Download Carbonic anhydrase isoenzymes CA I and CA II in the human

Carbonic Anhydrase Isoenzymes CA I and CA II
in the Human Eye
Per J. Wisfrand, Mono Schenholm, and Gudmar Lonnerholm
The distribution of carbonic anhydrase was studied in human donor eyes by the cobalt-phosphate histochemical method of Hansson and by immunofluorescence and immunoperoxidase techniques using
antisera specific against the human cytoplasmic isoenzymes CA I and CA II. Corneal endothelium
displayed specific immunological staining for CA I and CA II. Distinct enzyme activity was observed
histochemically in the plasma membranes and cytoplasm of the endothelium. In the ciliary processes
immunological evidence for the presence of CA II was found both in pigmented (PE) and in nonpigmented
(NPE) epithelium. Activity was observed in the cytoplasm and basolateral membranes of NPE, but only
in the basal membranes of PE. In the lens the plasma membranes of both the epithelium and fibers
displayed intense activity, whereas cytoplasmic enzyme activity was seen only in the epithelium. There
was no activity in the lens capsule. Immunofluorescence studies were difficult because of autofluorescence,
but the immunoperoxidase technique indicated the presence of both CA I and CA II in the lens. In the
central retina, Miiller cells stained for CA II. Histochemically, enzyme activity was seen in the cytoplasm
and at the plasma membranes. Activity was also observed in some but not all cones. Electron microscopy
revealed this to be located in the cristae and plasma membranes adjacent to the pigment epithelium.
Activity was also found in PE. Neurons and rods lacked both immunological staining and activity.
Endothelial cells of capillaries in ciliary processes and in the choroid stained for CA I and exhibited
histochemical activity, particularly those which faced neighboring epithelial cells containing the enzyme.
The isoenzyme CA III, which is resistant to inhibition by sulfonamides, did not appear to be present in
these ocular tissues, since the histochemical staining of enzyme activity was completely abolished by
1O~6 M acetazolamide. Invest Ophthalmol Vis Sci 27:419-428, 1986
Human tissues are known1 to contain three soluble
cytoplasmic isoenzymes of carbonic anhydrase (CA)
designated CA I, CA II, and CA III. CA II is a highactivity form (with respect to hydration of CO 2 and
dehydration of H2CO3) that is found in erythrocytes,
where it facilitates CO 2 transport.2 It is also present in
secretory epithelia, where it facilitates electrolyte secretion.3 CA I is a low-activity isoenzyme which occurs
in erythrocytes, in certain epithelia of the jejunum and
colon, and in the endothelium of capillaries in the lung,
kidney, and gastrointestinal tract.4 Its function is not
known. CA III, whose function is also unknown, is a
low-activity, sulfonamide-resistant form, found in red
fibers of skeletal muscle.5 Recently, a new hydrophobic
membrane-bound isoenzyme, designated CA IV, has
been detected in plasma membranes and microsomes
of human renal tubular cells. It appears to be involved
in the translocation of hydrogen or bicarbonate ions
across plasma membranes. 6
Carbonic anhydrase activity has been found in human ciliary processes, cornea, iris, and retina.7'8 CA II
has been demonstrated in ciliary processes by immunological and kinetic techniques, 910 and in the Miiller
cells in the retina by an immunocytochemical technique." 1 2 There is an obvious need for further studies
on the distribution of the various isoenzymes, so that
the role of carbonic anhydrase in the ocular tissues can
be elucidated. In the present study we have therefore
investigated the distribution of the various isoenzymes
in human donor eyes by histochemical and immunocytochemical techniques. The histochemical cobaltphosphate method of Hansson, 1314 a well-documented
and specific method for demonstrating carbonic anhydrase activity, was used here in a modified form as
described by Ridderstrale,15 which permitted studies
of semi-thin sections by electron microscopy. Immunolocalization of the enzymes was limited to the lightmicroscopic level and allowed identification of the sites
of the two main soluble cytoplasmic isoenzymes, using
From the Departments of Ophthalmology and Medical Pharmacology, University of Uppsala, Uppsala, Sweden.
Supported by the Swedish Medical Research Council (grants 2874
and 5413) and by the National Eye Institute, Bethesda, Maryland
(grant EY-0231 to Professor Ernst Barany, Uppsala).
