Comparison of tight junction protein expression in the ciliary epithelia of mouse, rabbit, cat and human eyes.

Tight junctions in the nonpigmented epithelium (NPE) of the ciliary processes and the iris vascular endothelium form the ocular blood aqueous barrier that prevents leakage of proteins, immune cells and non-immune cells of blood into the anterior chamber. We attempted to determine whether ultrastructural differences in tight junctions reported in earlier studies are reflected in the expression pattern of tight junction proteins (TJP) and whether the TJP in mice, rabbits and cats resemble those of humans. For immunohistochemistry, 10 µm thick cryosections were rehydrated in PBS and fixed in 50 mM ammonium chloride at room temperature. After rinses in PBS, the sections were incubated twice in 0.1% Triton X-100, 10% goat serum, specific primary antibody or in PBS. After rinses in PBS, the sections were incubated in FITC-conjugated secondary antibody. After rinses in PBS, the sections were mounted with Vectashield mounting medium with propidium iodide, examined and photographed using a confocal microscope. The expression patterns of TJP in ocular ciliary epithelium of human, rabbit, cat and mouse were similar. Occludin immunoreactivity was observed as a sharp line along the junction between pigmented epithelium (PE) and NPE, and along the apico-lateral surfaces of NPE. Very light staining of the ciliary stroma was observed in cat and mouse. Claudin-1 was expressed along the entire boundaries of NPE and was more distinct between PE and NPE in rabbit. The ciliary stroma showed faint staining in cat and mouse. ZO-1 showed staining between PE and NPE, and at the adjacent membrane. Moderate staining was seen in PE in cat and mouse, which suggests that claudin-1, occludin and ZO-1 are expressed along the junction between PE and NPE, and the apico-lateral border of NPE. Lack of major difference in the expression patterns among the different species is important for validating the use of rabbit, mouse and cat in studies of intraocular inflammation in humans.

Modulation of ocular inflammatory responses by EP1 receptors in mice.

The purpose of the study was to investigate the role of EP1 receptors in intraocular inflammation and to determine possible interplay between EP1, EP2 and EP4 receptors. The eyes of separate groups of EP1 receptor knockout and wild type mice were: 1) treated topically with prostaglandin E2 (PGE2) or the EP2 receptor selective agonist, butaprost; 2) given intravitreal injection of LPS; or 3) paracentesis performed. Another group of knockout mice were pretreated topically with an EP4 receptor selective antagonist prior to paracentesis or LPS treatment. Results demonstrated a significant increase (50% or more) in the protein levels of aqueous humor of the EP1 knockout mice in response to PGE2, paracentesis or LPS. The leukocyte infiltration in the aqueous humor of the knockout mice was 47% higher when compared with that in the wild type controls in response to LPS injection. No significant change was observed in the protein levels in response to butaprost. Pretreating the knockout mice with an EP4 receptor antagonist prior to paracentesis and LPS treatment substantially reduced the aqueous humor protein levels. Also, the leukocyte count in the aqueous humor of the knockout mice in response to LPS was reduced 4 fold after pretreatment with EP4 receptor antagonist when compared with the findings in knockout mice receiving LPS only. We concluded that EP1 receptor has no modulatory effect on EP2 receptors but there is definitely cross-talk between EP1 and EP4 receptors.

Ocular inflammatory responses in the EP2 and EP4 receptor knockout mice.

PURPOSE: To examine the role of EP2 and EP4 receptors in murine ocular inflammation. METHODS: Prostaglandin EP2 and EP4 receptor knockout and wild-type mice were treated topically with prostaglandin E2, SDF-1, and RANTES and lipopolysaccharide by intravitreal injection. Paracentesis was performed by puncturing the cornea. The increase in the level of aqueous humor protein and the number of leukocytes were measured and the vascular leakage of protein was visualized using fluorescein angiography. RESULTS: In the EP2 receptor knockout mice, there was significant inhibition of the disruption of the blood-aqueous barrier caused by lipopolysaccharides, paracentesis, prostaglandin E2, SDF-1, and RANTES. Reductions in the disruption in the blood-aqueous barrier and leukocyte infiltration after lipopolysaccharide injection and paracentesis were significant, but there was no increase in the aqueous humor protein level after prostaglandin E2 treatment in EP4 receptor knockout mice. CONCLUSIONS: The results of the present experiments suggest that EP2 and EP4 receptors partly mediate the disruption of the blood-aqueous barrier and leukocyte infiltration induced by prostaglandin E2, SDF-1, RANTES, and lipopolysaccharides.

