Ocular responses to leukotriene C4 and D4 in the cat

PROSTAGLANDINS

OCULAR RESPONSES TO LEUKOTRIENE

Johan

Stjernschantzx,

Department of sity, School

Truman

Ophthalmology of Medicine,

C4 AND D4

Sherk

and Visual New Haven,

and

IN THE CAT

Marvin

Science, Connecticut

Sears Yale Univer06510, U.S.A.

ABSTRACT The effects of leukotriene C4 (LTC4) and D4 (LTD4) on the iridial smooth muscles, intraocular pressure, blood-aqueous barrier and regional blood flow in the eye have been studied in cats. Both The test compounds were injected into the anterior chamber. LTC4 and LTD4 caused a dose-dependent constriction of the pupil, The effect on the iridial the agents being about equipotent. sphincter muscle was not dependent on nerve conduction, cycloMaximal constriction oxygenase products or muscarinic receptors. The smallest was achieved with 0.1-l pug of the test compounds. dose to induce a decrease in pupil diameter was 0.01 pg. After intracameral injection of 4 pug the miotic response was markedly delayed. This indicates that in high concentrations LTC4 and LTD4 probably also stimulate the iridial dilator muscle. The blood flow in the anterior uvea decreased after intracameral injection of 4 pg LTCq/LTDq. Smaller doses had no clear effect. There was no effect on the blood-aqueous barrier as judged from the aqueous humor protein concentration. The intraocular pressure decreased sl ightly after injection of the test compounds. INTRODUCTION Recently leukotriene C4 (LTC4) and D4 (LTD4) have been identias components of slow reacting substance (SRS and SRS-A) These agents have marked effect on certain smooth muscles, (1,2). particularly in the lung and vasculature, and may be imp1 icated in pathophysiologic conditions such as acute immunogenic reactions and inflammation (1,2,3,4,5). We have reported that LTC4 and D4 constrict the pupil in the cat after injection into the anterior chamber (6). In the present study we have characterized the action of LTC4 and D4 on the iridial smooth muscles in more detail as well as studied the effect on the vasculature in the eye, the bloodaqueous barrier and the intraocular pressure (IOP). fied

x)

Present address: Pharmaceutical Laboratories, P.O.Box 33, 33721

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1984 VOL. 27 NO. 1

Company Oy Star Ab, Tampere 72, Finland

Research

5

PROSTAGLANDINS

MATERIALS

AND METHODS

weighing 2.2-4.1 kg were used. They were Cats of either sex, anaesthetized with pentobarbital sodium 30-50 mg/kg, about half of which was administered intraperitoneally, and half intravenously. All the animals were pretreated with indomethacin 20 mg/kg. Half of this dose was administered intravenously go-120 min, and half 5-10 min before the experimental procedure. The animals also received atropine sulphate 0.2 mg/kg intravenously 5 min before injection of the test compound. Tetrodotoxin (TTX) in a dose of 30 pg per eye was injected into the anterior chamber 30 min before injection of the test compound. Rheomacrodex, mostly diluted 1:l with isotonic saline was given intravenously to avoid too low blood pressure after TTX. The animals were tracheotomized and artificial ly venti lated. PC02, PO2 and pH of the arterial blood were checked 5-10 min prior to the blood flow determination. A femoral vein and both femoral arteries were cannulated. The animals were thoracotomized and left heart ventricle was cannulated. The an i mals were kept warm by a heating pad. LTC4 and LTD4 were dissolved in water. The control eye received the same volume of water only.

1. Measurement of intraocular pressure, intracameral injection and sampling of aqueous humor. The anterior chambers of the eyes were cannulated with 27 G needles connected to pressure transducers by polyethylene tubing. The intraocular pressu’res were registered continuously. The test compounds were injected through the same needles. A volume of 5-20 ~11 was injected. This had only a small and transient effect on IOP. In experiments with TTX the anterior chamber of both eyes was cannulated with a second 27 G needle, used for the injection of TTX. A volume of 15 ,~l of the TTX solution was injected into the eyes. Aqueous humor was collected at the end of the experiment through a 22 G needle inserted into the anterior chamber. The samples were frozen and stored at -2OOC until assayed for protein concentration.

