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The Retinal Pigment Epithelium in Visual Function

Bereich Experimentelle Ophthalmologie, Klinik und Poliklinik fuer Augenheilkunde, Universitaetsklinikum Hamburg-Eppendorf, Hamburg, Germany

ABSTRACT

I. INTRODUCTION

II. EMBRYONIC ORIGIN OF THE RETINAL PIGMENT EPITHELIUM

A. First Phase: Establishment of Two Layers

B. Second Phase: Differentiation of RPE and Photoreceptors

III. ABSORPTION OF LIGHT AND PROTECTION AGAINST PHOTO-OXIDATION

A. The RPE Is Able to Absorb Light Energy Focused by the Lens Onto the Retina

B. Increasing Imbalance of Protective and Toxic Factors With Aging Leads to Retinal Degeneration

IV. TRANSEPITHELIAL TRANSPORT

A. The RPE Transports Nutrients and Ions Between Photoreceptors and the Choriocapillaris

1. Transport from subretinal space to blood

2. Transport from blood to the photoreceptors

B. Increases in Epithelial Transport Help to Treat Edema: Reductions in Epithelial Transport Cause Retinal Degeneration

V. SPATIAL BUFFERING OF IONS IN THE SUBRETINAL SPACE

A. Spatial Buffering of Ions in the Subretinal Space Maintains Excitability of Photoreceptors

B. Spatial Buffering Gives Rise to Wave Forms in the ERG: Monitoring of Metabolic Status

VI. VISUAL CYCLE OR RETINOID CYCLE

A. Exchange of Retinal Between Photoreceptors and RPE: Isomerization and Reisomerization Between 11-cis and all-trans

B. Inherited Retinal Degenerations Are Caused by Mutations in a Variety of Genes of the Visual Cycle

VII. PHAGOCYTOSIS

A. Photoreceptor Outer Segment Renewal: Phagocytosis of Shed Photoreceptor Membranes by the RPE

B. Failure of Photoreceptor Outer Segment Phagocytosis Leads to Retinal Degeneration

VIII. SECRETION

A. The RPE Secretes a Variety of Growth Factors

B. Changes in the Secretory Activity Are Associated With Proliferative Diseases in the Retina

IX. SUMMARY

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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The retinal pigment epithelium (RPE) is a monolayer of pigmented cells forming a part of the blood/retina barrier (72, 372, 492, 558). The apical membrane of the RPE faces the photoreceptor outer segments (Fig. 1). Long apical microvilli surround the light-sensitive outer segments establishing a complex of close structural interaction. With its basolateral membrane the RPE faces Bruch’s membrane, which separates the RPE from fenestrated endothelium of the choriocapillaris (Fig. 1).

View larger version (43K): [in this window] [in a new window]   FIG. 1. Summary of retinal pigment epithelium (RPE) functions. PEDF, pigment epithelium-derived growth factor; VEGF, vascular epithelium growth factor; Epithel, epithelium.

	II. EMBRYONIC ORIGIN OF THE RETINAL PIGMENT EPITHELIUM

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The development and differentiation of RPE and photoreceptors reflects the unique relationship between both tissues (372, 373, 484, 487, 492). First, the RPE is essential for the proper development of the retina (260, 565). For this purpose, the RPE was found to secrete factors that promote photoreceptor survival and differentiation (3, 126, 306, 531–534). Second, the development of both tissues occurs in concert with developmental steps where the RPE depends on the specific stages in photoreceptor development, and vice versa (373, 484, 487, 492). In the case of a selective loss of the RPE, neuronal retina and photoreceptors are the most affected tissues in the eye (50, 51, 339). Third, the proper development of the retina appears to be dependent on the genes involved in the proper development of the melanogenesis pathway in RPE cells (286). Human albino eyes show abnormal fovea and chiasmatic projections (159, 361).

A. First Phase: Establishment of Two Layers

The development of both tissues can be divided into two main events. In the first stage, the structure of the two layers, the prospective RPE and prospective neuronal retina, is established. After invagination of the optic cup, cells of the neuroepithelium which, on one hand, are designated to become RPE cells and, on the other hand, cells which are determined to become the neuronal retina are building two layers opposed to each other (Fig. 2) and separated by a thin remnant of lumen (487). The remnant of the lumen is filled by a new material, the interphotoreceptor matrix, the IPM (204, 206, 207). Before this stage, the RPE is a ciliated and pseudostratified epithelium (179). After the IPM is formed, the RPE starts to mature. At this stage both layers are still able to differentiate into RPE or neural retina if the IPM is disturbed (204, 206, 207, 224). Several factors were found to be essential for determination of RPE differentiation. The expression of the transcription factors OTX2 (homeodomain-containing transcription factor) and MITF (microphthalmia-associated transcription factor) appears to be critical initial steps of determination and differentiation of the RPE (384). In early development, before formation of the two layers, the region destined to form the anterior parts of the eye express cellular retinol binding protein (CRBP), cellular retinaldehyde binding protein (CRABP), and several enzymes in the retinal metabolism pathway. The embryonic retina anlage releases retinoic acid. RPE cells, in turn, express receptors for retinoic acid (RAR-₂), CRBP, and CRABP (see sect. VI for a more detailed description of these proteins) (396, 513, 515). In addition, it has been shown that retinoic acid alone can promote RPE differentiation (100, 145, 393, 396, 563), although an exchange of retinoic acid between the developing RPE and developing neural retina also seems to be of importance. This exchange and retinoic acid buffering are mediated by interphotoreceptor retinal binding protein (IRBP) (204). IRBP is present in the IPM from the onset of IPM formation, which is at embryonic day 19 in rodents, for example (110, 112, 156, 204, 206, 207, 562). IRBP is synthesized in cells destined to become photoreceptors and also those that will become RPE cells. Thus IRBP is produced long before it is needed in the visual cycle (see sect. VI) and must play an important role in development. This is supported by the finding that the IRBP concentration is much lower in the IPM of the developing eye than in the adult eye in which the visual cycle is fully functional. Onset of expression of the RPE specific protein of the visual cycle, RPE65, occurs in the same time frame as the synthesis of the IPM. RPE65 expression in the rat steadily increased from the first expression at embryonic day 18 to postnatal day 12, just before eye opening (364). Another important signaling pathway essential for the development of both tissues is the hedgehog signaling pathway (462, 623). Different hedgehog signaling pathways are active in the RPE and in the retina. As mentioned above, the selective differentiation of RPE cells mostly affects the development of photoreceptors and neuronal retina. However, the inactivation of the retina-specific hedgehog signaling leads to improper development of the retina (623). Thus the proper development of the retina is not solely dependent on the presence of the RPE.

