European Journal of Pharmacology, 182 (1990) 291-299 Elsevier

291

EJP 51369

Evidence for two leukotriene receptor types in the guinea-pig isolated ileum Phillip J. G a r d i n e r , T r e v o r S. A b r a m a n d N i g e l J. C u t h b e r t Bayer U.K. Limited, Pharmaceutical Business Group, Research Department, Stoke Court, Stoke Poges, Slough SL2 4L Y, U.K. Received 23 January 1990, accepted 3 April 1990

Leukotriene (LT) receptors in the guinea-pig ileum were characterized using LTB4, LTC4, LTD 4 and LTE 4 and the LT antagonists FPL 55712, ICI 198615 and (_+)SKF 104353. LTB4 was inactive but the other LTs induced concentration-related contractions. LTC4 responses differred to those induced by LTD 4 or LTE 4. Inhibitors of LT metabolism had no significant effects on any LT responses. LTD 4 contractions were inhibited by all three antagonists but a resistant response was apparent at concentrations of ICI 198615 > 10 -8 M. All three antagonists were weak/inactive against LTC 4. LTE4 was a partial agonist which antagonized LTD 4 responses but had little or no activity against LTC 4 or histamine. These results suggest that two distinct LT receptor types exist on guinea-pig ileum. One type is predominantly activated by LTD 4 and is antagonized by three structurally distinct LT antagonists and the partial agonist LTE 4. The second type is predominantly activated by LTC 4 and is insensitive to the LT antagonists. Leukotriene receptors; Ileum (guinea-pig); FPL 55712; ICI 198615; S K & F 103944

1. Introduction The sulphidopeptide leukotrienes (LTs) LTC4, LTD4 and L T E 4 and the 5, 12, d i H E T E leukotriene B4 are all metabolites of arachidonic acid which are specifically p r o d u c u e d b y a 5-1ipoxygenase enzyme (Samuelsson et al, 1980). LTC4, L T D 4 and L T E 4 are potent spasmogenic agents on a range of tissues and together they constitute the l o n g - k n o w n slow reacting substance of anaphylaxis (SRS-A) (Drazen et al., 1980; Dahlen et al., 1980; Krell et al., 1981). In contrast LTB 4 has little or no direct spasmogenic activity but instead is considered to be a p r o - i n f l a m m a t o r y agent (Ford H u t c h i n s o n et al., 1980; Bray et al., 1981; Dahlen et al., 1983). Following the discovery and synthesis of the leukotrienes a large

Correspondence to: N.J. Cuthbert, Bayer U.K. Limited, Pharmaceutical Business Group, Research Department, Stoke Court, Stoke Poges, Slough SL2 4LY, U.K.

a m o u n t of interest has centred a r o u n d their p u l m o n a r y and cardiovascular actions. A n u m b e r of studies have previously suggested that multiple leukotriene receptor types exist and that they occur either as a h o m o g e n o u s population as with h u m a n bronchial muscle or as an heterogenous population as with the guinea-pig trachea (Fleisch et al., 1982; Snyder and Krell, 1984; Buckner et al., 1986). The guinea-pig isolated ileum has been used for m a n y years in conjunction with the leukotriene antagonist F P L 55712 as a specific bioassay for SRS-A (Augstein et al., 1973). Although the leukotrienes are potent contractile agonists on this tissue it remains uncertain at present whether one or more specific leukotriene receptors are responsible for such activity. In general, the studies reported to date using the guinea-pig ileum either evaluated only one leukotriene or, when a range of leukotrienes were used, only one antagonist, usually F P L 55712, was tested over a narrow concentration range ( H o l m e et al., 1980; Denis et al.,

