholine (n-g/ml) on a log2 scale. Each open square represents the PC200 for individual animals treated with saline or BW755c and each solid diamond represents untreated animals. The horizontal lines signify the geometric mean PC200 for each group. and 2). BW755c did not significantly alter the PC200 of animals exposed to air (Figures 1 and 2). Five out of eight animals exposed to TDI after treatment with BW755c were more sensitive to acetylcholine than any of the untreated/saline treated animals exposed to TDI (Figure 2). Within each treatment group, baseline values of RL were not significantly different after exposure to TDI than after exposure to air. No drug caused a significant change in RL in comparison to the matched control (saline for piriprost and BW755c, no treatment for indomethacin). Animals treated with BW755c, piriprost, and saline had lower baseline values of RL than animals treated with indomethacin and animals receiving no treatment. This difference was probably due to the use of a larger bore tracheostomy cannula in the former groups.
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The mean TDI chamber concentration for all TDI-exposed groups was 3.03 + 0.24 ppm (mean + SD). The mean chamber temperature and relative humidity for all air and TDI exposures was 25.6 f 1.l “C and 20 * 7%, respectively. DISCUSSION The results of the present study suggest that TDI-induced airway hyperresponsiveness is not mediated by products of the cyclooxygenase or 5-lipoxygenase pathways of arachidonic acid metabolism. Neither indomethacin, a cyclooxygenase inhibitor, nor piriprost, a 5-lipoxygenase inhibitor, had any effect on the airway hyperresponsiveness induced by exposure to TDI. Treatment with BW755c, a mixed cyclooxygenase and lipoxygenase inhibitor, also did not inhibit TDI-induced airway hyperresponsiveness, confirming that this effect of TDI is not caused by arachidonic acid metabolites. However, BW755c potent&d TDI-induced airway hyperresponsiveness, a result that we did not anticipate. Since BW755c inhibits metabolic enzymes of arachidonic acid other than cyclooxygenase and 5-lipoxygenase (e.g., 12- or 15lipoxygenase enzyme) (14-18) we speculate that BW755c inhibits the production of an arachidonic acid metabolite that is normally produced during or after exposure to TDI and may serve to attenuate TDI-induced airway hyperresponsiveness. The results of the present study were surprising in light of the considerable evidence suggesting a causative role of arachidonate metabolites in the airway hyperresponsiveness induced by other inflammatory stimuli. For example, the increase in airway responsiveness to inhaled acetylcholine caused by exposure of dogs to ozone was inhibited by treatment with indomethacin (5) and by the specific thromboxane synthesis inhibitor OKY-046 (6). In dogs, increases in airway responsiveness caused by inhaled platelet activating factor (7) and by ragweed (in ragweed sensitized animals) were also inhibited by OKY-046 (8). However, the well established role of the cyclooxygenase pathway in these responses in dogs may not apply to other mammalian species. The ozone-induced increase in airway responsiveness was not inhibited by indomethacin in guinea pigs (9). Furthermore, maximally tolerated doses of indomethacin and aspirin do not appear to inhibit ozone-induced airway hyperresponsiveness in human subjects (Homer Boushey, personal communication). Like cyclooxygenase products, products of the 5-lipoxygenase pathway of arachidonate metabolism have been implicated in the increased airway responsiveness that accompanies acute inflammation. The maior evidence supportino a role for the products of this Dathway in causing airway hyperresponsiveness in vivb comesfrom studies done by Murlas and co- . workers examinina the effects of oiriorost. BW755c. and the leukotriene D4 antaaonist FPL 55712 on the increase in airway responsiveness tolntravenously administered acetylcholine in guinea pigs exposed to ozone (9). In these studies, all three drugs inhibited ozone-induced airway hyperresponsiveness, suggesting that this effect of ozone was mediated by a product of the 5-lipoxygenase pathway, presumably a sulfidopeptide leukotrlene. The results of the present study would appear to suggest that the increase in bronchoconstrictor responsiveness to inhaled acetylcholine caused by TDI in guinea pigs and the increase in responsiveness to intravenously administered acetylcholine caused by ozone in the same species occur by different mechanisms. These differences could be due to differences in the nature of the inflammatory response initiated by ozone and by TDI or to differences between responses to inhaled and intravenous acetylcholine. One other difference between the protocol used in the present study and in the studies of Murlas and co-workers is that in our study RL was measured through a tracheostomy in anesthetized and ventilated animals, whereas in Murlas’ studies specific conductance was measured through an intact upper airway in awake, spontaneously breathing animals. It is thus possible that the effects of acetylcholine examined by Murlas and co-workers were occurring primarily in the upper airways or that the effects of leukotrienes are somehow obscured by anesthesia. Resolution of the apparent differences in results must await studies directly examining the significance of each of these factors.