View larger version (34K): [in this window] [in a new window]   FIG. 2. Initiation of RPE and retina development: frontal section through the center of the optic cup (region of the optic fissure). The arrows indicate how neuroepithelium designated to become the RPE and neuroepithelium designated to become retina move into an opposed position. The space between the two layers will then be filled with interphotoreceptor matrix, the important interface for cross-talk between these two tissues and proper development. The ocular field of the neural plate shows the fate of various regions. [From Reichenbach and Pritz-Hohmeier (487), with permission from Elsevier.]

By now the RPE is a pigmented epithelium showing almost complete apical to basolateral polarity (372) but with short microvilli and small basolateral membrane infoldings. At this stage 75% of the RPE apical architecture has developed (372–374). The surface of the apical membrane is now three times greater than the one of the basolateral membrane. Na⁺-K⁺-ATPase, _V5-integrin (phagocytosis receptor), N-CAM-140 (a morphoregulator), and EMMPRIN (inducers of matrix metalloproteinase secretion) are now primarily localized in the apical membrane (265, 372, 374, 490, 493, 494, 582, 631). Later, the maturation of the apical surface is promoted by the presence of ezrin. Ezrin seems to be essential for the development of long apical microvilli (76, 78). In the developed RPE, ezrin and its associated proteins radixin and moesin might play a role in 11-cis-retinoid transport and, thus, in the visual cycle (see sect. VI). A key role is played by EBP50 [ERM (ezrin, radixin, moesin)-binding phosphoprotein 50], which interacts with CRBP (75, 425). This polar distribution of these proteins is thought to be achieved by a suppression of basolateral sorting mechanisms. This may be due to masking of a basolateral targeting signal by its serine/threonine phosphorylation (92, 116). Furthermore, active apically directed transcytosis adds to the suppression of basolateral sorting mechanisms. This was shown using the influenza hemagglutinin (77). A second mechanism may be the suppression of gene expression of basolateral sorting adaptors. If this is the case, the suppression modulates several different sorting mechanisms at different times. For example, N-CAM is already localized exclusively to the apical membrane, whereas EMMPRIN is still detectable in the basolateral membrane (372, 374). In addition to intracellular factors, several receptors interacting with extracellular signaling molecules such as integrins and cadherins also play a role in the formation of this special apical to basolateral polarity (25, 122, 125, 379, 394).

Another important factor for the establishment of the apical to basolateral polarity is the formation of tight junctions between RPE cells. Three stages of tight junction development have been described (34, 36, 314, 456, 481, 494, 634). In the early stage, key proteins for tight junction formation are expressed. Because these tight junction complexes are only rudimentary at this point, the RPE forms a leaky barrier at this stage. This corresponds with the stage of a partial apical to basolateral polarity. The tight junctional complexes define the apical and basolateral parts of the membrane because they decrease a free exchange of membrane proteins over the cell membrane (492). In this phase -tubulin was found in the apical membrane, whereas ₁₃-integrin is present in the basal membrane (494, 496). At the end of the early phase, defined as late early phase by Rizzolo (492), the Na⁺-K⁺-ATPase becomes apically polarized in the central region (493, 495) and apical microvilli start to elongate (89, 90). The second stage is designated the intermediate stage. During this stage tight junctions have constant tight junctional connections but develop a constant decrease in permeability. This is due to a major change in the isoform composition of tight junction proteins. Now tight junctions not only prevent free diffusion of membrane proteins over the cell membrane but also the underlying maturation of the cytoskeleton additionally defines the apical to basolateral polarity (492). In this phase a remodeling of the basal membrane occurs (490–492, 495). In the third or late stage of tight junction development, the composition of tight junction protein isoforms stabilizes and the tight junction permeability decreases to that characteristic of a tight epithelium. This is primarily due to an increase in the number and branching of tight junction strands. According to studies using chick RPE, mature tight junctions are composed of ZO-1, occludin, and the claudins 1, 2, 5, 12, and AL (481). The formation of tight junctions establishes the blood retina barrier and signifies coincident onset of epithelial transport. Thus, following completion of tight junctions, the RPE starts to express transporters like the glucose transporter, which is essential for transepithelial glucose transport (35).

B. Second Phase: Differentiation of RPE and Photoreceptors

With the differentiation properties acquired by the end of the first developmental phase, the RPE is prepared to interact with photoreceptors. Now primordial photoreceptors can start to differentiate (179). It is in this second developmental phase that the photoreceptors and RPE undergo the last differentiation events and become a functional unit. Primordial photoreceptors start to extend their outer segments. The RPE responds by elongating its apical microvilli into the subretinal space. The microvilli start to surround the growing outer segments of photoreceptors. By the end of differentiation, the RPE has developed two types of microvilli (84, 179): long microvilli (5–7 µm) that maximize the apical surface for epithelial transport (see sect. IV) and shorter microvilli that form photoreceptor sheaths for phagocytosis of photoreceptor outer segments (see sect. VII). Accompanying onset of microvilli growth at the apical membrane is the formation of deep basal infoldings in the basolateral membrane. Thus RPE and photoreceptors are interacting as they undergo their last maturation steps. This tight coordination of differentiation is also reflected in differential gene expression in these cells. For example, in the RPE, the expression of the visual cycle protein RGR opsin (591) and the putative Cl channel bestrophin is coordinated with the onset of the electrical activity in the retina including photoreceptors (33).

The coordinated maturation requires the adaption of RPE cells to different functional properties of the retina. Differentiated RPE cells in the macula are smaller with 14 µm in diameter and a height of 12 µm than cells in the periphery which have a diameter of 60 µm and which show a variable height (380, 580, 665). Together with a higher melanin content (625), the different cell morphology in the macula leads to organization of melanosomes, which is essential for a more efficient light absorption (85). This is believed to be the reason for the observation that the RPE in the macula appears darker (85, 86). For interaction with photoreceptors, RPE cells develop sheaths that closely surround photoreceptor outer segments (31, 555, 560). Because cones and rods are surrounded by different types of sheaths, the RPE cells in the macula have a different apical architecture (555, 560). Due to the higher number of photoreceptors per RPE cell in the macula, macular RPE cells (143, 194, 544) are adapted to a higher turnover rate of shed photoreceptor outer segments (595) (see sect. VI). For example, macular RPE cells display higher enzyme activities that are required for degradation processes (87, 93, 249).

