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Effect of inhibition of thromboxane production on the leukotriene D4-mediated bronchoconstriction in the guinea pig
PROSTAGLANDINS
EFFECT OF INHIBITION OF THROMBOKANE PRODUCTION ON THE LEUKOTRIENE D4-MEDIATED BRONCHOCONSTRICTION IN THE GUINEA PIG
Roseanna M. Muccitelli,
Ruth R. Osborn and Barry M. Weichman*
Department of Pharmacology, Smith Kline and French Laboratories, Philadelphia, PA 19101
ABSTRACT
Leukotriene D4 (LTD4) administered intravenously to anesthetized, spontaneously breathing guinea pigs elicited decreases in dynamic Lung compliance (Cdyn ) and airway conductance (GAW) with a maximal response achieved at 0.5 min. Simultaneously, plasma levels of the thromboxane metabolite, TxB2, and the prostacyclin metabolite, 6-keto-PGFL,, increased lo-fold over pre-LTD4 Levels. Pretreatment of the guinea pigs with meclofenamic acid delayed the onset of the LTD4-induced bronchoconstriction, antagonized the magnitude of the decreases in Cdyn and GAW, and blocked the increase in plasma TxB2 and 6-keto-PGFlc levels. The thromboxane synthetase inhibitor, UK 37,248, suppressed the LTD4-induced bronchoconstriction, while it completely blocked TxB2 production without significantly affecting 6-keto-PGFl,. The SRS-A end organ antagonist, FPL 55712, blocked both the LTD4induced bronchoconstriction and the production of the arachidonic acid metabolites. These results suggest that thromboxane A2 plays an important role in mediating part of the bronchoconstriction elicited by intravenously administered LTD4 in the guinea pig. INTRODUCTION The identification of the leukotrienes (LTC4, LTD4 and LTE4) as the active components of slow reacting substance of anaphylaxis (L-3) has prompted investigations into the mechanism of action of these agents. In isolated guinea pig lung , these leukotrienes have been shown to elicit the generation of cyclooxygenase-dependent metabolites of arachidonic acid, specifically thromboxane (4-6). In that the contraction of isolated Lung elicited by these Leukotrienes can at least be partially antagonized by inhibitors affecting the production of thromboxane, it can be suggested that part of the contractile effects of the Leukotrienes may be indirectly mediated (7,8). In vivo, the bronchoconstriction elicited by LTD4 has been re-ported to also be antagonized by cyclooxygenase inhibitors, e.g. aspirin and meclofenamic acid (8-10). However, this antagonism was *to whom correspondence
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should be addressed
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observed following intravenous administration of LTD4 but not following aerosol administration (8,lO). Recently, we demonstrated that TxB2 can be found in the plasma of guinea pigs following intravenous LTD4 but not following aerosol LTD4, thus suggesting the mechanism of action of LTD4 differs depending upon the route of administration (11). In the present investigation, we have studied the effects of UK 37,248,'a thromboxane synthetase inhibitor (12), on the LTD4 -induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs , while simultaneously monitoring the production of several arachidonic acid metabolites, For comparison, we also employed meclofenamic acid and FPL 55712 (13). Our goal was to ascertain if thromboxane plays an important role in the bronchoconstriction elicited by intravenous LTD4 in the guinea pig. MATERIALS AND METHODS Changes in dynamic lung compliance (Cdyn ) and airway conductance (GAN) following intravenous administration of LTD4 to urethane anesthetized, spontaneously breathing guinea pigs were quantitated as previously described using a Buxco Pulmonary Mechanics computer (8). Guinea pigs were pretreated i-v. with FPL 55712 (5 mg/kg, 30 set pretreat), meclofenamic acid (2 mg/kg, 2 min), UK 37,248 (5 mg/kg, 2 min) or vehicle control. Blood samples (0.5 ml) were withdrawn from the carotid artery using a syringe containing indomethacin (3!Jg;to prevent spontaneous cyclooxygenase product formation) and EDTA (2.7 pmo1e.s) in 0.1 ml. The plasma was utilized for the determination of TxB2, 6-keto-PGFls and PGF2s Statistical analyses of the -in vivo data were by radioimmunoassay. performed at each time point (0, 0.5, 1, 2 and 5 min) using the unpaired t-test with significance achieved at the WO.05 level. 5S,GR-LTD4 was synthesized at Smith Kline & French Materials. The molar concentration of stock solutions of LTD4 Laboratories. was determined by ultraviolet spectroscopy. UK 37,248 was kindly supplied by Pfizer, Groton, CT; meclofenamic acid by Warner-Lambert, Ann Arbor, MI; and FPL 55712 by Fisons, Ltd., Leicestershire, England. TxB2, 6-keto-PGFlu and PGF2, concentrations were determined by radioimmunoassay, using kits purchased from New England Nuclear, Boston, MA (TxB2 and 6-keto-PGFl,) and Seragen, Inc., Cambridge, MA (PGF2u). LTD4 did not crossreact with any of the antisera used in these studies. RESULTS Administration of synthetic LTD4 intravenously elicited bronchoconstriction as reflected by decreases in Cdyn and GAN (Fig. 1). The onset of the changes in the pulmonary parameters was rapid, achieving a peak response at 0.5 min following LTD4 administration followed by a decline toward baseline. The change in Cdynwas of greater magnitude and of longer duration than the decrease in GAN.
