Exp. Eye Res. (1990)

51, 585-590

Stimulatory Cyclase

Effects of Prostaglandin D, Analogues in Rabbit Iris-Ciliary Body Membrane YASUMASA

GOH”

AND

MASAYUKI

on Adenylate Fractions

NAKAJIMA”

Shionogi Research Laboratories, Osaka, and aOsaka Medical College, Takatsuki, Japan (Received 9 February 1990 and accepted in revised form 77 April 7990) Effectsof prostaglandin(PG)D, analogueson the adenylatecyclaseactivity in membranefractionsof the iris-ciliary body complexwere studied.PGD, dose-dependently activated the adenylatecyclasewith a maximal activity increaseof about 60 %. Theconcentrationrequiredto causea half-maximalstimulation (EC,,) was about 5 x lo-’ M. The stimulatory effect of PGD, wastotally dependenton GTPwith EC,, for GTPat about lo-’ M. The rank order of potency of PGD,analoguesfor stimulatingthe adenylatecyclase and BW245C (a selective PGD, agonist)> PGD, > PGD,> 9&PGD,. PGD, metabolitesand PGD, analogueswhich have little hypotensiveactivity were essentiallyineffectivein stimulatingthe adenylate cyclase.This rank order wasstrikingly similarto that reportedpreviously for their intraocular pressurelowering effects.One exceptionwas PGD, methylester.This compound,though reportedly effective in reducingIOP, failedto activate the adenylatecyclaseby itself, presumablybecauseits hypotensiveeffect is due to its hydrolyzed product, PGD,. Theseresultsindicate that the abilitiesof PGD, analoguesto stimulatethe adenylate cyclaseof the iris-ciliary body complexin GTP-dependentmanner are highly correlatedwith their ocular hypotensiveactivities, and suggestthat a PGD, receptor-stimulatoryGTPbinding protein-adenylate cyclasecomplexis involved in the PGD,-inducedocular hypotension. Key words: prostaglandinD, ; prostaglandinD, analogue; prostaglandinD, receptor: GTPregulatory protein ; intraocular pressure; adenylatecyclase: iris-ciliary body complex; rabbit.

1. Introduction Prostaglandins (PGs) have been shown to affect intraocular pressure (IOP) in many animal species (Waitzman and King, 196 7 ; Camras, Bito and Eakins, 1977; Stern and Bito, 1982; Bite, 1984; Lee, Podos

and Severin, 1984: Bite, Baroody arid Miranda, 1987). For example, topical applications of higher doses of PGE, and PGF,, have been reported to cause an initial transient elevation of IOP followed by longlasting reduction in rabbits (Camras, Bito and Eakins, 1977; Lee, Podos and Severin, 1984). The initial ocular hypertension and some of the side effects which are seen after these PGs, such as increases in aqueous humour protein, and iridal and conjunctival hyperaemia, are highly dependent on the dose and the animal species used (Camras, Bito and Fakins, 19 77 ; Lee, Podos and Severin, 1984). In fact, hypotensive doses of PGF,, and its isopropylester have been shown to induce no initial hypertension or no severe intraocular inilammations in cats (Bito, 1984 ; Bito, Baroody and Miranda, 1987), and these compounds

have been proposed to be a new therapeutic agent for glaucoma. Furthermore, recent reports (Giuffre’, 1985 ; Alm and Villumsen, 1986; Camras et al., 1989) showed that they are in fact effective in reducing IOP of normotensive as well as glaucomatous humans. This work has been previously present at the 91st annual meeting of the Society of Ophthalmology. Japan (Goh et al., 1987), and at the 1988 Ocular Drug Therapy Update held at Newport Reach, CA. * For correspondence at: Shionogi Research Laboratories, Fukushima-ku, Osaka 553. Japan.

