I t has been demonstrated in in vivo experiments

Transcription

1 Influence of pilocarpine on iodopyracet and iodide accumulation by rabbit ciliary body-iris preparations Per-Erik Walinder The simultaneous accumulation of iodopyracet labeled with 13J I and of J25 I by ciliary bodyiris preparations from albino and pigmented rabbits was studied in vitro. The ciliary processes ivere separated from the iris and the uptake of iodopyracet and iodide was shown to be mainly localized to the processes. Pilocarpine in low concentrations (10-7 to 10~ 5 M) tended to increase the iodopyracet accumulation in albino rabbits. In pigmented rabbits the increase was statistically significant. The accumulation of iodide was not affected by low concentrations of pilocarpine. High pilocarpine concentrations (10~ 3 to 10-2 M) inhibited the accumulation of both iodopyracet and iodide. The inhibition of iodopyracet accumulation was reversible and was not caused by an increased leakage from the preparation. I t has been demonstrated in in vivo experiments that iodopyracet (Diodrast) and iodide are actively transported out of the eye. n> 2 In vitro, this is seen as an active uptake by the ciliary processes in ciliary body-iris preparations of the rabbit. 1-2 Recent in vitro observations have indicated that pilocarpine inhibits the secretion of fluid of surviving rabbit ciliary processes. 3 The present investigation shows that pilocarpine also has an influence on the accumulation of iodopyracet and iodide by ciliary body-iris preparations in vitro. Method Albino rabbits weighing 1.5 to 3.0 kilograms and pigmented rabbits weighing 2.5 to 5.5 kilo- From the Departments of Pharmacology and Ophthalmology, University of Uppsala, Sweden. This investigation was in part supported by a Crant B 3060 to Professor E. Barany from the National Institute of Neurological Diseases and Blindness, United States Public Health Service. 378 grams were employed. They were fed on a diet of hay, oats, and water ad libitum. As in Becker's experiments, 1 ' 2 the rabbits were put to death painlessly by intravenous air injection, both eyes were immediately enucleated and opened posteriorly, and the lens and the vitreous were removed. The ciliary body-iris was excised as a complete ring, and immediately put into the incubation fluid. In some experiments, especially indicated under Results, the ring was halved. In the drug experiments, one preparation was incubated with pilocarpine hydrochloride, the other was incubated in the same medium but with pilocarpine omitted. In the experiments where temperature was varied, no control was used. The time interval from killing to start of incubation was 6 to 8 minutes. Iodopyracet (Diodrast) labelled with 131 I and iodide ( 1-5 I) supplied by the Radiochemical Centre, Amersham, where added to an incubation fluid of the following composition: NaCl, 8.00 Cm.; KCl, 0.20 Gm.; CaCL, 0.20 Gm.; MgCU + 6H 2 O, 0.10 Cm.; NaHJ 5 O 4 + 2H,O, 0.05 Cm.; NaHCC-3, 1.00 Cm.; glucose, 1.00 Gm.; and enough H 2 O to make 1,000 ml. The iodopyracet concentration in the incubation fluid was 2.5 x 10~ 5 M (0.01 to 0.5 nc per milliliter). Iodide was carrier-free (0.1 to 0.5 pc per milliliter).

