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Prostaglandin E2 binding site distribution and subtype classification in the rabbit iris-ciliary body
Prostaglandins
44:199-208,
1992
PROSTAGLANDIN E BINDING SITE DISTRIBUTION AND SUBTYPE CLASSIFICAT 3ON IN THE RABBIT IRIS-CILIARY BODY S. Csukas, P. Bhattacherjee,
L. Rhodes and C.A. Paterson
Department of Ophthalmology and Visual Sciences, Kentucky Lions Bye Reeearch Institute, Univereity of Louieville, School of Medicine 301 E. Muhammad Ali Boulevard Louisville, KY 40292 ABSTRACT The distribution and characteristics of specific binding sites for tritium labeled prostaglandin E2 (3H-PGE2) were examined in membrane preparations from rabbit iris-sphincter, iris and ciliary body. The majority of 3H-PGE2 specific binding sites were found in the ciliary body (46%) followed by the iris (37%) and the iris-sphincter muscle (5%). Scatchard analysis of saturable 3H-PGE2 binding sites in the ciliary body indicated a single binding site with a Kd of 2.81 nM and Bmax value of 84 fmoles bound/mg protein. Competition by agonists selective for the EPI, EP2 and EP3 receptor subtypes of the EP (PGE2) prostanoid receptor indicated that the majority of rabbit ciliary body 3H-PGE2 binding sites are of the EP2 subtype. Incomplete displacement of labeled 3H-PGE2 from its binding sites by the EP2 selective agonist 11-deoxy PGEI suggests the presence of additional EP or non-EP binding sites. There was essentially no binding to EPI receptor sites as defined by the dis lacement of 3H-PGE2 by 17-phenyl-trinor PGE2. A weak displacement of !H-PGE2 by the EP3/EPI specific agonist, sulprostone, may account for the presence of a small number of EP3 specific binding sites in this tissue. The predominant distribution of PGE2 binding sites in the ciliary body and their identification as EP2 selective, supports recent functional studies where topical application of prostanoids with EP2 but not EPI or EP3 agonist activity resulted in breakdown of the blood-aqueous barrier. INTRODUCTION An understanding of the relationship between eicosanoid receptors and the physiological effects of prostaglandins (PG's) is now emerging. Bovine iris-sphincter smooth muscle contraction is mediated through PGE2 (EP) receptors (1). PGF2, (FP) receptors mediate contraction of cat and dog iris-sphincter muscles (2-4). Yousufzai et al. (5) reported that PGF2& contracts bovine iris-sphincter muscle via a receptor mediated myo-inositol 1,4,5_triphosphate transduction pathway. In vivo, prostaglandins of the E-series and PGF2a elicit ocular responses ranging from vasodilation, to breakdown of the blood aqueous barrier (BAB), to reduction of intraocular pressure (4,6-11).
Copyright0 1992 Butterworth-Heinemann
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Prostaglandins
However, the linkage between the presence of a prostanoid in an ocular tissue and receptor mediated transduction into a specific response remains unclear. Classification of prostanoid receptors according to the type of prostanoid involved, PGE2 (EP), PGF2e (FP), PGD2 (DP), PG12 (IP), TXB2 (TP), initially described by Kennedy et al. (2), has progressed to an identification of receptor subtypes based upon their ability to mediate specific physiological responses (3,12,13). Three subtypes of the EP receptor have been described: EPI, EP2 and EP3 (14). EP2 receptors are thought to mediate BAB breakdown in the rabbit (15). In cultured rabbit cornea1 endothelial cells, the EP2 subtype, coupled to activation of adenylate cyclase (16), is important in the maintenance of endothelial morphology. Activation of presynaptic EP3 receptors in the whole iris-ciliary body inhibits the stimulated release of norepinepherine (17). Bhattacherjee et al. (18) report stimulation of adenylate cyclase by various EP-subtype selective agonists in the rabbit, cat and bovine iris-ciliary body. Although a significant body of information has accumulated concerning these receptor subtypes, the lack of highly selective EP2 and EP3 antagonists hampers the definitive determination of the activity of these EP receptor selective agonists. We have previously reported the presence and properties of PGE2-selective binding sites in the membrane preparations from both whole and individual components of bovine iris-ciliary body (19-20). In the present study, we examine the regional distribution of 3H-PGE2 specific binding sites in membrane preparations of separated rabbit iris-sphincter muscle, iris and ciliary body. In addition, we attempt to determine whether the rabbit ciliary body exhibits selectivity for agonists of the EP receptor subtype. MATERIALS AND METHODS Both a phosphate and a Tris buffer were used in these studies. The initial phosphate buffer used for distribution studies was later supplanted by a Tris buffer which provided improved specific binding of the labeled ligand, in competition and saturation studies. Both buffers provided consistent and reliable binding conditions. Membrane Preoaration. Rabbit eyes were harvested and immediately placed on ice for overnight delivery to our laboratory by Pel-Freez (Rooers. AR). Uoon arrival. the whole iris-ciliarv bodv was dissected cir&ferentiall) into its component parts: the ir"is-sphinctermuscle, the iris and the ciliary body. Tissues were maintained at 2-4"C, and the anatomical identity of separated tissues was verified in histological sections (not shown). The ciliary muscle content of the iris-ciliary body section was not controlled, and some ciliary muscle was lost during tissue recovery. As previously reported (19), each tissue was homogenized separately in 50 mM Tris or 50 mM phosphate buffer, pH 7.5, containing cyclooxygenase (flurbiprofen), protease (phenylmethyl-sulfonylfluoride) and soybean trypsin inhibitors. In addition to these inhibitors the Tris buffer also contained 1 mM EGTA. Membranes were obtained after a three-stage high speed centrifugation as previously described for bovine tissue (19).
