G, radual shrinkage of ciliary processes

Effect of parasympathomimetic and sympathomimetic drugs on secretion in vitro by the ciliary processes of the rabbit eye Lennart Berggren Secretion teas measured by determining differences in the area of an optical transverse section of the ciliary processes. Pilocarpine is an effective inhibitor of secretion in vitro at 10' 9 M, while acetylcholine, carbachol, and arecoline inhibited only at 10-5 M. Physostigmine as well as atropine had an inhibitory effect at 10~ 7 M and epinephrine depressed secretion at 10- J 'M; a combination of epinephrine and acetazolamide was not synergistic. The inhibitory effect of epinephrine could be abolished by blocking its metabolic effects by N-isopropyl methoxamine. G, radual shrinkage of ciliary processes in vitro was in previous experiments 1 - assumed to be a measure of transport across the ciliary processes and ultimately of aqueous humor production, and the results were similar to those observed with transport processes in other epithelial tissues. In the present study, different inhibitors of aqueous flow, such as epinephrine and acetazolamide, were studied, as were drugs that facilitate outflow. Methods Male albino rabbits weighing about kilograms and fed a diet of hay, oats, and water ad libitum were used. From the Departments of Pharmacology and Ophthalmology, University of Uppsala, Sweden. This study was supported by Research Grant B-30 from the Institute of Neurological Diseases and Blindness, U. S. Public Health Service, Bethesda, Md., and by the Swedish Medical Research Council. 91 The techniques and calculations were as described previously. 1 - The tissue fluid of the ciliary processes was replaced with a physiologic buffer solution by perfusion through a carotid artery in the living animal. Sections of iris-ciliary body were prepared and put into a bath containing the same buffer solution. Care was taken to prevent secretion as much as possible up to the incubation in vitro. The area of a transverse optical section of the processes was photographed intermittently during one hour and one to four processes were generally suitable for planimetry. The reduction in area of the different processes with time was calculated in per cent from the zero time value and averaged for each time. The single eye was treated as the statistical unit. Ciliary processes showing a shrinkage of 1 per cent or less at 5 minutes were considered to have impaired function, and those with a shrinkage of 15 per cent or more at 5 minutes to have too small a residual capacity. The remaining group is defined as a selected group. Drugs were added to the bath at 5 minutes after zero time. Blocking effects on secretion were considered to have occurred when the calculated mean curves describing the rate of shrinkage were above a line drawn from the point of 85 per cent remaining surface area at 5 minutes from zero time to the mean value plus

9 Berggren Investigative Ophthalmology February 1965 Carbachol Acetylcholine A Arecoline Ar - C10' 5 M A10" s M Ar10" 5 M A10" 7 M 10 0 30 40 50 MIN Fig. 1. Carbachol, acetylcholine, or arecoline added to the bath solution at arrow. Ordinate = calculation of transverse section area of ciliary processes in per cent. Zero value = per cent. Abscissa = time in minutes. Mean selected timeshrinkage curves. Carbachol 10* 5 M (4 animals, 10 processes). Arecoline 10' 5 M (3 animals, 9 processes). Acetylcholine 10" 5 M (4 animals, 8 processes), 10""M (4 animals, 1 processes). Continuous curve = mean selected standard timeshrinkage curve with Cole's solution, ph 7.4. Minimum slope line from 85 per cent at 5 minutes to mean standard value plus three standard deviations at minutes. Pilocorpine 3 standard deviations at minutes. This line is defined as the minimum slope line. Buffer solution. Cole's solution without glutamate was used as bathing medium, prepared from chemicals of analytical grade. Drugs. The following drugs were used: pilocarpine hydrochloride, physostigmine salicylate, acetylcholine chloride, carbamylcholine chloride or carbachol, arecoline hydrobromide, atropine sulfate, 1-epinephrine (as the commercial preparation, Eppy eyedrops, containing 10 mg. base per milliliter and stabilized with borate, Pharmacia, Sweden), sodium acetazolamide (Diamox, Lederle Laboratories) and BW 61-43 or N-isopropyl methoxamine (obtained through the courtesy of Wellcome Laboratories). All drug concentrations are given in moles per liter. Results Acetylcholine, carbachol, and arecoline. Two quaternary compounds, acetylcholine and carbachol, and one tertiary compound, arecoline, were used in a concentration of 10~ 5 M. The curves from experiments with all the drugs showed about the same course and followed the minimum slope line, and the inhibitory effects of the drugs are regarded as probable. Arecoline, which could be expected to penetrate more easily since it is a tertiary compound and only partly dissociated at ph 7.4, did not differ in its effects from the others. An acetylcholine concentration of 10~ 7 M was without effect (Fig. 1). Pilocarpine. This drug is treated separately since its effects differed from other parasympathomimetics with a direct action. Pilocarpine was tested in several concen- - 10 0 30 40 50 MIN Fig.. Pilocarpine added to the bath solution at arrow. Coordinates as in Fig. 1. Mean selected time-shrinkage curves; 10~ 3 M (1 animal, processes), 10" 6 M to 10~ 7 M (4 animals, 8 processes), 1O' S M to 10" 9 M (5 animals, 14 processes), 10-10 M ( animals, 4 processes). Mean selected standard curve and minimum slope line as in Fig. 1.