Submitted for publication: April 8, 1985.
Reprint requests: Per J. Wistrand, MD, Department of Medical
Pharmacology, Biomedicum, Box 593, S-751 24 Uppsala, Sweden.
419
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1986
antibodies against CAI and CAII. This technique also
served as a valuable control for the histochemical
method.
Materials and Methods
Normal human eyes intended for corneal transplantation were enucleated 12-24 hr postmortem. They
were obtained from the Department of Ophthalmology
of the Uppsala University Hospital. The eyes had been
stored in cold physiological saline solution for about 3
hr before fixation. Some eyes were dissected, and the
various tissues were taken for fixation. Others were
fixed by injection of 0.2 ml of 2.5% glutaraldehyde into
the vitreous body, whereupon the whole eye was placed
in the fixative.
Tissue Preparation
For histochemical studies the tissues werefixedwith
2.5% glutaraldehyde in 0.1 M phosphate buffer at pH
7.4, which gave excellent tissue preservation and good
histochemical staining. Thin slices were cut and immersed in thefixativefor 4-6 hr. After being rinsed in
0.2 M sucrose in 0.05 M phosphate buffer, the tissue
slices were embedded in the water-soluble resin JB 4
(Polysciences Inc.; Warrington, PA) or the Sorvall
embedding kit (Sorvall, Inc.; Washington, D.C.), sectioned for light and electron microscopy, and stained
for carbonic anhydrase as described.
For immunohistochemical studies the tissues were
immersed in different fixatives, namely 4% formaldehyde with 0.1% glutaraldehyde, Bouin's fluid, or Carnoy's fluid. Fixation in Bouin's fluid for 6 hr, followed
by dehydration through graded ethanols and embedding in paraffin, was found to yield the best material
for immunofluorescent staining, in the case of all tissues. Formaldehyde could not be used because of the
occurrence of unspecificfluorescencein the whole tissue. Carnoy's fluid gave good fluorescence but poor
tissue preservation. However, in the lens and cornea,
autofluorescence was encountered with all fixatives,
and for these tissues the pseudoperoxidase (PAP) technique16 was applied instead.
Histochemical Staining
A slightly modified version15 of the Hansson
method1314 was used for demonstrating CA activity.
The incubation medium invariably contained 3.5 mM
cobalt sulfate and the incubation time was 2 to 8 min,
since with longer times unspecific precipitation was
produced in the sections. For light microscopy, 1-2fim thick sections were used. Some sections were
Vol. 27
counterstained with hematoxylin and eosin. The sections for electron microscopy were about 0.1-0.3 /zm
thick. Reproducible staining could not be achieved with
thinner sections.
At least 50 sections of each eye were examined by
light microscopy. Adjacent sections from the same
block of tissue showed very similar staining.
The specificity of the staining reaction was checked
by incubating sections in the presence of 10 ixM acetazolamide (Diamox®, American Cyanamid Company;
Pearl River, NY), a specific inhibitor of carbonic anhydrase.17 At this concentration, acetazolamide completely abolished visible staining, whereas the presence
of the inactive control substance Cl 13850,17 an N5-tbutyl-analogue of acetazolamide (American Cyanamid
Company), at a concentration of 10 ^M, did not interfere with the staining.
CA III is a sulfonamide-resistant form of the enzyme,
but CA I, CA II, and CA IV are inhibited by low concentrations of acetazolamide (I50 = 0.2, 0.01 and 0.05
/xM, respectively). This inhibitor can therefore be used
to distinguish CA III activity from that of other isoenzymes in histochemical sections.
Immunolocalization
Sections 3 to 6 ^m thick of fixed tissues were put
onto gelatin-coated glass slides, deparaffinized and dehydrated through xylol and graded ethanols. Sections
6 ixm thick of unfixed tissue were cut in a cryostat at
-20°C, put onto slides, and fixed in 99% methanol at
4°C for 2 min. The sections were exposed to specific
or nonspecific (control) rabbit antiserum at room temperature (22°C). Antisera against the human carbonic
anhydrase isoenzymes CA I and CA II (anti-CA I and
anti-CA II) raised in rabbits were purchased from
Behring Institut, Marburg-Lahn, GFR. The specificities and affinities of the antisera for their respective
isoenzymes were determined in our laboratory by immunodiffusion and radioimmunosorbent techniques.18
The antisera were diluted in phosphate-buffered saline
(PBS; 0.8% NaCl in 0.01 M phosphate buffer at pH
7.2) and tested as serial dilutions from 1:10 to 1:2560.