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Pretreatment of the guinea pigs with FPL 55712 (5 mg/kg, i.v.) 30 set prior to LTD4 completely blocked the LTD4-induced bronchoconstriction (Fig. 1). In the FPL 55712-treated animals, both
............................................................... . G&W
Cdyn
IOO-
80
80.
**
I I
1
I
2
+FPL
I 3
55712
I
4
1 5
lime[min)
Fig. 1.
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Effect of meclofenamic acid and FPL 55712 on the bronchoconstriction elicited by i.v. LTD4. Anesthetized, spontaneously breathing guinea pigs received either meclofenamic acid (2 mg/kg; i-v.; O-O), FPL 55712 (5 mg/kg; i-v.; A-A) or saline vehicle (o-o) and then, LTD4 The bron(0.5 nmole/kg) was administered intravenously. chopulmonary responses were analyzed using a pulmonary mechanics computer. Plotted are the mean + S.E.M. of the LTDd-induced decreases in C!ayn (left) and C+,W (right) expressed as percentage changes from the pre-LTD4 values. Statistical significance was achieved at PcO.05 (*I, PO.01 (*+I or p
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Cayn and GAwwere not statistically different (p>O.O5) from baseline. In contrast, whereas meclofenamic acid (2 mg/kg, i.v.) pretreatment completely blocked the changes in the pulmonary parameters at 0.5 min following LTD4, decreases in Cayn and GAw were observed in these animals at 2-5 min following LTD4 (Fig. 1). While the pulmonary parameters were being monitored in these animals, blood samples were collected and the plasma was subsequently analyzed for TxB2, 6-keto-PGFLo and PGF2a. LTD& elicited a ten-fold elevation in plasma TxB2 and 6-ketoPGFla levels, while only slightly elevating PGF2c, relative to pre-LTD4 controls (Fig. 2). The maximal response was observed at 0.5 min, simultaneous with the maximal changes in the pulmonary parameters. Animals receiving saline vehicle control did not produce any TxB2, 6-keto-PGFl, or PGFa nor did Cayn or GAG change from baseline over the 5 min period. Pretreatment of the animals with meclofenamic acid blocked the LTD4-induced generation of TxB2 and 6-keto-PGFL,. FPL 55712, which by itself elevated baseline TxB2, 6-keto-PGF1, and PGF3 levels, also blocked the LTD4-induced increase in plasma TxB2 and 6-keto-PGFLa (Fig 2). Pretreatment of the guinea pigs with the thromboxane synthetase inhibitor, UK 37,248 (5 mg/kg, i-v.) suppressed but aid not completely abolish the LTD4 -induced bronchoconstriction (Fig. 3). LTD4 administered to control animals yielded 80 + 1% decreases in Cayn and 64 + 6% decreases in GAW , whereas in animals pretreated with UK 37,248, only 30 + 7% and 21 + 10% decreases in Cayn and GAw were observed, respectively. UK37,248 completely abolished the LTD4- induced elevation of TxB2 but did not significantly affect 6-keto-PGFL, levels (Fig. 4). DISCUSSION From the results of this study , the bronchoconstriction elicited by intravenous administration of LTD4 appears to depend partly on thromboxane. Concomitant with the bronchoconstriction elicited by LTD4 (as reflected by decreases in Cayn and GAw) were observed elevations in the plasma concentration of TxB2, a metabolite of TxA2, and 6-keto-PGFlp, a metabolite of prostacyclin. In the two series of experiments, the control animals for the UK 37,248 treated animals produced greater elevations in TxB2 than did the controls for the meclofenamic acid and FPL 55712-treated animals. This may reflect the higher baseline circulating Levels of TxB2 (and the other cyclooxygenase products) in the animals of the UK 37,248 experiment. The maximal percentage decreases in Cayn and GAw in both experiments were similar.
