The effects of topically applied epinephrine and timolol on intraocular pressure and aqueous humor cyclic-AMP in the rabbit

Ezp. E?/e R~.F.(1981) 32, 681-690

The Effects of Topically Applied Epinephrine Timolol on Intraocular Pressure and Aqueous Cyclic-AMP in the Rabbit* S. BOAS,? MARK

RICHARD

AND

J.

MESSENGER,~ THOMAS W.

and Humor MITTAG~$

M. PoDost

STEVEN

tDepartment of Ophthalmology and IDepartment of Pharmacology, Mount Sinai School of Medicine, 1, &stave L. Levy Place, New York, N. Y. 10029, l.:.S.A. (Received 20 May 1980 and accepted 7 October 1980, New

York)

Intraocular pressure and aqueous humor cyclic-AMP concentrations were measured in albino rabbits following topical treatment of one eye with a single dose of 2 ?b epinephrine alone. 0.5 ‘?,> timolol alone, or epinephrine after timolol pretreatment. Most animals demonstrated a significant hypotensive response 6 hr after epinephrine treatment, which lasted at least an additional 6 hr. However, aqueous humor c-AMP was significantly elevated 30 min after epinephrine treatment. peaked between 60 and 240 min, and declined to baseline by 6 hr before a significant ocular hypotensive response was noted. Timolol treatment alone had no significant effect on either intraocular pressure or aqueous c-AMP. However, in epinephrine-treated animals which were pre-treated with timolol, the c-AMP response was blocked, with no significant alteration of the hypotensive response. Previous reports in the literature suggested a causal relationship between elevated aqueous humor c-AMP and fall in IOP. On the basis of the present work, this relationship is questionable.

1. Introduction Epinephrine is an effective ocular hypotensive agent used in the therapy of glaucoma. The drug may affect both aqueous humor production and aqueous outflow mechanisms. The nature of these mechanisms has been extensively investigated using adrenergic drugs applied topically to the rabbit eye. Such studies have demonstrated the complex pharmacology of the ocular response to adrenergic agents. Treatment of the rabbit eye with adrenergic agonists can produce a biphasic pressure response - an initial hypertension followed by a prolonged hypotension. Both n-adrenergic and /3-adrenergic receptors and c-AMP appear to be involved in these responses but the role of c-AMP has not yet been clearly elucidated. It has been postulated (Neufeld, Chavis and Sears, 1973; Neufeld, Dueker, Vegge and Sears, 1975; Neufeld, Jampol and Sears, 1972; Neufeld and Sears, 1974) that epinephrine lowers intraocular pressure via a c-AMP-mediated mechanism. In our experiments, we sought to determine the temporal relationship between intraocular pressure and aqueous humor c-AMP in response to topical epinephrine. We also investigated the possible alteration of one or both of these effects when animals were pretreated with the /3-adrenergic blocking agent, timolol maleate.

2. Materials Studies were conducted of L-epinephrine to use. Timolol

0014-4835/81/060681+

on restrained,

bitartrate (Sigma, maleate was obtained

* Presented at the meeting Florida, 30 April 1979.

and Methods

awake 2 kg female albino rabbits. A 2 y/0 solution St Louis) was made in distilled water immediately prior as a05 yO solution (Merck, Sharpe & Dohme, West Point.

of the Association

10 $01.00/O

for Research

681

in Vision

CQ 1981 Academic

and Ophthalmology,

Press Inc. (London)

Sarasota,

Limited

6H:!

I(. s. IIOAS ET A4lI,.