2 Volume 5 'Number 4 Pilocarpine effect on iodopyracet, iodide accumulation 379 Before incubation the solution was oxygenated with 100 per cent O ; for from 8 to 10 minutes, pilocarpine was added to the test solution, and the ph was then adjusted to between 7.4 and 7.5 (glass electrode) by bubbling a mixture of 6.5 per cent CO 2 and 93.5 per cent O 2 through the solution. Paired closed flasks containing 5 ml. of medium and one preparation were gently shaken in a water bath at 37 C. for 60 minutes. The pilocarpine and control preparations were then blotted, put into closed polyethylene tubes, and weighed. These procedures lasted from 10 to 15 minutes and took place at room temperature. The preparations with the ciliary processes exposed were then put on pieces of polyvinyl foil on a block of ice to reduce the rate of drying out. Most of the ciliary processes were dissected free from the iris under 2x magnification. The dissections took about 2 minutes. The processes were put into closed polyethylene tubes and weighed. Fluid leaking during dissections was sucked up on filter paper and later assayed together with the iris preparation. Two or 3 samples of medium were obtained by dipping prevveighed strips of filter paper in the medium and then treating them as tissue samples. The samples were then dried at 75 C. overnight with the tubes unstoppered, assayed, and reweighed. The wet weight of the preparation remaining after the dissection ("iris") was obtained by subtracting the wet weight of the ciliary processes from that of the whole ciliary body-iris preparation. To reduce the influence of irrelevant factors during these procedures, the pilocarpine-treated eyes and the control eyes were handled in reversed order every second time. 131 I and 125 I were assayed with a two channel gamma spectrometer with a well-type crystal, one channel taking the 0.36 mev. peak of 131 I and the other the to mev. peaks of 12r >l. The latter is not counted in the 0.36 mev. channel and the counts due to 131 I in the low energy channel were subtracted with the use of data from standard samples containing only ]31 I. The counting error of the analysis of these samples was less than 2 per cent for each of the isotopes. The Diodrast ( 131 I) and iodide ( r - r 'I) accumulations in the samples were calculated as the ratio between the radioactivity expressed in counts per minute of I mg. wet weight of a tissue sample and 1 mg. of incubation fluid (DA = Diodrast accumulation ratio, IA = iodide accumulation ratio). The counts per minute of the whole ciliary body-iris preparation is calculated as the sum of the counts per minute of the ciliary processes and the counts per minute of the "iris." Results A. The Diodrast and iodide accumulation ratio of the ciliary processes and iris preparation. The accumulation ratios of Diodrast and iodide (DA and IA respectively) of the control eyes are presented in Table I. These values were obtained with a Diodrast concentration of 25 pm in Table I. Diodrast and iodide accumulation ratios in control eyes at 37 C. Wet weight (mg.) M ± S.E.M. SD M Dry weight (ing.) ± S.E.M. SD Accumulation Diodrast ratio Iodide M ± S.E.M. SD M± S.E.M. SD No. of animals Albino rabbits Ciliary processes 4.88 ± ± ± ± "Iris" 33.9 ± ± ± ± Total ciliary body-iris preparation 38.7 ± ± ± ± Pigmented rabbits Ciliary processes ± ± ± ± "Iris" 48.5 ± , ± ± 0^ ± Total ciliary body-iris preparation 63.8 ± ± ± ± Accumulation ratio = (counts per milligram of wet tissue): (counts per milligram of medium). Diodrast concentration was 25 jitm. Iodide was carrier free. S.E.M., Standard error of the mean; SD, standard deviation. Each animal contributed one eye.

3 380 Wdlinder Investigative Ophthalmology August 1966 the medium and with carrier-free iodide. In 5 experiments with % 0 of this Diodrast concentration and % 0 of the carrier free iodide, where halved ciliary body-iris preparations were used, the ciliary processes accumulated Diodrast to a ratio of ± 20.6 (M ± S.E.M.) compared with 30.4 ±1.8 (M ± S.E.M.) at the standard concentration, the iodide accumulation was little influenced: 4.73 ± 0.76 (M ± S.E.M.) compared with 4.0 ± 0.2 (M ± S.E.M.). Table I shows further that there was no great difference in DA and IA between albino and pigmented animals. The high DA and IA values in the ciliary processes (compared with the whole preparation) indicated that the Diodrast and iodide were especially localized to the ciliary processes. It seemed of interest to see if the two substances differ in this respect. The amount of substance in the processes in percentage of the total in the ciliary body-iris preparation was therefore calculated. In the albino series 33 per cent of the Diodrast and 23 per cent of the iodide are localized in the processes, in the pigmented group 62 and 37 