Prostaglandins
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Radioliqand Bindinq Assay. The binding of radioligand to reconstituted membrane was found to increase linearly with increasing membrane concentration up to approximately 400 1(g of membrane protein per assay tube. Specific binding to the membranes reached equilibrium after 15 min incubation. a. Distribution studies. The distribution of 3H-PGE2 specific binding sites in the iris-sphincter, iris and ciliary body was determined using a 50 mM phosphate buffer containing inhibitors. Membranes were separately reconstituted in buffer, and aliquots containing 50-250 pg of membrane protein were incubated with 18 nM 3H-PGE2 at 37°C for 15 min. A lOOO-fold excess of the unlabeled ligand was employed to define non-specific binding. At the end of the 15 min incubation period, free and membrane bound ligand were separated by rapid filtration through a type HA Millipore filter. The radioactivity bound to the membranes retained on the filter was quantitated in a Beckman LS3801 scintillation counter. Blank values for radioactivity retained by the filters in the absence of membrane were subtracted from all values. b. Competition and saturation studies on ciliarv bodv membranes. Ciliary body membranes were reconstituted in 50 mM Tris buffer with inhibitors and supplemented with 2.5 mM Mn++. Aliquots containing 50-250 fig of membrane protein were incubated with 6 nM 3H-PGE2 at 37°C for 15 min and recovered as in the distribution studies above. Competition studies were performed in the presence of varying concentrations of unlabeled ligands specific for receptors of the EP subtype: PGE2 (EP), 11-deoxy PGEL (EP2), sulprostone (EP3/EPT) and 17-shenyl trinor PGE2 (EPT). Saturation studies were performed using 6 nM H-PGE2 and O-1000 nM unlabeled PGE2. Scatchard analysis of competition data was carried out to determine the maximum number of PGE2 binding sites (Bmax) and equilibrium dissociation constant (kd) for the ciliary body membrane preparation. Data Analvsis. Data were analyzed using the analytical portion of the computer software program Sigma Plot Version 4.0 (Jandel Scientific) and EBDA (Biosoft). EBDA was used to perform the transformations necessary to analyze the Scatchard data. Sigma Plot was used to fit data by an iterative process to a four parameter logistic equation. This analysis produced a "best fit" to a sigmoidal curve. The analysis provided a value for the IC50 (concentration of unlabeled ligand that displaced 50 percent of specifically bound labeled ligand) of the competing ligand and a coefficient of cooperativity describing the relative shape of the sigmoidal function. Materials. Prostanoid compounds (Cayman Chemical, Ann Arbor, MI) were made up to a 14.2 mM concentration in 95% ethyl alcohol. Workino solutions contained less than 0.18% ethyl alcohol. Sulprostone was the generous gift of Schering AG (Germany). Labeled ligands were purchased from New England Nuclear (Boston, MA). All other compounds were obtained from Sigma Chemical Company (St. Louis, MO).
Prostaglandins
202 RESULTS
Distribution Studies. These studies were designed to determine the localization of jH-PGEp binding in isolated regions of iris-ciliary body. The density of specific binding of 3H-PGE2 in the irissphincter, iris and ciliary body membranes was 70.6, 68.4 and 39.2 fmoles bound per mg membrane protein, respectively (Table 1). The distribution of 3H-PGE2 binding found in each tissue (fmoles bound /tissue) was calculated as the product of the fmoles bound per mg membrane times the quantity (mg) of membrane recovered from each type of tissue. The majority of PGE2 specific binding sites are located in the ciliary body (46%), followed by iris (37%) and iris-sphincter (17%). Table 1. Regional distribution of PGE2 specific binding sites in the rabbit iris-ciliary body (ICB) membrane preparation.
