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Analysis of the antagonist profile of SK&F 88046 on guinea pig trachea
Prostaglandins Leukotrienes and Medicine 15: 167-175, 1984
ANALYSIS OF THE AKTAGGNIST PRGFILR OF SK&F 88046 ON GUINEA PIG TRACHEA Barry M. Weichman, Jeris F. DeVan, Roseanna M. Muccitelli. Stephanie S. Tucker, Lynne M. Vickery and Martin A. Wasserman Department of Pharmacology, Smith Kline h French Laboratories, Philadelphia, Pennsylvania 19101, USA (reprint requests to BMW) ABSTRACT SK&F 88046 preferentially antagonized the contractions elicited by the functional thromboxane (TX) AZ mimics and structural endoperoxide analogs, U-44069 and U-46619, on the guinea pig trachea. This concentration-dependent antagonism was described by pA2 values of 7.03 against U-46619 and 6.97 against U-44069: the slopes of both Schild plots were 0.9. Whereas SK&F 88046 did not antagonize the tracheal contractions elicited by leukotriene (LT) C!eor Da. carbachol or histamine, this agent did antagonize the contractions induced by carbocyclic thromboxane AZ (C!CAzland prostaglandin (EG) Fz- and Dz. At Ix~O-~M SK&F 88046, the antagonism was described by -log KB values of 5.9 for CTAz, 5.5 for PGFz,, and 6.4 for PGDt. Thus, SK&l?88046 was 3 to 20-fold more potent an antagonist of U-44069 and U-46619, suggesting that SK&F 88046 may function primarily as a thromboxanel endoperoxide antagonist on the guinea pig trachea. INTRODUCTION SK&F 88046 {N.N'-bis[7-~3-chlorobenzeneaminosulfonyl~-1,2,3,4-tetrahydroisoquinolyl]disulfonylimide}was initially identified as an antagonist of slow reacting substance of anaphylaxis and synthetic LTDI in several guinea pig systems (isolated guinea pig lung parenchymal strips and i.v.-LTDe-induced bronchoconstriction in the anesthetized guinea pig) (11. Subsequently, the mechanism by which LTDI functions in these systems was found to be dependent in part upon the actions of thromboxane (2-4). In contrast, the contractions elicited by LTD4 on the isolated guinea pig trachea were found to be directly mediated and independent of thromboxane action (41. On this tracheal preparation, SK&F 88046 was unable to antagonize the actions of LTD4, whereas on both lung parenchyma and trachea, SK&F 88046 antagonized the contractions elicited by the stable thromboxane analog, CTAZ. and by PGD2 and PGFz,, (5). Thus, it appears that the mechanism of action of SK&F 88046 in the guinea pig lung may be explained by an 167 PRoST-B
end organ antagonist action against the contractile prostanoids. Our goal in this study was to provide a quantitative analysis of the antagonist activity of SK&F 88046 and to define its specificity on the guinea pig trachea, a tissue very sensitive to the spasmogenic actions of TxA2 (6). MATERIALS AND METHODS Tracheal spiral strips prepared from adult, male Albino Hartley strain guinea pigs (400-600 g) were placed in jacketed lo-ml tissue baths and connected via silk suture to Grass model FTO3C force displacement transducers (Grass Instrument Co., Quincy, MA) for recording isometric tension. The tissues were equilibrated for 1 hr in modified Krebs' buffer of the following composition (mM): NaCl (118): KC1 (4.61, MgS04*7HzO (1.1): CaClz (1.8): NaHC03 (24.9); KHtP04 (1.0) and glucose (11.1). Then, the tissues were incubated for 30 additional min with SK&F 88046 or Krebs' buffer vehicle. The tracheas were also pretreated