Effects of Endothelin Receptor Antagonists on Anterior Chamber Inflammation Induced by Intravitreal Injection of Endothelin-1

Exp. Eye Res. (1999) 69, 437–444 Article No. exer.1999.0721, available online at http :\\www.idealibrary.com on

Effects of Endothelin Receptor Antagonists on Anterior Chamber Inflammation Induced by Intravitreal Injection of Endothelin-1 N O B U Y U K I S H O JI,a, * T E T S U R O O S H I KA,b S H I R O A M A NOb    K A N J I R O M A S U D Ac a

Department of Ophthalmology, University of Kitasato School of Medicine, Kanagawa 228-0055, Japan b Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo 113-8655, Japan and c Department of Ophthalmology, Kanto Rosai Hospital, Kanagawa 211-0021, Japan (Received Cleveland 1 December 1998 and accepted in revised form 9 June 1999) To investigate the role of endothelin receptors (ETA and ETB) in the inflammatory reaction induced by endothelin-1 (ET-1), the time course of changes in aqueous protein concentration (APC) after the intravitreal injection of ET-1 (10−%, 10−& ) was measured using a laser flare-cell meter in pigmented rabbit eyes. The effects of pre-treatment with specific ETA receptor antagonist (97-139) (10−", 10−#, 10−$, 10−% ), specific ETB receptor antagonist (BQ-788) (1n6i10−$ ), and vehicle solution were assessed. The influence of ETA receptor antagonist pre-treatment on aqueous prostaglandin E and leukotriene B # % concentrations was also evaluated. As a result, pre-treatment with ETA receptor antagonist blocked the APC increase induced by 10−%  ET-1 in a dose dependent fashion, while BQ-788 did not suppress the inflammatory reaction. The injection of ET-1 increased aqueous prostaglandin E concentration, which # was inhibited by pre-treatment with ETA receptor antagonist. Aqueous leukotriene B concentration was % not affected by ET-1 nor ETA receptor antagonist. In conclusion, ETA receptor mediates the increases in aqueous protein and prostaglandin E concentration induced by ET-1 injection, and this inflammatory # reaction occurs via the cyclooxygenase pathway of arachidonic acid cascade. # 1999 Academic Press Key words : Aqueous protein concentration ; endothelin-1 ; ETA receptor ; ETB receptor ; laser flare-cell meter ; leukotriene B ; prostaglandin E . % #

1. Introduction Three types of endothelin (ET) receptors have been identified : ETA with high affinity for ET-1 and ET-2 ; ETB having similar affinity for all ET isopeptides ; and ETC with high affinity for ET-3 (Arai et al., 1990 ; Sakurai et al., 1990 ; Hosoda et al., 1991 ; Sakamoto et al., 1991 ; Ogawa et al., 1991 ; Nakamuta et al., 1991 ; Kondoh et al., 1991 ; Karne et al., 1993). Among these, ETA and ETB receptors exist in the iris, ciliary body, choroid, and retina (Osborne et al., 1993 ; El-Mowafy et al., 1994 ; MacCumber et al., 1994). It has been shown that ETA receptor mediates the increase of aqueous prostaglandin E (PGE ) con# # centration after the injection of ET-1 (Sugiyama et al., 1995 ; Abdel-Latif et al., 1996). We previously reported that aqueous ET-1 concentration was raised in endotoxin induced uveitis of rabbit eyes (Shoji et al., 1997). We also found that the ET-1 injection into the eye increased aqueous protein concentration (APC) and these reactions were mediated by the cyclooxygenase pathway of the arachidonic acid cascade (Shoji et al., 1997 ; 1998). These results suggest that the increases of aqueous PGE (Abdel-Latif et al., 1991) # and protein concentration after the injection of ET-1

* Address correspondence to : Nobuyuki Shoji, Department of Ophthalmology, University of Kitasato School of Medicine, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa 228-8555, Japan. The authors have no commercial or proprietary interest in the product or company described in the current article.