Localization of EP(1) and FP receptors in human ocular tissues by in situ hybridization.

PURPOSE: To examine the expression and localization of EP(1) and FP receptor mRNAs in normal human ocular tissues by in situ hybridization. METHODS: Digoxigenin-labeled human EP(1) and FP receptor antisense and sense riboprobes were used for in situ hybridization on paraffin sections of normal human eye tissue. RESULTS: In situ hybridization revealed the presence of high levels of both EP(1) and FP receptor mRNA transcripts in the blood vessels of iris, ciliary body, and choroid. Both the endothelial and smooth muscle cells of blood vessels demonstrated intense hybridization signals corresponding to EP(1) receptor mRNA transcript. EP(1) receptor hybridization signals were present in all the muscle fibers of the ciliary body. In the retina, hybridization signals for EP(1) receptors were observed in photoreceptors and both nuclear layers and in ganglion cells. The hybridization signals corresponding to FP receptor transcript were similar to those of EP(1) receptors in the iris tissues. In the ciliary muscle, FP receptor mRNA transcript was predominantly present in the circular muscle and in the collagenous connective tissues; no hybridization signal for this receptor was observed in the retina. CONCLUSIONS: The wide distribution of EP(1) and FP receptor mRNAs in human ocular tissues appears to be localized in the functional sites of the respective receptor agonists. Selective localization of FP receptor mRNA in the circular muscles and collagenous connective tissues of the ciliary body suggests their involvement in the increased uveoscleral outflow of aqueous humor by PGF(2alpha).

Studies on receptor binding and signal transduction pathways of unoprostone isopropyl.

We examined the binding characteristics of unoprostone isopropyl and its metabolite, M1 (M1), in bovine corpus luteum membranes, mobilization of intracellular calcium in human ciliary muscle cells and cyclic AMP generation in rabbit iris-ciliary body. The ligand binding assay of 3H-unoprostone isopropyl and M1 did not demonstrate any specific binding of these compounds in the bovine corpus luteum membranes. However, there was a high specific binding of prostaglandin F2alpha. Competitive ligand binding studies showed that neither the docosanoid, unoprostone isopropyl, nor M1 binds to prostaglandin receptor sites. In human ciliary muscle cells that express EP1, EP2 and FP receptors, unoprostone isopropyl did not increase the mobilization of intracellular calcium nor was it able to generate cyclic AMP at low concentrations in rabbit iris-ciliary body. Similar observations were made with M1 on the above signal transduction pathways. From these results, it is concluded that unoprostone isopropyl and M1 do not bind to prostaglandin (PG) receptor sites in the bovine corpus luteum membranes and do not have affinity for PG receptors linked to intracellular calcium and cyclic AMP second messenger systems.

Responses of intraocular pressure and the pupil of feline eyes to prostaglandin EP1 and FP receptor agonists.

