Identification of Endothelin Receptor Subtypes in Rat Ciliary Body Using Subtype-Selective Ligands

Exp. Eye Res. (1998) 66, 69–79

Identification of Endothelin Receptor Subtypes in Rat Ciliary Body Using Subtype-Selective Ligands A I N H O A R I P O D AS, J O S E A. D E J U A N, F. J O S E M O Y A, A R T U R O F E R N A N D E Z-C R U Z    R A Q U E L F E R N A N D E Z-D U R A N G O* Diabetes, Hypertension, and Obesity Unit, Department of Internal Medicine, Hospital Universitario San Carlos, 28040 Madrid, Spain (Received Lund 12 May 1997 and accepted in revised form 2 September 1997 ) The endothelins are important vasoactive ocular peptides and there is some evidence that they may modulate intraocular pressure. We investigated the existence and localization of endothelin receptor subtypes using subtype selective ligands in rat ciliary body. Scatchard transformation of saturation binding experiments revealed that the KD and Bmax for ["#&I]ET-1 and ["#&I]ET-3 to membranes from ciliary body were 41±7³9 pM and 236³20 fmol mg−" protein and 37±8³0±4 pM and 160³2±0 fmol mg−" protein, respectively. Competitive experiments in the presence of cyclic pentapeptide BQ123 (selective for ETA receptors) and BQ3020 (selective for ETB receptors), demonstrated the existence of ETA and ETB receptors in a ratio of 35 : 65. Cross-linking of ["#&I]ET-1 and ["#&I]ET-3 to ciliary body membranes resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa, suggesting that ETA and ETB receptors have similar molecular mass. The 34 Kda band is a proteolytic degradation product of the 52 Kda band. Autoradiographic results show that specific ["#&I]ET-1 binding sites, displaced by BQ123 and BQ3020, are localized to the ciliary epithelium, supporting the idea that ETA and ETB subtype receptors exist in this tissue. # 1998 Academic Press Limited Key words : endothelin ; receptors ; ciliary body.

1. Introduction Endothelin-1 (ET-1) was originally isolated and identified from the culture medium of porcine aortic endothelial cells (Yanagisawa et al., 1988). Following the discovery of ET-1, several related peptides have also been identified which include ET-2 and ET-3. Genomic DNA analysis revealed the existence of three distinct genes, encoding the three isopeptides (Inoue et al., 1989). These three isoforms are widely distributed and mediate their biological actions by interacting with at least two receptors, named ETA and ETB, which have been cloned and well characterized (Sakurai et al., 1990 ; Arai et al., 1990). The endothelin A subtype (ETA) receptor which is distributed and preferentially expressed in vascular smooth muscle cells mediates potent vasoconstrictor actions and binds preferably the ET-1 isoform (Masaki et al., 1991). The endothelin B subtype (ETB) receptor is preferably expressed in vascular endothelial cells and binds equipotently all three ET isoforms, ET-1, ET-2 and ET-3. The ETB receptor is thought to mediate vasodilation through the release of nitric oxide and prostaglandins (Inoue et al., 1989 ; Masaki et al., 1991). A third ET receptor, designated ETC, cloned from amphibian dermal melanophores has a greater affinity for ET-3 than for ET-1 and ET-2 (Karne, Jayawickreme and Lesnez, 1993). Several subtype selective peptides are valuable tools in identifying the * Experimental Research Unit, Hospital Universitario San Carlos, 28040 Madrid, Spain.

0014–4835}98}010069­11 $25.00}0}ey970405

subtype of ET receptors in various tissues. Sarofotoxin 6c (S6c) and the BQ3020 are selective for ETB receptors (Williams et al., 1991a ; Karet, Kuc and Davenport, 1993). The cyclic pentapeptide BQ123 is a highly selective ETA antagonist (Ihara et al., 1992). Using both of these subtype-selective ligands, we demonstrated the presence of ETA and ETB receptor subtypes in rat retina (De Juan et al., 1995). In the eye, endothelin-1-like immunoreactivity is present in the iris-ciliary body, choroid, retina (MacCumber, Jampel and Snyder, 1991 ; Chakravarthy et al., 1994 ; De Juan et al., 1993), aqueous humor (Chakravarthy et al., 1994) and ciliary epithelium (Eichorn and Lutjen-Drecoll, 1993). Endothelin-3-like immunoreactivity is present in the retina (De Juan et al., 1995), ET-mRNA has been found in the rat iris (MacCumber, Ross and Snyder, 1990). ET-1 is a potent constrictor of iris sphincter of different mammalian species (Geppeti et al., 1989 ; Abdel-Latif, Zhang and Yourufzai, 1991 ; Osborne and Barnet, 1992). The presence of the ETA and ETB receptor subtypes was demonstrated in the rabbit iris sphincter (El-Mowafy et al., 1994). In this tissue, the ETA receptor subtype is coupled to the stimulation of phosphoinositide hydrolysis, and the ETB subtype to the stimulation of cAMP accumulation (El-Mowafy et al., 1994). In vivo, intracameral injection of ET-1 into rabbit eyes (Granstam, Wang and Bill, 1991) and cat eyes (Granstam, Wang and Bill, 1992) causes an indomethacin-sensitive breakdown of the blood-aqueous barrier, a rise in intraocular pressure (IOP), a # 1998 Academic Press Limited

