In comparison with iris muscle homogenates from the control eyes, those from the treated eyes demonstrated an eightfold increase in the number of receptor ...
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Permanent Alterations in Muscarinic Receptors and Pupil Size Produced by Chronic Atropinization in Kittens Earl L. Smith III,* Dianna A. Redburn,f Ronald 5. Harwerrh,* and Gregory W. Maguire* Chronic mydriasis was induced in six kittens (four monocular, two binocular) and two adult cats (both monocular) by the daily topical application of atropine. Both the kittens and the adult cats were atropinized for a 13-week period with the treatment regimen beginning at the time of eye opening for the kittens. Pupil size measurements, obtained 1 year after the atropinization were discontinued, revealed that, although the pupils of the adult cats were normal, the pupils of the kittens' treated eyes were consistently smaller than pupils in control eyes. The status of the muscarinic receptors in the kittens' irides was investigated using 3 H-QNB binding assays. In comparison with iris muscle homogenates from the control eyes, those from the treated eyes demonstrated an eightfold increase in the number of receptor binding sites. The results indicate that pupil size can be altered permanently by chronic mydriasis initiated early in the life of a kitten and that the permanent change in pupil size may result, in part, from a type of permanent supersensitivity response in the muscle following chronic blockade of muscarinic transmission by atropine. Invest Ophthalmol Vis Sci 25:239-243, 1984
Postdilation miosis is a reduction in pupillary size relative to pretreatment dimensions or nontreated control pupils following recovery from the primary action of a mydriatic agent. In humans, postdilation miosis has been observed following the topical administration of certain sympathomimetic agents12 (eg, phenylephrine and hydroxyamphetamine) and has been reported2 to persist for as long as 7 days after treatment. To our knowledge, the phenomenon of postdilation miosis has not been associated with the use of parasympatholytic agents in humans. However, in a recent study3 on the effects of chronic atropinization on visual acuity in kittens, it was noted that following recovery from mydriasis (approximately 3 wks posttreatment) the pupils of the kittens' treated eyes were consistently smaller than the pupils in the nontreated eyes. If confirmed, this observation would not only be clinically important, it would also provide insight into the physiology of the iris musculature. Moreover, since chronic atropinization is an experimental procedure sometimes used for depriving an eye of a clear retinal image, it would be potentially important in the interpretation of certain experimental results. Therefore, one of the purposes of the present study was to verify and document the presence of postdilation miosis in chronically atropinized kittens. In this respect, since the original observation was in kittens, it was important to
determine if the phenomenon was reversible and if it represented a developmental age-dependent process. A second purpose of the present study was to examine the mechanism by which an alteration in the tone of the iris muscles is maintained over extended periods of time following chronic atropinization. This phenomenon could result from a variety of changes in the iris including simple atrophy of certain iris muscles, an up-regulation of parasympathetic neuronal activity (ie, an increase in acetylcholine release from each nerve terminal or an increase in the number of terminals as seen in sprouting), or an up-regulation in muscle response (ie, an increase in muscarinic receptors). The in vitro binding of the irreversible muscarinic ligand, 3H-quinuclidinyl benzylate (QNB), which has been widely used as a measure of the number and affinity of muscarinic receptors in neuronal and muscle preparations, provides a way to examine the status of muscarinic receptors in the iris (for a review see reference 4). We therefore analyzed the QNB binding activity of iris from control and chronically atropinized eyes. Our results suggest that the number of muscarinic receptors is greatly increased in the treated iris and, therefore, may represent one of the mechanisms by which pupillary size is permanently altered after chronic atropinization in kittens. Materials and Methods. Eight kittens that were born in an isolated colony and 10 additional cats that were obtained as adults were used in the study. Six of the kittens were treated with a 1% atropine sulfate solution (four monocularly, right eye; two binocularly) on a daily basis from the time of eye opening («8-9 days of age) until 14 wks of age. Two of the adult cats had atropine topically applied to the right eye on a daily basis for 13 wks. In order to maintain a maximal mydriasis, it was necessary to instill atropine into the experimental animals' eyes three times each day. Two of the kittens that had coloration patterns similar to their binocularly treated littermates and the remaining eight adult cats were used as nontreated controls. Throughout the experiment, all of the animals were housed in a normally illuminated environment (12hr light/12-hr dark cycle). The sizes of the animals' pupils were measured from individual photographs. To help minimize distractions, the animals were held 150 cm from the camera at one end of a 50 X 50 X 150 cm white rectangular box. The interior of the box was illuminated uniformly by
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Fig. 1. Photographs taken 1 year posttreatment of a monocularly treated adult (A), a monocularly treated kitten (B), and a binocularly treated kitten (C, right) and its paired nontreated littermate (C, left).
variable intensity incandescent light sources positioned above the camera. The outlines of the pupils were traced from enlarged projections of the photographic negatives, and the horizontal dimensions of the pupils were measured to the nearest 0.5 mm with a handheld magnifier. A correction factor to compensate for the magnification of the camera and enlarger was derived from photographs of a calibrated scale positioned in the same plane as the animals' eyes. For a given background level of illumination, measurements for each pupil were obtained from a minimum of three photographs. For the treated animals, estimates of pupillary size were made periodically during a 1-year recovery period beginning 1 month after the atropine regimen was discontinued.
