Download Angiotensin-converting enzyme activity in ocular fluids.

Volume 20
Number 2
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7.
8.
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10.
rate atrophy of the retina. Biochem Biophys Res
Coinmun 79:396, 1977.
Shih VE, Berson EL, Mandell R, and Schmidt SY:
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Blazer-Yost B and Jezyk PD: Free amino acids in the
plasma and urine of dogs from birth to senescence.
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Jezyck P, Haskins ME, Patterson DF, Mellman WJ,
and Greenstein M: Mucopolysaccharidosis in a cat
with aiylsulfata.se B deficiency: a model of Maroteaux-Lamy syndrome. Science 198:834, 1977.
Valle D, Goodman SI, Applegarth DA, Shih VE,
and Phang JM: Type II hyperprolinemia: A'-pyrroline-5-carboxylic acid dehydrogenase deficiency in
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Valle D, Walser M, Brusilow SW, and KaiserKupfer M: Gyrate atrophy of the choroid and retina:
amino acid metabolism and correction of hyperornithinemia with an arginine-deficient diet. J Clin
Invest 65:371, 1980.
Angiotensin-converting enzyme activity in
ocular fluids. JOAO BRASIL VITA, JANET A.
ANDERSON, CHARLES D. HULEM, AND IRVING
H. LEOPOLD.
Angiotensin 11 is a biologically active octapeptide that is
formed by the action of angiotensin-converting enzyme
(ACE) on the inactive precursor, angiotensin 1. ACE activity was found in tears and aqueous humor from both
rabbit and human eyes. The activity was higher in tears
than aqueous humor. Enzyme activity was determined
fluorimetrically from the rate of breakdown of the substrate, hippuryl-L-histidyl-L-leucine. The enzyme activity
was further characterized by determining the effects of
inhibitors. There was a significant difference in ACE
levels in human tears when eye color was considered.
People with either green or brown eyes had a higher ACE
level than did blue-eyed individuals. The presence of this
enzyme activity in ocular fluids suggests that angiotensin
11 may play a role in normal ocular physiology.
Angiotensin II is a powerful vasopressor agent
that has been shown to lower intraocular pressure
in anesthetized rabbits1 and cats.2 Angiotensin II
is formed when the terminal dipeptide, histidylleucine, is removed from the relatively inactive
precursor molecule, angiotensin I, by the action of
angiotensin-converting enzyme (ACE).3 ACE also
catalyzes the breakdown of the vasodepressor
bradykinin by the sequential release of Phe-Arg
and Ser-Pro from the COOH terminus of bradykinin.3 ACE activity has been found in ocular tis-
Reports
Table I. ACE activity in ocular fluids
(nmol/min/ml)
Fluid
Source
Human
Rabbit
Tear
Aqueous humor
7.31 ± 4.62 (55)
3. 00 ± 1 . 2 4 (6)*
1.34 ± 0.26 (7)
1.68 ± 1.84(21)
Values are mean ± S.D.; number of samples tested in parentheses.
*Six pools of tears from 30 rabbits were analyzed.
sues of rabbit, cow, pig, and man.4 The present
study was undertaken to determine whether there
are enzymes present in the ocular fluids of rabbits
and humans that catalyze the formation of angiotensin II and whether this enzymatic activity is
due to ACE.
Materials and methods
Chemicals. Hippuryl-L-histidyl-L-leucine (HipHis-Leu), L-histidyl-L-leucine (His-Leu), EDTA,
angiotensin I, bradykinin, and o-phthaldialdehyde
were obtained from Sigma Chemical Co.; purified
HPLC-grade methanol was from Fisher Scientific
Co. Inorganic chemicals were all reagent grade.
Captopril (D - 2 - methyl - 3-mercaptopropanoyl-Lproline) was a gift from Doctor Z. P. Horovitz of
the Squibb Institute for Medical Research.
Sample collection. Rabbit tears were collected
from female New Zealand albino rabbits by using a
10 /x\ glass micropipet. Six tear pools were formed,
each containing tears from four to eight rabbits. A
total of 30 rabbits was tested. Human tears were
collected from 55 healthy donors. Rabbit aqueous
humor was collected from 21 defrosted Pel-Freeze
rabbit eyes by using a 30 gauge needle. Human
aqueous humor was collected from seven cataract
surgery patients. After the corneal-scleral incision
had been made, the aqueous humor was collected
in a tuberculin syringe and frozen until assayed.
Blood samples were collected from finger punctures into microcapillary pipettes and centrifuged
to remove red blood cells. Serum was kept frozen
until assayed. Enzyme assays before and after
freezing of samples showed no change of enzyme
activity.