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Fig.
2
0
h
--o
3.0.
4.0.
liii45
PGFzcx Control
o + MA A + FPL 55712
0
Effect of meclofenamic acid and FPL 55712 on the LTDq-induced changes in the plasma Blood samples were withdrawn via the carotid artery levels of cyclooxygenase products. The "0" time at the indicated times following LTD4 from the animals used in Fig. 1. The concentrations of TxB2, point was sampled just prior to the LTD4 administration. 6-keto-PGFlu and PGF2c in the plasma were determined by radioimmunoassay. Statistical significance was achieved at PcO.05 (*), Plotted is the mean + S.E.M. HO.01 (**) or pCO.001 (***).
5
-f-n-PO
***
Time (min)
I :
)
I
bketo-PGF,a
PROSTAGLANDINS
100
80 1
01
Time (min)
Fig.
202
3.
Effect of UK 37,248 on the bronchoconstriction elicited by i.v. LTD4. Anesthetized, spontaneously breathing guinea pigs received either UK 33,248 (5 mg/kg, i-v., o-o) or saline vehicle (o-e), then LTD4 (0.3 nmole/kg) was administered intravenously 2 min later. The bronchopulmonary responses were analyzed using a pulmonary mechanics computer. Plotted are the mean -+ S.E.M. of the LTD4-induced decreases in Cdyn (left) and GAG (right) expressed as percentage changes from the pre-LTD4 values. Statistical significance was achieved at PcO.05 (*), SO.01 (**) or PO.001 (***).
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1983 VOL. 26 NO. 2
Fig.
4.
Time (min)
18.0-
6-keto
PGFqa
6.0
18.0
PGFza
Control
o + UK 37,248
0
changes in the plasma levels of cyclooxygenase Effect of UK 37,248 on the LTD4 -induced Blood samples were withdrawn via the carotid artery at the indicated times products. The "0" time point was sampled just following LTD4 from the animals used in Fig. 3. The concentrations of TxB2, 6-ketoPGFla, prior to the LTD4 administration. Plotted is the mean + and PGF2,-, in the plasma were determined by radioimmunoassay. S.E.M. Statistical significance was achieved at PcO.05 (*I, P
18.01
TxB,
PROSTAGLANDINS
The cyclooxygenase inhibitor, meclofenamic acid, blocked the production of both TxB2 and 6-keto-PGFl,, while delaying in onset and suppressing the magnitude of the LTD4-induced decreases in 'dyn and GAW. The thromboxane synthetase inhibitor, UK 37,240, blocked only TxB2 production, and suppressed the magnitude of the changes in Cdyn and GAW but did not affect the time course profile of these changes. The difference in the effects of meclofenamic acid and UK 37,240 on the time course profile of the LTD4induced changes in the pulmonary parameters remains to be fully and GAW in meclofenamic acidexplained. The decreases in Cdyn treated animals seen 2-5 min following LTD4 is reminiscent of the time course profile observed following aerosol LTD4, which appears to represent a directly mediated LTD4 response (10,ll). Following aerosol LTD4 administration, no TxB2 is detected in the plasma and the bronchoconstriction is not antagonized, but rather slightly potentiated, by meclofenamic acid pretreatment. Thus, in these studies, the residual decreases in Cdyn and GAW observed at 2-5 min following i.v. LTD4 administration to meclofenamic acidtreated animals are not accompanied by elevations in plasma TxB2 levels, and thus, may also reflect direct LTD4-induced effects. In contrast, in terms of UK 37,240, either shunting of the endoperoxides (PGG2 or PGH2) to another bronchoactive agent (e.g., PGD2) or the action of these endoperoxides themselves (14), may be responsible for the bronchoconstriction (although diminished in magnitude) that was still observed in the absence of plasma TxB2 elevations. In these studies, FPL 55712, an end organ SRS-A antagonist (13), completely blocked the LTD4- induced decreases in GAW and Cdyn and prevented the LTD4-induced appearance of TxB2 and 6-keto-PGFlu The activity of FPL 55712 may either be attributed in the plasma. to antagonism of LTD4 directly, or perhaps to its other reported actions, namely cyclooxygenase or thromboxane synthetase inhibitory activity (15,161. It would be of interest to study a pure leukotriene antagonist to verify that the appearance of TxB2 in the plasma is caused directly by LTD4. As suggested by the use of the above pharmacologic agents , the LTD4-induced bronchoconstriction in guinea pigs as reflected by decreases in Cdyn and GAW is partially dependent upon the actions of thromboxane and partiallymediated directly. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Drs. M. Wasserman and J. Gleason for their helpful discussions and continued interest in this work and to Ms. S. Tucker for excellent technical contributions.
