Retinal Biochemistry- Ocular Biochemistry

Optometry

1. Retinal
Biochemistry
-BY SUNNY BISWAS

2. Introduction
•The retina consists of the nonneural pigment epithelium and the neural retina, organized
into ten layers.
•Photoreceptors in the outer layer detect light, while inner layers process signals.
•Blood supply varies, with photoreceptors relying on choroidal circulation and inner layers
on retinal circulation.
•Rod cells support dim light vision in peripheral areas, while cone cells in the central region
nablecolor vision at higher light intensities.
•The human eye contains 100 to 120 million rods and about 6 millioncones.

3. Fig:Schematic organization of the vertebrate retina.

4. Neural Retina

5. Intact
NeuralRetina
(Excluding
Lipids)
•This area maintain gradientsfor
electrolytes and amino acids, carry on
active aerobic and anaerobic
glycolysis, maintain protein synthesis,
and respond to photic stimulation.

6. Glucose:
The neural retina, especially in photoreceptor cells, undergoes high ratesof
glycolysis,withglucose primarily converted to lactate.
Carbohydrate metabolism is more pronounced in bicarbonate/C0
2buffer.
Glucose consumption in the retina is approximately 0.73 µmol/mg dry weight per hour.
Photoreceptors exhibit heightened glucose oxidation, and the pentosephosphatepathway
helps maintain glutathione levels in response to oxidative stress.
Besides glucose,mannose serves as an energy source.
The photoreceptor plasma membrane has a unique high Na+ permeability, crucial
forthe"dark current," maintained by ATP from mitochondria.
TheNa+,K+-ATPase pump in inner segments plays a role in sustaining the sodium gradient
necessary for the light response.

7. Amino
Acids:
The neural retina,contains a substantial pool of neuroactive
amino acids.
Taurine, a prominent free amino acid in the retina, is
implicated in maintaining photoreceptor structural integrity.
Despite being released in response to light, taurine's specific
role in visual excitation remains unclear.
The retina synthesizes neuroactive amino acids, including
GABA, glycine, glutamate, and aspartate, with glucose and
glutamine as key precursors.
Glutamate undergoes various metabolic reactions, including
conversion to GABA and glutamine.
Glutamate's have neurotoxityoverretinal tissues and iteffects
on glutamine synthetase activity.

8. Figure: Metabolic pathways of amino
acids and glucose through the
tricarboxylic acid cycle.
The encirclednumbers correspond to
the following enzymes:
•(I) glutamate transaminase;
•(2)glutamic dehydrogenase;
•(3)glutamine synthetase (GS), which
also has glutamine transferase (GT)
activity;
•(4) glutamic aciddecarboxylase
(GAD);
•(5) GABA aminotransferase;
•(6) aspartate transaminase; and
•(7) succinic semialdehyde
dehydrogenase.

9. Protein Synthesis:
Neural retina demonstrates active protein turnover and synthesis from
aminoacids.
Photoreceptor proteins undergo dynamic renewal, with synthesis occurring in
theinner segments' rough endoplasmic reticulum and transportation to
outersegments.
Protein synthesis in the retina is not directly light-dependent
Continuous darkness reduces leucine incorporation, indicating a decline in
overallmetabolic activity.

10. Nucleic
Acids:
The retina typically exhibits a high DNA/RNA ratio,
e.g., 11/3.5 mg/g in mice.
DNA and RNA localize to nuclei and inner
segments, with rapid RNA renewal observed after
[3H]cytidine injection.
Autoradiography indicates label concentration in
rod cell nuclei, shifting to inner segments after 1
h.
[3H]uridine studies reveal diurnal variation in RNA
incorporation in Rana pipiens. In vitro, rat retinas
exhibit localized RNA synthesis in photoreceptor
nuclei, with minimal synthesis in the inner retina.