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vasodilation in the anterior uvea, and an increase in the concentration of protein and prostaglandin E # (PGE ) in the aqueous humor. However, intravitreous # (i.v.) injection of ET-1 and ET-3 produced a reduction of IOP by 46 % and 45 % respectively, and did not return to normal for at least 5 days (MacCumber et al., 1991). Sugiyama et al. (1995) reported that i.v. injection of ET-1 produced an initial rise of IOP and a subsequent reduction lasting for more than 96 hours. They suggested that the initial ET-1-induced IOP increase is mediated by cycloxygenase products primarily through the stimulation of ETA receptor. In contrast, the prolonged decrease in IOP is mediated by ETB receptors and, in part, by ETA receptors, without involvement of cycloxygenase products. The precise mechanism of the IOP-lowering effect of endothelins is not yet elucidated. ET-1 induced contraction in bovine ciliary muscle and trabecular meshwork (Lepple-Wienhues et al., 1991). In studies with the perfused monkey eye, ET-1 increases outflow facility, probably by a direct effect on the ciliary muscle (Erickson-Lamy et al., 1991). Tanaguchi et al. (1994) also observed that i.v. injection of ET-1 in the rabbit eye produced an increase of outflow facility and a decrease of aqueous humor formation. It has been demonstrated that in the ciliary muscle, isolated from different mammalian species, ET-1 binds to ETA receptor subtype to activate phospholipase A and to # release arachidonic acid. The latter is then converted into prostaglandins (PG) which stimulate adenylyl cyclase (Abdel-Latif et al., 1996). In cultured human ciliary muscle cells, ET-1 activated phopholipase C, calcium mobilization, PGE , and cAMP production by # the ETA receptor subtype (Matsumoto et al., 1996). However, it is not clear the precise role that PG release and cAMP accumulation induced by ET-1 in ciliary muscle plays in the lowering of IOP. On the other hand, in the iris-ciliary processes, Osborne, Barnett and Luttmann (1993) demonstrated that endothelins increase the production of inositol phosphates and, using the polymerase chain reaction (PCR), they only found the ETB receptor subtype. However, they indicated the possible existence of another type of ET receptor since ET-1 reduces the forskolin elevated cAMP in these tissues. The present study was undertaken to identify and characterize the subtypes of ET receptors in rat ciliary body using subtype-selective ligands, such as S6c, BQ3020 and BQ123, and to localize the ET receptors by microautoradiography. These studies could throw more light on the mechanism of ET-1-lowering IOP.

2. Materials and Methods Materials Human}porcine endothelin ET-1, ET-3, BQ123 and S6c were obtained from Peninsula Laboratories Inc. (Merseyside, U.K.). BQ3020 was purchased

A. R I P O D A S E T A L.

from Neosystem Laboratoire (Strasbourg, France). ["#&I]ET-1 and ["#&I]ET-3 (2000 ci mmol−") and the LM1 photographic emulsion for autoradiography were obtained from Amersham International (Buckinghamshire, U.K.). Dissuccinimidyl suberate (DSS) and Coomassie blue were purchased from Pierce Chemical Co. (Rockford, U.S.A.). Standard proteins for electrophoresis (SDS-PAGE standards LMW) were from BioRad (Richmond, U.S.A.). Whatman GF}G filters were from Whatman International Ltd. (Maidstone, U.K.). Aprotinin, Phenylmethylsulfonylfluoride (PMSF), EDTA, pepstatin, poly--lysina, paraformaldehyde, and glutaraldehyde were obtained from Sigma Chemical Co. (St Louis, U.S.A.). The Sep-Pak C-18 cartridges were from Water Associates (Milford, U.S.A.). Developer D19 was purchased from Kodak and fixing liquid was purchased from Ilford limited (Cheshire, U.K.).

