Download Choline acetyltransferase in ocular tissues of rabbits, cats, cattle

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 epithelium of rabbit,
bovine, and human eyes, but little or none could be detected in that of cats. Feline retina
and pigment epithelium-choroid also contained far less choline acetyltransferase activity
than the same tissues in the other three species.
Key words: choline acetyltransferase, eye, ocular, cornea, iris, ciliary body,
aqueous humor, retina, choroid.
C
optic nerve. In recent years, the retinal and
corneal epithelial choline acetyltransferases
have attracted the most interest. Ross and
McDougal" have found that the inner plexiform layer contains high levels of choline
acetyltransferase activity. Van Alphen7 and
Williams and Coopers reported high concentrations of the enzyme in rabbit and
bovine corneal epithelium. The corneal epithelium contains none of those structures
normally associated with cholinergic activity, i.e., synapses, myoneural junctions,
or parasympathetic motor axons. The presence of choline acetyltransferase activity
in corneal epithelium, which consists primarily of epithelial cells and sensory nerve
endings, has elicited several theories attempting to link cholinergic activity with
corneal touch sensitivity0"11 and epithelial
ion transport.s> 12
Although the choline acetyltransferase of
specific tissues has interested different
workers, no general survey of ocular structures has been reported. This paper de-
holine acetyltransferase, the enzyme
responsible for acetylcholine synthesis, has
been assayed in a number of ocular tissues.1"s This enzyme is a more specific
marker of cholinergic activity than are the
ubiquitous cholinesterases. The first estimates of ocular choline acetyltransferase
activity appeared in 19461'2; low enzyme
levels were reported for canine and rabbit
From the Departments of Pharmacology and
Ophthalmology, Mount Sinai School of Medicine, City University of New York, and the
Bronx Veterans Hospital, New York, N. Y.
Supported in part by a Fight-for-Sight Grant-inAid and National Eye Institute Grant I RO1
EY01243.
"Chief of Ophthalmology, Bronx Veterans Administration Hospital. Dr. Mindel is the recipient of a Research Career Development
Award from the National Eye Institute.
Submitted for publication Feb. 12, 1976.
Reprint requests: J. S. Mindel, The Mount Sinai
School of Medicine, Fifth Avenue & 100th St.,
New York, N. Y. 10029.
808
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Volume 15
Number JO
scribes the distribution of choline acetyltransferase activity in the eyes of four
species: rabbit, cat, cattle, and man.
Materials and methods
Eyes of adult female Dutch Belt rabbits and
female mongrel cats, killed with parenteral pentobarbital or secobarbital, were studied. Bovine
eyes were supplied on ice by an abattoir within
5 hours of death. Human eyes were obtained
hours to days after death from the Eye Bank
for Sight Restoration, New York, N. Y.
Corneas were removed by trephine as full thickness buttons. In rabbits, the ciliary body is poorly
developed and both it and the iris were excised
and assayed together. In all four species, the
pigment epithelium and choroid were dissected
out and assayed together.
Choline acetyltransferase was assayed by a
modification of the methods of Schrier and
Sinister™ and McCaman and Hunt.11 Tissues
were homogenized on ice in pH 7.4 buffer solution of sufficient volume to give a concentration
of less than 10 per cent weight/volume. The
concentrations of buffer ingredients during incubation were 0.5 per cent Triton-X, 10 mM
ethylenediamine tetraacetic acid (EDTA), 300
mM sodium chloride, and 150 mM potassium
hydrogen phosphate. The assay was performed
in duplicate 6 by 50 mm. tubes containing 200
fil of homogenate and 20 <ul of a solution giving
a final concentration of 2 mM dithiothreitol, 0.1
mM physostigmine, 5 mM choline chloride, and
0.2 mM acetyl-coenzyme A. The last contained
sufficient acetyl-1-C1 '-coenzyme A to give approximately 1 c.p.m. per picomole. A third tube
served as blank and contained 0.1 mM naphthylvinylpyridinium hydroxyethyl bromidelr> but no
choline. After a 30 min. incubation at 37.5° C,
the reaction was terminated with 1 drop of
CuCl;, 2.5 per cent, per tube. The contents of
each tube were quantitatively transferred, using
1 ml. of H-O, to a 3 by 0.6 cm. column of BioRad AC1-X8, 200 to 400 mesh chloride form ion
exchange resin in water. Effluents were collected
directly in scintillation vials and counted in 10
ml. of Bray's solution. Protein content of tissues
was determined by the method of Lowry and
associates.1 <: Naphthylvinylpyridinium hydroxyethyl
bromide, dithiotreitol, choline chloride, and
acetyl-coenzyme A solutions were made up weekly
and stored frozen. Physostigmine solutions were
made up daily.
