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Choline Acetyltransferase in Ocular Tissues of Rabbits, Cats, Cattle, and Man

Choline Acetyltransferase in Ocular Tissues of Rabbits, Cats, Cattle, and Man

Choline acetyltransferase in ocular tissues of rabbits, cats, cattle, and man

Joel S. Mindel* and Thomas W. Mittag

The variation of choline acetyltransferase activity in ocular tissues of four mammalian species, rabbits, cats, cattle, and man, was determined. Enzyme activity of irides and ciliary bodies, i.e., parasympathetically innervated structures, tended to be similar in all four species. Two exceptions were bovine irides and human ciliary bodies; these two tissues had higlier enzyme activities. Choline acetyltransferase activity was present in the corneal of rabbit, bovine, and human , but little or none could be detected in that of cats. Feline and pigment epithelium- also contained far less choline acetyltransferase activity than the same tissues in the other three species.

Key words: choline acetyltransferase, , ocular, , , , aqueous humor, retina, choroid.

Choline acetyltransferase, the enzyme optic nerve. In recent years, the retinal and responsible for acetylcholine synthesis, has corneal epithelial choline acetyltransferases been assayed in a number of ocular tis- have attracted the most interest. Ross and sues. 1"s This enzyme is a more specific McDougal" have found that the inner plexi- marker of cholinergic activity than are the form layer contains high levels of choline ubiquitous cholinesterases. The first esti- acetyltransferase activity. Van Alphen7 and mates of ocular choline acetyltransferase Williams and Coopers reported high con- activity appeared in 19461'2; low enzyme centrations of the enzyme in rabbit and levels were reported for canine and rabbit bovine corneal epithelium. The corneal epi- thelium contains none of those structures normally associated with cholinergic ac- From the Departments of Pharmacology and tivity, i.e., synapses, myoneural junctions, Ophthalmology, Mount Sinai School of Medi- cine, City University of New York, and the or parasympathetic motor axons. The pres- Bronx Veterans Hospital, New York, N. Y. ence of choline acetyltransferase activity Supported in part by a Fight-for-Sight Grant-in- in corneal epithelium, which consists pri- Aid and National Eye Institute Grant I RO1 marily of epithelial cells and sensory nerve EY01243. endings, has elicited several theories at- "Chief of Ophthalmology, Bronx Veterans Ad- tempting to link cholinergic activity with ministration Hospital. Dr. Mindel is the re- corneal touch sensitivity0"11 and epithelial cipient of a Research Career Development ion transport.s> 12 Award from the National Eye Institute. Submitted for publication Feb. 12, 1976. Although the choline acetyltransferase of Reprint requests: J. S. Mindel, The Mount Sinai specific tissues has interested different School of Medicine, Fifth Avenue & 100th St., workers, no general survey of ocular struc- New York, N. Y. 10029. tures has been reported. This paper de- 808

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scribes the distribution of choline acetyl- on kinetic studies of rabbit tissues where transferase activity in the eyes of four the concentration of choline was varied species: rabbit, cat, cattle, and man. between 25 /xM and 10 mM and the con- centration of acetyl-coenzyme A was Materials and methods varied between 10 and 400 /xM. Using a Eyes of adult female Dutch Belt rabbits and double reciprocal plot by the method of female mongrel cats, killed with parenteral pento- Lineweaver-Burk, the Michaelis-Menton barbital or secobarbital, were studied. Bovine constants (Km's) were calculated from the eyes were supplied on ice by an abattoir within 5 hours of death. Human eyes were obtained extrapolated intercepts. The Km for choline hours to days after death from the Eye Bank was 500 /xM for all four ocular tissues, for Sight Restoration, New York, N. Y. corneal epithelium, iris-ciliary body, retina, were removed by trephine as full thick- and pigment epithelium-choroid. The Km ness buttons. In rabbits, the ciliary body is poorly for acetyl-coenzyme A was 20 ju,M for developed and both it and the iris were excised corneal epithelium, iris-ciliary body, and and assayed together. In all four species, the pigment epithelium and choroid were dissected pigment epithelium-choroid; the Km for out and assayed together. retinal acetyl-coenzyme A was 50 /xM. The Choline acetyltransferase was assayed by a ocular tissues from eight eyes were pooled modification of the methods of Schrier and and the specific activities determined (ex- 11 Sinister™ and McCaman and Hunt. Tissues pressed as nanomoles acetylcholine formed were homogenized on ice in pH 7.4 buffer solu- per hour per milligram [nmole ACh tion of sufficient volume to give a concentration of less than 10 per cent weight/volume. The formed/hr./mg.] of protein)—comeal epi- concentrations of buffer ingredients during in- thelial protein, 18.1; iris-ciliary body pro- cubation were 0.5 per cent Triton-X, 10 mM tein, 1.6; retinal protein, 58.4; and pigment ethylenediamine tetraacetic acid (EDTA), 300 epithelium-choroid protein, 6.7. The rates mM sodium chloride, and 150 mM potassium of reaction were found to be linear during hydrogen phosphate. The assay was performed in duplicate 6 by 50 mm. tubes containing 200 the incubation period used in the assay. fil of homogenate and 20