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The potentiating effect of BW755c on TDI-induced airway hyperresponsiveness was not anticipated and our protocol was thus not designed to examine its mechanism. Nonetheless, the lack of effect of piriprost and indomethacin suggest that this potentiation was not due to inhibition of the cyclooxygenase or 5-lipoxygenase pathways. It is noteworthy, therefore, that in addition to inhibiting these pathways, BW755c has been reported to inhibit enzymes involved in the 12- and 15-lipoxygenation of arachidonic acid (15-18). BW755c has been shown to inhibit these pathways in rat (18), guinea pig (18) and human tissues (15) and to inhibit the activity of the 15-lipoxygenase enzyme purified from soybeans (14). Thus, in our experiments, BW755c might have inhibited lipoxygenation products of arachidonic acid metabolism that would not have been inhibited by indomethacfn or pirlprost. We speculate that TDI exposure might stimulate secretion of a product of one of these pathways that normally attenuates acetylcholine-induced bronchoconstriction. Inhibition of this pathway by BW755c would then QQ&~Q& the increase in acetylcholine responsiveness caused by TDI. The presence of a “bronchodilator” lipoxygenase product has been suggested by other investigators (19-20). In a previous study, administration of BW755c (20 rng/kg intravenously) was found to potentiate the increase in pulmonary resistance caused by intravenous substance P in guinea pigs, whereas indomethacin had no effect (19). The authors therefore hypothesized the existence of a bronchodilator lipoxygenase product which might modify the in viva contraction of airway smooth muscle. Recently, a 15-lipoxygenase pathway metabolite of arachidonic acid has been described to have smooth muscle relaxant properties in vitro (20). Tracheal spirals from guinea pigs exhibited a decrease in tension after 15-HPETE was added to the muscle bath. However, neither these studies nor the present study rules out some other effect of BW755c independent of its known effects on arachidonic acid metabolism. In addition, since the drug treatments used in the present study did not block the effect (i.e., TDI-induced airway hyperresponsiveness), we cannot be certain that sufficient inhibition of each metabolic pathways occurred. Measurements of arachidonic acid metabolites were not performed. However, the doses we administered have previously been used to inhibit airways effects thought to be mediated by arachidonic acid metabolites (9,10,20-22). In summary, the present study shows that the increase in airway responsiveness that follows exposure of guinea pigs to TDI is not caused by the effects of known cyclooxygenase or lipoxygenase products of arachidonate metabolism. Rather, the observation that TDI-induced airway hyperresponsiveness is pg&bQ&d by BW755c suggests that if arachidonate metabolites play any role in this response it is an inhibitory one.
1. Holtzman MJ, Fabbri LM, O’Byme PM, Gold BD, Aizawa H, Walters EH, Albert SE, Nadel JA. Importance of airway inflammation for hyperresponsiveness induced by ozone in dogs. Am Rev Respir Dis 1983; 127686-690. 2. Gordon T, Sheppard D, McDonald DM, Distefano S, Scypinski LA. Toluenediisocyanate induced hyperresponsiveness and inflammation of guinea pigairway. Am Rev Respir Dis 1986; 132:1106-112. 3. Seltzer J, Bigby BG, Stulberg MS, Holtzman MJ, Nadel JA, Ueki IF, LeikaufGD, Goetzel EJ. Ozone-induced chanae in bronchial reactivitv and airwavinflammation in human _ subjects. J Appl Physiol 1986; 60:1321-1326. 4. O’Bvrne PM. Leikauf GD. Aizawa H. Bethel RA. Ueki IF. Holtzman MJ. NadelJA. Leukotriene 84 induces airway hyperresponsiveness in dogs. J ApplPhysiol 1985; 59:1941-1946. 5. O’Byrne PM, Walters EH, Aizawa H, Fabbri LM, Holtzman MJ, Nadel JA. lndomethacin inhibits the airway hyperresponsiveness but not theneutrophil influx induced by ozone in dogs. Am Rev Respir Dis 1984; 130:220-224.