0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

292 1982; Hoiroyde and Ghelani, 1983). In one notable study all of the naturally occurring leukotrienes were tested together with FPL 55712 over a wide concentration range (Feniuk et al., 1984). The experimental conditions used however, were unsuitable for the characterization of receptors. The ileum was superfused in a cascade system and the leukotrienes were administered as bolus injections. However, functional analysis of receptors requires that equilibrium conditions between drug and receptor be attained to provide estimates of drug constants such as affinity and efficacy (Kenakin, 1984). Such conditions are unlikely to occur using the cascade superfusion technique in which the drug rapidly washes over the tissue unless drugs are given by infusion. Characterization of receptors predominantly using rank orders of agonist potency is at best only a preliminary procedure, as factors such as drug diffusion, uptake, metabolism etc. can all produce misleading results (Kenakin, 1981). One such factor, metabolism, was shown to have a significant effect upon the contractile potency of LTC4 on the guinea-pig trachea (Snyder and Krell, 1984). This tissue possesss 7-glutamyltranspeptidase and aminopeptidase the enzymes specifically responsible for LTC 4 and L T D 4 metabolism respectively (Orning and H a m marstrom, 1980; Sok et al., 1980; Snyder et al., 1984). When L-serine borate and L-cysteine, selective inhibitors of these enzymes were used to prevent leukotriene metabolism, the potencies of LTC 4 and L T D 4 and the activity of F P L 55712 were significantly altered. Using F P L 55712, and two structurally distinct leukotriene antagonists, racemic SKF 104353 and ICI 198615 and inhibitors of leukotriene metabolism we sought to characterize the activity of all the naturally occurring leukotrienes on the guineapig ileum and to clarify whether one or more leukotriene receptor types are present.

3 cm length were taken and suspended with the lumen open in a 10 ml tissue bath containing Tyrode solution with indomethacin (3 x 10 -6 M) at 3 7 ° C gassed with 5% CO 2 in 02 . Tissue m o v e m e n t was monitored with a Harvard isotonic transducer with a load of 1 g. Each preparation was washed at 10 min intervals and allowed to equilibrate for 30 min to 1 h before the experiment began. Histamine 10 4-3 x 10 4 M was then added to the tissue on two separate occasions to obtain a maximal contractile response. All leukotriene contractile responses were expressed as a percentage of the second histamine response for each tissue. For the antagonist studies the responses of the test tissue were normalized as a percentage of the maximal leukotriene response in the paired control tissue as it was not possible to obtain more than one concentrationresponse curve to the leukotriene in each tissue.

2.1. Dosing procedure In preliminary experiments the leukotrienes were added to the tissue using two dosing procedures, sequential and cumulative. No significant difference was observed in m a x i m u m response or ECs0 values for any of the leukotrienes using either procedure. Consequently, cumulative dosing (e.g. each dose was added when the response to the previous one had levelled out) which was a more convenient and faster procedure was adopted throughout the studies. Only one leukotriene concentration-response curve was obtained on each tissue.

2.2. Cyclooxygenase inhibition Indomethacin (3 x 10 - 6 M) was routinely present in the bath fluid in all studies to avoid any possible interference by endogenous prostanoids.

2.3. Inhibition of leukotriene metabolism 2. Materials and methods

Male Dunkin Hartley guinea-pigs (400-500 g) were sacrificed, the abdomen opened and a length of terminal ileum at least 10 cm from the ileocaecal valve removed. Preparations of approximately

The inhibitors of LTC 4 and L T D 4 metabolism, L-serine borate and L-cysteine have been shown to effectively protect exogenous LTC 4 and L T D 4 in the guinea-pig ileum (Krilis et al., 1983). Generally these enzyme inhibitors contracted the tissue

293 on induced spontaneous contractions making further studies impossible. These problems were overcome by placing the tissues into Tyrode solution containing the enzyme inhibitor immediately after they were removed from the guinea-pig. In the present studies LTC 4, LTD 4 and LTE 4 (10-1°-10 -5 M) were all cumulatively added to paired ileal preparations. One preparation had enzyme inhibitors (L-serine borate 45 mM and L-cysteine 10 mM) present in the Tyrode and the other acted as a control with Tyrode solution only. 2.4. Antagonist studies

