The Function of the Retinal Pigment Epithelium




Introduction


The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the light-sensitive photoreceptor outer segments and the fenestrated endothelium of the choriocapillaris. On both sides, specialized extracellular matrices enable a close interaction of the RPE with its adjacent tissues. On the basolateral side the multilayered Bruch’s membrane combines barrier function and selective transport matrix as part of the blood–retina barrier. On the apical side of the RPE, the interphotoreceptor matrix provides an interface for interaction between the photoreceptor outer segments and the RPE. In this interaction, the RPE forms a functional unit with the photoreceptors ( Fig. 13.1 ). In humans, every RPE cell is in interaction with a mean of 23 photoreceptors. The formation of that functional unit is already of importance during the embryonic development. The RPE and the neuronal retina depend on each other in the process of differentiation and maturation. Mutations in genes expressed in the RPE can lead to primary photoreceptor degeneration and mutations in genes expressed in photoreceptors can lead to primary RPE degenerations. In this functional unit the RPE fulfills a multitude of tasks which are essential for visual function. The failure of one of these functions leads to retinal degeneration.




Figure 13.1


Summary of the different functions of the RPE by which a functional unit between photoreceptors is formed.

(Modified from Strauss O: The retinal pigment epithelium in visual function, Physiol Rev 85:845–81, 2005. Used with permission. )




Absorption of light


As a pigmented layer of cells the RPE covers the inner wall of the bulbus and absorbs scattered light to improve the optical quality. In the human eye light is focused by a lens onto the macula. This represents a high density of energy which is absorbed by the melanin granula of the RPE leading to an increase in the temperature of the RPE choroid complex. The heat is transported away by the bloodstream in the choriocapillaris. However, this functional arrangement bears the danger of photo-oxidative damage. , The relative blood perfusion of the choriocapillaris is higher than that of the kidney. , However, only small amounts of oxygen are extracted by the adjacent tissues and venous blood from the choroid still shows an oxygen saturation of more than 90 percent. Thus, there is an overflow of oxygen in combination with a large density of light energy. This leads to a large production of reactive oxygen species. The RPE is protected by various lines of defense against this intoxication: melanin of the melanosomes and the carotenoids lutein and zeaxanthin absorb light energy, ascorbate, α-tocopherol and β-carotene, and glutathione are non-enzymatic antioxidants and melanin itself can function as an anti-oxidant. This is supplemented by the cell’s natural ability to repair damaged DNA, lipids and proteins.




Transepithelial transport


The RPE cells form a part of the blood–retina barrier. The RPE is by its electrophysiological parameters a tight epithelium: the paracellular resistance is at least ten times larger than that of the transcellular resistance. , This barrier function is of great importance for the immune privilege of the eye (see below). Due to this tight barrier function, the complete exchange of molecules and ions between the bloodstream and the photoreceptor side relies on transepithelial transport through the RPE.


Transport from the blood side to the photoreceptor side


The transport from the blood to the photoreceptor side consists mainly of transport of nutrients such as glucose, ω3 fatty acids and retinal.


For glucose transport, the RPE contains an abundance of the glucose transporters GLUT1 and GLUT3 in both the apical and the basolateral membrane. , GLUT3 mediates the basic transport whereas GLUT1 is responsible for an inducible glucose transport to adopt the glucose transport to different metabolic demands.


All- trans retinol is taken up from the bloodstream via a receptor-mediated process involving a serum-retinol-binding protein/transthyretin complex. Retinol is formed to all- trans retinyl ester and then enters directly the visual cycle (see below).


Docosahexaenoic acid (a polyunsaturated ω-3 fatty acid; 22:6ω3) is essential for the renewal process of the photoreceptor outer segments and cannot be synthesized by the photoreceptors. The compound is synthesized in the liver from the precursor linolenic acid and transported to the eye in the blood bound to plasma protein. The RPE preferentially takes up docosahexaenoic acid in concentration-dependent manner. Docosahexaenoic acid is incorporated into glycerolipids for synthesis and storage.