PA). Intraocular pressures were measured with a manomrtrically calibratrcl Alt*cm pneumotonometer (values not corrected). Rabbits were killed with sodium pentoharbital at predetermined times and anterior chamber paracentesis performed immediately aftrrwarcl. The c-AMP assay method was based on the procedure of Ferrendelli. Rubin. Orr. Kinscherf and Lowry (1977). Samples were deproteinated with 10 9~ trichloroacetic acid. extracted four times with analytical grade anydrous ether. Iyophilized and stored at -2OY’. ImmediateI> before assay the samples were reconstituted m distilled water and acetylated. c-AMP was measured using a commercial radioimmunoassay kit (New England Nuclear, Boston. Mass.) with determinations made in triplicate. Animals in which intraocular pressure determinations were made were different from thosr in which c-AMP determinations were made. Intraocular pressure measurements were made on the same animals throughout the st,udy. However, the drugs in each part of the study were administered at least 1 week apart. For the study. animals were divided into three groups. Group A : One eye of each animal treated with 50 ,BI 2 “/; epinephrine bitartrate. other eye treated wit,h 50 ~1 distilled water. Group R: One rye of each animal treated with 50 ~1 @5 y0 timolol maleate, other eye treated with 50 ~1 distilled water. Group C: One eye of each animal pretreated with 50 ,~l 0.5% timolol maleate, the other with distilled water. Two hours later, experimental eye given 50 ~1 29,;) epinephrine bitartrate, the other given 50 ~1 distilled water. For each of the above experiment,al groups, intraocular pressure was measured at intervals over a 1%hr period, and c-AMP levels over a 24.hr period. c-AMP was expressed as the ratio of the experimental to the control eye to normalize changes in c-AMP, as there is a considerable variation in aqueous humor c-AMP in individual normal. untreated animals. (The values in control eyes ranged from 625 nM/l to 73.7 nM/l.) Results were analyzed in two forms: mean intraocular pressure and mean c-AMP levels in each experimental group of animals, and percentage of animals showing significant change in intraocular pressure and c-AMP compared to their control eye. Responders were defined as animals which, at the time under consideration, showed a pressure response outside the range of the mean intraocular pressure f 2 S.D. of the control eyes of animals treated with epinephrine alone. In the case of c-AMP, a responder is defined as an animal which had a c-AMP ratio outside the range of the mean ratio +2 S.D. of a group of control animals.

3. Results

Group A There was no significant variation in mean intraocular pressure in the untreated eyes over the full 1%hr period (Fig. 1). There was no significant change in intraocular pressure in the epinephrine-treated eyes until 6 hr post-treatment, when hypotension was noted, lasting the remaining 6 hr of the experiment. Figure 2 shows the percentage of intraocular pressure responders to epinephrine at given times. Approximately 10 y0 of animals exhibited a hypertensive response at 10 min. There were no hypotensive responders before 360 min. The percentage of responders was nearly unchanged for the remaining 6 hr though no more than 85 o/0 responded at any one time. Figure 3 shows the aqueous humor c-AMP ratio (treated/control) over a 24-hr period following epinephrine treatment. The rise in c-AMP peaked between 60 and 90 min, followed by a second peak at 240 min. c-AMP levels then declined and were not significantly elevated above the control eyes at or beyond 360 min. The percentage of animals showing an elevated c-AMP response increased 30 min after treatment, lasting through 240 min (Fig. 4). Figure 5 compares intraocular pressure and e-AMP changes, showing the percentage

EPINEPHRINE,

I

I”,

60

20

TIMOLOL,

I

120

I

,

180 240

I

300

IO

following

30

of IOP

C-AMP eye.

responders

over

AND

I

I

480

540

c-AMP

1

683

1

600

660

1

720

(min)

with

epinephrine b&&rate 24)o~. Experimental

60 120 180360420480540600660720 over

a 24.hr

(min)

a 12.hr

Time

FIG. 3. Aqueous humor expressed as exp. eye/cant

420

treatment

Time

FIQ. 2. Percentage bitartaratc 2.0 “ib.

I

360 Time

FIG. 1. IOP over a 12-hr period eye (o-0): control eye (O---e).

IOP

period

period

following

treatment

with

epinephrine

treatment

with

epinephrine

bitartrate.

(min)

following

100 $ 80 f 60 ,” g 40 20 0

0

IO

20

3Cl 45

60

90

Time

FIG. 4. Percentage bitartrate 2.0 y0

of c-AMP

2Ot

“In

0

IO

responders

over

120 (min)

IS0

a 24-hr

period

240

360

following

IL 30

60

!O

180

Time

FIG. 5. Comparison of percentages treatment with epinephrine bitertrate

of c-AMP 2.0 %.