11. Cyclic Nucleotides:
Cyclic
GMP
(cGMP)
Highly compartmentalized in the retina.
Rod-dominant retinas have 100 times more cGMP than any other neural tissue.
About 90% of cGMP is localized in photoreceptors.
Concentration increases with visual cell maturation.
Light exposure results in a rapid decrease in cGMP, correlating with light signal
amplification.
Controlled by guanylate cyclase and photoreceptor-specific cGMP phosphodiesterase
(ROS PDE).
Guanylate cyclase activity likely increased by light; ROS PDE activated by light.
Light-induced reduction in cGMP correlated with amplified light signals.

12. Cyclic
AMP
(cAMP)
Evenly distributed throughout retinal cell layers except for photoreceptors.
Lower total content compared to cGMP in rodent retina.
cAMP levels in outer plexiform and outer nuclear layers reduced after
illumination.
Modulated by adenylate cyclase, particularly in innerretinal layers.
Modulates glycogen synthesis and breakdown in Müller cells.
Activates protein kinases in the retina, influencing myosin phosphorylation.
Activates cAMP-dependent protein kinase phosphorylating tyrosine
hydroxylase.

13. Retinoid-Binding Proteins:
CRABP and CRBP: Bind all-trans retinoic acid and retinol, structurally
conserved.
CRABP identical in amino acid sequence in bovine retina and adrenal
gland.
CRBP found exclusively in Müller cell cytoplasm of rat, bovine, and
human neural retina.
CRALBP: 33-kDa protein in bovineretina, binds 11-cis retinal and 11-
cisretinol.
Complex with 11-cis retinal shows properties of visualpigments.

14. Low photosensitivity suggests CRALBP's role is to solubilizeand protect
11-cis retinal.
Highly stereoselective for 11-cis retinoids, likely associatedwith the
visual cycle.
CRALBPs in bovine retina and pigment epithelium (with 11-cis retinal)
purified to homogeneity.
CRALBP concentration in adult bovine retina:approximately 3 nmol
per eye, two-thirds in neural retina,one-third in pigment epithelium.

15. Lipids in
Neural Retina

16. General Composition of Retinal Lipids
Lipids constitute approximately 20% of the retina's dry weight, varying across layers.
Phospholipids dominate (65-75%), followed by cholesterol (10-12%), diglycerides,
triglycerides, free fatty acids (5-7%), and sphingolipids (2%).
Notably, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) make up nearly
80% of phospholipids across subcellular fractions.
The retina is distinct for its high content of unsaturated fatty acids, especially polyenoic
fatty acids, with 22:6(n-3) being a major component.

17. The sn-1 and sn-2 positions ofglycerolipidshost saturated and unsaturated fatty acids,forming
diverse molecular species. Additionally, "supraenes," containing over six doublebonds, and very
long-chain polyunsaturated fatty acids (VLCPUFA) have been identified.
Cholesterol dominates neutral lipids (40%), mainly in an unesterified form, whilediacylglycerol and
triacylglycerol constitute 10.2% and 7.0%, respectively.
Retinal glycolipids, particularly gangliosides, are present but at relatively low levels.

18. Metabolism of Retinal Lipids
•The retina's diverse lipid composition, constituting 20% of its dry weight, is characterized by
phospholipids (65-75%), saturated fatty acids, and high levels of polyenoic fatty acids like
22:6(n-3). Cholesterol dominates neutral lipids (40%).
oGlycerolipidbiosynthesis involves de novo synthesis and base-exchange
reactions.Enzymes, including acyltransferases, play crucial roles.
oPhospholipid metabolism invertebrae includes unique pathwaysfor
phosphatidylinositol (PI).
oGanglioside biosynthesis peaks in embryonic development.
oMinimal cholesterol biosynthesis suggests preformed cholesterol entry.
oDolichols, crucial for glycoprotein synthesis, follow distinct pathways.
oEnzymatic oxygenation of polyunsaturated fatty acids yields eicosanoids and
docosanoids, influencing retinal function.