Cross-reactivity between anti-CA I and anti-CA II was
sometimes observed, but only when the concentration
of the nonspecific antiserum was at least 20 times higher
than that of the specific antiserum.
Immunofluorescence Procedure
After 30 min of incubation with specific antiserum,
the sections were rinsed in PBS for 10 min and then
exposed to a 1:10 dilution of goat anti-rabbit immunoglobulin labeled with fluorescein isothiocyanate
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•y
Fig. 1. Cornea. Immunofluorescence staining with anti-CA I, 1:
100 dilution. The endothelium (arrowhead) shows specific fluorescence. S is unstained stroma (X260).
Fig. 2. Cornea. Immunofluorescence staining with anti-CA II, 1:
100 dilution. There is strong staining in the endothelium and also
some staining also in the corneal stroma (S) (X260).
Fig. 3, Cornea (same as Figs. 1-2). Immunofluorescence staining
with non-specific antiserum, 1:10 dilution (X260).
(Behring Institut; Marburg-Lahn, GFR) for 30 min.
After a final wash in PBS for 30 min, followed by rinsing in distilled water, the sections were mounted under
glass coverslips in PBS-glycerin (1 part PBS, 9 parts
glycerin). No counterstaining was used. The slides were
examined with use of incident-light excitation with a
filter system of type B 450-490/FT 510/LP 520 (Zeiss,
GFR, Carl Zeiss, Inc.; Oberkochen, West Germany).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Morch 1986
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Fig. 4. Ciliary processes. Immunofluorescence staining with anti-CA II, 1:20 dilution. The ciliary epithelium is intensely stained. Note the
absence of staining in the capillary walls (X260).
Fig. 5. Ciliary processes (same as Fig. 4). Immunofluorescence staining with anti-CA I, 1:20 dilution. The capillary walls are distinctly stained
(two are seen in the center). Arrowheads indicate heavily stained erythrocytes trapped within vessels. The ciliary epithelium (arrow) lacks
staining (X260).
Immunoperoxidase Procedure
In order to block endogenous peroxidase activity and
to reduce the nonspecific background staining, sections
were first treated for 15 min with methanolic hydrogen
peroxide, followed by treatment with 20% normal
swine serum. After 60 min of incubation with specific
antiserum, the sections were exposed to a 1:20 dilution
of swine anti-rabbit immunoglobulin, and then to a 1:
80 diluted peroxidase-antiperoxidase (PAP) complex
(Dakopatts A/S, Glostrup, Denmark) for 30 min. The
peroxidase activity was visualized by incubation for 15
min in 3-amino-9 ethylcarbazole in 0.02 M/l acetate
buffer containing dimethylsulfoxide. After each step
the sections were thoroughly washed in PBS. The sections were mounted under glass coverslips in glycerol
gelatin and examined by light microscopy.
Results
Cornea
Specific fluorescence for CA I and CA II was found
in the endothelium. (Figs. 1-3), but the detailed distribution of these enzymes could not be decided. Histochemically, strong activity was found both in the cytoplasm and all around the plasma membranes of the
endothelium. In the epithelium weak fluorescence and
histochemical activity was seen in some cells, particularly near the limbus. Streaky fluorescence for CA II
was also seen in the stroma (Fig. 2).
Ciliary Processes
Intense staining for CA II was observed primarily in
the nonpigmented epithelium (NPE), but there was also
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weak staining in the pigmented epithelium (PE) (Fig.
4). There was no clear difference between the epithelia
of the valley and crest of the pars plicata. The ciliary
epithelium lacked staining for CA I (Fig. 5). Histochemical activity was seen adjacent to the basolateral
membranes and in the cytoplasm of NPE (Fig. 6). The
basolateral membranes stained clearly, but there was
no or only weak cytoplasmic enzyme activity in PE.
