Download Identification of isoenzyme C as the principal carbonic

Volume 18
Number 8
Reports
867
of a primary infection, although examination of
rats in other experiments at this same time of infection has shown an increase in intestinal1' 6 and
lung7 mast cells. These observations suggest that
the stimulus to mast cell proliferation present during infection does not involve the eyes; possibly
the stimulating factor is only elaborated by tissues
that are in direct contact with larval or worm
products.
by measuring the degree of inhibition produced by
acetazolamide at two standard concentrations. Also, the
effect of incubating with iodoacetate was determined in
two pairs of these eyes. Only isoenzyme C was detected.
Accordingly, it seems that differences in reduction of intraocular pressure that are common among patients
treated for glaucoma with systemic carbonic anhydrase
inhibitors, despite uniform serum concentrations, are not
attributable to interindioidual variation of carbonic
anhydrase isoenzymes in the ciliary processes.
From the Departments of Ophthalmology and
Medicine, Harvard Medical School, the Department of
Cornea Research, Eye Research Institute of Retina
Foundation, and the Clinical Immunology and Allergy
Units, Medical Services, Massachusetts General Hospital, Boston, Mass. This work was supported by grants
EY-02099 and AM-20580 from the National Institutes of
Health, a grant from Bausch & Lomb, and grants from
the National and Massachusetts Chapters of the Arthritis
Foundation. Submitted for publication Dec. 4, 1978.
Reprint requests: Mathea R. Allansmith, M.D., 20
Staniford St., Boston, Mass. 02114.
Recent chronic dose-response studies of carbonic anhydrase inhibitors (CAIs) in patients with
glaucoma have indicated that among glaucoma patients there is marked variation from person to
person in the influence of CAIs on intraocular
pressure, despite similar dosages and serum levels
of the medications.1"2 Certain patients are clearly
"hyporesponders," despite high dosage therapy.1
The reason is not known.
There are in human red blood cells two carbonic
anhydrase (CA) isoenzymes, B and C. Acetazolamide is a much more effective inhibitor of isoenzyme C than of B. If there were differences in
isoenzymes in tissues that form aqueous humor
in different individuals, this could help explain the
variable interindividual effect of CAI therapy. Accordingly, we thought it important to examine the
type of CA isoenzymes present in a series of
human ciliary bodies.
Materials and methods. Human eyes enucleated post-mortem were stored at 4° C in a moist
chamber and dissected within 24 hr. The ciliary
processes, which are assumed to be the principal
portion secreting aqueous humor, were identified
and excised from 10 pairs of human eyes. The tissue from each pair was centrifuged with 5 ml of
distilled water for 5 min at 900 x g in a precooled
MSE bench-top centrifuge. The resulting pellet
was homogenized in 10 times the weight of distilled water and centrifuged in a Sorvall RC-2B
refrigerated centrifuge at 27K x g for 10 min. The
supernatant was retained and kept at 4° C, to be
assayed for CA activity.
CA activity of each sample was determined by
the "micro-method" of Maren.3 The amount of tissue required to provide 0.5 enzyme unit of activity
was determined.
The isoenzyme distribution in each sample was
determined by measuring the degree to which enzyme activity was inhibited by 25 and 50 ng of
acetazolamide, which differentially inhibits isoenzymes B and C.4 Standard curves were established
each day with pure isoenzymes obtained from
Sigma Chemical Co., St. Louis, Mo. Human pairs
1 to 4 were studied on day 1, and human pairs 5 to
10 on day 2.
Key words: histamine, immunology, anaphylaxis, mast
cells, allergy, inflammatory mediators
REFERENCES
1. Miller, H.R.: Immune reactions in mucous membranes. II. The differentiation of intestinal mast cells
during helminth expulsion in the rat, Lab. Invest.
24:339, 1971.
2. Bloch, K.J., and Wilson, R.J.: Homocytotropic antibody response in the rat infected with the nematode
Nippostrongylus brasiliensis. III. Characteristics of
the antibody, J. Immunol. 100:629, 1968.
3. Wilson, R.J., and Bloch, K.J.: Homocytotropic antibody response in rat infected with the nematode,
Nippostrongylus brasiliensis. II. Characteristics of
the immune response. J. Immunol. 100:622, 1968.
4. Mallory, F. B.: Unna's method for mast cells. In
Pathological Technique. New York, 1961, Hafner
Publ. Co., p. 175.
5. Levene, R.Z.: Mast cells and amines in normal ocular
tissues, INVEST. OPHTHALMOL. 1:531, 1962.