(0)

240

360

480

7201440

treatment

with

epinephrine

720

(min)

) responders

at various

times

after

of intraocular pressure and c-AMP responders together. From 30 to 240 min there was a significant number of c-AMP responders with no intraocular pressure responders. However, beginning at 360 min, only intraocular pressure responders were observed. Group

B

Figure 6 demonstrates that there was no significant variation in the mean intraocular pressure of either the treated or untreated eyes after topical 65 o/0 timolol maleate. The pressures recorded from these eyes did not differ from those recorded from the control eye intraocular pressure of animals which had one eye treated with epinephrine. The c-AMP levels were not significantly different in timolol-treated eyes from those of control eyes, throughout the time period in which pressure and c-AMP changes occur after epinephrine treatment (Table I). Group

c

Figure 7 shows the intraocular pressure epinephrine compared to eyes treated with

effect in eyes treated with epinephrine only (Epi-Exp)

timolol plus and control

EPIIiEPHRTNE.

i

-120

I

-60

II

TIMOLOL,

I”[

0 20

I

60

180

* 120

I

240

IOP

11

AND

t

300 360 Time (min)

L

420

480

FIG. 6. IOP over a t2-hr period following treatment with timolol (e--.-a). timolol, control (o-0); epinephrine, control (O----

TABLE

Aqueous

humor

c-AMP (explcont)

0

I

11%

I

I

I

I

0 20

60

120

180

240

300

I

FIG. maleate

(l

600

L

I

660

72(

0.5 9,0. Timolol.

experimental

I

with timolol

9

1~08+016

i+o6+0-07 0.79 + 0.08

I

360 420 Time (min)

7. IOP over a 1%hr period following treatment 0.5 y’ pretreatment. Timoloi-epinephrine, - 0) : epinephrine, experimental (m-0).

0.5 9,;

+S.E.X.

5 5

I

maleate

Mean c-AMP (exp/cont)

n’umber of animals

130 180

[I

540

maleatc 0).

after treatment

Time after timolol treatment (min)

685

v-AMP

I

480

I

540

I

I

I

600

660

720

with epinephrine bitartrate 2-O% with experimental ( l - . -0) ; epinephrine.

timolol control

It. S. SOAS

686

ET

AI,

eyes (no drugs). There was an irregular hypertensive phase after epinephrine treatment which lasted until 240 min. Intraocular pressure then rapidly fell. From 30 rnin until the end of the experiment, the hypotension did not appear to differ from that of eyes treated with epinephrine alone (Epi-Exp). However, it was noted that the control eye and experimental eye baseline pressures for this last group of animals (Group C) were an average of 2.5 mm above those of animals treated with epinephrine alone (Group B), although the animals used for both experiments were the same, one week apart. Figure 8 compares the percentage of intraocular pressure responders in the timolol and epinephrine group with responders in the group treated with epinephrine alone. The percentage of animals showing a hypotensive response was strikingly similar in the two groups over the time period from 360 to 720 min. The effect of timolol pretreatment on the aqueous humor c-AMP response to epinephrine was examined at 10 min before the rise in c-AMP occurs and at the peak period, 1 hr after epinephrine treatment. Table II shows that there was not a significant rise in c-AMP 60 min after epinephrine was given to timolol-pretreated

1004/

’

360

I

I

420

480

I

I

540

600

Time

FIG. 8. Percent IOP responders: epinephrine with timolol maleate @5 y0 pretreatment (

Aqueous

humor

c-AMP

I

720

(min)

bitartrate

TABLE

I

660

2.0 Y0 only (0)

vs. epinephrine

2.0 y0 with

Mean c-AMP (exp/cont) f S.E.M. after treatment with epinephrine bitartrate 2.0 % 10 min 60 min

Drug(s) Epinephrine bitartrate Epinephrine bitartrate with timolol maleate pretreatment

PO y0 2.0 y0 @5 o/0

to animals

e97+002 0.91 *@lo

treated

20 Ti,

II

(expplcont) : epinephrine 2-O y0 alone vs. epinephrine timolol maleate 0.5 y’ pretreatment

(n) = number of animals. * Denotes P < 005 compared

bitartrate

with

(10) (5)

epinephrine

320 + 0250 (4) 1.53fo.24 (8)*

bitartrate

alone,

at 60 min

EPINEPHRIKE,

TIMOLOL.

