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

Prostaglandin E2 and F2a Binding Sitesin the Bovine Iris Ciliary Body

Stephen Csukas, Parimal Bhattacherjee, Lori Rhodes, and Christopher A. Paterson

Purpose. To determine the regional distribution and selectivity of prostaglandin E2 (PCE2) andprostaglandin F2o (PGF2J specific binding sites in membrane preparations from bovine irisciliary body.

Methods. Bovine eyes were obtained fresh from a local abattoir. Whole irides were separatedinto sphincter muscle, ciliary body, and remaining iris tissue then homogenized in 50 raM trisbuffer, pH 7.5, containing cyclooxygenase, protease, and soybean trypsin inhibitors. Mem-branes were obtained after three-stage high-speed centrifugation then reconstituted in buffer.Aliquot portions were incubated with 3H-PGE2 or 3H-PGF2o at 37°C for 30 min in a finalvolume of 500 /tl. Competition studies were performed in the presence of up to 1000-foldexcess unlabeled ligand. At the end of a 30-min incubation period, free and membrane boundligand were separated by rapid nitration through a type HA millipore filter preequilibratedwith buffer. The radioactivity bound to the membranes retained on the filter was quanlitatedin a scintillation counter.

Results. The equilibrium dissociation constant and maximum number 3H-PGES saturable bind-ing sites were determined by Scatchard analysis. The data best fit to a single binding site modelfor all three membrane preparations. The majority of the 3H-PGEa specific binding sites werein sphincter muscle (65%), followed by iris (17%), and ciliary body (18%). 3H-PGF2a bindingsites were not measurable in either iris or ciliary body and PGF 2 Q was less competitive thanPGE2 in all tissues. The rank order of potency for agonist displacement by both 3H-PGE2 and3H-PGF2 in sphincter muscle membrane was PGE2 ) PGF2o ) PGD2 ) Iloprost. It was deter-mined that 3H-PGE2 and 3H-PGF2a binding in the bovine sphincter was primarily to EP not FP,DP, or IP prostaglandin receptor types.

Conclusions. The characteristics of PGEa and PGF2a specific binding sites in the bovinesphincter correlate with its in vitro contractile response to these prostanoids. The inability ofPGF2a to effectively compete with PGE2 for specific binding sites in the sphincter and lack ofhigh affinity PGF2o specific binding sites in the iris and ciliary body suggests that this prosta-glandin may exert its in vivo effects through PGE2 specific binding sites. Invest Ophthalmol VisSci 1993;34:2237-2245.

Jtrostaglandin E2 (PGE2) and prostaglandin F2a

(PGF2a) have well characterized physiologic and patho-

t'rom Kentucky Lions Eye Research Institute, University of Louisville School ofMedicine, Louisville, KY 40292Supported by USPHS Research Grant #EY06918, an unrestricted grant fromResearch to Prevent Blindness, Inc., and The Kentucky Lions Eye Foundation.Submitted for publication December 13, 1991; accepted August 28, 1992.Proprietary Interest, Category: N.Reprint requests: Stephen Csukas, Eye Institute, Medical College of Wisconsin,8700 West Wisconsin Avenue, Milwaukee, WI 53226.

physiologic roles in the eye. '"5 They elicit receptor-me-diated responses including: vasodilation, alteration ofintraocular pressure, and the constriction of irissphincter and ciliary muscles.5"7. Recent efforts havebeen directed at identifying the type and distributionof prostaglandin specific binding sites in ocular tis-sues.8"11. The current nomenclature classifies fivetypes of prostanoid receptors based on their affinityfor naturally occurring prostanoids,12>1S i.e., PGE2

(EP), PGF2a (FP), PGD2 (DP), thromboxane (TP), and

Investigative Ophth;Copyright © Associ;

ology & Visual Science, June 1993, Vol. 34, No. 7n for Research in Vision and Ophthalmology 2237

Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933173/ on 02/19/2018

2238 Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7

prostacydin (IP). In addition, the EP receptor hasbeen subclassified into at least three subtypes EP,, EP2

and EP3.14'16 This nomenclature was developed by

testing the functional responses of various tissues tonaturally occurring prostanoids and their analogs.1315'17

An alternate system of classification was developedbased on second messenger generation in response toprostanoid exposure.18 We have characterized specificprostanoid binding sites in membrane preparationsfrom bovine ocular tissue based on their affinity forselected prostanoid agonists and antagonists.