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6. Aizawa H, Chung KF, Leikauf GD, Ueki I, Bethel RA, O’Byrne PM, HoltzmanMJ, Nadel JA. Significance of thromboxane generation in ozone-inducedhyperresponsiveness in dogs. J Appl Physiol 1985; 59:1918-1923. 7. Chung KF, Aizawa H, Leikauf GD, Ueki IF, Evans TW, Nadel JA. Airwayhyperresponsiveness induced by platelet activating factor: role of thromboxane generation. J Pharmacol Exp Therap 1986; 286:580-584. 8. Chung KF, Aizawa H, Becker AB, Frick 0, Gold WM, Nadel JA. Inhibition ofantigen-induced airway hyperresponsiveness by a thromboxane synthetaseinhibitor (OKY-046) in allergic dogs. Am Rev Respir Dis 1986; 134:258-261. 9. Lee HK, Murlas C. Ozone-induced bronchial hyperreactivity in guinea pigsis abolished by EiW755c or FPL55712 but not by indomethacin. Am Rev RespirDis 1985; 132:10051009. 10. Murlas C, Lee HK. U-60,257 inhibits 03-induced bronchialhyperreactivity in the guinea pig. Prostaglandin 1985; 30:563-572. 11. Marcali K. Microdetennination of toluene diisocyanate in atmosphere.Anal Chem 1957; 29:552-558. 12. National Institute of Occupational Safety and Health. Criteria for a recommended standard. Occupational exposure to toluene diisocyanate.Report NIOSH-TR-041-73. U.S. Department of Commerce, National Technicallnformation Service, Springfield, VA. 13. Hulbert WC, McLean T, Hogg JC. The effect of acute airway inflammationon bronchial reactivity in guinea pigs. Am Rev Respir Dis 1985; 132:7-l 1. 14. Chang J, Skowronek MD, Cherney ML, Lewis AJ. Differential effects ofputative lipoxygenase inhibitors on arachidonic acid metabolism in cell-free and intact cell preparations. Inflammation 1984; 8:143-155. 15. Salari H, Braquet P, Borgeat P. Comparative effects of indomethacin,acetylenic acids, 15HETE, nordihydroguaiaretic acid and BW755c on themetabolism of arachidonic acid in human leukocytes and platelets.Prostaglandins, Leukotrfenes, and Med 1984; 13:5360. 16. Turk J, Wolf BA, Comens PG, Colca J, Jakschik B, McDaniel ML. Arachidonic acid metabolism in isolated pancreatic islets. IV. Negativeion-mass spectrometric quantitation of monooxygenase product synthesis byliver and islets. Biochim Biophys Acta 1985; 835:1-l 7. 17. Levine L. Inhibition of A-23187stimulated leukotriene and prostaglandinbiosynthesis of rat basophil luekemia (RBL-1) cells by nonsteroidal anti-inflammatory drugs, antioxidants, and calcium channel blockers. BiochemPharmacol 1983; 32:3023-3026. 18. Boughton-Smith NK, Whittle BJR. increased metabolism of arachidonic acidin an immune model of colitis in guinea pigs. Br J Pharmacol 1985; 86:439-446. 19. Stewart AG, Thompson DC, Fennessy MR. Modulation of substance Pinducedbronchoconstriction by lipoxygenase metabolites. J Pharm Pharmacol 1985;37:345-347. 20. Dahlen S-E, Raud J, Serhan CN, Bjork J, Samuelsson B. Biological actiivities of Lipoxin A include lung strip contraction and dilation of arterioles in viva Act Physiol Stand 1987; 130:643-647. 21. Bach MK, Griffin RL, Richards IM. Inhibition of the presumably leukotriene-dependent component of antigen-induced bronchoconstriction in the guinea pig by piriprost (U60,257). Int Arch Allergy Appl lmmunol 1985;77:264-266. 22. Garrett RC, Foster S, Thomas HM. Lipoxygenase and cyclooxygenase blockade by BW755c enhances pulmonary hypoxic vasoconstriction. J Appl Physiol 1987; 62:129133.
Editor:P.Piper
706
Received:lO-22-87
Accepted:3-15-88
MAY 1988 VOL. 35 NO. 5
ARACHIDONIC ACID METABOLITES DO NOT MEDIATE TOLUENE DIISOCYANATEINDUCED AIRWAY HYPERRESPONSIVENESS IN GUINEA PIGS’
Terry Gordon2-3, James E. Thompson2, and Dean Sheppard Cardiovascular Research Institute, Department of Medicine, Northern California Occupational Health Center, University of California, San Francisco, San Francisco, CA 94110
ABSTRACT Arachidonic acid metabolites have previously been demonstrated to mediate the airway hyperresponsiveness observed in guinea pigs and dogs after exposure to ozone. Guinea pigs were treated with indomethacin (a cyclooxygenase inhibitor), U-60,257 (pirfprost, a 5 lipoxygenase inhibitor), or BW755c (a lipoxygenase and cyclooxygenase inhibitor) and exposed to air or 3 ppm TDI. Airway responsiveness to acetylcholine aerosol was examined 2 h after exposure. In control animals, the provocative concentration of acetylcholine which caused a 200% increase in pulmonary resistance over baseline (PC2oo) was significantly less (~~0.05) after exposure to TDI (8.6 + 2.0 mg/ml, geometric mean + geometric SE, n=lO) than after exposure to air (23.9 + 2.5 mg/ml, n=14). The airway responsiveness to acetylcholine in animals treated with indomethacin or pirfprost and exposed to TDI was not different from that of control animals exposed to TDI. Treatment with BW755c enhanced the airway hyperresponsiveness observed in animals exposed to TDI without altering the PC2oo of animals exposed to air. The PC200 of animals treated with BW755c and exposed to TDI (2.3 + 0.8 mg/ml, n=8) was significantly lower than the PC200 of control animals exposed to TDI (PC 0.025). These results suggest that products of arachidonic acid metabolism are not responsible for TDI-induced airway hyperresponsiveness in guinea pigs. BW755c. however, appears to potentiate the TDI-induced airway hyperresponsiveness to acetylcholine by an as yet unidentified mechanism. INTRODUCTION Acute airway injury and inflammation are commonly associated with an acute increase in the Arachidonic acid metabolites are known to play important roles in acute inflammation. A number of these metabolites have direct effects on airway smooth muscle. Thus, several studies have examined the contributions of arachidonic acid metabolites to the acute increases in airway responsiveness that follow exposure to various inflammatory stimuli (4-6). In dogs, the increase in airway responsiveness caused by exposure to ozone (6) platelet activating factor (7). or inhaled antigen (8) can be attenuated by specific inhibitors of thromboxane synthesis, in viva responsiveness of airway smooth muscle to contractile stimuli (l-3).