The RPE is essential for the development of retinal structures partly because of its capability to secrete a variety of growth factors. Growth factors secreted by the RPE function in both endothelial cell differentiation and photoreceptor differentiation (3, 48, 94, 282, 306). This secretion of growth factors is maintained in the adult eye and helps to stabilize the structural integrity of the retina (described in detail in sect. VIII).

	III. ABSORPTION OF LIGHT AND PROTECTION AGAINST PHOTO-OXIDATION

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A. The RPE Is Able to Absorb Light Energy Focused by the Lens Onto the Retina

The RPE increases optical quality by forming a dark pigmented wall cover of the inner bulbus, which aids in absorption of scattered light. In addition, RPE pigmentation is essential for maintenance of visual function. Light enters the eye via the pupil and is focused onto the macula lutea by the lens. This concentrates light energy onto the retina. The outer retina is also exposed to an oxygen-rich environment. The blood perfusion of the choriocapillaris is 1,400 ml · min^–1 · 100 g tissue^–1, which is even higher than the perfusion in the kidney (17, 19). Venous blood from the choriocapillaris shows a 90% O₂ saturation, indicating that there is a negligible O₂ extraction during the passage through the choriocapillaris (16–19). By comparison, venous blood from retinal vessels shows a O₂ saturation of 45% (16–19). The retina is thought to float on the choriocapillaris, which appears to function as a bed of blood-filled vessels. This combination is ideal to allow photo-oxidation and subsequent oxidative damage. This photo-oxidative activity is increased by the load of reactive oxygen species produced by phagocytosis of shed photoreceptor outer segments (see sect. VII) (399). As elegantly reviewed by Boulton and Dayhaw-Barker (86), the RPE has three lines of defense against this damage and toxins. The first line is absorption and filtering of light. For this purpose, the RPE contains a complex composition of various pigments that are specialized to different wavelengths and the special wavelength-dependent risks (44–46). General light absorption occurs via melanin in melanosomes (85). This is supplemented by additional light absorption by photoreceptors. Photoreceptors contain as the most important pigments the carotenoids lutein and zeaxanthin (74, 244, 245, 332, 435). These pigments form a kind of biological sunglasses that absorb blue light (44–46). Blue light appears to be most dangerous for RPE cells in the adult eye because it permits the photo-oxidation of lipofuscin components to cell toxic substances (52, 53, 352, 503, 504, 547, 549, 550, 552–554). The exposure of the adult retina to ultraviolet light is rather low because the lens absorbs most of the ultraviolet light. However, melanosomes and blue light absorbing pigments are only responsible for the absorption of 60% of light energy (84, 85). This implies the presence of other pigments that have not been described yet. One of these pigments might be lipofuscin, which accumulates in the RPE during life (140, 625). As a light-absorbing pigment lipofuscin might first be beneficial for visual function. In the older eye, lipofuscin concentration seems to reach a toxic concentration for the RPE. The second line of defense is made by antioxidants. As enzymatic antioxidants, the RPE contains high amounts of superoxide dismutase (185, 399, 428, 447) and catalase (399, 594). As nonenzymatic antioxidants, the RPE accumulates carotenoids, such as lutein and zeaxanthin (45, 46), ascorbate, -tocopherol, and -carotene (45, 429). This is supplemented by glutathione and melanin, which itself can function as an antioxidant. The third line of defense is the cell’s physiological ability to repair damaged DNA, lipids, and proteins.

B. Increasing Imbalance of Protective and Toxic Factors With Aging Leads to Retinal Degeneration

A reduced capability to absorb light energy is an important factor in the cascade of events leading to age-related macular degeneration (AMD), the most common cause for blindness in industrialized countries (20, 45, 52, 84, 86, 261, 311, 659). An increase in oxidative stress due to a reduction in protective mechanisms or an increase in number and concentration of active photo-oxidative reaction species are believed to contribute to the pathogenesis of AMD (84, 86, 87). One starting point is the accumulation of lipofuscin in the RPE (139–141). The onset of this chain of events is based in age-dependent changes of the RPE. This includes a reduction in the cell density of RPE cells while the epithelial layer remains intact (143, 226, 415). The reduction in cell density itself may result from apoptosis, which is caused by accumulation of toxic substances. This is enhanced by an age-related reduction in one of the most important antioxidants, -tocopherol (186). Additional important age-related alterations are changes in pigmentation. These changes include age-dependent reduction of melanosomes as well as an increase in the number of lipofuscin granules (165, 166, 521, 625). New types of pigmented organelles can also be detected. These are melanolysosomes, which are a sign of melanin degradation and melanolipofuscin granules, which are a result of accumulation of lipofuscin in melanosomes (165). The increase in the amount of reactive oxygen species destabilizes intracellular membrane compartments such as lysosomes and mitochondria. The resulting decrease in metabolic efficiency produces more lipofuscin and reactive oxygen species. It has been reported that oxidative stress can be seen as the accumulation of advanced-glycation end products (AGEs) in Bruch’s membrane (243). These AGEs may also play an important role in the induction of choroidal neovascularization (CNV). The developing neovascular tissue contains high amounts of AGE and active receptors for AGEs (RAGE) expressed on RPE cells (242). RPE cells are able to secrete vascular endothelial growth factor (VEGF), the major angiogenic factor in CNV, in response to AGE exposure (360). One important constituent of lipofuscin is derived from the inability of the RPE to convert all all-trans-retinol into 11-cis-retinal (see sect. VI) (548, 550). An additional source is photoreceptor outer segments that form the precursor of this compound (see sect. VI) This precursor enters RPE cells by phagocytosis of photoreceptor outer membranes. The resulting compound, N-retinyl-N-retinylidene ethanolamine, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium or A2E, increases the sensitivity of the RPE to blue light and has several toxic effects on RPE cells (55, 56, 262, 523, 524, 548, 549, 552, 553). Coupled with oxygen, A2E is converted by light of the wavelength 430 nm into A2E-epoxides (53, 553). In this reaction, oxygen reacts with the carbon-carbon double bonds of A2E to form epoxides. Studies of a mouse model for Stargradt’s disease (see sect. VI) showed the light-dependent accumulation of A2E photoreactivity in the RPE (479). The light-dependent toxic effect of A2E was demonstrated in vitro when RPE cells were fed A2E (53, 503–505, 524, 553). This had a toxic effect on the RPE cell only when coupled to exposure to blue light. The active compound is able to destabilize mitochondrial membranes and lysosomal membranes (55, 56, 262, 523). In addition, A2E can inhibit cytochrome oxidase in a light-dependent manner which results in interruption of electron flow in the respiratory chain (529, 530, 586). This not only reduces efficiency of energy metabolism but also produces more reactive oxygen species. However, the importance of A2E photoreactivity for AMD in vivo is not entirely proven. Pawlak and co-workers (454, 455) reported that compared with other related compounds A2E showed a low photoreactivity that was insufficient to account for reactive oxygen species in the human RPE. Thus other components of lipofuscin A2E must be the principal photo-inducible generators of a range of reactive oxygen species (503, 505). Destabilization of lysosomal and mitochondrial membranes has also been shown without light exposure (261, 262, 523). Nonoxidized A2E was capable of inducing comparable effects in studies with isolated mitochondria and lysosomes (84, 87, 262, 523, 548, 586). An alternative toxic pathway for A2E was described by Finnemann et al. (172). In this study A2E-laden RPE cells did not show destabilized lysosomes, but the cells failed to complete the digestion of phagocytosed photoreceptor outer segments in 24 h. Because the phagocytosis (see sect. VII) is a circadian-regulated process, this would constantly increase nondegraded phospholipids, which represents a source for reactive oxygen species. The destabilization of mitochondria and the incomplete digest of proteins and lipids by destabilized lysosomes lead to a subsequent increase in accumulation of reactive oxygen species and free radicals. In a vicious cycle, these mechanisms further destabilize RPE cells leading to a loss of RPE cells, which denotes the beginning of the formation of Drusen (20, 226–228). Drusen is the most important symptom of age-related macular degeneration and consists of basal laminar deposits that are located between RPE and Bruch’s membrane and basal linear deposits that are located inside Bruch’s membrane. These deposits include metabolic end products such as lipoproteins and other hydrophobic materials (227). These might be a consequence of incomplete degradation of metabolic end products from both photoreceptors and RPE. However, a more detailed analysis of the protein composition of Drusen led to alternative theories of Drusen formation (128, 226, 519). In one theory, the formation of Drusen begins with loss of RPE cells that are removed by an inflammatory event. The resulting gap in the epithelial barrier is actively closed by adjacent RPE that secretes a new extracellular matrix (226). The major matrix component is vitronectin (228). The theory is supported by detection of active dendritic cells and active components of the complement system in Drusen (414, 415). The hydrophobic material and lipoproteins could be the debris left over by incomplete degradation of cells. The end stages of the disease are either geographic atrophy (GA), a loss of RPE and photoreceptors over large areas, or CNV with subsequent intraocular bleeding and formation of a disciform scar (20, 99, 659).