IOP

AND

687

VAMP

eyes. Thus, timolol had no significant effect, on c-AMP levels measured 10 min after epinephrine treatment, but almost completely blocked the rise in e-AMP at 1 hr. As noted above, an unexpected intraocular pressure response was observed in the ‘no drug’ eyes of animals treated in the other eye with timolol plus epinephrine. This effect is seen more clearly by comparing ‘no drug ’ eyes of these animals with the ‘no drug’ eyes of animals treated in the other eye with epinephrine only, as shown in Fig. 9. A significant drop in intraocular pressure occurred in the two hours following timolol treatment (also shown in Fig. 9) followed by a hypertensive phase peaking 60 min after

28

Epl Cont

-120

-60

FIG. 9. IOP of control eyes of animals treated (0-O).

0 20

60

120

180 240

300 360420 Time (min)

480

540

eyes of animals treated with epinephrine hitartrate with epinephrine bitartratc 2.0% with timolol

600

660

720

2.0 y0 ( l 0) vs. control maleate @5”/, pretreatment

treatment with epinephrine, and remaining significantly elevated for 300 min, above the mean intraocular pressure of ‘no drug’ control eyes of animals in which the experimental eye received epinephrine. Thereafter, the pressure was irregular until 480 min, after which intraocular pressure was about the same as that of epinephrine-treated animals’ control ‘no drug’ eyes.

4. Discussion Various authors have reported both a- and P-adrenergic components for the intraocular pressure effects of epinephrine. Sears and Barany showed in an early paper (1960) that both a-adrenergic and &?-adrenergic receptors were involved in the effects of epinephrine on the rabbit eye. The more recent studies of Langham and co-workers have carefully analyzed the pressure responses to epinephrine (Langham, Simjee and Josephs, 1973; Langham and Kieglstein, 1976). The pressure response in the rabbit eye is not usually biphasic except at high concentrations (Norton and Viernstein, 1972) or on repeated applications 24-hr apart. The hypertensive phase is blocked by phenoxybenzamine or thymoxamine (classical a-adrenergic blockers) but also partly by propranolol, showing the involvement of both a-adrenergic and P-adrenergic receptors in the hypertension. Similarly, by using selective antagonists, the hypotensive reponse was shown to have an a-adrenergic component and a smaller /Y-adrenergic component (Potter and Rowland,