Volume 4 Number 1 Effect of drugs on secretion by ciliary processes 93 trations and inhibited secretion of the ciliary processes in very low concentrations (Fig. and Table I). All experiments down to a concentration of 10~ 9 M showed inhibition, and the course of the curves was well above the minimum slope line. At a concentration of 10~ 10 M, pilocarpine did not change secretion (Fig. ). Physostigmine. An inhibitory effect was established with this choline esterase inhibitor. There was no striking difference between the results from 10~ 5 M or 10~ 7 M experiments (Fig. 3); 10~ s M was without effect. Atropine. The parasympatholytic agent, atropine, also significantly inhibited secretion when a 10~ r 'M solution was used, and possibly when the final concentration was as low as 10~ 7 M (Fig. 4). Epinephrine. Epinephrine experiments were performed at ph 7.0 instead of ph 7.4. The experiments were compared with a standard curve at ph 7.0 and a minimum slope line calculated similarly as for ph 7.4. The ph of 7.0 was chosen in order to be able to study later a combined use with acetazolamide. 1 The standard curves at ph 7.4 and 7.0 did not differ from each other (Table II). Epinephrine affected shrinkage of the processes down to a concentration of 10~ 4 M (Fig. 5). No effect could be established with a 10~ 5 M solution (Fig. 5). A recently developed blocking agent of the metabolic effects of epinephrine 3 ' 4 was also tested. The code name, BW 61-43, stands for N-isopropyl methoxamine. Concentrations of 10" 4 M and 10" 5 M did not Table I. Pilocarpine experiments. Standard buffer solution at ph 7.4. Mean percentage values of remaining transverse section area of the ciliary processes Pilocarpine Transverse section area of ciliary processes in per cent at different No. of concentration in time intervals selected group Rabbit measured the bath 0 5 7.5 JO 0 40 No. processes 10-3 M 89.7 91. 91. 9.6 88. 88. 87 10-oM 87.1 89.0 87.9 89.3 87.9 87. 89 1(HM 91.7 89.7 84. 76.9 71.1 79.5 98 1(HM 1(HM 91.3 9.8.7 9.8 88.5 9.9 8.9.0 8.7 89.5 79.3 87.1 99 10-sM 10-'JM 10- <J M 93.4 85. 88.1 93.4 85. 88.1 91.9 87.7 89.8.1 93.8 89.8 89.0.1 84.7 87.3 84.0 83.6 111 11 3 3 10-10 M 10-"M 10-»M 10-oM 88.7 88.5.6 89.6. 79.7 89.7. 85.0 74.0 87.8 87. 79..0 86.7 84.3 75.6 66.0 86.4.9 7.5 6.7 8.4 78.9 148 149 113 114 3 3 Zero time value = per cent. The animal (or single eye) is the statistical unit. Processes showing a shrinkage of 1 per cent or less or 15 per cent or more at 5 minutes were discarded. Pilocarpine added to the bath at 5 minutes. Table II. Standard experiments at ph 7.4 and ph 7.0 with Cole's solution. Mean percentage values of remaining transverse section area of the ciliary processes Buffer solution Cole's at ph 7.4 Cole's at ph 7.0 Transverse section area of ciliary processes in per cent at different time intervals selected group 0 5 1 10 0 40.7 78.9 73.8 67.7 64.8.6 8.0 76.0 69.7 64.0 Standard error of mean ± 1.3 ± 1.6 Standard deviation 4.0 4.8 No. of animals No. of measured processes 0 Zero time value = per cent. The animal (or single eye) is the statistical unit. Processes showing a shrinkage of 1 per cent or less or 15 per cent or more at 5 minutes were discarded.