0014–4835\99\100437j08 $30.00\0

might be blocked by pre-treatment with ETA receptor antagonist. It has been demonstrated that the intraocular pressure reduction induced by intravitreal injection of ET-1 was suppressed by pre-treatment with ETA receptor antagonist (Sugiyama et al., 1995). In another study, BQ-123, selective ETA receptor antagonist, was used to block the increase of APC and aqueous PGE concentration after the injection of low # doses of ET-1 (Haque et al., 1996). However, the concentration of ET-1 (10−& ) used in their study seems insufficient to induce a significant increase of APC. In our previous study on the ET-1 dosedependent effects on APC (Shoji et al., 1998), the increase of APC at 1 and 24 hr after the intravitreal injection of 10−&  ET-1 was not significant. Thus, it is necessary to investigate the effect of various concentrations of ETA receptor antagonist on the higher concentrations of ET-1. No studies have been available on the role of ETB receptor in the inflammatory reaction induced by ET-1. In this study, we investigated the influence of selective ETA and ETB receptor antagonists on the time course of APC after the injection of ET-1 into the vitreous cavity of pigmented rabbit eyes. Changes in aqueous PGE and leukotriene # B concentration were also assessed. % 2. Materials and Methods Experimental Animals Normal pigmented rabbits of either sex were used (body weight : 1n5–2n5 kg). All of our experimental # 1999 Academic Press

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prepared to 1n6i10−$ , approximately the maximum concentration in the solution. Experimental Procedure

F. 1. Chemical structure of 97-139, o27-O-3-[2-(3carboxy-acryloylamino)-5-hydroxyphenyl]-acryloyloxy myricerone, sodium saltq (Mihara et al., 1993).

After topical anesthesia, 10 µl of ET-1, ETA receptor antagonist, ETB receptor antagonist, or vehicle was injected into the core of the vitreous cavity using a 30gauge microsyringe needle inserted through the sclera at a position 2 mm away from the corneal limbus. Pretreatment with ETA receptor antagonist, ETB receptor antagonist or vehicle (Opeguard2 MA) was performed 1 hr before the injection of ET-1 solution. If the solution leaked from the injection site, the animal was excluded from the subject of this study. Aqueous protein concentration (APC) was measured with the laser flare-cell meter (FC-1000, Kowa, Tokyo, Japan). The obtained photon count was converted to albumin concentration (Oshika et al., 1989). Measurements were taken just prior to and 0n5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 168 hr after the injection of ET-1 or vehicle. Experiment 1 : The Influence of ETA Receptor Antagonist on the Time Course of Aqueous Protein Concentration

F. 2. Chemical structure of BQ-788, N-[N-[N-(2,6Dimethyl-1-piperidinyl) carbonyl]-4-methyl-L-leucyl]-1(methoxycarbonyl)-D-tryptophyl]-D-norleucine monosodium (Ishikawa et al., 1994).

procedures were performed in accordance with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. Before the procedures, rabbits were topically anesthetized with 0n4 % oxybuprocaine hydrochloride ophthalmic solution (Santen Pharmaceutical, Osaka, Japan).

The influence of pre-treatment with ETA receptor antagonist on APC changes after intravitreal ET-1 injection was assessed. The vehicle or ETA receptor antagonist solution was injected into the vitreous cavity 1 hr before the injection of 10−%  ET-1. The concentration of ETA receptor antagonist solution was 10−", 10−#, 10−$ or 10−%  (eight eyes in each group). APC was also measured in eyes receiving vehicle injection 1 hr after the pre-treatment with 10−" or 10−#  ETA receptor antagonist solution or vehicle (four eyes in each group). Measurement results of the ETA receptor antagonist pre-treatment group were compared with those of the vehicle pre-treatment group.