2. Measurement of ocular blood flow. Regional blood flow in the eye was determined with radioactively labelled microspheres (sphere size 15*1 pm) using the reference flow method (7). A total of 2.0-2.5 ml of a microsphere suspension containing approximately 3.2-4.0 x 106 spheres labelled with 141Ce was injected into the left heart ventricle during a 10 set period. From the start of the injection, for one minute, a reference blood sample was obtained from a cannulated femoral artery. The animal was then

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1984 VOL. 27 NO. 1

PROSTAGLANDINS

sacrificed

with an intracardial injection of saturated KCI The radiosolution. The eyes were enucleated and dissected. activity of the tissue and blood samples was counted in a gammaspectrometer and the regional blood flow was obtained by comparing the radioactivity in the tissue sample with that of the reference The blood flow was expressed as mg/min per blood sample (7). whole tissue. Measurement of pupil diameter and aqueous humor protein 3. concentration. The horizontal pupil diameter was measured with a mm ruler at about 1 cm distance from the eye, under ordinary Measurements were made with l-2 min intervals laboratory 1 ight. for the first lo-15 min after injection of the test compound. Thereafter measurements were made with 5 min intervals. The aqueous humor protein concentration was measured with the Coomassie Brilliant Blue protein dye-binding method (8). 4. Source of drugs. LTC4 and LTD4 were obtained from pentobarbital sodium and Dr. J. Rokach, Merck Frosst Laboratories, atropine sulphate from Abbot, Coomassie Brilliant Blue G-250, indomethacin and tetrodotoxin from Sigma Chemical Company, heparine from Kodak, Rheomacrodex from Pharmacia and radioactively label led microspheres from New England Nuclear. were used and divided into the Groups. A total of 13 cats fol lowinq qroups: 1)lniection of 4 uq LTC4/LTD4 - 4 animals, 2) Injeciion of 1 !lg LTC4/LTD4 - 2 animals, 3) Injection of.O.1 pg LTC4/LTD4 - 3 animals (control eye in one animal failed due to cannulation), 4) Injection of 0.01 pg LTC4/LTD4 - 2 animals, and 5) Injection of 0.2 pg into one eye and 4 pg into the other eye 2 animals. In group 5 only pupil diameter was measured.

5.

standard Student’s

The results are expressed error of mean and were t-test of pa;red data.

as the arithmetical mean value statistically analyzed with

+ -

RESULTS The effect of intracameral injection of LTC4 and LTD4 on the pupil diameter is shown in Fig. 1 and 2. All the animals were pretreated with indomethacin and atropine and those which received LTD4 were also given TTX. The smallest dose to induce a detectable change in pupil diameter both with LTC4 and LTD4 was 0.01 jtg (LTC4: 2.1 x lo-“Moles, LTD4: I .6 x 10-llMoles). Submaximal constriction of the pupil was achieved with intracameral injection 1.6 x lo-lOMoles) but large of 0.1 vg (LTC4: 2.1 x 10-lOMoles, LTD4:

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1984 VOL. 27 NO. 1

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PROSTAGLANDINS

LTC4

12K

4.0 WI a E 1.olJg t

1

IO

,

20

I

30

40 TIME

FIG.

50

I

60

I

70

(MINI

1.

Effect of intracameral injection of various amounts of LTC4 on pupil diameter. Each curve represents one experiment. The animals were pretreated with indomethacin and atropine. The pupil diameter of the control eye remained unchanged after atropine in the respective experiments. Note the slow induction of miosis after intracameral injection of 4 ,ug LTC4.

8

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1984 VOL. 27 NO. 1

PROSTAGLANDINS

LTD4

I

I I

10

I 1

I

I

,

I

40

50

60

70

1

20

30 TIME

FIG.

+ TTX

I

(MN)

2.

Effect of intracameral injection of various amounts of LTD4 on pupil diameter. Each curve represents one experiment. The animals were pretreated with indomethacin, TTX and atropine. The pupil diameter remained unchanged in the control eye after atropine, in the respective experiments. Note the slower induction of miosis after intracameral injection of 4 ,ug LTD4.

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1984 VOL. 27 NO. 1

PROSTAGLANDINS

LTC

4

a 6

1

rl

10

FIG.