‘This research was supported in part by a grant from the California Medical Research and Education Fund of the American Lung Association of California and by USPHS Grants No. HL35222 and HL-33259. 2Supported by the USPHS Pulmonary Faculty Training Grant No. HL-07185. 3Reprint requests to Terry Gordon, Chest Service, room 5K1, San Francisco General Hospital, 1001 Potrero Ave, San Francisco, CA, 94110.
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suggesting that these increases in airway responsiveness are mediated by the cyclooxygenase product thromboxane. In guinea pigs, however, the increase in airway responsiveness caused by ozone was not inhibited by indomethacin (9), suggesting that cyclooxygenase products such as thromboxane are not involved in that species. In related experiments, ozone-induced airway responsiveness in guinea pigs was attenuated by various inhibitors or antagonists of metabolites of the 5-lipoxygenase pathway of arachidonic acid metabolism, including piriprost, BW755c, and FPL-55712 (9,lO). We have been studying the acute increase in airway responsiveness in guinea pigs caused by exposure to toluene diisocyanate (TDI), a precursor of polyurethane that is a well known cause of occupational asthma. To determine the role of arachidonic acid metabolites in TDI-induced airway hyperresponsiveness, we examined the effects of three different inhibitors of arachidonic metabolism (indomethacin - a cyclooxygenase inhibitor, piriprost - a 5-lipoxygenase inhibitor, and BW755c - a mixed cyclooxygenase and lipoxygenase inhibitor) on airway responsiveness to inhaled acetylcholine in guinea pigs exposed to air or 3 ppm TDI. METHODS Male Hartley outbred guinea pigs (virus free, Charles River Breeding Laboratories, Wilmington, MA) weighing between 236 and 592 g were fed standard guinea pig chow (Purina Lab Chow, St. Louis, MO) and water ad /&i&m and housed in hanging steel mesh cages. Treatments Treatment of animals with various inhibitors of arachidonic acid metabolism was initiated prior to a 1h exposure to air or 3 ppm TDI. One group of animals received intraperttoneal (ip) injections of 10 mglkg indomethacin (lyophilized preparation dissolved in saline, a gift of Dr. Morton Rosenberg, Merck, Sharp, and Dohme, West Point, PA) twice per day for 2 days prior to exposure and 5 mg/kg at 30 min before and 30 min after exposure to air (n=6) or TDI (n=6). Another group of animals was treated with intra-arterial injections of 20 mg/kg BW755c (a gift of Dr. Salvador Moncada, Wellcome Research Laboratories, Beckenham, England) 15 min before and 15 min after exposure to air (n=5) or TDI (n=8). An additional group of animals was treated with intravenous injections of U60,257 (piriprost, a gift of Dr. Michael Bach, Upjohn Co., Kalamazoo, Ml) in 5 doses of 10 mglkg each at 30 min intervals beginning 5 min before exposure to air (n=5) or TDI (n=8) and ending approximately 30 min before the acetylcholine aerosol challenges. For intravascular drug administration, PElO or PE50 lntramedic tubing (dead space of 50 to 100 ul, Becton, Dickinson and Co., Parsippany, NJ) was surgically implanted into the jugular vein or carotid artery, respectively. Following each injection of drug, the catheter dead space was cleared with heparinized saline. Drug injections were given in a volume of 1 ml/kg. Airway responsiveness to acetylcholine was also determined in untreated control animals exposed to air (n=9) or TDI (n=6). To examine the possible effect of surgery and the placement of indwelling catheters, intravenous catheters were inserted into anesthetized animals prior to exposure to air (n=5) or TDI (n=4). Saline was given in 5 injections of 1 ml/kg in a manner identical to that used for the administration of piriprost. Animals were exposed to TDI in a 22 liter dynamic exposure chamber constructed of acrylic plastic and lined with a Teflon overlay. The TDI vapor was generated by passing dry filtered air through the head space of a flask containing TDI (80% 2,4 TDl/20% 2.6 TDI, Aldrich Chemical, Milwaukee, WI). The flask was maintained at 40°C in a water bath. The TDI vapor was diluted with filtered air in a glass mixing chamber before it entered the exposure chamber. The exposure and generation systems were enclosed in a fume hood and all tubing in contact with the gas mixture was made of glass or Teflon. TDI concentrations were monitored at IeaSt twice
