Documenta Ophthalmologica 44,2 : 421-434, 1977 PROSTAGLANDINS AND OCULAR INFLAMMATION*

D.W.R. HALL & I.L. B O N T A

(Delft~Rotterdam, The Netherlands) Keywords: Blood-aqueous barrier, Corticosteroids, Ocular inflammation, Ocular hypertension, Prostaglandins, Non-steroidal anti-inflammatory drugs ABSTRACT Evidence for prostaglandins as mediators of some inflammatory responses is briefly, but critically discussed. The hypothesis that prostaglandins are mediators of ocular inflammation is presented and discussed according to the tenets of Miller & Melmon (1970). Prostaglandins are released after mechanical stimulation, paracentesis, laser irradiation, (non)-immunogenic uveitis in experimental animals, and in patients with acute anterior uveitis. Prostaglandins were neither found after antidromic stimulation of the trigeminal nerve, oculomotor nerve stimulation, intracameral formaldehyde, topical nitrogen mustard, nor in patients with open angle glaucoma. Administering of prostaglandins produces transient ocular hypertension, increased vascular permeability and miosis. Ocular hypotension has been shown to follow the hypertensive response. Hypertension is considered to be due to a breakdown of the blood-aqueous barrier of the ciliary processes and iris. Prostaglandins of the E series are the most potent, and species differences occur. Prostaglandin E 1 potentiates the histamine-induced increase in vascular permeability of the conjunctiva, but not the urea. Polyphloretin phosphate aspeciflcally blocks the ocular actions of prostaglandins. Non-steroidal anti-inflammatory drugs inhibit prostaglandin synthetase and the ocular effects of arachidonic acid, its substrate. Prostaglandins are poorly catabolised by the eye, but are removed from ocular fluids by a transport mechanism in the anterior urea. This becomes inoperative in (non)-immunogenic uveitis in rabbits. allowing prostaglandins to accumulate. Anti-inflammatory steroids decrease the availability of arachidonic acid for prostaglandin synthetase. Intermediates formed in the biosynthesis of prostaglandins may well be more important in inflammation than the prostaglandins themselves. INTRODUCTION A l t h o u g h p r o s t a g l a n d i n s were i d e n t i f i e d in biological tissues m a n y years ago, o n l y in t h e last 15 years have t h e i r p r o p e r t i e s b e e n e x t e n s i v e l y s t u d i e d and reviewed (see B e r g s t r 6 m et al., 1968; K a r i m & Hillier, 1974; K a d o w i t z et al., 1975), and t h e i r i m p o r t a n c e in i n f l a m m a t i o n fully a p p r e c i a t e d . T h e criteria set o u t b y K o c h as m o d i f i e d b y Miller & M e l m o n ( 1 9 7 0 ) , have served as a y a r d - s t i c k f o r t h e c o n s i d e r a t i o n o f s u b s t a n c e s as m e d i a t o r s o f

* Part of this review was presented at the 171st Congress of the Dutch Ophthalmological Society.

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inflammation. These criteria were: 1. isolation of abnormally large quantities of the substance from an inflammatory site at appropriate times during the inflammatory process; 2. when the suspected mediator is administered as an exogenous substance it should reproduce at least part of the inflammatory process characteristic for that species; 3. the inflammatory response should be inhibited by specific peripheral antagonists which block or inactivate the substance, and 4. the inflammatory response should be suppressed by pretreatment with agents which deplete the animal of the chemical. The purpose of this review will be to survey the evidence for the pathophysiological role of prostaglandins as mediators of some forms of ocular inflammation, and further to delineate possible target mechanisms for the development of therapeutics for use in ophthalmology. The reader is also referred to the reviews of Waitzman (1970), Neufeld & Sears (1973) and Perkins (1975) for coverage of the possible physiological actions of prostaglandins on the eye.

