The Mode of Action of Pilocarpine on Outflow Resistance in...

The mode of action of pilocarpineon outflow resistance in the eye of a

primate (Cercopithecus ethiops)

Ernst H. Bdrdny

Facility of outflow in the eye of the vervet monkey Cercopithecus ethiops was studied byconstant-rate and by constant-pressure infusion. Facility increased greatly after the injectionof 2 to 3 fig of pilocarpine into the anterior chamber or after the application of 0.2 to 0.3 ing.to the cornea. The effect on facility consisted of 2 components. One component was re-versed in 3 to 5 minutes by 1 ing. per kilogram intravenous atropine, the time it took forthe atropine to relax pilocarpine-incluced accommodation. The other component was re-versed only slowly by intravenous or even intracameral atropine. The slowly reversed com-ponent toas thought to represent a pilocarpine action directly on the endothelial cells of theoutflow system, possibly a histamine-like action on the endothelial wall of Schlemm's canal.

-L ilocarpine is a curious drug. Amongophthalmologists it is thought of as amiotic, as a purely cholinergic substanceattacking the smooth muscle cells directly.However, things are more complicated.For instance, pilocarpine dilates the pupilof the rat1 and the guinea pig- (but notthat of cat, rabbit, or primate); it com-petes with other cholinergics3' 4>s; and itcan exert an atropine-like action undercertain conditions. Above all it does notlook like a cholinergic drug. I dare saythat no pharmacologist or pharmaceuticalchemist could have predicted a cholinergic

From the Department of Physiology, School ofMedicine, Makerere College, Kampala, Uganda,and the Department of Pharmacology, Uni-versity of Uppsala, Sweden (permanent ad-dress of author).

This study was supported by Research GrantB-3060 from the Institute of Neurological Dis-eases and Blindness, United States PublicHealth Service, Bethesda, Md.

action from the structure of pilocarpine(Fig. 1). One would have expected anaction related to histamine and, in fact,there are histamine-like effects of pilo-carpine.6 Apart from stimulating gangliaand the adrenal medulla in low concen-tration, it has vascular actions in the catand dog resembling those of histamine. Wewill return to this later.

Pilocarpine was first isolated in 1875.As a remedy in glaucoma it was first men-tioned in 1877 in the very last sentence ofa 91 page paper by Adolf Weber7 ofDarmstadt, the father of applanation to-nometry. The title of the paper was "DieUrsache des Glaucoms" but he spent thelast few pages warning against the dangerof eserine and ended up by saying, ineffect: "There is another drug, pilocarpine,which I can recommend much more warmly. . . and I hope that it will make iridectomyunnecessary in many cases and help otherswhere the effect of operation is not suffi-ciently marked."

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Volume 1Number 6 Action of pilocarpine on outflow resistance 713

In warning against eserine in glaucomaWeber certainly was a little too pessimistic.He had used it for several years in anamazing variety of ophthalmologic con-ditions including cornea! ulcers but hadnot published anything on it when a one-page preliminary note by Ludwig Laqueurs

from Strasbourg appeared in June, 1876.Laqueur, who suffered from the diseasehimself, enthusiastically proposed the useof 0.3 to 0.5 per cent serine in glaucoma.Weber immediately wrote a rather lengthyreport of his experience with the drug,0

warning against its indiscriminate use inglaucoma. This report appeared in thelast 1876 issue of v. Graefe's Archiv andalready in the next issue he proposed pilo-carpine.

Thus, the two drugs on which glaucomatherapy has rested for so long a time wereproposed almost simultaneously and formany years they reigned unopposed. Theirusefulness in glaucoma has usually beenconsidered to be due to their effect on theiris sphincter and the ciliary muscle. Thereis little doubt that the miotic action properis the important one in angle-closure glau-coma. But in open-angle glaucoma it isnot clear how pilocarpine or for that matterany drug improves outflow facility. Thatmiosis is not essential has been known fora long time. Armaly and co-workers1011

presented tonographic evidence that volun-tary ciliary muscle contraction by accom-modation improved facility in the humaneye, but there are also experiments sug-

CH3CH2

PILOCARPINE

1 CH2 CH.

HISTAMINE

NH2 CH2 — CH2

Fig. 1.

gesting that this is not the whole story.Shaffer1- has reported that facility is lessimproved by voluntary accommodationthan by the same amount of pilocarpine-induced accommodation, and Ballintine11

has repeatedly stressed his clinical impres-sion that there is a difference betweeneserine and pilocarpine; he believes thatwith eserine the effect on facility runsparallel with the effect on accommodationwhile with pilocarpine an effect is obtainedon facility in doses which affect accom-modation but little.

This is where the matter stands at themoment. It is possible but not provedthat pilocarpine acts at several differentlevels.

Evidently the matter was worth lookinginto from the ophthalmologic point of viewas well as from the pharmacologic, andsince the generosity of the United StatesPublic Health Service made it possible forme to work with large numbers of monkeysI decided to look into the mode of actionof pilocarpine in a primate species.