	IV. TRANSEPITHELIAL TRANSPORT

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A. The RPE Transports Nutrients and Ions Between Photoreceptors and the Choriocapillaris

1. Transport from subretinal space to blood

There is a large amount of water produced in the retina derived primarily as a consequence of the large metabolic turnover in neurons and photoreceptors. Furthermore, intraocular pressure leads to a movement of water from the vitreous body into the retina (236, 366, 369). This establishes the need for constant removal of water from the retina. Water in the inner retina is transported by Müller cells (411, 418), and water in the subretinal space is eliminated by the RPE. Furthermore, the water transport is required for close structural interaction of the retina with its supportive tissues in establishment of an adhesive force between RPE and the retina (308, 366, 370).

The RPE transports ions and water from the subretinal space or apical side to the blood or basolateral side (Fig. 3A) (268). Therefore, the RPE has the structural properties of an ion transporting epithelium. Tight junctions establish a barrier between the subretinal space and choriocapillaris (34, 313, 315, 434, 492). The paracellular resistance is 10 times higher than the transcellular resistance, classifying the RPE as a tight epithelium (404, 405). Furthermore, the RPE has an apical to basolateral polarity by structure, organization of organelles, and distribution of the membrane proteins (72, 107, 136, 189, 219, 290, 372–374, 477, 490–494). On the apical side, the RPE extends long microvilli, and on the basolateral side, the membrane is thrown into deep foldings. The majority of mitochondria are located near the basolateral side (372).

View larger version (31K): [in this window] [in a new window]   FIG. 3. Epithelial transport and pH regulation. A: transepithelial transport of ions across the RPE is shown. Cl– is predominantly transported from the subretinal space to the choriocapillaris. This transport drives water through aquaporins from the subretinal space to the choriocapillaris. The driving force for epithelial transport is provided by the apically located Na+-K+-ATPase. K+ transported by the ATPase recycles through the apical membrane and facilitates establishment of a Na+ gradient into the cell. The Na+ gradient serves to drive uptake K+ and Cl– on the apical side resulting in a high intracellular Cl– activity. Cl– is extruded from the cell on the basolateral side through Cl channels. The efficiency of Cl– extrusion from the cell is reduced by activity of a Cl–/HCO3– exchanger, which transports Cl– back into the cell. B: transport of lactic acid and pH regulation is also shown. Lactic acid is removed from the subretinal space by the lac–-H+ cotransporter. Through the basolateral membrane lactic acid leaves the cell using a different subtype of lac–-H+ cotransporter. The necessary pH regulation occurs using Na+/H+ exchanger and Na+/HCO3– exchanger in the apical membrane and a Cl–/HCO3– exchanger at the basolateral membrane. The activity of Cl–/HCO3– exchanger is supported by basolateral Cl channels, which result in a cycling of Cl– across the basolateral membrane. In the case of intracellular acidification, the Cl–/HCO3– exchanger is inhibited whereas Cl channels are activated. This increases the net water transport. ClC-2, voltage-dependent Cl channels of the ClC family; CFTR, cystic fibrosis transmembrane conductance regulator; Kir7.1, inwardly rectifying K+ channel 7.1; maxi-K, large-conductance Ca2+-dependent K+ channel; MCT1, monocarboxylate transporter 1; MCT3, monocarboxylate transporter 3.