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during the 1 h exposure by a modified Marcali method (11, 12). Animals were exposed to filtered air in an identical chamber under similar flow and exposure conditions. Animals treated with drugs or saline via indwelling intravenous or intraartenal catheters were placed in restrainers and exposed head only to air or TDI. All other animals were exposed unrestrained via whole body exposure in the air or TDI chambers. Following exposure, animals were anesthetized with intraperitoneal injections of chloralose (Sigma). Additional injections were given as necessary. A tracheal cannula was inserted after additional local lidocaine anesthesia and a 23 gauge polyethylene catheter was introduced into the right carotid artery for monitoring arterial pH and blood gases (the tracheal cannula set-up was changed part way through the study and is reflected by a lower baseline RL in animals treated with saline, piriprost, or BW755c than in untreated animals or animals treated with indomethacin). A water-filled polyethylene catheter (PE 90) with one side hole was inserted into the esophagus to measure esophageal pressure as an approximation of intrapleural pressure. The animal was then placed in a whole body plethysmograph and ventilated with a small animal respirator (model 683, Harvard Apparatus, South Natick, MA) at a frequency of 90 breaths/minute. Rectal temperature was maintained at approximately 37”- 38°C with a heat lamp. Tidal volume was adjusted to yield an artertal pH of 7.35 to 7.45. Transpulmonary pressure (the difference between airway and esophageal pressure) was measured with a Sanborn 2688 transducer (Hewlett-Packard, Palo Alto, CA). Tidal volume was measured by a differential pressure transducer (MP-45, Validyne, Northridge, CA) connected to the plethysmograph and was electronically differentiated to obtain flow. Pulmonary resistance (RL) was calculated with an analogue computer (model 6, Buxco, Sharon, CT) and recorded continuously. To determine airway responsiveness, a dose-response curve to an inhaled acetylcholine aerosol was constructedat 1 l/2 to 2 h after exposure to air or TDI. The aerosol was generated with a DeVilbiss 646 nebulizer fSomerset. PA) driven bv 8 Vmin of medical arade breathina air and was delivered via the tracheal cannula. The aerosol delivery system was a modificationof that described by Hulbert and co-workers (13) and has been described previously. A largebore 3-way stopcock distal to the cannula was used for rapid switching from the animal respirator to the aerosol delivery train (dead space from the ventilator to the animal was 0.83 ml). Pulmonary function measurements were monitored continuously throughout the experiment and an initial challenge of 5 tidal volume breaths of a saline aerosol was followed by successive doubling doses of acetylcholine aerosol (initial dose was 0.5 mglml) at 2 min intervals until RL increased 500% above the post-saline value. Acetylcholine-induced bronchoconstriction was rapid and RL values were chosen as the peak changes obtained during the 2 min intervals. mistical
Analv&
For comparison of airway responsiveness, the provocative concentration of acetylcholine which caused a 200% increase in RL above the baseline saline value (PC2oo) was calculated for each animal by logllinear interpolation between the doses immediately before and after the 200% increase in RL. A one-way analysis of variance and a Neumann-Keuls test were used to examine group differences in PC20o. Statistical comparfsons were made between treatment groups and their matched controls (i.e., untreated and unrestrained controls versus indomethacin animals and catheterized and restrained controls versus piriprost and BW755c animals. RESULTS As we have reported in previous studies, untreated/saline treated animals exposed to TDI had significantly lower values for PC200 than did untreated/saline treated animals exposed to air
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(8.6 + 2.0 mg/ml, geometric mean + geometric SE after TDI and 23.9 + 2.5 mg/ml after air, peO.005). As expected, treatment with saline had no effect on PC200 in either TDI or airexposed animals (PC200 was 6.3 + 2.9 mglml in TDI-exposed saline-treated animals and 10.6 + 3.1 mg/ml in TDI-exposed untreated animals, 22.3 + 2.3 mg/ml in air-exposed, saline-treated animals and 24.8 + 3.8 mglml in air-exposed untreated animals) (Figure 1). Treatment with
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Fiaure 1: A) The effect of treatment with saline, BW755c or piriprost on the airway responsiveness to acetylcholine aerosol in guinea pigs exposed to air or 3 ppm TDI. The ordinate represents the provocative concentration of acetylcholine (mglml) on a log2 scale. The columns represent the geometric mean concentration of acetylcholine calculated to cause a 200% increase in pulmonary resistance over baseline. The error bars represent the geometric SE. Animals were treated with saline (n=5 for air and n=4 for TDI), BW755c (n=5 for air and n=8 for TDI), or pirfprost (n=5 for air and n=8 for TDI). The asterisks indicate that the mean PC200 was significantly less (~~0.05) for each group of animals exposed to TDI than for the similarly treated group of animals exposed to air. The double asterisk indicates that the mean PC200 was significantly