- Email: [email protected]
Pilocarpine Uptake by Pigmented Uveal Tissue
P I L O C A R P I N E U P T A K E BY P I G M E N T E D UVEAL T I S S U E JONATHAN S. LYONS,
M.D., AND DAVID L. K R O H N ,
M.D.
New York, New York Pilocarpine has been shown to be more effective as an ocular hypotensive agent in lightly pigmented individuals than in heavily pigmented individuals.1'2 A study of normal subjects by Melikian, Lieberman, and Leo pold1 showed that a 4% solution of pilocar pine topically applied produced significant ocular hypotension and increased facility of outflow only in those who were lightly pig mented and had no effect on the more heav ily pigmented subjects. In glaucomatous pa tients of varying pigmentation, Harris and Galin2 showed that the drug's effectiveness in lowering intraocular pressure correlated with lightness of iris pigment. They did not note a corresponding increase in facility of outflow. They speculated that the pilocarpine administered to more heavily pigmented sub jects is inactivated either by direct binding to uveal pigment or by an enzyme present in the uveal tract. The purpose of this study was to confirm that pilocarpine is taken up or inactivated by pigmented uveal tissue to a much greater ex tent than by non-pigmented uveal tissue, to quantitate the amount taken up, and to inves tigate the relationship of this uptake or inactivation to enzyme activity. MATERIALS AND METHODS
Ciliary bodies and irides from heavily pig mented black and white Dutch rabbits and albino New Zealand rabbits were excised from freshly killed females weighing 1.6-2.8 kg. The iris was severed at its root. The ciliFrom the Department of Ophthalmology of the New York University School of Medicine and the Institute for Medical Research and Studies, New York. This study was supported in part by Public Health Service Grant CA 12523-01 and by National Institutes of Health Training Grant 7-1257-192-200 from the National Institutes of Health. Reprint requests to David L. Krohn, M.D., Insti tute for Medical Research and Studies, 220 East 23rd Street, New York, New York 10010.
ary body was then excised at the ora ciliaris retinae and separated from its vitreal adhe sions. Since ciliary processes in the rabbit extend variably beneath the iris, small ele ments of these are presumed to have been in cluded in iris samples. Each of the uveal tis sues was then incubated separately at 22°C in a test tube containing 0.5 ml pilocarpine nitrate, 500 μg/ml in physiologic saline. Af ter 30 minutes a 0.2 ml aliquot of the pilo carpine solution was withdrawn. The incu bated uveal tissue was then removed, airdried for 48 hours, and weighed. The 0.2 ml aliquots were analyzed for pi locarpine content by the Hestrin method.3'4 To each 0.2 ml sample, 0.4 ml of a solution containing equal volumes of 2 N hydroxylamine and 3.5 N NaOH was added. After vortex mixing, the solution was allowed to stand for two minutes. Then 0.2 ml of 3 N HC1 was added and the pH was adjusted to between 1.0 and 1.5. Finally, 0.2 ml of a so lution containing 0.37 M FeCl 3 in 0.1 N HC1 was added. Each sample was centrifuged for at least three minutes to remove any tissue debris and the supernatant was transferred to one of a pair of matched cuvettes. Optical density was read against a distilled water blank at 540 nm in a Zeiss spectrophotometer and converted to pilocarpine concentra tion in μg/ml by comparison with a stan dard curve. The concentration of pilocarpine remain ing in each sample was subtracted from the initial concentration of the incubating solu tion (500 μg/ml) in order to measure the amount of pilocarpine taken up by the tissue which had been incubated. Since 0.5 ml, con taining only 250 μg, was used, the resultant values were divided by two. As a correction to the measurement of up take, ciliary bodies and irides of pigmented and albino rabbits were incubated in saline without pilocarpine for one-half hour and