The Na⁺-K⁺-ATPase establishes a gradient for sodium from the extracellular space to the intracellular space. At the apical membrane this gradient facilitates uptake of HCO₃^– via the Na⁺-HCO₃^– cotransporter and uptake of K⁺ and Cl^– via the Na⁺-K⁺-2Cl^– cotransporter (5, 236, 267, 290, 295, 297, 298, 327, 346). Accumulation of Cl^– results in a high intracellular Cl^– activity of 20–60 mM (290, 325 , 402, 477, 632). The intracellular Cl^– activity is increased by coupling Cl^– transport to HCO₃^– transport for regulation of intracellular pH. For this purpose, a basolateral Cl^–/HCO₃^– exchanger extrudes HCO₃^– from the cell and increases the intracellular Cl^– concentration (150, 151, 295, 346, 347). Cl^– leaves the cell via Ca²⁺-dependent Cl channels (248, 475, 574, 577, 585, 607) and via ClC-2 channels (80, 248, 287, 629). In addition, immunohistochemical analysis shows the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the RPE, which might provide an additional efflux pathway for Cl^– over the basolateral membrane (71, 401, 482, 629, 635). CFTR is known to function as a Cl channel activated by protein kinase A (PKA) (2). The observation of a cAMP-dependent increase in transepithelial Cl^– transport indicates the functional role of CFTR in the RPE (71, 401). In the regulation of intracellular pH, the activity of the Cl^–/HCO₃^– exchanger and the activity of the Cl channels are combined resulting in a cycling of Cl^– over the basolateral cell membrane. In addition, the Cl^–/HCO₃^– exchanger also decreases the efficiency of extrusion of Cl^– from the cytosol and, thus, the efficiency of transepithelial Cl^– transport. Intracellular acidification results in a decrease in the transport activity of the Cl^–/HCO₃^– exchanger (150, 347). Thus intracellular acidification results in an increase in epithelial Cl^– transport, which might be important in elimination of water from the retina during increased metabolic activity. This is of importance because increased metabolic activity may cause intra- and extracellular edema in response to increased formation of lactic acid as well as increased uptake of glucose (80, 369).

K⁺ entering the RPE on the apical site by Na⁺-K⁺-ATPase and Na⁺-K⁺-2Cl^– cotransporter can leave the cell through the basolateral or the apical membrane via K⁺ channels. Normally, the basolateral K⁺ conductance is higher than the apical K⁺ conductance establishing the basis for the net transepithelial K⁺ transport from the subretinal space to the choroidal site (64, 189, 213, 250, 290, 328, 405, 477). Depending on changes in the K⁺ concentration in the subretinal space, this transport direction can be changed (see below) (328). The apical K⁺ conductance is primarily provided by inward rectifier K⁺ channels of the Kir 7.1 subtype (274, 276, 323, 528, 536, 571, 576, 593, 649). These K⁺ channels are located in the apical microvilli of the RPE (323, 536). Two main functions have been attributed to these inward rectifier K⁺ channels. The close colocalization of Kir 7.1 and Na⁺-K⁺-ATPase suggests that these act synergistically. Kir 7.1 promotes a cycling of K⁺ through the apical membrane (214, 215, 250, 273, 275, 276, 323, 436–438, 528, 536). The resulting decrease in intracellular K⁺ concentration together with decreasing gradient of K⁺ against which the Na⁺-K⁺-ATPase is transporting facilitates the Na⁺ transport out of the cell through the apical membrane. In this way, the Kir 7.1 increases the efficiency of Na⁺-K⁺-ATPase for transporting Na⁺ across the apical membrane. As mentioned above, the transepithelial transport of ions is linked to pH regulation. The inward rectifier K⁺ channels appear to be dependent on extra- and intracellular pH (657). Acidification results in an activation of these K⁺ channels and therefore facilitates Na⁺ transport by Na⁺-K⁺-ATPase, thereby transepithelial transport. With these properties, Kir channels can help the transepithelial transport adapt to increases in metabolic activity and pH regulation required for transport of lactic acid. A second function of Kir 7.1 is to react in response to changes in the subretinal K⁺ concentration, which will be described in detail in section V (276, 528). Channels responsible for efflux of K⁺ over the basolateral membrane have not been clearly identified. Candidates include the large-conductance Ca²⁺-dependent K⁺ channel (508, 592) or the M-type K⁺ channel (587). These K⁺ channels can both provide a K⁺ conductance over a broad voltage range, and they are coupled to second messenger signaling pathways making them good candidates for function in transepithelial transport of K⁺.

Epithelial transport of Cl^– and K⁺ drives epithelial transport of water. The transport rate of water was estimated between 1.4 and 11 µl · cm^–2 · h^–1 (123, 152, 271, 602, 604, 640). Because the RPE is a tight epithelium, water cannot pass through the paracellular transport route. Thus movement of water occurs mainly by transcellular pathways. In a recent publication, expression of aquaporin-1 was detected in the RPE. Furthermore, it was shown that the transepithelial transport of water is facilitated by the functional presence of aquaporin-1 (238, 556).

In the RPE’s role to support photoreceptor function, it is responsible for elimination of metabolic end products from the photoreceptors (237, 302, 330, 660). The most important metabolic end product seems to be lactic acid for which a subretinal concentration of 19 mM has been reported (4). Photoreceptor outer segments are known to produce lactic acid and might represent the major source for lactic acid (263, 637). A smaller part of lactic acid might come from inner segments, which react to transient changes in illumination with increases in lactate production (21, 67, 620). The transport of lactic acid by the RPE (Fig. 3B) requires an efficient regulation of intracellular pH (452). Lactic acid is removed from the subretinal space by the lactate-H⁺ cotransporter MCT1 (54, 345, 467) and the Na⁺-dependent transporter for organic acids (302). Protons are delivered to the subretinal space by the apically located Na⁺/H⁺ exchanger (150, 295, 658). Thus uptake of lactic acid by MCT1 is by tertiary active transport. Lactic acid is extruded through the basolateral membrane from the intracellular space to the choroid by MCT3 (467, 650) and the Na⁺/lac^– exchanger (302). The subretinal pH as well as intracellular pH of the RPE are regulated by transepithelial transport of HCO₃^– (295, 296, 346). In the apical membrane the Na⁺/HCO₃^– cotransporter transports HCO₃^– into the cell (267, 290, 295, 301, 326, 327, 329, 346). This is an electrogenic cotransporter which transports 1 Na⁺ with 2 HCO₃^– (326, 327, 329). Thus the transport direction is dependent on the apical transmembrane potential and intracellular HCO₃^– concentration. This enables the HCO₃^– transport system to regulate the transport direction in response to pH changes. At high intracellular and subretinal pH, HCO₃^– is taken into the RPE cells by the Na⁺/HCO₃^– cotransporter in the apical membranes and leaves the cell through the basolateral membrane in exchange with Cl^– mediated by the Cl^–/HCO₃^– exchanger (144). This results in a subretinal to choroid directed HCO₃^– transport. At low intracellular and subretinal pH, new driving forces for HCO₃^– are established. In this case HCO₃^– is taken up by the Cl^–/HCO₃^– exchanger in the basolateral membrane and leaves the cell through the Na⁺-HCO₃^– cotransporter in the apical membrane resulting in a HCO₃^– transport from the choroid to the subretinal space. The coupling of transepithelial HCO₃^– transport with pH regulation and Cl^– transport is explained above.