with meclofenamic acid (1~10-~M) in order to antagonize the compensatory release of dilator PG products during tracheal contraction (7). Then, cumulative concentration-response curves were generated for each tissue by successive increases in the bath concentration of each agonist according to the method of Van Rossum (8). Each concentration of agonist was added during the plateau of the contraction elicited by the preceding agonist concentration. Only one concentration-response curve per tissue was generated. In order to minimize intertissue variability, the contractile responses were normalized by expressing them as a percentage of the maximum response obtained to carbachol (1~10-~H). The concentration of agonist needed to elicit 30% of the carbachol maximum (ECHO) in the presence and absence of SK&F 88046 was employed to define the agonist potency and the SK&F 88046 antagonist activity. The Kz describing the antagonist activity of SK&F 88046 was calculated from the equation: KB
=
[Concentration of SK&F 880461 (EC30 + SK&F 88046/lC30 Control) - 1
From these data, a Schild plot was constructed when appropriate and the pA2 and slope determined by regression analysis (9). Statistical analysis was performed by an unpaired t-test with significance achieved at the p
168
RESULTS
Contraction of the isolated guinea pig trachea was elicited by a variety of spasmogens (Table 1). Due to the limited supply of several agents, the contraction of agonist needed to elicit 30% of the maximum contraction induced by carbachol (ECao) was employed as a reflective index of agonist potency. This EC30 was on the linear portion of the concentration-response curve for each agonist. The rank order of potency for the agonists was found to be: LTC+ > LTD~ > U-46619 = u-44069 = Carbachol > CTAZ = PGFza =
PGDz > Histamine The contractile activity of LTC4 was assessed in the presence of 45 &I .&'-serine borate complex, a transition state inhibitor of y-glutamyl transpeptidase (10). and thus, under conditions in which LTC4 metabolism to LTDI was prevented (11). In order to define the spectrum of the antagonist activity of SK&F 88046, initial experiments were performed using 1~10-~M of this agent. SK&F 88046 did not antagonize the contractions elicited by LTC4, LTD4, carbachol or histamine (Table 1). However, SK&F 88046 did antagonize the contractions elicited by U-46619 and U-44069 (Kz = 0.14 pM and 0.16 pM, respectively) as well as the contractions elicited by PGDz (KEI= 0.41 pM). CTAZ (Kg = 1.4 pMM)and PGF2,,(KE = 3.4 pM1M).
TABLE 1.
Agonist
Effect of SK&F 88046 (1x10-'Ml on Agonist-Induced Contractions of the Guinea Pig Trachea Control (-log Ecso)
LTC4 LTD4 U-46619 U-44069 Carbachol CTAz PGFzor PGD2 Histamine
a.70 8.09 1.74 1.56 7.48 6.46 6.37 6.19 5.82
2 + + k + + k k +
0.02 0.23 0.12 0.07 0.11 0.08 0.06 0.07 0.08
a
+SKW 88046
a.77 7.98 5.87 5.76 1.52 5.54 5.78 4.78 5.64
2 2 + k + + + + ;
0.05 0.11 0.19 0.10 0.03 0.10 0.11 0.13 0.02
b KB (lJM)
0.14 0.16 1.4 3.4 0.41
'The negative logarithm + S.E.M. of the concentration of agonist needed to achieve 30% of the contraction elicited by l~lO-~Eicarbachol in the absence (control) and presence of 1~10-~M SK&F 88046. bThe KB was calculated from the mean -log EC30 values obtained in the presence and absence of SK&F 88046 as described in the Methods. A dash indicates that significant antagonism by SK&F 88046 was not observed.