885
886
AMERICAN JOURNAL OF OPHTHALMOLOGY
0.2 ml of the solution taken as a sample. This was tested by the Hestrin method and the release of Hestrin method-reacting components (false-positives) was measured. These results were added to the pilocarpine uptake figures as a correction factor, since the presence of these compounds masked part of the pilocarpine uptake. The final up take of each sample was divided by its weight and expressed as μg uptake/mg tis sue. Two further groups of experiments were done to investigate the possibility of an en zymatic mechanism for uveal uptake of pilo carpine. 1. A series of uveal pilocarpine uptake measurements was run at 37°C for compari son with that at 22°C. 2. In addition, a series of measurements of uptake using the same method was done in the presence of ethylene-diamine-tetraacetic acid (EDTA) at 22°C, since EDTA
MAY, 1973
has been shown to inhibit pilocarpine inactivation by rabbit serum.3 RESULTS
Table 1 shows comparative uptake of pilo carpine by pigmented and albino uveal tissue at 22°C. The mean uptake of pilocarpine by pigmented irides was 8.1 μg/mg of tissue, whereas that of albino irides was 3.9 μg/mg of tissue. The difference is significant to a P-value between .05 and .02 by Student's ttest.5 Similarly, the mean uptake of pilocarpine by pigmented ciliary body was 10.7 μg/mg of tissue, significantly higher than the 4.6 μg / m g of tissue taken up by albino ciliary body. By Student's t-test, P was less than 0.001. The uptake of pilocarpine by each class of tissue at 37°C (Table 2) was the same as or lower than that of tissue incubated at 22°C (Table 1). There was no statistically signifi-
TABLE 1 UPTAKE* OF PILOCARPINE BY IRIS AND CILIARY BODY OF ALBINO AND PIGMENTED RABBITS AT 22°C
Albino Ciliary Body
Iris
Sample — Tissue (mg) 2.5 1 2.5 2 2.0 3 5.1 4 2.6 S 4.9 6 3.5 7 3.3 8 9 10 11 12 Mean (±SEM) 3.2
Pigmented
Uptake/ Tissue O^g/mg) 0.4 4.4 4.0 0.0 8.5 3.3 3.8 7.0
3.9±1.0
„. , *
T lssu
("tf 3.5 3.7 4.8 4.9 4.5 4.3 4.0 4.7
4.3
Uptake/ Tissue
G*/mg) 5.1 6.1 5.8 7.7 2.5 3.0 6.3 0.0
4.6±0.9
Iris Tissue (mg) 4.2 3.0 2.7 2.4 3.4 3.0 2.7 2.8 3.1 2.9 2.1 3.3 3.0
Ciliary Body
Uptake/ Tissue (Mg/mg) 3.1 14.7 14.0 10.0 6.8 7.7 15.5 8.1 5.9 5.5 1.5 4.5 8.1±1.3
Tissue X 103UC
,
(m
N
*) 9.8 5.4 4.5 5.0 5.0 5.5 6.4 3.8 4.3 4.8 5.3 9.7
5.8
U
Ptake/ rp·
1 issue Gig/mg) 6.6 15.3 12.0 11.0 7.6 11.6 9.4 11.8 12.6 12.7 10.4 7.8 10.7±0.7
* Uptake is determined by subtracting the final concentration (in /ig/ml) of a solution of pilocarpine, incu bated for one-half hour with a tissue sample, from the initial concentration of the solution. Since 0.5 ml aliquots of pilocarpine solution are used for the incubation, the apparent pilocarpine uptake is obtained by dividing by two. The numbers also take into account the fact that there is a liberation of a small amount of material from the uvea which reacts positively when using the Hestrin method for pilocarpine concentration. In a series of six tests, uveal tissues were found to release the equivalent of 13 /
887
PILOCARPINE UPTAKE
VOL. 75, NO. 5
TABLE 2 UPTAKE OF PILOCARPINE BY IRIS AND CILIARY BODY OF ALBINO AND PIGMENTED RABBITS AT 37°C Pigmented
Albino Iris
Ciliary Body
Iris
Ciliary Body
Sample —-
1 2 3 4 S 6 7 8 Mean (±SEM)
Tissue (mg)
Uptake/ Tissue 0*g/mg)
_. T 1SSU , * (mg)
Uptake/ Tissue O^g/mg)
2.4 2.6 2.8 3.9 2.1 2.3 2.9 3.2
0.8 5.0 3.6 3.3 4.8 0.9 0.7 0.0
4.6 4.2 3.6 5.7 3.3 2.7 3.1 3.0
0.0 8.3 5.0 2.8 5.2 0.0 3.7 0.7
3.6 3.0 4.0 4.5 5.5 4.4 5.8
2.8
2 . 4 + 0.7
3.7
3.2±1.0
4.4