Preparation of Ciliary Body Particulate Preparations Adult male Wistar rats (200–250 g) (n ¯ 150) were used in all experiments. The animals were kept in a temperature-controlled room on a 12-hr light}dark cycle and fed ad libitum. The eyes of the rats were removed immediately after decapitation. The ciliary bodies were removed, free of vitreous humor and retina. Ciliary body particulate preparations were prepared as previously described (Ferna! ndez-Durango et al., 1991). The ciliary bodies were homogenized in 5 m Tris–HCl buffer pH 7±4, containing 0±32  sucrose, 0±5 m PMSF, 0±2 m pepstatin, and 0±8 m aprotinin, then centrifuged at 40±000 g for 30 min. The pellets were resuspended in 50 m Tris–HCl buffer pH 7±4. The protein concentration was estimated by the method of Lowry et al. (1951). Tissue preparations were stored in aliquots at ®70°C and used within 3 months.

ET Binding Assay Ciliary body particulate preparations (10–25 µg of protein) were incubated for 240 min at 25°C in 300 µl (final volume) of 50 m Tris–HCl buffer pH 7±4 containing 0±5 m PMSF, 0±2 m pepstatin, 0±1 % BSA, and 0±03 % bacitracin. In the competitive experiments, the binding assay was performed with 10–25 pM of ["#&I]ET-1 or ["#&I]ET-3 and varying concentrations of unlabeled ET-1, or ET-3 (10−"$ to 10−) ). Increasing concentrations of labeled ET-1 or ET-3 (0±2–100 p) were used in the saturation experiments. The specific binding of ["#&I]ET-1 was calculated by subtracting non specific binding (["#&I]ET-1 bound in the presence of 0±1 µ ET-1) from total ["#&I]ET-1 binding (in the absence of unlabeled ET-1). The binding reaction was terminated by diluting the reaction mixture with 1 ml of 50 m Tris–HCl buffer pH 7±4, followed by rapid vacuum filtration through Whatman GF}C filters

ENDOTHELIN RECEPTORS IN RAT CILIARY BODY

previously soaked for 1 hr in 0±3 % vol}vol polyethylenimine solution. Each filter was washed three times with 3 ml of 50 m Tris–HCl buffer, dried, and removed for counting on a LKB gamma-counter with 75 % efficiency. Several unrelated peptides, including somatostatin, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), thyroid stimulating hormone (TSH), growth hormone (GH), or insulin, at the concentration of 1 m, were testing to inhibit binding of ["#&I]ET-1 at the same conditions as the competitive experiments. Association binding assay. Ciliary body tissue at a total of 20 µg of protein was incubated with 10 p ["#&I]ET-1 at 25°C. At selected times, the mixture was rapidly filtered through Whatman GF}C filter. Specific binding was calculated as the difference in binding in presence and absence of 0±1 µ ET-1. Dissociation binding assay. Rat ciliary body particulate preparations were incubated with 10 p of ["#&I]ET-1 for 240 min at 25°C. At the end of the binding incubations, the initial amount bound was determinated by filtration. At zero time, 1 µ unlabeled ET-1 were added to the incubation medium. Then, at selected times, aliquots of the mixture were rapidly filtered. Nonspecific binding was determined as described above. Affinity cross-linking studies. The labeled peptide, 38 p, was incubated with ciliary body particulate preparations (60 µg of protein) in a total volume of 300 µl of 10 m Hepes buffer pH 7±4, containing 0±5 m PMSF, 0±2 m pepstatin, 0±1 % BSA, and 0±03 % bacitracin, for 240 min at 25°C. The reaction was terminated by centrifugation at 40 000 g for 20 min at 4°C. The pellets were resuspended in 10 m Hepes buffer pH 7±4 and ["#&I]ET-1 or ["#&I]ET-3 were cross-linked on receptors with 0±5 m DSS. Reaction was quenched 15 min later by ammonium acetate 2 . Specificity of the binding was determined by the addition of 0±1 µ ET-1, ET-3, S6c or BQ123 in incubation mixture. Samples were denatured under reducing (2 % β-mercaptoethanol) and non-reducing conditions, before the analysis on polyacrylamide gel electrophoresis.

