Histamine, prostaglandins and ocular inflammation

Exp. Eye Res. (1977) 24, 299-305

Histamine, Prostaglandins and Ocular Inflammation K.E.

EAKINS

AND P. BHATTACHERJEE

Departments of Ophthalmology and Pharmacology, Collegeof Physicians and Surgeons, Columbia University, New York, N.Y. 10032, U.S.A. and Department of Experimental Ophthalmology, Institute of Ophthalmology, London University, U.K. (Received11 June 1976, New York) The interactions of different concentrations of prostaglandin E, and prostaglandin F,, with histamine were studied both inside the rabbit eye following intracameral injections and externally on the conjunctiva after topical administration. Clinical observation and [12sI]albumin measurements showed that the simultaneous administration of prostaglandin E, (but not prostaglandin F.J potentiated the edema-producing action of histamine in conjunctival tissue. Similar results were obtained in a limited number of experiments using prostaglandin E,. In addition, the mixture of hista,mine and prostaglandin E, (but not the individual components) resulted in the conjunctival area being hypersensitive to touch. Histamine alone, in doses up to 50 pg, produced very little effect when given by intracamera1 injection and in contrast to the results obtained in conjunctival tissue, neither the actions of histamine on intraocular prostaglandin E, nor prostaglandin F,, enhanced pressure or the protein content of aqueous humor following simultaneous intracameral administration. These results suggest that prostaglandins may play a direct role in internal ocular inflammation but that in the conjunctiva, prostaglandins may serve to amplify the inflammatory responses produced by other vasoactive amines (histamine in the present study). It is suggested that drugs which inhibit the action or synthesis of prostaglandins may prove useful in treating various symptoms of external ocular inflammation. Key ujorcls : prostaglandins ; histamine ; inflammation ; edema pain ; hyperaemia ; conjunctiva ; blood-aqueous barrier.

1. Introduction Prostaglandins are known to function as modulators of the inflammatory responseto other substances. For example, prostaglandins can potentiate both the edemaproducing actions of histamine and bradykinin and endogenous inflammatory reactions in the rat paw and guinea pig skin (Moncada, Ferreira and Vane, 1973; Williams and Morley, 1973). Similarly, in man, low concentrations of prostaglandins sensitize pain receptors to stimulation by other inflammatory mediators (Ferreira, 1972) and potentiate itching due to histamine (Greaves and McDonald-Gibson, 1973). In the present experiments we have studied the interactions of different prostaglandins with histamine, both inside the eye following intracameral injection and externally on the conjunctiva after topical administration, to determine whether prostaglandins may play a modulatory role in internal and external ocular inflammation. 2. Methods New Zealand white rabbits (a) Intraocular

weighing

between

2 and 3.5 kg were used in all experiments.

studies

The animals were anesthetized with l-2 g/kg urethane injected into a marginal ear vein as a, 25% solution in normal saline. One 26 gaugeneedlewasthen introduced through the

300

K.

E. EAKIhTS

AND

P. BHATTACHERJEE

cornea into the anterior chamber of each eye. The needle was connected via polyethylene tubing to a calibrated Agla (Burroughs Wellcome) micrometer syringe for the intracameral injections. In all experiments the intracameral injections were made in a standard volume of 10 ~1. Doses of histamine are expressed as the base. Pupil size was measured visually, in uniform artificial illumination, with a, transparent plastic millimeter ruler across the horizontal meridian. Quantitative calorimetric determinations of the protein in aqueous humor samples were made by the method of Lowry, Rosebrough, Farr and Randall (1951). (b) Extraocular

studies

Rabbits were divided into groups of 4-6 animals. Either histamine (Histamine ,lcid phosphate, Calbiochem) or prostaglandins E, or F,, (PGE, or PGF,,) dissolved in normal saline was instilled in a standard dose-volume of 20~1 onto the conjunctiva of one eye. The contralateral eye was treated with the same volume of a mixture of histamine and prostaglandin. A small number of experiments on the conjunctiva were also carried out using PGE,. Photographs of the eyes were taken on Tri-X film using a Nikon F with 200 mm medical Nikkor lens with Vivitar auto thyristor flash model 292. Measurement