19. Photoreceptor cells
•The outer segments of these cells contain the photosensitive visual pigments involved in
the capture of photons and the transduction of light energy into electrical impulses.
•Signals are transmitted through the synaptic body of the photoreceptor cells to the
bipolar cells, which then interact with second-order neurons before being transmitted to
the brain.
•The unique structural design and chemical organization of photoreceptor cells subserve
this central role in the visual process.

20. Fig: Schematic diagram of primate rod
and cone cells and theirrelationship to
the underlying pigment epithelial cell layer

21. PhotoreceptorCells
Chemical
Composition
•The rod outer segments
(ros) membranes,
representing around 50%
protein and 50% lipid, are
commonly used.
•Rhodopsin is the primary
protein, while
phospholipids,
constituting 80-90% of ros
lipids, are the
predominant lipid class.

22. Protein:
Rhodopsin is the major integral membraneprotein (glycoprotein) of ROS disks,
consisting of a single 40-kDapolypeptide chain, opsin, in combination with 11-
cis retinal chromophore.
The amino acid sequence of bovine rhodopsin has been elucidated, revealing
structural details such as the seven membrane-spanning hydrophobic alpha-
helices.
Several integral ROS disk membrane proteins, including a 220-kDa rim
glycoprotein and a 33-35 kDaprotein called peripherin, play roles in
maintaining structural integrity.
Peripheral and soluble proteins, such as transducin, phosphodiesterase (PDE),
and rhodopsin kinase, contribute to phototransduction.

23. The cGMP-sensitive channel, localized in the ROS plasma
membrane, isactivated bycGMP and plays a crucial role
in membrane conductance.
The ROS (rod outer segment) plasma membrane contains a
crucial 240-kDaspectrin-likeprotein, potentially forming a
unique cytoskeletal system.
This protein is anchored by the 54-kDa plasma membrane
channel protein.

24. Lipids:
ROS membranes exhibit high fluidity due to a
lowcholesterol/phospholipid ratioandabundant
polyunsaturatedfatty acids.
Phospholipids, especially
Phosphatidylcholine(PC) and
Phosphatidylethanolamine (PE), dominate ROS
lipidcomposition.
ROS membranes also contain neutral lipids such as
cholesterol,free fatty acids, and diglycerides.
The lipid composition is consistent across
vertebrates, withphospholipids constituting 85-
90mol%.
ROS membranes contain unique metabolism
forphosphatidylinositol (PI).

25. ROSmembranes are rich in polyunsaturated acids,
especiallydocosahexaenoate(22:6n-3).
Changes in fatty acid composition can affect vision.
Lighting conditions influence ROS membrane composition,
With higher illuminancedecreasing 22:6n-3 and increasing 20:4n-6,
cholesterol, and affecting rhodopsin packingdensity.
Environmental factors impact ROS length, suggesting adaptative
protection againstillumination-induced damage.

26. Metabolism of
Photoreceptor
cells
(Excluding
Phototransdu
ction)
•Photoreceptor cells, crucial for vision,
have compartmentalized outer and
inner segments.
•Exchange occurs through the
connecting cilium.
•Renewal involves assembling new disk
membranes at the base and shedding
old ones, with no mitosis.
•Opsin renewal relies on membrane
replacement, and phospholipids
undergo both membrane and
molecular replacement.

27. Renewal of
Outer Segments
The renewal process of rod outer segment membranes
involves the incorporation of radioactive amino acids in the
rough endoplasmic reticulum (RER), subsequent transfer
through the Golgi complex, and assembly into proximal basal
disks.
The radioactivity gradually moves distally along the rod outer
segment, leading to the shedding and phagocytosis of
terminal disks.
The synthesis and renewal of cone outer segments follow a
different pattern due to structural differences between rod
and cone outer segments.
Renewal patterns of lipids and proteins are initially similar, but
the mechanisms differ.

