Aqueous Outflow Pathway Complex
Carbohydrate Synthesis In Vitro
Paul A. Knepper, Jennifer A. Collins, Hymon G. Weinstein,* and Moira Breen*
A technique is described for examining the in vitro synthesis of glycosaminoglycans and glycoproteins
in the aqueous outflow pathway (AOP). New Zealand red rabbit eyes were maintained at near-physiologic
conditions and were infused by anterior chamber exchange with 30 MCI [3HJ glucosamine and 100 fid
[35S] sulfate. At 0.5, 1.0, and 2.0 hours, the anterior segment tissues were dissected, isolated, and
fractionated by gel filtration chromatography on Sephadex® G-50 columns. The elution profiles demonstrated an increase of labeled material with time in the glycosaminoglycan (GAG) fractions and in
the glycopeptide fractions. The relative rate of synthesis of labeled material was: central cornea > peripheral cornea > iris-ciliary body > AOP > anterior sclera. In order to characterize the profiles of
each tissue, the isolated material was analyzed for hexosamine, hexuronic acid, and sulfate. The AOP
cells synthesized a heterogenous mixture of GAGs and glycoproteins which biochemically appeared to
be distinct from other anterior segment tissues. In addition, the percent distribution of GAG polymers
in each tissue was determined by selective GAG degradative procedures and by gel filtration chromatography. The AOP cells synthesized four types of GAGs, and the percent distribution of the labeled
GAGs was different from the other tissues. The present results suggest that this technique may provide
a well-controlled method to probe the metabolic activity of complex carbohydrates in the AOP and in
the anterior segment. Invest Ophthalmol Vis Sci 24:1546-1551, 1983
Cells of the aqueous outflow pathway ie, trabecular
meshwork, synthesize and secrete a variety of glycosaminoglycans (GAGs) and glycopeptides.1 The relative
amounts of these carbohydrate components of the
aqueous outflow pathway (AOP) tissue vary from species to species2 and with age,3 and some evidence suggests that complex carbohydrates influence the rate of
aqueousflowthrough the outflow pathway.4 In general,
the carbohydrate chains are elongated by the stepwise
addition of monosaccharide units from the appropriate
UDP-sugars and specific glycosyltransferases to the
nonreducing ends of the nascent GAG chains.5 As the
chain is synthesized, the GAG moieties may be sulfated
at various positions by the transfer of sulfate groups
from 3'-phosphoadenyl 5'-phosphosulfate.
Studies with labeled sugars, sulfate, and amino acids
indicate that hyaluronic acid and other GAGs are subject to constant degradation and resynthesis at varying
rates that are characteristic of individual GAGs in specific tissues.6'7 The purpose of this study was to investigate the rate of synthesis of GAGs and glycoproteins, as indicated by the incorporation of the precursors [3H] glucosamine and [35S] sulfate in the
anterior segment. The results demonstrated that the
AOP cells synthesized a unique mixture of complex
carbohydrates that was distinct from those of the cornea, sclera, and iris-ciliary body.
Materials and Methods
Ten-week-old New Zealand red rabbits weighing
from 1.6 to 2.1 kg were sacrificed with an intracardiac
air embolism. The eyes were placed in a moist, gauzelined cavity within a paraffin block that was maintained
at 37 C by a heating module. The eyes were covered
with gauze to the limbal area of the globe, and the
surface of the globes and the gauze lining were moistened with warm buffer at regular intervals.
From the Laboratory for Oculo-Cerebrospinal Investigation, Children's Memorial Hospital, the Departments of Cell Biology and
Ophthalmology, Northwestern University Medical School, Chicago,
Illinois, and the Research-In-Aging Laboratory, Veterans Administration Medical Center, North Chicago, Illinois.*
Supported in part by NIH research grants EY-04175, RR-O537O,
the Illinois Society for Prevention of Blindness, and the Andrew
Foundation.
Presented in part as a poster at the Spring ARVO meeting in
Sarasota, Florida, 1982.
Submitted for publication: August 31, 1982.