PURPOSE: Previous studies suggested that FP receptors do not mediate the relaxation of the ciliary muscle and reduction of intraocular pressure in cats by prostaglandin (PG) F2alpha. The present study was undertaken to determine whether the reduction of intraocular pressure in cats induced by PGF2alpha is mediated by FP or other prostaglandin receptors. METHODS: One eye of each cat was treated topically with prostaglandin F2alpha, fluprostenol (FP receptor agonist), or 17-phenyl trinor PGE2 (EP1 receptor agonist) in a dose range of 12.5 to 50 microg. The effects of SC19220 and SC51089 (EP1 receptor antagonists), BWA868c, and SQ29548 (DP and TP receptor antagonists, respectively) on the intraocular response to PGF2alpha were also examined. At intervals up to 6 hours after treatment, intraocular pressure was measured with a pneumotonometer, and pupil diameters were measured with a millimeter ruler. RESULTS: In the dose ranges used, PGF2alpha and 17-phenyl trinor PGE2 decreased intraocular pressure and pupil diameter. The greatest reduction of intraocular pressure by 50.0 microg PGF2alpha was 5.0+/-1.4 mm Hg, whereas that by 50 microg 17-phenyl trinor PGE2 was 6.2+/-1.5 mm Hg. The isopropyl ester of PGF 2alpha at a dose of 1.25 microg reduced intraocular pressure by 3.75+/-0.25 mm Hg at 2 hours. At doses up to 100 microg, fluprostenol did not decrease intraocular pressure but did reduce pupil diameter. SC19220, a weak but selective EP1 receptor antagonist, inhibited the intraocular pressure response to both PGF2alpha and 17-phenyl trinor PGE2. The more potent EP1 receptor antagonist SC51089 had a greater inhibitory effect than SC19220 on the intraocular pressure response to PGF2alpha. Both of these antagonists had a small but non-dose dependent and statistically insignificant effect on the pupil response to PGF2alpha. These observations suggest that in cats, intraocular pressure and pupil responses to PGF2alpha, are mediated by EP1 and FP receptors, respectively. However, SC19220 significantly and dose-dependently inhibited the pupil response to 17-phenyl trinor PGE2alpha suggesting that EP1 receptors mediate pupil response to this agonist. DP and TP receptor antagonists at doses 5- to 20-fold greater than the IC50 values had no effect on the ocular hypotensive response to PGF2alpha. The concurrent administration of 12.5 microg of each of PGF2alpha and 17-phenyl trinor PGE2 did not produce an additive effect on intraocular pressure, indicating that in cats PGF2alpha and 17-phenyl trinor PGE2 act on the same receptor type. CONCLUSIONS: These results suggest that a significant proportion of the ocular hypotensive action of PGF2alpha in cats is mediated by EP1 but not by FP receptor. Evidence was also provided to show that 17-phenyl trinor PGE2 is an ocular hypotensive agent in cats.

Bovine iris-sphincter muscle lacks FP receptor binding sites.

PURPOSE: To determine the affinity, density, and specificity of the binding sites of tritium-labeled prostaglandin F2alpha in membrane preparations of bovine iris-sphincter muscle and corpus luteum. METHODS: Membrane preparations were incubated with 0.312-40.0 nM 3H-prostaglandin F2alpha in saturation experiments, or 8 nM 3H-prostaglandin F2alpha in competition studies, in the presence or absence of varying concentrations of unlabeled prostaglandin F2alpha or other prostaglandin receptor agonists. The affinity (Kd) and the density of the binding sites (Bmax) of 3H-prostaglandin F2alpha in the bovine iris-sphincter muscle were determined by Scatchard analysis. Reverse transcription polymerase chain reaction was performed with bovine iris-sphincter muscle and corpus luteum total RNA and the PCR products were hybridized with specific 32P-labeled probe for further confirmation of FP receptor expression. RESULTS: Specific binding sites of 3H-prostaglandin F2alpha in the membranes of bovine iris-sphincter muscle are saturable with an affinity of 9.5 nM and a density of 596 fmoles/mg of protein. Prostaglandin E2, 17-phenyl trinor prostaglandin E2, and GR63799 (EP, EP1, and EP3 receptor agonists, respectively) inhibited 3H-prostaglandin F2alpha binding with an IC50 of 0.0048 microM, 0.0038 microM, and 0.044 microM, respectively. Fluprostenol, a specific FP receptor agonist did not inhibit 3H-prostaglandin F2alpha binding. In contrast, prostaglandin F2alpha and fluprostenol effectively inhibited 3H-prostaglandin F2alpha binding in the bovine corpus luteum with an IC50 of 0.031 microM and 0.037 microM, respectively. CONCLUSIONS: Our results demonstrate that in the bovine iris-sphincter muscle, FP receptors are not expressed and 3H-prostaglandin F2alpha binds to EP1 and EP3 receptor sites. RT-PCR results demonstrated that FP receptor mRNA, which is present in bovine corpus luteum, is probably absent in iris-sphincter muscle.

Pharmacological validation of a feline model of steroid-induced ocular hypertension.

