Download in the ciliary body and iris of the rat

Investigative Ophthalmology & Visual Science, Vol. 32, No. 5, April 1991
Copyright © Association for Research in Vision and Ophthalmology
Ulfracytochemical Localization of the Erythrocyte/
HepG2-Type Glucose Transporter (GLUT1) in the
Ciliary Body and Iris of the Rat Eye
Kuniaki Takara,* Toshiko Kasahara,t Michihiro Kasahara,j- Osamu Ezaki4 and Hiroshi Hirano*
Aqueous humor, with its unique low concentration of proteins, is produced by the ciliary body and
isolated by the blood-aqueous barrier from the body fluid. Glucose in aqueous humor is a major source
of nutrients for lens and corneal cells, and is maintained near the plasma level, suggesting a specific
glucose transport mechanism in the blood-aqueous barrier. Using antibodies against erythrocyte/
HepG2-type glucose transporter (GLUTl), one isoform of the facilitated diffusion glucose transporters, the authors found immunocytochemically that GLUTl localizes in the epithelial cells of ciliary
body and iris. GLUTl is also found in the endothelial cells of blood vessels in the iris, whereas no
labeling is seen in the blood vessels in the ciliary body. In the ciliary body epithelium, the plasma
membranes of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells are positive
for GLUTl. By the colloidal gold particle counting, the basal infoldings of PE cells show approximately two-fold denser labeling than those of NPE cells. Since PE and NPE cells make up a functional
syncytium with numerous gap junctions, the authors suggest that glucose transport in the ciliary body
occurs in this manner: glucose diffuses out from blood vessels through the pores of fenestrated endothelial cells, is transported into PE cells by GLUTl in their plasma membrane, enters NPE cells through
gap junctions connecting PE and NPE cells, and is finally transported into the aqueous humor by
GLUTl of NPE cells. The higher density of GLUTl in PE cells may account for the consumption of
glucose by PE and NPE cells in addition to the transepithelial transport. Invest Ophthalmol Vis Sci
32:1659-1666,1991
The aqueous humor is a transparent, watery solution that is continuously produced by the ciliary body
of the eye and flows from the posterior chamber into
the anterior chamber.1 One of its physiologic roles is
to supply oxygen and nutrients to the lens and cornea.1 The composition of the aqueous humor is different from that of plasma in its low concentration of
plasma proteins and high concentration of ascorbic
acid.2 The ciliary body epithelium, the site of aqueous
humor production, is mainly responsible for the determination of the constituents of the aqueous humor.
This epithelium is made of two layers: the outer pigmented epithelium (PE) and the inner nonpigmented
epithelium (NPE). PE and NPE cells oppose at their
apical surfaces because of the embryologic invagination of the optic cup.3 Most of the hydrophilic substances are blocked by the plasma membrane of the
NPE cells connected by tight junctions, thus forming
the blood-aqueous barrier.4"9 In spite of the tight
barrier, the concentration of glucose in aqueous humor is kept at a level similar to that in plasma.2 Erythrocyte/HepG2-type glucose transporter (GLUTl),
one isoform of facilitated diffusion glucose transporters (GTs),10 is localized in the epithelial cells of the
ciliary body.1112 We show ultracytochemically that a
majority of GLUTl is localized at the plasma membranes of PE and NPE cells of the ciliary body. Our
observations indicate a possible glucose transport
pathway through the ciliary body epithelium.
From the *Department of Anatomy, Kyorin University School
of Medicine, Shinkawa, Mitaka, Tokyo, the "("Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, and the
^Division of Clinical Nutrition, National Institute of Health and
Nutrition, Toyama, Tokyo, Japan.
Supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan, and by grants from the Takeda
Science Foundation and Yazaki Memorial Foundation for Science
and Technology.
Submitted for publication: September 19, 1990; accepted November 28, 1990.
Reprint requests: Kuniaki Takata, PhD, Department of Anatomy, Kyorin University School of Medicine, Shinkawa, Mitaka,
Tokyo 181, Japan.