Transport from the retinal side to the blood side


The retina is the tissue with the highest density of cells. Neuronal cells show a high metabolic activity. This results in the production of large amounts of water and accumulation of lactic acid. Additional amounts of water are moved towards the retina by intraocular pressure from the vitreous. Since the RPE is a tight epithelium water cannot pass in the paracellular route. Water and lactic acid are eliminated from subretinal space by active transcellular transport by the RPE (see Box 13.1 ).



Box 13.1

Retinal Detachment


Retinal detachment is characterized by a separation of the retinal pigment epithelium from the photoreceptor layer of the neuroretina. This separation prevents close interaction between the two layers leading to reduction of visual function. After successful closure of the retinal break, preventing access of fluid from the vitreous to the subretinal space, remaining subretinal fluid reabsorbs spontaneously. Subretinal fluid is eliminated by the electrolyte and fluid pump of the retinal pigment epithelium.


RPE cells usually do not divide, but under certain circumstances they may become activated to play a role in wound repair. These wound repair mechanisms can also have devastating consequences by creating so called “proliferative vitreoretinopathy” (PVR). RPE cells migrating through retinal tears in the vitreous cavity and on the retinal surface may metaplastically transform to myofibroblasts, creating contracting membranes which cause complicated tractive forms of retinal detachment that are difficult to repair.



The transport of water is driven by an active transport of Cl from the retina to blood side ( Fig. 13.2 ). It is energized by the apically located Na + /K + -ATPase which uses the energy of ATP to transport Na + out of the cell in exchange with a transport of K + to the cytosol of the RPE. The K + ions recycle across the apical membrane through inward rectifier potassium channels which provide the large K + -conductance of the apical membrane. This recycling keeps the K + gradient across the apical membrane small to support the activity of the Na + /K + -ATPase. In the apical membrane a Na + /2Cl /K + -co-transporter uses the Na + gradient across the apical membrane to transport K + and Cl into the cytosol of the RPE. Since K + moves back through inward rectifier channels to the subretinal space the Na + /2Cl/K + -cotransporter accumulates Cl in the intracellular space of the RPE cells (40–60 mM). This high Cl concentration provides a driving force for Cl to leave the cell. The basolateral membrane displays a large conductance for Cl through which Cl leaves the cell from intracellular space to the blood side. This is the last step of transepithelial Cl transport from subretinal space to the blood side resulting in a basolateral negative transepithelial potential between −6 to −15 mV. ClC2 Cl channels seem to provide the major part of the basolateral Cl conductance. Other channels providing basolateral Cl conductance are Cl channels linked to intracellular second-messenger systems. Cl transport can be increased by rises in intracellular free Ca 2+ or by increases in cytosolic cAMP concentration resulting from either activation of Ca 2+ -dependent Cl channels or cAMP-dependently regulated Cl channels.




Figure 13.2


Transport of water and ions from subretinal space to blood side.

( A ) Using the energy of ATP hydrolysis the Na + /K + -ATPase establishes a gradient for Na + which drives the uptake of Na + , K + and Cl by the Na + /K + /2Cl cotransporter. K + recycles across the apical membrane through inward rectifier K + channels. The concerted transport activity of the three transport proteins results in the accumulation of Cl in the cytosol of the RPE. Cl leaves the cell through a variety of basolateral Cl channels. The net transport of Cl results in a basolateral negative transepithelial potential. The transport of Cl as osmolytes drives a transcellular transport of water through aquaporine water channels located in both the apical and the basolateral membrane. ( B ) Lactate is taken up from subretinal space via the activity of the MCT1 (monocarboxylate transporter-1) and accumulated in the cell. The driving force for this transport is large because the activity of retinal neurons produces large amounts of lactic acid (concentration in the subretinal space can be as high as 19 mM). Lactate leaves the cell through the basolateral membrane via the activity of the MCT3 transporter. The required pH regulation occurs at the apical membrane by the activity of sodium-dependent transporters: Na + /H + exchanger and the Na + /HCO 3 cotransporter which use the sodium gradient of the Na + /K + ATPase. At the basolateral membrane pH regulation occurs by the activity of Cl -dependent transport proteins which use the large intracellular Cl activity. In this part of the pH regulation a Cl /HCO 3 exchanger and the Cl channel ClC2 are involved.