fM Eoundlmg protein (18nM HOTPGEP)
n % ofwhole ICB membrane protein [
46 (
96 of whole ICE binding sites/tissue 1 %
1
Sphincter 70.59 25.47
Iris 88.44 7.04
Ciliaty Body 39.20 5.78
(9)
(9)
(10)
12%
1
28%
1
60%
1
17%
1
37%
1
48%
1
Competition Studies. The relative selectivity of several EP receptor subtype agonists to displace bound 3H-PGE2 from a ciliary body membrane preparation is shown by the displacement curves in Fig. 1. The curves for the non-selective EP receptor agonist PGE2 and the EP2 selective agonist 11-deoxy PGEI were similar, although 11-deoxy PGE2 effected a greater displacement of 3H-PGE2 at lower concentrations and was unable to displace all of the labeled PGE2. The displacement curve of sulprostone, an EP3/EPI selective agonist with somewhat greater EP3 selectivity, was shifted far to the right of the PGE2 and 11-deoxy PGEI curves. The displacement curve for 17-phenyl trinor PGE2 (EPI selective agonist) was also shifted to the far right indicating its significantly lesser ability to displace 3H-PGE2 from its binding sites. The relative ability of the EP agonists to displace 3H-PGE2 was expressed as the IC50 (concentration of agonist displacing 50 percent of the specific 3H-PGE2 binding). The IC50 values for each agonist are represented in Figure 1. The IC50 of 11-deoxy PGEI (5.7 nM) was only marginally greater than that of PGE2 (4.7 nM). The IC50 for sulprostone (143 nM) suggested a much lesser ability to compete with 3H-PGE2 for binding sites. The large IC50 for 17-phenyl trinor PGE2 (202.7 nM) suggests the absence of EPl selective binding sites in the ciliary body.
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203
Fig. 1. Competitive inhibition of 6 nM 3H-PGE2 specific binding to rabbit ciliary body membranes by unlabeled PGE2, II-deoxy PGEI, sulprostone and 17-phenyl trinor PGE2. Incubations in triplicate were performed for 15 min at 37.5"C in the presence or absence of various concentrations of unlabeled ligand. 100% specific binding is defined as the amount of bound 3H-PGE2 displaced in the presence 6 PM unlabeled PGE2 (n>4 for each point represented, values represent the mean + S.E.M.)
Prostaglandins
204
The equilibrium dissociation constant, Kd, and the Scatchard Analvsis. maximum number of 3H-PGE2 specific binding sites, Bmax, in the ciliary body were determined from saturation studies. Specific binding increased with increasing PGE2 concentration and approached saturation Specific binding represented approximately 56% of above 30 nM PGE2. total PGE2 binding. Scatchard analysis of saturation data revealed a single 3H-PGE2 binding site (Table 2). The Kd and Bmax values + SEM for PGE2 binding to 3H-PGE2 specific sites were 2.81 + 3.10 nM and 84.25 + 25.66 fmoles PGE2 bound/mg protein, repectively.
Table 2. Scatchard analysis rabbit ciliary body membrane
WV
of PGE2 specific preparation.
binding
sites
in the
pJ-zG&J
fmoles boundhngprotein
DISCUSSION
In the present study, we have demonstrated the relative distribution and binding characteristics of 3H-PGE2 specific binding sites in the membrane preparations of separated rabbit eye iris-sphincter muscle, iris and ciliary body. The density (fmoles bound per mg membrane protein) of PGE2 specific binding sites in the iris-sphincter muscle and iris were roughly equivalent and greater than that found in ciliary body. However, due to the large amount of membrane recovered from ciliary body versus iris and iris-sphincter, 46 percent of PGE2 binding sites were located in the ciliary body. This contrasts to data from the bovine eye (20), where only 18 percent of PGE2 specific iris-ciliary body binding sites are present in ciliary body. The presence of significant iridial ciliary process tissue in the rabbit which is not present in the bovine eye may account for this finding. Scatchard analysis of 3H-PGE2 binding in the ciliary body indicated the presence of a single binding site with a Kd of 2.81 nM. This binding constant is similar to that reported in other tissues such as baboon and rabbit uterine membranes (21), where the Kd's for PGE2 are in the range 3.3-13 nM. The bovine ciliary body Kd value of 17.8 nM for PGE2 (20) is several-fold higher than in rabbit; however, the ciliary body Bmax values for both species are nearly identical. The IC50 of 11-deoxy PGEI (5.7 nM) to displace rabbit ciliary body 3H-PGE2 was only marginally reater than that of PGE2 (4.7 nM) and accounted for the majority of Y H-PGE2 displacement, indicating that these PGE2 specific binding sites are primarily of the EP2 subtype. The IC50 values for sulprostone (143 nM) and 17-phenyl trinor PGE2 (202 nM) suggests a far lesser ability than 11-deoxy PGEI to compete with 3H-PGE2 for specific binding sites. These large Ic50 values suggest either the presence of few EP3/EPI selective binding sites and/or the non-specific displacement of bound 3H-PGE2. A definitive
Prostaglandins
205