THE ROLE OF PROSTAGLANDINS IN SYSTEMIC INFLAMMATORY RESPONSES

In the following paragraphs we will briefly survey the evidence for consideration of prostaglandins as mediators of some forms of systemic inflammation. Inflammation is characterised by five cardinal signs: erythema and oedema, pyrexia and algesia, and further loss of function. These characteristics have been recognized since Greek and Roman times. Inflammatory exudates from animals (Willis, 1969) and man (Greaves et al., 197 i) have been shown to contain large quantities of prostaglandins. Sustained erythema can be found after intradermal injection of prostaglandin E l , (PGE1) in nanogram concentrations in human skin (Solomon et al., 1968; Horton, 1963), and increased vascular permeability can be found after injection in the s k i n of experimental animals (Horton, 1963; Kaley & Weiner, 1968; Crunkhorn & Willis, 1969). Prostaglandins have been shown to' cause in vitro leukocyte migration, at least in the rabbit (Kaley & Weiner, 1971). There is evidence to suggest that prostaglandins mediate vasodilation in a variety of tissues (Lewis, 1971), the resulting hyperemia possibly leading to local temperature elevation. When PGE 1 was injected into the cerebral ventricles it was shown to be an extremely potent pyretic substance (Milton, 1973). Furthermore, concomitantly increased levels of E prostaglandins were found in the cerebrospinal fluid when fever was induced in cats by bacterial endotoxin (Feldberg & Saxena, 1972). On the other hand, although pain could not be produced when

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prostaglandins were applied to the human blister base (Horton, 1965) hyperalgesia was produced in combination with histamine or bradykinin (Ferreira, 1972). The oedema producing actions of histamine and bradykinin were potentiated by prostaglandins in the rat paw and the guinea-pig skin (Moncada et al., 1973; Williams & Morley, 1973). When prostaglandins are injected into the dog knee joint severe debilitating arthritis develops (Rosenthale et al., 1972). The biosynthesis of prostaglandins from arachidonic acid by prostaglandin synthetase has been shown by Vane (1973) to be inhibited by various non-steroidal anti-inflammatory drugs (e.g., acetylsalicylic acid, indomethacin), and this has been suggested to be the rationale of their anti-inflammatory activity. Depletion of the substrate of prostaglandin synthetase, arachidonic acid, by feeding rats on a diet containing a deficiency in essential fatty acids was found to reduce the rat paw carageenin oedema (Bonta et al., 1976) thus supporting the role of prostaglandins as proqnflammatory agents. Recently, the mode of action of antiqnflammatory steroids has been related to prostaglandin biosynthesis (Lewis & Piper, 1975; Gryglewski et al., 1975; Nijkamp et al., 1976). It is now thought that corticosteroids reduce the availability of arachidonic acid for cyclo-oxygenase, the initial enzyme in the prostaglandin synthetase complex, and that this might well be their primary anti-inflammatory activity. It is commonly believed that inhibition of prostaglandin synthetase by non-steroidals is the rationale of their anti-inflammatory activity in man. This is probably an oversimplification, and has received increasing criticism (Smith, 1975; Bonta et al., 1977). Moreover, although we have presented considerable evidence to suggest prostaglandins as pro-inflammatory agents there is a growing realization that they can also play an anti-inflammatory role (Willoughby & Dieppe, 1976; Bonta et al., 1977; Morley, 1977). The concept of a dual role for prostaglandins in the regulation of inflammation raises the critical question as to the desirability of suppression at the level of prostaglandin synthesis. Substantiation of this hypothesis will undoubtedly have considerable ramifications for the development of therapeutic agents.

ROLE OF PROSTAGLANDINS IN INFLAMMATORY CONDITIONS OF THE EYE

Evidence for the presence of prostaglandins One of the earliest studies to implicate prostaglandins in inflammation was carried out on the rabbit eye by Ambache and his colleagues in 1965. They showed that mechnical stimulation of the iris or paracentesis evoked release 423