00144835/90/110585+06

.$03.00/O

In our previous reports (Goh et al., 1986, 1988a, b) we demonstrated that PGD, in a wide range of dosages reduces rabbit IOP without causing the initial hypertension or any intraocular inflammatory responses, and we proposed that PGD, or its analogues may be a promising therapeutic agent for glaucoma. In fact, we subsequently found that PGD, is effective in

lowering human IOP without causing any intraocular inflammatory response (Goh et al., 1986). More detailed experiments using normal human volunteers have been recently conducted, and the result will be published elsewhere (Nakajima et al., in press). It has been repeatedly demonstrated that the ocular hypotension induced by PGF,, is due to increased uveoscleral outflow (Crawford and Kaufman, 19 8 7 ; Hayashi, Yablonski and Bite, 1987 ; Nilsson, Stjernschantz and Bill, 1987) which may be caused by the change in ciliary muscle tone. Our recent results suggested that. unlike after PGF,,, the reduction of rabbit IOP after PGD, is caused by the inhibition of aqueous humour flow rate through the anterior chamber (Goh et al., 1989). Recently, we further examined effects of PGD, analogues and metabolites on the rabbit IOP (Goh et al., 1988b). Among several PGD,-related compounds, BW245C, a specific PGD, agonist (Town, CasalsStenzel and Schillinger, 1983), PGD,, PGD,, 9p-PGDZ and PGD, methylester were found to be effective in reducing IOP, which has been subsequently confirmed (Woodward et al., 1990). An interesting finding in our previous study was that PGD, analogues that effectively reduce the IOP of rabbits are also reportedly 0 1990 AcademicPressLimited 41-2

Y. GOH

586

AND

M. NAKAJIMA

guanosine-5’-O-(3-thio-triphosphate) (GTPyS), 3-isobutyl-1-methylxanthine (IBMX), and forskolin were obtained from Sigma (St Louis, MO). All other chemicals were of reagent grade. Solutions

I 0

I 3

I 6 Incubation

I IO time (min)

I 15

J

FIG. 1. Time-dependent CAMP formation in membrane fractions of iris-ciliary body complex. Membrane fractions were incubated for the indicated times in the standard assay mixture with ( l ) or without 10 pM of PGD, (0). Each point shows the mean of three determinations.

effective in inhibiting ADP-induced human platelet aggregation (Bundy et al., 1983 : Narumiya and Toda, 1985). Therefore, we suggested that the PGD,-induced ocular hypotony is mediated by a specific PGD, receptor analogous to that found in human platelets (Goh et al., 1988b). In human platelets, it is known that PGD, binds to a specific PGD, receptor which is coupled with the adenylate cyclase-stimulating system (Siegl, Smith and Silver, 1979 ; Cooper and Ahern, 19 79). Furthermore, all human platelet-type PGD, receptors, such as those found in rat mast cells and rabbit stomach strips, were also shown to be coupled with adenylate cyclase, similar to that reported in human platelets (Holgate et al., 19 80 ; Narumiya and Toda, 1985). These observations suggest that IOP reduction after PGD, application may also be coupled to an adenylate cyclase-stimulating system. In the present study, we investigated effects of PGD, analogues on the adenylate cyclase of rabbit irisciliary body membrane fractions, and found that the adenylate cyclase-stimulating activities of PGD, analogues are highly correlated with their ocular hypotensive activities in rabbits. 2. Materials

and Methods

Chemicals

PGs were kindly supplied by Ono Pharmaceutical Company. BW245C [(S-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxy-propyl) hydantoin)], a potent PGD, agonist, was a gift from Dr S. Narumiya of Kyoto University. These compounds were dissolved in ethyl alcohol and stored at - 2O’C. Adenosine-S’-triphosphate (ATP), guanosine-5’-triphosphate (GTP).