Five preparations were obtained from each tissue. One acted as a control and the others received various concentrations of the antagonist 30 min prior to the cumulative addition of leukotriene. The antagonist effects of FPL 55712 against LTC 4, L T D 4 and LTE 4 responses were determined in the absence of the inhibitors of leukotriene metabolism. Preliminary experiments demonstrated that the activity of FPL 55712 against LTC 4 or L T D 4 was unaffected by L-serine borate or L-cysteine nevertheless these enzyme inhibitors were used in the remaining studies with the other antagonists. Racemic SKF 104353, ICI 198615 and LTE 4 were evaluated for possible antagonism of LTC4 or L T D 4 responses. Racemic SKF 104353 was also evaluated as an antagonist of LTE 4. 2.5. Materials

The following drugs or reagents were used: histamine acid phosphate and boric acid (BDH Chemicals Ltd.), indomethacin (Sigma); L-cysteine and L-serine (SAS Pharmaceuticals). Leukotriene (LT) B4, C 4, D 4 and E 4 were all synthesised by Dr. T.S. Abram and colleagues (Bayer UK, Stoke Court). The purity was 90-97% determined by analytical reverse-phase HPLC. Racemic SKF 104353 (SKF 103944) was synthesised by Dr. H. Kluender (Bayer AG, Wuppertal), ICI 198615 was generously supplied by Dr. R. Krell (Stuart Pharmaceuticals, Delaware). A stock solution of indomethacin (3 × 10-2 M) was prepared fresh each day using absolute ethanol. Racemic SKF 104353 and ICI 198615

(]0 -2 M) were dissolved in dimethyl sulphoxide (DMSO). Ten fold dilutions were prepared in DMSO as required. Leukotriene stock solutions (10 -3 M) and further dilutions were all prepared fresh each day using phosphate buffered saline pH 7.2 and were kept on ice during the experiment. Test drugs were added to the 10 ml bath in a volume not exceeding either 0.1 ml, or 0.01 ml for those dissolved in DMSO. Experiments in which inhibition of -/-glutamyltranspeptidase and aminopeptidase was studied involved the addition to the Tyrode solution (5 1) of 6 g of L-cysteine (final concentration 10 mM), 23.6 g L-serine and 13.9 g boric acid (final concentration L-serine borate complex 45 mM). 2.6. Statistics

In the agonist studies the leukotriene responses were all expressed as a percentage of the maximal histamine response. As none of the leukotrienes achieved a 100% response the ECs0 values were expressed as the concentration which produced 50% of the maximal LT response. In the antagonist studies the ECs0 values for tissues tested with LT plus antagonist were expressed as the concentration which produced 50% of the maximal contraction induced by that LT in the appropriate paired control tissue. Concentration-response slopes for each leukotriene were analysed to produce an ECs0 (the concentration which produces a 50% maximal response for that leukotriene) value with 95% confidence limits using a least squares linear regression line analysis. Analysis of antagonism and the subsequent determination of pA 2 values was performed using the Schild analysis (Arunlakshana and Schild, 1959). On those occasions where antagonism proved by Schild analysis to be noncompetitive a p K B value (negative logarithm of the dissociation constant of an antagonist) was determined for each tissue at each concentration of antagonist and mean + S.E. values calculated. Both of these procedures were performed using a computer statistics package developed by Tallarida and Murray (1981). An approximate pKp (negative logarithm of the dissociation constant

294 for a p a r t i a l agonist) was d e t e r m i n e d using the dose ratios d e r i v e d f r o m ECs0 values.

