Metabolic and Molecular Pathways and Processes in the Eye
Production of Aqueous Humor
The aqueous humor is secreted into the posterior chamber of the eye by a part of the ciliary epithelium situated close to the region where the zonula fibers are attached (Fig. 1b). The composition of aqueous humor is relatively similar to blood plasma. However, its protein concentration is low (less than 1 %), whereas ascorbate is up to 50 times higher than in blood plasma. Oxygen is derived by diffusion from the cornea and from the vasculature of the iris . The aqueous humor flows from the posterior chamber through the pupil into the anterior chamber (Fig. 1b) and drains away at the angle between the cornea and iris, where it passes through a porous tissue – the trabecular meshwork – into a collecting channel (Schlemm's canal), which empties into veins and thus into the bloodstream. In the healthy eye, the delicate balance between aqueous fluid production, circulation, and drainage must be maintained in order to keep the intraocular pressure at a constant level. Slow drainage of aqueous humor or overproduction may lead to an increase in intraocular pressure that can result in the death of retinal ganglion cells – a disease called glaucoma (see chapter “Glaucoma”).
The retina is inverse, i.e., before light reaches the photoreceptors, it has to pervade the different retinal layers: three layers of somata (termed ganglion cell layer, inner nuclear layer, and outer nuclear layer), separated by two synaptic layers (i.e., inner and outer plexiform layer). The ganglion cells are the output neurons of the retina. Their axons form the optic nerve (Fig. 1a). As all neurons, retinal cells conduct information by generating electrical signals at their plasma membrane (see chapter “Overview” under part “Brain”). Inside the cell, there is a high concentration of K+ ions and large polyanions (such as proteins and nucleic acids), while outside the Na+ ion concentration is high resulting in a resting membrane potential of −70 mV.
Photoreceptor cells can be divided into two compartments (Fig. 2a). The inner compartment comprises the cell body, the axon with the synaptic region, as well as the biochemical machinery with the mitochondria and ribosomes for routine cell metabolism. The outer segment harbors all proteins to absorb light, to amplify the signal, and to generate an electrical signal in response to light.
Rod outer segments are effective light catchers. The outer segment of a human rod contains a stack of up to 800 flat, hollow membrane compartments,
called discs (Fig. 2b). The latter contain a high concentration of the photopigment rhodopsin in their membranes (Fig. 2c). Altogether 50–150 million rhodopsin molecules are found within a photoreceptor cell. Rhodopsin belongs to the family of G-protein-coupled receptors (see below). It consists of a protein part (the opsin) and a light-absorbing cofactor, the aldehyde form of vitamin A, retinal (Fig. 2c, d).
Retinal can exist in two different conformations. The folded 11-cis-retinal is covalently bound within the opsin molecule (Fig. 2c). Absorption of a light quantum causes 11-cisretinal to switch to the elongated all-trans-retinal (Fig. 2d), inducing a conformational change in the rhodopsin molecule. This conformational change activates the G-protein transducin, which in turn activates phosphodiesterase 6 (PDE6). The latter hydrolyzes cGMP, which acts as a signal transducer, amplifier, and molecular switch. In the dark, due to an elevated cGMP concentration, cGMP-dependent ion channels (called cyclic nucleotide-gated ion channels) in the outer segments are open, leading to an influx of Na+ and Ca2+, depolarization of the membrane potential, and transmitter release. Upon illumination and cGMP hydrolysis via PDE6, cGMP-dependent ion channels close. As fewer Na+ and Ca2+ enter the cell, the membrane potential becomes more negative. Thus, during illumination, the cells are more hyperpolarized, and fewer transmitter molecules are released from the synapse. This kind of membrane potential modulation, also termed graded potentials, controls the activity of photoreceptor cells, like that of most retinal cells. Graded potentials induce variations in the site of membrane potential in contrast to the “all-ornothing” action potentials. To shut off the signaling cascade, rhodopsin becomes phosphorylated  and then sealed by arrestin, which ultimately stops the interaction with transducin.