OBJECTIVE: To validate pharmacologically the feline model of steroid-induced ocular hypertension. METHODS: Serial studies were conducted in domesticated adult female cats trained to accept topical ocular drug administration and pneumotonometry. To establish intraocular pressure (IOP) values for each study, measurements were performed at the same time of day for 6 consecutive days. Beginning on day 7, cats received either steroid or vehicle administered topically to both eyes three times a day for approximately 28 days. The IOP measurements were performed daily. RESULTS: After 5 to 7 days of treatment with 0.1% dexamethasone or 1.0% prednisolone acetate, IOP began to increase, reaching peak values within 2 weeks. These values were sustained throughout dosing but declined rapidly to baseline upon cessation of treatment. Maximum IOPs for the dexamethasone- and prednisolone-treated groups averaged 4.5 +/- 0.3 mm Hg (n = 12) greater than the mean IOP value obtained in vehicle-treated cats. Cats treated with 0.25% fluorometholone, 1.0% loteprednol etabonate, and 1.0% rimexolone exhibited increases of 0.6, 1.2, and 1.7 mm Hg, respectively. These values were significantly lower than those observed following treatment with dexamethasone or prednisolone. CONCLUSIONS: The ocular hypertensive effects of selected anti-inflammatory topical ocular steroids in this model are consistent with clinical findings. CLINICAL RELEVANCE: This feline model is a useful tool for assessing the potential IOP liability of novel anti-inflammatory steroids.

Expression of prostaglandin receptors EP4 and FP in human lens epithelial cells.

PURPOSE: To examine the expression of prostaglandin (PG) receptors EP2, EP4, and FP in a human lens epithelial cell line (HLE-B3) at molecular and pharmacologic levels. METHODS: Reverse transcription-polymerase chain reactions (RT-PCR) were performed with total RNA preparations from HLE-B3 cells using sense and antisense primers for each of the three prostaglandin receptors. The PCR products were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. At the pharmacologic level, the expression of EP4 receptors was determined by measuring intracellular cyclic adenosine monophosphate (cAMP) formation in response to PGE2 (EP1, EP2, EP3, and EP4 agonist) and the EP4 receptor-selective antagonist AH23848. The expression of FP receptors in HLE-B3 cells was explored by measuring intracellular [Ca2+]i mobilization. RESULTS: RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors. Hybridization of the PCR products with specific 32P-labeled probes and restriction digestion of the PCR products further confirmed that they were generated from the respective EP2, EP4, and FP mRNAs. The EP receptor agonist PGE2 significantly increased the cAMP level in HLE-B3 cells. The formation of cAMP by PGE2 was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. Stimulation of HLE-B3 cells by the FP receptor agonist fluprostenol increased [Ca2+]i in a time-dependent manner. CONCLUSIONS: The results of the molecular biologic and pharmacologic experiments showed conclusively the presence of EP4 and FP receptor messenger RNAs and proteins, respectively, in HLE-B3 cells.

Intracellular calcium mobilization following prostaglandin receptor activation in human ciliary muscle cells.

PURPOSE: To investigate the presence of specific prostaglandin receptors in primary cultures of human ciliary muscle cells by measuring agonist-stimulated intracellular calcium mobilization. METHODS: The ciliary muscle cells, cultured from postmortem human ciliary muscle explants, were first characterized by anti-desmin and anti-smooth muscle alpha-actin antibodies. Increase in intracellular calcium concentrations, in fura 2-AM loaded human ciliary muscle cells stimulated by prostaglandin receptor agonists, were determined with a digital fluorescence imaging system. RESULTS: The resting intracellular calcium concentration in the fura 2-AM loaded cells was 60.0 +/- 6.0 nM. The threshold concentration of PG receptor agonists needed to increase the concentration of intracellular calcium was 10(-8) M. The stimulation of these cells by PGF2 alpha, 17-phenyl trinor PGE2, and U46619, the FP, EP1, and TP receptor agonists, resulted in the dose-dependent increase of intracellular calcium. CONCLUSION: The results of the present study suggest that EP1, FP, and TP receptors are present in human ciliary muscle cells.