of con&&al

vascular permeability

Animals received 8 $i/kg of [ls51]b ovine serum albumin (sp. act. 6-60 Ci/mmole, New England Nuclear Corporation, Boston, Mass.) intravenously immediately prior to treatment with histamine, prosta,glandins or their mixture. Forty-five min later the animals were killed with an overdose of pentobarbitone and the eonjunctiva dissected free and counted in a Packard model 30001 Autogamma spectrometer.

3. Results (a) Intraocular Compared

studies. to the prostaglandins

studied,

histamine

was found to have little

ability

to increasevascular permeability in the rabbit eye. Intracameral injections of 5-50 ,LL~ produced only a small increase of the protein content of aqueous humor (Table I) which was not dose-dependent. These doses of histamine were also only weakly miotic resulting in decreasesin pupil diameter of less than 1 mm at the dose of 50 pg histamine. In contrast, low quantities (lo-50 ng) of PGE, or somewhat higher doses (So-100 ng) of PGF,,, injected into the anterior chamber of the rabbit eye, produced the dose-dependentincreasesin aqueoushumor protein levels accompanied by dose-dependent increases in intraocular pressure that have been described previously

(Beitch

and Eakins,

1969).

These

changes

were

accompanied

by only

slight pupillary constriction, again lessthan 1 mm at the highest dose levels of the prostaglandins. Two doses of each prostaglandin which produced significant, but submaximal increasesin the protein levels were then chosen for the next series of experiments,

the results

of which

are shown

in Table

1. The lowest

concentration

of

histamine (5 pg) was mixed with either PGE, (10 and 50 ng) or PGFa’,,(50 and 100 ng) and injected into the anterior chamber. These mixtures of histamine and PGE, produced increasesin protein levels which were substantially the sameas the sum of the responses to either agent alone. Similar histamine and PGF,,. No potentiation of the actions of histamine PGE, or PGF,.

results on pupil

were

obtained

diameter

with

mixtures

were seen with

of

either

HISTAMINE,

PROSTAGLANDINS

AND

INFLAIvIXATION

301

TABLEI

w

Interactiolzs

OCULAR

of histamine

pemeability

CWKI? prostnglundi*ru

m

of the blood-apeous

Aqueous humort,if protein (ma%)

Dose (ng)*

DlUg

Histamine

5000 10 000 50 000 10 5000

PGE, Histamine + PGE, PGE, Histamine + PGE, PGF,, Histamine + PGF’,a PGII’,, Histamine i PGF,:,

the

bawier

16S,j1142 (5) 158151 (4) 169,t36 (4) 720*102 (11)

G 50 5000 + 50 10 5000 Ih 100 5000

793&161

(10)

1650&300

(5)

16875146

(4)

97*27

(4)

168&50

(4)

703268

(4)

G46i2OO

(4)

lC&l

* All doses injected in a volume of JO ~1 into the anterior &umber f Results expressed as Mean+s.z.x (n.). $ Aqueous humor samples removed 30 min after the injection.

(b) l?ztra.ocular

studies

Various doses of histamine (20, 40, 100, 200 and 400 pg) were applied topically to bhc conjunctiva in a dose-volume of 20 pl. Only the higher doses (200 and 400 pg) produced a mild edema and vasodilatation. Low doses (20 and 40 pg) produced only mild vasodilation of conjunctival vessels without any sign of edema. Similarly

B#ect

of topical

application

of postaglandin~

the response of rabbit cowjumtivn Treat,ment

None Histamine (40 pg) Prostaglandin E, (200 ng) Histamine (40 pg) + P1,nstaglandin E, (200 IV)c

Co7mts/min/m~

E,

022,

to l~ista~~~i~~e wet weight

5.0*2.3 40.0+4.9 10~9+4.0

(4) (5) (4)

743*%?