cant difference in uptake of pilocarpine at 37°C compared with 22°C. However, the pat tern of statistically greater uptake by pig mented tissue found at 22°C is present at 37°C as well. (Student's t-test for uptake by pigmented iris compared with albino iris at 37°C gave a P-value between 0.01 and 0.001. For uptake by pigmented and albino ciliary body at 37°C, P was also between 0.01 and 0.001.) No significant decrease of uptake of pilo carpine by pigmented iris or ciliary body oc curred in the presence of EDTA. Mean pig mented ciliary body uptake of pilocarpine in this experiment was 9.9 μg/mg of tissue as compared with 10.7 pg/mg of tissue
Tissue (rag)
Uptake/ Tissue Og/mg)
Tissue (mg)
Uptake/ Tissue (Mg/mg)
10.2 5.1 4.7 6.2 6.3 6.5 5.6
3.6 7.7 11.5 7.3 8.6 6.4 4.3 7.1±1.0
6.7 13.3 9.6 9.0 5.6 7.7 6.4
6.4
8.3 + 1.0
without EDTA. Mean pigmented iris uptake was 8.8 μg/mg of tissue with EDTA com pared to 8.1 μg/mg of tissue without E D T A (Table 3 ) . Student's t-test compar ing both ciliary body uptake and iris uptake with and without EDTA gives P values be tween 0.5 and 0.8, indicating the similarity of data in the two experiments. DISCUSSION
The data presented here suggest the hy pothesis that the pigment present in the uveal tissue of pigmented rabbits is the agent responsible for most of the uptake or inactivation of pilocarpine, but that the mechanism is probably not enzymatic. This is supported
TABLE 3 UPTAKE OF PILOCARPINE BY IRIS AND CILIARY BODY OF PIGMENTED RABBITS INCUBATION WITH EDTA (3.6 mg/ml)
Sample
—
Ciliary Body
Iris Tissue (mg)
μξ u p t a k e / m g tissue
Tissue (mg)
Mg uptake/mg tissue
1 2 3 4 5 6 7
3.9 3.8 4.2 4.0 4.1 3.2 3.4
3.4 6.8 10.7 8.8 8.5 11.9 11.2
4.7 4.5 5.8 6.0 6.2 6.2 6.2
7.9 7.7 8.8 7.5 11.6 12.6 12.6
Mean ( ± S E M )
3.8
8.8±1.0
5.7
9.8±0.9
885
888
AMERICAN JOURNAL OF OPHTHALMOLOGY
by the following findings : 1. There is a positive correlation between density of pigmentation and pilocarpine up take ability. Pigmented uveal tissue takes up significantly more pilocarpine than albino uveal tissue. 2. An enzyme mechanism for pilocarpine inactivation would be expected to be much more active at 37° than at 22° in fresh tis sue. Sample mean uptake values were actu ally slightly lower at 37° relative to those at 22°, but this difference is not statistically significant because of the dispersion in rela tion to the number of samples. The data do, however, suggest the absence of an enzyme system to explain the uptake. 3. EDTA had no effect on the uptake of pilocarpine by pigmented rabbit uveal tissue. The supposition was made by Schonberg and Ellis3 that an enzyme present in rabbit serum is responsible for inactivation of pilocarpine. This was based in large part on the fact that EDTA at a concentration of 3.6 mg/ml pre vented inactivation by serum, presumably by binding to a co factor. An enzyme hypothesis in the case of uveal tissues would require the assumption that a second enzyme with dif ferent properties and a different co factor must be present. These findings suggest that in the clinical situation some of the pilocarpine adminis tered is bound by uveal pigmented tissue, probably pigment itself, and prevented from reaching sites where it could produce phar macologie activity. This would explain the decreased effectiveness of pilocarpine in heavily pigmented patients found by Melikian, Lieberman, and Leopold1 and by Har ris and Galin.2 It is also possible to explain the interest ing findings of Tornqvist 6 on this basis. Working with monkeys, he found that 100 times more pilocarpine was required to pro duce one-half maximal change in refraction than was required to produce one-half maxi mal pupillary constriction when the medica tion was given topically. However, when the medication was given systemically, the amount of pilocarpine required to produce
MAY. 1973