Materials and Methods
Anti-GLUTl antibodies were raised in rabbits
against the synthetic peptides corresponding to amino
acids 480-492 (c-terminus) of the deduced amino
acid sequence of HepG2-GT13, using the peptide-
1ASQ
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1991
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keyhole limpet hemocyanin conjugates,1415 or against
purified human erythrocyte GT. 1617
Male Sprague-Dawley 4-6 week-old rats (Nippon
Bio-Supply Center, Tokyo, Japan) were fed Clea CE-2
rat chow (Clea Japan, Tokyo, Japan), and were anesthetized with ether. All animals were treated in accordance with the ARVO Resolution on the Use of Animals in Research. The ciliary body was taken with the
iris and immunoblotted according to the procedure described earlier. "8 For light microscopic localization of
GLUT1, eye specimens were fixed in 3% formaldehyde-phosphate-buffered saline (PBS) at room temperature for 2 hr and embedded in paraffin wax. Six-^mthick sections were cut, deparaffinized, and treated
with methanol and H 2 O 2 to quench endogenous peroxidase activity.19 The sections were blocked with 5%
normal goat serum for 10 min, then covered with either of the anti-GLUTl antibodies for 1 hr, 10 jug/ml
biotinylated goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) for 40 min, and avidin-biotinylated peroxidase complex (ABC, Vector Laboratories, Burlingame, CA) for 40 min. The specimens were
200K-
116K9 7K~
66K4 3K-
31K-
Vol. 32
Fig. 2. Light microscopic localization of GLUT 1 by the immunoperoxidase method. Positive staining for GLUT1 is seen in the
epithelia of ciliary body (CB) and iris (I). Blood vessels in the iris
show positive staining for GLUT I (arrows). PC, posterior chamber;
Bar = 50 (im.
examined after the diaminobenzidine-H2O2 reaction.
For the ultrastructural localization of GLUT 1, specimens were fixed in 3% formaldehyde-0.5% glutaraldehyde-PBS, dehydrated through a series of graded ethanols, and embedded in LR White (London Resin, Basingstoke, UK). Ultrathin sections were cut and
mounted on nickel grids. The grids were floated on 5%
normal goat serum for 10 min, anti-GLUTl antibodies for 60 min, PBS for 10 min, and then goat-anti-rabbit IgG-10 nm colloidal gold conjugate [prepared according to Slot and Geuze20 and DeMey21] for 60 min.
After being washed with PBS, the grids were floated on
2.5% glutaraldehyde-PBS for 10 min, washed with
deionized water, stained with uranyl acetate and lead
citrate, and examined with a JEOL JEM-1200EX
(JEOL, Tokyo, Japan) transmission electron microscope. Immunocytochemical stainings in this report
were obtained with the use of antibodies against the
synthetic c-terminus peptide of HepG2 GT. AntiGLUTl antibody raised against purified human erythrocyte GT showed no appreciable difference in immunocytochemical staining or in immunoblotting.
To quantify the anti-GLUTl antibody binding in
NPE and PE cells, the number of colloidal gold particles within the range of 20 nm from the plasma membrane was measured. We calculated the density of colloidal gold particles per l-/mi length of plasma membrane on electron micrographs with a Nikon Cosmozone-lS image analyzer (Nikon, Tokyo, Japan).
Results
A
B
Fig. 1. Immunoblotting of ciliary body and iris with anti-GLUT 1
antibodies. Ten micrograms of homogenate was applied to SDSpolyacrylamide gel electrophoresis and subjected to immunoblotting with antibody raised against the c-terminal peptide of HepG2
• (A), or with antibody against human erythrocyte GT (B).
When the ciliary body-iris homogenate was immunoblotted with anti-GLUTl antibody raised
against the c-terminus peptide of HepG2 GT, a 46-kD
protein was detected (Fig. 1A). The broad profile of
the band was characteristic of GLUT I.17 The antibody raised against the purified human erythrocyte
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No. 5
GLUT1 IN CILIARY BODY / Tokoro er ol
PC
1661
PC
NPE
N
L _^
cc
PE
BV
Fig. 4. Enlargement of the base of the NPE cell (rectangle) in
Figure 3. Colloidal gold particles representing GLUT 1 are localized
along the plasma membrane of the basal infoldings. PC, posterior
chamber; N, Nucleus of the NPE cell; Bar = 0.1 fim.