Reprint requests: Paul A. Knepper, MD, PhD, Laboratory for
Oculo-Cerebrospinal Investigation, 2300 Children's Plaza, Chicago,
IL 60614.
Labeling Method
Three needles were inserted into the globe to maintain intraocular pressure (IOP) and to introduce the
labeled precursors, D-[6-3H] glucosamine (30 Ci/mmol,
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AQUEOUS OUTFLOW PATHWAY COMPLEX CARBOHYDRATE SYNTHESIS / Knepper er ol.
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New England Nuclear, Boston, MA) and carrier-free
[35S] sulfate (New England Nuclear, Boston, MA). At
the onset of the labeling procedure, a specialflowneedle8 was inserted into the anterior chamber and allowed
for free communication between the posterior chamber, anterior chamber, and a perfusion reservoir. The
IOP was maintained at 7.5 mmHg by a perfusion reservoir that contained 25 mM Hepes-Ringer's lactate
solution, pH 7.4. This IOP setting in the rabbit eye
results in an aqueous flow of 1.78 ^1/min.4 A mixing
needle9 was inserted into the anterior chamber. After
a 20-minute control infusion, a 25-gauge needle that
was connected to a 50-MI pre-loaded Hamilton syringe
containing 30 jiCi of [3H] glucosamine and 100 fid
of [35S] sulfate was inserted into the anterior chamber.
All needles were sealed to the cornea with one drop
of cyanoacrylate glue to prevent leakage of the radioisotopes and aqueous. Through the flow needle port,
50 fi\ of aqueous humor were withdrawn and the precursors were injected. After 5 minutes, the perfusion
reservoir was reopened and the anterior chamber was
stirred periodically with the mixing needle to distribute
the radioisotopes. At 30,60, and 120 minutes following
the injection of the labeled precursors, the anterior
segment was dissected into thefivetissue specimens—
central cornea, peripheral cornea, anterior sclera, irisciliary body, and AOP—by a method described previously.1'3 Fifty, 42, and 26 eyes were labeled for
30-, 60-, and 120-minute labeling periods, respectively.
The tissue specimens from each time period were
pooled and stored at —20 C.
Isolation of Complex Carbohydrate Fraction
The complex carbohydrate fraction was isolated by
methods that have been described by our laboratory.13
In brief, the pooled specimens were delipidated with
several rinses of chloroform and methanol to remove
any glycolipids or unincorporated label. The fat-free
tissue was dried in vacuo. Protein was removed by
digestion with Pronase B (Calbiochem., La Jolla, CA)
and precipitated with trichloroacetic acid. Precipitates
contained negligible radioactivity and were discarded,
The complex carbohydrate fraction was isolated by
75% ethanol precipitation, rinsed, and dried in vacuo.
The isolated, labeled complex carbohydrates were redissolved in 0.075 M NaCl and eluted in 0.1 M ammonium acetate in 20% (v/v) ethanol6 from Sephadex
G-50fine(Pharmacia, Uppsala, Sweden) columns (1.6
X 22 cm) previously equilibrated with 0.1 M ammonium acetate and standardized by the elution of
blue dextran (MW = 2 X 106; Sigma, St. Louis, MO)
and reference standard GAGs. Fractions of 1 ml were
collected and 0.1-ml aliquots were analyzed for hexosamine,10 hexuronic acid,11 sulfate,12 and radioactivity.
20
30
Fraction Number
Fig. 1. Separation of complex carbohydrates of the isolated anterior
segment tissue on Sephadex G-50. Fractions (1 ml) were monitored
for hexosamine, hexuronic acid, and sulfate. (A) Profile of central
cornea. (B) Profile of anterior sclera. (C) Profile of the iris-ciliary
body. (D) Profile of the AOP.
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / December 1983
2 -
d
20
40
Fraction Number
roooocx/
20
i
40
"ooo
60
20
40
Fraction Number
Fraction Number
Fig. 2. Separation of total labeled GAGs from labeled glycopeptides by chromatography on Sephadex G-50. Fractions of 1 ml were collected.