Drug Preparation The nonpeptide ETA receptor-specific antagonist, 97–139 (MW ; 774) (Fig. 1) (Mihara et al., 1994), was obtained from Shionogi pharmaceutical (Osaka, Japan) for this experiment. The peptide ETB receptorspecific antagonist, BQ-788 (MW ; 663n7) (Fig. 2) (Ishikawa et al., 1994 ; Karaki et al., 1994), was used (Research Biochemicals International, MA, U.S.A.) in the second experiment. The intraocular irrigating solution (Opeguard2 MA, Senju Pharmaceutical, Osaka, Japan) was used to dissolve ET-1 (MW ; 2491n9) (Human) (Peptide Institute, Osaka, Japan), ETA receptor antagonist, and ETB receptor antagonist. The ET-1 solution was adjusted to the maximum concentration of ET-1 dissolved in the solution (10−% ) and 10−& . The concentration of ETA receptor antagonist solution (maximum solubility was about 200 mg ml−" in the solution) was 10−", 10−#, 10−$, and 10−% , the ETB receptor antagonist solution was

Experiment 2 : The Influence of ETB Receptor Antagonist on the Time Course of Aqueous Protein Concentration The influence of pre-treatment with ETB receptor antagonist on APC changes after intravitreal ET-1 injection was assessed. The vehicle or ETB receptor antagonist solution was injected into the vitreous cavity 1 hr before the injection of 10−%  or 10−&  ET-1 (six eyes in each group). The concentration of ETB receptor antagonist solution was 1n6i10−$ . Measurement results of the ETB receptor antagonist pre-treatment group were compared with those of the vehicle pre-treatment group. Experiment 3 : Aqueous Prostaglandin E and # Leukotriene B Measurements % In another 136 eyes of 68 rabbits, 200 µl aqueous humor was collected to determine the concentration

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F. 3. Aqueous protein concentration after the pre-treatment with ETA receptor antagonist and subsequent injection of 10−%  ET-1 (eight eyes in each group). $ Pre-treatment with vehiclej10−%  ET-1. # Pre-treatment with 10−%  ETA receptor antagonistj10−%  ET-1. Pre-treatment with 10−$  ETA receptor antagonistj10−%  ET-1. = Pre-treatment with 10−#  ETA receptor antagonistj10−%  ET-1. 7 Pre-treatment with 10−"  ETA receptor antagonistj10−%  ET-1.

of prostaglandin E (PGE ) and leukotriene B (LTB ) # # % % using the radioimmunoassay technique (Powell et al., 1980 ; Lewis et al., 1982 ; Kawano et al., 1987 ; Kurimoto et al., 1990). None of the eyes were used repeatedly. Depending on the combination of pretreatment (vehicle or ETA receptor antagonist) and subsequent intravitreal injection (vehicle or ET-1), as well as the timing of measurements, eyes were randomly divided into the following 17 groups (eight eyes in each group). Group N : normal controls. Group V-V-4, V-V-24 : vehicle (pre-treatment) – vehicle (intravitreal injection) (measurements at 4 or 24 hr after the intravitreal injection). Group A-V-4, A-V-24 : 10−"  ETA receptor antagonist – vehicle (4 or 24 hr). Group V-E-4, V-E-24, V-E-48 : vehicle – 10−%  ET-1 (4, 24, or 48 hr). Group A1-E-4, A1-E-24, A1-E-48 : 10−"  ETA receptor antagonist – 10−%  ET-1 (4, 24, or 48 hr). Group A2-E-4, A2-E-24, A2-E-48 : 10−#  ETA receptor antagonist – 10−%  ET-1 (4, 24, or 48 hr). Group A3-E-4, A3-E-24, A3-E-48 : 10−$  ETA receptor antagonist – 10−%  ET-1 (4, 24, or 48 hr). APC was measured with the laser flare-cell meter just prior to the aqueous humor collection.