169
Quantitation of the antagonist activity of SK&F 88046 was made at multiple concentrations of antagonist. SK&F 88046 exhibited a concentration-dependent antagonism of the contractions elicited by U-44069 and U-46619 (Fig. 11. Significant antagonism was observed at concentrations as low as 1x10-'M SK&F 88046. Analysis of the antagonist activity on a Schild plot yielded pA2 values of 6.97 and 7.03 vs. U-44069 and U-46619, respectively (Figure 1). In both cases the slope of the Schild plot was 0.9, suggestive of competitive antagonism. SK&F 88046 exhibited less antagonist activity against the contractions elicited by CTA2, PGD2 and PGFza. Against these agonists, concentration-dependent antagonism by SK&F 88046 was observed: however, the lowest concentration of SK&F 88046 showing significant antagonist activity was 1~10-~M (Figure 21. DISCUSSION SK&F 88046 preferentially antagonized the contractions elicited by U-44069 and U-46619 in an apparently competitive manner with pAz values of 6.97 and 7.03, respectively. These agonists are structural cyclic endoperoxide analogs of PGH2 and appear to mimic the actions of thromboxane AZ in isolated smooth muscle preparations (12). However, the lack of tool agents has precluded a conclusive demonstration that U-44069 and U-46619 function only via the activation of thromboxane receptors. Thus, antagonism of U-44069- and U-46619-induced contractions of the trachea by SK&F 88046 could reflect antagonism of a thromboxane receptor and/or antagonism of a PGH2 receptor (which may be one and the same on the trachea). Since SK&F 88046 showed a weaker antagonism of the contractions elicited by CTAz, PGF2, and FGDz and did not antagonize the contractions elicited by LTG, LTD4, carbachol or histamine, these results suggest that SK&F 88046 functions primarily as a thromboxane/endoperoxide antagonist on the trachea. However, it was surprising that SK&F 88046 was a weaker antagonist of CTAz vis-a-vis U-44069 and U-46619. Even though CTA2, a stable analog of thromboxane AZ, is lo-fold weaker in agonist activity relative to the endoperoxide analogs, the difference in the apparent affinity of SK&F 88046 as an antagonist of CTAz vis-a-vis U-44069 and U-46619 would imply that these agonists elicit contraction via different receptor mechanisms on the trachea. Furthermore, the similar potency of SK&F 88046 against CTAp, pGFZ, (and possibly PGD21 would suggest that CTA2 interacts with a PG receptor. However, the limited supply of CTA2 prevented us from constructing full concentration-response curves, and thus, it is difficult to draw any firm conclusions about the relative mechanisms of CTA2 and U-44069/ U-46619. The construction of a full concentration-response curve for CTAs is of importance, because this agent has been reported to inhibit U-46619-induced platelet aggregation (13). Thus, CTA2 may not be functioning as a full agonist on the trachea, resulting in a lowering of the apparent affinity of SK&F 88046. It will be of interest to compare two other recently identified thromboxane/endoperoxide antagonists, EP 045 (14) and SQ 27,427 (15) versus the contractions elicited by CTAZ and U-44069/U-46619. On the guinea pig trachea, EP 045 an-
170
loo
/-
I-
1 80 1
100
0
a -B 1.0
7
2.0.
5
_\ -log
7
W&F85046 Corm 1MJ
6
.
h
u46619@----q p4 =7.03 mc0.9 r=0.98
rr0.99
ix-~ .
U-@-4
FIGURE 1. Antagonismof the U-44069- and U-46619-inducedcontractions by SK&F 88046. Tracheas were greincubatedwith SK&F 88046 (1x10-'M,a: 1~10-~W,Cl:1x10- M: 0) or buffer control (0) for 30 min. then, cumulative-concentration responsecurves using U-44069 (A) or U-46619 (B) were constructed. Each point representsthe mean percentageof carbacholcontraction _tS.E.M. of 3 to 6 tissues. A Schild plot of the SK&F 88046 antagonism is plotted (Cl.
0 resmtKIF8m46
?la-rms&wrrur ?
A lo-7M SK&? me46
??control
C
50
??
0
0
A
FIGURE 2.
log CTA2
to-%
5K&F 0
1
too
100cootconelM1
50 i
too
log PGF2, Cone [Ml
Effect of SK&F 88046 on the contractions elicited by CTAz (A), PGD, (B) and PGFz, (C). Details are described in the legend to Figure 1. Each point represents the mean percentage of the carbachol contraction + S.E.M. of 6 tissues.