Plan of investigation

The simple plan of the experiments wasbased on the fact known to every phar-macologist, that the cholinergic smooth-muscle effects of pilocarpine are quicklyreversed by a sufficient dose of atropine.Thus, take an eye under the influence ofpilocarpine. Record facility of outflow.Give a huge dose of atropine intravenously.If the effect of pilocarpine is due to itsinfluence on the ciliary muscle or onsmooth muscle in vessels carrying blood,the pilocarpine effect on facility will dis-appear within a few minutes. If it doesnot, pilocarpine has another point of attackon facility.

Choice of primate species

Pilocarpine is a miotic in all monkeysmentioned below, but different closes areneeded to affect facility in different species.Thus in the green vervet Cercopithecusethiops, in the baboon Papio anubis, in themangabey Cercocebus albigena and in the


714 Barony Investigatioe OphthalmologyDecember 1962

| MOTOR L - ^ ^ ^ S 3

<ZH2 1 I—MICROMETER

f—1 WEIGHT 1

—IPRESSUREI

4 CHANNELSTRIP CHARTRECORDER

SECONDJEYE

Fig. 2. Diagram of infusion system used for facility determinations. Appropriate stopcocksallow either constant-pressure infusion from reservoir or constant-rate infusion from motor-driven syringe. Drugs are injected from micrometric syringe.

redtail Erythrocebus patas, all of themAfrican, a few micrograms of pilocarpineinjected into the anterior chamber have amarked effect on facility. This is not so inthe two macaques tested, the Indian rhesus,Macaca mulatta, and the Javanese cyno-molgus monkey, Macaca irus. In 6 individ-ual rhesus monkeys I found no certaineffect of doses up to 12 fig in spite ofmaximal miosis and in the cynomolgus thedose of 20 /ig was needed to obtain amoderate effect. The bulk of my experi-ments were done in the green vervet and,when nothing else is expressly stated, thedata to be presented here derive fromabout 130 individuals of this species. Theconstant-rate infusions were made in EastAfrican monkeys, the constant-pressureones in West African animals.

Methods

The monkeys were anesthetized with intrave-nous veterinary Nembutal, 25 to 30 mg. perkilogram body weight, and kept warm with anelectric heating pad. Under these conditionsmean blood pressure was about 80 to 100 mm.Hg. Quite a number of experiments were lostbecause of nystagmus. Other anesthetics weretried but were no better. The only remedy wasto keep anesthesia really deep. The anteriorchambers were cannulated with branched needles,0.45 mm. outer diameter, by means of the needlegun described by Sears.14 Sometimes topicalanesthesia with lidocaine was used. One of thebranches of each needle was connected bynarrow polythene tubing to an Agla micrometric

syringe for drug or saline injections. The otherbranch was connected by similar tubing to amanifold leading to a strain gauge pressurerecorder and to sources for infusion fluid. Twotypes of infusions were made to record facility:constant rate and constant pressure. For constantrate, Agla syringes actuated by a constant ratemotor were used. They were set to deliver iden-tical amounts to both eyes, as a rule close to 5 /*1per minute. The increase in steady-state pressurein millimeters of mercury per microliter perminute inflow rate was a measure of outflow resist-ance. The pressure rise due to needle resistancewas deducted. For constant-pressure infusion asmall reservoir, a polythene "thimble" suspendedon a force transducer, could be set at differentlevels above its eye. A narrow polythene tubingconnected the thimble with the manifold. Theweight of the thimble with its content of remain-ing infusion fluid was recorded and facilityestimated from the difference in rate of inflow attwo different levels. Appropriate corrections forthe resistance of the perfusing needle and tubingwere made. The infusion fluid was 0.9 per centNaCl unless otherwise stated. Pilocaq^ine hydro-chloride and atropine sulfate were used and areidentified without mentioning the anion in thefollowing. The doses given refer to the salts.

Fig. 2 is a diagram showing the arrangement.

Results

1. Constant-rate infusions. Fig. 3 showsa constant-rate infusion experiment demon-strating the effect of 2 fxg of pilocarpineinjected into the anterior chamber. Firstan infusion lasting 13 minutes was giveninto both untreated eyes. Then the pilo-carpine in 10 JX\ of saline was injected into


Volume 1Number 6

Action of pilocarpine on outflow resistance 715

Table I. Effect of 2monkeys

pilocarpine HC1 into the anterior chamber of vervet

ExperimentNo.

Bodyweight(Kg.)

Resistance ratio, experimental eye/control eye

A. Before pilocarpine \ B. After pilocarpineRatioB/A

8149563558555036

1.23.23.62.41.94.82.54.8

1.440.900.861.061.730.880.810.89

0.390.340.330.470.900.54

< 0.44°< 0.60°

0.270.370.380.440.520.62

< 0.55< 0.67

•Pressure in pilocarpine eye still falling when infusion was stopped.

the experimental eye and the same volumeof saline into the control eye. Two laterinfusions showed that the resistance of theexperimental eye was reduced to abouthalf and that it was still low one hour afterthe pilocarpine.