3. Results

3.1. Response characteristics and concentration-response curves LTB 4 (10-~°-10 4 M) was inactive o n the ileum in the presence or a b s e n c e of i n d o m e t h a c i n (3 × 1 0 - 6 M ) . T h e highest c o n c e n t r a t i o n of LTB 4 was also e v a l u a t e d for the p o t e n t i a t i o n , or the a n t a g o n i s m of, L T C 4 or L T D 4 responses. N o interactions were observed. LTC4, L T D 4 a n d L T E 4 all c o n t r a c t e d the ileum in a c o n c e n t r a t i o n - r e l a t e d m a n n e r . As r e p o r t e d b y others the characteristics of the L T C 4 r e s p o n s e s differed significantly to the o t h e r leukotrienes (Krilis et al., 1983). LTCa-ind u c e d c o n t r a c t i o n s were slow in onset ( a p p r o x . 30 s r e f r a c t o r y p e r i o d ) relative to L T D 4 a n d L T E 4 a n d the time to reach a m a i m a l r e s p o n s e for each c o n c e n t r a t i o n was m u c h longer ( a p p r o x . 1-2 min) t h a n that for L T D 4 or L T E 4 ( a p p r o x . 10 s). T h e m a x i m a l responses i n d u c e d b y L T C 4, L T D 4 a n d L T E 4 were 64, 53 a n d 41% respectively of histam i n e m a x i m a l response. D e s p i t e the differences in their r e s p o n s e characteristics L T C 4 a n d L T D 4 h a d very similar c o n c e n t r a t i o n - r e s p o n s e curves (responses m e a s u r e d as % m a x i m a l response) with c o m p a r a b l e ECs0 values of 2.9 x 1 0 - 9 M (95% c o n f i d e n c e limits, 2.3-3.8) a n d 3.4 x 1 0 - 9 M (2.44.9) respectively (fig. 1). In c o n t r a s t L T E 4 was a p p r o x i m a t e l y 10 X less p o t e n t t h a n the o t h e r l e u k o t r i e n e s with an ECs0 value of 2.4 x 10 - 8 M (1.0-5.5). In a d d i t i o n to differences in the onset time a n d time to reach m a x i m a l r e s p o n s e the d u r a t i o n of the L T C n r e s p o n s e was m o r e p r o l o n g e d t h a n L T D 4 or L T E a.

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Fig. 1. Cumulative concentration-response curves for LTC 4 (O), LTD4 ([3) and LTE 4 (zx) in guinea-pig isolated ileum. Segments of ileum were bathed in Tyrode solution containing indomethacin (3 X 10 -6 M). Points indicate the means_+ S.E.M. for at least 10 observations.

sponses to L T C 4 a n d L T D 4 were also slightly increased. H o w e v e r , the l a t t e r effect was n o t selective for the l e u k o t r i e n e s in that the c o n t r a c t i l e responses to h i s t a m i n e were also i n c r e a s e d a n d the c o n c e n t r a t i o n - r e s p o n s e curve m o v e d 10 fold to the left. T h e e n z y m e i n h i b i t o r s d i d n o t alter the characteristics of the c o n t r a c t i l e r e s p o n s e s - L T C 4 responses r e m a i n e d slower in o n s e t a n d d u r a t i o n t h a n either L T D 4 or L T E 4.

3.3. Antagonist studies It was d e m o n s t r a t e d in a series of p r e l i m i n a r y studies that LTC4, L T D 4 a n d L T E 4 r e s p o n s e s were u n a f f e c t e d b y a r a n g e of p h a r m a c o l o g i c a l a n t a g o n i s t s (atropine, m e p y r a m i n e , m e t h y s e r g i d e p h e n o x y b e n z a m i n e or p r o p r a n o l o l ) .

3.2. Effects of L-serine borate and L-cysteine

3.3.1. F P L 55712

L T C 4, L T D 4 a n d L T E 4 were retested in the p r e s e n c e of L-serine b o r a t e 45 m M a n d L-cysteine 10 m M . In the p r e s e n c e of the inhibitors, L T C 4, L T D 4 a n d L T E 4 p r o d u c e d c o n t r a c t i l e responses which were similar to those o b s e r v e d using p a i r e d c o n t r o l s b u t each l e u k o t r i e n e h a d a slightly longer d u r a t i o n of action. T h e m a x i m a l c o n t r a c t i l e re-