identification of these binding sites as EP2 or EP3 subtypes or an as yet unidentified EPx site, awaits the development of highly selective EP receptor subtype antagonists. The rabbit eye is generally considered to be more responsive to E-series prostanoids than other species, including man. Prostaglandin E2 levels are highly elevated in rabbit aqueous humor following inflammatory insult (22) and the rabbit ciliary epithelia produce several prostanoids, including PGE2, following incubation with 14Carachidonic acid (23). A variety of ocular responses (vasodilation of iridial vessels, pupilary constriction, altered intraocular pressure and breakdown of the BAB have been reported following E-series prostanoid release into the anterior segment (6-7, 24-27). Only prostanoid mediated constriction of sphincter and ciliary muscle have been closely examined both in vivo and in vitro. We are particularly interested in the actions of prostanoids as they relate to loss of BAB integrity during ocular inflammatory episodes. Our ciliary body data have led us to consider whether the sensitivity of the rabbit BAB to prostaglandins of the E-series is correlated with the relative distribution, affinity and subtype classification of PGE2 binding sites in this tissue. A mechanism specifically linking the presence of E-series prostanoids to receptor mediated BAB breakdown has not been established. Earlier studies (24-26) demonstrated that exposure to a sufficient quantity of these prostanoids resulted in iridial vasodilation, hyperemia, ciliary process edema and alteration of the junctional complexes of the ciliary epithelia. It is unknown whether the primary event triggering BAB breakdown is altered iridial blood flow, direct action at the ciliary epithelia or some other as yet undescribed event. Recent work by Protzman and Woodward (15) demonstrated that rabbit BAB breakdown was mediated by EP2 but not EPT or EP3 selective agonists. At least one other species, the cow, exhibits a completely different EP binding site profile than rabbit, where binding sites are predominantly of the EPT subtype and heavily localized in the iris-sphincter muscle (20). Therefore, whether the current findings correlate to human ocular tissue is as yet unknown and awaits the localization of EP receptor sites to regions of the ciliary body such as the non-pigmented epithelium, vascular endothelium or ciliary muscle in these species in order to provide a better understanding of the site of action of the E-series prostaglandins. Our finding concerning the relative abundance of PGE2 binding sites in the rabbit ciliary processes and their significant EP2 character combined with the capacity of rabbit ocular tissue to produce PGE2 helps to explain the highly responsive nature of the rabbit anterior segment to PGE2. ACKNOWLEDGEMENT This study was supported by USPHS research grant number EY-06918, the Kentucky Lions Eye Research Foundation, and an unrestricted grant from Research to Prevent Blindness. The authors thank Mary Henning for her assistance in manuscript preparation.
206
Prostaglandins
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and N.H. Wilson. Prostaglandin E receptor Dong, Y.J., R.L. Jones, subtypes in smooth muscle: Agonist activities of stable prostaglandin analogues. Br. J. Pharmacol. 87, 97-107 (1986).
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Kennedy, I., R. Coleman, P.P.A. Humphrey, G.P. Levy and P. Lumley. Studies on the characterization of prostanoid receptors: A proposed classification. Prostaglandins 24, 667-89 (1982).
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Coleman, R.A., P.P.A. Humphrey, I. Kennedy, and P. Lumley. Prostanoid receptors - the development of a working classification. TIPS 5, 303-6 (1984).
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Bito, L.Z., C.B. Camras, G.C. Gum, and B. Resul. The ocular hypotensive effects and side-effects of prostaglandins on the eyes of experimental animals. In "The Ocular Effects of Prostaglandins and other Eicosanoids." (Eds Bito, L.Z. and Stjernschantz, J.). Alan R. Liss, Inc.: New York, 1989, Pp. 349-68.
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Yousufzai, S.Y.K., S.D. Tachado, K.D. Carter, and A.A. Abdel-Latif. Short term densitization of prostaglandins F2o receptors increases cyclic AMP formation and reduces inositol phosphates accumulation and contraction in the bovine iris sphincter. Curr. Eye Res. 8, 1211-20 (1989).
6.
Cole, D.F. and W.G. Unger. Prostaglandins as mediators for the responses of the eye to trauma. Exp. Eye Res. 17, 357-68 (1973).
7.
Eakins, K.E. (1977). Prostaglandins and non-prostaglandin mediated breakdown of the blood-aqueous barrier. Exp. Eye Res. 25, 483-98 (1977).
8.
Rahi, A.H., P. Bhattacherjee, and R.N. Misra. Release of prostaglandins in experimental immunocomplex endophthalmitis and phacoallergic uveitis. Br. J. Ophthalmol. 62, 105-9 (1978).
9.
Bhattacherjee,
10.
Camras, C.B., L.Z. Bito, and K.E. Eakins. Reduction of intraocular pressure by prostaglandins applied topically to the eyes of conscious rabbits. Invest. Ophthalmol. Vis. Sci. 16, 1125-34 (1977).
11.
Kaufman, P.L. and K. Crawford. Aqueous humor dynamics: How PGF2@ lowers intraocular pressure. In "The Ocular Effects of Prostaglandins and Other Eicosanoids." (Eds Bito, L.Z. and Stjernschantz, J.). Alan R. Liss, Inc.: New York, 1989, Pp. 387-416.
P. The role of arachidonate metabolites in ocular inflammation. In "The Ocular Effects of Prostaglandins and other Eicosanoids." (Eds Bito, L.Z. and Stjernchantz, J.). Alan R. Liss, Inc. : New York, 1989, Pp. 211-27.
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Eglen, R.M. and R.L. Whiting. The actions of prostanoid receptor agonists and antagonists on smooth muscles and platelets. Br. J. Pharmacol. 94, 591-601 (1988).
13.
Coleman, R.A. (1987). Methods in prostanoid receptor classification. In "Prostaglandins and Related substances. A Practical Approach." (Eds Beneditto, C., McDonald-Gibson, R.G., Nigam, S. and Slater, T.F.). IRL Press Ltd.: Oxford, U.K., 1987, Pp. 167-303.
14.