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Microautoradiography of ["#&I]ET-1 binding. The eyes of the rats, immediately removed after decapitation, were immersed in 2-methylbutane on liquid nitrogen. Twelve-micrometer sections were cut in a cryostat at ®28°C, thaw-mounted onto poly--lysine treated slides, and dried for 4 hr in a desiccator under vacuum at 4°C. The sections were then preincubated in 50 m Tris–HCl buffer pH 7±4 containing 50 m NaCl, 0±5 m PMSF, 2 m pepstatin, 10 000 U.I.C. aprotinin, and 0±1 % BSA for 15 min at 25°C. Sections were then incubated with 35 p ["#&I]ET-1 in the same buffer for 60 min at room temperature. Specific binding was determined by displacing the radioligand with 0±1 µ of ET-1, ET-3, BQ3020 or with 1 µ of BQ123. After incubation, the slides containing the sections were washed three times with fresh buffer for 20 min, rinsed in fresh bidistillate water and fixed in 4 % paraformaldehyde and 1 % glutaraldehyde in phosphate buffer 100 m for 4 hr. The sections were covered with LM1 Amersham emulsion, stored in the dark at 4°C for 10 days and developed with Kodak D19 developer. Slides were counterstained with toluidine and photographed with both light and dark-field microscopy. Data Analysis The binding data for the determination of the density and affinity of binding sites were evaluated by computer-assisted non-linear regression analysis with the LIGAND program (Munson and Rodbard, 1980) after preliminary treatment of data with the EBDA program (MacPherson, 1985). The inhibition constant (ki) was calculated according to the method of Cheng and Prussof (1973). The values are presented as the mean³... values. The curves were designed using the computer program Sigma Plot Scientific Graphing System (version 4.10) by Jandel Corporation. 3. Results ["#&I]ET-1 binding to rat ciliary body membranes was linear in the range of 5–50 µg of protein and was also specific since it could be displaced by the addition of 0±1 µ ET-1, ET-3, S6c, BQ3020 or 1 µ BQ123, but not by unrelated hormones. Specific binding relative to the total binding was greater than 85 % for the labeled hormones.

Sodiumdodecylsulfate (SDS)-polyacrylamide Gel Electrophoresis

Saturation Studies

Gel electrophoresis was performed according to the method of Laemmli (1970), using 10 % SDS-polyacrylamide gels. Samples were run for 1 hr 45 min at 125 V. After electrophoresis, proteins were visualized by Coomassie blue staining, the gels were then dried and exposed to a X-OMAT RP6 Kodak film with intensifying screen at ®70°C. Developed autoradiograms were analysed on a Pharmacia LKB Image Master densitometer.

Specific ["#&I]ET-1 and ["#&I]ET-3 binding to rat ciliary body membranes were saturable in a concentration-dependent manner. Scatchard analysis of saturation binding data of ["#&I]ET-1 ad ["#&I]ET-3 indicated the presence of a single class of high-affinity binding sites. KD and Bmax values evaluated from ["#&I]ET-1 saturation binding data by non-linear regression analysis were 41±7³9 p and 236³20 fmol mg−" of protein (n ¯ 4), respectively

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A. R I P O D A S E T A L.

F. 1. Scartchard plots of saturable binding of ["#&I]ET-1 and ["#&I]ET-3 to rat ciliary body. Inset : Saturation curves. The linear plots obtained are compatible with a one-site model. Data points are means of duplicate measurements in a representative experiment. (A) : The KD and Bmax values (mean³...) obtained using ["#&I]ET-1 from four independent experiments performed in duplicated were 41±7³9±02 p and 236³20 fmol mg−" of protein, respectively. (B) : And the KD and Bmax values (mean³...) obtained using ["#&I]ET-3 from three independent experiments performed in duplicated were 37±8³0±4 p and 160³2±0 fmol mg−" of protein, respectively.

F. 2. Association of ["#&I]ET-1 binding to rat ciliary body particulate preparations. Data points are means of duplicate measurements in a representative experiment. Similar values were obtained in three independent experiments performed in duplicate.

F. 3. Dissociation of ["#&I]ET-1 binding to rat ciliary body particulate preparations. Data points are means of duplicate measurements in a representative experiment. Similar values were obtained in three independent experiments performed in duplicate.

[Fig. 1(A)] and Hill coefficient was 0±95³0±03 (n ¯ 4). KD and Bmax values evaluated from ["#&I]ET-3 saturation binding data by the same method were 37±8³0±4 p and 160³2±0 fmol mg−" of protein (n ¯ 3), respectively [Fig. 1(B)] and Hill coefficient was 0±99³0±01 (n ¯ 3). ET receptors were saturated at approximately a ligand concentration of 80–90 p for both labeled hormones.

by 200–240 min and remaining constant through 280 min (Fig. 2). In dissociation studies, at 25°C unlabeled ET-1 caused only a small displacement of specifically bound ["#&I]ET-1, with more than 90 % binding remaining even after 300 min (Fig. 3).