20

30

40

50

TIME

(FAIN)

60

70

3.

injection of a low and high dose of Effect of intracameral The animal was prein the same animal. on pupi 1 d iameter Note the difference and atropine. treated wi th indomethacin in favour of the low dose. in pupil d iameter

10

90

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LTC4

1984 VOL. 27 NO. 1

PROSTAGLANDINS

amounts, induced (Figs.

4 jig (LTC4: constriction l-3).

0.84 of

x 10-8Moles, the

pupil

LTD4: 0.64 x a much slower

with

10-8Moles) time course

Injection of 0.01-l pg LTC4/LTD4 into the anterior chamber had little if any effect on ocular blood flow. Injection of 4 /lg of the test compounds caused a tendency toward vasoconstriction in the anterior uvea (Table I), however, statistically not significant. There was no change in blood flow in the choroid, retina or sclera. The mean arterial blyod pressure at the time of injection of the microspheres was 104-13 mmHg. Table I. Regional of 4 lrg LTC4 or LTD4

eye 55-84 min chamber (n=4).

blood flow in the into the anterior

Tissue

injection

Eye Experimental (mg/min)

Ret ina Choroid Cil iary Iris Sclera

after

body

26

j4

657 ‘59 9 23

i +

‘33 26 3 12

Control (mg/min) 32

+

623 235 23 ‘7

+ + + +

8 85 29 9 5

The intraocular pressure of the whole material (eyes injected with 0.01-4 rrg, LTC4: 2.’ x lo-” - 0.84 x ‘0-8 Moles or LTD4: 1.6 x to-” - 0.64 x ‘o-8 Moles) was 15.6+1.4 mmHg and 17.1+1 .6 mmHg in the experimental and control eyes (n=lO), respectively, at+the time of blood flow determination. The difference of 1.5-0.4 mmHg was statistically significant at the pt0.005 level. The protein concentration of the aquyous humor 60-132 min after injection of the leukotrienes was 32-Z mg/lOO ml in the experimental as well as control eyes (n=lO). The mean arterial blood pressure before the intraocular administration of TTX was 117+13 mmHg (n=6). After TTX it dropped to a level of 5bf5 mmHg (n=6). It was raised by iv infusion of Rheomacrodex. DISCUSSION In the present anterior chamber of the of the iris and probably trabecular meshwork and

work the leukotrienes were injected into the eye. Thus, the smooth muscles and vasculature the ciliary body as wel 1 as the outflow channels of aaueous humor have

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PROSTAGLANDINS

been well exposed to the test substances. However, only traces of the test substances may have reached the choroid and the retina. It should be pointed out that the leukotrienes have been administered It is possible that from the adventitial side of the blood vessels. intraluminal administration may have stronger or even qua1 itatively different effects on the vasculature. Earlier studies have indicated that the effects of LTC4 and LTD4 on several parameters are qualitatively as well as in many cases quantitatively similar One reason for this may be that LTC4 is or almost similar (1). In the present study TTX, causing a strong metabol i zed to LTD4. drop in blood pressure even after intracameral injection, was given only to the animals in the LTD4-group. All the animals (n = 13) reacted with a decrease in pupil diameter after injection’of LTC4/LTD4 into the anterior chamber. Thus, it is clear that in the cat the predominant effect of LTC4/ is stimulation of the sphincter. LTD4 on the iridial smooth muscles It is also clear that maximal or near maximal effect can be achieved with amounts around 0.1-0.2 pg (n = 4) since 1 lug (n = 2) did not appreciably increase the response and 0.01 ,ug (n = 2) The fact caused a barely detectable decrease in pupil diameter. an intense and longlasting that 0.1-l pug LTC4/LTD4 ( n = 6) induced miosis considerably faster than large amounts (4 pg) (n = 6), l-3) indicates that in large doses these (see also Figs. leukotrienes probably also stimulate the dilator muscle of the iris. It is likely that when the concentration of the leukotrienes, due to inactivation with time, falls, the effect on the sphincter muscle becomes predominant and therefore at a later stage, even after large doses, only miosis is observed. This was recently verif ied in another study (6). Conventional prostaglandins have in a similar way been shown to stimulate both the sphincter and the dilator muscle of the cat iris (9). Another possibility that cannot be excluded is that LTC4/LTD4 in high concentrations causes a release of a secondary substance e.g. some unknown autacoid stimulating the dilator muscle or causing relaxation of the sphincter muscle, The fact that indomethacin administered intravenously in a dose of 20 mg/kg was completely ineffective in blocking the pupillary response to LTC4/LTD4 indicates that cyclooxygenase products are unlikely to mediate the response. Such an interaction between leukotrienes and cyclooxygenase products has recently been described in the cardiovascular system of the guinea pig (lo), and indomethacin in a dose of 5 mg/kg completely prevented the involvement of prostaglandins in the smooth muscle response to leukotrienes.