Results
The choline acetyltransferase activities
of all four species were measured with a
standardized assay. This assay was based
Ocular choline acetyltransferase 809
on kinetic studies of rabbit tissues where
the concentration of choline was varied
between 25 /xM and 10 mM and the concentration of acetyl-coenzyme A was
varied between 10 and 400 /xM. Using a
double reciprocal plot by the method of
Lineweaver-Burk, the Michaelis-Menton
constants (Km's) were calculated from the
extrapolated intercepts. The Km for choline
was 500 /xM for all four ocular tissues,
corneal epithelium, iris-ciliary body, retina,
and pigment epithelium-choroid. The Km
for acetyl-coenzyme A was 20 ju,M for
corneal epithelium, iris-ciliary body, and
pigment epithelium-choroid; the Km for
retinal acetyl-coenzyme A was 50 /xM. The
ocular tissues from eight eyes were pooled
and the specific activities determined (expressed as nanomoles acetylcholine formed
per hour per milligram [nmole ACh
formed/hr./mg.] of protein)—comeal epithelial protein, 18.1; iris-ciliary body protein, 1.6; retinal protein, 58.4; and pigment
epithelium-choroid protein, 6.7. The rates
of reaction were found to be linear during
the incubation period used in the assay.
Enzyme activities of individual tissues
were then assayed and calculated on a per
milligram of protein basis and/or a per
whole tissue basis.
Variation in ocular choline acetyltransferase activity of rabbit, cat, cattle, and
human ocular tissues (per milligram of
protein). The corneal epithelium of cats,
unlike the other three mammalian species,
had little or no detectable choline acetyltransferase activity (Tables I to IV).
Rabbit, bovine, and human corneal epithelia had high levels of enzyme activity.
The corneal stroma and endothelium of all
four species had little or no choline acetyltransferase activity.
The enzyme activity of human, rabbit,
and cat iris was, on the average, approximately 2 nmole ACh formed/hr./mg. of
protein. However, the average bovine iris
contained four to five times this activity.
The ciliary bodies of cattle, rabbits, and
cats formed 2 to 3 nmole ACh/hr./mg. of
protein, whereas the average human ciliary
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Investigative Ophthalmology
October 1976
810 Minclel and Mittag
Table I. Distribution of choline
acetyltransferase activity (per milligram
of protein) in the ocular tissues of Dutch
Tissue
Average*
Conjunctiva
0.3
Cornea:
Epithelium
20.6
Stroma-endo0.0f
thelium
Iris-ciliary body
2.6
Lens
0.0$
Vitreous
0.5
Retina
26.1
Pigment epithelium3.4
choroid
Optic nerve
0.0
o.o§
Sclera
°Nanomoles ACh formed/hr./mg.
Range*
_
Std.
Dev.
Table III. Distribution of choline
acetyltransf erase activity (per milligram
of protein) in the ocular tissues of cattle
Tissue
n
0.0
3
13.6
0.0
21
8
1.7
0.0
0.0- 2.1 0.7
5.8-94.4 20.5
0.2-12.4
3.1
5i
13
g
46
47
0.0- 0.1 0.0
0.0
—
of protein.
g
8
3.9-41.2
0.2- 4.9
Average*
Range*
Std.
Dev.
Cornea:
46.5 11.6-89.2 27.5
Epithelium
0.4
Stroma-endo0.0- 1.4 0.6
thelium
IrJ
10.8
1.6-22.2 6.5
s
Ciliary body
1.9
0.3- 6.9 2.1
0.1
—
Lens
o.ot
Vitreous
0.9
0.5- 1.3 0.6
9.8
Retina
13.0
4.0-31.4
4.5
Pigment epithelium4.6
0.0-10.9
choroid
—
Optic nerve
0.3
—
Sclera
1.8
—
—
°Nanomoles ACh formed/hr./mg. of protein
f-0.15.
n
8
8
10
10
2
2
10
10
1
1
t< 0.036.