28. Opsinsynthesis, glycosylation, and acylation by palmitic acid are detailed, with
evidencesuggesting that 11-cis retinal is attached to opsin in the RER.
Phospholipids, including phosphatidylcholine (PC), undergo renewal through bothmembrane
and molecular replacement processes, with rapid turnover and dispersionthroughout the
outer segments, possibly facilitated by acyl exchange reactions.
In bovine rod outer segment (ROS) membranes, the biosynthesis of phospholipids,particularly
phosphatidylcholine (PC), involves acyl-CoA synthetase anddocosahexaenoyl-CoA :lyso-PC
acyltransferase.
Pro-oxidizingconditions and light exposure stimulate both acylation anddeacylationof ROS
phospholipids.

29. Figure: Diagram illustrating protein
renewal in vertebrate rod outer
segments. Autoradiographic
studiesshow that -
•Newly synthesized protein is first
concentrated in the rough
endoplasmic reticulum and
shortlyafterward passes into the
Golgi complex.
•After posttranslational
glycosylation, the protein is
transferred vectorially in vesicles
through the inner segment to the
connecting cilium, where it is
incorporated into newlyformed
disk membranes.
•They are displaced distally, and,
on reaching the distal tip, packets
of outer segmentdisks are shed
and phagocytized by the pigment
epithelium.

30. Monensin disrupts Golgi apparatus, demonstrating partial dissociation of lipid
and protein synthesis in ROS.
Disk shedding, a rhythmic event, is controlled by an endogenous circadian
oscillator within the eye, possibly influenced by melatonin.
Excitatory amino acids induce disk shedding, and melatonin synthesis may play
a role in this process.
Enzymes like phospholipase A2 and phospholipase C, regulated by transducin,
participate in phospholipid turnover in photoreceptors.

31. Figure:Enzymatic
conversion of serotonin to
melatonin and to 5-
methoxytryptophol in the
pineal and in
the retina.
•The first step in the
enzymatic synthesis of
melatonin from serotonin
is acylation of the
terminalamino group;
this step is mediated by
NAT, an enzyme that is
under diurnal control.
•The final synthesis of
the two methoxyindoles
is catalyzed by HIOMT, an
enzyme that shows no
circadian regulation.

32. Phosphoinositide
Turnover and
Protein Kinase C
In bovine rod outer segment (ROS) membranes, despite constituting
less than 2% of total lipids, phosphatidylinositol (PI) exhibits a higher
synthesis rate than other phospholipids.
PI metabolism involves kinases, and light enhances PI turnover, linked
to receptor-mediated responses. The "phosphoinositide effect" in
vertebrate retinas involves phospholipase C activation, producing IP3
and diacylglycerol (DG).
Light stimulates the breakdown of PIP2, leading to second messenger
formation.
Phospholipase C activity in ROS is under G protein control, with light-
induced disinhibition. Light-stimulated PIP2 breakdown may contribute
to phototransduction.
Cytidine nucleotides, glycine, and acetylcholine influence PI turnover.
Light-mediated PIP2 breakdown activates Ca2+/phospholipid-
dependent protein kinase C, involved in rhodopsin phosphorylation
and potential connections to visual processes.

33. Exocytosis
(lnterphotoreceptor
Matrix) and
Endocytosis
Photoreceptors exhibit both endocytotic and exocytotic
activities, involving inner segment cytoplasmic vesicles
implicated in opsin transport.
Interphotoreceptor retinol-binding protein (IRBP), a major
glycolipoprotein in the interphotoreceptor matrix, is
synthesized and secreted primarily by rod photoreceptors.
Studies using labeled probes and in situ hybridization
techniques confirm substantial IRBP mRNA in rod neurons.
Bovine retinas in vitro synthesize and secrete IRBP into the
medium, with photoreceptor cells identified as the source.
Endocytotic activity is observed in bovine and frog retinas,
internalizing IRBP-coated particles into vesicles within rod and
cone inner segments.
The inner segment may play a crucial role in internalizing and
degrading components of the interphotoreceptor matrix,
including IRBP.

