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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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933391/ on 06/17/2017 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1991 1660 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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933391/ on 06/17/2017 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. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933391/ on 06/17/2017 1662 Vol. 32 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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933391/ on 06/17/2017 GLUT1 IN CILIARY BODY / Takara er ol No. 5 PC 1663 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. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933391/ on 06/17/2017 1664 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. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933391/ on 06/17/2017 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. References 1. Stamper RL: Aqueous humor: Secretion and dynamics. In Physiology of the Human Eye and Visual System. Records RE, editor. Hagerstown, MD, Harper & Row, 1979, pp. 156-182. 2. Cole DF: Ocular fluid. In The Eye. Vol. 1A Vegetative Physiology and Biochemistry. Davson H, editor. Orlando, Academic Press, 1984, pp. 269-390. 3. Scars ML: The aqueous. In Adlcr"s Physiology of the Eye. 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Invest Ophthalmol Vis Sci 29:606, 1988. Martin-Vasallo P. Ghosh S, and Coca-Prados M: Expression of Na, K-ATPasc alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells. J Cell Physiol 141:243, 1989. Fain G L Smolka A, Gilluffo MC, Fain MJ ; Lee DA, Brecha NC, and Sachs G: Monoclonal antibodies to the H + -K + ATPase of gastric mucosa selectively stain the non-pigmented cells of the rabbit ciliary body epithelium. Invest Ophthalmol Vis Sci 29:785. 1988. ' Fischbarg J, Kuang K, Vera JC. Arant S, Silvcrstcin SC, Loike J. and Rosen OM: Glucose transporters serve as water channels. Proc Natl Acad Sci U S A 87:3244. 1990. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933391/ on 06/17/2017