The endothelium of capillaries near PE showed specific fluorescence for CA I (Fig. 5) and strong histochemical staining. Erythrocytes trapped in vessels displayed fluorescent staining for CA I (Fig. 5) and CA II
and also heavy histochemical staining.
Lens
Owing to autofluorescence it was difficult to discern
specific fluorescence for CA I and CA II, but when the
pseudoperoxidase method was used instead, both isoenzymes seemed to be present in the epithelium and
the lens fibers. Histochemically, strong activity was seen
in the epithelium, particularly at the plasma membranes (Fig. 7). The cytoplasm was only weakly stained.
Lens fibers showed high activity along the outer cell
membranes. No definite cytoplasmic staining was observed. There was no difference in enzyme activity between the different parts of the lens. The lens capsule
did not stain histochemically (Fig. 7).
Fig. 7. Lens. Histochemistry. Enzyme stain is seen in the epithelium
(arrows) and lens fibers (to the right). C is unstained capsule. Incubation time 8 min. Hematoxylin-eosin (X875).
Retina
Fig. 6. Ciliary epithelium. Histochemistry. Pigmented epithelium
(PE) shows staining only at the basolateral cell membranes, while in
nonpigmented epithelium (NPE) the cytoplasm is also stained. Incubation time 5 min (X7000).
Immunocytochemically, CA II was found to be
evenly distributed in the Miiller cells and possibly also
in PE, but not in the neurons (Figs. 8-9). CA I was not
found in any part of the retina. With the histochemical
method strong activity was seen in the cytoplasm and
cell membranes of Muller cells (Fig. 10). PE exhibited
enzyme activity in the apical and basal membranes,
but there was only weak staining in the cytoplasm (Fig.
10). Histochemically, activity was observed in some
cones (Figs. 10-11). Findings with the PAP technique
also indicated that CA II was only present in certain
cones (Fig. 12). Electron microscopic examination revealed that the activity in these cones (Fig. 11) occurred
particularly in the part of the photoreceptor bridging
across to the pigmented epithelium. The mitochondriarich part of these cones, as well as intracellular (cristae)
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / March 1986
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Fig. 8. Retina. Immunofluorescence staining with anti-CA II, 1:10 dilution. Miiller cells are intensely stained. V is vitreous body (X260).
Fig. 9. Retina (same as Fig. 8). Immunofluorescence staining with nonspecific antiserum, 1:10 dilution (X260).
and outer membranes was also intensely stained (Fig.
11). None of the rods exhibited any staining (Fig. 10).
The endothelium of the choroid (Fig. 10) stained for
CA I. Histochemically the activity was seen to be adjacent to the plasma membranes.
Discussion
General
Fig. 10. Retina. Histochemistry. There is heavy staining in Miiller
cells (to the left). Some staining is also seen in the pigment epithelium
(lower right) and in some cones (arrows). Arrowhead indicates stained
endothelium of choroid vessel. Incubation time 5 min (X1200).
The distribution of carbonic anhydrase was similar
in all eyes tested, and there was good accordance between the histochemical and immunocytochemical
findings. The only exception was the corneal epithelium, where the histochemical staining was variable
and always considerably weaker than the immunofluorescence. The reason for this discrepancy is not clear
at present.
With the histochemical method, staining for carbonic anhydrase activity was found in the endothelium
of the cornea, in the pigmented and nonpigmented
epithelium of the ciliary processes, in the epithelium
and fibers of the lens, in the Miiller cells and certain
cones in the retina, and in the endothelium of capillaries of the choroid and ciliary processes. With the
immunolocalization techniques CA II was found in
the same cells of the cornea, ciliary processes, lens, and
retina. Moreover, CA I was observed together with CA
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CARBONIC ANHYDRASES IN THE HUMAN EYE / Visrrond er ol.
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II in the endothelium of the cornea, and as the only
isozyme in the endothelium of capillaries of ciliary
processes and choroid. CA III was probably not present
in any tissue, since all histochemical staining was inhibited by 10 nM acetazolamide (see Methods). It
should be noted that the immunological method does
not detect the membrane-bound CA IV, which is immunologically different6 from the cytoplasmic forms.