There is a small amount of transepithelial Na⁺ transport by the RPE, and the pathway controlling this is not fully understood. Na⁺ is extruded from the cell through the apical membrane via Na⁺-K⁺-ATPase, but it enters the cell primarily through the Na⁺-2Cl^–-K⁺ cotransporter. Therefore, most of the transported Na⁺ recycles through the apical membrane. However, for the small amount of transepithelial transported Na⁺ which leaves the RPE cell through the basolateral membranes, the basolateral transport mechanism is not clear. This transmembrane transport is coupled to other metabolites and has to occur against a Na⁺ gradient. A Na⁺-coupled lactic acid transporter and an additional Na⁺-HCO₃^– cotransporter have been proposed to mediate this function in bovine RPE (301, 302). Using the Na⁺/HCO₃^– cotransporter seems unlikely because the other HCO₃^– transport mechanisms in the basolateral membrane should not support the establishment of a large enough HCO₃^–-dependent driving force to transport Na⁺ against its concentration gradient.

RPE-mediated removal of water from the retina and the subretinal space is coordinated with the neuronal retina. The regulation of water and ion transport by neuropeptide Y and serotonin imply such a coordination. Neuropeptide Y stimulates both transport of Cl^– and water via stimulation of Ca²⁺-dependent Cl channels (23). Serotonin increases transepithelial potential, which mainly results from transport of Cl^– from the subretinal space to the choroid (91, 269–272, 424, 449). Furthermore, the stimulating effect of epinephrine on fluid absorption suggests that there is a sympathetic influence (49, 289, 478, 511). At least purinergic stimulation of RPE cells results in activation of several types of ion channels as well as in an enhancement of fluid absorption (363, 406, 508).

2. Transport from blood to the photoreceptors

In one direction, the RPE transports electrolytes and water from the subretinal space to the choroid, and in the other direction, the RPE transports glucose and other nutrients from the blood to the photoreceptors. To transport glucose, the RPE contains high amounts of glucose transporters in both the apical and the basolateral membranes. Both GLUT1 and GLUT3 are highly expressed in the RPE (35, 54, 582). GLUT3 mediates the basic glucose transport while GLUT1 is responsible for inducible glucose transport in response to mitogens or oncogenes and can, thus, adapt glucose transport to different metabolic demands.

Another important function of the RPE is the transport of retinol to ensure the supply of retinal to the photoreceptors. The bulk of the retinal is exchanged between RPE and photoreceptors during the visual cycle in which all-trans-retinol is taken up from photoreceptors, isomerized to 11-cis-retinal, and redelivered to photoreceptors (30). The vitamin A (all-trans-retinol) uptake from the bloodstream through the basolateral membrane constitutes a smaller, additional supply to this process. The uptake of vitamin A occurs in a receptor-mediated process with recognition by a serum retinol-binding protein/transthyretin (RBP/TTR) complex (466, 476, 615). Within the RPE cell, all-trans-retinol binds to CRBP (513–515) and enters the isomerization and oxidation steps of the visual cycle (30). The subsequent reactions are described in detail in section VI. Once vitamin A is converted to 11-cis-retinal, it is transported to the photoreceptors where it binds to opsin and can serve in its function to initiate the phototransduction cascade (30). The direction of transepithelial transport of vitamin A from the blood to the photoreceptors is achieved by binding retinal to specific binding proteins that ensure absence of free retinol inside the cell (254). In addition, the rapid transfer through the visual cycle maintains a constant gradient of retinol from inside the RPE cell and the bloodstream and drives the directed transport (392, 512).

The space between the RPE and photoreceptors, the IPM, forms an important interface for interaction between RPE and photoreceptors. The IPM mediates adhesion between the RPE and photoreceptor layer, phagocytosis by the RPE (described in more detail in section VII), and nutrient exchange between RPE and the photoreceptors (204, 224). The IPM is a complex structure containing IRPB, growth factors such as basic fibroblast growth factor (bFGF), hyaluronan and hyaluronan binding proteoglycans, sulfated glycosaminoglycans, and matrix metalloproteases (1, 225, 253, 258, 259, 320). The IPM changes its structure between light and dark (610). In spite of its importance, the exchange of retinal and retinol between RPE and the photoreceptors is not understood (204). A very promising candidate to mediate exchange of retinal and retinol was IRBP (72, 107, 111, 113, 114, 206, 207, 254, 532, 562). IRBP functions to solubilize retinal and retinol, which are otherwise insoluble in water and mediates targeting of these compounds and defines the transport direction (107, 108, 130, 445, 446, 457, 458, 518). This role for IRBP is further supported by the observation that IRBP is not only present in the IPM but also in endosomes of the RPE (131). The transport direction is then defined by the rapid turnover of IRBP between the IPM and the RPE. However, this model was recently challenged by investigation of IRBP–/– mice (453). These mice still have an intact visual cycle and a normal regeneration of retinal that can be interpreted in one of two ways: either there is an efficient alternative retinoid transport pathway between RPE and photoreceptors which can compensate for the loss of the IRBP-dependent pathway, or IRBP has a function other than transport of retinal and retinol between RPE and photoreceptors. Even though this alternative function has not been established, detailed structure/binding correlation studies support its existence. IRBP isolated from dark-adapted retinas carries larger quantities of 11-cis-retinal and 11-cis-retinol than from light-adapted retinae (348). This suggests that IRBP has a 11-cis-retinal buffering function and that it protects this essential compound from oxidative damage. With this buffering function, photoreceptors could take up 11-cis-retinal from a pool and would therefore not be dependent on the numerous reactions of the visual cycle pathway to supply 11-cis-retinal, which would be much slower. This would result in faster regeneration of rhodopsin after the onset of light.

Delivery of docosahexaenoic acid to photoreceptors is a third kind of transport of importance for visual function (41). Membranes of neurons and photoreceptors as well as photoreceptor disk membranes are selectively built from phospholipids that are highly enriched with docosahexaenoic acid, a 22:63 fatty acid (26, 29, 178, 566, 621, 633). This compound cannot be synthesized by neuronal tissue. Thus neuronal tissue is dependent on the delivery of 22:63. New photoreceptor outer segments that are rebuilt from the base, the inner segment of photoreceptors, selectively require 22:63 fatty acid (26, 29, 566, 621, 633). This compound is synthesized from the precursor, linolenic acid, in the liver and transported in the blood bound to plasma lipoprotein (26, 29, 566, 621, 633). The RPE preferentially takes up docosahexaenoic acid in a concentration-dependent manner (41–43, 208, 209). In the RPE this fatty acid is incorporated into glycerolipids in de novo synthesis reactions for storage and connection into the recycling pathways during the phagocytosis process (see sect. VII)(499).