    but no choline. After a 30 min. incubation at 37.5° C, human ocular tissues (per milligram of the reaction was terminated with 1 drop of protein). The corneal epithelium of cats, CuCl;, 2.5 per cent, per tube. The contents of unlike the other three mammalian species, each tube were quantitatively transferred, using had little or no detectable choline acetyl- 1 ml. of H-O, to a 3 by 0.6 cm. column of Bio- transferase activity (Tables I to IV). Rad AC1-X8, 200 to 400 mesh chloride form ion exchange resin in water. Effluents were collected Rabbit, bovine, and human corneal epi- directly in scintillation vials and counted in 10 thelia had high levels of enzyme activity. ml. of Bray's solution. Protein content of tissues The corneal stroma and of all was determined by the method of Lowry and four species had little or no choline acetyl- 1 <: associates. Naphthylvinylpyridinium hydroxyethyl transferase activity. bromide, dithiotreitol, choline chloride, and acetyl-coenzyme A solutions were made up weekly The enzyme activity of human, rabbit, and stored frozen. Physostigmine solutions were and cat iris was, on the average, approxi- made up daily. mately 2 nmole ACh formed/hr./mg. of protein. However, the average bovine iris Results contained four to five times this activity. The choline acetyltransferase activities The ciliary bodies of cattle, rabbits, and of all four species were measured with a cats formed 2 to 3 nmole ACh/hr./mg. of standardized assay. This assay was based protein, whereas the average human ciliary

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    Table I. Distribution of choline Table III. Distribution of choline acetyltransferase activity (per milligram acetyltransf erase activity (per milligram of protein) in the ocular tissues of Dutch of protein) in the ocular tissues of cattle

    Aver- Std. Aver- Std. Tissue age* Range* Dev. n Tissue age* Range* Dev. n Cornea: 0.3 _ 0.0 3 Epithelium 46.5 11.6-89.2 27.5 8 Cornea: Stroma-endo- 0.4 0.0- 1.4 0.6 8 Epithelium 20.6 3.9-41.2 13.6 21 thelium Stroma-endo- 0.0f 0.0 8 IrJs 10.8 1.6-22.2 6.5 10 thelium Ciliary body 1.9 0.3- 6.9 2.1 10 Iris-ciliary body 2.6 0.2- 4.9 1.7 5i o.ot — 0.1 2 Lens 0.0$ 0.0 13 Vitreous 0.9 0.5- 1.3 0.6 2 Vitreous 0.5 0.0- 2.1 0.7 g Retina 13.0 4.0-31.4 9.8 10 Retina 26.1 5.8-94.4 20.5 46 Pigment epithelium- 4.6 0.0-10.9 4.5 10 Pigment epithelium- 3.4 0.2-12.4 3.1 47 choroid choroid Optic nerve 0.3 — — 1 Optic nerve 0.0 0.0- 0.1 0.0 g 1.8 — — 1 Sclera o.o§ — 0.0 8 °Nanomoles ACh formed/hr./mg. of protein °Nanomoles ACh formed/hr./mg. of protein. f-0.15. t< 0.036. }< 0.003. 5< 0.010. Table IV. Distribution of choline acetyltransf erase activity (per milligram Table II. Distribution of choline of protein) in the ocular tissues of man acetyltransf erase activity (per milligram Aver- Std. of protein) in ocular tissues of cats Tissue age* Range* Dev.