per cent, respectively. In 4 pigmented eyes (groups at 10~ 3 and lo'^m. in Table II) dissected immediately after incubation (and not 10 to 15 minutes later, as in the rest) 64 per cent of the Diodrast and 41 per cent of the iodide were localized in the processes. In eight pigmented halved preparations handled in the same way the corresponding values were 54 per cent of Diodrast and 34 per cent of iodide. Thus in all groups relatively more Diodrast than iodide was localized to the processes. The difference between albino and pigmented animals is explained by the fact that in the pigmented eye the dissected processes occupied a greater part of the total preparation than in the albinos (see Table I). There was a positive correlation between the degree of accumulation of Diodrast and iodide in the albino ciliary processes; the coefficient of correlation was (N = 40; p < 0.001). This coefficient is influenced by the common factor of dissection. In the total preparation the corresponding coefficient was (N = 40; p < 0.01). B. The effect of cold. Six eyes incubated at +4 C. showed very little accumulation of Diodrast and iodide. DA and IA of the ciliary processes were 1.76 ± 0.06 (M ± S.E.M.) and 1.24 ± 0.03 (M ± S.E.M.) re- Table II. Influence of pilocaqiine on Diodrast accumulation ratio (DA) and iodide accumulation ratio (IA). Pilocarpine concentration Albino rabbits 10-2 M 10-3 M 10-* M 10-5 M 10-«M 10-7 M 10- s M Pigmented rabbits 10-2 M (DA, pilocarpine):(da, control) Ciliary processes ± "Iris" ± Ciliary body-iris 0.22 ± ± ± ± (IA, pilocarpine) :(l A, control) Ciliary processes ± ± »M x 10- M ± ± ± Values are mean ± standard error of the mean (S.E.M.) except where only two values are given. Each animal contributed one drug and one control preparation "Iris" 0.75 ± ± ± ± ± Ciliary body-iris 0.74 ± ± ± No. of animals

4 Volume 5 Number 4 Pilocarpine effect on iodopyracet, iodide accumulation 381 spectively, and the corresponding values in the ciliary body-iris preparation were 1.18 ± 0.02 and 1.07 ± 0.04 respectively. C. Experiments with pilocarpine. 1. The effect of pilocarpine. The influence of pilocarpine on the accumulation of Diodrast and iodide was studied in 92 eyes of 46 albino animals and 28 eyes of 14 pigmented animals. The concentration was varied from 10~ s to 10-2 M. In all experiments and for each preparation DA of the treated eye was divided by DA of the control eye. The results are presented in detail in Table II and summarized in Fig. 1. DA of the ciliary body and iris preparations was clearly reduced by high concentrations of pilocarpine with 50 per cent inhibition by 10~ 3 M. Low concentrations (10" 7, 10", and 10' 5 M) increased DA somewhat, but in no single group was the increase statistically significant. The influence of low concentrations of pilocarpine was also studied in 20 eyes from 10 adult pigmented rabbits and Table II shows that 2 x 10- (! M pilocarpine increased DA and that the increase was statistically significant (p < 0.01). The inhibitory effect of higher concentrations was of about the same order in the pigmented animals as in the albinos. In the presence of 10~-M pilocarpine the value of DA for ciliary processes increased only slightly with the degree of accumulation in the control eye. Thus the inhibitory effect was much more evident if the control accumulation was high (Fig. 2). The "stimulatory" effect of pilocarpine was not correlated with the degree of accumulation (Fig. 2). High concentration (10~ 2 M) pilocarpine reduced IA moderately but low concentrations did not influence IA in a detectable t.» 0.5- diodrast whole prep diodrast processes - -- iodide whole prep. - O---O iodide processes i i i "ON \ ^ \ \ \ Fig. 1. Influence of pilocarpine concentration on Diodrast and iodide accumulation in rabbit ciliary body-iris preparations. Abscissa, molarity of pilocarpine in medium; ordinate, accumulation ratio with: without pilocarpine ACCUMULATION RATIO CONTROL EYE Fig. 2. Pilocarpine effect on accumulation ratio in rabbit ciliary processes. In each experiment accumulation ratio in the pilocarpine eye is plotted against accumulation ratio in the control eye. Diodrast concentration was 25 p.m in all experiments except those (plotted as solid black squares) where 2.5 i*m was used. Processes from a whole ciliary body-iris were used, except in those (plotted as solid black triangles and solid black squares) where processes from half a preparation were taken. Clear circles, 10~ s M pilocarpine; clear triangles, 10-7 M pilocarpine; clear squares, lo- (i M pilocarpine; half-black circles, 10" 5 M pilocarpine; half-black triangles, 1(HM pilocarpine; half-black squares, 10~ 3 M pilocarpine; solid black circles, 10-'-M pilocarpine; solid black triangles, lo-'-m pilocarpine; solid black squares, 10~ 2 M pilocarpine.