94 Berggren Investigative Ophthalmology February 1965 Physostigmine 10 0 30 40 50 MIN Fig. 3. Physostigmine added to the bath solution at arrow. Coordinates as in Fig. 1. Mean selected time-shrinkage curves; 1CHM (4 animals, 6 processes), 10~ 7 M (4 animals, 11 processes), 10~ s M (3 animals, 9 processes). Mean selected standard curve and minimum slope line as in Fig. 1. influence secretion in vitro by the ciliary processes (Fig. 6). A combination of epinephrine and the metabolic blocking agent, BW 61-43, showed that it was possible to abolish totally the inhibitory effect of 10" 4 M epinephrine (Fig. 7). The effect on shrinkage of the processes by the simultaneous use of epinephrine and acetazolamide was also studied. The previous results with acetazolamide 1 were confirmed. There was an inhibitory effect at 10- G M but not at 10" 7 M (Fig. 8). It was not possible to inhibit secretion further by a combination of epinephrine and acetazolamide (Fig. 9). Discussion The action of parasympathomimetic drugs on the regulation of intraocular pressure is often defined in terms of increasing outflow facility and vasodilation. These effects do not preclude a direct action on aqueous secretion, but such an action is not easily discovered in in vivo experiments because of difficulties in eliminating influences on the secretory rate of an increased outflow facility and/or vasodilation. An in vitro preparation of the secreting region, on the other hand, though no doubt less physiologic, might be a better object for the study of the effects on secretion. With the present technique, there was only a moderate effect on secretion by the quaternary compounds acetylcholine and carbachol in doses known to be effective in other respects. The tertiary parasympathomimetic, arecoline, had similar effects. Pilocarpine, on the other hand, which is also tertiary, inhibited secretion in exceedingly small doses. The blocking effect was also established with the reversible choline esterase inhibitor, physostigmine. The exact nature of the action of this enzyme inhibitor is unknown, but it has been found to inhibit - 10 0 30 40 50 MIN Fig. 4. Atropine added to the bath solution at arrow. Coordinates as in Fig. 1. Mean selected time-shrinkage curves; 10~ 5 M (4 animals, 11 processes), 10~ 7 M (4 animals, 11 processes). Mean as in Fig. 1.

Volume 4 Number 1 Effect of drugs on secretion by ciliary processes 95 50 Epinephrine 10' 5 M sodium transport in the gills of the crab. 5 Choline esterases were also demonstrated in this organ. In the ciliary processes, choline esterase has been demonstrated histochemically between the two layers of epithelium. Koelle and Friedenwald 0 suggested that the enzyme might play some role in the secretory process. An inhibitory effect on active sodium transport in the frog skin has been found for choline esterase inhibitors, whereas pilocarpine was a stimulant when added from the outside but not from the inside. 7 ' s Pilocarpine possibly facilitates diffusion of sodium through the outward facing membrane of the frog skin cells. 9 An inhibitory effect on secretion by pilocarpine has not been described previously. This effect points to differences between the transport mechanism of the ciliary epithelial layers and that of the frog skin. In clinical investigations there is evidence that the effects of pilocarpine cannot wholly be explained by its influence on outflow facility. 10 Atropine had an inhibitory effect on secretion at 10-5 M, while at 10' 7 M it had a probable effect. In frog skin experiments, atropine has been described as having the same effect as pilocarpine when added to the outside but it could not be elicited in all frog species. 7 ' s It is thought that the action of atropine in the frog skin does not involve competition with acetylcholine. The same effect was obtained with pilocarpine. In the present experiments the effects of pilocarpine and atropine were in the same direction but opposite to their effects in frog skin. 10 0 30 40 50 MIN Fig. 5. Epinephrine added to the bath solution at arrow. Coordinates as in Fig. 1. Mean selected time-shrinkage curves; 10~ 3 M (8 animals, 3 processes), 10" 4 M (4 animals, 10 processes), 10-5 M (4 animals, 10 processes). Continuous curve = mean selected standard time-shrinkage curve with Cole's solution, ph 7.0. Minimum slope line from 85 per cent at 5 minutes to mean standard value plus three standard deviations at minutes. - \ BW 61-43 - 50 0 10 0 30 40 50 MIN Fig. 6. BW 61-43 added to the bath solution at arrow. Coordinates as in Fig. 1. Mean selected time-shrinkage curves; 10~*M ( animals, 3 processes), 10" r 'M (3 animals, 3 processes). Mean as in Fig. 5.