Coleman, R.A., I. Kennedy, R.L.G. Sheldrick and I.Y. Tolowinska. Further evidence for the existance of three subtypes of PGE2 sensitive (EP-) receptors. Br. J. Pharm. 91, 407P (1990).
15.
Protzman, E.E. and D.F. Woodward. Prostanoid-induced blood-aqueous barrier breakdown in rabbits involves the EP2 receptor subtype. Invest. Ophthalmol. Vis. Sci. 31, 2463 (1990).
16.
Jumblatt, M. and C.A. Paterson. PGE2 effects on cornea1 endothelial cyclic AMP synthesis and cell shape are mediated by a receptor of the EP2-subtype. Invest. Ophthalmol. Vis. Sci. 32, 360 (1991).
17.
Ohia, S.E. and J.E. Jumblatt. Prejunctional inhibitory effects of prostanoids on sympathetic neurotransmission in the rabbit iris-ciliary body. J. Pharmacol. Exp. Therap. 255, 11-16 (1990).
18.
Bhattacherjee, P., L. Rhodes and C.A. Paterson. Iris-ciliary body prostaglandin receptors coupled to adenylate cyclase. Exp. Eye Res. (Submitted).
19.
Bhattacherjee, P., S. Csukas and C.A. Paterson. Prostaglandin E2 binding sites in bovine iris-ciliary body. Invest. Ophthalmol. Vis. Sci. 31, 1109 (1990).
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Csukas,
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Hodam, J.R., M.C. Snabes, T.J. Kuehl, M.A. Jones, and M.J.K. Harper. Characterization of prostaglandin E2 binding to uterine membranes from baboon, rabbit and tree shrew (Tupaia belangeri). J. Molec. Endocrinol. 3, 33-42 (1989).
22.
Csukas, S., C.A.Paterson, K. Brown, and P. Bhattacherjee. Time course of rabbit ocular inflammatory response and mediator release after intravitreal endotoxin. Invest. Ophthalmol. Vis. Sci. 31, 382 (1990).
23.
King, K.L., N.A. Delamere, S.C. Csukas, and W.M. Pierce Jr.. Metabolism of arachidonic acid by isolated ciliary epithelium. Exp. Eye Res. (in press).
S., P. Bhattacherjee, L. Rhodes and C.A. Paterson. Prostaglandin E2 and F2o binding sites in the bovine iris ciliary body. Invest. Ophthalmol. Vis. Sci. (Submitted).
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Oesantis Jr, L.L. and V.L. Sallee. Comparison of the effects of prostaglandins and their esters on blood aqueous barrier integrity and intraocular pressure in rabbits. In "The Ocular Effects of Prostaglandins and Other Eicosanoids" (Eds Bito, L.Z. and Stjernschantz, J.) Alan R. Liss, Inc: New York, 1989, Pp 379-86.
25.
Neufeld A.H. and M.L. Sears. The site of action of prostaglandin E2 on the disruption of the blood aqueous barrier in the rabbit eye. Exp. Eye Res. 17, 445 (1973).
26.
Wilkie D.A. Control of ocular inflammation. Vet. Clin. N. America: Small Animal Practice 20, 693 (1990).
27.
Vegge T., A.H. Neufeld and M.L. Sears. Morphology of the breakdown of the blood-aqeous barrier in the ciliary processes of the rabbit eye after prostaglandin E2. Invest. Ophthalmol. Vis. Sci. 14, 33 (1975).
Editor:
F. Coceani
Received:
1-21-92
Accepted:
3-23-92
44:199-208,
1992
PROSTAGLANDIN E BINDING SITE DISTRIBUTION AND SUBTYPE CLASSIFICAT 3ON IN THE RABBIT IRIS-CILIARY BODY S. Csukas, P. Bhattacherjee,
L. Rhodes and C.A. Paterson
Department of Ophthalmology and Visual Sciences, Kentucky Lions Bye Reeearch Institute, Univereity of Louieville, School of Medicine 301 E. Muhammad Ali Boulevard Louisville, KY 40292 ABSTRACT The distribution and characteristics of specific binding sites for tritium labeled prostaglandin E2 (3H-PGE2) were examined in membrane preparations from rabbit iris-sphincter, iris and ciliary body. The majority of 3H-PGE2 specific binding sites were found in the ciliary body (46%) followed by the iris (37%) and the iris-sphincter muscle (5%). Scatchard analysis of saturable 3H-PGE2 binding sites in the ciliary body indicated a single binding site with a Kd of 2.81 nM and Bmax value of 84 fmoles bound/mg protein. Competition by agonists selective for the EPI, EP2 and EP3 receptor subtypes of the EP (PGE2) prostanoid receptor indicated that the majority of rabbit ciliary body 3H-PGE2 binding sites are of the EP2 subtype. Incomplete displacement of labeled 3H-PGE2 from its binding sites by the EP2 selective agonist 11-deoxy PGEI suggests the presence of additional EP or non-EP binding sites. There was essentially no binding to EPI receptor sites as defined by the dis lacement of 3H-PGE2 by 17-phenyl-trinor PGE2. A weak displacement of !H-PGE2 by the EP3/EPI specific agonist, sulprostone, may account for the presence of a small number of EP3 specific binding sites in this tissue. The predominant distribution of PGE2 binding sites in the ciliary body and their identification as EP2 selective, supports recent functional studies where topical application of prostanoids with EP2 but not EPI or EP3 agonist activity resulted in breakdown of the blood-aqueous barrier. INTRODUCTION An understanding of the relationship between eicosanoid receptors and the physiological effects of prostaglandins (PG's) is now emerging. Bovine iris-sphincter smooth muscle contraction is mediated through PGE2 (EP) receptors (1). PGF2, (FP) receptors mediate contraction of cat and dog iris-sphincter muscles (2-4). Yousufzai et al. (5) reported that PGF2& contracts bovine iris-sphincter muscle via a receptor mediated myo-inositol 1,4,5_triphosphate transduction pathway. In vivo, prostaglandins of the E-series and PGF2a elicit ocular responses ranging from vasodilation, to breakdown of the blood aqueous barrier (BAB), to reduction of intraocular pressure (4,6-11).