Void volume equaled 16 ml. GAGs eluted in the excluded fractions. The material in fractions 12 to 28 was combined and constituted the
total GAG pool. Glycopeptides eluted in a broad zone after the excluded pool. Panels A, B, and C represent the AOP profile at incubation
periods of 30, 60, and 120 minutes, respectively.
Assay for Glycosaminoglycans
In order to identify the type of GAGs in each of
the tissue specimens, the labeled material emerging in
the excluded fractions (total GAG pool) from the initial
passage on the Sephadex G-50 column was pooled,
lyophilized to dryness, and subjected to specific GAG
degradative procedures. Aliquots of 0.1 ml were treated
with 1 unit of hyaluronate lyase,13 0.3 unit of chondroitin ABC lyase,14 0.3 unit of endo-/?-galactosidase,15
or with nitrous acid.16 After completion of each degradative procedure, the aliquot was rechromatographed
on Sephadex G-50. Fractions of 1 ml were collected
and analyzed for radioactivity. In each procedure the
amount of radioactivity in the included fraction represented the amount of susceptible GAG material. This
amount of radioactivity was expressed as a percentage
of the radioactivity present in the total labeled GAG
pool. Measurements of radioactivity were made in a
Beckman® model 6800 liquid scintillation counter, using Ready Solv EP® scintillation cocktail (Beckman
Instruments, Irvine, CA). Background was subtracted
from each sample. Spillover of [35S] into [3H] was set
at 22%. The spillover of [3H] and [35S], correction for
[35S] decay, and integrations of CPM in the excluded
peaks and included peaks were calculated on a microcomputer.
Results
Gel Filtration Chromatography
The isolated, unlabeled complex carbohydrate material of each of the anterior segment tissue components
was separated by gel nitration chromatography into
two peaks. The first peak, which was in the excluded
volume as determined by calibration of the Sephadex
G-50 column with blue dextran and reference hyaluronic acid, contained the GAG constituents, hexosamine and hexuronic acid, and sulfate. The profile
of the GAG constituents of the AOP in the first peak
was distinct from the other anterior segment tissues.
It contained more hexuronic acid and sulfate than the
iris-ciliary body and anterior sclera (See Fig. 1). Similarly, the profile of the constituents of the AOP in the
included fractions contained hexosamine and some
sulfate and was considered to consist of glycopeptide
material. The hexosamine determinations indicated a
heterogenous mixture of glycopeptide material in the
AOP. The separation of hexosamine containing glycopeptide material into a distinct second peak was
more uniform in the cornea and to some extent in
iris-ciliary body. Interestingly, the sulfate determination
in the AOP, iris-ciliary body, and cornea tissues indicated the presence of some sulfated glycopeptide
material (See Fig. 1).
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AQUEOUS OUTFLOW PATHWAY COMPLEX CARBOHYDRATE SYNTHESIS / Knepper er ol.
Fig. 3. Incorporation of
GAG precursors into the total GAG pool. The radioactivity in the excluded volume
was measured at each time
period to determine the
amount of [3H] glucosamine
(panel A) and [35S] sulfate
(panel B) incorporated into
the total GAG pool for each
of the anterior segment tissue.
30
60
90
Time (minutes)
120
60
90
Time (minutes)
O
•
A
•
D
Incorporation of [3H] Glucosamine and [3SS] Sulfate
The incorporation of [3H] glucosamine and [35S]
sulfate into complex carbohydrates during the different
labeling periods was investigated by gel nitration chromatography. As the labeling period increased from 30
to 120 minutes, the elution profiles of each of the
anterior segment tissue components indicated a progressive increase of labeled material in both the excluded fractions, the GAGs, and in the included fractions, the glycopeptide material. The elution profile of
the AOP is illustrated in Figure 2.
A measure of the radioactivity incorporated into the
total labeled GAGs during the time course of the labeling periods for each anterior segment tissue component is shown in Figure 3. The incorporation of
both precursors increased over time. The relative rate
of incorporation of [3H] and [35S] per mg dry-defatted
weight for the tissue components was: central cornea
> peripheral cornea > iris-ciliary body > AOP > anterior sclera.