each of these changes was equal. On the basis of our findings we propose that topically applied pilocarpine comes into contact first with iris pigment, iris muscle receptors, and ciliary body pigment with sig nificant absorption before reaching ciliary muscle. Systemically administered pilocar pine probably has direct access to ciliary body muscle receptors without previous pig ment inactivation, thus producing its effect on ciliary muscle with much smaller doses. It would seem likely that the difference be tween topical and systemic administration would be much less striking in albinos. SUMMARY
The uptake of pilocarpine by pigmented and albino rabbit uveal tissue was compared. It was found that pigmented irides and cili ary bodies took up two to three times more pilocarpine than did the albino tissues. Uveal uptake of pilocarpine is unaffected by incubation with EDTA, which has been shown to prevent pilocarpine inactivation by serum. This suggests that the pigment re lated uveal uptake is dependent on a differ ent mechanism. No significant difference between uptake at 22°C and 37°C was found. ACKNOWLEDGMENT
We thank Mrs. Theresa Reilly for her technical assistance. REFERENCES
1. Melikian, H. E., Lieberman, T. W., and Leo pold, I. H. : Ocular pigmentation and pressure out flow responses to pilocarpine and epinephrine. Am. J. Ophth. 72:70, 1971. 2. Harris, L. S., and Galin, M. A. : Effect of ocu lar pigmentation on hypotensive response to pilocar pine. Am. J. Ophth. 72:923, 1971. 3. Schonberg, S. S., and Ellis, P. P. : Pilocarpine inactivation. Arch. Ophth. 82:351, 1969. 4. Hestrin, S. : The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application. J. Biol. Chem. 180:249, 1949. 5. Bancroft, H. : Introduction to Biostatistics, 1st ed. New York, Hoeber Medical Division of Harper and Row, 19S7. 6. Tornqvist, G. : Comparative studies of the effect of pilocarpine on the pupil and on the refrac tion in two species of monkey (Cercopithecus ethiops and Macaca irus). Invest. Ophth. 3 :388, 1964.
M.D., AND DAVID L. K R O H N ,
M.D.
New York, New York Pilocarpine has been shown to be more effective as an ocular hypotensive agent in lightly pigmented individuals than in heavily pigmented individuals.1'2 A study of normal subjects by Melikian, Lieberman, and Leo pold1 showed that a 4% solution of pilocar pine topically applied produced significant ocular hypotension and increased facility of outflow only in those who were lightly pig mented and had no effect on the more heav ily pigmented subjects. In glaucomatous pa tients of varying pigmentation, Harris and Galin2 showed that the drug's effectiveness in lowering intraocular pressure correlated with lightness of iris pigment. They did not note a corresponding increase in facility of outflow. They speculated that the pilocarpine administered to more heavily pigmented sub jects is inactivated either by direct binding to uveal pigment or by an enzyme present in the uveal tract. The purpose of this study was to confirm that pilocarpine is taken up or inactivated by pigmented uveal tissue to a much greater ex tent than by non-pigmented uveal tissue, to quantitate the amount taken up, and to inves tigate the relationship of this uptake or inactivation to enzyme activity. MATERIALS AND METHODS
Ciliary bodies and irides from heavily pig mented black and white Dutch rabbits and albino New Zealand rabbits were excised from freshly killed females weighing 1.6-2.8 kg. The iris was severed at its root. The ciliFrom the Department of Ophthalmology of the New York University School of Medicine and the Institute for Medical Research and Studies, New York. This study was supported in part by Public Health Service Grant CA 12523-01 and by National Institutes of Health Training Grant 7-1257-192-200 from the National Institutes of Health. Reprint requests to David L. Krohn, M.D., Insti tute for Medical Research and Studies, 220 East 23rd Street, New York, New York 10010.