Phosphate-buffered saline (PBS) consisted of 10 mM sodium phosphate (pH 7.4) and 140 mM NaCl. Buffer A for the tissue homogenization contained O-25 M sucrose, 50 mM Tris HCl (pH 74), 5 mM MgCl,, 5 mM dithiothreitol (DTT), and 20 ,UM indomenthacin. Buffer B for the resuspension of membrane pellets consisted of 25 mM Tris HCl (pH 7.4) 5 mM MgCl,, 1 mM ethylene glycol-bis(~-aminoethyester)-N,N,N’N’-tetraacetic acid (EGTA). 1 mM DTT, and 20 FM indomethacin. Buffer B also served as the basic assay mixture of the adenylate cyclase. Preparation of Membrane Fraction

Male Japanese white rabbits (2.5-3 kg) were killed by overdosing with pentobarbital, and their eyes were quickly enucleated and washed with PBS. The irisciliary body complex was removed and homogenized with 10 ml of buffer A using a Potter homogenizer. After centrifugation at 600 g for 1 min, the supernatant was further centrifuged at 6000 g for 10 min. The resulting pellet was then resuspended with buffer A and centrifuged again at 6000 g for 10 min. The final pellet was resuspended in buffer B and used for the adenylate cyclase assay. Typically, the final suspension contained about 1 mg ml-’ of protein. Adenylate Cyclase Assay

The standard assay mixture contained 1 mM ATP, 0.5 mM IBMX, 10 ,UM GTP, O-1 % bovine serum albumin, and an ATP-regenerating system (5 mM creatine phosphate and 30 units ml creatine kinase-‘) in buffer B. When PGs were added, the final ethanol concentration was adjusted to be less than 0-l % (v/v). The assay was started with the addition of membrane (20 ,ul of membrane suspension) to the prewarmed assay mixture (80 ~1). After incubation at 37’C, the reaction was stopped by the addition of 100 ,ul of 0.2 N HCl and subsequent cooling in an ice bath. After centrifugation at 3000 g for 10 min. an aliquot of each supernatant was diluted and used for triplicate determinations for adenosine-3’, 5’-cyclic monophosphate (CAMP) by radioimmunoassay using an [1251]CAMP assay kit (Amersham). Protein was determined according to the method described by Lowry et al. 1951) using bovine serum albumin as the standard. 3. Results

When membrane fractions of the iris-ciliary body complex were incubated in the standard assay mix-

STIMULATION

OF

OCULAR

ADENYLATE

CYCLASE

BY PGD,

587

TABLE I

Effects of PGD,, PGE,, PGF,, andforskolin on the adenylate cyclaseactivity in membranefractions of the iris--diary body complex

Addition

Amount (PM)

n

Activity (pm01 min-’ mg protein-‘)

Relative activity (% to control)

None PGD, PGE, PGF,, Forskolin

10 IO 10 100

5 5 7 6 4

26.5k2.1 41-6 & l-6* 36-6&O-8* 32.1k2.5 245.8 +45.4*

100 157 138 121 928

* P < 0.05 by Duncan’s multiple range test.

tin-e, CAMP was time-dependently formed. The rate of CAMP formation was linear for at least 15 min of incubation time in the control as well as PGD,stimulated (10 ,UM) samples (Fig. 1). The basal activity of the membrane adenylate cyclase was 26.5 + 2.1 pmol min-’ mg protein-’ (Table I). When stimulated with 10 ,LLM of PGD,, PGE, or PGF,,, the activity increased to 41.6, 36.6 or 32.1 pmol min-’ mg-l, respectively. Among the three PGs, PGD, was the most effective. With 100 ,UM of forskolin, the activity increased by about ninefold. By using iris sphincter muscle of rabbits, PGF,, isopropylester and PGA, were reported to activate the formation of CAMP by about three- and twofold, respectively (Yousutiai, Chen and Abdel-Latif, 1988). Although the magnitude of the facilitation appears to be slightly lower in the present experiment, these observations agree that PGs, though not strong, do stimulate the formation of CAMP in the rabbit iris-ciliary body complex. Figure 2 indicates the dose-dependent stimulation of the adenylate cyclase activity by PGD,. The stimulatory effect of PGD, increased approximately linearly within the dose range from 1Oms to 10m5 M and saturated at around 10e4 M, resulting in a sigmoidal dose-response curve. The concentration required to cause half-maximal effect (EC,,) was about 5 x lo-’ M. This value was comparable to the EC,, value observed in the PGD,-induced adenylate cyclase stimulation in human platelet membranes (0.3-0.7 PM; Cooper and Ahern, 1979; Schafer et al., 1979) the I& value of PGD, binding in intact human platelets (0.4 ,UM; Siegl, Smith and Silver, 1979), and the EC,, value in the PGD,-induced relaxation of rabbit stomach strips (0.1 ,UM; Narumiya and Toda, 1985) but higher than the IC,, value for inhibiting the ADP-induced human platelet aggregation (32 nM; Whittle, Moncada and Vane, 1978). The effect of PGD, was seen only in the presence of GTP as shown in Fig. 3. The increase in the enzyme activity due to the addition of 10 PM of PGD, reached a maximum at approx. 10m6M of GTP: higher concentrations of GTP did not further facilitate the stimulatory effect of PGD, (Fig. 3, inset). The addition of a hydrolysis-resistant GTP analogue, GTPyS was