of a substance they termed 'iNn' into the anterior chamber. Mechanical irritation of the iris produces miosis, increased vascular permeability and elevation of intraocular pressure (IOP) in rabbits. Irin had already been shown to be a normal constituent of rabbit iris (Ambache, 1957), and was later identified as PGE2 and PGF2a (/~nggard & Samuelsson, 1964; Ambache et al., 1 9 6 6 ; Ambache & Brummer, 1968). Since the pioneering studies of Ambache, the presence of prostaglandins has been confirmed both indirectly and directly, after scratching the rabbit iris (Neufeld et al., 1972a; Cole & Ungar, 1973) and after paracentesis (Miller et al., 1973). In a recent study, ruby laser irradiation of the iris, used in the treatment of certain types of glaucoma and known to produce transient increases in lOP, was accompanied by a release of prostaglandins in the rabbit eye (Neufeld et al., 1972a; Ungar et al., 1974). The presence of large amounts of E prostaglandins in the aqueous humour has also been detected in immunogenic uveitis induced by BSA (Eakins et al., 1972a) and in non-immunogenic uveitis induced by Shigella endotoxin (Bhattacharjee, 1975). Surprisingly, substantial quantities of PGE1 while only small amounts of PGE2 were found. Normal or inflamed iris or ciliary bodies of rabbits contains only PGE 2 and PGF2a (Ambache & Brummer, 1968). On the other hand, inflammation induced bY paracentesis resulted in lower levels of PGE 2 with no PGEI (Miller et al., 1973). The possibility was advanced that PGEI had been produced by white blood cells invading the inflamed anterior chamber (Eakins et al., 1972a). It is known that white blood ceils (polymorphonuclear leukocytes) can enter an inflamed eye (Meyers & Pettit, 1974). A further explanation of the exceedingly high levels of prostaglandins in immunologically induced uveitis may be a deficiency in the prostaglandin transport system from the eye. Bito & Salvador (1972) established the existence of an active transport system for the rapid removal of p~ostaglandins from intraocular fluids, and that during BSAinduced uveitis in rabbits this transport mechanism no longer functions (Bito, 1974) thus allowing prostaglandins to accumulate. Substantially raised levels of E prostaglandin have been found in the aqueous humour of patients having acute anterior uveitis (Eakins et al., 1972b). In contrast, only slightly raised prostaglandin levels were found in one study in patients with o p e n angle glaucoma (Wyllie & Wyllie, 1972), while no increase was observed in another study (Podos et al., 1972). Chiang (1974) reported some preliminary observations where plasma prostaglandin levels were elevated in patients with narrow or open angle glaucoma. Although prostaglandins have been identified in some ocular inflammatory conditions there are a number of ophthalmic situations where it is by no means certain that prostaglandins have a role to play. Mechanical stimula424

tion of the intracranial portion of the trigeminal nerve, the so-called antidromic stimulation, produced a reaction similar to that produced by mechanical stimulation (e.g. scratching of the iris, cornea or paracentesis) and chemical stimulation (e.g. formaldehyde, alkali, nitrogen mustard). These reactions are characterised by local vasodilatation, ocular hypertension, miosis, and an increase in the protein content of the aqueous humour. It is ironic that the first studies on the eye to implicate prostaglandins in inflammation report a release after antidromic stimulation (~nggard & Samuelsson, 1964; Ambache et al., 1965), yet recent work indicates that there is no increase in the prostaglandin levels of the aqueous humour of the rabbit eye (Neufeld et al., 1972a; Cole & Ungar, 1973). Miosis produced by antidromic stimulation of rabbit eye was not inhibited by the prostaglandin synthesis inhibitor, acetylsalicylic acid (Dellow & Miles, 1975). The role of prostaglandins in antidromic stimulation warrants further investigation. Recent indirect evidence with a prostaglandin biosynthesis inhibitor, indomethacin, suggested that oculomotor nerve stimulation, which produces increased blood flow in the anterior urea and breakdown of the bloodaqueous barrier, was also not mediated by prostaglandins (Stjernschantz et al., 1977). Although Cole & Ungar (1973) found no increase in prostaglandin levels after intracameral injections of formaldehyde, Bethel & Eakins (1971) concluded that prostaglandins are released after topical application. Their evidence was based on antagonism of prostaglandin and formaldehyde responses by polyphloretin phosphate (PPP), a prostaglandin antagonist, This conclusion is suspect since both high and low molecular weight fractions of PPP block the formaldehyde response while only the low molecular weight fraction blocks the prostaglandin response. Other mechanisms are obviously possible. Alkali burns have been produced by the application of 2N sodium hydroxide to the rabbit cornea (Paterson & Pfister, 1975) though increased prostaglandin levels were only found when 20 /~1 was applied, and not when 50 or 100/~1 were used. Massive tissue destruction and alkali hydrolysis were thought to be the reason for the lack of prostaglandins. The ocular inflammatory response to topical nitrogen mustard is n o t mediated by prostaglandins (indirect evidence with a prostaglandin biosynthesis inhibitor, acetylsalicylic acid) (Neufeld et al., 1972a) though it is dependent on an intact sensory innervation (Jampol et al., 1975). F r o m the results that we have just presented it might be concluded that the nervous response of the eye to a chemical stimulus apparently plays a greater role than the prostaglandin-mediated pathway.