There are several features of this experi-ment which merit discussion. The restingpressure was low even for an anesthetizedmonkey. Under our conditions the averagepressure in 135 eyes was 8.9 mm. Hgls (asagainst about 20 mm. in light ether anes-thesia10). The rise on the first infusion of4.77 p\. per minute (as it happens to bein this experiment) corresponded to a re-sistance of 1.93 and 2.14 mm. Hg//xl x

in the experimental and controlmm.'eyes, respectively, and was slightly higherthan the geometric mean 1.63 derived from116 eyes.15

As usual the resistance of the control eyediminished during the course of the ex-periment, and the rise caused by successiveinfusions decreased. This drop was smallerthan average in the experiment illustrated.In 35 eyes, where a second infusion wasmade without any intervening treatmentof the eye but after varying amounts offirst infusion, the mean resistance obtainedduring the first infusion was 1.91 ± 0.15mm. Hg/jul x minr1 as against 1.46 ± O.Iduring the second. The coefficient of cor-relation between first and second was 0.81and the coefficient of regression of thesecond on the first was 0.56. In some ex-periments resistance dropped very mark-

edly during the infusions, down to below50 per cent of the original value.

The degree of pilocarpine effect duringthis experiment was rather typical for thedose. In * 8 experiments with 2 /ig, per-formed in a similar manner, the values

mm Hg

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Vervet, 3.2 kg Infusion 5jjl/min.

Fig. 3. Effect of intracameral pilocarpine on out-flow resistance demonstrated by constant-rate in-fusion. The black bars indicate periods of infusioninto both eyes. The ordinates indicate intraocularpressure.


716 Bdrdny Investigative OphthalmologyDecember 1962

-VERVET•-BABOON

b - 0.86r = 0.73

0 ~ 1 2 3 ARESISTANCE mm Hg/iul.min

Fig. 4A. Correlation between outflow resistanceand Tyju, the time taken for half the pressure in-crease to take place on start of the infusion.Normal eyes of vervets and baboons.

shown in Table I were obtained. The eyesillustrated in Fig. 3 belonged to No. 49.

The time course of the pressure riseswhen infusion starts were not strictlyexponential but can be approximatelycharacterized by the time needed for halfthe rise to take place: 2.7 and 3.3 minutes,respectively, in the first period. Unex-pectedly, but regularly, the half-time forreturn to resting pressure after the infusionwas much shorter, 1.3 and 1.1 minutes,respectively, in the first period. Now, ifthe volume compliance of the globe andthe outflow resistance had been constantin the pressure range covered, the curveswould have been strict exponentials andtheir half-times would have been propor-tional to the volume compliance and theoutflow resistance and equal for the ascend-ing and descending phases. There was agood correlation (r = 0.73) between re-sistance and half-time for the ascendingphase (Fig. 4A) but the correlation be-

tween resistance and half-time of thedescending phase looked quite different(Fig. 4B). This remains unexplained butindicates the presence of some complicat-ing factor probably affecting the timecourse of the descending phase.

The importance of the time constantbecomes evident if one regards a typicalatropine experiment (Fig. 5). After thefirst infusion of saline into both eyes, 3 ^gof pilocarpine is given into one anteriorchamber. The effect on resting pressure isnot significant but that on resistance isdramatic. Intravenous atropine in the veryhigh dose of 1 mg. per kilogram is thengiven. It reverses the pilocarpine effect andthe pressure in the pilocarpine-treated eyerises gradually after a few minutes oflatency.

Let us consider how the time course ofthis latter rise would have looked underthe simplest assumptions, that atropinerapidly and completely reverses the effectof pilocarpine after a certain latency, andthat volume compliance and outflow re-sistance are pressure independent.

As soon as the reversal has taken place,then, the eye is back in its untreated con-dition. Under continued infusion the pres-sure will move toward its old steady stateand this movement will be governed bya time constant characteristic of the un-treated eye (Fig. 6). Comparing Figs. 5

1 2RESISTANCE mm Hg//ul,min

Fig. 4B. Correlation between outflow resistance,Tv,u, the time taken for half the pressure dropto take place on cessation of infusion. Samevervet eyes as in Fig. 4A.


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mmHg26

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1 mg/kg i.v.ATR

3Mg ,PIL

' £ . . 8 8

- Vervet 3.8 kg. Infusion 5 pl/min

10 20 30 40 50 60 70 80TIME IN MINUTES

90 100

Fig. 5. The effect of pilocarpine and its reversal by intravenous atropine demonstrated byconstant-rate infusion.

Fig. 6. The expected time course of pressure change in a constant-rate infusion experimentsimilar to that of Fig. 5, if volume compliance and outflow resistance were pressure independentand if atropine reversed pilocarpine suddenly and completely. Abscissa, time; ordinate,intraocular pressure; both in arbitrary units.

and 6 we see that the real time course ofrecovery of pressure after atropine looksdifferent from the simple quasi-exponentialone would have expected from the simpli-fied assumptions and the actual behaviorof the control eye. The probable explana-tion is that pilocarpine effect is not reversedrapidly and completely by the atropinebut more gradually. However, the dis-crepancy is not large enough to make thisquite evident and the experiment istypical in not proving anything one wayor the other.