Generation second messengers by prostanoids in the iris-sphincter and ciliary muscles of cows, cats and humans.

We have examined the generation of second messengers after stimulation of feline, bovine, human iris-sphincter and ciliary muscles by selected prostaglandins (PGs). The tissues, labeled or unlabeled with 3H-myo-inositol, were stimulated by a range of concentrations of 16,16-dimethyl PGE2, 11-deoxy PGE1, 17-phenyl trinor PGE2 and PGF2alpha. In both tissues of all three species, 16,16-dimethyl PGE2 and 11-deoxy PGE1 stimulated the formation of cyclic AMP. Butaprost, an EP2 receptor agonist, which was tested only in feline ciliary muscle, generated cyclic AMP. In the feline iris-sphincter and in bovine and feline ciliary muscles, 17-phenyl trinor PGE2, an EP1 receptor agonist, significantly increased inositol phosphate turnover. The FP receptor agonist, PGF2alpha stimulated inositol phosphate turnover in the bovine, feline, and human iris-sphincter muscles and in human ciliary muscles. Feline and bovine ciliary muscles did not respond to PGF2alpha. These results suggest that EP1 receptors are present in feline iris-sphincter muscle and in bovine and feline ciliary muscles. The EP2 receptors exist in both tissue. These results also suggest the presence FP receptors in bovine, feline, and human iris-sphincter and in human ciliary muscles. Bovine and feline ciliary muscles do not appear to express FP receptors.

Detection of EP2, EP4, and FP receptors in human ciliary epithelial and ciliary muscle cells.

We have examined the expression of three prostaglandin (PG) receptors, EP2, EP4, and FP, in a nonpigmented ciliary epithelial cell line (ODMCl-2) and in human ciliary muscle (HCM) cells. Total RNA preparations from either ODMCl-2 or HCM cells were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with sense and antisense primers for each of the three PG receptors. The RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors in both ODMCl-2 and HCM cells. PCR products corresponding to each receptor were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. Pharmacological studies with the EP2 receptor-selective agonist butaprost resulted in a significant increase in the cyclic AMP level in ODMCl-2 cells. The stimulation of cyclic AMP in ODMCl-2 cells by PGE2 and 11-deoxy PGE1, the respective EP1/EP2/EP3/EP4 and EP2/EP3/EP4 receptor agonists, was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. These results conclusively demonstrate the presence of both mRNA and protein for EP2, EP4, and FP receptors in ODMCl-2 and HCM cells.

Comparative studies on prostanoid receptors in human non-pigmented ciliary epithelial and mouse fibroblast cell lines.

To examine the expression of functional prostanoid receptors in the human non-pigmented ciliary epithelial (ODMC1-2) and mouse fibroblast cell lines (NIH 3T3) we have measured the generation of the second messengers, cyclic AMP, inositol phosphates and the mobilization of intracellular calcium ([Ca2+]i) following stimulation by prostaglandin receptor agonists. The amount of cyclic AMP generated was measured by a protein binding method. Radiolabeled inositol phosphates were separated using ion exchange columns and quantified by counting the radioactivity. For intracellular calcium measurements, Fura 2-AM loaded cells were stimulated by PG receptor agonists and the calcium activated fluorescence was measured in a spectrofluorometer. In the ODMC1-2 cell line, the formation of cyclic AMP was stimulated by prostaglandin E2, butaprost and 11-deoxy-prostaglandin E1. The stimulation of cyclic AMP production by prostaglandin E2 was partially inhibited by the EP4 receptor antagonist AH23848. Prostaglandin E2 and 11-deoxy-prostaglandin E1 stimulated the formation of cyclic AMP in NIH 3T3 cells. In ODMC1-2 cells, total inositol phosphate turnover was not increased by 17-phenyl-trinor-prostaglandin F2 alpha, 17-phenyl-trinor-prostaglandin E2 or 11-deoxy-prostaglandin E1. In contrast, all these agonists, with the exception of 11-deoxy-prostaglandin E1, significantly increased total inositol phosphates in NIH 3T3 cells. In the NIH 3T3 cell line, only prostaglandin F2 alpha and 17-phenyl-trinor-prostaglandin F2 alpha increased [Ca2+]i in a dose-dependent manner; in ODMC1-2 cells, neither these agonists nor 17-phenyl-trinor-prostaglandin E2 increased [Ca2+]i. The present studies suggest that in ODMC1-2 cells, EP2 and EP4 receptors but neither EP1 nor FP receptors are expressed; these studies also imply, NIH 3T3 cells express EP2 and FP receptors, while EP1 receptors appear to be absent in this cell line.