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REFERENCES Murphy,
S. Hammarstrcm and B. Samuelsson. Leukotriene C, R.C., a slow reacting substance (SRS) from mouse mastocytoma cells. Proc. Natl. Acad. Sci., U.S.A. 76:4275, 1979. Morris, H.R., G.W. Taylor, P.J.Piper, M.N. Samhoun, and J.R. 2) Tippins. Slow reacting substances (SRSs): The structure identification of SRSs from rat basophilic leukemia (RBL-1) cells. Prostaglandins 19: 185, 1980. Lewis, R.A., S.F. Austec J.M. Drazen, D.A. Clark, A. Marfat and 3) E.J. Corey. Slow reacting substances of anaphylaxis: Identification of leukotrienes C-l and D from human and rat sources. Proc. Natl. Acad. Sci., U.S.A. 77:3710, 1980. of action of 4) Piper, P.J. and M.N. Samhoun. The mechanzm leukotriene C4 and D4 in guinea pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat. Prostaglandins 21:793, 1981. 5) Schiantarelli, p., S. Bongrani and G. Folco. Bronchospasm and pressor effects induced in the guinea pig by leukotriene C4 Eur. J. are probably due to release of cyclooxygenase products. Pharmacol. 73~363, 1981. 6) Folco, G., G. Hansson and E. Grastr%m. Leukotriene C4 stimulates TxA2 formation in isolated sensitized guinea pig lungs. Biochem. Pharmacol. -30:2493, 1981. 7) Piper, P.J. and M.N. Samhoun. Stimulation of arachidonic acid metabolism and generation of thromboxane A2 by leukotrienes B4r C4 and D4 in guinea pig lung in vitro. Br. J. Pharmacol. 77:267, 1982. 8) Weichman, Bx., R.M. Muccitelli, R.R. Osborn, D.A. Holden, J.G. Gleason and M.A. Wasserman. In vitro and in vivo mechanisms of leukotriene-mediated bronchoconstriction in the guinea pig. J. Pharmacol. Exp. Ther. 222:202, 1982. R.C. Murphy. Inhibition by 9) Vargaftig, B.B., J. LeforGnd aspirin of bronchoconstriction due to leukotrienes C4 and D4 in the guinea pig. Eur. J. Pharmacol. 72~417, 1981. 10) Hamel, R., P. Masson, A.W. Ford-Hutchinson, T.R. Jones, G. Brunet and H. Piehuta. Differing mechanisms for leukotriene D4-induced bronchoconstriction in guinea pigs following intravenous and aerosol administration. Prostaglandins -24:419, 1982. 11) Weichman, B.M., R.M. Muccitelli, S.S. Tucker, R.R. Osborn, J.G. Gleason and M.A. Wasserman. Relationship between prostaglandin and thromboxane production and the bronchoconstrictive property of LTD4. In: Leukotrienes and Other Lipoxygenase Products. (P. Piper, ea.) John Wiley & Sons, Ltd., Chichester, England, 1983. In press. 1)
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12) Randell, M.J., M.J. Parry, E. Hawkeswood, P.E. Cross and R.P. UK-37,248, a novel, selective thromboxane synthetase Dickinson. inhibitor with platelet anti-aggregatory and anti-thrombotic activity. Thrombosis Research 23:145, 1981. 13) Augstein, J., J.B. Farmer, T.B.yee, P. Sheard and M.L. Selective inhibitor of slow reacting substance of Tattersall. anaphylaxis. Nature New Biol. 245: 215, 1973. 14) Wasserman, M. Bronchopulmonary pharmacology of some prostaglandin endoperoxide analogs in the dog. Eur. J. Pharmacol. 36: 103, 1976. 15) Krell, R.D.,R. Osborn, L. Vickery, K. Falcone, M. O'Donnell, J. Gleason, J. Kinzig and D. Bryan. Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4. Prostaglandins 22:387, 1981. 16) Welton, A-F., WT. Hope, L.D. Tobias and J.G. Hamilton. Inhibition of antigen-induced histamine release and thromboxane Biochem. synthetase by FPL 55712, a specific SRS-A antagonist? Pharmacol 30:1378, 1981.
Editor: P.J.