ment results in groups V-V-4, V-V-24, A-V-4 and A-V24 were compared with those in group N using the Student’s t-test. Data in groups V-E, A1-E, A2-E and A3-E were compared among the groups using the Kruskal-Wallis test. Specific differences in mean values between groups were tested for significance with the Scheffe multiple comparison test. 3. Results Experiment 1 Aqueous protein concentration (APC) did not change by the pre-treatment with ETA receptor antagonist and subsequent vehicle injection. The time courses of APC in the 10−%  ET-1 groups are shown in Fig. 3. Results of statistical comparison between the vehicle pretreatment group and the ETA receptor antagonist pretreatment groups are given in Table 1. The pretreatment with ETA receptor antagonist suppressed the APC increase induced by ET-1 injection in a dosedependent fashion. APC in the 10−%  ETA receptor antagonist pre-treatment group was significantly lower than that of the vehicle pre-treatment group up to 48 hr after the ET-1 injection. The pre-treatment with 10−$  ETA receptor antagonist blocked the APC increase until 72 hr after the ET-1 injection. The 10−# and 10−"  ETA receptor antagonist pre-treatment completely prevented the APC increase.

Statistical Analysis The results were presented as meanpstandard error. In experiment 1 and 2, the Student’s t-test was used to assess the inhibitory effects of ETA and ETB receptor antagonists. In experiment three, measure-

Experiment 2 The pre-treatment with ETB receptor antagonist did not suppress the APC increase induced by 10−%  (Fig. 4) and 10−&  ET-1 injection (Fig. 5).

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T I Statistical analysis for the inhibitory effect of ETA receptor antagonist on aqueous protein concentration increase after injection of 10−% M ET-1 (n l 8, each) Hours after the injection of 10−%  ET-1 Pre-treatment with 97-139

0n5

1

2

4

6

8

12

24

48

72

168

10−%  10−$  10−#  10−" 

n.s. n.s. n.s. n.s.

* * * *

* * * *

* * * *

* * * *

* * * *

* * * *

* * * *

** * * *

n.s. * * *

n.s. n.s. * *

Mean values were compared with those of vehicle pre-treatment group. * P

0n01, ** P

0n05, n.s. : not significant (Student’s t-test).

F. 4. Aqueous protein concentration after the pre-treatment with ETB receptor antagonist and subsequent injection of 10−%  ET-1 (six eyes in each group). $ Pre-treatment with vehiclej10−%  ET-1. # Pre-treatment with 1n6i10−$  ETB receptor antagonistj10−%  ET-1.

F. 5. Aqueous protein concentration after the pre-treatment with ETB receptor antagonist and subsequent injection of 10−&  ET-1 (six eyes in each group). Pre-treatment with vehiclej10−&  ET-1. Pre-treatment with 1n6i10−$  ETB receptor antagonistj10−&  ET-1.

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T II Aqueous protein, prostaglandin E and leukotriene B concentration # % Pre-treatment Normal controls Group V-V-4 (4 hr*) Group V-V-24 (24 hr) Group A-V-4 (4 hr) Group A-V-24 (24 hr) Group V-E-4 (4 hr) Group V-E-24 (24 hr) Group V-E-48 (48 hr)

none vehicle vehicle 10−"  ETA receptor antagonist 10−"  ETA receptor antagonist vehicle vehicle vehicle

Injection

Protein (mg dl−")

Prostaglandin E # (pg ml−")

Leukotriene B % (pg ml−")

none vehicle vehicle vehicle

44n5p4n2 32n0p3n7 35n4p3n2 51n2p5n3

50n0p3n5 40n3p2n7 55n3p2n9 62n4p5n8

1126n8p86n1 1191n8p37n4 1168n8p63n6 1222n5p85n2

vehicle

48n4p5n1

62n3p5n1

1123n1p81n9

10−%  ET-1 10−%  ET-1 10−%  ET-1

2522n9p148n3** 1892n3p76n9** 5382n2p314n4**

2529n4p123n1** 2271n9p120n1** 3852n5p98n5**

Meanpstandard error. * Hours between injection and measurements. ** Significantly different from normal controls (P t-test).