68X

K. s. I30 .t4q FT 2 11 41

1978). Thus. because epinephrine causes mixed receptor activations, the observecl pressure responses may be considered as a combination of the responses observed when a selective /3-adrenergic agonist or a-adrenergic agonist is used individually. The hypotensive response to adrenergic agonists in the rabbit eye is due primaril? to an increased outflow facility. This rise in aqueous outflow is mediated principalI\ by a-adrenergic receptors when norepinephrine is epinephrine are employed (Sears and Barany, 1960: Sears and Sherk, 1964). The involvement of a-adrenergic receptors in the increased outflow can be shown unequivocally by phenylephrine, which is much less potent than norepinephrine but is selective for cc-adrenergic receptors (Becker. 1956; Lorenzetti, 1971). The smaller P-adrenergic receptor component to outflow facility is seen with the selective /?-adrenergic agonists isoproterenol and salbutamol. Recent studies by Neufeld and co-workers and by Langham and co-workers have provided strong evidence that the increase in facility of outflow is mediated by cyclic: adenosine 3’,5’-monophosphate (c-AMP). Cyclic AMP and several analogs such as dibutyryl c-AMP and 8 methyl thio 3’,5’-AMP cause marked increases in outflow when injected into the rabbit anterior chamber (Neufeld and Sears, 1974). The administration of adrenergic agonists was also shown to raise aqueous humor levels of (*-AMP concomitant with a fall in intraocular pressure (Neufeld, Jampol and Sears 197%; Neufeld. Chavis and Sears, 1973; Radius and Langham, 1973). These authors showed that the c-AIMP of aqueous humor in the normal rabbit eye approximated the concentration found in the plasma (about 20 nM) but was increased by 3 to 5-fold b> epinephrine or isoproterenol. The pressure rise and the rise in (:-AMP were largely blocked by phenoxybenzamine when agonists with a-adrenergic receptor activity were used (epinephrine and norepinephrine). The experiments wit.h isoproterenol gave unexpected results which were found in studies by Neufeld and by Langham. The fall in ocular pressure and rise in c-AMP elicited by isoproterenol were not blocked by propranolol. The present results are in general agreement with those of previous workers. These experiments show that there is a qualitative variability in the intraocular pressure responses of rabbits to epinephrine. The initial hypertensive response was observed in only about lO-15% of rabbits given an initial dose of epinephrine. while a hypotensive response occurring between 4 and 12 hr after treatment was observed in about 85 y;, of animals. Thus, some rabbits appear to be completely insensitive t,o epinephrine-induced ocular hypotension. Topical treatment with timolol alone has little effect on intraocular pressure or on levels of aqueous humor c-AMP (Fig. 6, Table I), consistent with the results of Neufeld and Page (1977). Timolol blocks the epinephrine-induced rise in aqueous humor c-AMP which occurs within 4 hr of treatment, without causing much change in the pressure response to epinephrine. However, timolol may have lessened the hypotension to some extent. but this is difficult to quantitate because the baseline intraocular pressure in the contralateral ‘no-drug’ controls for timolol+ epinephrine-treated eyes were somewhat higher than those in the corresponding ’ no-drug ’ eyes of epinephrine-treated animals. These effects indicate that topical timolol may have systemic effects which become manifest when epinephrine is given subsequently. These results also suggest that epinephrine has effects on the contralateral eye which are normally not observed unless a /?-adrenergic blocking drug is also used. Overall, the present results confirm that timolol behaves as a typical P-adrenergic blocker, preventing the rise of c-AMP but with little effect at this dose on epinephrineinduced ocular hypotension. The dissociation of aqueous c-AMP levels and hypotension,

EPIKEPHRIKE,

TIMOLOL.

IOP

Ah‘])

c-AMP

6X9

both in time and more significantly by selective blockade, seems to rule out a cause-and-effect relationship between these two parameters. The results provide additional confirmation that under these conditions the principal mechanism for epinephrine-induced hypotension in rabbits is a-adrenergic receptor-mediated and probably does not involve c-AMP. However, the present experiments did not distinguish the smaller p-adrenergic receptor component to the ocular pressure effects of epinephrine in the rabbit. In primates, where the P-adrenergic receptor-mediated mechanisms may be as important as a-adrenergic mechanisms or may predominate, there may be a temporal correlation between aqueous humor c-AMP levels and the hypotensive effect of epinephrine. However, by analogy with the above experiments. topical timolol would probably prevent theepinephrine-induced rise in aqueous c-AMP without blocking the pressure response. In fact, timolol lowers pressure by itself in primates and is synergistic (additive) when given together with epinephrine direct. Thus, c-AMP levels in the aqueous humor may have no relationship to changes in ocular pressure and may merely be an associated phenomenon of treatment with adrenergic agonists. ACKNOWLEDGMENTS This research was supported EY 02619.

by National

Eye Institute

Grants EY 01661, EY 01867 and

REFERENCES Becker,

B. (1956). Annual

reviews:

glaucoma,

19551956.

A.M.A.

Arch.

OphthaZmoZ.

56.

898-956.

(!onquet, Ph. and Vareilles, mediated elevation of Ferrendelli, J. A., Rubin, Measurement of cyclic Analyt.

Biochem.

P. (1978). Effects of ocular hypotensive agents on prostaglandinintraocular pressure. Ophdhal. Res. 10, 202-l 1. E. H., Orr, H. T., Kinscherf. D. A. and Lowry, 0. H. (1977). nucleotides in histologically defined samples of brain and retina.

78, 252-9.