cone [Ml
Control to-‘MsK&Foun6 to~suLF~
1
too
tagonized the U-46619-induced contractions with a ~Az value of 7.5, making it 3-fold more potent than SK&F 88046 (14). [In these latter studies, EP 045 had pA2 values.of 7.2 and 6.3 on the dog saphenous vein and rabbit aorta, respectively, vs. U-46619.1 Like SK&F 88046, EP 045 and SQ 21,421 antagonized the contractions elicited by PGFz, and PGDz on the trachea (14,15). SK&F 88046 did not antagonize the contractions induced by LTCI and LTD4 on the guinea pig trachea, a tissue in which LT action is directly mediated and independent of thromboxane (4). In contrast, the actions of LTD4 on the guinea pig lung parenchyma are mediated both directly and in part indirectly via the actions of thromboxane. Thus, on the lung parenchyma, it is not surprising that SK&F 88046 antagonizes LTD4-induced contractions, presumably via antagonism of the indirect thromboxane-dependent component (5). However, recently Burke et al. (16) demonstrated that SK&F 88046 antagonized the LTGinduced decreases in contractile force and coronary flow rate of isolated guinea pig heart. Since the LTs do not appear to release thromboxane A2 or prostaglandins from guinea pig heart (171, it remains questionable how SK&F 88046 is functioning in the heart. Two possibilities are: i) Low levels of thromboxane, below the level of detection by available techniques, are released following LT stimulation of the heart and account for at least part of the observed effects on contractile force and coronary flow rate. ii) Tissue differences in leukotriene receptors may exist, such that SK&F 88046 does not interact with the airway LT receptor but does with the cardiac LT receptor. This latter explanation will undoubtedly be addressed following the identification and characterization of LT receptors using r3H]LTC4 and r3H]LTD4 in both tissues. In suxxnary,SK&F 88046 appears to preferentially antagonize the actions of U-44069 and U-46619 in the guinea pig trachea, thus suggesting its principal designation as a thromboxane/endoperoxide antagonist. Thus, SK&F 88046 may find clinical application in those disorders whose pathogenesis involves the actions of thromboxane A2 or prostaglandin HZ. ACKNOWLEDGEMENT We would like to thank Dr. K.C. Nicolaou for generously making available the carbocyclic thromboxane AZ. REFERENCES 1.
Gleason, J.G., Krell, R.D., Weichman. B.M., Ali, F.E.. Berkowitz, B. Comparative pharmacology and antagoniom of synthetic leukotrienes on airway and vascular smooth muscle. Advances in Prostaglandin, Thromboxane and Leukotriene Research 9:243, 1982.
173
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UPA
‘6
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-SZ)SOld
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‘S
‘OJq?A UT za pue TequauJlJadx3 pue d6o~oc%?uuayd 30 ~ewnoy ‘Dz~ surpueT6eqsocid pue auexoqw~y~ “a aua?JrqoynaT dq pampuT uoyz+~rJ~suo~oqwo~q 30 7sy106e7ue ~+Tooeuueyd anbyn ‘3.r ‘uoseaT=) “\z”A ‘UwlJasseM “R.8 ‘UwJlqoTaM Y :9b088 d’S)IS
'9
auexoqumq~
‘186T ‘E6,t:Tz surPueT6eWoJd ‘z)cl pue z):qqe1 ‘6Td eaurn6 30 sd?lyz TwulycwaJed pue 6~11 pasn3Jad paqelos: 6yd eaurnfi UT +a pm *3 saua?Jq -0ynaT 30 uo~~m 30 ursruey~am aqJ ‘N’H ‘UIIOIJUIPS ’ ‘r’d ‘Jadrd
‘2
12 .
Coleman, R.A., Huaqhrey, P.P.A., Kennedy, I., Levy, G.P.. Lumley, P. Comparison of the actions of U-46619, a prostaglandin Hz analog, with those of prostaglandin HZ and thromboxane AZ on some isolated smooth muscle preparations. British Journal of Pharmacology 73:773, 1981.
13 .
Lefer, A.M., Smith, E.F. III, Araki. H., Smith, J.B., Aharony, D. Claremon, D.A., Magolda, R.L., Nicolaou, K.C. Dissociation of vasoconstrictor and platelet aggregatory activities of thromboxane by carbocyclic thromboxane AZ, a stable analog of thromboxane A2. Proceedings of the National Academy of Sciences, U.S.A. 77:1706, 1980.
14.
Jones, R.L., Peesapati, V., Wilson, N.H. Antagonism of the thromboxane-sensitive contractile systems of rabbit aorta, dog saphenous vein and guinea pig trachea. British Journal of Pharmacology 76:423, 1982.
15.
Steinbacher. T., Greenberg, R., O'Keefe, E. Thromboxane receptor antagonism in isolated smooth muscle. Pharmacologist 25:234, 1983.