6. Keller, R., Cottier, H., and Hess M.W.: Mast cell
responses in mesenteric lymph nodes to infection of
rats with the nematode, Nippostrongylus brasiliensis,
Immunology 27:1039, 1974.
7. Wells, P. D.: Nippostrongylus brasiliensis: mast cell
populations in rats, Exp. Parasitol. 30:30, 1971.
Identification of isoenzyme C as the principal carbonic anhydrase in human ciliary
processes. PAULA C. DOBBS, DAVID L. E P STEIN, AND P. JOHN ANDERSON.
Carbonic anhydrase was extracted from the excised processes of the ciliary bodies of 10 pairs of enucleated
human eyes, and the isoenzyme composition was assayed
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Invest. Ophthalmol. Visual Sci.
August 1979
Reports
Table I. Comparison of the reaction times* of CA from 10 pairs of human ciliary processes and pure
isoenzymes B and C in the presence of inhibitors
Dayl
B
C
1
2
3
4
Uninhibited
445 ± 10
447 ± 6
447 ± 6
447 ± 12
443 ± 12
450 ± 10
Acetazolamide:
0.141 /xM
0.282 /*M
493 ± 12
530 ± 10
533 ±12
530 ± 10
520 ± 20
523 ± 6
Sample
Iodoacetate,
10 miM
* Units are minutes x 103 ± S.D. All values are means of 3 determinations.
Uncatalyzed times were 652 ± 12 and 637 ± 6 for day 1 and day 2, respectively.
0.55-
053-
D
D
V*
051-
•
049-
\
047-
A
•
045040
042
0.44
048
UNINHIBITED TIME IN MINUTES
Fig. 1. Plot of inhibited vs. uninhibited times for
CA assay in the presence of acetazolamide. Conditions are as in Table I. For day 1 and day 2,
respectively: o, • = carbonic anhydrase from
pairs of human ciliary processes; A, A = isoenzyme, B; o, • = isoenzyme C.
Also, the isoenzyme composition of two of the
pairs of eyes was tested by preincubating for 20 hr
in 10~2 M iodoacetate, which inhibits isoenzyme B
but not isoenzyme C,4> 5 and then assaying for
enzyme activity.
Results. Results of the CA activity assay for pure
isoenzymes B and C and for the ciliary processes of
10 pairs of human eyes are given in Table I. The
acetazolamide data were analyzed by a one-way
analysis of variance. 6 The results demonstrated
that the human samples, pure B, and pure C do
not form a homogeneous population (p < 0.01,
day 1; p < 0.05, day 2) as might be expected if the
human enzymes were mixtures of B and C in various proportions. Selecting isoenzyme B as the
likely "odd man out" and testing against the remainder demonstrated a highly significant difference (p < 0.005, both days). No significant differences were found within the remaining groups.
These relations are clearly seen in Fig. 1 where
the inhibited times (0.141 JUM acetazolamide)
are plotted against the uninhibited times of the
reaction.
The inhibitor constant Ki was obtained by plotting the reciprocal of the enzyme rate vs. the inhibitor concentration. Kms and CO 2 concentration
were taken from Maren et al.4 The mean value of
the 10 ciliary process samples was 0.102 ± 0.013
)LtM; for isoenzyme C (mean of day 1 and day 2),
0.093 ± 0.002 fxM; and similarly for isoenzyme B,
0.169 ± 0.013 fxM. Errors are expressed as standard deviations.
In two samples, there was enough material left
to test the differential inhibition of isoenzyme B
and C with iodoacetate. The result (Table I) accords closely with the acetazolamide findings.
Discussion. Since, as determined by this method, the CA of human ciliary processes from the
eyes of 10 people has been indistinguishable from
pure isoenzyme C, it seems that variation in
amount of isoenzyme B and C does not account for
the observed variation in reduction of intraocular
pressure in different patients in response to CAI
therapy. Variability in response from person to
person has been common, with essentially no response in about one out of six glaucomatous patients. We would expect that a difference in isoen-
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Volume 18
Number 8
Reports
869
Day 2
B
C
5
6
7
8
9
10
427 ± 6
423 ± 6
420 ± 17
420 ± 10
423 ± 6
423 ± 12
423 ± 6
413 ± 12
467 ± 15
483 ± 6
507 ± 6
523 ± 6
497 ± 6
530 ± 10
510 ± 17
513 ± 15
497 ± 6
523 ± 6
507 ± 12
520 ± 10
500 ± 17
527 ± 6
497 ± 15
517 ± 12
610 ± 10
420 ± 10
zymes might have shown up at least once among
our 10 human control donors if this were a factor.