In Fig. 7 where one can also see therapid drop after pilocarpine, a slow re-covery after atropine is more clear-cut. Itis seen to have continued between the firstand second infusions. In Fig. S where theresistance of the control eye is not sus-tained under continuous infusion, thereversal of the pilocarpine effect evidentlyis quite slow. The same can be seen inFig. 9, a quick effect of a huge dose ofpilocarpine and a very slow recovery afteratropine.

Clear-cut slowness of this kind was not


718 Barony Investigative OphthalomologyDecember 1962

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kg. Infusion 5 /jl/min

-4 0 10 20 30 40 50 60 70TIME IN MINUTES

Fig. 7. Slow reversal of pilocarpine effect by intravenous atropine.

80

the rule in our experiments. Of about 20experiments with this protocol only 7showed such slowness of recovery that thepresence of a gradual effect of atropinewas suggested. In the remaining cases,recovery was not markedly slower thanwould be expected from the time constantof the untreated eye. In the 6 experimentswhere recovery was slowest relative to thetime constant of the eye, the average re-sistance was 2.3 mm. Hg/^,1 x minr1, inthe 6 fastest the average resistance was1.0 mm. Hg/jul x minr1. We will return tothis difference later.

We thus had evidence suggesting thatat least part of the pilocarpine effect onfacility could be reversed only slowly byatropine, the process taking many minutes.However, this slow part was not demon-strable in every experiment.

From general pharmacologic experienceone would not expect antagonism betweenatropine and pilocarpine to develop soslowly. In ordinary experiments with iso-lated smooth-muscle strips from cat orrabbit intestine a contact time of 3 minuteswas ample for atropine to exert its relaxingeffect against spasm induced by a 50 times

higher concentration of pilocarpine. Itseemed probable that relaxation of theciliary muscle by atropine or the contrac-tion of pilocarpine-dilated vessels shouldbe equally prompt.

To make doubly sure, however, I havetried to see directly how fast pilocarpine-induced accommodation was relaxed by in-travenous atropine. Since this was difficultto do in eyes with a needle in the cornea(without excluding cornea] refraction byimmersion), it was necessary to introducethe pilocarpine through the intact cornea.Preliminary experiments similar to thatshown in Fig. 10 indicated that the dose of200 fig contained in 10 /J. and applied tothe cornea about one hour before the ex-periment has approximately the same effecton facility as 2 fxg injected into the anteriorchamber. In 6 experiments with constant-rate infusion, the average resistance on theside treated with 200 /xg was 48 per centof that on the control side (range 24 to 70per cent) and in 4 experiments with con-stant-pressure infusion the values were 28to 36 per cent. Consequently this dose wasapplied to 9 intact eyes and after one hour1 mg. per kilogram atropine was given


Volume 1Number 6


intravenously. The refraction was followedby retinoscopy. The pupil widened suf-ficiently within about 1 minute. Since itwas difficult to do the retinoscopy fastenough during the phase where refractionchanged quickly, the individual curveswere rather irregular, but when the 9experiments were averaged, as shown inFig. 11, it became evident that the ciliarymuscle behaved similarly to other smoothmuscles: pilocarpine was quickly antag-onized by a sufficient dose of atropine.Within 3 to 5 minutes the pilocarpine

effect on accommodation was reversed.There was no reason to believe that theeffect of pilocarpine on vascular smoothmuscle would behave very differently.

Pilocarpine applied to the cornea 1 hourbefore the infusion had the opportunity toact on intraocular structures a much longertime than in our regular experiments. It wastherefore of interest to study what hap-pened to facility after intravenous atropinein eyes treated with pilocarpine in thismanner. Fig. 12 shows the results.

The lowermost curve represents 5 eyes

mmHg

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Fig. 10. Effect of pilocarpine applied to thecornea demonstrated by constant-rate infusion.Verbet, 4.6 kilograms. Infusion 5 fi\ per minute.Experimental, 200 Mg pilocarpine at -60 minuteson the cornea.

in 5 monkeys. Four of these had received0.2 and one 0.3 mg. pilocarpine on thecornea one hour before the experiment. In4 of them retinoscopy was successfullyperformed on the contralateral, not cannu-lated eye during the pressure recording,and these values were included in theaverage refraction curve. Atropine wasinjected intravenously at zero time and thecurve shows the average pressure duringinfusion of 5 /x\. per minute.

The middle curve is the refraction curvealso shown in Fig. 11, illustrating howaccommodation was relaxed.