}< 0.003.
5< 0.010.
Table IV. Distribution of choline
acetyltransf erase activity (per milligram
of protein) in the ocular tissues of man
Table II. Distribution of choline
acetyltransf erase activity (per milligram
of protein) in ocular tissues of cats
Tissue
Average*
Cornea:
Epithelium
0.1
Stroma-endo0.0f
thelium
Full thickness
0.0
Iris
2.0
Ciliary body
2.9
Lens
0.0$
Vitreous
0.0§
Retina
0.7
Pigment epithelium0.5
choroid
°Nanomoles ACh formed/hr./mg.
t < 0.010.
| < 0.029.
5< 0.013.
Tissue
Range*
Std.
Dev.
n
0.0-0.6
—
0.2
0.0
8
4
0.0-0.3
0.3-5.5
0.5-6.6
—
—
0.4-1.2
0.2-0.7
0.1
1.5
2.2
0.0
0.0
0.3
0.2
12
16
16
4
4
12
6
of protein
body had five to eight times this level of
activity.
The vitreous humor contained small
amounts of choline acetyltransferase activity. Aqueous humor choline acetyltransferase was assayed in 14 rabbit eyes. In
13 of the 14, no enzyme activity was detectable. In one eye, an activity of 0.1
nmole ACh formed/hr./ml. was found.
Two pooled samples of bovine aqueous
humor, one from 40 eyes and the other
Average*
Range*
Std.
Dev.
Cornea:
11.2
Epithelium
—
—
Strnma-enrio0.4
thelium
1.8
0.1- 5.3
2.5
Iris
0.1-38.0 14.3
16.8
Ciliary body
—
0.0
0.0$
Lens
—
—
2.2
Vitreous
0.9-23.1 6.7
6.1
Retina
4.5
0.6-11.0
Pigment epithelium5.1
choroid
—
—
0.7
Optic nerve
1.2
—
Sclera
—
"Nanomoles ACh formed/hr./m{%. of protein.
tPooled sample of 12 eyes.
t < 0.027.
t
|
10
11
6
t
10
10
t
t
from eight eyes, were assayed and their
average enzyme activity was 1.4 nmole
ACh formed/hr./ml. Aqueous humor choline acetyltransferase in seven human eyes
ranged in value from 5.4 to 10.1 nmole
ACh formed/hr./ml., with an average
value of 7.8 ± 2.8.
The retinal choline acetyltransferase activities of rabbits and cattle were, on the
average, similar whereas human and, especially, feline enzyme activities were far
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Ocular choline acetyltransferase 811
Volume 15
Number 10
Table V. Distribution of choline acetyltransferase activity (per whole tissue)
in ocular tissues*
Tissue
Cornea
Iris
Ciliary body
Retina
Pigment epithelium—choroid
Rabbit
< 1-235 (100)t
10 A(\ (AA\
60-160 (47)
< 1 - 15 (42)
Cat
Bovine
0 (8)
27-60 (4)
26-48 (4)
9-26 (4)
26-50 (4)
130-1,600 (10)
28- 539 (5)
9- 239 (5)
157- 944 (5)
3- 533 (5)
Human
0- 10 (6)
1- 35 (5)
0-221 (5)
22-105 (4)
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,
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 activity that indicated considerable individual variation.
Variation in ocular choline acetyltransferase activity of rabbit, cat, cattle, and
human ocular tissues (per whole tissue).
Table V shows the range of ocular choline
acetyltransferase activities in the four species studied, calculated on a per whole
tissue basis. Sufficient rabbit eyes were assayed to evaluate the distribution of enzyme activities for this species. Values for
rabbit corneal choline acetyltransferase
(Fig. 1) did not cluster around a central
value as they did for the other three tissues:
62 per cent of iris-ciliary body samples fell
in the range of 20 to 30 nmole ACh
formed/hr. per whole tissue, 68 per cent of
retinal samples fell in the range of 80 to
120 nmole ACh formed/hr. per whole tissue, and 60 per cent of pigment epithelium-choroid samples fell in the range of
5 to 10 nmoles ACh formed/hr. per whole
tissue.