Kinetic Analysis Kinetic analysis showed that the association of ["#&I]ET-1 was time dependent, reaching steady state

Competition Studies Competitive binding experiments were performed using a concentration of 10 p of ["#&I]ET-1 or ["#&I]ET3 as labeled hormones (Fig. 4, Fig. 5 and Table I). Unlabeled ET-1, ET-3, S6c and BQ123 competed for specific ["#&I]-ET-1 binding sites in rat ciliary body

ENDOTHELIN RECEPTORS IN RAT CILIARY BODY

F. 4. Competitive inhibition of ["#&I]ET-1 binding to membranes from rat ciliary body particulate preparations : by unlabeled ET-1 (D), ET-3 (E), BQ123 (y), S6c (x) and BQ3020 (*). The specific binding of ["#&I]ET-1 in absence of competitor was normalized to 100 % (Bo). Nonspecific binding was calculated as ["#&I]ET-1 binding in presence of 0±1 µ ET-1 in all cases (0 % of specific binding). The results (B}Bo) are expressed as the percentage of ["#&I]ET-1 binding in the presence of competitor. Data points are means of duplicate measurements in a representative experiments. Similar values were obtained in three independent experiments performed in duplicate.

F. 5. Competition for ["#&I]ET-3 binding sites in rat ciliary body particulate preparations by unlabeled ET-1 (D), ET-3 (E), S6c (x) and BQ3020 (*). The specific binding of ["#&I]ET-1 in absence of competitor was normalized to 100 % (Bo). Nonspecific binding was calculated as ["#&I]ET-3 binding in presence of 0±1 µ ET-3 in all cases (0 % of specific binding). The results (B}Bo) are expressed as the percentage of ["#&I]ET-1 binding in the presence of competitor. Data points represent means of duplicate measurements in a representative experiment. Similar values were obtained in three independent experiments performed in duplicate.

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membranes in a dose-dependent manner (Fig. 4). The competition profiles of ["#&I]ET-1 binding sites by ET-1, S6c, and BQ123 were monophasic and Hill coefficient values were not different from unity, however ET-3 displaced ["#&I]ET-1 in a biphasic manner. Analysis of the competition curves showed that ET-3 competed by ["#&I]ET-1 binding sites with high affinity (Ki ¯ 176³30 p ; Table I) and low affinity (Ki ¯ 950³120 p ; Table I). These results suggested the presence of ET receptor subtypes in this tissue. On the other hand, S6c at doses higher than 0±1 µ was able to displace only the 82 % of the specific ["#&I]ET-3 binding (Fig. 5). For this reason, we tested the ETBligand BQ3020, linear truncated peptide analogue of ET-1 (Karet et al., 1993). This analogue competed for specific ["#&I]ET-1 binding sites in ciliary body membranes in a dose-dependent manner (Fig. 4) and displaced 100 % of the specific ["#&I]ET-3 binding at doses of 0±1 µ (Fig. 5). Analysis of the competition curves showed that BQ3020 competed for approximately 65 % of the ["#&I]ET-1 specific binding sites, whereas S6c competed for only 35 % the ["#&I]ET-1 specific binding sites. In addition BQ3020 competed by ["#&I]ET-1 with higher affinity (Ki ¯ 758³130 p) than S6c (Ki ¯ 2921³230 p) (Fig. 4 ; Table I). On the other hand, BQ123 competed for approximately 35 % of the ["#&I]ET-1 binding sites present in the ciliary body (Fig. 4). In order to better quantify the proportion of ETA and ETB subtypes, we designed the following experiments. Competition experiments were performed in the presence of high concentration of BQ3020 (0±1 µ) to block all ETB receptors or BQ123 (1 µ) to block all ETA receptors (Fig. 6). The presence of 0±1 µ BQ3020 produces the following changes in comparison with their absence : (1) a decrease of ["#&I]ET1 binding by 65 %, (2) a shift to the right of the ET-3 curve (Ki ¯ 910³116 p ; Table I), and (3) BQ123 displaced 100 % of ["#&I]ET-1 binding [Fig. 6(A) ; Table I]. These results suggest that when ETB receptors are blocked by BQ3020, ET-3 binds to ETA receptors with low affinity. On the other hand, the addition of 1 µ BQ123 produces : (1) a decrease of ["#&I]ET-1 binding by 35 %, (2) a shift to the left of the ET-3 curve (Ki ¯ 145³22 p), and (3) BQ3020 displaced 100 % of ["#&I]ET-1 binding [Fig. 6(B) ; Table I]. These results suggest that when ETA receptors are blocked by BQ123, ET-1 and ET-3 bind to ETB receptors with similar affinity.