The characteristics of ocular eicosanoid receptorsare only now being examined. PGE2 receptors in cul-tured rabbit corneal endothelial cells are reported tobe of the EP2 subtype, linked to activation of adenylatecyclase and important in the maintenance of endothe-lial morphology.8 EP2 receptors apparently mediatebreakdown of the rabbit blood-aqueous barrier9 andactivation of EP3 receptors in the whole iris ciliarybody can inhibit adrenergic neurotransmission.10

PGF2a is believed to contract feline and canine irissphincter muscle via a distinct FP receptor.1920 In thebovine sphincter, PGE2 is a more potent contractileagent than PGF2a

2122; however, the receptor responsi-ble for sphincter contraction is unclear. It has beensuggested that iris sphincter contractile state is con-trolled by means of stimulation of a receptor mediatedmyo-inositol 1,4,5-triphosphate (IP3) transductionpathway.23 Stimulation of the FP receptor has beendemonstrated to result in IP3 formation.'6182426

We have previously reported the properties ofPGE2-selective binding sites in the membrane prepara-tions of whole bovine iris ciliary body.". In the currentstudy the kinetic characteristics and regional distribu-tion of 3H-PGE2 and 3H-PGF2a specific binding sites inmembrane preparations of separated bovine sphinctermuscle, iris and ciliary body are examined. Our goal isto determine whether both PGE2 and PGF2a specificbinding sites are present, their regional distributionwithin the iris ciliary body, and, whether our findingscan help interpret data from functional studies.

MATERIALS AND METHODS

Membrane Preparation

Fresh bovine eyes were obtained from a local abattoirand transported on ice to the laboratory. Whole irisciliary body was dissected circumferentially into threecomponent parts: the sphincter muscle, the ciliarybody and processes, and the remaining iris tissue. Thesphincter was first dissected free then the ciliary bodywith attached ciliary processes was dissected. The re-maining iris consisted of vasculature and dilator mus-cle with a minor contamination from sphincter muscle

and ciliary body fragments. Membranes were pre-pared from these three isolated tissues. This investiga-tion adhered to the "ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research."

The tissues were maintained at 2-4°C and the ana-tomic identity of separated tissues was verified in histo-logic sections (not shown). Each tissue was homoge-nized separately in 50 mM tris buffer, pH 7.5, contain-ing cyclooxygenase (1 nM flurbiprofen), protease(0.43 mM PMSF), and 0.01% soybean trypsin inhibi-tors. In addition the homogenization buffer also con-tained 1 mM EGTA. Membranes were obtained afterthree-stage high-speed centrifugation was done as pre-viously reported.11 Tissue from 80 eyes yielded 3.1,4.5, and 9 mg membrane protein27 from sphincter, irisand ciliary body, respectively.

Radioligand Binding AssayThe homogenization buffer was supplemented with2.5 mM manganese (Mn++) and 0.2% bovine serumalbumin for both competition and saturation studies.Membranes were reconstituted in buffer and aliquotportions containing 50-300 ng of membrane proteinwere incubated with 8 nM 3H-PGE2 (200 Ci/mmol) or3H-PGF2a (209 Ci/mmol) at 37°C for 30 min in a finalvolume of 500 ii\. Competition studies were per-formed in the presence of varying concentrations ofunlabeled ligand as described previously.11. A 1000-fold excess of the unlabeled ligand was employed todefine the level of nonspecific binding. Nonspecificbinding represented 30-40% of total binding in thesphincter and ciliary body membranes. The iris mem-branes displayed the highest percentage (70%) of non-specific binding. At the end of a 30-min incubationperiod, free and membrane bound ligand were sepa-rated by rapid filtration through a type HA inilliporefilter preequilibrated with buffer. The radioactivitybound to the membranes retained on the filter wasquantitated in a scintillation counter. Blank values forradioactivity retained by the filters in the absence ofmembrane were subtracted from all values.

Scatchard analysis for the determination of maxi-mum number of binding sites (Blnax) and equilibriumdissociation constant (KJ was performed in sphinctermuscle, iris, and ciliary body preparations using 8 nM3H-PGE2 and zero-1000 nM unlabeled ligand.

Data AnalysisData were analyzed using the analytic portion of thecomputer software program Sigma Plot Version 4.0(Jandel Scientific) and EBDA/Ligand (Biosoft). EBDAwas used to perform the transformations necessary toanalyze the Scatchard data. Sigma Plot was used to fitdata by an iterative process to the four-parameter lo-gistic equation. This analysis produced a "best fit" to a


Prostaglandin Binding Sites in Uveal Tissues 2239

sigmoidal curve. The analysis provided a value for theIC50 (concentration of unlabeled ligand that displaced50% of specifically bound labeled ligand) of the com-peting ligand and a coefficient of cooperativity de-scribing the relative shape of the sigmoidal function.

Materials

Prostaglandins (Cayman Chemical, Ann Arbor, MI)were made up to a 14.2 mM concentration in 95%ethanol. Working solutions contained less than 0.18%ethanol. BWA868C was donated by Wellcome Re-search Laboratories, United Kingdom. Iloprost wasdonated by Schering AG, Germany. Labeled ligandswere purchased from New England Nuclear (Boston,MA). All other compounds were obtained from SigmaChemical Company (St. Louis, MO).