Copyright0 1992 Butterworth-Heinemann
200
Prostaglandins
However, the linkage between the presence of a prostanoid in an ocular tissue and receptor mediated transduction into a specific response remains unclear. Classification of prostanoid receptors according to the type of prostanoid involved, PGE2 (EP), PGF2e (FP), PGD2 (DP), PG12 (IP), TXB2 (TP), initially described by Kennedy et al. (2), has progressed to an identification of receptor subtypes based upon their ability to mediate specific physiological responses (3,12,13). Three subtypes of the EP receptor have been described: EPI, EP2 and EP3 (14). EP2 receptors are thought to mediate BAB breakdown in the rabbit (15). In cultured rabbit cornea1 endothelial cells, the EP2 subtype, coupled to activation of adenylate cyclase (16), is important in the maintenance of endothelial morphology. Activation of presynaptic EP3 receptors in the whole iris-ciliary body inhibits the stimulated release of norepinepherine (17). Bhattacherjee et al. (18) report stimulation of adenylate cyclase by various EP-subtype selective agonists in the rabbit, cat and bovine iris-ciliary body. Although a significant body of information has accumulated concerning these receptor subtypes, the lack of highly selective EP2 and EP3 antagonists hampers the definitive determination of the activity of these EP receptor selective agonists. We have previously reported the presence and properties of PGE2-selective binding sites in the membrane preparations from both whole and individual components of bovine iris-ciliary body (19-20). In the present study, we examine the regional distribution of 3H-PGE2 specific binding sites in membrane preparations of separated rabbit iris-sphincter muscle, iris and ciliary body. In addition, we attempt to determine whether the rabbit ciliary body exhibits selectivity for agonists of the EP receptor subtype. MATERIALS AND METHODS Both a phosphate and a Tris buffer were used in these studies. The initial phosphate buffer used for distribution studies was later supplanted by a Tris buffer which provided improved specific binding of the labeled ligand, in competition and saturation studies. Both buffers provided consistent and reliable binding conditions. Membrane Preoaration. Rabbit eyes were harvested and immediately placed on ice for overnight delivery to our laboratory by Pel-Freez (Rooers. AR). Uoon arrival. the whole iris-ciliarv bodv was dissected cir&ferentiall) into its component parts: the ir"is-sphinctermuscle, the iris and the ciliary body. Tissues were maintained at 2-4"C, and the anatomical identity of separated tissues was verified in histological sections (not shown). The ciliary muscle content of the iris-ciliary body section was not controlled, and some ciliary muscle was lost during tissue recovery. As previously reported (19), each tissue was homogenized separately in 50 mM Tris or 50 mM phosphate buffer, pH 7.5, containing cyclooxygenase (flurbiprofen), protease (phenylmethyl-sulfonylfluoride) and soybean trypsin inhibitors. In addition to these inhibitors the Tris buffer also contained 1 mM EGTA. Membranes were obtained after a three-stage high speed centrifugation as previously described for bovine tissue (19).