The topmost curve is obtained from 7other monkeys. These eyes had not re-ceived any treatment except the topicallidocaine for cannulation. It is seen thatthe normal eye did not change its resist-ance despite the sudden cycloplegia. Butalso the pilocarpine-treated eye showedonly a small pressure rise as accommodationrelaxed, corresponding to a resistance riseof 0.15 unit. The main part of the pres-sure difference between the two groups

of eyes remained. Part of this differenceadmittedly could have been due to thefact that the eyes belonged to differentgroups of monkeys. But all of it could notbe due to this cause. If the pilocarpineeffect had been completely reversed by theatropine, it would mean that its size wouldhave been only about 0.15 unit. Since thedose of pilocarpine reduced the resistanceby an average of 60 per cent, the averageresistance of this group of 7 eyes wouldhave been about 0.4 unit. This is lower thanany single eye of 116 I have studied,15

their range being from 0.73 to 4.09. It isquite certain therefore that a considerablepart of the pilocarpine effect remained un-reversed even at the end of the 12 minutesof observation in these experiments.

A slow reversal of the pilocarpine effectcould also be seen if atropine was injecteddirectly into the anterior chamber. In Fig.13 the enormous dose of 15 /xg per eye isseen not to affect the control eye and onlyvery slowly to bring the pilocarpine-treated eye up to control level. Similar,very slow pressure rises were seen in 2more experiments with this protocol, whilein 2 experiments the rise was no slowerthan could be expected from the half-time of the eye and in 3 experiments theresult was questionable.

Fig. 11. Reversal by intravenous atropine of ac-commodation induced by 0.2 m^. pilocarpineplaced on the cornea about 60 minutes earlier.Data from 8 monkeys, 1 contributing both eyesat 5 day interval. Experimental values for eachanimal were plotted and values for whole minutesderived by interpolation. The curve shows theaverage of these interpolated values. Refractionzero has arbitrarily been assigned to the asymptote.


Volume 1Number 6


mm Hg

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1 mg/kgATR

^*—•"

__o—-o o—o^

^ * — * — * — • — • —

Av. pressurea 7 untreated eyes

_>^ during continuous- ^ infusion of 5/jl/min

Av. refract ion^*-~~* 9 pilocarpine eyes

Av. pressure_^_^ 5 pilocarpine eyes

during continuousinfusion of 5/ul/min

10 15 M I N

Fig. 12. Eye pressure during constant-rate infusion and refraction changes after intravenousatropine. The refraction curve is that of Fig. 11. Note rapid disappearance of myopia withonly small increase in resistance after atropine.

mm HQ

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EXPTL 200 ug pilocarpine on cornea at -69 min•CONTROL „

15ugATR

ATR

Vervet 3.1kg. Infusion 5pl/min

20 30TIME IN MINUTES

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Fig. 13. Slow effect of 15 Mg atropine injected into the anterior chamber of both eyes onresistance of pilocarpine-treated eye.

Experiments like Fig. 13 exclude theextremely improbable possibility that theslowly reversible action of pilocarpine ison smooth muscle not accessible from thebloodstream.

2. Constant-pressure infusions. Until nowwe have concentrated on evidence for thepresence of a slowly reversible componentin the pilocarpine response. This has beenclearly demonstrated in some experimentsbut may be present in many more, hiddenbehind the intrinsic slowness of eye pres-sure as an index of outflow resistance. Andif a slow response can hide, this must bedoubly true of any quickly reversible com-

ponent, for instance, due to pilocarpine-induced accommodation. A hint of thepresence of such a component was seen inFig. 12 where pressure rose a little as ac-commodation relaxed. In order to dis-entangle the situation, however, we need amethod to study quick changes in resist-ance. Constant-pressure infusion is sucha method.

The advantage of constant-pressure in-fusion in certain situations depends on thefact that the viscoelastic time constant ofthe eye is determined by the product ofvolume compliance and a resistance whichcomprises the ordinary outflow resistance in



A*'270

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VERVET 2.7 kg.

• EXPTL

° CONTROL

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-2 A 6 8 10 12TIME IN MINUTES

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Fig. 14. Latency of action of intracameral pilocarpine dose recorded withconstant-pressure infusion. Ordinate, microliters of saline left in reservoir.The lower of the two pressure levels was 5 mm. Hg above original restingpressure.

parallel with the resistance of the per-fusion system Rp. In constant-rate infusionR,, is virtually infinite, the machine deliversat a rate independent of the counterpres-sure in the eye. In this case the propertiesof the eye itself govern the situation. Inconstant-pressure infusion, R,,, the resist-ance between the fluid container and theeye can be made quite low and the timeconstant correspondingly short. Thus, withconstant-pressure infusion, quick changesin outflow resistance should be resolvable.It seems that a limiting factor with thissystem is the imperfect elasticity of thesclera, which in our monkeys takes about1 minute to adjust its volume to a newpressure. Whether or not blood volumechanges in the uvea also occur in this firstminute is not clear.

To demonstrate the time resolution at-tainable with constant-pressure perfusion,Figs. 14 and 15 show the latency of anintracameral pilocarpine effect and an in-travenous atropine effect, respectively. Inthese experiments the drugs were givenduring perfusion at 12.5 mm. Hg above

the original resting level of the eye. Fig.14 shows that already 2 minutes after thebeginning of the injection of 1.5 /.ig pilo-carpine the rate of inflow started to ac-celerate. In Fig. 15 there is a slowing ofthe inflow beginning about 4 minutes afterintravenous atropine.