96 Berggren Investigative Ophthalmology February 1965 - - - Epincphrlne E W'M e 10" 5 M BW 61- A3 B10**M b 10' 5 M 0 10 0 30 40 50 MIN Fig. 7. Epinephrine and BW 61-43 added to the bath solution at arrow. Coordinates as in Fig. 1. Mean selected time-shrinkage curves. Epinephrine 1(WM plus BW 61-43 KHM (3 animals, 7 processes), epinephrine 10~ l M plus BW 61-43 10~ s M (4 animals, 9 processes), epinephrine 10" 5 M plus BW 61-43 KHM (4 animals, 10 processes). Mean as in Fig. 5. of bisulfite, which is added as an antioxidant but also has inactivating effects on epinephrine. 11 ' la It is not known, however, if the complex properties are the same in extreme dilutions (such as in the present experiments). If the chelate of epinephrine and borate was broken up, the epinephrine might be more easily inactivated. The inhibitory action of epinephrine in this "avascular" in vitro preparation must have been due to interference with the metabolic pathways in the ciliary processes. N-isopropyl methoxamine, which blocks the metabolic effects of epinephrine, such as hyperglycemia, hyperlactacidemia, and elevation of free fatty acids, H ' 4 also blocked the effects of epinephrine on secretion in the ciliary processes. The clinical synergism between epinephrine and acetazolamide on aqueous flow could not be reproduced in Acctazolamide The intraocular pressure drop by epinephrine is interpreted as a decrease of aqueous flow, and cannot be explained solely by a vasoconstrictive action, though exact details are unknown. The present results show that secretion in the ciliary processes was inhibited by epinephrine. The minimum effective concentrations were much larger than those generally able to produce true sympathomimetic ("vascular") responses. It is, however, not possible to know if the solution used is per cent active. Epinephrine hydrochloride was found unsatisfactory in preliminary experiments because of its instability at a neutral ph. A complex compound of epinephrine base and boric acid (manufactured in a 1 per cent solution as Eppy eyedrops), which is stable at a neutral ph, was used. This complex is also stable in the presence - - 0 10 0 30 40 50 MIN Fig. 8. Acetazolamide added to the bath solution at arrow. Coordinates as in Fig. 1. Mean selected time-shrinkage curves; 10~ G M (4 animals, 9 processes), 10-"M (4 animals, 14 processes). Mean as in Fig. 5.

Volume 4 Number 1 Effect of drugs on secretion by ciliary processes 97 7, - Epinephrine E lo^m e 10" 5 M Acetozolamide A 10"! M a 10-'M ^ e a 10 0 30 40 50 MIN Fig. 9. Epinephrine and acetazolamide added to the bath solution at arrow. Coordinates as in Fig. 1. Mean selected time-shrinkage curves. Epinephrine 10~' ( M plus acetazolamide 10~ G M (8 animals, 14 processes), epinephrine 10~ 4 M plus acetazolamide 10~ 7 M (3 animals, 9 processes), epinephrine KH'M plus acetazolamide 10~ 7 M (4 animals, 11 processes). Mean selected standard curve and minimum slope line as in Fig. 5. the present system. This might be interpreted in different ways. With the drugs tested in tenfold steps of concentration, a combination of the highest ineffective concentration of epinephrine and acetazolamide was not inhibitory, and the lowest effective concentration of either drug gave an almost maximal inhibition. On the other hand, the effect of epinephrine on in vivo flow might not be exclusively a metabolic effect but a vasoconstrictive one as well. REFERENCES 1. Berggren, L.: Direct observation of secretory pumping in vitro of the rabbit eye ciliary processes. Influence of ion milieu and carbonic anhydrase inhibition, INVEST. OPHTH. 3: 66, 1964.. Berggren, L.: Effect of composition of medium and of metabolic inhibitors on secretion in vitro by the ciliary processes of the rabbit eye, INVEST. OPHTH. 4: 83, 1965. 3. Salvador, R. A., Colville, K. I., and Burns, J. J.: Control of fatty acid mobilization with the N-isopropyl derivative of methoxamine (BVV 61-43), Biochem. Pharmacol. 1: suppl. 194, 1963. 4. Salvador, R. A., Colville, K. I., Lindsay, L. A., and Burns, J. J.: Ability of the N-isopropyl derivative of methoxamine (BW 61-43) to block the metabolic effects of epinephrine, Fed. Proc. : 508, 1963. 5. Koch, H. J.: Cholinesterase and active transport of sodium chloride through the isolated gills of the crab Eriocheir sinensis, in Kitching, J. A., editor: Recent developments in cell physiology, London, 1954, Butterworth & Co., Ltd., pp. 15-31. 6. Koelle, G. B., and Friedenwald, J. S.: The histochemical localization of cholinesterase in ocular tissues, Am. J. Ophth. 33: 53, 1950. 7. Kirschner, L. B.: Effect of cholinesterase inhibitors and atropine on active sodium transport across frog skin, Nature 17: 348, 1953. 8. Kirschner, L. B.: The effect of atropine and the curares on the active transport of sodium by the skin of Rana esculenta, J. Cell. & Comp. Physiol. 45: 89, 1955. 9. Ussing, H. H.: Handbuch der experimentellen Pharmakologie, XIII, Berlin, 19, Springer-Verlag, pp. 1-195. 10. Krill, A. E., and Newell, F. W.: Effects of pilocarpine on ocular tension dynamics, Am. J. Ophth. 57: 34, 1964. 11. Riegelman, S., and Fischer, E. Z.: Stabilization of epinephrine against sulfite attack, J. Pharmacol. Sc. 51: 06, 196. 1. Riegelman, S., and Fischer, E. Z.: Effect of boric acid and bisulfite on the rate of oxidation of epinephrine, J. Pharmacol. Sc. 51: 10, 196.