Prostaglandins
201
Radioliqand Bindinq Assay. The binding of radioligand to reconstituted membrane was found to increase linearly with increasing membrane concentration up to approximately 400 1(g of membrane protein per assay tube. Specific binding to the membranes reached equilibrium after 15 min incubation. a. Distribution studies. The distribution of 3H-PGE2 specific binding sites in the iris-sphincter, iris and ciliary body was determined using a 50 mM phosphate buffer containing inhibitors. Membranes were separately reconstituted in buffer, and aliquots containing 50-250 pg of membrane protein were incubated with 18 nM 3H-PGE2 at 37°C for 15 min. A lOOO-fold excess of the unlabeled ligand was employed to define non-specific binding. At the end of the 15 min incubation period, free and membrane bound ligand were separated by rapid filtration through a type HA Millipore filter. The radioactivity bound to the membranes retained on the filter was quantitated in a Beckman LS3801 scintillation counter. Blank values for radioactivity retained by the filters in the absence of membrane were subtracted from all values. b. Competition and saturation studies on ciliarv bodv membranes. Ciliary body membranes were reconstituted in 50 mM Tris buffer with inhibitors and supplemented with 2.5 mM Mn++. Aliquots containing 50-250 fig of membrane protein were incubated with 6 nM 3H-PGE2 at 37°C for 15 min and recovered as in the distribution studies above. Competition studies were performed in the presence of varying concentrations of unlabeled ligands specific for receptors of the EP subtype: PGE2 (EP), 11-deoxy PGEL (EP2), sulprostone (EP3/EPT) and 17-shenyl trinor PGE2 (EPT). Saturation studies were performed using 6 nM H-PGE2 and O-1000 nM unlabeled PGE2. Scatchard analysis of competition data was carried out to determine the maximum number of PGE2 binding sites (Bmax) and equilibrium dissociation constant (kd) for the ciliary body membrane preparation. Data Analvsis. Data were analyzed using the analytical portion of the computer software program Sigma Plot Version 4.0 (Jandel Scientific) and EBDA (Biosoft). EBDA was used to perform the transformations necessary to analyze the Scatchard data. Sigma Plot was used to fit data by an iterative process to a four parameter logistic equation. This analysis produced a "best fit" to a sigmoidal curve. The analysis provided a value for the IC50 (concentration of unlabeled ligand that displaced 50 percent of specifically bound labeled ligand) of the competing ligand and a coefficient of cooperativity describing the relative shape of the sigmoidal function. Materials. Prostanoid compounds (Cayman Chemical, Ann Arbor, MI) were made up to a 14.2 mM concentration in 95% ethyl alcohol. Workino solutions contained less than 0.18% ethyl alcohol. Sulprostone was the generous gift of Schering AG (Germany). Labeled ligands were purchased from New England Nuclear (Boston, MA). All other compounds were obtained from Sigma Chemical Company (St. Louis, MO).
Prostaglandins
202 RESULTS
Distribution Studies. These studies were designed to determine the localization of jH-PGEp binding in isolated regions of iris-ciliary body. The density of specific binding of 3H-PGE2 in the irissphincter, iris and ciliary body membranes was 70.6, 68.4 and 39.2 fmoles bound per mg membrane protein, respectively (Table 1). The distribution of 3H-PGE2 binding found in each tissue (fmoles bound /tissue) was calculated as the product of the fmoles bound per mg membrane times the quantity (mg) of membrane recovered from each type of tissue. The majority of PGE2 specific binding sites are located in the ciliary body (46%), followed by iris (37%) and iris-sphincter (17%). Table 1. Regional distribution of PGE2 specific binding sites in the rabbit iris-ciliary body (ICB) membrane preparation.
fM Eoundlmg protein (18nM HOTPGEP)
n % ofwhole ICB membrane protein [
46 (
96 of whole ICE binding sites/tissue 1 %
1
Sphincter 70.59 25.47
Iris 88.44 7.04
Ciliaty Body 39.20 5.78
(9)
(9)
(10)
12%
1
28%
1
60%
1
17%
1
37%
1
48%
1
Competition Studies. The relative selectivity of several EP receptor subtype agonists to displace bound 3H-PGE2 from a ciliary body membrane preparation is shown by the displacement curves in Fig. 1. The curves for the non-selective EP receptor agonist PGE2 and the EP2 selective agonist 11-deoxy PGEI were similar, although 11-deoxy PGE2 effected a greater displacement of 3H-PGE2 at lower concentrations and was unable to displace all of the labeled PGE2. The displacement curve of sulprostone, an EP3/EPI selective agonist with somewhat greater EP3 selectivity, was shifted far to the right of the PGE2 and 11-deoxy PGEI curves. The displacement curve for 17-phenyl trinor PGE2 (EPI selective agonist) was also shifted to the far right indicating its significantly lesser ability to displace 3H-PGE2 from its binding sites. The relative ability of the EP agonists to displace 3H-PGE2 was expressed as the IC50 (concentration of agonist displacing 50 percent of the specific 3H-PGE2 binding). The IC50 values for each agonist are represented in Figure 1. The IC50 of 11-deoxy PGEI (5.7 nM) was only marginally greater than that of PGE2 (4.7 nM). The IC50 for sulprostone (143 nM) suggested a much lesser ability to compete with 3H-PGE2 for binding sites. The large IC50 for 17-phenyl trinor PGE2 (202.7 nM) suggests the absence of EPl selective binding sites in the ciliary body.
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203
Fig. 1. Competitive inhibition of 6 nM 3H-PGE2 specific binding to rabbit ciliary body membranes by unlabeled PGE2, II-deoxy PGEI, sulprostone and 17-phenyl trinor PGE2. Incubations in triplicate were performed for 15 min at 37.5"C in the presence or absence of various concentrations of unlabeled ligand. 100% specific binding is defined as the amount of bound 3H-PGE2 displaced in the presence 6 PM unlabeled PGE2 (n>4 for each point represented, values represent the mean + S.E.M.)
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204
The equilibrium dissociation constant, Kd, and the Scatchard Analvsis. maximum number of 3H-PGE2 specific binding sites, Bmax, in the ciliary body were determined from saturation studies. Specific binding increased with increasing PGE2 concentration and approached saturation Specific binding represented approximately 56% of above 30 nM PGE2. total PGE2 binding. Scatchard analysis of saturation data revealed a single 3H-PGE2 binding site (Table 2). The Kd and Bmax values + SEM for PGE2 binding to 3H-PGE2 specific sites were 2.81 + 3.10 nM and 84.25 + 25.66 fmoles PGE2 bound/mg protein, repectively.