GT gave similar results (Fig. IB). Preimmune serum
showed no detectable band.
When paraffin sections were stained for GLUT1 by
the immunoperoxidase method, ciliary body epithelium was positively stained (Fig. 2). In the iris, where
the epithelium is a continuation of the ciliary body
epithelium, the epithelial cells also exhibited positive
staining for GLUT1 (Fig. 2). In addition, blood vessels in the iris stroma were positive for GLUT1.
. To examine the ultrastructural localization of
GLUT 1, we labeled ultrathin sections of specimens
embedded in LR White by the immunogold method.
Figure 3 shows the ciliary body epithelium. Both PE
and NPE cells were positively labeled for GLUT 1. In
the NPE cells, which faced the posterior chamber, the
colloidal gold label was evident at the plasma membrane of the basal infoldings (Figs. 3, 4). In the outer
Fig. 3. Ultrastructural localization of GLUT 1 in the ciliary body
by the immunogold method. Colloidal gold particles representing
GLUT1 are seen along the plasma membranes of both NPE cells
(NPE) and PE cells (PE). PC, posterior chamber; CC, ciliary channel; BV, blood vessel. Rectangles indicate the areas shown in Figures 4-6. Bar = 0.5 ^m.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1991
portion of the ciliary body epithelium, basal infoldings of PE cells were densely labeled for GLUT 1 (Figs.
3, 5). The label was also seen along the lateral and
apical plasma membranes of both NPE and PE cells
(Figs. 3, 6). Fenestrated capillaries were seen beneath
the PE cells.9 However, we did not detect any positive
staining in the endothelial cells of these capillaries
(Figs. 3,5). The apexes of PE and NPE cells were connected by well-developed gap junctions,6-7'9'22"24
where a small number of gold particles was seen. Ciliary channels exhibited positive staining for GLUT 1
(Figs. 3, 6).
In the iris, the epithelium is made of two layers of
cells that are a continuation of the ciliary body epithelium. Colloidal gold particles representing GLUT1
were seen along the entire aspect, except for junctional regions, of the plasma membrane of epithelial
cells in both layers (Fig. 7). Capillaries in the iris
stroma are of the nonfenestrated' continuous type.9
The endothelial cells, connected by tight junctions,
serve as a part of the blood-aqueous barrier.9'25 Posi-
PE
NPE
PE
GC
Fig. 6. The apical sides of NPE and PE cells (rectangle) in Figure
3. Well-developed gap junctions connect the apexes of NPE and PE
cells (arrow), and labeling is sparse along these gap junctions. Colloidal gold particles representing GLUT1 are seen in the ciliary
channel (CC). Bar = 0.1 jim.
tive staining for GLUT1 was seen along the plasma
membranes of both luminal and contraluminal sides
in these endothelial cells (Fig. 8).
No significant labeling was seen in the cytoplasmic
organelles or nuclei of any of the cells, examined. Pretreatment of sections with methanol and H 2 O 2 19 effectively quenched the pseudoperoxidase activity of hemoglobin of erythrocytes. No positive staining was
seen when diaminobenzidine-H 2 0 2 reaction was performed in the sections treated similarly but without
ABC. When anti-GLUTl antibody was replaced with
preimmune serum, none of the positive staining described was detected, confirming the specificity of the
labeling.
The basal infoldings of the PE cells were more
heavily labeled with colloidal gold particles for
GLUT1 than those of NPE cells (Figs. 3-5). As summarized in Table 1, the gold particles at the basal infoldings of PE cells showed approximately two-fold
higher labeling density than those at the basal infoldings of NPE cells. The difference was statistically significant according to the Cochran-Cox test.
Discussion
Fig. 5. Enlargement of the base of PE cells (rectangle) in Figure 3.
Colloidal gold particles representing GLUT 1 are localized along the
plasma membrane of the basal infoldings of PE cells (PE). The
density of the label is higher than that of the NPE cell shown in
Figure 4. The endothelial cell (E) of the blood vessel is negative for
GLUT1. Bar = 0.1 Mm.