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PROSTAGLANDINS

Both atropine and TTX were ineffective This pupillary response to leukotrienes. is not mediated by muscarinic receptors.

in blocking the indicates that the Nor is it dependent

response on

intact function of sensory or other nerves, since blockade of nerve This is clearly different from conduction was without effect. the mechanism of action of exogenous prostaglandins in the eye, because prostaglandins exert much of their effect through sensory nerves (11). The dose of atropine used (0.2 mg/kg) should have been adequate for blocking muscarinic receptors, since this dose blocks The dose miosis during cholinergic stimulation in the cat (12). of TTX (30 /lg per eye) for blocking nerve conduction should also have been adequate since even the systemic blood pressure dropped it is highly likely severely after intracameral injection. Thus, that the concentration in the anterior segment of the eye was high enough to stop all nerve conduction. was a tendency toward decrease in blood flow of uvea in animals that received 4 ilg LTC4 or LTD4 Smaller amounts did not have consistent effect. Thus, (Table I). Topical the overall effects on the vasculature were small. application of LTC4/LTD4 on the vascular network in the hamster cheek In vitro, strips of human pouch also causes vasoconstriction (13). pulmonary artery and vein (3) as well as rabbit coronary artery (14) and strips of guinea pig pulmonary arteries (15) have been reported Thus it would seem that LTC4/LTD4 in many to contract to LTC4/LTD4. In the present study there vascular beds causes vasoconstriction. was no indication of an increased vascular permeability with plasma extravasation in the eye after administration of the leukotrienes. This is based on the low concentration of protein in the aqueous humor reflecting a normal integrity of the blood-aqueous barrier. Contrary to this, SRSA, LTC4 and LTD4 have been shown to induce plasma leakage in the rat (16) and guinea pig skin (17) and in the hamster cheek pouch (13). However, it is possible that there was some plasma extravasation in the present study too, which remained undetected since only a more marked extravasation would be reflected in an increased protein concentration of the aqueous humor. the

There anterior

The reason for the slight but statistically significant difference in IOP between eyes treated with LTC4 and LTD4 and It is possible that there was those not treated is not clear. a decrease in outflow resistance of aqueous humor or that the However, the decrease in episcleral venous pressure decreased. was so slight that it is of little significance in practice. It is not clear whether the miotic and vascular responses in the eye to LTC4/LTD4 are unique only for the cat. In previous studies it has been demonstrated that rabbits and guinea pigs do not respond appreciablv to intracameral injection of these leukotrienes (6,18). However, it is possible that too high doses thus leading to a considerable have been employed in these studies,

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PROSTAGLANDINS

stimulation of the dilator muscle in addition to that of the Consequently the change in pupil diameter could sphincter muscle. It is not inconceivable that LTCb/LTD4 is involved be very small. in immunogenic states of the eye such as uveitis.

ACKNOWLEDGEMENTS We would like to thank Dr. J. Rockach Laboratories for providing us with LTC4 and gation was supported in part by USPHS grants the Connecticut Lions Eye Research Foundation to Prevent Blindness Inc.

at Merck Frosst LTD4. This investiEY-00237, EY-00785, Inc., and Research

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Editor:

ished

Brendan

Paul. Leucot r i enes, of PCA. Agents and

Hammond, Products Prostagl.

Whittle

1984 VOL. 27 NO. 1

W.

J. A. Salmon on Leukocyte and Thromb.

Received: 4-25-83

ion

SRS-A and Actions.

and K. E. Eakins. Accumulation in Res. 2:325,

Accepted: 11-9-83

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