Glycosaminoglycan Analysis
In order to analyze the type of labeled GAG material
emerging in the excluded fractions, the total GAG
pool from the initial separation on the Sephadex G50 column was pooled, lyophilized to dryness, and
suspended in 0.075 M NaCl. Aliquots of the isolated
total labeled GAG pool were subjected to degradative
procedures, each of which specifically degrades one
type of GAG chain, and the treated aliquots were rechromatographed on Sephadex G-50. The amount of
radioactivity in the included fractions was compared
1549
120
Central Cornea
Peripheral Cornea
Sclera
Iris
Aqueous Outflow Pathway
to the amount of total radioactivity and was expressed
as the percent degraded by the procedure. As shown
in Tables 1 and 2, the AOP labeled GAGs were hyaluronic acid, chondroitin sulfate-dermatan sulfate,
keratan sulfate, and heparan sulfate. The percentage
of radioactivity distributed among the four classes of
GAGs in each of the other anterior segment tissues is
also shown in Table 1 for [3H] and in Table 2 for [3SS].
The percent distribution profile of labeled GAGs in
the AOP was most similar to that of the iris-ciliary
body, but contained more labeled hyaluronic acid and
less of the labeled chondroitin sulfates.
Discussion
The results of this in vitro study demonstrate that
the AOP and anterior segment cells are metabolically
active and incorporate labeled GAG precursors under
these conditions. The gel filtration chromatography
profiles of the labeled material indicated that labeled
GAGs were present in all tissues of the anterior segment. The rate of synthesis of the AOP GAGs, on a
per mg dry-defatted weight basis, was greater than the
adjacent anterior sclera and considerably less than the
iris-ciliary body. The excluded material contained the
recently synthesized long-chain GAGs, and the included material contained the newly formed precursors
of GAGs and other glycopeptides. The metabolic viability of the anterior segment cells over the 2-hour
time course was demonstrated by the increase in labeled
GAGs and glycopeptides. Significantly, the tritiated
glucosamine was converted readily to galactosamine,
as determined by the incorporation of the tritium label
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1983
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Vol. 24
Table 1. Percentage* of degradable activities of [3H] glucosamine incorporated into glycosaminoglycans
in vitro after 30, 60, and 120 minutes
Hyaluronate lyase
Chondroitin ABC
lyase
Endo-P-D
galactosidase
Nitrous acid hydrolysis
Time (min)
Time (min)
Time (min)
Time (min)
Tissue
30
60
120
30
60
120
30
60
120
30
60
120
Central cornea
Peripheral cornea
Anterior sclera
Iris-ciliary body
Aqueous outflow pathway
0
27
0
10
36
0
0
0
16
33
0
0
4
11
30
43
43
58
44
83
41
40
35
61
74
57
40
52
63
62
34
37
8
10
NDf
32
27
9
4
10
30
28
21
9
20
13
11
3
15
NDf
2
6
0
16
3
3
13
7
8
26
* Percentage of radioactivity of [3H] glucosamine-labeled total GAG pool
was calculated by: [(CPM in included fraction)/(CPM in all the fractions)]
X 100 following a degradative procedure. Note: hyaluronate lyase degrades
hyaluronic acid; chondroitin ABC lyase degrades hyaluronic acid, the chon-
droitin sulfates and dermatan sulfate; endo-0-D-galactosidase degrades most
of keratan sulfate polymer, nitrous acid treatment degrades heparin and heparan
sulfate.
t ND: not determined.
in the galactosamine-containing GAGs, the chondroitin sulfates, and dermatan sulfate, in each of the
labeling periods. To our knowledge, this is the first
demonstration of the types and amounts of GAGs
synthesized in an anatomically and biochemically defined AOP.
The results of this study are consistent with previous
reports of GAG synthesis by cultured trabecular meshwork cells. Francois and Victoria-Troncoso17 observed
that cultivated trabecular cells contained alcian blue
positive granules or GAG material sensitive to exogenous hyaluronidase. Schactschabel et al18 demonstrated uptake of [35S] sulfate and [14C] glucosamine
by monolayer cultures of cells from explanted trabecular tissue, and determined by electrophoresis on cellulose acetate strips that the cultured medium contained labeled hyaluronic acid (60-85%), chondroitin
sulfate (6-33%), and dermatan sulfate (3-31%). Hassell
et al19 also demonstrated synthesis and secretion of
labeled GAGs by trabecular meshwork explants in culture and identified the labeled GAGs as hyaluronic
acid and a chondroitin sulfate-containing proteoglycan.