Discussion
In 1946, two papers appeared giving the
first estimates of choline acetyltransferase
activity in ocular tissues. Feldberg and
Mann1 reported 0 to 15 /xg ACh formed/
hr./gram of acetone-dried powdered canine optic nerve and 400 /xg ACh formed/
hr./gram of acetone-dried powdered ca-
20 n
50 65 80 95 110 125 140155
7 14 21 28
nanomoles ACh FORMED/hr/CORNEA
190 235
Fig. 1. Distribution of choline acetyltransferase
activity in the corneal epithelia of 100 Dutch
Belt rabbits (per cent of total number of eyes
assayed).
nine retina. Nachmansohn and Bermanreported the same year that rabbit optic
nerve produced 13 to 21 fxg of ACh/hr./
gram of whole tissue. De Roetth4 reported
a much higher enzyme activity for rabbit
optic nerve, 100 to 300 /xg of ACh formed/
hr./gram of acetone-dried powder. Hebb5
studied ocular choline acetyltransferase activity by an improved enzyme assay that
was more likely to ensure that the acetylcoenzyme A substrate remained at saturating levels. A marked species variation in
retinal choline acetyltransferase of dog,
rabbit, chicken, and pigeon was found, and
far lower optic nerve enzyme activities
than de Roetth had reported earlier were
noted. Hebb found a material present in
ocular tissues that interfered with bioassay
determinations of acetylcholine.
The conditions of enzyme assay in the
present study were chosen so as to be well
above the Km's determined for rabbit ocular choline acetyltransferase. The corneal
epithelial, iris-ciliary body, and pigment
epithelium-choroid Km's were 500 tiM for
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Investigative Ophthalmology
October 1976
812 Minclel and Mittag
choline and 20 /.LM for acetyl-coenzyme A.
The Km's for retina were 500 fiM for choline and 50 /iM for acetyl-coenzyme A.
These were of the same order of magnitude as those reported for calf and human
brain. White and Cavallito17 found the
Km's for calf brain choline acetyltransferase to be 800 /JM for choline and 16 pM
for acetyl-coenzyme A. White and Wu ls
found the Km's for human choline acetyltransferase to be 510 /xM for choline and
11 [xM for acetyl-coenzyme A.
Choline acetyltransferase activities were
calculated in two different manners. For
the purpose of comparing different species
whose ocular structures vary in size, enzyme activities were calculated on a per
milligram of protein basis. However, the
enzyme activities of the corresponding tissues of the two eyes of an individual
animal agreed better if the data were calculated on a per whole tissue basis. The
reasons this occurred will be dealt with in
a separate publication. This is an important consideration when comparisons of
enzyme activity between the two eyes are
made following experimental manipulations
to one eye with the other serving as control.
The corneal epithelium showed the most
marked species variation in choline acetyltransferase activity (Tables I to IV and
Fig. 1). In contrast to the very high choline
acetyltransferase activities of some rabbit
(20.6 nmole ACh formed/hr./mg. of protein), bovine (46.5 nmole ACh formed/
hr./mg. of protein), and human (11.2
nmole ACh formed/hr./mg. of protein)
corneal epithelia, that of the cat contained
very little enzyme activity (0.1 nmole ACh
formed/hr./mg. of protein). The value for
bovine corneal epithelial choline acetyltransferase activity, 46.5 nmole ACh
formed/hr./mg. of protein, agrees well
with that of Williams and Cooper,8 33
nmole ACh formed/hr./mg. of protein.
Van Alphen7 found a lower level of rabbit
corneal epithelial enzyme activity than is
reported here but he used a high concentration of cysteine in his extraction procedure and this was subsequently found to
inhibit enzyme activity.19 Howard and
Wilson'-0 and Howard, Wilson, and Dunn21
found an upper value of rabbit comeal epithelial choline acetyltransferase activity,
123.1 nmole ACh formed/hr./mg. of protein, approximately three times that reported here. Since there is normally great
variation in the corneal enzyme activity
of different rabbits (Fig. 1), these authors
may have used animals with higher choline
acetyltransferase activities. A second possibility is that since their assay did not use
a specific choline acetyltransferase inhibitor, other acetylated cationic molecules,
such as acetylcarnitine, may have contributed to falsely elevated values. The variation in rabbit corneal choline acetyltransferase activity (Fig. 1) is much greater
than that described for other rabbit ocular
tissues. The corneal epithelium is the only
ocular tissue directly in contact with the
external environment and several authors
have suggested1-' -1 that the environment
influences this tissue's choline acetyltransferase activity.