Assay of angiotensin converting enzyme. ACE
activity was determined from the amount of HisLeu formed by enzymatic breakdown of HipHis-Leu or angiotensin I. A modification5 of the
procedure originally described by Yang and Neff6
was followed. A 10 ^tl volume of tears, aqueous
humor, or serum was added to 240 ^tl of substrate
(final concentration 5 mM) in 0.1M potassium
phosphate, pH 8.3, plus 0.3M NaCl and incubated
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255
256
Invest. Ophthalmol. Vis. Sci.
February 1981
Reports
Table II. Human tear ACE activity
No. of
samples
Male
Female
Age (yr)
(x ± S.D.)
Brown
Green
Brown and
green
26
7
33
16
3
19
10
4
14
25 ± 4
28 ± 4
26 ± 4
Blue
Total
22
55
5
24
17
32
28 ± 8
27 ± 6
Eye color
ACE activity
(nmol/min/ml)
(x ± S.D.)
7.69 ± 3.88*
11.93 dt 7.24
8.59 2t 4.97t
5.38 :t 3.27*-t
7.31 ± 4.62
Significantly different from each other by Student t test: *p < 0.05; tp s 0.010.
Table III. His-Leu peptidase activity
Fluids
His-Leu
hydrolyzed in
15 min (/xM)
Final concentration
of His-Leu produced in ACE
assay* (fiM)
Rabbit AH
Rabbit tears
Human AH
Human tears
1.24
0.92
0
0.20
1.01
0.74
0.82
3.56
AH = aqueous humor.
* Measured after breakdown of Hip-His-Leu by the indicated
fluids in the ACE assay.
for 15 min at 37°. The reaction was stopped by the
addition of 1.45 ml of 0.28M NaOH. A fluorescent
adduct of o-phthaldialdehyde and the product was
made and measured in a FS 970 L.C. fluorometer
(Schoeffel). Enzymatic activity, in nanomoles of
His-Leu per minute per milliliter, of tear, aqueous
humor, or serum was calculated by method 2 of
Friedland and Silverstein.5
Inhibition studies. Captopril, EDTA, and bradykinin were tested for their effect on the enzymatic activity in human tears. Fifty microliters of
aqueous solution of captopril, EDTA, or bradykinin were substituted for 50 fjd of water in the
Friedland-Silverstein assay.5
Histidyl-leucine peptidase activity. Tear and
aqueous humor samples were tested for the presence of peptidase activity which might hydrolyze
the ACE product, His-Leu. Ten microliters of
tears or aqueous humor were incubated for 15 min
at 37° with 3.22 /u,M His-Leu in the assay buffer.
The amount of His-Leu remaining was determined fluorometrically.
Results. ACE activity was found in all tear and
aqueous humor samples (Table I). In both human
and rabbit tear samples, activity was higher than
in aqueous humor samples. Comparison of enzyme activity in eyes of different color shown in
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Table II indicated significant differences in enzyme activity with eye color. A pool of human
tears collected from 25 to 30 donors showed ACE
activity both with the artificial substrate (4.60
nmol/min/ml) and with angiotensin I as substrate
(1.95 nmol/min/ml). Peptidase activity capable of
degrading the His-Leu product of ACE hydrolysis
of Hip-His-Leu was found to be high in rabbit
ocular fluids, but in human ocular fluids was very
low or absent (Table III). Because of product
breakdown, the ACE activity in rabbit tissues
could be underestimated by nearly 50% in our
assay system. Human ACE activity would not be
substantially underestimated in this assay system.
ACE serum levels were tested in 19 of the 55
tear donors. The average concentration (mean ±
S.D.) was 32.79 ± 11.02 nmoles/min/ml. This result compares well with the previously reported
concentration of 32.2 ± 9.87. 5 The serum levels
were higher in brown-eyed individuals (35.51 ±
11.66 nmoles/min/ml) than in blue-eyed (26.38 ±
10.91), but the differences were not significant by
the Student t test.
Inhibition of human tear enzyme activity was
examined with selected concentrations of known
inhibitors of ACE, 8 X 10~5M EDTA and 3 x
10~5M bradykinin. 7 Captopril, a specific inhibitor
of ACE, 8 was found to inhibit tear enzyme activity
at concentrations from 5 x 10"4M to 5 X 10"7M
(Table IV) with degree of inhibition dependent on
inhibitor concentration. 8
Discussion. Enzymatic activity capable of converting angiotensin I to angiotensin II was shown
to be present in all ocular fluids tested. Known
inhibitors of ACE were shown to inhibit the activity at appropriate concentrations. 7 The sensitivity
of the enzyme activity to captopril, a specific ACE
inhibitor, 8 also indicates that the activity is due to
ACE.