At the end of the 1 -year recovery period, the treated kittens were sacrificed by decapitation. The eyes were removed and hemisected. Muscles of the iris and ciliary body were obtained by gently exerting pressure on the attachments along the outer circumference of the iris with a blunt probe inserted through an incision in the center of the cornea. Iris and ciliary muscles were separated with scissors. Each tissue sample was frozen overnight at -20°C. By a modified filtration assay described by Yamamura and Snyder,5 3H-QNB binding was determined. Frozen pieces of tissue from the irides of the treated and nontreated eyes of the atropinized kittens were thawed, placed in 0.1 M Na-K phosphate buffer (pH 7.4) and homogenized for 30 sec with a Brinkman
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polytron at setting 6. The tissue suspensions then were diluted with Na-K phosphate buffer (0.05 M,finalconcentration) to a final volume containing 0.3-0.5 mg protein/ml for the 3H-QNB assay. Fifty microliters of 3 H-QNB solution (final concentrations were 0.25 to 5.0 nM; New England Nuclear, Boston, MA) and 1 ml of phosphate buffer were placed in each assay tube. Twenty microliters of 10 fiM atropine (final concentration 0.1 /xM; Sigma Chemical Co., St. Louis, MO) were placed in selected assay tubes to determine nonspecific binding. One milliliter of tissue suspension was added to each assay tube for a 1-hr incubation period at room temperature (25 °C). Incubation was terminated by filtration on GF/C filters (Whatman, England) followed by three 5-ml washes with cold buffer. The filters were removed, placed in vials, and allowed to dry. A toluene base counting solution containing Triton X-100 was added, and the filters were counted on a Searle Mark III liquid scintillationcounter. Results. Pupil Size: There were no obvious behavioral differences between the treated and nontreated kittens, either during or after the treatment period. Casual inspection of the atropinized animals revealed that the responsiveness of the treated pupils to changes in illumination (both direct and consensual) increased gradually after the atropinization had been discontinued and that all of the treated pupils responded in a normal manner within 3 wks of the termination of the atropine treatment. Measurements of pupil size obtained 1 month posttreatment indicated that the pupils of the atropinized adult cats were essentially
equal in size (ie, the differences in pupil size between the right and left eyes were within the range of size differences for the 8 nontreated controls). However, the differences in pupil size for all four monocularly atropinized kittens were outside the range for normals, the pupils of the treated eyes being consistently smaller than the pupils of the nontreated eyes. In addition, the pupils of the two binocularly treated kittens were essentially equal in size but smaller than the pupils of their paired nontreated littermates. The normal pupil sizes observed in the atropinized adult cats and the postdilation miosis demonstrated by the treated eyes of the kittens remained unchanged over a 1-year recovery period. The relative miotic nature of the kittens' treated pupils is illustrated in Figure 1, where photographs taken 1 year posttreatment of a monocularly treated adult (A), a monocularly treated kitten (B), and a binocularly treated kitten and its paired nontreated littermate (C) are shown. The ratios of pupil sizes (right eye/left eye) obtained for a moderate level of illumination (2.2 log lux) at the end of the 1year recovery period are illustrated for each animal in Figure 2A. For the nontreated controls, the difference in the horizontal dimensions of the pupils never exceeded 6.0% and, as can be seen, the size ratios for both the monocularly treated adults and the binocularly treated kittens fall within the range of differences for the controls (dashed line). In contrast, the horizontal dimensions of the treated pupils of the monocularly atropinized kittens were between 12% and 27% smaller than those for the nontreated pupils. Similarly, both the pupils of the two binocularly treated kittens were
A.
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0.7 NORMAL CONTROLS
MONOCULARLY TREATED ADULTS
BINOCULARLY TREATED KITTENS
MONOCULARLY TREATED KITTENS
-1.0
0
1.0
2.0
3.0
LOG LUMINANCE (LUX)
REARING CONDITION Fig. 2A. Ratio of pupil sizes (right eye/left eye) obtained at a moderate background illuminance (2.2 log lux) plotted as a function of the rearing condition for each animal (filled circles). The open squares represent the ratios of the pupils of the binocularly treated kittens compared to their paired nontreated controls. B. Horizontal dimensions of the treated (filled circles) and nontreated pupils (open circles) of a monocularly atropinized kitten plotted as a function of background illuminance.
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3
A. IRIS (CONTROL)
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BOUND (fmoles/mg protein) Fig. 3. Scatchard analysis of specific 3 H-QNB binding in control (A) and atropine-treated (B) cat iris. The tissue was incubated in 0.05 M Na-K phosphate buffer (pH 7.4, 1 hr) containing different concentrations of ligand, and specific binding was obtained by subtracting the amount of radioactivity not displaced by 0.1 fiM atropine from the total 3H-QNB bound. K