Infusion rate at only one pressure levelcan give quite misleading results if rate ofsecretion in the eye changes. Therefore,for really quantitative studies, we haveused alternating periods of 2 pressures, 7.5mm. Hg apart, usually placing the lowerone 5 mm. Hg above the original restinglevel of the eye. The minimum durationat each pressure was 4 minutes. The dif-ference between the inflow rate at the twopressures gives a facility, A flow/A pres-sure. Thus each period of infusion in eacheye affects 2 facility values, one precedingand one following the period. The methodpresupposes constant conditions over the8 minutes of each pair of periods. Evi-dently it is only a simplification of theslope-plotting method of Becker and Con-stant.17


Volume 1Number 6 Action of pilocarpine on outflow resistance 723

Fig. 16 shows a section of a record withthe two-pressure method. The figures arethe facilities derived from the just preced-ing breaks in the curves. It is seen that,when pressure was lowered, 7 minutesafter pilocarpine, the slope of the pilo-carpine-treated eye became nearly nil. Theslope difference was large, the facilitydefinitely increased, but the drug had in-creased secretion so that actually inflowfrom the perfusion system at a low pres-sure was smaller than it was before thedrug was given. Evidently it is not quitesafe to use single-pressure methods. Thetwo-pressure method moreover allowed anextra check: The resting pressure whereslope would be zero could be calculatedby extrapolation from the two slopes at thetwo pressures. In successful experimentsit was reasonably constant.

When the two-pressure method wasapplied in 16 vervet experiments withintracameral injection of 1.5 to 3 /xg ofpilocarpine, a quick pilocarpine effect onfacility was seen in 15 of them. Intra-venous atropine caused a quick reversalin almost all. Only in 2 of them was thereversal virtually complete, however (Fig.17). The incompleteness in the majority ofexperiments (Fig. 18) might have beendue to the presence of a slowly reversiblecomponent. But, as a rule, no actual slowdecline in facility was discernible. Instead,on continued infusion, the experimentaland the control eye usually showed grad-ually increasing facility, sometimes endingwith facilities several times larger thanthe starting values. Changing the 0.9 percent NaCl as infusion fluid to Ringer'ssolution containing 24 mg. per 100 ml.CaCl2 and 42 mg. per 100 ml. KC1 did notimprove matters.

Only when we changed to Ringer's solu-tion containing 75 mg. glucose per 100 ml.solution and shortened the period of in-fusion by preapplication of 200 /xg pilo-carpine to the cornea one hour before theexperiment did we succeed in demon-strating the slow component in reversalwith this method too (Fig. 19). A sys-

tematic study of the influence of the com-position of the infusion fluid evidentlywould be worthwhile.

Discussion

Two components in the pilocarpine effecton facility in normal vervet eyes have beendemonstrated. The one component dis-appears in a few minutes after intravenousatropine. This time course is similar to thatof the disappearance of accommodativespasm caused by pilocarpine. There is littlereason to doubt that this component ofpilocarpine action is due to an effect ofthe drug on the ciliary muscle and onlyindirectly on the trabecular meshwork. Theother component is only slowly reversibleby intravenous atropine. Thus it veryprobably is due to a direct pilocarpineeffect on the structures forming the out-

240

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• 1.5/ug PIL at -15 mins.

° Control

ATR 1 mg/kg i.v.

VERVET 1.8 kg

-i, -2 0 2 4 6 8TIME IN MINUTES

10 12

Fig. 15. Latency of action of intravenous atropinerecorded with constant-pressure infusion. Ordinate,microliters of saline left in reservoir. Both eyeskept at 12.5 mm. Hg above resting level. Thefacility of the control eye had increased during theearly part of the experiment but did not reactto atropine.



270

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• EXPTL

• CONTROL

• * * • • • • 0 6 6 I" 0.64 *• o ' 1

'•'.'•., i1 0.66*«.#°o

-

Infusion alternatingly at twopressure levels 75 mm Hg apart.Figures indicate facility values.

-

VERVET 3.0 kg.

0.65

1.63

-10 - 8 - 6 -4 - 2 0 2 4 6TIME IN MINUTES

10 12 14 16

Fig. 16. Part of constant-pressure infusion experiment. Ordinate, microliters of saline left inreservoir.

flow resistance. The slowness with whicheven intracameral atropine reverses theeffect indicates that smooth muscle is notresponsible for this effect. The only otherpossibility seems to be the endothelial cells,most importantly those forming the wall ofSchlemm's canal.

If pilocarpine reduces the resistance ofthe endothelial wall of the canal, the effectof this component of its action on over-allfacility evidently will depend on the frac-tion of total resistance residing in the endo-thelium from the start. If the major resist-ance is in the fibrous meshwork, changesin the endothelial resistance will have littleover-all effect. Thus, if in prolonged per-fusion the endothelial wall has reactedwith leakiness even before pilocarpine wasgiven, its resistance will be low and themajor part of the resistance perhaps residein the fibrous meshwork. Pilocarpine willthen induce a clear quickly reversibleeffect by ciliary muscle action on this latterstructure but no noticeable slowly revers-ible one. The findings in Section 1 thatwhen a slowly reversible effect was seen

the resistance was more than twice ashigh as when it was not seen may have thisexplanation.