RESULTS

Analysis of PGE2- and PGF2a Binding Site Data

In a typical experiment, using membrane from sepa-rated iris, after subtraction of the filter blanks, we find5588 DPM (disintegrations per minute) captured onfilters from the total binding tubes and 3379 DPMfrom the nonspecific binding tubes, allowing a maxi-mum of 1708 DPM for specific binding calculations.In sphincter muscle, where the greatest binding oc-curred, there were 23,122 DPM from the total bindingtubes and 6128 from the nonspecific binding tubesleaving 16594 DPM as specific binding. These data areconverted to femtomoles (fmol) bound per milligramof membrane protein and analyzed by the Ligand/EBDA binding program for estimation of kinetic pa-rameters. The program allows for the determinationof whether the data from one or several saturationstudies best fit to a single- or multiple-site model.

SH-PGE2 saturation data for sphincter, iris, andciliary body revealed Hill plot values for these tissuesof 0.75, 0.81, and 0.88, respectively. The K^ and Bmax

values for sphincter, iris, and ciliary body were: 3.1,28, and 17.8 nM, and 842, 154, and 82 fmoles bound/

mg protein, respectively. The data analysis indicated abest fit to a single-site model for each tissue. PGF2a

binding sites in sphincter were not sufficiently satu-rated by 8nM PGF2a to report meaningful scatchardanalysis data. No significant specific PGF2c, binding toiris or ciliary body membranes was measured.

These data are summarized in Table 1.

Distribution of PGE2 Binding Sites

The distribution of PGE2 specific binding sites withineach separated region of the iris-ciliary body was cal-culated. Bmax values from Scatchard analysis data wereused to determine the percentage of binding sites lo-cated within each tissue type. Based on the quantity ofmembrane protein recovered for each region of theiris ciliary body, the majority of PGE2 binding siteswere determined to be located in the sphincter musclefollowed by the ciliary body and the iris (Table 1). Al-though the sphincter represented 19% of the mem-brane protein, 65% of the PGE2 binding sites werelocated within this tissue. Ciliary body, representing54% of the recovered membrane protein, contained18% of the PGE2 specific binding sites.

Competition Studies3H-PGE2 Binding Sites in Sphincter, Iris, and CiliaryBody. The relative selectivity of unlabeled PGE2 andPGF2a to displace bound 8 nM 3H-PGE2 is shown inFigure 1. The IC50 values for displacement of 3H-PGE2

specific binding by PGE2 from membranes were as fol-lows: sphincter, 7.5 nM; iris, 10.5 nM; and ciliarybody, 7.9nM. In all three membrane preparations,PGF2a was 30-100-fold less effective than PGE2 atcompeting for the 3H-PGE2 binding site. The IC50 val-ues for displacement of 3H-PGE2 specific binding byPGF2a from membranes were as follows: sphincter,792 nM; iris, 304 nM; and ciliary body, 608 nM.

iH-PGF2a Binding Sites in Sphincter, Iris, ami CiliaryBody. The relative affinities of unlabeled PGF2a orPGE2 to displace 8 nM 3H-PGE2 or sH-PGF2a bindingfrom sites in the sphincter are shown in Figure 2. TheIC50 values for displacement of 3H-PGF2<( were 0.8 nM

TABLE l. Scatchard Analysis of the Regional Distribution of PGE2Binding Sites in Separated Bovine Iris-Ciliary Body MembranePreparations

Sphincter Iris Ciliary Body

PGE.,nmol/lfmol bound/mg protein

% of whole ICB membrane proteinfmol bound/tissue (%)

3.1 ± 1.2 28 ± 14 17.8 ± 7.0842 ± 5 1 154 ± 4 3 82 ± 1 4

1965

2717

5418

Values are mean ± SEM.



SPHINCTER IRIS CILIARY BODY

11 10 9 8

LIGAND CONCENTRATION (-L0G[M])

• PGEg o PGF2fl

FIGURE l. Competitive inhibition of 8 nM 3H-PGE2 (200 Ci/mmol) specific binding to bovinesphincter, iris, and ciliary body membranes by unlabeled PGE2 and PGF2a. Incubations wereperformed in a 500 jitl final volume, for 30 min at 37.5°C in triplicate, in the presence orabsence of various concentrations of unlabeled ligand. 100% specific binding is defined asthe amount of bound 3H-PGE2 displaced in the presence ] ^M unlabeled PGE2 (n = 6 foreach PGE2 and n = 3 for each PGF2a point represented, values represent the mean ± SEM).Calculated IC60 values (nM) are shown next to each curve.

SPHINCTER SPHINCTER

to•J

1

O

2;

(X,

s

100

90

80

70

60

50

40

30

20

10

n

}17

7.5 /

{

V Jr h

/A "Y 792-9

7

/

LIGAND CONCENTRATION (-L0G[M])

• PGE,

FIGURE 2. Comparison of competitive curves for inhibition of 8 nM 3H-PGE2a (left panel) and8 nM sH-PGF2n (209 Ci/mmol; right panel) specific binding to bovine sphincter muscle mem-branes by unlabeled PGF2(, and PGE2. Incubation conditions were the same as those in Fig. J(n = 3 or more for each ligand, values represent the mean ± SEM). Calculated IC50 values(nM) are shown next to each curve.



for PGE2 and 14.82 nM for PGF2a, an 18.5-fold higheraffinity for PGE2, indicating that PGE2 actually has agreater affinity for 3H-PGF2a binding sites. Iris andciliary body membranes were not examined for com-petition studies with PGF2a because they did not signif-icantly bind this ligand.