Cross-linking ["#&I]ET-1 and ["#&]ET-3 (38 p) were used to identify the apparent molecular weight of the ET receptors in rat ciliary body membranes. Affinity labeling of these membranes by cross-linking with ["#&I]ET-1 [Fig. 7(A)] and ["#&I]ET-3 [Fig. 7(B)], using disuccinimidyl suberate (DSS), indicated the presence of two specific

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A. R I P O D A S E T A L.

T I Ki ( pM) values for ET-1, ET-3, S6c, BQ123 and BQ3020 in competing for ["#&I ]ET-1 and ["#&I ]ET-3 binding to rat ciliary body membranes Conditions ["#&I]ET-1 Without saturating

In the presence of 1 µ de BQ123 In the presence of 0±1 µ de BQ3020 ["#&I]ET-3 Without saturating

Unlabeled ligands

Ki values

ET-1 ET-3 BQ123 S6c BQ3020

121³25 176³19 1191³200 2921³230 756³130

ET-1 ET-3 BQ3020

134³15 145³22 1100³300

ET-1 ET-3 BQ123

320³50 910³160 1460³156

ET-1 ET-3 S6c BQ3020

115³15 120³20 2650³243 1060³163

950³120

Binding experiments were performed as described in Material and Methods. The data presented are the mean³... of at least three determinations performed in duplicate. The Ki was calculated according to the formula of Cheng and Prussof (1973).

bands with molecular weights of 52 and 34 kDa. The labeling of these binding proteins was specifically inhibited by the presence of an excess nonradioactive ET-1 or ET-3 (0±1 µ) indicating that the labeling of the two bands was specific. When 0±1 µ S6c or 1 µ BQ123 were used as competitor for binding sites, the labeling patterns obtained with ["#&I]ET-1 was different from those obtained with ["#&I]ET-3. In the labeling of ["#&I]ET-1 binding, S6c reduced the intensity of the 52 kDa band by 31 % and the intensity of the 34 kDa band by 28 %, however BQ123 reduced the 52 kDa and 34 kDa band by 18 % and 15 %, respectively (as calculated from densitometric analysis the autoradiogram). When ["#&I]ET-3 was used as the labeled hormone, S6c completely abolished the bands and BQ123 did not affect the labeling. Similar results were obtained under reducing conditions (2 % βmercaptoethanol, data not shown). Microautoradiography A significant amount of the ["#&I]ET-1 specific binding was observed in the rat ciliary body. The silver grains are localized on the epithelium and stroma. These results are shown in Fig. 8(A) (total binding) and Fig. 8(C) (non-specific binding). Unlabeled ET-1 displaced almost completely the radioactive ET1. The ETA ligand BQ123 and the ETB ligand BQ3020 displaced radioactive ET-1 binding. These results demonstrated that the ["#&I]ET-1 binding in rat ciliary body is specific and that ETA and ETB receptor subtypes are present in this tissue.

4. Discussion This study demonstrates for the first time, the existence of the two endothelin receptor subtypes in the rat ciliary body and localizes autoradiographically the two subtype receptors in this tissue. The ["#&I]ET1 binding to ciliary body particulate preparations exhibits a very slow kinetic dissociation at 25°C in the presence of a large excess of cold ligand. This finding was observed in many other tissue types (Kanse, Ghatei and Bloom, 1989 ; Fischli, Clozel and Guilly, 1989 ; De Juan et al., 1993) and might reflect the peculiar long-lasting effect of ET-1 in vivo. Scatchard analysis derived from saturation binding data using ["#&I]ET-1 revealed the presence of a single class of ET binding sites with a KD of 41±7³9±02 p and a Bmax of 236³20 fmol mg−" protein. The KD value calculated from ["#&I]ET-3 saturation binding experiments was similar to that obtained for ["#&I]ET1, however the Bmax was approximately 65 % of that obtained for ["#&I]ET-1. These data suggest the presence of more than one ET receptor subtype in ciliary body although detection of two sites in the saturation curves using ["#&I]ET-1, over the concentration-range used, was not achieved. The KD values obtained from ["#&I]ET-1 and ["#&I]ET-3 saturation binding experiments were similar to those obtained in human cerebral cortex (Ferna! ndez-Durango et al., 1994) and in rat retina (de Juan et al., 1995). To identify the subtypes of ET receptors present in rat ciliary body membranes we used S6c and BQ3020 as selective ETB ligands and BQ123 as selective ETA