(5)

* Conjunctival samples removed 45 min after 7 Results expressed as mean&s.z.x. (n).

topical

of tissue”;~

pretreatment,

302

K.

E. EAKlP;B

AND

P. BHATTACHEl%JEE

PGE, or PGF,, in doses of 200 ng-1 pg (200 ng, 400 ng and 1 pg) failed to produce conjunctival edema, although some vasodilatation (mild to moderate) was observed with the higher doses (400 ng and 1 pg). In contrast a mixture of histamine (4Opg) and PGE, (200 ng) applied to the

FIG. 1. Actions and interactions of prostaglandin X1 (PGE,) and histamine applied topically to the rabbit eye. (a) Histamine alone (40 /~g in 20 pl saline) ; (b) PGE, alolie (200 ng in 20 ~1 saline); (c) mixture of histamine (40 pg) plus PGE, (200 ng) in 20 ~1 saline. Photographs taken 45 min after apphcation of the drugfi. Note the severe edema following application of the histamine-prostagla~din mixture in (c).

HISTAMINE,

PROSTAGLANDINS

AND

OCULAR

INFLAMRIATION

303

conjunctiva produced severe edema and vasodilatation (Fig. 1). This conjunctival reaction appeared within 5-10 min; reached a maximum between 30-60 min and subsided gradually until only slight residual effects remained 15-24 hr later. Increasing the dose of histamine in the mixture to 100 ,ug resulted in a greater, and decreasing the dose to 2Opg in the mixture resulted in a lesser conjunctival reaction. Similar responses were obtained in a limited number of experiments using POE,. No reaction was observed using similar mixtures of histamine (either 20 or 4Opg) with PGF,, (200 ng). Increasing the dose of PGF,, to 400 ng, in the drop applied to the conjunctiva, failed to produce the synergistic response seen with PGE, previously. These clinical observations on the development of edema were confirmed when the vascular permeability was measured quantitatively using intravenous [1251]albun3in (Table II). In each case, the total radioactivity of the conjunctival sample taken from the eyes pretreated with the mixture of histamine and PGE, was considerably higher than the sum of the effects produced by each separate component of the mixture. Finally, we observed that the animals receiving the histamine-prostaglandin mixture invariably showed discomfort, in each case the treated eye was closed and hypersensitive to touch. In contrast, the eyes treated with either histamine or POE, alone did not exhibit these signs. 4. Discussion In the present experiments PGE, (and PGE, but not PGF,,) potentiated the edema-producing action of histamine in rabbit conjunctival tissue when the drugs were given by topical application. In contrast, neither PGE, nor PGF,, enhanced the effect of histamine when administered into the anterior chamber of the eye. There is now much evidence linking prostaglandins with inflammatory reactions of the uveal tract. In the experimental animal, prostaglandins are released during internal ocular inflammatory reactions produced immunogenically (Eakins, Whitelocke, Perkins, Bennett and Unger, 1972) or mechanically (Miller, Eakins and Atwal, 1973; Cole and Unger, 1973), or following laser irradiation of the iris (Unger, Perkins and Bass, 1974) and after application of strong alkali to the cornea (Paterson and Pfister, 1975). Furthermore, topica, application or intracameral injection, particularly of E-type prostaglandins can produce certain of the clinical signs of ocular inflammation such as local vasodilation and increased vascular permeability in several species (Beitch and Eakins, 1969; Eakins, 1970; Keley and Starr, 1971). The failure of histamine, in doses up to 50 pg, to increase the permeability of the blood-aqueous barrier in the present experiments was unexpected. The rabbit eye is particularly sensitive to irritation, characteristically responding with a breakdown of the blood-aqueous barrier leading to exudation of proteins into the normally protein-free aqueoushumor. Prostaglandins E, or E, given by intracameral injection increasethe protein content of aqueoushumor in dosesas low as 5-10 ng (Beitch and Eakins, 1969). Bradykinin produces similar effects in dosesas low as l-2 ng (Eakins, Stier, Bhatiacherjee and Greenbaum, 1975). The present results obtained with histamine are not in agreement with those of Cole (1974) who observed that infusions of histamine into the anterior chamber produced a marked increasein the permeability of the blood-aqueous barrier to both proteins and fluorescein. In the present experiments the histamine was injected in a small (10 ~1) dose volume immediately after cannulation into an otherwise normal eye, whereas in the previous experiment.s, the anterior chamber was perfused with Kreb’s solution at 31 $/min at a pressure head

304

K.