Selectivity Study of Sphincter PGF2a Binding Sites. Alimited study was performed to determine whetherPGF2a (Table 2) was binding to EP or FP binding sites(Fig. 3). No difference was observed in the ability of4-4000 nM of the highly selective FP agonist, 17-phenyl trinor PGF2a (Table 2) versus the less selectivePGF2a (Table 2) to displace 4 nM sH-PGF2a or 3H-PGE2 when compared to the displacement by 4-400nM of PGF2a. In addition, 4-400 nM PGE2 was moreeffective than either PGF2a and 17-phenyl trinorPGF2a at displacing either labeled ligand.

Displacement of3H-PGE2 or 3H-PGF2a by EP, FP, DPand IP Agonists and a DP Antagonist in the SphincterMuscle. The selectivity of 3H-PGE2 and 3H-PGF2a bind-ing sites was examined by testing their displacement byEP, FP, DP, and IP selective agonists as well as a DPselective antagonist. A single concentration (400 nM)of each agonist, equivalent to the dose of PGE2 thatproduces 80% displacement of labeled PGE2 andPGF2a (see Fig. 3) was used. The selectivity of theseagonists and antagonists is detailed in Table 2.

Displacement of 3H-PGE2

Figure 4 illustrates the ability of IP, EP, FP, and DPselective agonists, and a DP selective antagonist to dis-place 4 nM 3H-PGE2 from its specific binding sites inthe bovine sphincter membrane preparation. The IPagonist Iloprost (Table 2) marginally displaced 3H-PGE2, indicating that the radioligand was not boundto IP sites. The DP agonist PGD2 and the DP selective

TABLE 2. Prostanoid Receptor Agonists andAntagonists Used to Displace RadiolabeledPGE2 or PGF2a From the SphincterMembrane PreparationCompound

Agonists (all 400 nM)PGE.,17-Phenyl trinor PGE.,,

11-DeoxyPGE,PGF2a1 7-Phenyl tiinor PGF2a

PGD2[loprost

Antagonist (4 ^M)BWA868C

Selectivity

EPEP1

EP2 > EPSFP > EPFPDP > TPIP > EP1

DP

Reference

31, 32, 3628, 29, 3327-30,

32,3914, 3129, 3229, 3032, 3332, 36

28

• * 2 0 -

40nM 400nM 4000nM

PGF. PGE2 17-phenyl

The relative selectivity of each compound is indicated.

4nM 40nM 400nM 4000nM

FIGURE 3. Comparison of the selectivity of PGF2tt, 1 7-phenyltrinor PGF2a and PGE2 for displacement of either 4 nM 3H-PGF2a (upperpanel) or 3H-PGE2 (lowerpanel) specific bindingto bovine sphincter muscle membranes. Incubation condi-tions were the same as those in Fig. 1 (n = 3 for each ligand).

antagonist BWA868C28 displaced some of the boundradioligand (Table 2), although significantly less thanthat displaced by unlabeled PGE2. The highly selectiveantagonist, BWA868C, displaced even less radioligandthan PGD2 indicating that DP receptors are probablynot involved. PGF2n and 17-phenyl trinor PGF2a dis-placed approximately equivalent amounts of the ra-dioligand, although the FP selective 17-phenyl trinorPGF2a displaced less 3H-PGE2 than did PGF2a. Thislack of selectivity in displacement suggests that 3H-PGE2 is not bound to FP sites.

The nonselective EP agonist PGE2 (Table 2) dis-placed more 3H-PGE2 than any other agonist or antag-onist. The EP2/EP3 (11-deoxy PGE,) and EP, (17-phenyl trinor PGE2) selective agonists (Table 2) werenearly as effective as unlabeled PGE2 and significantly



more effective than PGF2a or 17-phenyl PGF2a at dis-placing 3H-PCE2. This evidence suggests that the ra-dioligand 3H-PGE2 is highly selective for EP bindingsites in bovine sphincter. Because the Kj value forPGE2 in sphincter muscle is 3.1 nM (Table 1), the con-centration of EP selective agonists used (400 nM) werenot chosen to demonstrate whether EP2 or EP, sitespredominate but to demonstrate 3H-PGE2 selectivityfor EP versus non-EP binding sites.

Displacement of 3H-PGF2

Figure 5 illustrates the ability of IP, EP, FP, and DPselective agonists and a DP selective antagonist to dis-place 4 nM sH-PGF2a from its binding sites in the bo-vine sphincter membrane preparation. Iloprost wasunable to displace any of the 3H-PGF2a, indicating thatit was not bound to IP sites. The DP agonist PGD2 andthe selective antagonist BWA868C did displace someof the bound radioligand, although significantly lessthan that displaced by unlabeled PGF2a. The highlyselective DP antagonist displaced even less radioligandthan PGD2, indicating that DP receptors may be onlymarginally involved.