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The inflammatory actions o f prostaglandins on the eye

In 1967, Waitzman & King examined for the first time pure PGEI and PGE 2 on the rabbit eye after administration into the anterior chamber. They found a prolonged rise in IOP and a breakdown of the blood-aqueous barrier, which was later substantiated by Beitch & Eakins (1969)9 Miosis was only occasionally associated with the prostaglandin response9 Topical application of PGE1 has also been shown to lead to an increase in lOP in conscious rabbits (Kass et al., 1972). In 1971, Starr reported that PGE~ and PGFa not only increased IOP in the rabbit but there followed a long-lasting hypotensire effect after intracameral or intravenous administration. Long-term infusion of either PGE1 or PGFa into the anterior chamber of the rabbit lead first to a hypertensive response, which was invariably reversed to a hypotensive response during the course of infusion (Green & Kim, 1975). In a recent paper, Bhattacherjee & Hammond (1977) give preliminary results of a pure hypotensive response to a low concentration of PGE1 given intracamerally in the rabbit. It might be concluded that in the rabbit PGEs can exhibit biphasic, hyper- and hypotensive ocular responses. The mechanism of the IOP increase appears to be a breakdown in the blood-aqueous barrier, indicated by entry of large amounts of plasma proteins into the aqueous humour (Waitzman & King, 1967; Beitch & Eakins, 1969; Whitelocke & Eakins, 1973). The breakdown of the barrier was primarily located in the posterior chamber, and the increase in vascular permeability in the ciliary processes and their iridial extensions (Cole, 1974; Szalay et al., 1976). Possible mechanisms for barrier breakdown are vascular leakage leading to stromal oedema, and the concomitant mechanical stress caused by distension of the ciliary processes resulting in disruption of the barrie_r epithelium. Another possibility is that vasodilator drugs, e.g. prostaglandin El, act directly on the ciliary process epithelium as well as on the vascular endothelium. Further research is needed to clarify these mechanisms. The mechanism for the prostaglandin-induced hypotensive response is unknown though Bhattacherjee & Hammond (1977) have speculated that a prostaglandin-stimulated increase in cyclic adenosine-3,5 -monophosphate (cyclic AMP) might mediate the response. It is well known that catecholamines reduce ocular tension, and, as PGE1, they further increase the levels of cyclic AMP in ocular tissues (Waitzman & Woods, 1971; Neufeld et al., 1972b; Neufeld & Sears, 1974). Cyclic AMP was also found to lower ocular tension and increase outflow facility (Neufeld et al., 1972b; Neufeld et al., 1975). Prostaglandins of the E series have been examined in other species besides the rabbit. PGE1 and PGE2 were found by Waitzman & King (1967) to 9

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cause miosis but were without effect on the IOP in the cat after intracameral injections of large quantities. On the other hand, Eakins (1970) found variable increases in IOP accompanied by vasodilation and miosis with large doses of PGE1 of PGF52 (1 pg). With smaller doses only miosis could be observed. In the monkey neither high concentrations of PGE 1 nor PGE2 at high affected pupil diameter though lOP was raised, and there was a breakdown of the blood-aqueous barrier (Kelly & Starr, 1971). These observations are in complete contrast to those of Waitzman (1968), who found miosis without lOP changes. Recently, Casey (1974) unmasked the miotic effect of prostaglandins by pretreatment of monkeys with atkopine. There is little information "available concerning the pharmacological effects of prostaglandins on the human eye though Karim & Filshie (1970) reported blurring of vision in one female patient after intravenous injection of a high dose of PGE 2. The accidental instillation of prostaglandin into a human eye was reported to cause very long-lasting ocular inflammation (3 days) (Kelly & Starr, 1971). Although E prostaglandins are remarkably potent in the rabbit at causing increases in IOP, breakdown of the blood-aqueous barrier and miosis, those of the A, B, and F series were far less potent (Waltzman & King, 1967; Beitch & Eakins, 1969; Chiang, 1974). The metabolites of PGE1, 15-keto PGE1 and 13,14-dihydro-15-keto PGE1, were also much less potent than PGE 1 at increasing IOP in the rabbit (Chiang, 1974). Recently, 19-hydroxy PGE1 and 19-hydroxy PGE 2 have been isolated from human semen in very large quantities (Taylor & Kelly, 1974) though their function is unknown. These too possess very weak inflammatory activity after topical administration to the rabbit eye (Hall & Jaitly, 1977). Prostaglandins have now been shown to potentiate the actions of other substances on the eye (Eakins & Bhattacherjee, 1977). In their study, they found that topical administration of PGE 1 or PGE2, but not PGFza, potentiated the histamine4nduced oedema of the rabbit conjunctiva and synergistically increased vasodilation whereas none of these prostaglandins affected the histamine-induced ocular responses after intracameral injection. They suggest that in conjunctival inflammation prostaglandins may amplify the effects of other vasoactive substances while in uveal inflammation prostaglandins may play a more direct role. Hypersensitivity to the touch was also found after topical application of histamine and PGE1. It is possible that these reactions are involved in the itching that often accompanies conjunctival inflammation.