5 382 W Minder Investigative Ophthalmology August 1966 way, neither in the albino animals nor in the pigmented ones. 2. Reversibility of pilocarpine inhibition. In ten eyes the ciliary body-iris preparations were cut into two halves. One halfpreparation from each animal was assigned to one of four groups, A,B,C, or D which were treated in different ways. Group A and C were incubated without pilocarpine, B and D with 10~ 2 M pilocarpine for 1 hour. No radioactivity was present at this stage. All preparations were then washed for 15 minutes with incubation fluid. Then all groups were reincubated with 131 I-Diodrast in the solution, Groups A and B without pilocarpine and Groups C and D with 10" a M pilocarpine. The DA's in Groups B, C, and D were expressed in percentage of the control (A). The mean DA in Group B (p < 0.01) is significantly larger than in C and D, which indicates that the inhibitory action of 10~ 2 M pilocarpine is reversible by washing (see Fig. 3). 3. Influence of pilocarpine on the runout of Diodrast. Reduced accumulation could be due to an inhibited inward transport or to an accelerated outward transport 100 % 50-- B T 1 C T 1 D (5) (7) (3) (5) Fig. 3. Reversibility of pilocarpine inhibition of Diodrast accumulation. Group A, no pilocarpine at any time; Group B, only prewash pilocarpine; Group C, only post-wash pilocarpine; Group D, pre- and post-wash pilocarpine. Values are mean accumulation ratios in per cent of those of Group A, ± the standard error of the mean. Figures in parentheses are numbers of observations. For further explanation, see the text. (active or passive). To choose between these possibilities, the influence of pilocarpine on run-out of Diodrast was studied. The preparation was incubated for 1 hour in a solution which contained KU I- Diodrast (2.5 x 10~ 5 M), and then placed for 25 minutes in a fluid ("run-out fluid") which contained 4 x lo-'m unlabelled Diodrast. Since the volume of the preparation (weight 30 to 50 mg.) was small compared with the run-out fluid (20 ml.) the mechanism which transported Diodrast into the preparation would be almost entirely occupied by the transport of unlabelled Diodrast and the reaccumulation of 131 I-Diodrast (which had left the preparation) could be neglected. Pilocarpine was added to run-out fluid and, as in the previous experiments, the pilocarpine eye was compared with the control eye where pilocarpine was omitted. The influence of pilocarpine on the runout of Diodrast was indicated by the ratio between the radioactivity (expressed in counts per minute per milligram of wet weight) of the pilocarpine preparation and that of the control preparation at the end of the run-out period. Pilocarpine in concentration of 2 x 10~ M gave a ratio of 0.99 ± 0.10 (M ± S.E.M., 6 animals) and 10-M pilocarpine a ratio of 1.26 ± 0.11 (M ± S.E.M., 5 animals). These results do not give evidence of any accelerating influence of pilocarpine on the run-out of Diodrast, if anything, 10~ 2 M pilocarpine had an action in the opposite direction. 4. The effect of pilocarpine on the tissue water content. In 40 albino control eyes incubated at 37 C., the water content of the ciliary processes was 80.0 per cent ± 0.4 (M ± S.E.M.) and 83.5 per cent ± 0.1 of the whole ciliary body-iris preparation. In 6 eyes incubated at 4 C. the corresponding values were 83.4 per cent ± 0.8 and 86.4 per cent The water content of nonincubated processes was 86.0 per cent ± 1.2 (M±S.E.M.,N = 8). 5 At low concentrations of pilocarpine (10" 7 to 10" 5 M) in both the pigmented and the albino series, the water content of

6 Volume 5 Number 4 Pilocarpine effect on iodopyracet, iodide accumulation 383 the ciliary processes was somewhat lower than in the control preparation. However, the differences were small and not statistically significant except for the pigmented group at 2 x 10" M. There the water content was 1.5 per cent lower (0.01 < p < 0.02). No effect on the tissue water was observed in the whole ciliary body-iris preparation. High concentrations of pilocarpine (10" 2 M) increased the water content both of the ciliary processes and of the whole preparation. The increase was statistically significant (p < 0.01) in the 2 hour experiments (Group D of Fig. 3). Discussion In Becker's experiments/' 2 the concentration of Diodrast and iodide in the tissue water was compared with that in the incubation medium to determine the degree of accumulation, while, in the present experiments, the accumulation was expressed as a ratio between the concentration in the whole sample and that in the incubation fluid. Since the water contents of the preparations were determined, the degree of accumulation could be calculated as in Becker's experiments. The figures then become: for the ciliary body-iris preparations, Diodrast