Unlabeled PGF2n and 17-phenyl trinor PGF2tt dis-placed equivalent amounts of the 3H-PGF2a. The EPsubtype selective agonists 11-deoxy PGE, and 17-phenyl trinor PGE2 were nearly as effective as PGF2a atdisplacing radioligand. The nonselective EP agonistPGE2, displaced more radioligand than any other ago-

SELECTIVITY OF 3H-PGEO MEMBRANE BINDING

SELECTIVITY OF H-PGF, MEMBRANE BINDING

FIGURE 4. Comparison of the ability of IP, EP, FP, and DPselective agonists and antagonist to displace 4 nM 3H-PGE2

from its specific binding sites in the bovine sphincter mem-brane preparation. Concentration of all agonists are 400nM. The concentration of the antagonist BWA868C is 4 ftM.Incubation conditions were the same as those in Fig. 1 ex-cept the membranes receiving antagonist were preincubatedfor 10 minutes with antagonist before addition of radioli-gand (n = 3). Values are the mean ± SEM.

FIGURE 5. Comparison of the ability of IP, EP, FP, and DPselective agonists and antagonist to displace 4 nM 3H-PGF2a

from its specific binding sites in the bovine sphincter mem-brane preparation. Concentration of all agonists are 400nM. The concentration of the antagonist BWA868C is 4 fM.Incubation conditions were the same as those in Fig. 1 ex-cept the membranes receiving antagonist were preincubatedfor 10 min with antagonist before addition of radioligand (n= 3). Values are mean ± SEM.

nist or antagonist. The lack of selectivity in displace-ment of 3H-PGF2 a combined with the effectiveness ofEP selective agonist suggests that, 3 H-PGF2 a does notbind selectively to FP sites but nonselectively to other,predominantly EP sites.

DISCUSSIONIn the current study we have demonstrated the re-gional distribution and binding characteristics of 3H-PGE2 and 3H-PGF2a binding sites in membrane prepa-rations of separated bovine sphincter muscle, iris, andciliary body. Our previously reported K,, and Bmax val-ues for PGE2 in the whole iris ciliary body were 13 nMand 162 fmoles/mg protein, respectively.11 Data fromthe present study indicate a lower K,, (3.1 nM) and ahigher Bmax (842 fmoles bound/mg protein) in the iso-lated sphincter muscle, whereas iris and ciliary body Kjvalues are higher and Bmax values are lower than wholetissue values. Iris sphincter receptor density is fivefoldhigher than iris and tenfold higher than ciliary body.

For comparison, the Kj for PGE2 in bovine lutealcells is 2.4 nM.34 In baboon and rabbit uterine mem-branes,35 the Kj values for PGE2 are in the range of3.3-13 nM; similar to the 3.1-28 nM range we foundin separated ocular tissues. In rat kidney,36 the K̂ andBmax values for PGE2 and PGF2a were 9.37 and 9.17nM, and 2150 and 440 fM bound/mg protein, respec-tively. The regional distribution of 3H-PGE2 bindingsites was examined in the human kidney.37 Kj values



for PGE2 ranged from 3.7 nM in the cortex to 6.2 nMin the outer medulla; and Bmax values ranged from 143fM bound/mg protein in the cortex to 335 fM bound/mg protein in the outer medulla. The affinity of PGE2

for its binding sites does not appear to vary greatlybetween species or tissues. However, the relatively lowBmax values for these binding sites and the lipophiliccharacter of the agonist used served to increase theerrors in measurement and rendered collection of di-rect binding data more difficult.

Another focus of the current study was to examinethe selectivity of EP and FP binding sites in bovinesphincter muscle. In human kidney37 PGE2 is 51 timesmore effective than PGF2a at displacement of specifi-cally bound SH-PGE2; the rank order of potency ofselected prostanoid agonists is PGE2 ) PGF2a ) PGI2

= PGD2. Our data using 17-phenyl PGF2a demon-strated that sites selective for FP verses EP agonistsmay not exist in the iris sphincter (Fig. 3). In addition,the data in Figure 4 indicate the same order of potencyfor prostanoid agonist displacement of 3H-PGE2 inour bovine sphincter membrane preparation as re-ported for human kidney EP sites. The rank order ofpotency of prostanoid agonist displacement of SH-PGF2n (Fig. 5) from bovine sphincter muscle was PGE2

= PGF2a > PGD2 > PGI2. PGF2a has been reported tocompete with PGD2 for DP binding sites30 and the datasuggest (Fig. 5) the possibility of a small contributionof 9H-PGF2tt bound to DP sites. Overall, these resultsdemonstrate that 3H-PGF2(I was predominately boundto EP rather than FP sites and raises the possibility thatthe bovine iris does not contain a significant numberof FP receptor sites.