also effective in demonstrating the effect of PGD, (data not shown). With GTP#, the basal as well as the PGD,-stimulated adenylate cyclase activity increased more than twofold when compared with GTP, but the relative stimulatory effect of PGD, was unchanged. The maximal stimulatory effect of GTPyS was observed at around lo-’ M. This concentration was about one order less than that with GTP, which is presumably due to the hydrolization of GTP by the endogenous GTPase activity. The concentrations of GTP and GTPyS to induce half-maximal effects were approximately lo-’ and 2 x lo-” M, respectively, and are similar to that required to obtain the half-maximal adenylate cyclase stimulation with PGE, in human platelet membranes (about lo-’ M for GTP; Jakobs, Bauer and Watanabe, 198 5) or with other hormones in adenylate cyclase-stimulating systems (see Gilman, 1987, for a review). Figure 4 illustrates effects of various PGD,-related compounds on the adenylate cyclase activity together with their ocular hypotensive activities in rabbits which have been reported previously (Goh et al., 1988b). PGD, analogues effective in stimulating the adenylate cyclase were BW245C, PGD,, PGD, and 9& PGD,. These analogues were also effective in reducing IOP with the same descending order of potency. PGD, metabolites, i.e. PGF,, (9a,l l/3-PGF,), A12-PGJ, (9deoxy-Ae* 12-13,14diiydro PGD,), 13,14dihydro- 15 keto PGD,, and PGJ, (9-deoxy-Ag-PGD,), and a PGD, analogue, PGD,, which have little hypotensive activity, were all essentially ineffective in stimulating the adenylate cyclase activity. One exception was PGD, methylester, which is highly hypotensive, but caused only a weak stimulation of the adenylate cyclase. PGD, methylester has been shown to be quickly hydrolyzed by the endogenous esterase into PGD, in the blood circulation of rats (Suzuki et al., 1987). Furthermore, PGF,, esters have been reported to be degraded into PGF,, within 1 hr in the rabbit eye after topical application (Bito and Baroody, 1987). Therefore, we tentatively conclude that the hypotension observed after PGD, methylester is caused by its hydrolyzed product, PGD,. Thus, these results indicate a high correlation between the ocular hypotensive and

Y. GOH

PGD,

concentration

AND

M. NAKAJIMA

(M)

FIG. 2. Effect of PGD, concentration on the adenylate cyclase activity in membranefractions of the iris-ciliary body complex. Membrane fractions were incubated for 10 min with the indicated concentration of PGD, in the standard assay mixture. Each point shows the mean (+ s.E.M.)of five experiments.