Kass, M. A., Podos, S. M., Moses, R. A. and Becker, B. (1972). Prostaglandin E, and aqueous humor dynamics. Invest. Ophthalmol. 11, 1022-7. Lamble, J. W. (1973). Some factors affecting the ocular hypotensive and mydriatic responses of the rabbit to topically applied L-(-)adrenaline bitartrate. Exp. Eye Res. 17, 473-82. Lamble,J. W. (1974).Someresponsesoftherabbiteye totopicallpadministeredr,-(-)adrenaline bitartrate. Exp. Eye Res. 19, 41S34. Lamble, J. W. (1977). Some effects of topically applied L-(-)adrenaline bitartrate on the rabbit eye. Exp. Eye Res. 24, 12943. Langham, M. E. (1977). The equeous outflow system and its response to autonomic receptor antagonists. Exp. Eye Res. 25, Suppl.: 311-22. Langham, M. E. and Diggs, E. (1974). Beta-adrenergic responses in the eyes of rabbits. primates, and man. Exp. Eye Res. 19, 281-95. Langham, M. E. and Kriegelstein, G. K. (1976). The biphasic intraocular pressure response of rabbits to epinephrine. Invest. Ophthalmol. 15, 119-27. Langham, M. E., Simjee, A. and Josephs, S. (1973). The alpha and beta adrenergic responses to epinephrine in the rabbit eye. Exp. Eye Res. 15, 75-84. Lorenzetti, 0. J. (1971). Dose-dependent influence of topically instilled adrenergic agents on intraocular pressure and outflow in the rabbit. Exp. Eye Res. 12, 8&7. Neufeld, A. H., Chavis, R. M. and Sears, M. L. (1973). Cyclic-AMP in the aqueous humor. the effects of repeated topical epinephrine administration and sympathetic denervation. Exp. Eye Res. 16, 265-72. Neufeld, A. H., Dueker, D. K., Vegge, T. and Sears, M. L. (1975). Adenosine 3’.5’monophosphate increases the outflow of aqueous humor from the rabbit eye. Znwst. Ophthulmol. 14, 40-2.

690

K. S. BOAS

F,T Al,.

Neufeld. A. H., *Jampol, L. M. and Sears, M. I,. (1972). Clyclic-AMP in the aqueous humor: The effects of adrenergic agents. Exp. EYP Res. 14. 242-50. Neufeld, A. H. and Page, E. D. (1977). In vitro determination of the ability of drugs t’o bind to adrenergic receptors. Invest. Opthalmol. 16, 1118-24. Neufeld, A. H. and Sears, M. L. (1974). Cyclic-AMP in ocular tissues of the rabbit. monkq~. and human. Invest. Ophthalmol. 13, 475-7. Norton. A. L. and Viernstein, L. J. (1972). The effect of adrenergic agents on ocular dynamirs as a function of administration site. Exp. Eye Res. 14, 154-63. Potter, D. E. and Rowland, J. M. (1978). Adrenergic drugs and intraocular pressure: effects of selective beta-adrenergic agonists. Exp. Eye Res. 27, 615-25. Radius, R. and Langham, M. E. (1973). Cyclic-AMP and the ocular responses to norepinephrine. Exp. Eye Res. 17, 21%29. Sears, Marvin L. and Barany, E. H. (1960). Outflow resistance and adrenergic mechanisms: effects of sympathectomy, N-(2-chlorethyl) dibenzylamine hydrochloride (dibenamine) and dichloroisoproterenol on the outflow resistance of the rabbit eye. A.M.A. Arch. Ophthalmol. 64, 83S48. Sears, M. L. and Sherk, T. E. (1964). The trabecular effect of noradrenaline in the rabbit eye. Invest. Ophthalmol. 3. 157-63. Vareilles, P., Silverstone, D., Plazonnet, B., Le Douarec, J., Sears, M. L. and Stone, C. A. (1977). Comparison of the effects of timolol and other adrenergic agents on intraocular .pressure in the rabbit. Invest. Qphthalmol. 16, 987-96. Waitzman, M. B. and Woods, W. D. (1971). S ome characteristics of an adenyl cyclase preparation from rabbit ciliary process tissue. Exp. Eye Res. 12, 99-l Il.