It was easier to demonstrate the slowlyreversible effect if pilocarpine was appliedto the cornea 60 minutes before the in-fusion than if it was injected into thechamber only shortly before atropine wasgiven. There are two factors which mightbe of importance here. The preapplicationexperiments involved much shorter in-fusions, with presumably better conserva-tion of endothelial starting resistance, andthe drug had much more time to act. It ispossible that the slowly reversible effecttakes time to develop fully.

The nature of the slowly reversiblechange caused by pilocarpine is not clear.It could be a general shrinking or swellingof cells, affecting the passage of fluid inseveral places, presumably most efficientlyin the transcellular passages of Holm-berg.18 But it is also possible that pilo-carpine has a histamine-like action hereand that it opens new passages altogetherby producing a "leaky canal of Schlemm,"


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The fact that the slow effect is reversibleby atropine does not militate against ahistamine-like action. Atropine in highconcentrations is quite unspecific.

Histamine affects vascular endotheliumin two ways. It may cause the cells to swelland it causes them to separate.19 That thisseparation is actually the basis of thevascular leakiness caused by histamine hasrecently been beautifully shown by Majnoand Palade.-0 If pilocarpine were to causea swelling of the endothelial cells alongthe route of the aqueous, resistance couldhardly be lowered. But if the drug were tocause separation between the cells of thewall of Schlemm's canal (which after allis a small vein) an increase in facilitymight result.

There is some tonographic evidence fromthe human eye compatible with such anaction on the endothelial wall.

Scheie and collaborators-1 have pub-lished a diagram which indicates that theuse of miotics (presumably mainly pilo-carpine) increases facility in patients whohave chronic simple glaucoma by the

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0.5

FACILITY• EXPTL

o CONTROL

PIL3/jg

ATR VERVET 3.0 kg.


60 70

Fig. 17. Results of constant-pressure infusion ex-periment of Fig. 16 plotted in terms of facility.Rapid and complete disappearance of pilocarpineeffect on intravenous atropine, 1 mg. per kilo-gram.

3.0

2.0

1.0

FACILITY1.5/ug

PIL ATR

VERVET 2.7 kg.


K0

Fig. 18. Typical constant-pressure infusion ex-periment illustrating continuous increase in facilityof control eye, rapid elfect of pilocarpine, andincomplete or slow reversal by 1 mg. per kilogramintravenous atropine. A quick phase of atropineaction is also present.

same amount, independent of the startingvalue.

Linner-- has recently compared the addi-tional facility induced by pilocarpine in 80normal eyes (mean C = 0.27) and in 32eyes with chronic simple glaucoma (meanC = 0.11). Pilocarpine, 3 per cent, wasgiven twice with an interval of one hour;tonography was done one hour after thelast instillation. Facility increase was some-what lower in glaucoma—0.026 ± 0.0071—than in normal eyes—0.048 ± 0.0095. Thedifference is not statistically significant.

It is interesting to note that similar find-ings have been made by Becker-* with twostrong cholinesterase inhibitors, echothio-phate and demecarium.

If miotics open new pathways across theendothelium independent of the old onesand if endothelial resistance dominatesover all other resistance components inthe majority of cases, constant additionof facility is what one would expect.

Thus we are back to the question: Howlarge is the relative contiibLition of endo-thelial resistance? There is probably nosingle answer, either in normals or in



2.5

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FACILITY • 200 yug PIL at - 6 0

•CONTROL

GLUCOSE-RINGER

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- VERVET 2.55 kg.


60

Fig. 19. Gradual reversal of the effect of pilo-carpine applied to the cornea by 1 mg. per kilo-gram intravenous atropine. Constant-pressure in-fusion with Ringer's solution containing 75 mg.per 100 ml. glucose. A rapid atropine effect isalso seen.

glaucoma. To begin with glaucoma, theremay perhaps be a majority of eyes withchronic simple glaucoma in which endo-thelial resistance constitutes the dominantpart, but this is not necessarily so in caseswhere considerable hyalinization of thefibrous trabeculae has occurred. And inthe normal eye it seems possible that thehomeostatic mechanism regulating eyepressure plays on endothelial resistancesince it cannot very well play on accom-modation. If this is so one would expectconsiderable variation in the relative im-portance of endothelial resistance even innormal eyes.

A hint of the size of endothelial resist-ance can possibly be gained from the sizeof the very marked lowering of resistancesometimes observed during infusion, espe-

cially in constant-pressure infusions. Itseems improbable that homeostasis isresponsible for this resistance drop, butsince, whatever its mechanism, it cannotvery well be due to shifting ciliary musclepull, the size of the drop indicates the im-portance of the state of the endotheliumfor resistance.