FIG. 4. The adenylate cyclase-stimulating(bottom) and the intraocular pressure-decreasing(top) effects of PGD, and PGD,-relatedcompounds.Bottom, Effect on the adenylate cyclase activity in membranefractions of the rabbit iris-ciliary body complex. Each bar showsthe mean ( ~s.E.M.) of more than four experiments. Top, Effect on rabbit IOP. The figure wasdrawn from the data reported previously (Goh et al., 198813).The magnitude of IOP reduction is dependenton the initial (or control) IOP level and the IOP generallycannot get lower than the episcleral venous pressure.Therefore, to compare the hypotensive activities obtained from animal groups with different IOP levels,eachIOPdatumwasexpressed asa ratio (R)according to the following equation: R = (IOP,-IOP,)/(IOP, - lo), whereIOP, andIOP, areIOP levelsof control andPG-treated eye, respectively. The episcleralvenous pressurehas been reported to be about 10 mmHg in rabbits (Brubaker, 1967). PGD,-Me= PGDJmethylester; DHK-PGD,= 13,14dihydro-15keto PGD,.

the adenylate cyclase stimulatory analogues.

effect of PGD,

4. Discussion 0 ”

10-e

IO-’ GTP

lo-6

IO-

IO+

(M)

FIG. 3. Effect of GTP concentration on the adenylate cyclase activity in membranefractions of the iris-ciliary body complex. Main panel. Membrane fractions were incubated for 10 min in the assaymixture containing the indicatedconcentrationof GTPwith (a) or without 10 pM PGD,(0). Inset showthe differencein the adenylatecyclase activity in pm01mg-r min-’ (activity in the presenceminus that in the absence of PGD,, ordinate) at indicated concentrationof GTP(abscissa). Eachpoint showsthe mean of three determinations.

In the present study we found that PGD, stimulates the adenylate cyclase activity in membrane fractions of the rabbit lris-ciliary body complex. A similar observation has previously been reported with PGF,, isopropylester and PGA, by using iris sphincter muscles of rabbits (Yousufzai, Chen and Abdel-I&if, 1988). In this study we found that the PGD,-induced adenylate cyclase activation was dependent on the presence of GTP. It is well known in a hormone-

STIMULATION

OF

OCULAR

ADENYLATE

CYCLASE

induced adenylate cyclase stimulation that the hormone binds to a receptor, which then, in the presence of GTP, dissociates a stiiulatory guanine nucleotide-binding protein (Gs protein) into its active subunits, and the adenylate cyclase is finally stimulated by the active subunits. Therefore, the present result indicates that the stimulatory effect of PGD, on the adenylate cyclase is not due to a non-specific effect of PGD,, but suggests that a specific receptor complex containing the Gs protein is involved in the PGD,induced adenylate cyclase stimulation. This is consistent with the observation that the effective concentration ranges of PGD, and GTP for the adenylate cyclase activation is similar to those reported in other systems mediated by the human platelet-type PGD, receptor and by Gs proteins (see Results). The most interesting observation of the present study is that the rank order of potency of PGD, analogues for stimulating the adenylate cyclase is highly correlated with that previously reported for reducing the rabbit IOP (Goh et al., 1988b). Because the iris-ciliary body complex is one of the sites involved in IOP regulation, these observations suggest that the PGD,-induced ocular hypotony may be caused by adenylate cyclase stimulation in a certain part of the iris-ciliary body complex. It has been shown that the stimulation of ocular adenylate cyclase, such as with forskolin and cholera toxin, reduces IOP by reducing aqueous humour flow rate (Gregory et al., 1981; Caprioli and Sears, 1983; Burstein, Sears and Mead, 1984; Sears, 1985). In these systems, it has been assumed that CAMP generated in the ciliary epithelium inhibits the formation of aqueous humour (Sears, 1985). Because PGD, has also been reported to cause ocular hypotension by suppressing aqueous flow rate in rabbits (Goh et al., 1989), it is possible that PGD, binds to the receptor located in the ciliary epithelium, and thereby activates the adenylate cyclase, inhibiting the aqueous secretion. One discrepant observation for this assumption is that, in spite of the large difference in the adenylate cyclase-stimulating effect, the hypotensive activity does not differ much between forskolin and PGD, (cf. Caprioli and Sears, 1983 vs. Goh et al., 1988b). However, because the iris-ciliary body complex is composed of many functional units, all of which may contain adenylate cyclase but are likely to respond differently to PGD,, it is not unreasonable to expect that the direct adenylate cyclase stimulator forskolin causes much stronger overall activation than does PGD,. Thus, we hypothesize that PGD,. by binding to the human platelet-type PGD, receptor at the ciliary epithelium, stimulates the formation of CAMP and then inhibits the aqueous formation. However, to prove unequivocally our hypothesis and to identify the