    Aver- Cornea: Std. 11.2 Tissue age* Range* Dev. n Epithelium — — t Strnma-enrio- 0.4 | Cornea: thelium Epithelium 0.1 0.0-0.6 0.2 8 Iris 2.5 0.1- 5.3 1.8 10 Stroma-endo- 0.0f — 0.0 4 Ciliary body 16.8 0.1-38.0 14.3 11 thelium Lens 0.0$ — 0.0 6 Full thickness 0.0 0.0-0.3 0.1 12 Vitreous 2.2 — — t Iris 2.0 0.3-5.5 1.5 16 Retina 6.1 0.9-23.1 6.7 10 Ciliary body 2.9 0.5-6.6 2.2 16 Pigment epithelium- 5.1 0.6-11.0 4.5 10 Lens 0.0$ — 0.0 4 choroid Vitreous 0.0§ — 0.0 4 Optic nerve 0.7 — — t Retina 0.7 0.4-1.2 0.3 12 Sclera 1.2 — — t Pigment epithelium- 0.5 0.2-0.7 0.2 6 "Nanomoles ACh formed/hr./m{%. of protein. choroid tPooled sample of 12 eyes. °Nanomoles ACh formed/hr./mg. of protein t < 0.027. t< 0.010. |< 0.029. 5< 0.013.

    body had five to eight times this level of from eight eyes, were assayed and their activity. average enzyme activity was 1.4 nmole The vitreous humor contained small ACh formed/hr./ml. Aqueous humor cho- amounts of choline acetyltransferase ac- line acetyltransferase in seven human eyes tivity. Aqueous humor choline acetyltrans- ranged in value from 5.4 to 10.1 nmole ferase was assayed in 14 rabbit eyes. In ACh formed/hr./ml., with an average 13 of the 14, no enzyme activity was de- value of 7.8 ± 2.8. tectable. In one eye, an activity of 0.1 The retinal choline acetyltransferase ac- nmole ACh formed/hr./ml. was found. tivities of rabbits and cattle were, on the Two pooled samples of bovine aqueous average, similar whereas human and, es- humor, one from 40 eyes and the other pecially, feline enzyme activities were far

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    Table V. Distribution of choline acetyltransferase activity (per whole tissue) in ocular tissues*

    Tissue Rabbit Cat Bovine Human Cornea < 1-235 (100)t 0 (8) 130-1,600 (10) 0- 10 (6) Iris 10 A(\ (AA\ 27-60 (4) 28- 539 (5) 1- 35 (5) Ciliary body 26-48 (4) 9- 239 (5) 0-221 (5) Retina 60-160 (47) 9-26 (4) 157- 944 (5) 22-105 (4) Pigment epithelium—choroid <1- 15 (42) 26-50 (4) 3- 533 (5) 16-103 (4) •Nanomoles ACh formed/hr. per whole tissue. t Numbers in parentheses = number of subjects assayed.

    less. The average values of rabbit, cattle, 20 n and human pigment epithelium-choroid choline acetyltransferase activities (3 to 5 nmole ACh formed/hr./mg. of protein) were five to seven times that of cat. Underlying all these mean values was a wide range in choline acetyltransferase ac- tivity that indicated considerable individ- 7 14 21 28 50 65 80 95 110 125 140155 190 235 ual variation. nanomoles ACh FORMED/hr/CORNEA Variation in ocular choline acetyltrans- Fig. 1. Distribution of choline acetyltransferase activity in the corneal epithelia of 100 Dutch ferase activity of rabbit, cat, cattle, and Belt rabbits (per cent of total number of eyes human ocular tissues (per whole tissue). assayed). Table V shows the range of ocular choline acetyltransferase activities in the four spe- cies studied, calculated on a per whole nine retina. Nachmansohn and Berman- tissue basis. Sufficient rabbit eyes were as- reported the same year that rabbit optic sayed to evaluate the distribution of en- nerve produced 13 to 21 fxg of ACh/hr./ zyme activities for this species. Values for gram of whole tissue. De Roetth4 reported rabbit corneal choline acetyltransferase a much higher enzyme activity for rabbit (Fig. 1) did not cluster around a central optic nerve, 100 to 300 /xg of ACh formed/ value as they did for the other three tissues: hr./gram of acetone-dried powder. Hebb5 62 per cent of iris-ciliary body samples fell studied ocular choline acetyltransferase ac- in the range of 20 to 30 nmole ACh tivity by an improved enzyme assay that formed/hr. per whole tissue, 68 per cent of was more likely to ensure that the acetyl- retinal samples fell in the range of 80 to coenzyme A substrate remained at saturat- 120 nmole ACh formed/hr. per whole tis- ing levels. A marked species variation in sue, and 60 per cent of pigment epithe- retinal choline acetyltransferase of dog, lium-choroid samples fell in the range of rabbit, chicken, and pigeon was found, and 5 to 10 nmoles ACh formed/hr. per whole far lower optic nerve enzyme activities tissue. than de Roetth had reported earlier were noted. Hebb found a material present in Discussion ocular tissues that interfered with bioassay In 1946, two papers appeared giving the determinations of acetylcholine. first estimates of choline acetyltransferase The conditions of enzyme assay in the activity in ocular tissues. Feldberg and present study were chosen so as to be well Mann1 reported 0 to 15 /xg ACh formed/ above the Km's determined for rabbit oc- hr./gram of acetone-dried powdered ca- ular choline acetyltransferase. The corneal nine optic nerve and 400 /xg ACh formed/ epithelial, iris-ciliary body, and pigment hr./gram of acetone-dried powdered ca- epithelium-choroid Km's were 500 tiM for