B. Increases in Epithelial Transport Help to Treat Edema: Reductions in Epithelial Transport Cause Retinal Degeneration

In diabetic retinopathy or in some forms of inherited macular degeneration, a clinically important complication is the formation of macular edema (369). Macular edema is most likely caused by damage to the blood/retina barrier, which is formed by the RPE and the endothelium of retinal vessels. In the case of diabetic retinopathy, the site of damage is in the endothelium of retinal vessels. Macular edema is successfully treated by administration of inhibitors of carbonic anhydrase (119, 176, 369, 561, 639, 641). This results in a reduction of intracellular pH and intracellular HCO₃^– concentration (180, 293, 371, 404, 604, 648). As a result, transport activity of the Cl^–/HCO₃^– exchanger is reduced, and the uptake of Cl^– via the basolateral membrane is reduced. This increases the efficiency of Cl^– release through the basolateral membrane and enhances transepithelial Cl^– transport. Epithelial transport of Cl^– drives the transport of water and eliminates the fluid of the edema.

Epithelial transport of Cl^– was found to be essential for visual function. This was shown in a transgenic mouse model with a disrupted gene for ClC-2 Cl channels (80, 287). The resulting phenotype has a retinal degeneration comparable to retinitis pigmentosa. The disease is caused by a lack of epithelial transport of Cl^–, the RPE shows no transepithelial potential. It is likely that this results in the inability to extrude lactic acid from the photoreceptor side leading to subsequent metabolic stress and loss of photoreceptors (80). However, the exact mechanism for the retinal degeneration is not fully understood and needs further evaluation. A second example of retinal degeneration associated with defective Cl^– transport is Best’s vitelliform macular degeneration (12, 32, 98, 129, 157, 198, 210, 358, 375, 377, 378, 416, 464, 470, 567, 624). In this disease the retinal degeneration is caused by degeneration of RPE (198). This results in the formation of a bull’s eye shaped lesion that resembles an egg yolk. The shape of the lesion gave rise to the name of this disease. The bull’s eye lesion primarily contains extracellular fluid, suggesting there is a reduction in epithelial Cl^– transport (624). The leading symptom for diagnosis of Best’s vitelliform macular degeneration is a reduction in the light peak-to-dark ratio in the patient’s electro-oculogram (EOG) (198). The light peak results from the activation of basolateral Cl^– conductance (188, 190, 191, 193). Light-dependent stimulation of the retina leads to release of a light peak substance from the inner retina or photoreceptors (188). The light peak substance diffuses to the RPE and leads to activation of basolateral Cl^– channels via activation of intracellular second messenger pathways (192, 193). Because it is likely that the underlying second messenger pathway results in an increase in intracellular Ca²⁺, the basolateral Cl^– conductance is provided by Ca²⁺-dependent Cl channels. The as yet unidentified light peak substance is very likely ATP, which is known to activate the inositol 1,3,4-trisphosphate (InsP₃)/Ca²⁺ second messenger system. Furthermore, an increase in intracellular InsP₃ increases the concentration of cytosolic free Ca²⁺ and subsequently Ca²⁺-dependent Cl channels (573, 577). Thus the reduction of the light peak in patients’ EOG points to a reduction in epithelial Cl^– transport, which might be the cause for the pathology of the disease. The gene responsible for Best’s vitelliform macular degeneration has been isolated and identified as VMD2 gene (378, 464). The VMD2 gene product is named bestrophin (32, 98, 157, 358, 378, 416, 464, 567). Recent data suggest on several lines of evidence that bestrophin itself represents a new family of Ca²⁺-dependent Cl channels (175, 473–475, 585, 605). This elegantly explains the cause for the light peak reduction in the patient’s EOG. Heterologous expression studies showed for 15 different bestrophin mutations a loss of Cl channel function (585). Thus reduction in the light-peak amplitude in the patients’s EOG results from reduction in basolateral Cl^– conductance. However, studies searching for more bestrophin mutations described patients with bestrophin mutations that show normal light peaks or an onset of the light-peak reduction that occurs later than the onset of macular dystrophy (12, 157, 319, 358, 525, 617). In addition, in a recently published rat model for Best’s disease, overexpression of wild-type bestrophin did not change light-peak amplitude but desensitized luminance response while two investigated mutant bestrophins could generate the expected decrease in light-peak amplitude (376). These observations cannot be easily explained by the theory that Best’s disease is caused by a lack of Cl channel function.

Another example of Cl^– transport alteration in the RPE that might result in disease is cystic fibrosis (287, 482). Patients with cystic fibrosis have reduced amplitudes of the fast oscillation in the EOG (71, 629). Like the light peak, the fast oscillation represents a signal in the EOG that is generated by activation of Cl channels in the basolateral membrane of the RPE. The expression of CFTR has been established in RPE cells. Furthermore, part of the transepithelial transported Cl^– is dependent on cAMP-activated Cl conductance (269–272, 321, 325, 342, 401, 403, 422). Thus patients with cystic fibrosis have additional reduction in the transepithelial Cl^– transport by the RPE. However, patients with cystic fibrosis do not develop retinal degeneration perhaps because there is a compensatory activation of other Cl channels, such as the Ca²⁺-dependent Cl channel, in the RPE.

Alteration of transepithelial transport may function in the pathogenesis of age-related macular degeneration. As described above, the development of Drusen is required for diagnosis of this disease (20). In 20–25% of patients, Drusen become large and confluent (311) and establish large diffusion barriers between blood vessels and the RPE. This may lead to development of areas with reduced supply of oxygen and glucose, mimicking hypoxia (183, 360). These regions of hypoxic stress may cause degeneration of adjacent photoreceptors and subsequent loss of vision in areas of Drusen. A further consequence of metabolic stress might be the induction of choroidal neovascularization, the most severe complication in the etiology of age-related macular degeneration (311). Hypoxia reduces the secretion of pigment epithelium-derived growth factor (PEDF) by the RPE (134). PEDF is a potent antiangiogenic factor. The development of choroidal neovascularization can lead to intraocular bleeding, which is the main cause for vision loss (20). This theory is supported by the observation that choroidal neovascular membranes contain high amounts of advanced glycation end products that are generated by reactive oxygen species during reduced metabolism (242).