ENDOTHELIN RECEPTORS IN RAT CILIARY BODY

F. 6. Competitive inhibition of ["#&I]ET-1 binding to membranes from rat ciliary body. The specific binding of ["#&I]ET-1 in absence of competitor was normalized to 100 % (Bo). Nonspecific binding was calculated as ["#&I]ET-1 binding in the presence of 0±1 µ ET-1 in all cases (0 % of specific binding). The results (B}Bo) are expressed as the percentage of ["#&I]ET-1 binding in the presence of competitor. (A) Competition by unlabeled ET-1 (D), ET-3 (E), and BQ123 (y) for ["#&I]ET-1 binding sites in rat ciliary body membranes in the presence of 0±1 µ BQ3020. (B) Competition by unlabeled ET-1 (D), ET-3 (E), and BQ3020 (*) for ["#&I]ET-1 binding sites in rat ciliary body membranes in the presence of 1 µ BQ123. Data points represent means of duplicate measurements in a representative experiment. Similar values were obtained in three independent experiments performed in duplicate.

ligand, respectively. Analysis of the competition binding experiments showed that this tissue has a single class of high affinity binding sites for ET-1 and two different affinity sites for ET-3. We demonstrated that ET-1 binds to ETA and ETB receptor with similar

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F. 7. Autoradiogram of ["#&I]ET-1 and ["#&I]ET-3 crosslinked to their receptors on rat ciliary body. (A) Membranes were incubated with ["#&I]ET-1 in the absence (lane 1) or presence of 0±1 µ ET-1 (lane 2), 0±1 µ ET-3 (lane 3), 0±1 µ S6c (lane 4), 1 µ BQ123 (lane 5). (B) Membranes were incubated with ["#&I]ET-3 in the absence (lane 1) or presence of 0±1 µ ET-3 (lane 3), 0±1 µ ET-1 (lane 2), 0±1 µ S6c (lane 4), 1 µ BQ123 (lane 5). Similar results were found under reducing conditions (data not shown).

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A. R I P O D A S E T A L.

A

B

C

D

E

F

G

H

F. 8. Autoradiographical localization of specific ["#&I]ET-1 binding sites in rat ciliary body. Dark-field photomicrographs showing silver grains and their correspondent bright-field photomicrographs of rat ciliary body sections stained with toluidine blue. Cryostat sections were incubated with radioactive ET-1 in the absence (A, B) or in the presence of either 0±1 µ unlabeled ET-1 (C, D), 1 µ unlabeled BQ123 (E, F), or 0±1 µ unlabeled BQ3020 (G, H). It can be seen that the excess unlabeled ET1 displaced almost completely the radioactive binding of ET-1. Both ETA and ETB ligands displaced ["#&I]ET-1 specific binding in rat ciliary body.

ENDOTHELIN RECEPTORS IN RAT CILIARY BODY

affinity, whereas ET-3 binds to ETB receptor with higher affinity than to ETA receptor. On the other hand, the competition binding experiments showed that S6c, at doses of 0±1 µ, is not able to displace the total binding of ["#&I]ET-3 to ciliary body membranes, however BQ3020 displaced 100 % of the binding. In addition, BQ3020 competed for ["#&I]ET-3 with higher affinity than S6c. These results suggest that this linear truncated peptide analogue of ET-1, is a highly selective ETB-ligand and more suitable valuable tool than S6c in identifying the ETB receptor subtype in rat ciliary body. It has been shown that BQ3020 also binds selectively to ETB receptors in human kidney (Karet, Kuc and Davenport, 1993), human heart (Molenaar et al., 1993), in the media from aorta and coronary arteries (Davenport et al., 1993) and in canine lung (Nambi et al., 1995). However, this ETB-ligand competes with low affinity in canine spleen (Nambi et al., 1995) and in porcine and rat heart (Peter et al., 1996). These discrepancies in the binding affinity for BQ3020, in addition with data from functional experiments (Shetty et al., 1993 ; Gardiner et al., 1994), have led to the suggestion that ETB receptors may be modified according to the tissues in which they are expressed within the same animal or between different species (Nambi et al., 1995 ; Peter and Davenport, 1996). The binding affinity that we obtained in rat ciliary body for BQ3020 is in the nanomolar region and similar to that obtained in the tissues with high affinity for this ligand. These results suggest that the ETB receptor present in all these tissues might belong to the same subtype of ETB receptors and differs from those present in canine spleen (Nambi et al., 1995) and in rat and pig heart (Peter and Davenport, 1996). On the other hand, our binding studies demonstrated that BQ123 is highly selective for the ETA subtype in rat ciliary body. The affinity of BQ123 for the ETA receptor in this tissue is in the nanomolar region, similar to the affinity reported for a number of tissues including cardiac muscle (Molenaar et al., 1993), kidney (Karet, Kuc and Davenport, 1993), rat retina (de Juan et al., 1995) and rat heart (Peter and Davenport, 1996). The utilization of BQ123 and BQ3020 allowed us to demonstrate the existence in rat ciliary body of ETA and ETB receptor subtypes in a ratio of 35 : 65, respectively. In agreement with our results, ETB receptors are more abundant than ETA receptors in rat and human hippocampus (Williams et al., 1991a ; 1991b), human kidneys (Karet, Kuc and Davenport, 1993) and in rat retina (de Juan et al., 1995). In contrast, ETA predominates in other tissues such as left ventricle (Molenaar et al., 1993), human coronary artery and aorta (Bacon and Davenport, 1996), smooth muscle in the human vasculature (Davenport, O’Reilly and Kuc, 1995) and rat cardiac tissues (Peter and Davenport, 1996). Affinity labeling of rat ciliary body membranes with