The metabolic activity in the retina results in large subretinal concentrations of lactic acid which is transported away by the RPE. , This transport requires a tight regulation of the intracellular pH. , Lactic acid is removed from subretinal space by the monocarboxylate transporter 1 (MCT1) which transports H + in cotransport with lactic acid (can be as high as 19mM). This transport is driven by the activity of the Na + /H + -exchanger which eliminates H + from cytosol of the RPE cells using the gradient for Na + provided by the activity of the Na + /K + -ATPase. Across the basolateral membrane lactic acid leaves the cell via the activity of the monocarboxylate transporter 3 (MCT3) which is an H + /lactic acid cotransporter too. Intracellular pH is stabilized by the transport of bicarbonate. , HCO 3 is transported into the cell across the apical membrane by the Na + /2HCO 3 cotransporter which uses the Na + gradient established by the Na + /K + -ATPase. HCO 3 leaves the RPE cell across the basolateral membrane by the activity of the HCO 3 exchanger.


Production of metabolic water and accumulation of lactic acid are linked to each other. Therefore, transport of water and pH regulation are also coupled. An increase in the transport of lactic acid leads to intracellular acidification which inhibits the transport activity of the Cl /HCO 3 exchanger. Since the Cl / HCO 3 exchanger transports Cl in the opposite direction to the Cl channels the exchanger decreases the Cl transport efficiency in resting conditions. The inhibition of the HCO 3 exchanger, thus, increases the transepithelial transport of Cl and water. This is further increased by the ClC2 channels which are activated by a decrease in extracellular pH. Extracellular acidification would result from the increased transport of lactic acid by the MCT3 in the basolateral membrane.




Capacitative compensation of fast changes in the ion composition in the subretinal space


In the dark cGMP-dependent cation channels in the photoreceptor outer segments generate an inward current of Na + and Ca 2+ which is counterbalanced by a K + outward current at the inner segments. In the light, cGMP-dependent cation channels close and the K + current is decreased resulting in a decrease of the K + -concentration in the subretinal space from 5 to 2 mM. The decrease of the Na + conductance in the outer segments leads to an increase in the subretinal Na + concentration. Furthermore, illumination of the retina results in an increase in the subretinal volume. Both changes are compensated for in the moment when they occur by modulation of the ion transport by the RPE.


Both, compensation for changes in the K + concentration and in extracellular volume are linked to the potassium transport. The apical membrane of the RPE displays a large K + conductance. Decrease in the subretinal K + concentration leads to hyperpolarization of the apical membrane which corresponds to the c-wave in the electroretinogram. The decrease in the potassium concentration decreases the activity of the Na + /K + /2Cl cotransporter which subsequently decreases the intracellular Cl activity and hyperpolarizes the basolateral membrane. In the electroretinogram this can be seen as the delayed hyperpolarization. The hyperpolarization of the apical membrane activates inward rectifier K + channels which generate an efflux of K + into the subretinal space to compensate for the light-induced decrease in the subretinal K + concentration. At the same time the apical hyperpolarization decreases the activity of the Na + /HCO 3 cotransporter which is an electrogenic transporter by its stoichiometry. The subsequent intracellular acidification increases the transepithelial Cl and water transport as described above.


The light-induced increase in subretinal Na + concentration is compensated for by the activity of the Na + /K + /2Cl cotransporter and by the Na + /H + exchanger. , The Na + /K + -ATPase seem to uptake Na + to compensate for the increase in the subretinal Na + concentration during the transition from light to dark. It is believed that this task is the reason for its localization in the apical membrane of the RPE.