ary body was then excised at the ora ciliaris retinae and separated from its vitreal adhe sions. Since ciliary processes in the rabbit extend variably beneath the iris, small ele ments of these are presumed to have been in cluded in iris samples. Each of the uveal tis sues was then incubated separately at 22°C in a test tube containing 0.5 ml pilocarpine nitrate, 500 μg/ml in physiologic saline. Af ter 30 minutes a 0.2 ml aliquot of the pilo carpine solution was withdrawn. The incu bated uveal tissue was then removed, airdried for 48 hours, and weighed. The 0.2 ml aliquots were analyzed for pi locarpine content by the Hestrin method.3'4 To each 0.2 ml sample, 0.4 ml of a solution containing equal volumes of 2 N hydroxylamine and 3.5 N NaOH was added. After vortex mixing, the solution was allowed to stand for two minutes. Then 0.2 ml of 3 N HC1 was added and the pH was adjusted to between 1.0 and 1.5. Finally, 0.2 ml of a so lution containing 0.37 M FeCl 3 in 0.1 N HC1 was added. Each sample was centrifuged for at least three minutes to remove any tissue debris and the supernatant was transferred to one of a pair of matched cuvettes. Optical density was read against a distilled water blank at 540 nm in a Zeiss spectrophotometer and converted to pilocarpine concentra tion in μg/ml by comparison with a stan dard curve. The concentration of pilocarpine remain ing in each sample was subtracted from the initial concentration of the incubating solu tion (500 μg/ml) in order to measure the amount of pilocarpine taken up by the tissue which had been incubated. Since 0.5 ml, con taining only 250 μg, was used, the resultant values were divided by two. As a correction to the measurement of up take, ciliary bodies and irides of pigmented and albino rabbits were incubated in saline without pilocarpine for one-half hour and
885
886
AMERICAN JOURNAL OF OPHTHALMOLOGY
0.2 ml of the solution taken as a sample. This was tested by the Hestrin method and the release of Hestrin method-reacting components (false-positives) was measured. These results were added to the pilocarpine uptake figures as a correction factor, since the presence of these compounds masked part of the pilocarpine uptake. The final up take of each sample was divided by its weight and expressed as μg uptake/mg tis sue. Two further groups of experiments were done to investigate the possibility of an en zymatic mechanism for uveal uptake of pilo carpine. 1. A series of uveal pilocarpine uptake measurements was run at 37°C for compari son with that at 22°C. 2. In addition, a series of measurements of uptake using the same method was done in the presence of ethylene-diamine-tetraacetic acid (EDTA) at 22°C, since EDTA
MAY, 1973
has been shown to inhibit pilocarpine inactivation by rabbit serum.3 RESULTS
Table 1 shows comparative uptake of pilo carpine by pigmented and albino uveal tissue at 22°C. The mean uptake of pilocarpine by pigmented irides was 8.1 μg/mg of tissue, whereas that of albino irides was 3.9 μg/mg of tissue. The difference is significant to a P-value between .05 and .02 by Student's ttest.5 Similarly, the mean uptake of pilocarpine by pigmented ciliary body was 10.7 μg/mg of tissue, significantly higher than the 4.6 μg / m g of tissue taken up by albino ciliary body. By Student's t-test, P was less than 0.001. The uptake of pilocarpine by each class of tissue at 37°C (Table 2) was the same as or lower than that of tissue incubated at 22°C (Table 1). There was no statistically signifi-
TABLE 1 UPTAKE* OF PILOCARPINE BY IRIS AND CILIARY BODY OF ALBINO AND PIGMENTED RABBITS AT 22°C
Albino Ciliary Body
Iris
Sample — Tissue (mg) 2.5 1 2.5 2 2.0 3 5.1 4 2.6 S 4.9 6 3.5 7 3.3 8 9 10 11 12 Mean (±SEM) 3.2
Pigmented