and iodide 14.0 and 2.9 respectively and, for the ciliary processes, 39 and 4.8 respectively. The figures for the whole ciliary body-iris preparation are in good agreement with Becker's finding 12.2 and 2.4 for the same concentration of Diodrast and iodide in the medium. Becker suggested that the Diodrast concentration of the tissue water in the ciliary processes should be at least 50 to 75 times that in the medium. The figure obtained in the present experiments, 39, is in agreement with this suggestion, since it can be assumed that some Diodrast left the ciliary processes before and during the dissections. If Diodrast concentration in the medium was reduced, very high accumulation ratios, 100 to 200, were found. The accumulation of Diodrast in the whole ciliary bodyiris preparation differed considerably from one animal to another (Fig. 2). It is not clear what caused this variation but there seemed to be some increase in Diodrast accumulation with the weight of the ciliary body-iris preparation; the coefficient of correlation between DA and weight was (0.05 < p < 0.1; N = 40). Iodide accumulation did not seem to be correlated with the weight of the ciliary body-iris preparation; the coefficient of correlation was (N = 40). The albino ciliary processes contained 33 per cent of the total uptake of Diodrast in the ciliary body-iris preparation but only 23 per cent of the total uptake of iodide. The corresponding values in the pigmented series were 62 and 37 per cent. These figures indicate that the Diodrast uptake is more localized to the processes than that of iodide (the differences between albino and pigmented animals will be discussed below). However the values as given exaggerate this phenomenon because there occurs an unspecific accumulation in the preparations. This accumulation can be estimated in at least two different ways: Alternative 1. If Diodrast and iodide are supposed to be evenly distributed in the tissue water, the unspecific accumulation for both substances is 0.8. Alternative 2. As the cold experiments are set up, the unspecific accumulation can be estimated to 1.2 for Diodrast and 1.1 for iodide. To correct for these unspecifically accumulated amounts they have been subtracted from the amounts found by analysis. The distribution of the remaining amounts of Diodrast and iodide then become: in the first alternative 35 per cent of Diodrast and 25 per cent of iodide in the albino processes and 65 and 43 per cent, respectively, in the main group of pigmented processes. The second alternative gives corresponding values of 36 and 28 per cent in the albinos and 68 and 48 per cent in the pigmented. Also, in 4 whole and 8 halved pigmented preparations, dissected immediately after incubation, there was a difference between Dio-

7 384 Wdlinder Investigative Ophthalmology August 1966 drast and iodide. In the first four, 68 per cent of Diodrast and 51 per cent of iodide was localized to the processes; in the eight halved, 62 per cent of Diodrast and 48 per cent of iodide was localized (values calculated according to Alternative 2). In every single animal, the percentage of the Diodrast localized to the processes was greater than that of the iodide. This was true of all groups and according to both alternatives. However, it is probable that iodide diffuses somewhat faster than Diodrast. Thus the difference in degree of localization to the processes between Diodrast and iodide may at least partly be due to diffusion from the processes to the tissue at their base. This would have to occur mainly during the incubation period, since little difference did arise during the time interval between end of incubation and separation of processes from iris. The other and more intriguing possibility which would yield the same type of result is that the uptake of Diodrast is more sharply localized to the processes than the uptake of iodide. Despite the different figures given above for percentage localized to the processes, there is no real difference in the sharpness of localization between albinos and pigmented animals. If the fraction of Diodrast or iodide found in the processes is divided by the fraction of the total weight which the processes represent, the difference between albinos and pigmented animals disappears almost completely. Pilocarpine in high concentrations reduced Diodrast accumulation (DA) in the ciliary body and the run-out experiments indicated that the effect could not be explained by an increased rate of outflow of Diodrast. This shows that the pilocaqdine effect was to reduce the transport of Diodrast into the ciliary processes. This effect of pilocarpine was reversible. The findings, that low concentrations of pilocarpine increased DA in the ciliary body, indicate that it either stimulated the Diodrast uptake into