16.
Burke, J.A., Levi, R., Gleason, J.G. Antagonism of the cardiac effects of leukotriene C4 by compound SK&&'88046: Dissociation of effects on contractility and coronary flow. Journal of Cardiovascular Pharmacology 6:122, 1984.
17.
Burke, J.A., Levi, R., Guo. Z.-G., Corey, E.J. Leukotrienes C4, D4 and E4: Effects on human and guinea pig cardiac pteparations in vitro. Journal of Pharmacology and Experimental Therapeutics 221:235, 1982.
175
ANALYSIS OF THE AKTAGGNIST PRGFILR OF SK&F 88046 ON GUINEA PIG TRACHEA Barry M. Weichman, Jeris F. DeVan, Roseanna M. Muccitelli. Stephanie S. Tucker, Lynne M. Vickery and Martin A. Wasserman Department of Pharmacology, Smith Kline h French Laboratories, Philadelphia, Pennsylvania 19101, USA (reprint requests to BMW) ABSTRACT SK&F 88046 preferentially antagonized the contractions elicited by the functional thromboxane (TX) AZ mimics and structural endoperoxide analogs, U-44069 and U-46619, on the guinea pig trachea. This concentration-dependent antagonism was described by pA2 values of 7.03 against U-46619 and 6.97 against U-44069: the slopes of both Schild plots were 0.9. Whereas SK&F 88046 did not antagonize the tracheal contractions elicited by leukotriene (LT) C!eor Da. carbachol or histamine, this agent did antagonize the contractions induced by carbocyclic thromboxane AZ (C!CAzland prostaglandin (EG) Fz- and Dz. At Ix~O-~M SK&F 88046, the antagonism was described by -log KB values of 5.9 for CTAz, 5.5 for PGFz,, and 6.4 for PGDt. Thus, SK&l?88046 was 3 to 20-fold more potent an antagonist of U-44069 and U-46619, suggesting that SK&F 88046 may function primarily as a thromboxanel endoperoxide antagonist on the guinea pig trachea. INTRODUCTION SK&F 88046 {N.N'-bis[7-~3-chlorobenzeneaminosulfonyl~-1,2,3,4-tetrahydroisoquinolyl]disulfonylimide}was initially identified as an antagonist of slow reacting substance of anaphylaxis and synthetic LTDI in several guinea pig systems (isolated guinea pig lung parenchymal strips and i.v.-LTDe-induced bronchoconstriction in the anesthetized guinea pig) (11. Subsequently, the mechanism by which LTDI functions in these systems was found to be dependent in part upon the actions of thromboxane (2-4). In contrast, the contractions elicited by LTD4 on the isolated guinea pig trachea were found to be directly mediated and independent of thromboxane action (41. On this tracheal preparation, SK&F 88046 was unable to antagonize the actions of LTD4, whereas on both lung parenchyma and trachea, SK&F 88046 antagonized the contractions elicited by the stable thromboxane analog, CTAZ. and by PGD2 and PGFz,, (5). Thus, it appears that the mechanism of action of SK&F 88046 in the guinea pig lung may be explained by an 167 PRoST-B
end organ antagonist action against the contractile prostanoids. Our goal in this study was to provide a quantitative analysis of the antagonist activity of SK&F 88046 and to define its specificity on the guinea pig trachea, a tissue very sensitive to the spasmogenic actions of TxA2 (6). MATERIALS AND METHODS Tracheal spiral strips prepared from adult, male Albino Hartley strain guinea pigs (400-600 g) were placed in jacketed lo-ml tissue baths and connected via silk suture to Grass model FTO3C force displacement transducers (Grass Instrument Co., Quincy, MA) for recording isometric tension. The tissues were equilibrated for 1 hr in modified Krebs' buffer of the following composition (mM): NaCl (118): KC1 (4.61, MgS04*7HzO (1.1): CaClz (1.8): NaHC03 (24.9); KHtP04 (1.0) and glucose (11.1). Then, the tissues were incubated for 30 additional min with SK&F 88046 or Krebs' buffer vehicle. The tracheas were also pretreated with meclofenamic acid (1~10-~M) in order to antagonize the compensatory release of dilator PG products during tracheal contraction (7). Then, cumulative concentration-response curves were generated for each tissue by successive increases in the bath concentration of each agonist according to the method of Van Rossum (8). Each concentration of agonist was added during the plateau of the contraction elicited by the preceding agonist concentration. Only one concentration-response curve per tissue was generated. In order to minimize intertissue variability, the contractile responses were normalized by expressing them as a percentage of the maximum response obtained to carbachol (1~10-~H). The concentration of agonist needed to elicit 30% of the carbachol maximum (ECHO) in the presence and absence of SK&F 88046 was employed to define the agonist potency and the SK&F 88046 antagonist activity. The Kz describing the antagonist activity of SK&F 88046 was calculated from the equation: KB
=
[Concentration of SK&F 880461 (EC30 + SK&F 88046/lC30 Control) - 1
From these data, a Schild plot was constructed when appropriate and the pA2 and slope determined by regression analysis (9). Statistical analysis was performed by an unpaired t-test with significance achieved at the p
168
RESULTS
Contraction of the isolated guinea pig trachea was elicited by a variety of spasmogens (Table 1). Due to the limited supply of several agents, the contraction of agonist needed to elicit 30% of the maximum contraction induced by carbachol (ECao) was employed as a reflective index of agonist potency. This EC30 was on the linear portion of the concentration-response curve for each agonist. The rank order of potency for the agonists was found to be: LTC+ > LTD~ > U-46619 = u-44069 = Carbachol > CTAZ = PGFza =
PGDz > Histamine The contractile activity of LTC4 was assessed in the presence of 45 &I .&'-serine borate complex, a transition state inhibitor of y-glutamyl transpeptidase (10). and thus, under conditions in which LTC4 metabolism to LTDI was prevented (11). In order to define the spectrum of the antagonist activity of SK&F 88046, initial experiments were performed using 1~10-~M of this agent. SK&F 88046 did not antagonize the contractions elicited by LTC4, LTD4, carbachol or histamine (Table 1). However, SK&F 88046 did antagonize the contractions elicited by U-46619 and U-44069 (Kz = 0.14 pM and 0.16 pM, respectively) as well as the contractions elicited by PGDz (KEI= 0.41 pM). CTAZ (Kg = 1.4 pMM)and PGF2,,(KE = 3.4 pM1M).
TABLE 1.
Agonist
Effect of SK&F 88046 (1x10-'Ml on Agonist-Induced Contractions of the Guinea Pig Trachea Control (-log Ecso)
LTC4 LTD4 U-46619 U-44069 Carbachol CTAz PGFzor PGD2 Histamine
a.70 8.09 1.74 1.56 7.48 6.46 6.37 6.19 5.82
2 + + k + + k k +
0.02 0.23 0.12 0.07 0.11 0.08 0.06 0.07 0.08
a
+SKW 88046
a.77 7.98 5.87 5.76 1.52 5.54 5.78 4.78 5.64
2 2 + k + + + + ;
0.05 0.11 0.19 0.10 0.03 0.10 0.11 0.13 0.02
b KB (lJM)
0.14 0.16 1.4 3.4 0.41
'The negative logarithm + S.E.M. of the concentration of agonist needed to achieve 30% of the contraction elicited by l~lO-~Eicarbachol in the absence (control) and presence of 1~10-~M SK&F 88046. bThe KB was calculated from the mean -log EC30 values obtained in the presence and absence of SK&F 88046 as described in the Methods. A dash indicates that significant antagonism by SK&F 88046 was not observed.