This a n t a g o n i s t was e v a l u a t e d over the conc e n t r a t i o n range 1.5 x 10-7-1.5 × 10 -5 M a g a i n s t LTC4, LTD4, L T E 4 a n d h i s t a m i n e . A l t h o u g h F P L 5 5 7 1 2 h a d n o direct effect u p o n the tissue, it a n t a g o n i z e d LTC4, L T D 4 a n d L T E 4 r e s p o n s e s to differing degrees (fig. 2a,b,c). It h a d no effect u p o n h i s t a m i n e - i n d u c e d c o n t r a c t i o n s e x c e p t at the highest c o n c e n t r a t i o n which d e p r e s s e d the maxi-

295 M a x i m a l a n t a g o n i s m o c c u r r e d at 4.5 x 10 - 6 M with a higher c o n c e n t r a t i o n 1.5 x 10 -5 M, fairing to p r o d u c e a further shift (fig. 2a). Schild analysis of these responses demonstrated that the a n t a g o n i s m was n o n - c o m p e t i t i v e as the slope differed significantly f r o m u n i t y ( - 1 . 6 9 ) . N e v e r t h e less the following p K B values were d e t e r m i n e d , 6.5 (_+0.15) at 1.5 × 10 - 6 M, 6.8 (--+0.16) at 4.5 _+ 10 - 6 M a n d 6.0 (.+_0.31) at 1.5 × 10 -5 M.

m a l h i s t a m i n e r e s p o n s e b y 20%. L T D 4 - i n d u c e d c o n t r a c t i o n s were a n t a g o n i s e d in a c o m p e t i t i v e m a n n e r b y a l i m i t e d c o n c e n t r a t i o n range of F P L 55712 (1.5 x 1 0 - 7 - 4 . 5 X 10 - 6 M ) (fig. 2b). Alt h o u g h the highest c o n c e n t r a t i o n of F P L 55712 (1.5 x 10 -5 M) also a n t a g o n i s e d L T D 4, the shift in the c o n c e n t r a t i o n - r e s p o n s e curve was n o n p a r a l l e l a n d the m a x i m a l r e s p o n s e was depressed. Schild analysis of the three lower c o n c e n t r a t i o n s o f F P L 55712 gave a slope of u n i t y i n d i c a t i v e of c o m p e t i t i v e a n t a g o n i s m with a p A 2 of 6.9 + 0.3 ( m e a n + 1 S.E.). U n f o r t u n a t e l y higher c o n c e n t r a tions of L T D 4 c o u l d n o t b e e v a l u a t e d due to the large a m o u n t s of m a t e r i a l required. L T E 4 - i n d u c e d c o n t r a q t i o n s were also a n t a g o n i z e d b y F P L 55712 with a p a r a l l e l r i g h t w a r d shift in the c o n c e n t r a t i o n - r e s p o n s e curve (fig. 2c). T h e slope of the Schild p l o t differed significantly f r o m u n i t y ( - 1 . 4 8 ) p r e v e n t i n g the c a l c u l a t i o n of a m e a n i n g ful p A 2 value. H o w e v e r the following p K B values ( m e a n + S.E.) were o b t a i n e d 6.7 (-+0.25), 7.0 ( + 0 . 1 2 ) , 7.2 ( + 0 . 2 2 ) a n d 7.4 ( + 0 . 3 9 ) at conc e n t r a t i o n s of 4.5 X 10 -7, 1.5 × 10 -6, 4.5 X 10 - 6 a n d 1.5 × 10 -5 M respectively. A l t h o u g h F P L 55712 shifted the L T C 4 conc e n t r a t i o n - r e s p o n s e curve to the right it was never as effective as it was against L T D 4 o r L T E 4.