less (pcO.025) in BW755ctreated animals exposed to TDI than in saline-treated or piriprost-treated animals exposed to TDI. B) The effect of treatment with indomethacin on the airway responsiveness to acetylcholine aerosol in guinea pigs exposed to air or 3 ppm TDI. The left hand columns show the values for untreated guinea pigs exposed to air (n=9) or TDI (n=6). The right hand columns show the values for indomethacin-treated guinea pigs exposed to air (n=6) or TDI (n=6). The asterisks indicate that the mean PC200 was significantly less (~~0.05) in untreated or indomethacin-treated animals exposed to TDI than in similarly treated animals exposed to air. indomethacin and with piriprost also had no effect on the PC2cc of animals exposed to either TDI or air (7.0 + 4.3 mg/ml and 22.0 + 2.8 mgml in animals treated with indomethacin and
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exposed to TDI and to air, respectively; 10.2 + 3.5 n-g/ml and 30.3 + 5.5 mg/ml in animals treated with piriprost and exposed to TDI and to air). Thus, PC200 was significantly decreased after TDI exposure in both treatment groups (p
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E&.uuQ: The effect of treatment with BW755c on the airway responsiveness to acetylcholine aerosol in guinea pigs exposed to air or 3 ppm TDI. The ordinate represents the provocative concentration of acetylcholine (n-g/ml) on a log2 scale. Each open square represents the PC200 for individual animals treated with saline or BW755c and each solid diamond represents untreated animals. The horizontal lines signify the geometric mean PC200 for each group. and 2). BW755c did not significantly alter the PC200 of animals exposed to air (Figures 1 and 2). Five out of eight animals exposed to TDI after treatment with BW755c were more sensitive to acetylcholine than any of the untreated/saline treated animals exposed to TDI (Figure 2). Within each treatment group, baseline values of RL were not significantly different after exposure to TDI than after exposure to air. No drug caused a significant change in RL in comparison to the matched control (saline for piriprost and BW755c, no treatment for indomethacin). Animals treated with BW755c, piriprost, and saline had lower baseline values of RL than animals treated with indomethacin and animals receiving no treatment. This difference was probably due to the use of a larger bore tracheostomy cannula in the former groups.
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The mean TDI chamber concentration for all TDI-exposed groups was 3.03 + 0.24 ppm (mean + SD). The mean chamber temperature and relative humidity for all air and TDI exposures was 25.6 f 1.l “C and 20 * 7%, respectively. DISCUSSION The results of the present study suggest that TDI-induced airway hyperresponsiveness is not mediated by products of the cyclooxygenase or 5-lipoxygenase pathways of arachidonic acid metabolism. Neither indomethacin, a cyclooxygenase inhibitor, nor piriprost, a 5-lipoxygenase inhibitor, had any effect on the airway hyperresponsiveness induced by exposure to TDI. Treatment with BW755c, a mixed cyclooxygenase and lipoxygenase inhibitor, also did not inhibit TDI-induced airway hyperresponsiveness, confirming that this effect of TDI is not caused by arachidonic acid metabolites. However, BW755c potent&d TDI-induced airway hyperresponsiveness, a result that we did not anticipate. Since BW755c inhibits metabolic enzymes of arachidonic acid other than cyclooxygenase and 5-lipoxygenase (e.g., 12- or 15lipoxygenase enzyme) (14-18) we speculate that BW755c inhibits the production of an arachidonic acid metabolite that is normally produced during or after exposure to TDI and may serve to attenuate TDI-induced airway hyperresponsiveness. The results of the present study were surprising in light of the considerable evidence suggesting a causative role of arachidonate metabolites in the airway hyperresponsiveness induced by other inflammatory stimuli. For example, the increase in airway responsiveness to inhaled acetylcholine caused by exposure of dogs to ozone was inhibited by treatment with indomethacin (5) and by the specific thromboxane synthesis inhibitor OKY-046 (6). In dogs, increases in airway responsiveness caused by inhaled platelet activating factor (7) and by ragweed (in ragweed sensitized animals) were also inhibited by OKY-046 (8). However, the well established role of the cyclooxygenase pathway in these responses in dogs may not apply to other mammalian species. The ozone-induced increase in airway responsiveness was not inhibited by indomethacin in guinea pigs (9). Furthermore, maximally tolerated doses of indomethacin and aspirin do not appear to inhibit ozone-induced airway hyperresponsiveness in human subjects (Homer Boushey, personal communication). Like cyclooxygenase products, products of the 5-lipoxygenase