BY PGD,

589

active site of PGD,, a more detailed differentiation of the iris-ciliary body complex is obviously necessary. The PGF,- or PGA,-induced IOP reduction is reported to be due to the facilitation of uveoscleral outflow (Hayashi et al., 1987; Nilsson, Stjemschantz and Bill, 1987). Since these PGs have been known to cause a relaxation of precontracted ci!iary muscles (van Alphen, Wilhelm, and Elsenfeld, 1977) and pilocarpine which induces a contraction of the muscle antagonizes the IOP reduction induced by PGF,, (Crawford and Kaufman, 1987), it has been assumed that the uveoscleral flow is facilitated as a result of the reduction of tone in the ciliary muscle or an effect on the connective tissue matrix between the ciliary muscle bundles (Hayashi, Yablonski and Bito, 1987). Using iris sphincter muscles, Yousufxai, Chen and Abdel-Latif (1988) have found that the CAMP formed by PGs is inversely correlated with the muscle contraction, and suggested that CAMP acts as a relaxant for the sphincter. Therefore, it is possible that CAMP formed in the ciliary muscle causes relaxation of the muscle and then increases the uveoscleral outflow. However, since the ciliary muscle, unlike the sphincter, normally responds to PGs with a relaxation, the role of CAMP in this muscle is yet unclear. As previously shown (Goh et al., 1986, 1988b), PGD, is a rather weaker hypotensive agent among the three classical PGs, i.e. PGD,, PGE, and PGF,,. Supposing that our hypothesis is correct, it is conceivable that the simultaneous inhibition of phosphodiesterase, such as by theophylline, may potentiate the hypotensive activity of PGD,, and thereby makes PGD, more useful as an anti-glaucoma agent. In conclusion, we demonstrated that the activities of PGD, analogues in stimulating the adenylate cyclase of the iris-ciliary body complex in a GTP-dependent manner are highly correlated with their ocular hypotensive activities. Since the agonist specificity in these two systems is strikingly similar to that reported for the specific PGD, receptor in human platelets (Bundy et al., 1983; Narumiya and Toda, 1985; Goh et al., 1988b), we suggest that a human platelet-type PGD, receptor coupled with the Gs protein and adenylate cyclase, is involved in the PGD,-induced ocular hypotension in rabbits. Acknowledgments We are grateful to Drs 0. Hay&hi of Osaka Bioscience Institute and I. Azuma of Osaka Medical College for their helpful discussions and encouragements during the study. Thanks are also due to Ms. M. Akao for her secretarial assistance. References Alm, A. and Villumsen, J. (1986). Intraocular response and ocular side effects after prostagiandin F,, eye drops. A single dose response study in humans. Proc. Int. Sot. Eye Res.4, 14.

Bito, L. 2. (1984). Comparison of the ocular hypotensive

590

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efficiency of eicosanoids and related compounds. Exp. Eye Res. 38, 181-94.