One may make correlations between these bindingdata and the functional response of bovine iris ciliarybody to prostaglandins. PGE2 and PGF2(V are potentagonists for contractile responses in isolated irissphincter muscle.21'23 Dong et al21 reported that theequimolar ratio for contraction of bovine sphincter byPGF2a versus PGE2 to be 53:1. The addition of the TPreceptor antagonists EP045 and EP092 had little ef-fect on these molar ratios. The potent IP receptor ago-nist ZK96480 exhibited weak contractile activity, sug-gesting that the bovine sphincter contractile responseto Iloprost is caused by partial agonist activity at EPsites. Suzuki and Kobayashi22 demonstrated that con-traction of bovine iris occurred as a direct result ofprostaglandin binding to muscle rather than to nerve;indicating the presence of PG receptor sites associatedwith intraocular muscle membranes. These same au-thors examined the contractile response of isolatedbovine iris sphincter segments to PGE,, PGE2, andPGF2a. Dose-response curves for PGE2 and PGE, ex-posure began at 1 nM and are maximal below 10 fiM.The response to PGF2a begins at 100 nM and by 10 /nMreaches only 10% of the maximal PGE2 contractile re-

sponse. Although the bovine iris sphincter respondedto both PGE2 and PGF2a, PGE2 was two orders of mag-nitude more effective than PGF2a; data that corre-spond well to binding data from our current competi-tion studies (Fig. 1). Data from the current study indi-cating that the Kj for PGE2 in bovine sphincter is 3.1nM correlate well to a functional response curve thatbegins at lnM.

Yoshitomi and Ito19 and van Alphen et al20 demon-strated that dog and cat iris sphincter responses toPGE2 and PGF2M were opposite those of bovine irissphincter, and were 100-fold more sensitive to PGF2n

than to PGE2. In addition, contractile response curvesto PGF2a begin at less than 0.1 nM, suggesting a verylow K.J value for PGF2n in these tissues. Bovine irissphincter muscle responsiveness to PGF2a-ester expo-sure lies between the more responsive cat and dog andthe less responsive rabbit, pig, guinea pig, and hu-man.38 Dog dilator muscle contracts to PGF2(, expo-sure; with only a marginal response to PGE2 andPGE,.19 We found no appreciable specific PGF2ff bind-ing to the membrane preparation derived from bovineiris containing dilator muscle fibers. Suzuki andKobayashi22 report that the bovine iris dilator muscleresponds to PGF2a but not PGE2 or PGE,; however, intheir study, 1 fiM PGF2a was required to produced asmall change in the electrically evoked contraction ofiris dilator muscle and had no effect on ciliary muscle.This may help explain why only marginal PGF2o bind-ing to these tissues was observed in our study. Alter-nately, there may be no FP receptors present in thesetissues.

Woodward et al, 198929 demonstrated that PGF2a

induced ocular hypertension in cats was negativelycorrelated to FP receptor stimulation and positivelycorrelated to iris sphincter contraction. They con-clude that PGF2a may alter intraocular pressure bybinding to a prostanoid receptor other that those ofthe FP or TP classification. Their study demonstratesthat prostanoids introduced into the anterior chambermay not exert a single discrete physiological responsein this tissue. Our finding of a lack of measurable FPreceptor sites in bovine iris and ciliary body may ruleout a functional role for these receptors. We cannotdirectly correlate the EP binding sites we identifiedwith functional receptors responsible for muscle con-traction. However, it is generally accepted that IPS re-lease and the subsequent intracellular Ca++ elevationare closely involved in sphincter muscle contraction.38

In the cat and dog iris sphincter PGF2a apparentlybinds to an FP receptor12 stimulating the release ofIP3

38 with the subsequent contraction of the muscle. Ifwe postulate a similar mechanism for the bovine irissphincter PGF2a or PGE2 would bind to an EP recep-tor to contract the muscle. EP2 and EP3 receptor sub-type activation results in a stimulation or inhibition,



respectively, of cyclic adenosine monophosphate re-lease, The release of IP3 after EPj receptor subtypestimulation may be responsible for sphincter contrac-tion. Yousafzai38 suggested that the predominance ofthe IP3 second messenger pathway in the cat and dogiris sphincter explains a high sensitivity to PGF2a. Inrabbits, humans, and other species they suggest an in-verse relationship between the action of the IP3 andcyclic adenosine monophosphate second messengerpathways on contraction and relaxation, which resultsin a sphincter muscle that is nonresponsive to bothPGF2a and PGE2. Perhaps bovine contractile re-sponses are mediated by an EPj not an FP receptor.The result is a sphincter muscle that is responsive toPGE2 but exhibits an intermediate sensitivity to PGF2a

that falls between the more sensitive dog and cat, andthe less sensitive human, rabbit, and guinea pigsphincter muscle.

Our current study has determined the localizationand kinetic parameters of PGE2 and PGF 2 Q bindingsites in the bovine eye; however, these findings pointout how little is understood about the outcome ofprostanoid interaction with their receptors in oculartissue.