There are other possible factors to consider,
which include the following. We do not know
whether there are variations from person to person in the total amount of CA present in ciliary
processes. There is still the possibility of differences in isoenzymes in glaucoma eyes not present
in normal eyes. There may be variations among
different patients in the contributions of secretion
and ultrafiltration in production of aqueous humor. There may be barriers between the blood
supply and the site of CA in the tissue. There may
be nonspecific binding of acetazolamide in some
ciliary processes, interfering with the action of the
drug on the enzyme. There is a possibility that
isoenzyme B is present in quantities too small for
us to detect, yet may have an influence on aqueous
humor secretion. In addition, recent studies have
indicated that CA may also be present in the pars
plana of the ciliary body. 7 If CA in the pars plana
has a role in production of aqueous humor, it will
be important to measure the amount of enzyme
and its isoenzyme composition in this tissue.
We would, of course, like to assay CA isoenzyme in glaucomatous eyes, especially in eyes
from patients with clinically determined different
degrees of responsiveness to CA inhibitors, but
practically it is extremely difficult to get the appropriate tissues.
In this study, not enough material was available
from single pairs of eyes to carry out a full doseresponse study. This precluded our calculating an
I50 for acetazolamide as published by Maren and
coworkers.4' 8 However, by making a mechanistic
assumption, we were able to calculate a Ki for
acetazolamide which should be roughly comparable to an I50. The model chosen was the classic
competitive inhibition,9 which yields a straight
427 ± 6
413 ± 6
line when the reciprocal rate is plotted vs. the
enzyme concentration. It was evident from our
data that the model fitted rather poorly. Given
these limitations, the agreement of our Ki value
for isoenzyme B and the I50 of Maren et al. 8 is
excellent (17 and 25 x 1(T8M, respectively). The
agreement for isoenzyme C is less good (9.3 and
1.1 x 10~8M, respectively). Reassuringly, the plot
of reciprocal rate vs. inhibitor concentration for
pure C closely matches that for the human ciliary
body enzyme (slope/intercept = 15 and 16 X
10~8M, respectively). Therefore the Ki for the
human enzyme was computed with the Km for
pure C.
We are grateful for the assistance of Jennifer J. Anderson, who performed the analysis of variance.
From the Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical
School, Boston, Mass. Supported in part by the
Abraham Silver Memorial Fund of Fight for Sight, Inc.,
New York, N. Y., and National Eye Institute research
grants RO1 EY01894, RO1 EY00002, and TO1 EY00089.
Submitted for publication March 2, 1979. Reprint requests: David L. Epstein, M.D., Howe Laboratory, 243
Charles St., Boston, Mass. 02114.
Key words: carbonic anhydrase, ciliary processes, acetazolamide, isoenzymes, aqueous formation
REFERENCES
1. Dahlen, K., Epstein, D.L., Grant, W.M., Hutchinson, B.T., Prien, E.L., and Krall, J.M.: A repeated
dose-response study of methazolamide in glaucoma,
Arch. Ophthalmol. 96:2214, 1978.
2. Berson, F.G., Epstein, D.L., Grant, W.M., Hutchinson, B.T., and Dobbs, P.C.: A dose-response study
of acetazolamide sequels and tablets in the treatment
of
glaucoma,
INVEST.
OPHTHALMOL.
VISUAL
SCI.
17(ARVO Suppl.):121, 1978.
3. Maren, T.: A simplified micromethod for the deter-
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870
4.
5.
6.
7.
Invest. Ophthalmol. Visual Sci.
August 1979
Reports
mination of carbonic anhydrase and its inhibitors, J.
Pharmacol. Exp. Ther. 130:26, 1960.
Maren, T., Rayburn, C , and Liddell, N.: Inhibition
by anions of human red cell carbonic anhydrase B:
physiological and biochemical implications, Science
191:469, 1976.
Coleman, J.: Metal ions and enzymatic catalysis. In
Kaiser, E., and Kezdy, F., editors: Progress in
Bioorganic Chemistry, New York, 1971, John E.
Wiley and Sons, Inc., p. 282.
Goldstein, A.: Biostatistics, An Introductory Text,
New York, 1964, The MacMillan Co., pp. 63-87.
Lutjen-Drecoll, E., Lonnerholm, G., and Ober, M.:
Carbonic anhydrase activity in the monkey anterior
ocular segment. INVEST. OPHTHALMOL. VISUAL SCI.
17(ARVO Suppl.):162, 1978.