Bito, L. 2. and Baroody.R. A. (1987). The ocular pharmacokintics of eicosanoidsand their derivatives. 1. Comparison of ocular eicosanoidpenetration and distribution following the topical applicationof PGF,,, PGF,,1-methylesterand PGF,,-l-isopropylester.Exp. Eye Res. 44, 217-26. Bito, L. Z., Baroody, R. A. and Miranda, 0. C. (1987). Eicosanoids asa new classof ocular hypotensiveagents. 1. The apparent therapeutic advantagesof derived prostaglandinsof the A and B type as comparedto primary prostaglandinsof the E,F and D type. Exp. Eye Res.44, 825-37. Brubaker, R. F. (1967). Determinationof episcleralvenous pressurein the eye. Arch. Ophthuhnol.77, 110-4. Bundy, G. L., Morton, D. R., Peterson,D. C., Nishizawa,E.E. and Miller, W. L. (1983). Synthesisand platelet aggregation inhibiting activity of prostaglandinD analogues.J. Med. Chem. 26, 790-9. Burstein, N. L., Sears,M. L. and Mead, A. (1984). Aqueous flow in human eyes is reducedby forskolin, a potent adenylatecyclaseactivator. Exp. Eye Res. 39, 745-9. Camras,C. B., Bito, L. Z. and Eakins.K. E.(19 77). Reduction of intraocular pressure by prostaglandins applied topically to the eyes of conscious rabbits. Invest. Ophthalmol.Vis. Sci. 16, 1125-34. Camras,C. B., Sieblod,E. C.. Lustgarten, J. S., Serle, J. B., Frisch, S.C., Podos. S. M. and Bito, L. Z. (1989). Maintained reduction of intraocular pressureby prostaglandin F,,- 1-isopropylesterappliedin multipledosesin ocular hypertensive and glaucomapatients. OphthalmoZogy 96, 1329-37. Caprioli, J. and Sears,M. (1983). Forskolin lowers intraocular pressurein rabbits,monkeys,and man. Lanceti. 958-9. Cooper,B. and Ahern, D. (1979). Characterizationof the platelet prostaglandinD, receptor. J. Clin. Invest. 64, 586-90. Crawford, K. and Kaufman, P. L. (1987). Pilocarpine antagonizesprostaglandm F,,-induced ocular hypotensionin monkeys.Arch. Ophthalmol.105, 1112-6. Gilman, A. F. (1987). G proteins: transducersof receptorgeneratedsignals.Annu. Rev. Biochem.56, 61549. Giuffre, G. (1985). The effectsof prostaglandinF,, in the human eye. Albrecht von Gruefe’s Arch. Klin. Exp. Ophthalmol.222. 13941. Goh,Y., Araie, M., Nakajima,M., Azuma, I. and Hayaishi,0. (1989). Effect of topical PGD, on the aqueoushumor dynamics in rabbits. Albrecht von Gruefe’s Arch. Klin. Exp. Ophthdmol. 227, 476-81. Goh, Y., Nakajima, M., Azuma, I. and Hayaishi. 0. (1986). ProstaglandinD, reducesintraocular pressure:a possible therapeutic agent for glaucoma.Proc. Int. Sot. Eye Res. 4, 14.

Goh, Y., Nakajima,M.. Azuma, I. and Hayaishi, 0. (1987). High correlation of ocular hypotensive and anterior uveal adenylate cyclase-stimulatoryactivity of prostaglandin D,-related compounds.Actu Sot. OphthulmoI. Jpn. 91 (Suppl.), 181 (in Japanese). Goh, Y., Nakajima, M., Azmna, I. and Hayaishi,I. (1988a). ProstaglandinD, reducesintraocular pressure.Br. J. Ophthulmol. 72, 4614. Goh, Y., Nakajima,M.. Azuma, I. and Hayaishi,0. (1988b). Effects of prostaglandin D, and its analogues on intraocular pressurein rabbits. Jpn. J. Ophthulmol. 32, 471-80. Gregory, D., Sears,M., Bausher,L., Mishima, H. and Mead, A. (1981). Intraocular pressureand aqueousflow are decreasedby cholera toxin. Invest. Ophthalmol. Vis. Sci. 20. 371-81.

AND

M. NAKAJIMA

Hayashi, M., Yablonski. M. E. and Bito, L. Z. (1987). Eicosanoids asa new classof ocularhypotensiveagents. Invest. Ophthulmol.

Vis. Sci. 28, 163943.