Finally, reduction in the delivery of retinal to photoreceptors was found to induce a form of photoreceptor dystrophy (526). This rare disease was described as a symptom of the retinol binding protein deficiency syndrome. Here, patients show mutations in the retinol binding protein 4, RBP4, gene. This protein appears to be essential for uptake of retinol into the RPE cells. Because of the reduced retinol uptake, patients have no detectable rod function in dark adaption or in their scotopic ERG. However, these patients do not have a very severe degenerative phenotype, suggesting that there must be an alternative tissue source for vitamin A.

	V. SPATIAL BUFFERING OF IONS IN THE SUBRETINAL SPACE

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A. Spatial Buffering of Ions in the Subretinal Space Maintains Excitability of Photoreceptors

The RPE not only stabilizes the ion homeostasis in the subretinal space by transepithelial transport of ions, but it is also able to compensate for fast occurring changes in the ion composition in the subretinal space (558). This function is comparable to the ability of glia cells for spatial buffering of ions. In fact, a major part of the spatial buffering is enabled by Müller glia cells (427, 486, 644). Another part is controlled by the RPE. Stimulation of photoreceptors by light reduces the dark current, which consists of an influx of Na⁺ via open cGMP-gated cation channels in the outer segments which is counterbalanced by an efflux of K⁺ via K⁺ channels in the inner segment (39). The reduction of the K⁺ efflux results in a decrease of the subretinal K⁺ concentration from 5 to 2 mM (144, 558, 559). This leads to a change in the equilibrium potential for K⁺, which in turn disturbs the phototransduction cascade in the outer segments of the photoreceptors. The RPE compensates for these changes in the following manner: the decrease in the subretinal K⁺ concentration hyperpolarizes the apical membrane of the RPE (436–438), which causes activation of inward rectifier K⁺ channels (276, 528, 536, 649, 657). These inward rectifier channels respond with a mild rectification and an increase in activity in response to a decrease in the extracellular K⁺ concentration (276). The activation of inward rectifier K⁺ channels then causes a change in the ratio of apical and basolateral K⁺ conductance (328). Normally, the basolateral K⁺ conductance is higher than the apical conductance which results in directed epithelial transport of K⁺ from the subretinal space to the choroidal site (328). The rise in apical K⁺ conductance increases the portion of K⁺ that is transported back to the subretinal space. As a result, K⁺ is secreted to the subretinal space and compensates for the light-induced decrease of subretinal K⁺. Application of K⁺ channel blocker can prevent the hyperpolarization-induced decrease in intracellular K⁺ (215, 273). The compensation for the light-induced decrease in subretinal K⁺ is supported by the Na⁺-K⁺-2Cl^– cotransporter that appears to work in the early phase of apical membrane hyperpolarization in the opposite direction, leading to an additional K⁺ outflow of the cell (64, 65). In addition, the decrease in subretinal K⁺ concentration changes the Cl^– transport (64, 151, 213, 214). The decrease in subretinal K⁺ concentration decreases the uptake of Cl^– via the Na⁺-K⁺-2Cl^– cotransporter in the late phase of apical membrane hyperpolarization. The subsequent decrease in intracellular Cl^– activity changes the equilibrium potential for Cl^– through the basolateral membrane which has a high Cl^– conductance (64, 65). This results in a hyperpolarization of the basolateral membrane and decreases the driving force for Cl^– to leave the cell via the basolateral membrane. These compensatory mechanisms can be monitored in the electroretinogram (ERG) as the c-wave and delayed hyperpolarization (214, 436, 558). The c-wave results from the hyperpolarization of the apical RPE cell membrane, and the delayed hyperpolarization results from hyperpolarization of the basolateral membrane. Light-dependent activation of photoreceptors closes cation channels in the outer segments and thus increases the subretinal Na⁺ concentration, which is compensated for by the Na⁺/H⁺ exchanger and by the Na⁺-K⁺-2Cl^– cotransporter in the apical membranes of the RPE. The subsequent decrease in the subretinal Na⁺ concentration after closing of cation channels during the transition from light to darkness is compensated for by Na⁺-K⁺-ATPase in the photoreceptor’s inner segments and by the Na⁺-K⁺-ATPase of the RPE (255). It is believed that this might be why the Na⁺-K⁺-ATPase is localized to the apical membrane of the RPE.

The light-induced changes in ion transport do not only maintain ion homeostasis in the subretinal space. The changes in the transport direction also imply light-dependent changes in water transport (268). This effect is based on the fact that the activity of the apical Na⁺-HCO₃^– cotransporter is dependent on the membrane potential (326). Light-induced hyperpolarization of the apical membrane results in a decrease of its transport activity, which subsequently leads to intracellular acidification (301, 344, 346). This increases Cl^– efflux through the basolateral membrane (see sect. IV) and results in an increase in Cl^– and water transport from subretinal space to choroid. The light-induced increase in water absorption seems to be of importance to control subretinal space volume during changes in illumination. With the use of measurements of the concentration of tetramethylammonium (TMA^–) ions by a TMA^–-sensitive electrode, it was shown that illumination leads to transient volume increases in the retina that were most pronounced in the region between outer nuclear layer and subretinal space (266, 341). It is likely that changes in the extracellular volume of the inner retina are compensated for by Müller cells, whereas the RPE compensates for the changes in the subretinal space. In the dark, the apical membrane of the RPE is depolarized. Now the activity of the Na⁺-HCO₃^– cotransporter rises causing intracellular alkalinization (267, 346). In consequence, less Cl^– leaves the cell through the basolateral membrane. This reduces fluid absorption in the dark (301). Studies on epithelial transport indicate a more severe consequence (65, 603). A decreased efflux of Cl^– out of RPE cells increases intracellular Cl^– concentration providing a driving force for Cl^– to leave the cell through the apical membrane. Indeed, an apical Cl^– secretion was observed when K⁺ concentration at the apical site was increased from 2 to 5 mM (151, 347). Because apical Cl channels have not yet been described, the Cl^– efflux mechanism through the apical membrane is unclear. However, the postulated Cl^– secretion in the dark should result in a fluid secretion in the dark into the subretinal space. Indeed, a corresponding fluid movement from the basolateral side to the apical side has been observed in transepithelial transport studies but is not proven in vivo. The physiological significance of a fluid secretion into the subretinal space in the dark is not clear. However, the secretion likely occurs only for a short time until the increase in subretinal K⁺ concentration has been compensated and the apical membrane is no longer depolarized.

B. Spatial Buffering Gives Rise to Wave Forms in the ERG: Monitoring of Metabolic Status

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