8. Maren, T.H., Haywood, J.R., Chapman, S.K., and
Zimmerman, T.J.: The pharmacology of methazolamide in relation to the treatment of glaucoma. INVEST. OPHTHALMOL. VIS. SCI. 16:730, 1977.
9. Walter, C : Steady-State Applications in Enzyme Kinetics. New York, 1965, The Ronald Press Co., pp.
104 et. seq.
Aqueous humor dynamics following total
iridectomy in the cynomolgus monkey.
PAUL L. KAUFMAN.
Twenty-two cynomolgus monkeys underwent unilateral
total iridectomy in vivo. Several weeks to several months
postoperatively, intraocular pressure (IOP) was determined bilaterally by manometry under pentobarbital anesthesia (15 monkeys), by a minified Goldmann applanation tonometer under Cl-744 anesthesia (16 monkeys),
and by a minified Draeger applanation tonometer under
light phencyclidine catalepsia (4 monkeys). Mean IOP in
aniridic eyes was about 0.3 mm Hg, or about 3%, lower
than in opposite eyes. lOPs of aniridic and opposite eyes
of the individual monkeys were highly correlated in all
groups. In 11 monkeys, resting total outflow facility and
the facility response to intravenous pilocarpine were determined 6 to 9 weeks postoperatively by two-level
constant-pressure perfusion under pentobarbital anesthesia. There were no significant differences between
mean resting facility, postpilocarpine facility, or facility
response to pilocarpine of aniridic and opposite eyes.
Resting facility, postpilocarpine facility, and facility response to pilocarpine in aniridic and opposite eyes of the
individual monkeys were each highly correlated. Total
iridectomy in the cynomolgus monkey apparently has little, if any, effect on IOP, outflow facility, or facility
response to intravenous pilocarpine, and the iris plays
little, if any, role in mediating the facility response to
intravenous pilocarpine.
In vervet monkey (Cercopithecus ethiops) and
cynomolgus monkey (Macaca fasdcularis), the
entire iris may be removed at its root via a small
limbal incision in vivo.1 The anterior segments
of surgically aniridic eyes, including trabecular
meshwork, Schlemm's canal, and ciliary body, appear normal by biomicroscopic, gonioscopic, and
light and electron microscopic examination.1' 2
Physiologically, these eyes exhibit, under pentobarbital anesthesia, resting intraocular pressures, 3 outflow facilities,3"5 and facility responses
to intravenous and intracameral pilocarpine 3 and
to intracameral cytochalasin B 5 which are within
the range seen in normal eyes of these species.
Nonetheless, since surgically aniridic monkey
eyes are used in experimental studies of aqueous
humor outflow, ciliary body physiology, and druginduced cataractogenesis, it seemed important to
more rigorously define the effect of total iridectomy on aqueous humor dynamics.
Materials and methods. Twenty-two adolescent
and adult cynomolgus monkeys of both sexes,
weighing 1.6 to 4.9 kg, were studied. Each monkey underwent total iridectomy1 in one eye; the
opposite eye was untouched.
Intraocular pressure (IOP) studies. The monkeys were grouped according to type of anesthesia
and method of IOP measurement.
Group 1 consisted of 13 monkeys, seven with
the right eye aniridic and six with the left eye
aniridic. Five weeks postoperatively, the monkeys
were anesthetized with intramuscular CI-744, 5
mg/kg, 6 and placed prone in a head holder. IOP
was measured in both eyes (right first) with a slit
lamp-mounted Goldmann applanation tonometer
minified and calibrated manometrically for cynomolgus eyes (P. L. K., unpublished).
Group II consisted of three monkeys, all with
aniridic right eyes. Both eyes of these monkeys
had undergone two-level constant-pressure perfusion of the anterior chamber 6 weeks postoperatively to determine outflow facility.3' 7 Ten weeks
postoperatively and 4 weeks after anterior chamber perfusion, the monkeys were anesthetized,
and IOP was determined as in Group. I.
Group HI consisted of the 16 monkeys of
Groups I and II combined.
Group IVa consisted of four monkeys, three
with aniridic right eyes and one with an aniridic
left eye. Both eyes of these monkeys had undergone anterior chamber perfusion at 1- to 2-month
intervals postoperatively; at each perfusion, both
eyes received the same dose of the same drug intracamerally. Five weeks after the last perfusion,
and 7.5 to 9.5 months postoperatively, the monkeys were tranquilized with intramuscular phencyclidine HC1, 0.6 mg/kg, and held upright by an
assistant. One drop of 0.4% benoxinate HC1 was
0146-0404/79/080870+06300.60/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc.
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