Holgate, S.T., Lewis,R. A., Maguire, J. F., RobertsII, L. J.. Oates, J. A. and Austen, K. F. (1980). Effects of prostaglandmD, on rat serosalmastcells: discordance betweenimmunologicmediatorreleaseand cyclic AMP levels.J. Immunol. 125, 1367-73. Jakobs.K. H., Bauer, S. and Watanabe, Y. (1985). Modulation of adenylate cyclase of human platelets by phorbol ester.Eur. I. Biochem. 151, 42 5-30. Lee, P., Podos, S.M. and Severin, C. (1984). Effect of prostaglandin F,, on aqueous humor dynamics of rabbit, cat, and monkey.Invest. Ophthulmol. Vis. Sci. 25, 1087-93. Lowry, 0. H., Rosebrough,N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurementwith Folin phenol reagent.1. Biol. Chem. 193. 265-75. Nakajima.M., Goh, Y., Azuma, I. and Hayaishi,0. Effectsof prostaglandinD, and its analogue,BW245C in human eyes. Invest. Ophthulmol. Vis. Sci. (in press). Narumiya, S.and Toda, N. (1985). Differentresponsiveness of prostaglandinD,-sensitivesystemsto prostaglandin D, and its analogues.Br. J. Phurmucol. 85, 367-75. Nilsson,S. F. E., Stjernschantz,J. and Bill, A. (1987) PGF,, increasesuveoscleraloutflow. Invest. Ophthulmol. Iris. Sci. 28 (Suppl.),284. Schafer,A. I., Cooper,B., O’Hara,D. and Handin.R. (1979). Identification of platelet receptorsfor prostaglandinI, and D,. J. Biol. Chem. 254, 2914-7. Sears,M. L. (1985). Regulationof aqueoustlow by adenylate cyclasereceptorcomplexin the ciliary epithelium.Am. 1. Ophthulmol. 100, 194-8. Siegl, A. M., Smith, J. B. and Silver, M. J. (1979). Specific binding sitesfor prostaglandinD, on human platelets. Biochem. Biophys. Res. Commun. 90, 291-6. Stern, F. A. and Bito, L. Z. (1982). Comparisonof the hypotensiveand other ocular effectsof prostaglandinE, and F,, on cat and rhesus monkey eyes. Invest. Ophthulmol.

Vis. Sci. 22, 588-98.

Suzuki, F., Hayashi, H., Ito. S. and Hayaishi, 0. (1987). Methyl esterof prostaglandinD, asa delivery system of prostaglandinD, into brain. Biochim. Biophys. Actu 917. 224-30.

Town, M. H., Casals-Stenzel, J. and Schillinger, E. (1983). Pharmacologicaland cardiovascular properties of a hydantoin derivative, BW245C, with high affinity and selectivityfor PGD,receptors.Prostuglundins 25, 13-28. van Alphen, G. W. H. M.. Wilhelm, P. B. and Elsenfeld, P. W. (1977). The effect of prostaglandinson the isolated internal muscles of the mammalian eye, including man. Dot. Ophthulmol. 42, 397-415. Waitzman, M. B. and King. C. D. (1967). Prostaglandin influenceson intraocular pressureandpupil size.Am. J. Physiol. 212, 329-34.

Whittle, B. J. R., Moncada, S. and Vane, J. R. (1978). Comparisonof the effectsof prostacyclin (PGI,), prostaglandin E, and D, on platelet aggregationin different species.Prostuglundins 16, 3 73-88. Woodward, D. F., Hawley, S. B., Williams, L. S., Ralston, T. R., Protzman. C. E., Spada, C. S. and Nieves. A. L. (1990). Studieson the ocular pharmacologyof prostaglandin D,. Invest. Ophthdmol. Vis. Sci. 31, 13846. Yousufzai, S. Y. K., Chen, A. L. and Abdel-Latif, A. A. (1988). Speciesdifferencesin the effects of prostaglandinson inositoltrisphosphateaccumulation,phosphatidic acid formation, myocin light chain phosphorylation and contraction in iris sphincter of the mammalianeye: interaction with the cyclic AMP. 1. Pharmacol. Exp. Ther. 247. 1064-72.