Experiment 3 In groups V-V (vehicle pre-treatment – vehicle injection) and A-V (10−"  ETA receptor antagonist – vehicle), there were no significant increases in aqueous protein, PGE , and LTB concentrations at all measure# % ment points (Table II). Aqueous protein and PGE # concentrations in group V-E (vehicle – 10−%  ET-1) were significantly higher than those of normal controls, but LTB concentration was not affected % (Table II). The influence of pre-treatment with ETA receptor antagonist is shown in Fig. 4(A)–(C). The pre-treatment with ETA receptor antagonist suppressed the increases of aqueous protein [Fig. 6(A)] and PGE [Fig. 6(B)] # concentration induced by the 10−%  ET-1 injection. These inhibitory effects were dose-dependent and observed on all measurement points. The LTB % concentration was not affected [Fig. 4(C)]. 4. Discussion There are two sub-types of ETA receptor antagonists ; a nonpeptide ETA receptor antagonist, 97-139 ; and a selective peptide ETAreceptor antagonist, BQ-123 and FR139317 (Ihara et al., 1992 ; Sogabe et al., 1993). The 97-139 has been reported to have high affinity and selectivity for the ETA receptor (Mihara et al., 1994). The in vivo potency of 97-139 was similar to that of BQ-123, and the potencies of 97-139 in binding assays and in vitro functional assays were about one order of magnitude higher than those of BQ123. The 97-139 showed also good solubility in aqueous solution, and thus we could make high concentration of 97-139 solution (10−" ) in this study. Recently, another highly specific nonpeptide ETA receptor antagonist (S-0139) (Mihara et al., 1998) has been developed, but its detailed information is not yet available. As for the effective concentration of 97-139, Mihara et al. showed that the 10−(  97-139 solution

1370n0p72n3 1282n5p73n8 1251n9p68n1 0n01, Student’s

completely abolished the increase in the intracellular Ca++ level induced by 10−*  ET-1 (Mihara et al., 1994). Sugiyama et al. demonstrated that 97-139 (10−#  and 10−$ ) significantly blocked the ocular hypotensive effects of ET-1 (10−&  and 10−' ) (Sugiyama et al., 1995). In the current study, the 97139 solution suppressed the APC increase in a dose dependent fashion and 10−"  97-139 completely blocked the inflammatory reaction caused by ET-1. We also investigated the influence of ETB receptor antagonist on the inflammatory induction effects of ET-1. The BQ-788 is a commercially available ETB receptor antagonist and has been reported to be a potent and selective antagonist of ETB receptor with weak antagonistic effect on ETA receptor (Ishikawa et al., 1994 ; Karaki et al., 1994). It has been demonstrated that 10−&  BQ-788 inhibited the contraction of rat aorta induced by ET-1 (up to 10−( ) (in vitro). As shown in the results, 1n6i10−$  BQ-788, which was the highest concentration we could dissolve, did not prevent the increase of APC induced by intravitreal injection of 10−%  and 10−&  ET-1. It has been known that prostaglandin E (PGE ) # # causes breakdown of the blood aqueous barrier (Green et al., 1975 ; Camras et al., 1977) and increases APC (Granstam et al., 1991). We previously reported that aqueous PGE concentration significantly increased # after the intravitreal injection of ET-1 (Shoji et al., 1998). In the current study, we investigated the effects of pre-treatment with ETA receptor antagonist on the time course of changes in PGE concentration after the # ET-1 injection. We first confirmed that vehicle and high concentration of ETB receptor antagonist (10−"  97-139) did not affect APC and PGE concentration, # and that 10−%  ET-1 significantly increased aqueous PGE concentration at 4, 24 and 48 hr after injection # (Table II). We then showed that the pre-treatment with ETA receptor antagonist effectively blocked the increases in aqueous PGE concentration as well as # that of APC in a dose dependent fashion (Fig. 6). These