E. EAKIKS

AKD

P. BHATTACHERJEE

of 20 cm H,O. These differences in experimental procedures may be responsible for the different results. Furthermore, there was no significant difference in protein levels even when mixtures of histamine and prostaglandins were injected together or separately. Thus in a situation where the E-type prostaglandins are highly active in increasing vascular permeability, no potentiation of the effect of histamine was observed, suggesting that the prostaglandins play a more direct role in internal ocular inflammation. In contrast to the results obtained following intracameral injections: PGE, clearly potentiated the edema-producing action of histamine, after topical application to the eonjunctiva. No such potentiation was seenwith PGF,,. The dosesof histamine and PGE, used to demonstrate potentiation did not produce edemawhen given separately and were approximately 1/5th-l/lOth the concentrations required to produce any detectable edema when given alone. Previous studies have shown that microsomal fractions of rabbit conjunctival tissues are able to generate prostaglandin-like activity from arachidonic acid, this activity of conjunctival tissue being higher than in other ocular tissue (Bhattacherjee and Eakins, 1974). The role of the prostaglandins in the conjunctiva may be to amplify the inflammatory responseproduced by other vasoaetive substances(histamine in the present study). The synergism between histamine and E-type prostaglandins observed in this study may also extend to the itching which frequently accompanies inflammation of the conjunctiva, suggesting that drugs which inhibit the action or synthesis of prostaglandins may prove useful in treating various symptoms of external ocular inflammation. The present experiments indicate that prostaglandins may participate in the inflammatory responseof the conjunetiva in a different manner than in the anterior uvea. Such a differential participation of prostaglandins in the inflammatory response, dependent on the site of inflammation and the stimulus used to induce it, has been previously suggested(Ferreira, Moncada and Vane, 1974). BCBNOWLEDGMEBTS

This work was supported in part by grants from the Medical ResearchCouucil (U.K.) and the U.S.P.H.S. (EY 00457). We thank Brian Hammond for excellent technical assistance glandins

and Dr John Pike used in this study.

of the

Upjohn

Company

for

generously

supplying

the

prosta-

REFERENCES Beitch,

B. R. and Eakins, K. E. (1969). The effect of prostaglandins on the intraocular pressure of the rabbit. Br. J. Pharmacol. 37, 158-67. Bhattacherjee, P. and Eakins, K. E. (1974). Inhibition of prostaglandin synthetase systems in ocular tissues by indomethacin. Br. J. Pharmncol. 50, 227-30. Cole, D. F. (1974). The site of breakdown of the blood-aqueous barrier nnder the influence of vasodilator drugs. Exp. Eye Res. 19, 591-607. Cole, D. F. and Unger, W. G. (1973). Prostaglandins as mediators of the responses of the eye to trauma. Exp. Bye Res. 17, 357-368. Eakins, K. E. (1970). Increased intraocular pressure produced by prostaglandin E, and E, in t.he cat eye. Exp. Eye Res. 10,87-92. Eakins, K. E. Stier, C., Bhattacherjee, P. and Greenbaum, L. M. (1975). Actions and interactions of bradykinin, prostaglandins and nonsteroidal anti-inflammatory agents on the eye. I@ammation 1, 117-25. Eakins, K. E., Whitelocke, R. A. F., Perkins, E. S., Bennett, A. and Unger. W. G. (1972). Release of prost.aglandin in ocular inflammation in the rabbit. Natiuve, brew Biol. 239, 248-9.

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PROSTAGLAKDIXS Prostaglandins,

AND aspirin-like

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c