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Antagonism of the ocular actions of prostagIandins Polyphloretin phosphate (PPP) (when administered intravenously or into the lingual artery of the rabbit), is able to reduce the increase in IOP and the increase in vascular permeability that normally accompanies paracentesis or chemical irritation (Cole, 1961). Close arterial infusion, subconjunctival or intravitreal injection of PPP antagonized the IOP increase due to intracameral PGE1 (Beitch & Eakins, 1969; Bethel & Eakins, 1971; Starr, 1971). The low molecular weight fraction of PPP appears to display the major prostaglandin blocking activity. PPP was unable to prevent the ocular effects of PGE1 in the monkey (Kelly & Starr, 1971). Podos & Becker (1974) reported that the prostaglandin analogue, 7-oxa-13-prostynoic acid, administered topically in a high dose did not block the IOP increase after PGEa, whereas antagonism of the smooth muscle effects of prostaglandins has been found. It is also known that imidazole blocks the PGEl-induced increase in IOP (Zink et al., 1973) though the mode of action is unknown.

Antagonism of the biosynthesis of prostaglandins It is well known that non-steroidal anti-inflammatory drugs (e.g., acetylsalicylic acid, indomethacin) inhibit the synthesis of PGE2 and PGF2a from arachidonic acid in a variety of tissues (see Vane, 1973; Flower, 1974). In 1974, the presence of prostaglandin synthesizing enzymes was demonstrated in ocular tissues using arachidonic acid as substrate (Bhattacherjee & Eakins, 1974a). The descending order of microsomal activity was conjunctiva, anterior uvea, retina, and cornea. Recently, it has been shown that the prostaglandin synthesizing capacities of microsomal preparations from inflamed iris-ciliary processes were several times greater than preparations from normal-rabbit eyes (Bhattacherjee, 1977). Activation or induction of prostaglandin synthetase might be an explanation of this phenomenon. Although it is clear that the eye has the synthesizing capacity for prostaglandins Eakins and his co-workers (1974) demonstrated that the iris-ciliary body, conjunctiva, and lens metabolized PGE1 only very slov~ly when compared to lungs or kidney. The metabolite formed corresponded to 15-keto PGE1. There thus appears to be very little prostaglandin dehydrogenase in the rabbit eye. This lack of prostaglandin catabolic activity in the eye stresses the importance of the anterior uveal prostaglandin transport mechanism to remove prostaglandins from the aqueous humour, originally discovered by Bito & Salvador (1972). Its failure during ocular inflammatory conditions (Bito, 1974) might explain why prostaglandins accumulate in the aqueous humour. 428