Key Words

PGE2, PGF2a, receptor, binding sites, bovine, sphincter, iris-ciliary body

Acknowledgments

This study was supported by USPHS research grant numberEY-06918, ihe Kentucky Lions Eye Research Foundation,and an unrestricted grant from Research to Prevent Blind-ness. The authors thank Dr. Marcia Jumblatt, MarilynMcLendon, Mary Henning, and Hollis Brunner for theirassistance in the preparation of this manuscript.

References

1. Cole DF, linger WG. Prostaglandins as mediators forthe responses of the eye to trauma. Exp Eye Res.1973;17:357-368.

2. Eakins KE. Prostaglandins and non-prostaglandin me-diated breakdown of the blood-aqueous barrier. ExpEye Res. 1977;25:483-498.

3. Rahi AH, Bhattacherjee P, Misra RN. Release of pros-taglandins in experimental immunocomplex en-dophthalmiiis and phacoallergic uveitis. BrJ Ophthal-mol. 1978;62:105-109.

4. Bhattacherjee P. The role of arachidonate metabolitesin ocular inflammation. In: Bito LZ, Stjernchantz J,eds. The Ocular Effects of Prostaglandins and Other Eico-sanoids. New York: Alan R. Liss; 1989:211-227.

5. Bito LZ, Camras CB, Gum GG, Resul B. The ocularhypotensive effects and side-effects of prostaglandinson the eyes of experimental animals. In: Bito LZ,Stjernchantz J, eds. The Ocular Effects of Prostaglandinsand Other Eicosanoids. New York: Alan R. Liss;1989:349-368.

6. Camras CB, Bito LZ, Eakins KE. Reduction of intraoc-ular pressure by prostaglandins applied topically tothe eye of conscious rabbits. Invest Ophthalmol Vis Sci.1977;16:1125-1134.

7. Kaufman PL, Crawford K. Aqueous humor dynamics:how PGF2a lowers intraocular pressure. In: Bito LZ,Stjernchantz J, eds. The Ocular Effects of Prostaglandinsand Other Eicosanoids. New York: Alan R. Liss;1989:387-416.

8. Jumblatt M, Paterson CA. PGE2 effects on corneal en-dothelial cyclic AMP synthesis and cell shape are me-diated by a receptor of the EP2-subtype. Invest Ophthal-mol VisSci. 1991;32:360-365.

9. Protzman EE, Woodward DF. Prostanoid-inducedblood-aqueous barrier breakdown in rabbits involvesthe EP2 receptor subtype. Invest Ophthalmol Vis Sci.1990;31:2463-2466.

10. Ohia SE, Jumblatt JE. Prejunctional inhibitory effectsof prostanoids on sympathetic neurotransmission inthe rabbit iris-ciliary body. / Pharmacol Exp Ther.1990;255:ll-16.

11. Bhattacherjee P, Csukas S, Paterson CA. Prostaglan-din E2 binding sites in bovine iris-ciliary body. InvestOphthalmol Vis Sci. 1990;31:1109-1113.

12. Kennedy I, Coleman R, Humphrey PPA, Levy GP,Lumley P. Studies on the characterization of prostan-oid receptors: a proposed classification. Prostaglan-dins 1982; 24:667-689.

13. Coleman RA, Humphrey PPA, Kennedy I, Lumley P.Prostanoid receptors—the development of a workingclassification. TIPS 1984;5:303-306.

14. Armstrong RA, Matthews JS, Jones RL, Wilson NH.Characterization of PGE2 receptors mediating in-creased vascular permeability in inflammation. In: Sa-muelsson B, Paoletti R, Ramwell PW, eds. Advances inProstaglandin, Thromboxane, and Leukotriene Research,Vol. 21. New York: Raven; 1990:375-378.

15. Coleman RA, Kennedy I, Sheldrick RLG, TolowinskaIY. Further evidence for the existence of three sub-types of PGE2 sensitive (EP-) receptors. BrJ Pharma-col. 1990;91:407P.

16. Giles H. More selective ligands at eicosanoid receptorsubtypes improve prospects in inflammatory and car-diovascular research. TIPS 1990; 11:301-304.

17. Coleman RA, Kennedy I, Sheldrick RLG. New evi-dence with selective agonists and antagonists for thesubclassification of PGE2-sensitive (EP) receptors. In:Samuelsson B, Paoletti R, Ramwell PW, eds. Advancesin Prostaglandin, Thromboxane, and Leukotriene Re-search, Vol. 17. New York: Raven; 1987:467-470.

18. Muallem S, Merrill BS, Green J, Kleeman CR, Yama-guchi DT. Classification of prostaglandin receptorsbased on coupling to signal transduction systems. Bio-chemj. 1989;263:769-774.

19. Yoshitomi T, Ito Y. Effects of indomethacin and pros-taglandins on the dog iris sphincter and dilator mus-cles. Invest Ophthalmol Vis Sci. 1988;29:127-132.

20. van Alphen GWHM, Angel MA. Activity of prostaglan-din E, F, A and B on sphincter, dilator and ciliarymuscle preparations of the cat eye. Prostaglandins.1975;9:157-166.