169
Quantitation of the antagonist activity of SK&F 88046 was made at multiple concentrations of antagonist. SK&F 88046 exhibited a concentration-dependent antagonism of the contractions elicited by U-44069 and U-46619 (Fig. 11. Significant antagonism was observed at concentrations as low as 1x10-'M SK&F 88046. Analysis of the antagonist activity on a Schild plot yielded pA2 values of 6.97 and 7.03 vs. U-44069 and U-46619, respectively (Figure 1). In both cases the slope of the Schild plot was 0.9, suggestive of competitive antagonism. SK&F 88046 exhibited less antagonist activity against the contractions elicited by CTA2, PGD2 and PGFza. Against these agonists, concentration-dependent antagonism by SK&F 88046 was observed: however, the lowest concentration of SK&F 88046 showing significant antagonist activity was 1~10-~M (Figure 21. DISCUSSION SK&F 88046 preferentially antagonized the contractions elicited by U-44069 and U-46619 in an apparently competitive manner with pAz values of 6.97 and 7.03, respectively. These agonists are structural cyclic endoperoxide analogs of PGH2 and appear to mimic the actions of thromboxane AZ in isolated smooth muscle preparations (12). However, the lack of tool agents has precluded a conclusive demonstration that U-44069 and U-46619 function only via the activation of thromboxane receptors. Thus, antagonism of U-44069- and U-46619-induced contractions of the trachea by SK&F 88046 could reflect antagonism of a thromboxane receptor and/or antagonism of a PGH2 receptor (which may be one and the same on the trachea). Since SK&F 88046 showed a weaker antagonism of the contractions elicited by CTAz, PGF2, and FGDz and did not antagonize the contractions elicited by LTG, LTD4, carbachol or histamine, these results suggest that SK&F 88046 functions primarily as a thromboxane/endoperoxide antagonist on the trachea. However, it was surprising that SK&F 88046 was a weaker antagonist of CTAz vis-a-vis U-44069 and U-46619. Even though CTA2, a stable analog of thromboxane AZ, is lo-fold weaker in agonist activity relative to the endoperoxide analogs, the difference in the apparent affinity of SK&F 88046 as an antagonist of CTAz vis-a-vis U-44069 and U-46619 would imply that these agonists elicit contraction via different receptor mechanisms on the trachea. Furthermore, the similar potency of SK&F 88046 against CTAp, pGFZ, (and possibly PGD21 would suggest that CTA2 interacts with a PG receptor. However, the limited supply of CTA2 prevented us from constructing full concentration-response curves, and thus, it is difficult to draw any firm conclusions about the relative mechanisms of CTA2 and U-44069/ U-46619. The construction of a full concentration-response curve for CTAs is of importance, because this agent has been reported to inhibit U-46619-induced platelet aggregation (13). Thus, CTA2 may not be functioning as a full agonist on the trachea, resulting in a lowering of the apparent affinity of SK&F 88046. It will be of interest to compare two other recently identified thromboxane/endoperoxide antagonists, EP 045 (14) and SQ 27,427 (15) versus the contractions elicited by CTAZ and U-44069/U-46619. On the guinea pig trachea, EP 045 an-
170
loo
/-
I-
1 80 1
100
0
a -B 1.0
7
2.0.
5
_\ -log
7
W&F85046 Corm 1MJ
6
.
h
u46619@----q p4 =7.03 mc0.9 r=0.98
rr0.99
ix-~ .
U-@-4
FIGURE 1. Antagonismof the U-44069- and U-46619-inducedcontractions by SK&F 88046. Tracheas were greincubatedwith SK&F 88046 (1x10-'M,a: 1~10-~W,Cl:1x10- M: 0) or buffer control (0) for 30 min. then, cumulative-concentration responsecurves using U-44069 (A) or U-46619 (B) were constructed. Each point representsthe mean percentageof carbacholcontraction _tS.E.M. of 3 to 6 tissues. A Schild plot of the SK&F 88046 antagonism is plotted (Cl.
0 resmtKIF8m46
?la-rms&wrrur ?
A lo-7M SK&? me46
??control
C
50
??