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["#&I]ET-1 and ["#&I]ET-3 indicated, in both cases, the presence of two specific bands with approximated molecular weights of 52 and 34 kDa. While 52 kDa band represents the intact binding polypeptide, the 34 kDa band is a proteolytic degradation product of the 52 kDa protein (Schwartz, Ittoop and Hazum, 1991). The fact that S6c reduced the labeling of the two bands by ["#&I]ET-1, suggests the existence of ETB receptor and at least another type of ET receptor. Together with this, BQ123 diminished the intensity of 52 kDa and 34 kDa bands indicating the presence of ETA receptor. On the other hand, the labeling with ["#&I]ET-3 was totally inhibited by the presence of S6c, whereas BQ123 did not affect the labeling. These results suggest that ["#&I]ET-1 binds to the two receptor subtypes, while ["#&I]ET-3, at the concentrations used, binds only to the ETB receptor, in good accordance with the data obtained from the competitive experiments described above and with the data about the molecular weight of the receptors obtained by cloning (Arai et al., 1990 ; Sakurai et al., 1990). In agreement with the binding studies, our cross-linking experiment results also revealed the presence of the two subtypes of ET receptor in rat ciliary body. Autoradiographic data showed specific binding of ["#&I]ET-1 in the rat ciliary body, although do not indicate the exact cellular localization of the endothelin receptors i.e. whether being associated with pigmented or non pigmented epithelia and}or stroma. The finding that BQ123 and BQ3020 displaced the specific binding of ["#&I]ET-1, supports the fact that ETA and ETB subtype receptors are present in this tissue. In contrast with our results, Osborne et al. (1993), using the (PCR), only detected the ETB mRNA subtype receptor in iris-ciliary body. However, as they found that ET-1 was about three times more effective than ET-3 in stimulating inositol phosphates and that ET-1 reduced the forskolin elevated cAMP levels in the irisciliary processes, they suggested that more than one type of ET receptor is present in these tissues. From a physiological point of view, there is evidence linking endothelins to the control of IOP (Granstam et al., 1991, 1992 ; MacCumber et al., 1991 ; Taniguchi et al., 1994 ; Sugiyama et al., 1995). Interestingly, endothelin immunoreactivity has been found in the ciliary epithelium and aqueous humor (MacCumber et al., 1991 ; Chakravarthy et al., 1994) and it has been reported that endothelins inhibit forskolin-stimulated cAMP production in the rabbit (Osborne et al., 1993 ; Bausher, 1995a) and in human ciliary processes (Bausher and Horio 1995b), suggesting the existence of endothelin receptors linked to inhibition of cAMP levels in ciliary processes. As it is well known that cAMP generated by the ciliary epithelium plays an important role in the regulation of aqueous formation, all the above cited data together with our findings suggest that one of the mechanisms by which endothelins and their receptors may affect the IOP

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could occur through the modulation of the aqueous humor formation. In conclusion, we demonstrated the presence of both ETA and ETB receptor subtypes in rat ciliary body in a ratio of 35 : 65, respectively. Our autoradiographical results showed that both receptor subtypes are localized in the ciliary processes. These data suggest that endothelins may regulate the IOP in the eye through both receptor subtypes.

Acknowledgements This work was supported by grant PB92-0737 from the DGICYT.

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