pathway of arachidonate metabolism have been implicated in the increased airway responsiveness that accompanies acute inflammation. The maior evidence supportino a role for the products of this Dathway in causing airway hyperresponsiveness in vivb comesfrom studies done by Murlas and co- . workers examinina the effects of oiriorost. BW755c. and the leukotriene D4 antaaonist FPL 55712 on the increase in airway responsiveness tolntravenously administered acetylcholine in guinea pigs exposed to ozone (9). In these studies, all three drugs inhibited ozone-induced airway hyperresponsiveness, suggesting that this effect of ozone was mediated by a product of the 5-lipoxygenase pathway, presumably a sulfidopeptide leukotrlene. The results of the present study would appear to suggest that the increase in bronchoconstrictor responsiveness to inhaled acetylcholine caused by TDI in guinea pigs and the increase in responsiveness to intravenously administered acetylcholine caused by ozone in the same species occur by different mechanisms. These differences could be due to differences in the nature of the inflammatory response initiated by ozone and by TDI or to differences between responses to inhaled and intravenous acetylcholine. One other difference between the protocol used in the present study and in the studies of Murlas and co-workers is that in our study RL was measured through a tracheostomy in anesthetized and ventilated animals, whereas in Murlas’ studies specific conductance was measured through an intact upper airway in awake, spontaneously breathing animals. It is thus possible that the effects of acetylcholine examined by Murlas and co-workers were occurring primarily in the upper airways or that the effects of leukotrienes are somehow obscured by anesthesia. Resolution of the apparent differences in results must await studies directly examining the significance of each of these factors.
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The potentiating effect of BW755c on TDI-induced airway hyperresponsiveness was not anticipated and our protocol was thus not designed to examine its mechanism. Nonetheless, the lack of effect of piriprost and indomethacin suggest that this potentiation was not due to inhibition of the cyclooxygenase or 5-lipoxygenase pathways. It is noteworthy, therefore, that in addition to inhibiting these pathways, BW755c has been reported to inhibit enzymes involved in the 12- and 15-lipoxygenation of arachidonic acid (15-18). BW755c has been shown to inhibit these pathways in rat (18), guinea pig (18) and human tissues (15) and to inhibit the activity of the 15-lipoxygenase enzyme purified from soybeans (14). Thus, in our experiments, BW755c might have inhibited lipoxygenation products of arachidonic acid metabolism that would not have been inhibited by indomethacfn or pirlprost. We speculate that TDI exposure might stimulate secretion of a product of one of these pathways that normally attenuates acetylcholine-induced bronchoconstriction. Inhibition of this pathway by BW755c would then QQ&~Q& the increase in acetylcholine responsiveness caused by TDI. The presence of a “bronchodilator” lipoxygenase product has been suggested by other investigators (19-20). In a previous study, administration of BW755c (20 rng/kg intravenously) was found to potentiate the increase in pulmonary resistance caused by intravenous substance P in guinea pigs, whereas indomethacin had no effect (19). The authors therefore hypothesized the existence of a bronchodilator lipoxygenase product which might modify the in viva contraction of airway smooth muscle. Recently, a 15-lipoxygenase pathway metabolite of arachidonic acid has been described to have smooth muscle relaxant properties in vitro (20). Tracheal spirals from guinea pigs exhibited a decrease in tension after 15-HPETE was added to the muscle bath. However, neither these studies nor the present study rules out some other effect of BW755c independent of its known effects on arachidonic acid metabolism. In addition, since the drug treatments used in the present study did not block the effect (i.e., TDI-induced airway hyperresponsiveness), we cannot be certain that sufficient inhibition of each metabolic pathways occurred. Measurements of arachidonic acid metabolites were not performed. However, the doses we administered have previously been used to inhibit airways effects thought to be mediated by arachidonic acid metabolites (9,10,20-22). In summary, the present study shows that the increase in airway responsiveness that follows exposure of guinea pigs to TDI is not caused by the effects of known cyclooxygenase or lipoxygenase products of arachidonate metabolism. Rather, the observation that TDI-induced airway hyperresponsiveness is pg&bQ&d by BW755c suggests that if arachidonate metabolites play any role in this response it is an inhibitory one.