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3.3.2. I C I 198615 A l t h o u g h this c o m p o u n d a n t a g o n i z e d L T D 4 c o n t r a c t i l e responses this d i d n o t o c c u r in a c o m petitive m a n n e r (slope for Schild p l o t was n o t unity) (fig. 3b). A t 10 -10, 10 9 a n d 10 - 8 M it p r o d u c e d a large r i g h t w a r d shift of the c o n c e n t r a t i o n - r e s p o n s e curve. D u e to the large a m o u n t of m a t e r i a l r e q u i r e d we were u n a b l e to d e t e r m i n e w h e t h e r the L T D 4 m a x i m a l r e s p o n s e was dep r e s s e d or not. T h e p K a values were 11.5 (_+0.41) at 10 - H M, 12 ( + 0 . 3 5 ) at 10 -1° M, 11.5 ( + 0 . 9 5 ) at 10 - 9 M a n d 11.1 (_+0.44) at 10 - 8 M. In a limited n u m b e r of studies, I C I 198615 at 10 - 7 a n d 10 - 6 M failed to p r o d u c e a f u r t h e r r i g h t w a r d shift in the L T D 4 c o n c e n t r a t i o n - r e s p o n s e curve. T h e s a m e c o n c e n t r a t i o n range of I C I 198615 (10 -11

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Fig. 2. The effect of FPL 55712 on the concentration-response curves to LTC4 (panel a), LTD4 (panel b) and LTE 4 (panel c). Cumulative concentration-response curves to the leukotrienes were constructed either alone (© LTC4; [] LTD4; zx LTE4) or in the presence of FPL55712 1.5×10 -7 M (~); 4.5X10 -7 M (0); 1.5X10 -6 M ( , ) ; 4.5×10 -6 M (1) and 1.5×10 -5 M (a). All preparations were bathed in Tyrode solution containing indomethacin (3 x 10 -6 M). Some lines omitted for clarity. Points indicate the means+ S.E. for n = 8 observations.

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LTC4 conc. {M) Fig. 3. The effect of ICI 198615 on the concentration-response curves to the LTC 4 (panel a) and LTD 4 (panel b). Cumulative concentration-response curves to the leukotrienes were constructed either alone (© LTC4; [] LTD4) or in the presence of ICI198615 10 -11 M ( ~ ) , 10 - l ° M (O), 10 9 M ( ~ ) or 10 -8 M (I). All preparations were bathed in Tyrode solution containing indomethacin (3 X 10 - 6 M), L-serine borate (4.5 x 10 -2 M) and L-cysteine (10 -z M). Points indicate the means_+ S.E. for n = 8 observations.

10

8 M) had no significant effect on LTC 4 con-

t r a c t i l e a c t i v i t y (fig. 3a).

3.3.3. Racemic S K F 104353 This antagonist was evaluated over the concentration range (10-9-10 -6 M) against LTC 4 and

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L T D 4 (fig. 4 a , b ) . T h e t w o h i g h e s t c o n c e n t r a t i o n s o f ( _ + ) S K F 1 0 4 3 5 3 , 10 7 a n d 10 - 6 M a n t a g o n i z e d LTD 4 in what appeared to be a competitive manner however the slope for the Schild plot was n o t u n i t y a n d t h e p K B v a l u e s r a n g e d f r o m 7.6 a t 10 7 M t o 8.4 a t 10 - 6 M . T h e r e w a s n o s h i f t a t

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Fig. 4. The effect of racemic SKF 104353 on the concentration-response curves to LTC 4 (panel a) and LTD 4 (panel b). Cumulative concentration-response curves to the leukotrienes were constructed either alone ( o LTC4; [] LTD4) or in the presence of Racemic SKF 104353 10 - 7 M (A) or 10 -6 M ( ~ ) . All preparations were bathed in Tyrode solution containing indomethacin (3 × 10 - 6 M), L-serine borate (4.5 x 10 -2 M) and L-cysteine (10 -2 M). Points indicate the means_+ S.E. for n = 8 observations.

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Fig. 5. The effect of LTE 4 on the concentration-response curves to LTC 4 (panel a), LTD 4 (panel b) and histamine (panel c). Concentration-response curves for LTC 4 (0), LTD 4 (rq) or histamine (