The rather striking differences between
feline corneal enzyme activity and those of
rabbit, cattle, and human eyes were also
found for retina and pigment epitheliumchoroid. The average retinal choline acetyltransferase activity of the cat was only 0.7
nmole ACh formed/hr./mg. of protein
compared to values of 26.1, 13.0, and 6.1
nmole ACh formed/hr./mg. of protein
in rabbit, bovine, and human eyes, respectively. HebbV reliance on a regional difference in myelinization to explain why
pigeon central retina had lower choline
acetyltransferase activity than peripheral
retina would not seem to explain this interspecies variation. The rabbit retina is more
highly myelinated2- than those of the other
three species yet its enzyme activity is
higher. Ross and McDougal0 have presented evidence that the inner plexiform,
inner nuclear, and ganglion cell layers have
significant amounts of choline acetyltransferase activity. They found that these
retinal layers had less activity in cats than
in monkeys, mice, or rabbits. For example,
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Volume 15
Number 10
feline inner plexiform layer activity averaged only 3.61 mmol ACh formed/hr./kg.
dry weight whereas that of rabbit averaged 56.4 mmol ACh formed/hr./kg. dry
weight. This species difference was similar
to that reported here for whole retina: 0.7
nmole ACh formed/hr./mg. of protein in
cats and 26.1 in rabbits (Tables I and II).
Ross and McDougal believed their data
supported the view that the amacrine cells
were primarily responsible for retinal choJinergic activity.
The finding of choline acetyltransferase
activity in the pigment epithelium-choroid
samples was somewhat unexpected. Although neurons pass between sclera and
choroid, some of which may terminate on
the choroidal vasculature, there has been
little evidence suggesting a cholinergic system in the choroid. Kovacik,23 using a bioassay, has detected acetylcholine in the
choroid.
Another unexpected finding was that although rabbit sclera did not contain choline
acetyltransferase activity, human and
bovine sclera did. Perhaps this reflected
differences in the numbers of parasympathetic neurons penetrating the sclera of
cattle and human eyes, whose irides and
ciliary bodies were well developed, as opposed to those of rabbit eyes, whose irides
and ciliary bodies were relatively poorly
developed. Another possibility was that the
enzyme activity reflected contamination
from adjacent tissue autolysis in the same
way as suggested for aqueous and vitreous
humors in the ensuing discussion.
Those structures with parasympathetic
innervation, the iris and ciliary body,
tended to have a more uniform interspecies
distribution of choline acetyltransferase
than was found for cornea, retina, and pigment epithelium-choroid. The two exceptions were the bovine iris and human
ciliary body, both of which had considerably higher enzyme activities than those
found in the other species.
The amount of choline acetyltransferase
activity found in the aqueous and vitreous
humors correlated well with the length of
Ocular choline acetyltransferase 813
time between death and the removal of
these fluids; for example, human eyes,
which were received up to 96 hours after
death, and bovine eyes, which were received up to 5 hours after death, had
higher enzyme activities than aqueous and
vitreous humors of freshly killed rabbits
and cats. De Roetth24 attributed a similar
postmortem increase in cholinesterase activities of aqueous and vitreous humors to
autolysis of tissues bordering the ocular
fluids; a breakdown of iris, ciliary body,
and retina could release choline acetyltransferase into the aqueous and vitreous
humors. Alternatively, vitreous samples
could be contaminated with small pieces
of adjacent retina due to vitreoretinal adhesions. Both explanations assume that the
sources of aqueous and vitreous humor
choline acetyltransferase activities were the
adjacent tissues.
The authors wish to thank Patrick Freyne and
The Eye Bank for Sight Restoration, Inc., New
York, for generously supplying human ocular tissue. Art work was provided by the Medical Illustration Service of the Bronx Veterans Administration Hospital.
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Investigative Ophthalmology
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