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    choline and 20 /.LM for acetyl-coenzyme A. inhibit enzyme activity.19 Howard and The Km's for retina were 500 fiM for cho- Wilson'-0 and Howard, Wilson, and Dunn21 line and 50 /iM for acetyl-coenzyme A. found an upper value of rabbit comeal epi- These were of the same order of magni- thelial choline acetyltransferase activity, tude as those reported for calf and human 123.1 nmole ACh formed/hr./mg. of pro- brain. White and Cavallito17 found the tein, approximately three times that re- Km's for calf brain choline acetyltrans- ported here. Since there is normally great ferase to be 800 /JM for choline and 16 pM variation in the corneal enzyme activity for acetyl-coenzyme A. White and Wuls of different rabbits (Fig. 1), these authors found the Km's for human choline acetyl- may have used animals with higher choline transferase to be 510 /xM for choline and acetyltransferase activities. A second pos- 11 [xM for acetyl-coenzyme A. sibility is that since their assay did not use Choline acetyltransferase activities were a specific choline acetyltransferase inhibi- calculated in two different manners. For tor, other acetylated cationic molecules, the purpose of comparing different species such as acetylcarnitine, may have contrib- whose ocular structures vary in size, en- uted to falsely elevated values. The varia- zyme activities were calculated on a per tion in rabbit corneal choline acetyltrans- milligram of protein basis. However, the ferase activity (Fig. 1) is much greater enzyme activities of the corresponding tis- than that described for other rabbit ocular sues of the two eyes of an individual tissues. The corneal epithelium is the only animal agreed better if the data were cal- ocular tissue directly in contact with the culated on a per whole tissue basis. The external environment and several authors reasons this occurred will be dealt with in have suggested1-' -1 that the environment a separate publication. This is an impor- influences this tissue's choline acetyltrans- tant consideration when comparisons of ferase activity. enzyme activity between the two eyes are The rather striking differences between made following experimental manipulations feline corneal enzyme activity and those of to one eye with the other serving as control. rabbit, cattle, and human eyes were also The corneal epithelium showed the most found for retina and pigment epithelium- marked species variation in choline acetyl- choroid. The average retinal choline acetyl- transferase activity (Tables I to IV and transferase activity of the cat was only 0.7 Fig. 1). In contrast to the very high choline nmole ACh formed/hr./mg. of protein acetyltransferase activities of some rabbit compared to values of 26.1, 13.0, and 6.1 (20.6 nmole ACh formed/hr./mg. of pro- nmole ACh formed/hr./mg. of protein tein), bovine (46.5 nmole ACh formed/ in rabbit, bovine, and human eyes, respec- hr./mg. of protein), and human (11.2 tively. HebbV reliance on a regional dif- nmole ACh formed/hr./mg. of protein) ference in myelinization to explain why corneal epithelia, that of the cat contained pigeon central retina had lower choline very little enzyme activity (0.1 nmole ACh acetyltransferase activity than peripheral formed/hr./mg. of protein). The value for retina would not seem to explain this inter- bovine corneal epithelial choline acetyl- species variation. The rabbit retina is more transferase activity, 46.5 nmole ACh highly myelinated2- than those of the other formed/hr./mg. of protein, agrees well three species yet its enzyme activity is with that of Williams and Cooper,8 33 higher. Ross and McDougal0 have pre- nmole ACh formed/hr./mg. of protein. sented evidence that the inner plexiform, Van Alphen7 found a lower level of rabbit inner nuclear, and ganglion cell layers have corneal epithelial enzyme activity than is significant amounts of choline acetyltrans- reported here but he used a high concen- ferase activity. They found that these tration of cysteine in his extraction proce- retinal layers had less activity in cats than dure and this was subsequently found to in monkeys, mice, or rabbits. For example,