Identification of prostanoid receptors in rabbit non-pigmented ciliary epithelial cells.

Preliminary ligand binding studies demonstrated that the membrane preparations of the rabbit nonpigmented ciliary epithelial cell line have 3H-prostaglandin E2 binding sites. The binding sites were specific for 3H-prostaglandin E2 as demonstrated by competition with unlabeled prostaglandin E2. The IC50 of prostaglandin E2 for the inhibition of 3H-prostaglandin E2 binding was 435 nM. The stimulation of adenylyl cyclase and phospholipase C by prostanoid receptor agonists, in rabbit non-pigmented ciliary epithelial cells resulted in the formation of either cyclic AMP or inositol phosphates. Prostaglandin E2 and 16-16-dimethyl prostaglandin E2 (both are EP1, EP2, EP3 and EP4 receptor agonists). 11-deoxy prostaglandin E1 (EP2, EP3 and EP4 receptor agonist), butaprost (EP2 receptor agonist), and prostaglandin D2 (DP receptor agonist) stimulated the formation of cyclic AMP in a dose-dependent manner. Maximal stimulation occurred between 1.25 and 2.5 microM for prostaglandin E2 and 16,16-dimethyl prostaglandin E2 and between 10 and 20 microM for 11-deoxy prostaglandin E1 and prostaglandin D2. Prostaglandin E2 and 16,16-dimethyl prostaglandin E2 were more potent (EC50 of 0.25 microM and 0.42 microM respectively) than 11-deoxy prostaglandin E1, butaprost or prostaglandin D2. The formation of cyclic AMP by prostaglandin D2 was inhibited by BW868C, a highly selective DP receptor antagonist. 17-phenyl trinor prostaglandin E2, prostaglandin F2 alpha and U46619, the EP1, FP and TP receptor agonists, respectively stimulated phospholipase C (as measured by the formation of total inositol phosphates) in a dose-dependent manner. The agonists 11-deoxy prostaglandin E1 and butaprost coupled to adenylyl cyclase via guanine nucleotide binding protein, G8, did not increase the turnover of inositol phosphates. The results of the present study suggest that rabbit non-pigmented ciliary epithelial cells express EP1, EP2, DP, FP and TP receptors.

Studies on prostanoid receptors in ocular tissues.

Cyclooxygenase products of arachidonic acid, for example prostaglandins (PGs), prostacyclin, and thromboxane A2, mediate a wide range of physiological actions. Vasodilation, increased vascular permeability, platelet aggregation and its inhibition, and immunomodulation are some of the important biological actions of cyclooxygenase products (1). Depending on type and dose, PGs cause vasodilation, increase or decrease intraocular pressure, and disrupt the blood-aqueous barrier (2, 3). These actions also vary qualitatively and quantitatively with the animal species. Prostaglandins, like any biological molecule, must act by binding with their specific receptors. Coleman and coworkers (4, 5), from a series of studies with PG agonists and antagonists in vascular and non-vascular smooth muscle preparations, classified PG receptors. This classification led to a greater appreciation of the relationship between PG actions and specific PG receptors in various tissues. Ocular actions of PGs linked with specific PG receptors are far from being clear. In this communication we will review our work on PG binding sites in ocular tissues and PG receptors coupled to adenylyl cyclase or phosphoinositidase C signal transduction pathways in ocular tissues of various animal species.

Prostaglandin E2 and F2 alpha binding sites in the bovine iris ciliary body.