Arachidonic acid administered topically or subconjunctivally produced increases in lOP and vascular permeability that were antagonized by indomethacin (Jaffe et al., 1973; Podos et al., 1973; Conquet et al., 1975; Bhattacherjee & Eakins, 1975). Topical administration of arachidonic acid affected the vascular integrity of the iris and, to a lesser extent, the ciliary processes, whereas paracentesis affected mainly the ciliary processes (Bhattacherjee & Hammond, 1975). Indomethacin, indoxole, and pirprofen prevented the breakdown of the blood-aqueous barrier much more effectively after arachidonic acid than after paracentesis, which was moreover incompletely blocked (Bhattacherjee & Hammond, 1975). It seems likely that more than one mediator is involved in paracentesis, especially since acetylsalicylic acid and indomethacin have also been found not to inhibit the protein increase in the secondary aqueous humour (Miller et al., 1973; Ungar et al., 1975). In vitro studies using prostaglandin synthetase preparations from ocular tissues revealed that indoxole was 100 times as potent as indomethacin on a preparation from the anterior uvea while only 25 times as p o t e n t on a preparation from the conjunctiva. Acetylsalicylic acid, paracetamol, and dexamethasone had little to no activity (Bhattacherjee & Eakins, 1974b). On the other hand, topical application of these same anti4nflammatory agents reduced the arachidonate-induced increase in IOP and increased vascular permeability with indomethacin 2-4 times as potent as indoxole or pirprofen (Bhattacherjee & Eakins, 1975). A number of non-steroidal anti4nflammatory drugs have been examined on the arachidonic acid-induced increase in IOP after topical application (Podos & Becket, 1976). Of the substances examined, meclofenamic acid, flurbiprofen, and indoxole plus polysorbate 80 were more p o t e n t than indomethacin. Indoxole administered without polysorbate 80 was about 10 times less potent in their study, and 4 times less potent in the study of Bhattacherjee & Eakins (1975). It is clear that other important factors must be taken into consideration when evaluating the efficacy of a drug, e.g., penetration, protein binding, metabolism, and that evaluation by in vitro means alone might well lead to misleading conclusions. In recent years, the anti4nflammatory activity of corticosteroids has been linked with prostaglandin synthesis. Lewis & Piper ( t 9 7 5 ) c o n c l u d e d that corticosteroids inhibited the release of prostaglandins but not their formation. This was disputed by Gryglewski and his colleagues (1975), who favoured a blockade of the release of arachidonic acid, the substrate for cyclo-oxygenase (the initial enzyme in the formation of prostaglandins, which forms PGG2). This hypothesis has been developed by Nijkamp et al. (1976), who have shown that the release of arachidonic acid induced by a

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small peptide is antagonized by corticosteroids. The peptide, known as rabbit aorta-contracting substance releasing factor, has been found in the perfusates of guinea-pig lungs during anaphylaxis. The effects of corticosteroids have been studied in the rabbit eye using the paracentesis and endotoxin-induced uveitis models (Floman & Zor, 1977). Triamcinolone was found to reduce the accumulation of PGE in the aqueous humour, and the signs of inflammation after paracentesis. Hydrocortisone or Millicorten (100 /~g/ml) blocked the release of PGE from slices of iris and ciliary body of E.coli endotoxin-induced inflamed eyes. The blocking action of hydrocortisone on the PGE release was suppressed by arachidonic acid. These findings are in accordance with the clinical results of Eakins et al. (1972b), who found undetectable prostaglandin levels in two patients suffering from anterior uveitis receiving corticosteroid therapy. The studies of Floman & Zor (1977) give support to the hypothesis that corticosteroids reduce the availability of arachidonic acid for prostaglandin synthetase. It is curious that topical therapy with corticosteroids can often result in ocular hypertension. This might point to a regulating role for prostaglandins in the control of ocular tension. Kuehl and his colleagues (1977) have suggested that the cyclic endoperoxide intermediate, PGG2, formed during the biosynthesis of PGFa and PGF2a, may play a pivotal role in inflammation. PGE2 and PGF2a might thus be considered to be degradation products of this central compound. It will be interesting to see if a similar situation exists in the eye. At present, it is also not known whether ocular tissues can form thromboxanes from PGG2 as has been found in other organs (Hamberg et al., 1975). The ocular effects of thromboxanes are unknown. Advances to date in the prostaglandin field have given a rational basis for the use of many drugs previously used on an empirical basis. It is to be hoped that the knowledge that has been acquired can be used for the design of more meaningful models of inflammation, and hence the development of more selective therapeutics. REFERENCES Ambache, N. Properties of irin, a physiologicalconstituent of the rabbit's iris. J. Physiol. London 135:114-132 (1957). Ambache, N. & H.C. Brummer. A simple chemical procedure for distinguishing E from F prostaglandins, with application to tissue extracts. Brit. J. Pharmacol. 33: 1621 70 (1968). Ambache, N., H.C. Brummer, J.G. Rose & J. Whiting. Thin layer chromatography of spasmogenic unsaturated hydroxy-acids from various tissues. J. Physiol. London 185 : 7 7 - 7 8 P (1966). Ambache, N., L. Kavanagh & J. Whiting. Effect of mechanical stimulation on rabbit's eyes: release of active substance in anterior chamber perfusates. J. Physiol. London 176:378-408 (1965).

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Authors' addresses: D. W. R. Hall Research & Development Gist-Brocades N.V. P.O. Box 1 Delft The Netherlands

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I.L. Bonta Dept of Pharmacology Faculty of Medicine Erasmus University P.O. Box 1738 Rotterdam The Netherlands