1. Holtzman MJ, Fabbri LM, O’Byme PM, Gold BD, Aizawa H, Walters EH, Albert SE, Nadel JA. Importance of airway inflammation for hyperresponsiveness induced by ozone in dogs. Am Rev Respir Dis 1983; 127686-690. 2. Gordon T, Sheppard D, McDonald DM, Distefano S, Scypinski LA. Toluenediisocyanate induced hyperresponsiveness and inflammation of guinea pigairway. Am Rev Respir Dis 1986; 132:1106-112. 3. Seltzer J, Bigby BG, Stulberg MS, Holtzman MJ, Nadel JA, Ueki IF, LeikaufGD, Goetzel EJ. Ozone-induced chanae in bronchial reactivitv and airwavinflammation in human _ subjects. J Appl Physiol 1986; 60:1321-1326. 4. O’Bvrne PM. Leikauf GD. Aizawa H. Bethel RA. Ueki IF. Holtzman MJ. NadelJA. Leukotriene 84 induces airway hyperresponsiveness in dogs. J ApplPhysiol 1985; 59:1941-1946. 5. O’Byrne PM, Walters EH, Aizawa H, Fabbri LM, Holtzman MJ, Nadel JA. lndomethacin inhibits the airway hyperresponsiveness but not theneutrophil influx induced by ozone in dogs. Am Rev Respir Dis 1984; 130:220-224.
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6. Aizawa H, Chung KF, Leikauf GD, Ueki I, Bethel RA, O’Byrne PM, HoltzmanMJ, Nadel JA. Significance of thromboxane generation in ozone-inducedhyperresponsiveness in dogs. J Appl Physiol 1985; 59:1918-1923. 7. Chung KF, Aizawa H, Leikauf GD, Ueki IF, Evans TW, Nadel JA. Airwayhyperresponsiveness induced by platelet activating factor: role of thromboxane generation. J Pharmacol Exp Therap 1986; 286:580-584. 8. Chung KF, Aizawa H, Becker AB, Frick 0, Gold WM, Nadel JA. Inhibition ofantigen-induced airway hyperresponsiveness by a thromboxane synthetaseinhibitor (OKY-046) in allergic dogs. Am Rev Respir Dis 1986; 134:258-261. 9. Lee HK, Murlas C. Ozone-induced bronchial hyperreactivity in guinea pigsis abolished by EiW755c or FPL55712 but not by indomethacin. Am Rev RespirDis 1985; 132:10051009. 10. Murlas C, Lee HK. U-60,257 inhibits 03-induced bronchialhyperreactivity in the guinea pig. Prostaglandin 1985; 30:563-572. 11. Marcali K. Microdetennination of toluene diisocyanate in atmosphere.Anal Chem 1957; 29:552-558. 12. National Institute of Occupational Safety and Health. Criteria for a recommended standard. Occupational exposure to toluene diisocyanate.Report NIOSH-TR-041-73. U.S. Department of Commerce, National Technicallnformation Service, Springfield, VA. 13. Hulbert WC, McLean T, Hogg JC. The effect of acute airway inflammationon bronchial reactivity in guinea pigs. Am Rev Respir Dis 1985; 132:7-l 1. 14. Chang J, Skowronek MD, Cherney ML, Lewis AJ. Differential effects ofputative lipoxygenase inhibitors on arachidonic acid metabolism in cell-free and intact cell preparations. Inflammation 1984; 8:143-155. 15. Salari H, Braquet P, Borgeat P. Comparative effects of indomethacin,acetylenic acids, 15HETE, nordihydroguaiaretic acid and BW755c on themetabolism of arachidonic acid in human leukocytes and platelets.Prostaglandins, Leukotrfenes, and Med 1984; 13:5360. 16. Turk J, Wolf BA, Comens PG, Colca J, Jakschik B, McDaniel ML. Arachidonic acid metabolism in isolated pancreatic islets. IV. Negativeion-mass spectrometric quantitation of monooxygenase product synthesis byliver and islets. Biochim Biophys Acta 1985; 835:1-l 7. 17. Levine L. Inhibition of A-23187stimulated leukotriene and prostaglandinbiosynthesis of rat basophil luekemia (RBL-1) cells by nonsteroidal anti-inflammatory drugs, antioxidants, and calcium channel blockers. BiochemPharmacol 1983; 32:3023-3026. 18. Boughton-Smith NK, Whittle BJR. increased metabolism of arachidonic acidin an immune model of colitis in guinea pigs. Br J Pharmacol 1985; 86:439-446. 19. Stewart AG, Thompson DC, Fennessy MR. Modulation of substance Pinducedbronchoconstriction by lipoxygenase metabolites. J Pharm Pharmacol 1985;37:345-347. 20. Dahlen S-E, Raud J, Serhan CN, Bjork J, Samuelsson B. Biological actiivities of Lipoxin A include lung strip contraction and dilation of arterioles in viva Act Physiol Stand 1987; 130:643-647. 21. Bach MK, Griffin RL, Richards IM. Inhibition of the presumably leukotriene-dependent component of antigen-induced bronchoconstriction in the guinea pig by piriprost (U60,257). Int Arch Allergy Appl lmmunol 1985;77:264-266. 22. Garrett RC, Foster S, Thomas HM. Lipoxygenase and cyclooxygenase blockade by BW755c enhances pulmonary hypoxic vasoconstriction. J Appl Physiol 1987; 62:129133.
Editor:P.Piper
706
Received:lO-22-87
Accepted:3-15-88
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