Uptake/ Tissue O^g/mg) 0.4 4.4 4.0 0.0 8.5 3.3 3.8 7.0
3.9±1.0
„. , *
T lssu
("tf 3.5 3.7 4.8 4.9 4.5 4.3 4.0 4.7
4.3
Uptake/ Tissue
G*/mg) 5.1 6.1 5.8 7.7 2.5 3.0 6.3 0.0
4.6±0.9
Iris Tissue (mg) 4.2 3.0 2.7 2.4 3.4 3.0 2.7 2.8 3.1 2.9 2.1 3.3 3.0
Ciliary Body
Uptake/ Tissue (Mg/mg) 3.1 14.7 14.0 10.0 6.8 7.7 15.5 8.1 5.9 5.5 1.5 4.5 8.1±1.3
Tissue X 103UC
,
(m
N
*) 9.8 5.4 4.5 5.0 5.0 5.5 6.4 3.8 4.3 4.8 5.3 9.7
5.8
U
Ptake/ rp·
1 issue Gig/mg) 6.6 15.3 12.0 11.0 7.6 11.6 9.4 11.8 12.6 12.7 10.4 7.8 10.7±0.7
* Uptake is determined by subtracting the final concentration (in /ig/ml) of a solution of pilocarpine, incu bated for one-half hour with a tissue sample, from the initial concentration of the solution. Since 0.5 ml aliquots of pilocarpine solution are used for the incubation, the apparent pilocarpine uptake is obtained by dividing by two. The numbers also take into account the fact that there is a liberation of a small amount of material from the uvea which reacts positively when using the Hestrin method for pilocarpine concentration. In a series of six tests, uveal tissues were found to release the equivalent of 13 /
887
PILOCARPINE UPTAKE
VOL. 75, NO. 5
TABLE 2 UPTAKE OF PILOCARPINE BY IRIS AND CILIARY BODY OF ALBINO AND PIGMENTED RABBITS AT 37°C Pigmented
Albino Iris
Ciliary Body
Iris
Ciliary Body
Sample —-
1 2 3 4 S 6 7 8 Mean (±SEM)
Tissue (mg)
Uptake/ Tissue 0*g/mg)
_. T 1SSU , * (mg)
Uptake/ Tissue O^g/mg)
2.4 2.6 2.8 3.9 2.1 2.3 2.9 3.2
0.8 5.0 3.6 3.3 4.8 0.9 0.7 0.0
4.6 4.2 3.6 5.7 3.3 2.7 3.1 3.0
0.0 8.3 5.0 2.8 5.2 0.0 3.7 0.7
3.6 3.0 4.0 4.5 5.5 4.4 5.8
2.8
2 . 4 + 0.7
3.7
3.2±1.0
4.4
cant difference in uptake of pilocarpine at 37°C compared with 22°C. However, the pat tern of statistically greater uptake by pig mented tissue found at 22°C is present at 37°C as well. (Student's t-test for uptake by pigmented iris compared with albino iris at 37°C gave a P-value between 0.01 and 0.001. For uptake by pigmented and albino ciliary body at 37°C, P was also between 0.01 and 0.001.) No significant decrease of uptake of pilo carpine by pigmented iris or ciliary body oc curred in the presence of EDTA. Mean pig mented ciliary body uptake of pilocarpine in this experiment was 9.9 μg/mg of tissue as compared with 10.7 pg/mg of tissue
Tissue (rag)
Uptake/ Tissue Og/mg)
Tissue (mg)
Uptake/ Tissue (Mg/mg)
10.2 5.1 4.7 6.2 6.3 6.5 5.6
3.6 7.7 11.5 7.3 8.6 6.4 4.3 7.1±1.0
6.7 13.3 9.6 9.0 5.6 7.7 6.4
6.4
8.3 + 1.0
without EDTA. Mean pigmented iris uptake was 8.8 μg/mg of tissue with EDTA com pared to 8.1 μg/mg of tissue without E D T A (Table 3 ) . Student's t-test compar ing both ciliary body uptake and iris uptake with and without EDTA gives P values be tween 0.5 and 0.8, indicating the similarity of data in the two experiments. DISCUSSION
The data presented here suggest the hy pothesis that the pigment present in the uveal tissue of pigmented rabbits is the agent responsible for most of the uptake or inactivation of pilocarpine, but that the mechanism is probably not enzymatic. This is supported
TABLE 3 UPTAKE OF PILOCARPINE BY IRIS AND CILIARY BODY OF PIGMENTED RABBITS INCUBATION WITH EDTA (3.6 mg/ml)
Sample
—
Ciliary Body
Iris Tissue (mg)
μξ u p t a k e / m g tissue
Tissue (mg)
Mg uptake/mg tissue
1 2 3 4 5 6 7
3.9 3.8 4.2 4.0 4.1 3.2 3.4
3.4 6.8 10.7 8.8 8.5 11.9 11.2
4.7 4.5 5.8 6.0 6.2 6.2 6.2
7.9 7.7 8.8 7.5 11.6 12.6 12.6
Mean ( ± S E M )
3.8
8.8±1.0
5.7
9.8±0.9
885
888
AMERICAN JOURNAL OF OPHTHALMOLOGY