Visual cycle


Vision starts in photoreceptor outer segments with absorption of a photon by the chromophore of rhodopsin, 11- cis retinal, which undergoes a conformational change from 11- cis retinal to all- trans retinal. For the absorption of the next photon, rhodopsin replaces all- trans retinal to a new 11- cis retinal. To maintain visual function, a re-isomerization of all- trans to 11- cis retinal takes place in the RPE ( Fig. 13.3 ). All- trans retinal leaves the disc membrane as N-retinylidine-phosphatidylethanolamine (N-retinylidine-PE) by the transport activity of the ABC (ATP-binding cassette protein) transporter ABCR4. Released from N-retinylidine-PE, all- trans retinal is reduced to all- trans retinol by RDH (all- trans retinol dehydrogenase), leaves the photoreceptor outer segments into subretinal space, is loaded onto the IRPB (interphotoreceptor matrix retinal binding protein) and transported to the RPE. In the cytosol of the RPE it binds to the CRBP (cellular retinol binding protein) as entry into a reaction cascade catalyzed by a protein complex composed of LRAT (lecithin : retinol transferase), RPE65 (RPE specific protein 65 kDa, function as isomerase ) and 11cRDH (11- cis -retinol dehydrogenase or RDH5) which performs the re-isomerization. After re-isomerization 11- cis retinal loaded to CRALBP (cellular retinaldahyde binding protein), leaves the cell and is transported back to photoreceptors by IRBP. This pathway accounts for the re-isomerization of retinal from rods. Cones might have an additional secondary pathway involving Müller cells.




Figure 13.3


Cycle of retinal (visual cycle).

The process of vision is started by the conformational change of the chromophore of rhodopsin from 11- cis retinal to all- trans retinal. All- trans retinal is re-isomerized to 11- cis in the RPE. The first step into this cycle is the reduction of all- trans retinal into all- trans retinol. all- trans retinol leaves the photoreceptor and binds to IRPB and is transported to the RPE. Inside the RPE all- trans retinol binds to CRBP and enters with this step the re-isomerization pathway. The reaction is catalyzed by a protein complex consisting of LRAT, RPE65 and RDH5. The reaction product, 11- cis retinal binds to CRALBP, leaves the cell where it binds to IRPB again to be transported to the photoreceptors. RPE65 modulation adapts this cycle to different light intensities. In the light it is palmotylated and membrane bound with isomerase function. In the dark it is freely diffusible in the cytosol and serves as retinal pool when the visual cycle has a lower turn-over rate (CRBP = cellular retinol binding protein; CRALBP = cellular retinaldehyde binding protein; IPM = interphotoreceptor matrix; IRBP = interstitial retinal binding protein; LRAT = lecithin retinol acyltransferase; RDH = retinol dehydrogenase; RPE 65 = retinal pigment epithelium specific protein 65 kDa; RDH5 = 11- cis retinol dehydrogenase; Rho = rhodopsin; SER = smooth endoplasmic reticulum).


Since the visual cycle maintains the excitability of the photoreceptors (see Box 13.2 ) it needs to be adapted to the different photoreceptor activities between light and darkness. In the light there is a fast turnover of retinal whereas in the dark the turnover occurs much slower. Retinal which is not needed in the state of a slower turnover must be quickly available for the transition from darkness to light and vice versa. The different retinal binding proteins represent connected pools for retinal. In the transition from darkness to light IRPB represent the first pool which delivers retinal. This pool cannot be depleted because it refills retinal from the pool of CRALBP proteins. CRALBP in turn can recruit retinal from RPE65. Thus, with increasing light intensity retinal can be recruited from the different pools and in the transition from light to darkness retinal can be stored away. Of importance are IRBP which can carry in the dark larger amounts of retinal than in the light. Another key function is the modulation of the RPE65 function. In the dark RPE65 is water soluble and freely diffusible in the cytosol. With this configuration RPE65 functions as a retinal store. In the light RPE65 is palmotylated and bound to intracellular membranes. In this configuration, RPE65 predominantly catalyze the re-isomerization of all- trans retinol to 11- cis retinol.


Jan 23, 2019 | Posted by in OPHTHALMOLOGY | Comments Off on The Function of the Retinal Pigment Epithelium
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