206
Piper
Received: 3-4-83
Accepted: 5-3-83
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1983 VOL. 26 NO. 2
EFFECT OF INHIBITION OF THROMBOKANE PRODUCTION ON THE LEUKOTRIENE D4-MEDIATED BRONCHOCONSTRICTION IN THE GUINEA PIG
Roseanna M. Muccitelli,
Ruth R. Osborn and Barry M. Weichman*
Department of Pharmacology, Smith Kline and French Laboratories, Philadelphia, PA 19101
ABSTRACT
Leukotriene D4 (LTD4) administered intravenously to anesthetized, spontaneously breathing guinea pigs elicited decreases in dynamic Lung compliance (Cdyn ) and airway conductance (GAW) with a maximal response achieved at 0.5 min. Simultaneously, plasma levels of the thromboxane metabolite, TxB2, and the prostacyclin metabolite, 6-keto-PGFL,, increased lo-fold over pre-LTD4 Levels. Pretreatment of the guinea pigs with meclofenamic acid delayed the onset of the LTD4-induced bronchoconstriction, antagonized the magnitude of the decreases in Cdyn and GAW, and blocked the increase in plasma TxB2 and 6-keto-PGFlc levels. The thromboxane synthetase inhibitor, UK 37,248, suppressed the LTD4-induced bronchoconstriction, while it completely blocked TxB2 production without significantly affecting 6-keto-PGFl,. The SRS-A end organ antagonist, FPL 55712, blocked both the LTD4induced bronchoconstriction and the production of the arachidonic acid metabolites. These results suggest that thromboxane A2 plays an important role in mediating part of the bronchoconstriction elicited by intravenously administered LTD4 in the guinea pig. INTRODUCTION The identification of the leukotrienes (LTC4, LTD4 and LTE4) as the active components of slow reacting substance of anaphylaxis (L-3) has prompted investigations into the mechanism of action of these agents. In isolated guinea pig lung , these leukotrienes have been shown to elicit the generation of cyclooxygenase-dependent metabolites of arachidonic acid, specifically thromboxane (4-6). In that the contraction of isolated Lung elicited by these Leukotrienes can at least be partially antagonized by inhibitors affecting the production of thromboxane, it can be suggested that part of the contractile effects of the Leukotrienes may be indirectly mediated (7,8). In vivo, the bronchoconstriction elicited by LTD4 has been re-ported to also be antagonized by cyclooxygenase inhibitors, e.g. aspirin and meclofenamic acid (8-10). However, this antagonism was *to whom correspondence
AUGUST
should be addressed
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observed following intravenous administration of LTD4 but not following aerosol administration (8,lO). Recently, we demonstrated that TxB2 can be found in the plasma of guinea pigs following intravenous LTD4 but not following aerosol LTD4, thus suggesting the mechanism of action of LTD4 differs depending upon the route of administration (11). In the present investigation, we have studied the effects of UK 37,248,'a thromboxane synthetase inhibitor (12), on the LTD4 -induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs , while simultaneously monitoring the production of several arachidonic acid metabolites, For comparison, we also employed meclofenamic acid and FPL 55712 (13). Our goal was to ascertain if thromboxane plays an important role in the bronchoconstriction elicited by intravenous LTD4 in the guinea pig. MATERIALS AND METHODS Changes in dynamic lung compliance (Cdyn ) and airway conductance (GAN) following intravenous administration of LTD4 to urethane anesthetized, spontaneously breathing guinea pigs were quantitated as previously described using a Buxco Pulmonary Mechanics computer (8). Guinea pigs were pretreated i-v. with FPL 55712 (5 mg/kg, 30 set pretreat), meclofenamic acid (2 mg/kg, 2 min), UK 37,248 (5 mg/kg, 2 min) or vehicle control. Blood samples (0.5 ml) were withdrawn from the carotid artery using a syringe containing indomethacin (3!Jg;to prevent spontaneous cyclooxygenase product formation) and EDTA (2.7 pmo1e.s) in 0.1 ml. The plasma was utilized for the determination of TxB2, 6-keto-PGFls and PGF2s Statistical analyses of the -in vivo data were by radioimmunoassay. performed at each time point (0, 0.5, 1, 2 and 5 min) using the unpaired t-test with significance achieved at the WO.05 level. 5S,GR-LTD4 was synthesized at Smith Kline & French Materials. The molar concentration of stock solutions of LTD4 Laboratories. was determined by ultraviolet spectroscopy. UK 37,248 was kindly supplied by Pfizer, Groton, CT; meclofenamic acid by Warner-Lambert, Ann Arbor, MI; and FPL 55712 by Fisons, Ltd., Leicestershire, England. TxB2, 6-keto-PGFlu and PGF2, concentrations were determined by radioimmunoassay, using kits purchased from New England Nuclear, Boston, MA (TxB2 and 6-keto-PGFl,) and Seragen, Inc., Cambridge, MA (PGF2u). LTD4 did not crossreact with any of the antisera used in these studies. RESULTS Administration of synthetic LTD4 intravenously elicited bronchoconstriction as reflected by decreases in Cdyn and GAN (Fig. 1). The onset of the changes in the pulmonary parameters was rapid, achieving a peak response at 0.5 min following LTD4 administration followed by a decline toward baseline. The change in Cdynwas of greater magnitude and of longer duration than the decrease in GAN.
