Ординатура / Офтальмология / Английские материалы / Basic Sciences in Ophthalmology_Velayutham_2009

DHA is an important component of retinal membrane and it is presumed to function as a spring, helping to expand the membrane to facilitate the opening of the active state of rhodopsin. The polyunsaturated fatty acids of the retina are vulnerable to destruction by oxidative processes in the retina. But there is sufficient Vit E in the retina to prevent such destruction by absorbing the free radicals. Lipid soluble Vit A has an important dynamic role in visual excitation and perception described as Vit A cycle or wald's visual cycle.

Tay-Sachs Disease

Glycolipids storage disease due to deficiency of the enzyme hexosaminidase A that normally catalyze the breakdown of gangliosides as new ones are synthesized. So, accumulation of GM2 in retina, degeneration of ganglion cells and formation of cherry - red spot in macular region and blindness in early age. Gangliosides are glycolipids predominantly located in the membranes and have negatively charged sugar, sialic acid. Tay-Sachs ganglioside, GM2 is partially degraded ganglioside. The complete in situ, ganglioside, known as GM1 has a galactose attached to N- acetyl galactosamine which is detached by betagalactosidase in neural and ocular cell lysosomes. In Tay-Sachs disease N- acetyl galactosamine (which would be normally be hydrolysed next) is not removed due to deficiency of the enzyme Hexosaminidase A.

Sandhoff disease (GM2 gangliosidosis type II)

Due to Hexosaminidase A and B deficiency is also a lipid storage disorder forming macular cherry red spot.

The proteins of retina are

1.Visual pigments of 4 classes one in rod i.e., Rhodopsin - visual transduction protein of rod photoreceptors functional at lower light levels. And 3 colour sensitive visual pigments i.e., cone photoreceptor proteinsfor day light vision and colour vision of 3 types : blue, green, and red light absorbing or long wavelength sensitive (570 nm) (red sensitive) mid wavelength sensitive (540 nm) (green sensitive) short wavelength sensitive (440 nm) (blue sensitive).

All 4 calsses of protein have the same prosthetic group or chromophore viz. 11-cis retinal dehyde and all are derived from a common ancestral gene. Only the interaction of chromophore with the aminoacids of protein decides the spectral characterstics of each pigment or the capacity to absorb different wavelength.

Clinical aspect; colour blindness or colour deficiency and retinitis pigmentosa are caused by absence or mutation of visual pigments.

2.Transducin - mediates signal coupling for phototransduction.

3.Enzyme proteins - phosphodiesterase, rhodopsin kinase, guanylate cyclase, phospholipase C, protein kinase, phosphoprotein phosphatase, retinal dehydrogenase.

Fig 13.21

Schiff base linkage (i.e. Retinal – CH = NH+ protein). A Schiff base has the characterstic of being less permanent or less stable than many covalent bonds. This is useful for easy detachment of Vit A upon light stimulation.

Rhodopsin is prevented from migrating around the ends of the discs by other proteins such as peripherin, rdsROM -1 present at the rim of each disc.

Peripherin contributes to the integrity of the disc structure and controls the population of rhodopsin molecules, on each side of the disc.

Clinical aspect: mutation in peripherin genes gives rise to retinitis pigmentosa.

Synthesis and Turnover of Photoreceptor Outer Segments

Rods and cones visual cells in the retina do not undergo cell division. Instead, the old membrane are shed from the apical tips of both rods and cones, phagocytosed by the retinal pigment epithelium and new membrane material is added at the junction between inner and outer segments.

Renewal of the Outer Segment

Lipids and opsin are synthesized in the endoplasmic reticulum of the inner segment, and transported to the outer segment in vesicles or through exchange protein. Lipids and cone opsins diffuse throughout the entire outer segment. The phospholipids are hydrolysed by phoapholipase A2 and C. Rhodopsin is acylated with 2 moles of palmitic acid on adjacent cysteines near C terminus. There is no denovo synthesis of lipids in ROS (rod outer segment).

for free radicals generated by photochemical events in RPE. A peroxidase is associated with the melano-lysosome complex to detoxify the peroxides produced in these reactions. Melanin is synthesized from tyrosine, through many enzyme catalysed reactions starting with tyrosinase, a copper dependent enzyme. There is a slow and steady renewal of melanin in RPE throughout life.

Clinical aspects: deficiency of tyrosinase results in albinism, poor visual acuity and nystagmus in children—Albinism is of 2 types - oculo-cutaneous and ocular.

Ocular is autosomal recessive inheritance and also X linked trait. There is mosaic pattern of pigment distribution.

Oculo-cutaneous albinism—is also of autosomal recessive inheritance. It is of 2 subtypes tyrosinase positive and negative.

In tyrosinase positive - some visible pigment is present.

In tyrosinase negative - there is lack of all visual pigments, profound visual loss, photophobia, nystagmus, absent fundal pigmentation and foveal reflex. Autophagy and phagocytosis are two of the important functions of RPE.

1)Autophagy: Under physiologic conditions, RPE is a non-mitotic tissue. So, in order to replace and repair its various constituents and organelles, the post-mitotic RPE undergoes autophagy (autophagocytosis). The old organelles are destroyed by autophagocytosis and this stimulates the biosynthesis of new membrane, ribosomes, lysosomes, peroxisomes, mitochondria and melanosomes. Autophagic vacuoles form from the fusion of lysosomes with organelles. A small amount of RPE plasma membrane is lost, each time the tip of an outer segment of photoreceptor is internalized.