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    feline activity aver- time between death and the removal of aged only 3.61 mmol ACh formed/hr./kg. these fluids; for example, human eyes, dry weight whereas that of rabbit aver- which were received up to 96 hours after aged 56.4 mmol ACh formed/hr./kg. dry death, and bovine eyes, which were re- weight. This species difference was similar ceived up to 5 hours after death, had to that reported here for whole retina: 0.7 higher enzyme activities than aqueous and nmole ACh formed/hr./mg. of protein in vitreous humors of freshly killed rabbits cats and 26.1 in rabbits (Tables I and II). and cats. De Roetth24 attributed a similar Ross and McDougal believed their data postmortem increase in cholinesterase ac- supported the view that the amacrine cells tivities of aqueous and vitreous humors to were primarily responsible for retinal cho- autolysis of tissues bordering the ocular Jinergic activity. fluids; a breakdown of iris, ciliary body, The finding of choline acetyltransferase and retina could release choline acetyl- activity in the pigment epithelium-choroid transferase into the aqueous and vitreous samples was somewhat unexpected. Al- humors. Alternatively, vitreous samples though neurons pass between sclera and could be contaminated with small pieces choroid, some of which may terminate on of adjacent retina due to vitreoretinal ad- the choroidal vasculature, there has been hesions. Both explanations assume that the little evidence suggesting a cholinergic sys- sources of aqueous and vitreous humor tem in the choroid. Kovacik,23 using a bio- choline acetyltransferase activities were the assay, has detected acetylcholine in the adjacent tissues. choroid. The authors wish to thank Patrick Freyne and Another unexpected finding was that al- The Eye Bank for Sight Restoration, Inc., New though rabbit sclera did not contain choline York, for generously supplying human ocular tis- acetyltransferase activity, human and sue. Art work was provided by the Medical Illus- bovine sclera did. Perhaps this reflected tration Service of the Bronx Veterans Administra- differences in the numbers of parasympa- tion Hospital. thetic neurons penetrating the sclera of REFERENCES cattle and human eyes, whose irides and 1. Feldberg, W., and Mann, T.: Properties and ciliary bodies were well developed, as op- distribution of the enzyme system which posed to those of rabbit eyes, whose irides synthesizes acetylcholine in nervous tissue, and ciliary bodies were relatively poorly J. Physiol. 104: 411, 1946. developed. Another possibility was that the 2. Nachmansohn, D., and Berman, M.: Studies on choline acetylase. III. On the preparation enzyme activity reflected contamination of the coenzyme and its effect on the en- from adjacent tissue autolysis in the same zyme, J. Biol. Chem. 165: 551, 1946. way as suggested for aqueous and vitreous 3. de Roetth, A., Jr.: Choline acetylase activity humors in the ensuing discussion. in ocular tissues, Arch. Ophthalmol. 43: 849, Those structures with parasympathetic 1950. 4. de Roetth, A., Jr.: Role of acetylcholine in innervation, the iris and ciliary body, nerve activity, J. Neurophysiol. 14: 55, 1951. tended to have a more uniform interspecies 5. Hebb, C. O.: Choline acetylase in mam- distribution of choline acetyltransferase malian and avian sensory systems, Q. J. Exp. than was found for cornea, retina, and pig- Physiol. 40: 176, 1955. ment epithelium-choroid. The two excep- 6. Ross, C. D., and McDougal, D. B., Jr.: The distribution of choline acetyltransferase ac- tions were the bovine iris and human tivity in vertebrate retina, J. Neurochem. 26: ciliary body, both of which had consider- 521, 1976. ably higher enzyme activities than those 7. van Alphen, G. W. H. M. V.: Acetylcholine found in the other species. synthesis in corneal epithelium, Arch. Oph- thalmol. 58: 449, 1957. The amount of choline acetyltransferase 8. Williams, J. D., and Cooper, J. R.: Acetyl- activity found in the aqueous and vitreous choline in bovine corneal epithelium, Bio- humors correlated well with the length of chem. Pharmacol. 14: 1286, 1965.

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