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Pretreatment of the guinea pigs with FPL 55712 (5 mg/kg, i.v.) 30 set prior to LTD4 completely blocked the LTD4-induced bronchoconstriction (Fig. 1). In the FPL 55712-treated animals, both
............................................................... . G&W
Cdyn
IOO-
80
80.
**
I I
1
I
2
+FPL
I 3
55712
I
4
1 5
lime[min)
Fig. 1.
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Effect of meclofenamic acid and FPL 55712 on the bronchoconstriction elicited by i.v. LTD4. Anesthetized, spontaneously breathing guinea pigs received either meclofenamic acid (2 mg/kg; i-v.; O-O), FPL 55712 (5 mg/kg; i-v.; A-A) or saline vehicle (o-o) and then, LTD4 The bron(0.5 nmole/kg) was administered intravenously. chopulmonary responses were analyzed using a pulmonary mechanics computer. Plotted are the mean + S.E.M. of the LTDd-induced decreases in C!ayn (left) and C+,W (right) expressed as percentage changes from the pre-LTD4 values. Statistical significance was achieved at PcO.05 (*I, PO.01 (*+I or p
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Cayn and GAwwere not statistically different (p>O.O5) from baseline. In contrast, whereas meclofenamic acid (2 mg/kg, i.v.) pretreatment completely blocked the changes in the pulmonary parameters at 0.5 min following LTD4, decreases in Cayn and GAw were observed in these animals at 2-5 min following LTD4 (Fig. 1). While the pulmonary parameters were being monitored in these animals, blood samples were collected and the plasma was subsequently analyzed for TxB2, 6-keto-PGFLo and PGF2a. LTD& elicited a ten-fold elevation in plasma TxB2 and 6-ketoPGFla levels, while only slightly elevating PGF2c, relative to pre-LTD4 controls (Fig. 2). The maximal response was observed at 0.5 min, simultaneous with the maximal changes in the pulmonary parameters. Animals receiving saline vehicle control did not produce any TxB2, 6-keto-PGFl, or PGFa nor did Cayn or GAG change from baseline over the 5 min period. Pretreatment of the animals with meclofenamic acid blocked the LTD4-induced generation of TxB2 and 6-keto-PGFL,. FPL 55712, which by itself elevated baseline TxB2, 6-keto-PGF1, and PGF3 levels, also blocked the LTD4-induced increase in plasma TxB2 and 6-keto-PGFLa (Fig 2). Pretreatment of the guinea pigs with the thromboxane synthetase inhibitor, UK 37,248 (5 mg/kg, i-v.) suppressed but aid not completely abolish the LTD4 -induced bronchoconstriction (Fig. 3). LTD4 administered to control animals yielded 80 + 1% decreases in Cayn and 64 + 6% decreases in GAW , whereas in animals pretreated with UK 37,248, only 30 + 7% and 21 + 10% decreases in Cayn and GAw were observed, respectively. UK37,248 completely abolished the LTD4- induced elevation of TxB2 but did not significantly affect 6-keto-PGFL, levels (Fig. 4). DISCUSSION From the results of this study , the bronchoconstriction elicited by intravenous administration of LTD4 appears to depend partly on thromboxane. Concomitant with the bronchoconstriction elicited by LTD4 (as reflected by decreases in Cayn and GAw) were observed elevations in the plasma concentration of TxB2, a metabolite of TxA2, and 6-keto-PGFlp, a metabolite of prostacyclin. In the two series of experiments, the control animals for the UK 37,248 treated animals produced greater elevations in TxB2 than did the controls for the meclofenamic acid and FPL 55712-treated animals. This may reflect the higher baseline circulating Levels of TxB2 (and the other cyclooxygenase products) in the animals of the UK 37,248 experiment. The maximal percentage decreases in Cayn and GAw in both experiments were similar.