Labeling studies of cells grown in culture, however,
are dependent on cell density and viability,16'2021 and
it has been demonstrated that cultured trabecular cells
frequently alter both the pattern and amount of synthesized GAGs.2223
Our previous reports have demonstrated that hyaluronic acid and chondroitin sulfate-dermatan sulfate
are the major components of the AOP in human2 and
rabbit eyes,1 whereas keratan sulfate and heparan sulfate are present in smaller amounts. The results of the
present study indicate a similar GAG profile, that is,
the presence of four types of GAG polymers. The
amount of labeled hyaluronic acid, as determined by
hyaluronate lyase degradation, was greatest in the AOP,
considerably less in the iris-ciliary body, and essentially
absent in the sclera and cornea. The presence of a
significant percentage of tritiated hyaluronate lyasesensitive material in the peripheral cornea after the
30-minute labeling period was an unexpected result,
although rabbit stromal cells24 and endothelia25 in culture synthesize hyaluronic acid. The presence of hyaluronic acid in the peripheral cornea may result from
a nonspecific effect of the hyaluronate lyase enzyme,
or may indicate that hyaluronic acid is present in the
Table 2. Percentage* of degradable activities of [35S] incorporated into glycosaminoglycans
in vitro after 30, 60, and 120 minutes
Hyaluronate lyase
Chondroitin ABC
lyase
Endo-0-D
galactosidase
Nitrous acid hydrolysis
Time (min)
Time (min)
Time (min)
Time (min)
Tissue
30
60
120
30
60
120
30
60
120
30
60
120
Central cornea
Peripheral cornea
Anterior sclera
Iris-ciliary body
Aqueous outflow pathway
0
0
0
2
0
0
0
0
4
15
0
0
0
2
0
39
37
68
51
60
41
39
74
84
81
54
44
86
89
87
22
20
8
3
NDf
31
27
13
36
10
34
27
13
11
32
9
9
7
32
NDf
4
5
10
33
27
3
3
* Percentage of radioactivity of [33] sulfate-labeled total GAG pool was
calculated by: [(CPM in included fraction)/(CPM in all the fractions)] X 100
following a degradative procedure. Note: hyaluronate lyase degrades hyaluronic
acid; chondroitin ABC lyase degrades hyaluronic acid, the chondroitin sulfates
10
19
19
and dermatan sulfate; endo-/3-D-galactosidase degrades most of keratan sulfate
polymer, nitrous acid treatment degrades heparin and heparan sulfate.
t ND: not determined.
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AQUEOUS OUTFLOW PATHWAY COMPLEX CARBOHYDRATE SYNTHESIS / Knepper er ol.
early phase of GAG synthesis in peripheral cornea.
The amount of labeled chondroitin sulfate-dermatan
sulfate in the anterior segment components, as determined by chondroitin ABC lyase degradation, was essentially constant at each of the time points measured
in this study. Although there were variations in the
amount of labeled heparan sulfate and keratan sulfatelike material in the labeling periods, there was no
marked trend in the accumulation or disappearance
of any specific GAG in the time course of this in vitro
study.
Because of the ease of labeling the anterior segment
with GAG precursors, dissecting the AOP and isolating
the complex carbohydrate fraction, this in vitro technique may prove to be more useful and reliable than
autoradiographic26*27 or tissue culture methods for investigating the synthesis of the four types of GAG
polymers in the AOP. By the use of this technique,
the effects of various drugs, eg, steroids administered
to animals prior to short-term studies of metabolic
inhibitors, during the labeling period, can be examined
on complex carbohydrate synthesis in vitro.
Key words: aqueous outflow pathway, glycosaminoglycans,
glycoproteins, [3H] glucosamine, [35S] sulfate
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