Ciliary body epithelium is the site of aqueous humor secretion into the posterior chamber. The composition of aqueous humor is different from that of
blood plasma; it contains low concentrations of
plasma proteins, and a 20- to 60-fold higher level of
ascorbate.2'3 The glucose concentration in aqueous
humor is maintained near the plasma level.2'3 The
rate of glucose diffusion into posterior chamber is
high, and only specific sugars, such as glucose and
galactose, diffuse rapidly into the posterior chamber.2
A transporter for glucose is found in the ciliary
body.11-12
GLUT 1 is one of five isoforms of the facilitated
diffusion GT family (GLUT 1-GLUTS) that mediate
the transport of glucose down its chemical gradient
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GLUT1 IN CILIARY BODY / Takara er ol
No. 5
PC
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across the plasma membrane. 10 GLUT1 has been
found in a variety of tissues and cells such as the erythrocyte, kidney, brain, and placenta.1017 We have
found that GLUT1 is concentrated in the cells of
blood-tissue barriers12 and in this report, we have
shown ultracytochemically that GLUT1 is localized
at the plasma membranes of both types of epithelial
cells, ie, PE and NPE cells, in the rat ciliary body. Our
findings are in good agreement with the report on the
light microscopic11>12 and our preliminary electron microscopic12 observations of GLUT 1 in rat ciliary body
and iris.
The NPE cells are connected by tight junctions and
serve as a diffusion barrier between blood and
aqueous humor. 4 Intravenously injected horseradish
peroxidase passed through the fenestrated endothelial
cells, and easily reached the intracellular spaces
around PE cells and intercellular spaces between
PE and NPE cells, including the ciliary channels. It
was blocked by the tight junctions connecting NPE
cells.4"7'9 GLUT1 at the basal infoldings of NPE cells,
which face the posterior chamber, may serve as a transport machinery for the exit of glucose from the NPE
cells to the aqueous humor. A large area of basal
plasma membrane with dense GLUT 1 is provided by
the highly developed infoldings of the NPE cells,
which may function for the exit of glucose. On the
other hand, a small area of plasma membrane with a
relatively straight contour was seen at the apexes of
the NPE cells (Figs. 3,6),26 and only modest GLUT1
staining was noted there. These observations suggest
that GLUT1 at the apical plasma membrane of NPE
cells may not be a major site of glucose uptake by
NPE cells.
Gap junctions are ubiquitous in the ciliary body
epithelium, connecting PE-to-PE, NPE-to-NPE,
and PE-to-NPE cells. 6 - 7922 - 24 Well-developed gap
junctions connect the cytoplasm of PE and NPE cells
at their apical plasma membranes. Ciliary body epithelium, although it is made of two distinct layers, is a
functional syncytium. 924 Since intercellular gap junctions are permeable to spheroid molecules at least as
large as 900-1000 daltons,27'28 glucose may freely pass
between PE and NPE cells through these gap junctions. The PE cells have well-developed basal infoldings rich in GLUT1, which suggests that glucose may
enter the PE cells there.
Fig. 7. GLUTl in the iris epithelium. In a posterior epithelial cell
(P), colloidal gold particles representing GLUTl are seen along the
plasma membrane of the basal infoldings (arrows). In an anterior
epithelial cell (A), the positive labeling is seen along the interdigitating basolateral plasma membrane (arrowheads). PC, posterior
chamber; S, iris stroma. Bar = 0.1 fim.
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Aprii 1991
Vol. 32
Fig. 8, GLUT1 in capillary endothelial cells of the iris. Colloidal gold particles representing GLUT1 are seen along
the luminal (arrowheads) and contraluminal (arrows) plasma membranes of
the endothelial cell (E). R, red blood cell.
Bar = 0.1 urn.
Because of these ultrastructural features and the
distribution of GLUT 1, we suggest the following as a
major pathway for the transport of glucose through
the ciliary body epithelium (Fig. 9): (1) Glucose passes
the fenestrated endothelial cells through the pores
into the extracellular space; (2) Glucose is transported
into the cytoplasm of PE cells by GLUT1 located in
the basal infoldings of their plasma membrane; (3)
Glucose enters the NPE cell cytoplasm by passing
through the gap junctions that connect the apical
plasma membranes of PE and NPE cells; (4) Glucose
leaves the cytoplasm of NPE cells by the action of
GLUT 1 at the infolded basal plasma membrane and
thus passes into the aqueous humor. The present
model of the transepithelial glucose transport shows
that the transport of glucose is a facilitated diffusion.