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(B)

(C)

F. 6. The effect of pre-treatment with ETA receptor antagonist on aqueous protein (A), prostaglandin E (B), and leukotriene # B (C) concentration after the injection of ET-1 (eight eyes in each group). Pre-treatment with vehiclej10−%  ET-1. Pre% − − − treatment with 10 $  ETA receptor antagonistj10 %  ET-1. Pre-treatment with 10 #  ETA receptor antagonistj10−%  ET-1. Pre-treatment with 10−"  ETA receptor antagonistj10−%  ET-1. * P 0n05, ** P 0n01 (Scheffe test).

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results indicate that increases in aqueous PGE and # protein concentration after ET-1 injection are mediated by ETA receptor. The leukotriene B (LTB ) evokes both eosinophils % % and neutrophils chemotaxis and participated in ocular inflammatory reactions. It has been reported that the intracameral injection of LTB increases white blood % cells (Bhattacherjee et al., 1981 ; Stjernschantz et al., 1983 ; Fujiwara et al., 1988), and that LTB mediates % vascular permeability in the presence of a vasodilator such as PGE (Bray et al., 1981). In the current study, # LTB concentration was not affected by either vehicle, % ET-1 or ETA receptor antagonist. According to the current and previous results (Shoji et al., 1997 ; 1998), both the intracameral and the intravitreal injection of ET-1 induce the inflammatory reaction in rabbit eyes, and cyclooxygenase inhibitor, diclofenac sodium, blocks these reactions. Thus, it is indicated that ET-1 plays a role on the upper cascade of the cyclooxygenase pathway, and the lipoxygenase pathway is not involved in this process. It has been reported that ET-1 receptors exist in the iris, ciliary body, choroid, and retina (Osborne et al., 1993 ; El-Mowafy et al., 1994 ; MacCumber et al., 1994), and intravitreal injection of ET-1 caused breakdown of the blood aqueous barrier (BAB) in the ciliary body (Chen et al., 1996). We previously reported that ET-1 injection into the anterior chamber of rabbit eyes induced similar degree of inflammatory reaction as in the current study. Although we did not investigate histological changes in the current study, it appears that the ciliary body is the site where BAB breakdown occurred and the antagonist exerted its effect. In conclusion, ETA receptor mediates the increases in aqueous protein and PGE concentration induced # by ET-1, and this inflammatory reaction occurs via the cyclooxygenase pathway of arachidonic acid cascade. The ETB receptor and the lipoxygenase pathway are not involved in this reaction. References Abdel-Latif, A. A., Zhang, Y. and Yousufzai, S. Y. K. (1991). Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in rabbit iris sphincter smooth muscle : activation of phospholipase A . Curr. Eye Res. # 10, 259–65. Abdel-Latif, A. A., Yousufzai, S. Y. K., El-Mowafy, A. M. and Ye, Z. (1996). Prostaglandins mediate the stimulatory effects of endothelin-1 on cyclic adenosine monophosphate accumulation in ciliary smooth muscle isolated from bovine, cat, and other mammalian species. Invest. Ophthalmol. Vis. Sci. 37, 328–38. Arai, H., Hori, S., Aramori, I., Ohkubo, H. and Nakanishi, S. (1990). Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348, 730–2. Bhattacherjee, P., Hammond, B., Salmon, J. H., Stepney, R. and Eakins, K. E. (1981). Chemotactic response to some arachidonic acid lipoxygenase product in the rabbit eye. European J. Pharmacol. 73, 21–8. Bray, M. A., Cunningham, F. M., Davidson, E. M., Ford-