Table 2. Scatchard analysis rabbit ciliary body membrane
WV
of PGE2 specific preparation.
binding
sites
in the
pJ-zG&J
fmoles boundhngprotein
DISCUSSION
In the present study, we have demonstrated the relative distribution and binding characteristics of 3H-PGE2 specific binding sites in the membrane preparations of separated rabbit eye iris-sphincter muscle, iris and ciliary body. The density (fmoles bound per mg membrane protein) of PGE2 specific binding sites in the iris-sphincter muscle and iris were roughly equivalent and greater than that found in ciliary body. However, due to the large amount of membrane recovered from ciliary body versus iris and iris-sphincter, 46 percent of PGE2 binding sites were located in the ciliary body. This contrasts to data from the bovine eye (20), where only 18 percent of PGE2 specific iris-ciliary body binding sites are present in ciliary body. The presence of significant iridial ciliary process tissue in the rabbit which is not present in the bovine eye may account for this finding. Scatchard analysis of 3H-PGE2 binding in the ciliary body indicated the presence of a single binding site with a Kd of 2.81 nM. This binding constant is similar to that reported in other tissues such as baboon and rabbit uterine membranes (21), where the Kd's for PGE2 are in the range 3.3-13 nM. The bovine ciliary body Kd value of 17.8 nM for PGE2 (20) is several-fold higher than in rabbit; however, the ciliary body Bmax values for both species are nearly identical. The IC50 of 11-deoxy PGEI (5.7 nM) to displace rabbit ciliary body 3H-PGE2 was only marginally reater than that of PGE2 (4.7 nM) and accounted for the majority of Y H-PGE2 displacement, indicating that these PGE2 specific binding sites are primarily of the EP2 subtype. The IC50 values for sulprostone (143 nM) and 17-phenyl trinor PGE2 (202 nM) suggests a far lesser ability than 11-deoxy PGEI to compete with 3H-PGE2 for specific binding sites. These large Ic50 values suggest either the presence of few EP3/EPI selective binding sites and/or the non-specific displacement of bound 3H-PGE2. A definitive
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205
identification of these binding sites as EP2 or EP3 subtypes or an as yet unidentified EPx site, awaits the development of highly selective EP receptor subtype antagonists. The rabbit eye is generally considered to be more responsive to E-series prostanoids than other species, including man. Prostaglandin E2 levels are highly elevated in rabbit aqueous humor following inflammatory insult (22) and the rabbit ciliary epithelia produce several prostanoids, including PGE2, following incubation with 14Carachidonic acid (23). A variety of ocular responses (vasodilation of iridial vessels, pupilary constriction, altered intraocular pressure and breakdown of the BAB have been reported following E-series prostanoid release into the anterior segment (6-7, 24-27). Only prostanoid mediated constriction of sphincter and ciliary muscle have been closely examined both in vivo and in vitro. We are particularly interested in the actions of prostanoids as they relate to loss of BAB integrity during ocular inflammatory episodes. Our ciliary body data have led us to consider whether the sensitivity of the rabbit BAB to prostaglandins of the E-series is correlated with the relative distribution, affinity and subtype classification of PGE2 binding sites in this tissue. A mechanism specifically linking the presence of E-series prostanoids to receptor mediated BAB breakdown has not been established. Earlier studies (24-26) demonstrated that exposure to a sufficient quantity of these prostanoids resulted in iridial vasodilation, hyperemia, ciliary process edema and alteration of the junctional complexes of the ciliary epithelia. It is unknown whether the primary event triggering BAB breakdown is altered iridial blood flow, direct action at the ciliary epithelia or some other as yet undescribed event. Recent work by Protzman and Woodward (15) demonstrated that rabbit BAB breakdown was mediated by EP2 but not EPT or EP3 selective agonists. At least one other species, the cow, exhibits a completely different EP binding site profile than rabbit, where binding sites are predominantly of the EPT subtype and heavily localized in the iris-sphincter muscle (20). Therefore, whether the current findings correlate to human ocular tissue is as yet unknown and awaits the localization of EP receptor sites to regions of the ciliary body such as the non-pigmented epithelium, vascular endothelium or ciliary muscle in these species in order to provide a better understanding of the site of action of the E-series prostaglandins. Our finding concerning the relative abundance of PGE2 binding sites in the rabbit ciliary processes and their significant EP2 character combined with the capacity of rabbit ocular tissue to produce PGE2 helps to explain the highly responsive nature of the rabbit anterior segment to PGE2. ACKNOWLEDGEMENT This study was supported by USPHS research grant number EY-06918, the Kentucky Lions Eye Research Foundation, and an unrestricted grant from Research to Prevent Blindness. The authors thank Mary Henning for her assistance in manuscript preparation.
206
Prostaglandins
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Editor:
F. Coceani
Received:
1-21-92
Accepted:
3-23-92