It is different from that of the proposed ascorbate
transport system, which is made up of the combination of Na+-dependent active and passive transport.29
A high density of the label, about two-fold compared with the basal plasma membrane of NPE cells,
was seen along the basal plasma membrane of PE
cells, whose area was enlarged by the highly developed
basal infoldings. PE and NPE cells had a similar surface area of their respective basal infoldings.26 These
results indicate that the total number of GLUT 1 in
Table 1. Density of colloidal gold particles
representing GLUT1 at the plasma membrane of the
basal infolding of the ciliary body epithelial cells
Type of epithelial cells
Number of colloidal
gold particles/] \im
of plasma membrane*
Nonpigmented epithelial (NPE) cell
Pigmented epithelial (PE) cell
3.8 ± I.If (10)
9.3 ± 2.2f (12)
* Mean ± standard deviation. Numbers of cells examined are shown in
parentheses. In each cell examined, colloidal gold density was calculated by
counting colloidal gold particles along a 20-50 urn length of plasma membrane.
f Statistically significant {P < 0.05) by the Cochran-Cox test.
the basal plasma membrane of PE cells is about twofold higher than that of NPE cells. A high rate of glucose metabolism by the tricarboxylic acid cycle and
posterior chamber
tight
junction
fenestrated
endothelial
cell
glucose
blood vessel
Fig. 9. Schema showing the route of glucose transport in the
ciliary body epithelium. Glucose passes the endothelial cell through
its pores, is transported into the PE cell by GLUT! in the basal
infoldings, moves into the NPE cell through gap junctions between
PE and NPE cells, and is transported into the posterior chamber by
GLUT 1 in the basal infoldings of the NPE cell. NPE cells connected
by tight junctions serve as the structural basis of the blood-aqueous
barrier.
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GLUT1 IN CILIARY BODY / Tokoro er ol
No. 5
glycolysis was seen in the bovine30 and pig ciliary
body.31 Since ouabain, the inhibitor of Na+ • K+-ATPase, inhibited the oxidation of exogenous glucose by
50% in the ciliary body, it was suggested that a significant portion of glucose from the blood might be metabolized by the ciliary body epithelial cells in supplying ATP for Na+ • K+-ATPase.31 Energy-consuming
active transport systems, such as Na+ • K+-ATP+
+
34
and active ascorbate
ase26,32,33 H • K -ATPase,
29
transport system , have been identified in the ciliary
body epithelial cells. The higher density of the label
for GLUT1 in PE cells may be related to the consumption of glucose by PE and NPE cells, in addition
to the transepithelial transport.
When mRNA encoding GLUT1 was injected into
Xenopus laevis oocytes, an increase in osmotic water
permeability was seen.35 This suggested that GLUT1
also served as a water channel.35 Abundant GLUT 1 in
the ciliary body epithelium, therefore, may play a role
in water permeation through the ciliary epithelium as
well, thus, contributing to the regulation of osmotic
pressure.
The physiologic significance of GLUT 1 in the iris
epithelium is not clear. Since moderately developed
basal infoldings are seen in the epithelial cells,
GLUT1 may contribute to the secretion of glucose to
the posterior chamber. It is possible that GLUT 1 may
facilitate the absorption of glucose by the iris stroma,
since bidirectional glucose transport is possible with
the aid of facilitated diffusion GTs. In the iris stroma,
continuous-type endothelial cells connected by tight
junctions function as a permeability barrier.9-25
GLUT1 at the plasma membrane of these cells may
allow the selective permeation of glucose from the
blood into the iris stroma and the anterior chamber,
thus conforming to our observations that GLUT1 is
in the limiting membranes of blood-tissue barriers.12
Key words: ciliary body. iris, glucose transporter, GLUT1,
blood-aqueous barrier
Acknowledgments
The authors thank M. Fukuda, R. Nakamura, and M.
Kanai of the Kyorin University School of Medicine for
technical assistance.
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