We still have to consider the action ofpilocarpine on the resistance of the fibrousmeshwork. It is tempting to ascribe theeasily demonstrated, quickly reversiblecomponent in pilocarpine action to changesin this structure brought about by ciliarymuscle pull. If a considerable resistanceresides in the fibrous meshwork, contrac-tion of the ciliary muscle could changeit by pulling the scleral spur inward andseparating the trabecular lamellae. Butsince the endothelial wall of the canalcannot disengage from the rest of the mesh-work, ciliary muscle pull will also affectthis layer. It is therefore not yet permis-sible to estimate the relative importanceof endothelial wall resistance and fibrousmeshwork resistance from the relativesizes of the slowly reversible and thequickly reversible pilocarpine effects in thesingle case. Part of the quickly reversibleeffect may be an action on endothelial re-sistance. But if this should prove not to bethe case, very interesting conclusions as tothe site of resistance in individual cases ofglaucoma could be drawn from the differ-ence or lack of difference in effect betweenpilocarpine and accommodation or betweenpilocarpine and a miotic without endo-thelial action. Whether such a miotic existsis still an open question.

I wish to thank Professor C. P. Luck and hiscolleagues at the Department of Physiology atMakerere for kind hospitality, Miss Ingalill Dal-hagen for expert assistance, and the WellcomeTrust, London, for animal facilities donated toProf. Luck's department. Without these thepresent work could not have been undertaken.

REFERENCES1. Waddell, J. A.: The action of pilocarpin on

the rat's pupil, J. Lab. & Clin. Med. 12: 232,1926-1927.


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2. Koppanyi, T.: Comparative studies on pupil-lary reaction in tetrapods. V. Action of pilo-carpine on the pupil of the guinea pig, J.Pharmacol. & Exper. Therap. 36: 179, 1929.

3. Swan, K. C, and Gehrsitz, L.: Competitiveaction of miotics on the iris sphincter, A. M.A. Arch Ophth. 46: 477, 1951.

4. van Rossum, J. M.: A new view on an olddrug: Pilocarpine, Experientia 16: 373, 1960.

5. van Alphen, C. W. H. M., Robinette, S. L.,and Macri, F. J.: Drug effects on ciliarymuscle and choroid preparations in vitro,A. M. A. Arch. Ophth. 68: 81, 1962.

6. Burn, J. H.: The principles of therapeutics,Oxford, 1957, Blackwell Scientific Publica-tions, pp. 56-57.

7. Weber, A.: Die Ursache des Glaucoms, vonCraefes Arch. f. Ophth. 23: I, 1, 1877.

8. Laqueur, L.: Ueber eine neue therapeutischeVerwendung des Physostigmin, Zentralbl. f.med. Wissensch. 14: 421, 1876.

9. Weber, A.: Ueber Calabar und seine thera-peutische Verwendung, von Graefes Arch. f.Ophth. 22: IV, 215, 1876.

10. Armaly, M. F., and Burian, H. M.: Changesin the tonogram during accommodation, A. M.A. Arch. Ophth. 60: 60, 1958.

11. Armaly, M. F., and Jepson, N. C.: Accommo-dation and the dynamics of the steady-stateintraocular pressure, Invest. Ophth. 1: 480,1962.

12. Shaffer, R. N.: in Newell, F. W., editor:Glaucoma, Tr. of the Fifth Conference, NewYork, 1961, Josiah Macy, Jr. Foundation,pp. 234-237.

13. Ballintine, E.: In Newell, F. W., editor:Glaucoma, Tr. of the Fifth Conference, NewYork, 1961, Josiah Macy, Jr. Foundation, pp.246-247.

14. Sears, M. L.: Miosis and intraocular pressurechanges during manometry, A. M. A. Arch.Ophth. 63: 707, 1960.

15. Barany, E., and Rohen, J. W.: Glaucoma inmonkeys (Cercopithecus aethiops), A. M. A.Arch. Ophth. (in press).

16. Tornquist, G.: Personal communication, 1962.17. Becker, B., and Constant, M. A.: Species

variation in facility of aqueous outflow, Am.J. Ophth. 42: 189, 1956.

18. Holmberg, A.: The fine structure of theinner wall of Schlemm's canal, A. M. A.Arch. Ophth. 62: 956, 1959.

19. Zweifach, B. W.: Functional behavior of themicrocirculation, Springfield, 111. 1961,Charles C Thomas, Publisher.

20. Majno, G., and Palade, G. E.: Studies oninflammation. I. The effect of histamine andserotonin on vascular permeability: An elec-tron microscopic study, J. Biophys. &Biochem. Cytol. 11: 571, 1961.

21. Scheie, H. G., Spencer R. W., and Helmick,E. D.: Tonography in the clinical managementof glaucoma, A. M. A. Arch. Ophth. 56: 797,1956.

22. Linner, E.: Personal communication, 1962.23. Becker, B.: In Newell, F. W., editor: Glau-

coma, Tr. of the Fifth Conference, NewYork, 1961, Josiah Macy, Jr. Foundation, p.239.


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