With the histochemical method, on the other hand, all
isoenzymes, including the membrane-bound form, are
detected.
Cornea
We report here for the first time the presence of both
CA I and CA II in the corneal endothelium. Previously
these two isoenzymes have been found together in the
single human erythrocyte19 and in the surface epithelial
cells of the human colon.4 The function of CA II in
the human corneal endothelium, 20 which electrophysiologically behaves like that of the rabbit cornea, is
probably to facilitate fluid and bicarbonate secretion.
Thus, it has been shown that inhibition of carbonic
anhydrase activity in the rabbit cornea causes a reduction in potential across the epithelium (aqueous negative to stroma) and flux of bicarbonate and fluid.21
The role of CA I in the cornea as well as in erythrocytes
and colon cells needs to be elucidated, however.
The epithelium of the human cornea, however, primarily transports sodium chloride to the tear side,20
and there would appear to be no need for the enzyme
for this transport. Using a sensitive biochemical technique, Silverman and Gerster22 also detected no carbonic anhydrase activity in the stroma or epithelium
of rabbit cornea. Our data were inconclusive in this
respect.
11
Fig. 11. Retina. Histochemistry. Intensely (right) and weakly (left)
stained cones are seen. The staining is particularly heavy in the mitochondria-rich part of the cone (C). Electron microscopy. Incubation
time 5 min (X27,000).
Ciliary Processes
The presence of CA II in nonpigmented epithelium
of the ciliary processes is in accordance with the well
established theory23 that carbonic anhydrase plays a
role in the transfer of sodium bicarbonate and fluid
into the aqueous humor. The levels of CA II in the
human ciliary processes910 have been found to be similar to those in renal proximal tubular cells,3 which
constitute another bicarbonate-transporting epithelium. The presence of CA II in the pigmented epithelium suggests that this epithelium is also involved in
electrolyte secretion, the nature of which has yet to be
determined.
Lens
Carbonic anhydrase has been demonstrated in the
lens of many species by use of biochemical, immu-
12
Fig. 12. Retina. Immunoperoxidase staining with anti-CA II, I:
200 dilution. Staining is seen only in Muller cells and in some of the
cones (arrow) (X875).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Morch 1986
nochemical, and histochemical methods.24 These investigations have shown that the enzyme activity varies
greatly between different kinds of animals. Thus, the
hundred-fold difference between the activity in the lens
of the pig and cattle is striking. Very low activity has
been found in the human lens, from which both CA I
and CA II have been chromatographically separated
(Wistrand, unpublished). It has been suggested25 that
carbonic anhydrase, by its catalysis of the hydration of
metabolic CO2 and subsequent production of HCO 3 "
in the lens fibers, enhances the loss of CO 2 by "facilitated" diffusion. The enzyme in the lens epithelium is
probably also involved in translenticular electrolyte
transport, since in the human lens this transport has
been found to depend on the presence of Cl" and
HCXV. 20
Retina
The present results confirm the finding of carbonic
anhydrase in the primate retina (see Introduction) and
add further details concerning the type and distribution
of the enzyme in Miiller cells, pigment cells, and cones.
In Miiller cells, the enzyme is located both in the cytoplasm and in the membranes. The function of CA
II in the Miiller cells might be the same as that attributed to CA II in the glial cells of the brain,26 that is, to
convert CO2 formed in the neurons to bicarbonate and
hydrogen ions for use by the glial cells for exchange
reactions to maintain an optimal electrolyte environment around the neurons for their activity.
The pigmented epithelium displayed enzyme activity
primarily in the membranes. The epithelium of the
frog retina has been shown to transport bicarbonate
ions toward the choroidal side (or hydrogen ions toward
the apical (retinal) side).27 This transport is inhibited
by 10~4 M acetazolamide, suggesting that carbonic anhydrase is involved.
Rods did not stain, as reported previously,8 but in
contrast to the previous finding8 that all cones showed
activity, only certain cones were found to be stained.