2)Phagocytosis: RPE phagocytose the tips shed from the constantly renewing photoreceptor outer segments. First, the pseudopodial processes of the RPE envelope the shed tips of rods and cones and internalize them into the cell. Then the lysosomes of the cell fuse with the ingested tips to form the phagolysosome. The degradative, digestive enzymes of the lysosome digest the phagolysosome and leave a residual body. This residual body accumulates throughout life and fuse to form lipofusin granules noticed along the basal margin in older eyes. Actin filaments and microtubules of RPE are involved in phagocytosis. Each photoreceptor completely renews its outer segments every 10 days and so phagocytic load for RPE is high.

Clinical aspects: Defects in rds locus leads to lack of recognition of the material to be digested and hence retinal degeneration.

3)The RPE plays a role in maintaining the proper ionic and fluid environment by acting as part of bloodretinal barrier. The apical membrane of RPE has the transporters and channel proteins. They are bicarbonate transporter, carbonic anhydrase enzyme and Na+/K+ATPase. These help in the transport of Na+ towards subretinal space and K+ in the opposite direction (Fig 13.22).

Fig 13.22

4)RPE along with photoreceptors helps in maintaining the subretinal space (interphotoreceptor matrix IPM) by synthesizing and secreting some of

the proteoglycans and GAGs of IPM.

IPM - interphotoreceptor matrix or sub retinal space: Lies between RPE and photoreceptors. Proteins, GAG, glycoproteins and proteoglycans make up the major part of IPM. Interphotoreceptor retinoid binding protein is the glycoprotein and is soluble. But the predominant chondroitin sulfate proteoglycan is insoluble. The proteoglycan around the rod outer segment has sialyl conjugates also. The rods and cones associated IPM domains are tubular structures surrounding the outer segment and extend from the external limiting membrane of retina beyond the distal tip of outer segment to terminate at the apical surface of RPE.

Functions of IPM

1.Retinal attachment by promotion of adhesion between RPE and photoreceptors cells.

2.Visual pigment chromophore exchange.

3.Transport of metabolites between the RPE and rod outer segment.

Fig 13.23.

Clinical aspect:

Defects in IPM manifests as retinal detachment, macular degeneration and retinitis pigmentosa.

Fig 13.23

Wald's visual cycle and photo transduction

In 1958 George Wald demonstrated that light isomerizes 11 cis retinal, the chromopore in rhodopsin to ‘All trans retinal’. Thus, absorption of a photon by a visual pigment molecule is the initial event in visual excitation. This is followed by many reactions that transmit the visual message to the brain.

The cis form of Vit A (11–cis retinal) is folded on itself to form a compact molecule (Fig 13.24). On isomerisation it unfolds and straightens (disrupting the conformation of protein which also unfolds) to form the trans form (i.e., the double bonds in trans position) occupying more space in membrane provided by changes in DHA Fig. 13.25. The isomerisation reaction creates an energitcally unstable molecule that rapidly proceeds through several intermediate forms with different spectral activities in milliseconds as shown below (Fig. 13.26).

Fig 13.24: 11-cis-retinal

Fig 13.25: All trans retinal

Fig 13.26

Fig 13.28: Processing of transport of Vit A in RPE and ROS

Processing and transport of vit A to its target cells (Fig. 13.29)

Vitamin A is stored in liver as retinyl ester and it may be hydrolysed to retinol and free fatty acids. The retinol combines noncovalently with serum retinol binding protein (SRBP) with a molecular mass of 21 KD. This small complex is bound to a larger protein, prealbumin in serum. As the choroid capillaries are fenestrated this RBP prealbumin complex (76KD) penetrates Bruch's membrane and interacts with specific receptors on the basal side of the RPE. The protein, RBP does not enter the cell but delivers the retinol (vit A) to the membrane for transport inside the cell. The receptor now shows reduced

Fig 13.29: Processing of Transport Vit A to its target cells

affinity for RBP as the vitamin A is separated and can be replaced by another RBP with vitamin A.

Clinical aspect: An early effect of vitamin A deficiency is loss of night vision. Light transduction is a cGMP mechanism.

It is the coupling of absorption of a photon by rhodopsin in disc membrane with the closure of cation channels in the plasma membrane of outer segment.

It is similar to the mechanism of hormone action. Light replaces hormone. Rhodopsin is the receptor protein. 'G' protein is transducin. The activated enzyme here is phosphodiesterase. Transducin and phosphodiesterase are peripheral membrane proteins. Transducin has 3 subunits, alpha, beta, gamma. Presence of gamma keeps it inactive. It is bound to GDP in the dark.

Phosphodiesterase is a tetramer with 1 alpha, 1 beta and 2 gamma subunits. Here, also presence of gamma subunits keep the enzyme in inactive state. The action of phosphodiesterase is to hydrolyse cGMP into GMP.

Guanylate cyclase Phosphodiesterase

GTP …………………….. cGMP…………………..….. 5´GMP

On the cytoplasmic side of each disc, rhodopsin (receptor protein), transducin (G protein) and guanylate phosphodiesterase are located in close proximity on the disc membrane.

When light strikes a rhodopsin molecule, the molecular rearrangement of vitamin A (to an unprotonated Schiff base) and the protein conformation of opsin portion (to Metarhodopsin II) causes close contact with transducin.

The activated rhodopsin interacts with transducin causing the transducin to incorporate GTP with the removal of GDP. The alpha subunit of transducin gets detached and diffuses to the inactive phosphodiesterase. The alpha subunit of transducin, now binds to the gamma subunit (inhibitor) of phosphodiesterase causing it to be released from the enzyme. This release activates the enzyme, bringing about the catalytic hydrolysis of cGMP to GMP. So, the concentration of cGMP in the cytoplasm of photoreceptor outer segment is lowered. The above said events are shown in the figures 13.30 and 13.31.

Fig 13.30