PURPOSE: To determine the regional distribution and selectivity of prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) specific binding sites in membrane preparations from bovine iris ciliary body. METHODS: Bovine eyes were obtained fresh from a local abattoir. Whole irides were separated into sphincter muscle, ciliary body, and remaining iris tissue then homogenized in 50 mM tris buffer, pH 7.5, containing cyclooxygenase, protease, and soybean trypsin inhibitors. Membranes were obtained after three-stage high-speed centrifugation then reconstituted in buffer. Aliquot portions were incubated with 3H-PGE2 or 3H-PGF2 alpha at 37 degrees C for 30 min in a final volume of 500 microliters. Competition studies were performed in the presence of up to 1000-fold excess unlabeled ligand. At the end of a 30-min incubation period, free and membrane bound ligand were separated by rapid filtration through a type HA millipore filter preequilibrated with buffer. The radioactivity bound to the membranes retained on the filter was quantitated in a scintillation counter. RESULTS: The equilibrium dissociation constant and maximum number 3H-PGE2 saturable binding sites were determined by Scatchard analysis. The data best fit to a single binding site model for all three membrane preparations. The majority of the 3H-PGE2 specific binding sites were in sphincter muscle (65%), followed by iris (17%), and ciliary body (18%). 3H-PGE2 alpha binding sites were not measurable in either iris or ciliary body and PGF2 alpha was less competitive than PGE2 in all tissues. The rank order of potency for agonist displacement by both 3H-PGE2 and 3H-PGF2 in sphincter muscle membrane was PGE2 > PGF2 alpha > PGD2 > Iloprost. It was determined that 3H-PGE2 and 3H-PGF2 alpha binding in the bovine sphincter was primarily to EP not FP, DP, or IP prostaglandin receptor types. CONCLUSIONS: The characteristics of PGE2 and PGF2 alpha specific binding sites in the bovine sphincter correlate with its in vitro contractile response to these prostanoids. The inability of PGF2 alpha to effectively compete with PGE2 for specific binding sites in the sphincter and lack of high affinity PGF2 alpha specific binding sites in the iris and ciliary body suggests that this prostaglandin may exert its in vivo effects through PGE2 specific binding sites.

Prostaglandin receptors coupled to adenylyl cyclase in the iris-ciliary body of rabbits, cats and cows.

Stimulation of adenylyl cyclase by prostaglandin (PG) receptor agonists was examined in the iris-ciliary body of rabbits, cats and cows. Iris-ciliary bodies were incubated with or without agonists in Krebs pyruvate buffer containing protease and cyclooxygenase inhibitors. The formation of cAMP in a 15-min period in response to the agonists was measured. In the rabbit iris-ciliary body, all PG receptor agonists at concentrations of 0.062-10.0 microM with the exception of sulprostone stimulated adenylyl cyclase. In this species, 17-phenyl trinor PGE2 and PGF2 alpha stimulated adenylyl cyclase but only at high concentrations (> 5 microM). The order of potency of the agonists was dimethyl PGE2 > PGE2 > 11-deoxy PGE1 = PGD2 > iloprost > PGF2 alpha. The DP receptor antagonist BWA868C had no effect on EP agonist responses, indicating that PGE2, dimethyl PGE2 and 11-deoxy PGE1 were not stimulating DP receptors. In the cat, PGE2, dimethyl PGE2 and 11-deoxy PGE1, were all equally potent in stimulating adenylyl cyclase. The order of potency in the cow was PGE2 > dimethyl PGE2 > 11-deoxy PGE1. However, PGD2, iloprost, 17-phenyl trinor PGE2 and PGF2 alpha were inactive in this species. The results of this study indicate that EP receptor agonists stimulate adenylyl cyclase in the iris-ciliary body of the three species; stimulation by other agonists depends on the species. In the rabbit, the EP2 receptor subtype appears to predominate; such a predominance of EP2 receptors was not observed in the cat and cow.

Prostaglandin E2 binding site distribution and subtype classification in the rabbit iris-ciliary body.

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 EP1, 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 PGE1 suggests the presence of additional EP or non-EP binding sites. There was essentially no binding to EP1 receptor sites as defined by the displacement of 3H-PGE2 by 17-phenyl-trinor PGE2. A weak displacement of 3H-PGE2 by the EP3/EP1 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 EP1 or EP3 agonist activity resulted in breakdown of the blood-aqueous barrier.