The differential staining of cones and pigmented epithelial cells seems clear, even though the apical processes of the pigmented epithelium are known to be
closely associated with the cones.28 We have not been
able to identify the type of the stained cones, but since
they constituted about 10% of the cones in the parafoveal area, we suggest that they are identical to the
blue-sensitive ones.29 The function of carbonic anhydrase in the cones needs investigation. It is known that
light stimulation of photoreceptors will cause a decrease
in their intracellular pH. 30 The enzyme might therefore
have a buffering function in these cells. Moreover, it
Vol. 27
is known that carbonic anhydrase is involved in the
exchange mechanism for chloride and bicarbonate in
several tissues,31 and it has been shown32 that the electrical response of cones, but not of rods, to light is
dependent on the chloride concentration in the medium surrounding the receptor. The role of the enzyme
could be to furnish bicarbonate ions for this exchange
mechanism. Anyhow, it would be interesting to determine whether the function of the blue-sensitive cones
is altered by inhibition of their carbonic anhydrase.
Capillary Endothelium
The endothelium of the stromal capillaries of the
ciliary processes and choroid showed specific fluorescence for CA I. Carbonic anhydrase activity in the capillary endothelium has been demonstrated in skeletal
muscle and in lung tissue.33 Moreover, in the capillaries
of the mucosa of the gastrointestinal tract4 and of the
human kidney34 the enzyme has been shown to be CA
I. There is also functional evidence for the presence in
the capillary endothelium of a membrane-bound enzyme with its active site facing the blood.6 In skeletal
muscle and the lung, the enzyme is thought to facilitate
the transport of CO 2 after dehydration of H 2 CO 3 . 6
However, the enzyme in the endothelium of capillaries
facing secretory cells might not have the same purpose
as that in capillaries of the lung and skeletal muscle.
The spatial arrangement, with the enzyme activity facing the secretory cells of pigmented epithelium in the
retina and of ciliary epithelium, suggests that it is involved in the transport of ions (H + or bicarbonate)
from the epithelium to the blood vessel, or in the reverse
direction. Why this should be performed by CA I and
not CA II, however, is not clear.
Membrane-Associated Enzyme Activity
Carbonic anhydrase activity was found to be associated with the plasma membranes in all tissues.
Whether this activity originates from a membranebound carbonic anhydrase such as CA IV of the kidneys
is not known. Biochemical studies have indicated that
most of the activity, more than 99%, derives from soluble CA II of the ciliary epithelium9 and lens.24 In these
tissues only small amounts of activity were particlebound after homogenization. This contrasts with the
histochemical finding of strong activity in the plasma
membranes and relatively weak activity in the cytoplasm of many cells. In a previous histochemical study
of the primate eye, frozen tissues showed stronger cytoplasmic staining than tissues which had been resinembedded as in the present study.7 This might be due
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CARBONIC ANHYDRASES IN THE HUMAN EYE / Wisrrond er ol.
to the better preservation of cytoplasmic carbonic anhydrase activity in frozen tissues.
Carbonic Anhydrase Inhibition
Inhibitors of carbonic anhydrase play an important
role in ophthalmology, where they are used to reduce
elevated intraocular pressures. Apart from transient
myopia and blurred vision, no adverse reactions from
the eye have been described in spite of long-term treatment of patients, often for years.35 This is surprising
in view of the presence of carbonic anhydrase in several
ocular tissues. Acetazolamide is the inhibitor mostly
used, and the reason for its lack of effect on tissues
other than ciliary processes might be that its concentration in the aqueous humor, lens, and vitreous is low
as a result of active transport out of ocular tissues, as
has been found in rabbits.36 Acetazolamide is also 20
times less active against CA I than against CA II.2
However, this does not seem to explain the lack of
toxicity in the eye since the other available clinical inhibitors are all more lipid-soluble17 than acetazolamide
and therefore attain higher concentrations in the eye.
Moreover, they are all equally active against CA I and
CA II. One of them, methazolamide, has been used
clinically for almost as long a time as acetazolamide
and yet does not seem to have a higher toxicity in the
eye.35 Whether inhibition of carbonic anhydrase in
cornea, lens, and retina has any functional significance,
therefore, remains to be elucidated.
Key words: carbonic anhydrase, cornea, ciliary body, iso-enzymes, lens, retina, glaucoma
Acknowledgments
The authors wish to thank their colleagues at the Department of Ophthalmology for providing the eyes, and Professor
Ernst Barany, Uppsala University, for helpful discussions.
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