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Fig.
2
0
h
--o
3.0.
4.0.
liii45
PGFzcx Control
o + MA A + FPL 55712
0
Effect of meclofenamic acid and FPL 55712 on the LTDq-induced changes in the plasma Blood samples were withdrawn via the carotid artery levels of cyclooxygenase products. The "0" time at the indicated times following LTD4 from the animals used in Fig. 1. The concentrations of TxB2, point was sampled just prior to the LTD4 administration. 6-keto-PGFlu and PGF2c in the plasma were determined by radioimmunoassay. Statistical significance was achieved at PcO.05 (*), Plotted is the mean + S.E.M. HO.01 (**) or pCO.001 (***).
5
-f-n-PO
***
Time (min)
I :
)
I
bketo-PGF,a
PROSTAGLANDINS
100
80 1
01
Time (min)
Fig.
202
3.
Effect of UK 37,248 on the bronchoconstriction elicited by i.v. LTD4. Anesthetized, spontaneously breathing guinea pigs received either UK 33,248 (5 mg/kg, i-v., o-o) or saline vehicle (o-e), then LTD4 (0.3 nmole/kg) was administered intravenously 2 min later. The bronchopulmonary responses were analyzed using a pulmonary mechanics computer. Plotted are the mean -+ S.E.M. of the LTD4-induced decreases in Cdyn (left) and GAG (right) expressed as percentage changes from the pre-LTD4 values. Statistical significance was achieved at PcO.05 (*), SO.01 (**) or PO.001 (***).
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1983 VOL. 26 NO. 2
Fig.
4.
Time (min)
18.0-
6-keto
PGFqa
6.0
18.0
PGFza
Control
o + UK 37,248
0
changes in the plasma levels of cyclooxygenase Effect of UK 37,248 on the LTD4 -induced Blood samples were withdrawn via the carotid artery at the indicated times products. The "0" time point was sampled just following LTD4 from the animals used in Fig. 3. The concentrations of TxB2, 6-ketoPGFla, prior to the LTD4 administration. Plotted is the mean + and PGF2,-, in the plasma were determined by radioimmunoassay. S.E.M. Statistical significance was achieved at PcO.05 (*I, P
18.01
TxB,
PROSTAGLANDINS
The cyclooxygenase inhibitor, meclofenamic acid, blocked the production of both TxB2 and 6-keto-PGFl,, while delaying in onset and suppressing the magnitude of the LTD4-induced decreases in 'dyn and GAW. The thromboxane synthetase inhibitor, UK 37,240, blocked only TxB2 production, and suppressed the magnitude of the changes in Cdyn and GAW but did not affect the time course profile of these changes. The difference in the effects of meclofenamic acid and UK 37,240 on the time course profile of the LTD4induced changes in the pulmonary parameters remains to be fully and GAW in meclofenamic acidexplained. The decreases in Cdyn treated animals seen 2-5 min following LTD4 is reminiscent of the time course profile observed following aerosol LTD4, which appears to represent a directly mediated LTD4 response (10,ll). Following aerosol LTD4 administration, no TxB2 is detected in the plasma and the bronchoconstriction is not antagonized, but rather slightly potentiated, by meclofenamic acid pretreatment. Thus, in these studies, the residual decreases in Cdyn and GAW observed at 2-5 min following i.v. LTD4 administration to meclofenamic acidtreated animals are not accompanied by elevations in plasma TxB2 levels, and thus, may also reflect direct LTD4-induced effects. In contrast, in terms of UK 37,240, either shunting of the endoperoxides (PGG2 or PGH2) to another bronchoactive agent (e.g., PGD2) or the action of these endoperoxides themselves (14), may be responsible for the bronchoconstriction (although diminished in magnitude) that was still observed in the absence of plasma TxB2 elevations. In these studies, FPL 55712, an end organ SRS-A antagonist (13), completely blocked the LTD4- induced decreases in GAW and Cdyn and prevented the LTD4-induced appearance of TxB2 and 6-keto-PGFlu The activity of FPL 55712 may either be attributed in the plasma. to antagonism of LTD4 directly, or perhaps to its other reported actions, namely cyclooxygenase or thromboxane synthetase inhibitory activity (15,161. It would be of interest to study a pure leukotriene antagonist to verify that the appearance of TxB2 in the plasma is caused directly by LTD4. As suggested by the use of the above pharmacologic agents , the LTD4-induced bronchoconstriction in guinea pigs as reflected by decreases in Cdyn and GAW is partially dependent upon the actions of thromboxane and partiallymediated directly. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Drs. M. Wasserman and J. Gleason for their helpful discussions and continued interest in this work and to Ms. S. Tucker for excellent technical contributions.