0
0
A
FIGURE 2.
log CTA2
to-%
5K&F 0
1
too
100cootconelM1
50 i
too
log PGF2, Cone [Ml
Effect of SK&F 88046 on the contractions elicited by CTAz (A), PGD, (B) and PGFz, (C). Details are described in the legend to Figure 1. Each point represents the mean percentage of the carbachol contraction + S.E.M. of 6 tissues.
cone [Ml
Control to-‘MsK&Foun6 to~suLF~
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tagonized the U-46619-induced contractions with a ~Az value of 7.5, making it 3-fold more potent than SK&F 88046 (14). [In these latter studies, EP 045 had pA2 values.of 7.2 and 6.3 on the dog saphenous vein and rabbit aorta, respectively, vs. U-46619.1 Like SK&F 88046, EP 045 and SQ 21,421 antagonized the contractions elicited by PGFz, and PGDz on the trachea (14,15). SK&F 88046 did not antagonize the contractions induced by LTCI and LTD4 on the guinea pig trachea, a tissue in which LT action is directly mediated and independent of thromboxane (4). In contrast, the actions of LTD4 on the guinea pig lung parenchyma are mediated both directly and in part indirectly via the actions of thromboxane. Thus, on the lung parenchyma, it is not surprising that SK&F 88046 antagonizes LTD4-induced contractions, presumably via antagonism of the indirect thromboxane-dependent component (5). However, recently Burke et al. (16) demonstrated that SK&F 88046 antagonized the LTGinduced decreases in contractile force and coronary flow rate of isolated guinea pig heart. Since the LTs do not appear to release thromboxane A2 or prostaglandins from guinea pig heart (171, it remains questionable how SK&F 88046 is functioning in the heart. Two possibilities are: i) Low levels of thromboxane, below the level of detection by available techniques, are released following LT stimulation of the heart and account for at least part of the observed effects on contractile force and coronary flow rate. ii) Tissue differences in leukotriene receptors may exist, such that SK&F 88046 does not interact with the airway LT receptor but does with the cardiac LT receptor. This latter explanation will undoubtedly be addressed following the identification and characterization of LT receptors using r3H]LTC4 and r3H]LTD4 in both tissues. In suxxnary,SK&F 88046 appears to preferentially antagonize the actions of U-44069 and U-46619 in the guinea pig trachea, thus suggesting its principal designation as a thromboxane/endoperoxide antagonist. Thus, SK&F 88046 may find clinical application in those disorders whose pathogenesis involves the actions of thromboxane A2 or prostaglandin HZ. ACKNOWLEDGEMENT We would like to thank Dr. K.C. Nicolaou for generously making available the carbocyclic thromboxane AZ. REFERENCES 1.
Gleason, J.G., Krell, R.D., Weichman. B.M., Ali, F.E.. Berkowitz, B. Comparative pharmacology and antagoniom of synthetic leukotrienes on airway and vascular smooth muscle. Advances in Prostaglandin, Thromboxane and Leukotriene Research 9:243, 1982.
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12 .
Coleman, R.A., Huaqhrey, P.P.A., Kennedy, I., Levy, G.P.. Lumley, P. Comparison of the actions of U-46619, a prostaglandin Hz analog, with those of prostaglandin HZ and thromboxane AZ on some isolated smooth muscle preparations. British Journal of Pharmacology 73:773, 1981.
13 .
Lefer, A.M., Smith, E.F. III, Araki. H., Smith, J.B., Aharony, D. Claremon, D.A., Magolda, R.L., Nicolaou, K.C. Dissociation of vasoconstrictor and platelet aggregatory activities of thromboxane by carbocyclic thromboxane AZ, a stable analog of thromboxane A2. Proceedings of the National Academy of Sciences, U.S.A. 77:1706, 1980.
14.
Jones, R.L., Peesapati, V., Wilson, N.H. Antagonism of the thromboxane-sensitive contractile systems of rabbit aorta, dog saphenous vein and guinea pig trachea. British Journal of Pharmacology 76:423, 1982.
15.
Steinbacher. T., Greenberg, R., O'Keefe, E. Thromboxane receptor antagonism in isolated smooth muscle. Pharmacologist 25:234, 1983.
16.
Burke, J.A., Levi, R., Gleason, J.G. Antagonism of the cardiac effects of leukotriene C4 by compound SK&&'88046: Dissociation of effects on contractility and coronary flow. Journal of Cardiovascular Pharmacology 6:122, 1984.
17.
Burke, J.A., Levi, R., Guo. Z.-G., Corey, E.J. Leukotrienes C4, D4 and E4: Effects on human and guinea pig cardiac pteparations in vitro. Journal of Pharmacology and Experimental Therapeutics 221:235, 1982.
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