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Hutchinson, A. W. and Smith, M. J. H. (1981). Leukotriene B : A mediator of vascular permeability. Br. J. % Pharmacol. 72, 483–6. Camras, C. B., Bito, L. Z. and Eakins, K. E. (1977). Reduction of intraocular pressure by prostaglandins applied topically to the eyes of conscious rabbits. Invest. Ophthalmol. Vis. Sci. 16, 1125–34. Chen, H., Yamabayashi, S., Ou, B. and Tsukahara, S. (1996). Morphological changes in rabbit ciliary epithelium and blood-aqueous barriers after intravitreal 10−&  endothelin-1. Exp. Eye Res. 62, 605–12. El-Mowafy, A. M. and Abdel-Latif, A. A. (1994). Characterization of iris sphincter smooth muscle endothelin receptor subtypes which are coupled to cyclic AMP formation and polyphosphoinositide hydrolysis. J. Pharmacol. Exp. Ther. 268, 1343–51. Fujiwara, H., Katayama, T., Nakata, K., Kurozumi, S. and Hazato, A. (1988). Inhibitors of leukotriene B (LTB ) % % and their effects on ocular tissues. J. Eye 5, 143–7. Granstam, E., Wang, L. and Bill, A. (1991). Effects of endothelins (ET-1, ET-2 and ET-3) in the rabbit eye ; role of prostaglandins. European J. Pharmacol. 194, 217–23. Green, K. and Kim, K. (1975). Pattern of ocular response to topical and systemic prostaglandin. Invest. Ophthalmol. 14, 36–40. Haque, M. S. R., Sugiyama, K., Taniguchi, T. and Kitazawa, Y. (1996). Effects of BQ-123, an ETA receptor-selective antagonist, on changes of intraocular pressure, bloodaqueous barrier and aqueous prostaglandin concentrations caused by endothelin-1 in rabbit. Jpn. J. Ophthalmol. 40, 26–32. Hosoda, K., Nakano, K., Arai, H., Suga, S., Ogawa, Y., Mukoyama, M., et al. (1991). Cloning and expression of human endothelin-1 receptor cDNA. FEBS Lett. 287, 23–6. Ihara, M., Noguchi, K., Saeki, T., Fukuroda, T., Tsuchida, S., Kimura, S., et al. (1992). Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor. Life Sci. 50, 247–55. Ishikawa, K., Ihara, M., Noguchi, K., Mase, T. and Mino, N. (1994). Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788. Proc. Natl. Acad. Sci. USA 91, 4892–6. Karaki, H., Sudjarwo, S. A. and Hori, M. (1994). Novel antagonist of endothelin ETB and ETB receptors, BQ" # 788 : Effects on blood vessel and small intestine. Biochem. Biophys. Res. Commun. 205, 168–73. Karne, M., Jayawickreme, C. K. and Lerner, M. R. (1993). Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J. Biol. Chem. 25, 19126–33. Kawano, K., Sugita, M., Oka, M. and Tabata, N. (1987). A sample, rapid and simultaneous extraction of thromboxane B , 6-Ket-prostaglandin F α, and prostaglandin # " E . Jpn. J. Inflammation 7, 511–5. # Kondoh, M., Miyazaki, H., Uchiyama, Y., Yanagisawa, Y., Masaki, T. and Murakami, K. (1991). Solubilization of two types of endothelin receptor, ETA and ETB from rat lung with retention of binding activity. Biomed. Res. 12, 417–23. Kurimoto, F. (1990). Leukotriene. Nippon Rinsho 48 (suppl.), 213–6. Lewis, R. A., Mencia-Huerta, J. M., Soberman, R. J., Hoover, D., Marfat, A., Corey, E. J. and Austen, K. F. (1982). Radioimmunoassay for leukotriene B . Proc. Natl. Acad. % Sci. USA 79, 7904–08. MacCumber, M. W. and Salvatore, A. D. (1994). Endothelin receptor-binding subtypes in the human retina and choroid. Arch. Ophthalmol. 112, 1231–5.

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