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REFERENCES Murphy,
S. Hammarstrcm and B. Samuelsson. Leukotriene C, R.C., a slow reacting substance (SRS) from mouse mastocytoma cells. Proc. Natl. Acad. Sci., U.S.A. 76:4275, 1979. Morris, H.R., G.W. Taylor, P.J.Piper, M.N. Samhoun, and J.R. 2) Tippins. Slow reacting substances (SRSs): The structure identification of SRSs from rat basophilic leukemia (RBL-1) cells. Prostaglandins 19: 185, 1980. Lewis, R.A., S.F. Austec J.M. Drazen, D.A. Clark, A. Marfat and 3) E.J. Corey. Slow reacting substances of anaphylaxis: Identification of leukotrienes C-l and D from human and rat sources. Proc. Natl. Acad. Sci., U.S.A. 77:3710, 1980. of action of 4) Piper, P.J. and M.N. Samhoun. The mechanzm leukotriene C4 and D4 in guinea pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat. Prostaglandins 21:793, 1981. 5) Schiantarelli, p., S. Bongrani and G. Folco. Bronchospasm and pressor effects induced in the guinea pig by leukotriene C4 Eur. J. are probably due to release of cyclooxygenase products. Pharmacol. 73~363, 1981. 6) Folco, G., G. Hansson and E. Grastr%m. Leukotriene C4 stimulates TxA2 formation in isolated sensitized guinea pig lungs. Biochem. Pharmacol. -30:2493, 1981. 7) Piper, P.J. and M.N. Samhoun. Stimulation of arachidonic acid metabolism and generation of thromboxane A2 by leukotrienes B4r C4 and D4 in guinea pig lung in vitro. Br. J. Pharmacol. 77:267, 1982. 8) Weichman, Bx., R.M. Muccitelli, R.R. Osborn, D.A. Holden, J.G. Gleason and M.A. Wasserman. In vitro and in vivo mechanisms of leukotriene-mediated bronchoconstriction in the guinea pig. J. Pharmacol. Exp. Ther. 222:202, 1982. R.C. Murphy. Inhibition by 9) Vargaftig, B.B., J. LeforGnd aspirin of bronchoconstriction due to leukotrienes C4 and D4 in the guinea pig. Eur. J. Pharmacol. 72~417, 1981. 10) Hamel, R., P. Masson, A.W. Ford-Hutchinson, T.R. Jones, G. Brunet and H. Piehuta. Differing mechanisms for leukotriene D4-induced bronchoconstriction in guinea pigs following intravenous and aerosol administration. Prostaglandins -24:419, 1982. 11) Weichman, B.M., R.M. Muccitelli, S.S. Tucker, R.R. Osborn, J.G. Gleason and M.A. Wasserman. Relationship between prostaglandin and thromboxane production and the bronchoconstrictive property of LTD4. In: Leukotrienes and Other Lipoxygenase Products. (P. Piper, ea.) John Wiley & Sons, Ltd., Chichester, England, 1983. In press. 1)
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12) Randell, M.J., M.J. Parry, E. Hawkeswood, P.E. Cross and R.P. UK-37,248, a novel, selective thromboxane synthetase Dickinson. inhibitor with platelet anti-aggregatory and anti-thrombotic activity. Thrombosis Research 23:145, 1981. 13) Augstein, J., J.B. Farmer, T.B.yee, P. Sheard and M.L. Selective inhibitor of slow reacting substance of Tattersall. anaphylaxis. Nature New Biol. 245: 215, 1973. 14) Wasserman, M. Bronchopulmonary pharmacology of some prostaglandin endoperoxide analogs in the dog. Eur. J. Pharmacol. 36: 103, 1976. 15) Krell, R.D.,R. Osborn, L. Vickery, K. Falcone, M. O'Donnell, J. Gleason, J. Kinzig and D. Bryan. Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4. Prostaglandins 22:387, 1981. 16) Welton, A-F., WT. Hope, L.D. Tobias and J.G. Hamilton. Inhibition of antigen-induced histamine release and thromboxane Biochem. synthetase by FPL 55712, a specific SRS-A antagonist? Pharmacol 30:1378, 1981.
Editor: P.J.
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Received: 3-4-83
Accepted: 5-3-83
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1983 VOL. 26 NO. 2