Significant differences in human ACE activity
relative to eye color is an unusual finding. How-
Volume 20
Number 2
Reports
257
Table IV. Inhibition of human tear breakdown of Hip-His-Leu
Enzymatic activity (nmol/min/ml)
Agent
EDTA
Bradykinin
Captopril
Concentration
8
3
5
5
5
5
5
X 10" M
X 10"5 M
x 10' 4 M
x 10"5 M
x 10"6 M
x W7 M
Control
With inhibitor
% inhibition
14.77
12.19
4.23
4.23
4.23
4.23
1.08
0.25
93
98
100
100
99
90
0
0
0.03
0.43
ever, other differences in ocular physiology have
been shown to correlate with eye color. Millodot9
found that blue-eyed people had greater corneal
sensitivity than brown-eyed people, and Lee and
Robinson10 found that the rate of corneal metabolism of pilocarpine was at least 2 orders of magnitude greater in pigmented than in albino rabbits.
In this paper we have reported the presence of
an enzyme activity in ocular fluids capable of
catalyzing the formation of angiotensin II. These
observations and those of other investigators showing ACE activity in ocular tissues4 suggest that
there may be a physiological function for this octapeptide in the eye.
7. Lanzillo JJ and Fanburg BL: Angiotensin I converting enzyme from human plasma. Biochemistry
16:5491, 1977.
8. Ondetti MA, Rubin B, and Cushman DW: Design of
specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive
agents. Science 196:441, 1977.
9. Millodot M: Do blue-eyed people have more sensitive corneas than brown-eyed people? Nature 255:
151, 1975.
10. Lee VH and Robinson JR: Corneal metabolism of
From the Department of Ophthalmology, California
College of Medicine, University of California, Irvine.
J. B. V. is the recipient of a postdoctoral scholarship from
Allergan Pharmaceuticals. Submitted for publication
March 6, 1980. Reprint requests: Janet A. Anderson,
Ph.D., Department of Ophthalmology, California College of Medicine, University of California, Irvine, Calif.
92715.
Naturally occurring strabismus in monkeys
(Macaca nemestrina). LYNNE KIORPES AND
Keywords: angiotensin converting enzyme, angiotensin,
bradykinin, tears, aqueous humor
REFERENCES
1. Eakins KE: Effect of angiotensin on intraocular
pressure. Nature 202:813, 1964.
2. Macri FJ: The action of angiotensin on intraocular
pressure. Arch Ophthalmol 73:528, 1965.
3. Soffer RL: Angiotensin-converting enzyme and the
regulation of vasoactive peptides. Annu Rev Biochem 45:73, 1976.
4. Igic R and Kojovic V: Angiotensin I converting enzyme (Kininase II) in ocular tissues. Exp Eye Res
30:299, 1980.
5. Friedland J and Silverstein E: A sensitive fluorimetric assay for serum angiotensin-converting enzyme. Am J Clin Pathol 66:416, 1976.
6. Yang H-YT and Neff NH: Distribution and properties of angiotensin converting enzyme of rat brain. J
Neurochem 19:2443, 1972.
pilocarpine in pigmented rabbits. INVEST OPHTHAL-
MOL Vis SCI 19:210, 1980.
RONALD G. BOOTHE.
Seven naturally strabismic monkeys (Macaca nemestrina)
were identified. Five of these monkeys were examined by
ophthalmologists. No ophthalmoscopically obvious cause
for the squint was found in any case. Of thosefiveanimals, two were tested behaviorally on visual responsiveness and visual acuity. The acuity of both eyes of both
monkeys was somewhat poorer than normal. In addition,
an amblyopia of 0.8 octaves was found for one monkey
and 0.6 octaves for the other. The existence of naturally
strabismic monkeys supports the utility of the macaque
as an animal model for studying strabismus and
amblyopia.
Clinical data have established that there is a
close relationship between strabismus and amblyopia in humans.1' 2 Strabismus sometimes develops secondarily to amblyopia as in cases of organic
visual impairment. On the other hand, amblyopia
often results from the presence of strabismus.
There are questions about the development of
strabismus and amblyopia that have not been answered by clinical studies. Some of these questions, e.g., those concerning the physiological or
neuroanatomical bases for strabismus and ambly-
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