The photoreceptor information is relayed synaptically via the bipolar cells to the ganglion cells. Along this path, lateral neuronal interactions and feedback loops are provided by horizontal cells in the outer and by amacrine cells in the inner plexiform layer. As retinal output neurons, ganglion cells communicate via action potentials – short
Fig. 2 Rod photoreceptor and rhodopsin. (a) Schematic overview of a rod photoreceptor and its surrounding layers. The blood capillaries of the choroid provide nourishment and oxygen to the photoreceptors. The retinal pigment epithelium (RPE) cells fulfill many functions (e.g., retinal metabolism and blood-retinal barrier). Note that the center of the eye is towards the bottom. The outer segments of the photoreceptor cells contain lightabsorbing discs. The inner segments contain cell body and normal metabolic machinery. (b) Shows an electron micrograph of the region indicated in (a), showing an outer segment of a photoreceptor cell with its membranous discs. (c) Schematic of a disc membrane harboring the light-sensitive pigment rhodopsin, a G-proteincoupled receptor with seven transmembrane helices (left). Two helices are removed to reveal the retinalbinding pocket (right). (d) Chemical structure of free 11-cisand all-trans-retinal. Light converts 11-cis-retinal into all-trans-retinal
(1–2 ms) stereotyped changes in membrane potential that propagate in an all-or-none fashion along the axons of neurons (chapter “Overview” under part “Brain”).
The physiology of photoreceptors is extraordinary in the sense that a large ionic current in the dark is switched off in the light. Therefore, the energy consumption of the retina is four times higher in the dark than during illumination . In the dark, about 50 % of the energy is used by the Na+/K+-ATPase to pump out excess Na+ that enter the photoreceptors through open cGMPdependent ion channels in the outer segment . As during illumination the ion flux into the photoreceptor outer segment decreases, energy consumption by the Na+/K+-ATPase drops. However, the energy expenditure for the subsequent phosphorylation of rhodopsin and the regeneration of 11-cis-retinal increases.
Photoreceptor cells produce ATP from glucose mainly via oxidative phosphorylation and to a lesser extent via glycolysis , and thus large mitochondria are found densely packed in the photoreceptor inner segments. Glucose and oxygen are supplied from the capillary network of the choroid that lies close to the photoreceptors (Figs. 1a and 2a). The oxygen consumption of the retina is appr. 20 % higher than the oxygen consumption reported for the brain .
The Role of the Retinal Pigment Epithelium
Together with the retina, the RPE is among the most metabolically active tissues in the body. The RPE serves many functions. First, melanin in RPE cells absorbs light that has passed through the retina in order to prevent light scattering and to reduce light-induced damage. Second, the tight junctions between RPE cells form a barrier between the choroid and photoreceptors . Analogous to the blood-brain barrier, this bloodretinal barrier controls exchange between blood and retina and thus maintains the specialized environment of the photoreceptors. The barrier function of the RPE is physically supported by Bruch's membrane that acts as a semipermeable molecular sieve . At the same time, RPE cells utilize glucose transporters that allow passive transport of glucose from the choroid to the photoreceptors . Third, the RPE is involved in the regeneration of 11-cis-retinal. All-transretinal (see above) detaches from the opsin and is enzymatically reduced to all-trans-retinol. Retinoid binding proteins shuttle the retinol from the outer segments to the RPE, where the rest of the retinal metabolism takes place. Here, alltrans-retinol is first esterized with palmitate, then isomerized to 11-cis-retinol, and finally, oxidized to 11-cis-retinal. The latter is transported back to the outer segment, where it spontaneously reacts with opsin to regenerate rhodopsin, thus completing the visual cycle.
Finally, the RPE is of utmost importance for the regeneration of the photoreceptor outer segments. The strong illumination of the outer segments in a high-oxygen environment can lead to photochemical damage. If the energy from a photon is transferred from a light-absorbing molecule to oxygen, reactive oxygen species (e.g., singlet oxygen) can be created. Those can break molecular bonds or induce photooxidation. Accumulation of such events can ultimately lead to damage of the outer segments. Therefore, photoreceptor outer segments must be constantly renewed. Around sunrise, the tips of the outer segments are shed off and are phagocytosed by RPE cells, while new discs are added at the bases of the outer segments . Complete renewal of the rod outer segment takes approximately 10 days. Due to the high amount of material, phagocytosis in RPE cells is metabolically demanding. Some of the breakdown products may be recycled, while others are discharged into the choriocapillaries. Some undigested proteins, lipids, and retinoids remain as an aggregation complex called lipofuscin . Chemical reactions between these components lead to the formation of retinoid-lipid complexes in lipofuscin, the socalled bisretinoids . Lipofuscin can absorb light, shows autofluorescence, and is susceptible to photochemical changes. Lipofuscin accumulation is thought to contribute to the development of age-related macular degeneration (see chapter “Age-related macular degeneration”).