the ciliary processes or that it reduced the rate of outflow from the processes. The run-out experiments were too few to permit any decisions between these alternatives. In studies on in vivo secretion by ciliary processes, 3 ' 4 Berggren has observed that ciliary processes exposed to pilocarpine (10" to 10" 3 M) shrink more slowly than controls. If it is assumed that the processes shrink evenly in all directions, his data suggest that after 1 hour the volume of the ciliary processes under pilocarpine is about 15 to 20 per cent larger than that of the controls. In the present study, very high concentrations of pilocarpine (10~ 2 M) produced statistically significant increases of tissue water content in the processes and in the whole ciliary-body iris preparation. Low concentrations (10~ 7 to 10~ r 'M) showed a tendency to decrease tissue water content in the processes but there was no effect on the whole preparation. In Berggren's experiments, the ciliary processes are kept loaded with fluid until the start by perfusing in vivo with saline, while in the present experiments they may have pumped out a considerable part of their interstitial fluid before the start of the incubation and this might explain the different results. Becker has tested different metabolic inhibitors, and his data demonstrate a difference in sensitivity of the Diodrast and iodide transports. Metabolic inhibitors gave 50 per cent inhibition of iodide transport only at 10 to 200 times the concentration which gave 50 per cent inhibition of the Diodrast transport. The present experiments indicate that similarly the Diodrast transport is more sensitive to the inhibitory influence of pilocarpine than that of iodide. Fig. 1 shows that 10" 3 M pilocarpine gave 50 per cent inhibition of DA while 10'-M did not give more than 30 per cent inhibition of I A. Even if these values are corrected for "unspecific accumulation" (Alternative 2), there is 50 per cent inhibition of IA only at 10 times the pilocarpine concentration which gives 50 per cent inhibition of DA. It is of interest to note the findings by

8 Volume 5 Number 4 Pilocarpine effect on iodopyracet, iodide accumulation 385 Miiller, and associates 0 that pilocarpine in high concentration (10~ 3 M and more) gives a marked inhibition of the O, uptake by the lens but that low concentrations (less than 10~'M) often increase the uptake temporarily. In the present experiments inhibition and "stimulation" occurred in these same ranges. Pilocarpine at 10" 3 M (0.025 per cent) is a high concentration but, according to observations by Miiller and associates, 0 ' 7 the application of 2 drops of 2 per cent pilocarpine in the conjunctival sac of human eyes gives a concentration of 10~ 2 M pilocarpine in the anterior chamber after 20 minutes. Pilocarpine HC1 1 mg. per kilogram of body weight (intramuscularly) gives an increase in outflow facility, 53 accommodation, and contraction of the iris sphincter in the monkey. 9 If it is assumed that the drug is distributed equally throughout the body, the concentration is about 4 x 10~ M in the tissue. This concentration also contracts the ciliary muscle of both monkey and rabbit in vitro, 0 and in the present experiments it increased the Diodrast accumulation in rabbit ciliary body-iris preparations. It is not yet possible to state whether the pilocarpine effect studied in the present experiments is related to the cholinergic action of the drug or not. REFERENCES 1. Becker, B.: The transport of organic nnions by the rabbit eye, Am. J. Ophth. 50: 862, Becker, B.: Iodide transport by the rabbit eye, Am. J. Physiol. 200: 804, Berggren, L.: Effect of parasympathomimetic and sympathomimetic drugs on secretion in vitro by the ciliary processes of the rabbit eye, INVEST. OPHTH. 4: 91, Berggren, L.: Direct observation of secretory pumping in vitro of the rabbit eye ciliary processes, INVEST. OPHTH. 3: 266, Bill, A.: Personal communication. 6. Miiller, H. K., Kleifeld, U., Hockwin, O., and Dardenne, U.: Der Einfluss von Pilocarpin und Mintacol auf den Stoffwechsel der Linse, Ber. deutsch. ophthal. Ces. 60: 116, Hockwin, U., Miiller, H. K., and Bliiser, U.: Nachweiss von Pilocarpin in Kammerwasser von Kaninchenaugen mit Hilfe der Polarographie, von Craefes Arch. Ophth. 167: 459, Barany, E. H.: Dissociation of accommodation effects from outflow effects of pilocarpine, in Paterson, C, editor: Drug mechanisms in glaucoma, London, 1966, J. & A. Churchill, Ltd. 9. Tornqvist, C: Comparative studies of the effect of pilocarpine on the pupil and on the refraction in two species of monkey (Cercopithecus ethiops and Macaco, irus), INVEST. OPHTH. 3: 388, van Alphen, G. W. H. M., Robinette, S. L., and Macri, F. J.: Drug effects on ciliary muscle and choroid preparations in vitro, Arch. Ophth. 68: 81, 1962.