by the following findings : 1. There is a positive correlation between density of pigmentation and pilocarpine up take ability. Pigmented uveal tissue takes up significantly more pilocarpine than albino uveal tissue. 2. An enzyme mechanism for pilocarpine inactivation would be expected to be much more active at 37° than at 22° in fresh tis sue. Sample mean uptake values were actu ally slightly lower at 37° relative to those at 22°, but this difference is not statistically significant because of the dispersion in rela tion to the number of samples. The data do, however, suggest the absence of an enzyme system to explain the uptake. 3. EDTA had no effect on the uptake of pilocarpine by pigmented rabbit uveal tissue. The supposition was made by Schonberg and Ellis3 that an enzyme present in rabbit serum is responsible for inactivation of pilocarpine. This was based in large part on the fact that EDTA at a concentration of 3.6 mg/ml pre vented inactivation by serum, presumably by binding to a co factor. An enzyme hypothesis in the case of uveal tissues would require the assumption that a second enzyme with dif ferent properties and a different co factor must be present. These findings suggest that in the clinical situation some of the pilocarpine adminis tered is bound by uveal pigmented tissue, probably pigment itself, and prevented from reaching sites where it could produce phar macologie activity. This would explain the decreased effectiveness of pilocarpine in heavily pigmented patients found by Melikian, Lieberman, and Leopold1 and by Har ris and Galin.2 It is also possible to explain the interest ing findings of Tornqvist 6 on this basis. Working with monkeys, he found that 100 times more pilocarpine was required to pro duce one-half maximal change in refraction than was required to produce one-half maxi mal pupillary constriction when the medica tion was given topically. However, when the medication was given systemically, the amount of pilocarpine required to produce
MAY. 1973
each of these changes was equal. On the basis of our findings we propose that topically applied pilocarpine comes into contact first with iris pigment, iris muscle receptors, and ciliary body pigment with sig nificant absorption before reaching ciliary muscle. Systemically administered pilocar pine probably has direct access to ciliary body muscle receptors without previous pig ment inactivation, thus producing its effect on ciliary muscle with much smaller doses. It would seem likely that the difference be tween topical and systemic administration would be much less striking in albinos. SUMMARY
The uptake of pilocarpine by pigmented and albino rabbit uveal tissue was compared. It was found that pigmented irides and cili ary bodies took up two to three times more pilocarpine than did the albino tissues. Uveal uptake of pilocarpine is unaffected by incubation with EDTA, which has been shown to prevent pilocarpine inactivation by serum. This suggests that the pigment re lated uveal uptake is dependent on a differ ent mechanism. No significant difference between uptake at 22°C and 37°C was found. ACKNOWLEDGMENT
We thank Mrs. Theresa Reilly for her technical assistance. REFERENCES
1. Melikian, H. E., Lieberman, T. W., and Leo pold, I. H. : Ocular pigmentation and pressure out flow responses to pilocarpine and epinephrine. Am. J. Ophth. 72:70, 1971. 2. Harris, L. S., and Galin, M. A. : Effect of ocu lar pigmentation on hypotensive response to pilocar pine. Am. J. Ophth. 72:923, 1971. 3. Schonberg, S. S., and Ellis, P. P. : Pilocarpine inactivation. Arch. Ophth. 82:351, 1969. 4. Hestrin, S. : The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application. J. Biol. Chem. 180:249, 1949. 5. Bancroft, H. : Introduction to Biostatistics, 1st ed. New York, Hoeber Medical Division of Harper and Row, 19S7. 6. Tornqvist, G. : Comparative studies of the effect of pilocarpine on the pupil and on the refrac tion in two species of monkey (Cercopithecus ethiops and Macaca irus). Invest. Ophth. 3 :388, 1964.