21. Dong YJ, Jones RL, Wilson NH. Prostaglandin E re-ceptor subtypes in smooth muscle: agonist activities ofstable prostaglandin analogues. Br J Pharmacol.1986;87:97-107.

22. Suzuki R, Kobayashi S. Response of bovine intra-ocu-lar muscles to transmural stimulation in the presenceof various prostaglandins. Exp Eye Res. 1989; 36:789-798.

23. Yousufzai SYK, Tachado SD, Carter KD, Abdel-LatifAA. Short-term densitization of prostaglandin F2 re-ceptors increases cyclic AMP formation and reducesinositol phosphate accumulation and contraction inthe bovine iris-sphincter. Curr Eye Res. 1989; 8:1211 —1220.

24. Macphee CH, Drummond AH, Otto AM, de Asua LJ.Prostaglandin F2a stimulates prophatidylinositol turn-over and increases the cellular content of 1,2-diacyl-glycerol in confluent resting Swiss 3T3 cells. J CellPhysiol. 1984;119:35-40.

25. Woodward DF, Fairbairn CE, Goodrum DD, KraussAH-P, Ralston TL, Williams LS. Caa+ transientsevoked by prostanoids in Swiss 3T3 cells suggest anFP-receptor mediated response. In: Samuelsson B,Paoletti R, Ramwell PW, eds. Advances in Prostaglan-din, Thromboxane, and Leukotriene Research, Vol. 21.New York: Raven; 1990:367-370.

26. Black FM, Wakelam MJO. Activation of inositol phos-pholipid breakdown by prostaglandin F2a without anystimulation of proliferation in quiescent NIH-3T3 fi-broblasts. BiochemJ. 1990;266:661-667.

27. Lowry DH, Rosenbrough NJ, Farr AL, Randall RJ.Protein measurement with the Folin phenol reagent./BiolChem. 1951; 193:265-275.

28. Giles H, LeffP, Bolofo ML, Kelly MG, Robertson AD.The classification of prostaglandin DP-receptors inplatelets and vasculature using BWA868c, a novel, se-lective and potent competitive antagonist. Br J Phar-macol. 1989;96:291-300.

29. Woodward DF, Burke JA, Williams LS, et al. Prosta-glandin F2a effects on intraocular pressure negativelycorrelate with FP-receptor stimulation. Invest Ophthal-mol VisSci. 1989;30:1838-1842.

30. Miller WL, Weeks JR, LauderdaleJW. Biological activ-

ities of 17-Phenyl, 18,19,20- trinorprostaglandins.Prostaglandins 1975;9:9-19.

31. Eglin RM, Whiting RL. Classification of prostanoidreceptors. In: Willis AL, ed. CRC Handbook of Eicosan-oids: Prostaglandins and Related Lipids, Vol. 2. Boca Ra-ton, Florida: CRC Press, Inc.; 1989:273-289.

32. Coleman RA, Kennedy I, Humphrey PPA, Bunce K,Lumley P. Prostanoids and their receptors. In: Han-seh C, Sammes PG, Taylor JB, eds. Comprehensive Me-dicinal Chemistry, Vol 3. Oxford: Pergamon Press;1990:643-714.

33. Ito S, Negishi M, Sugama K, Okuda-Ashitaka E,Hayaishi O. Signal transduction coupled to PGD2. In:Samuelsson B, Paoletti R, Ramwell PW, eds. Advancesin Prostaglandin, Thromboxane, and Leukolriene Re-search, Vol. 21. New York: Raven Press; 1990:371-374.

34. Lin MT, Rao Ch V. Selected properties of[3H] prostaglandin E, binding to dispersed bovine lu-teal cells. Mol Cell Endocrinol. 1978;9:311-320.

35. Hodam JR, Snabes MC, Kuehl TJ, Jones MA, HarperMJK. Characterization of prostaglandin E2 binding touterine membranes from baboon, rabbit and treeshrew (Tupaia belangeri). J Mol Endocrinol. 1989;3:33-42.

36. Limas C, Limas CJ. Prostaglandin receptors in rat kid-ney. Arch Biochem Biophys. 1984; 233:32-42.

37. Eriksson LO, Larsson B, Hedlund, H, Andersson KE.Prostaglandin E2 binding sites in human renal tissue:characterization and localization by radioligand bind-ing and autoradiography. Ada Physiol Scand.1990; 139:393-404.

38. Yousufzai SYK, Chen A, Latif AA. Species differencesin the effects of prostaglandins on inositol triphos-phate accumulation, phosphatidic acid formation,myosin light chain phosphorylation and contraction iniris sphincter of the mammalian eye: interaction withthe cyclic AMP system. J Pharmacol Exp Therap.1988;27:1064-1072.

39. Lawrence RA, Jones RL, Wilson NH. Characteriza-tion of receptors involved in the direct and indirectactions of prostaglandins E and I on the guinea-pigileum. Br J Pharmacol. 1992; 105:271-278.


Documents

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