34. Insulin and IGF-1
Receptors
Insulin-like growth factor (IGF-1), a 70-kDa
polypeptide with 49% structural homology to
insulin, shares similar receptors with insulin in most
cell types.
These receptors are heterotetramers comprising
135-kDa alpha subunits and 90-95-kDa beta
subunits, arising from distinct genes.
Both hormones stimulate tyrosine-specific protein
kinase activity upon binding to the receptors,
leading to autophosphorylation of the beta
subunit and phosphorylation of substrates.
Although insulin and IGF-1 receptors were initially
recognized in nonneural tissues, they have
recently been identified in neural tissues, including
the retina.

35. Both ligands can stimulate a common pool of tyrosine kinases in bovine rod
outer segment(ROS) membranes, with insulin acting through the IGF-1
receptor.
Despite lower binding capacity, insulin is more potent in activating tyrosine
kinase.
Bovine retina membrane preparations exhibit one IGF-1 and two insulin binding
sites, withinsulin receptors having higher affinity for IGF-1 after purification.
Insulin receptors in bovineretina show heterogeneity in alpha subunit size, with
133-kDaand 120-kDa proteins, possibly related to tissue-specific differences.

36. Lipid
Peroxidation,
FreeRadicals,
LightDamage,
andProtective
Mechanisms
•Photoreceptor membranes, rich in polyunsaturated
fatty acids (PUFAs), are susceptible to lipid
peroxidation.Chronic light exposure and a well-
developed retinal circulation provide oxygen,
predisposing the retina to free radical production.
•Free radicals, generated in photoreceptor disk
membranes, initiate peroxidative chain reactions in
PUFAs.
•Iron toxicity, seen in clinical cases, leads to retinal
degeneration. Fenton-type reaction involving
ferrous iron (Fe2+) and hydrogen peroxide (H2O2)
produces highly reactive hydroxyl radicals
(OH·).Hydroxyl radicals can be damaging,
affecting retinal function and morphology.
•Light damage primarily affects the outer segment,
and rhodopsin-mediated mechanisms are
implicated. Vitamin E deficiency may exacerbate
light-induced retinal damage.

37. Ascorbic Acid (Vitamin C) Role:Ascorbic acid is present at high concentrations
in the retina and protects against light-induced injury.
Glutathione and Enzymes:Glutathione (GSH) gets elevated under oxidative
stress, and helps in reducing hydroperoxide levels.
Superoxide Dismutase (SOD) and Catalase:Retinal SOD, mainly cytosolic Cu-Zn
type, varies in localization among species.
Vitamin E (α-Tocopherol):Vitamin E, an antioxidant, protects against lipid
peroxidation.
Deficiency leads to extensive disruption of photoreceptors.
Vitamin E levels vary with age and dietary sources.

38. Macular Pigments (Carotenoids):Zeaxanthin and lutein, macular
pigments, attenuate blue light and may protect againstphototoxicity.
It's levels remain relatively stable withage.
Melanin:Melanin, a complex polymer, may act as both a prooxidant
and anantioxidant.
Role of retinal pigment epithelium (RPE) melanin in protecting
photoreceptors is still uncertain.

39. Interphotoreceptor
matrix
•The interphotoreceptor matrix (IPM) is an extracellular matrix comprising proteins,
glycoproteins, and proteoglycans situated between the retinal pigment epithelium (RPE),
photoreceptors, and external limiting membrane.
•Explored since the 1950s, the IPM's chemical composition, physical properties, and
biological functions, particularly in visual process development, have garnered attention.
•Interphotoreceptor retinol-binding protein (IRBP) acts as a transporter in the visual cycle.
The IPM exhibits heterogeneity, with specialized domains like cone matrix sheaths.
•Glycoconjugates, especially in cone matrix sheaths, contribute to adhesion between
photoreceptors and the RPE.
•Studies on rod-associated matrix domains also indicate potential adhesion roles

40. Glycosaminoglycans, Proteins, and
Glycoproteins (Excluding IRBP)
•Early IPM investigations used histochemical methods to identify soluble "mucosubstances."
Biochemical characterization became possible with matrix rinsing or extraction methods.
•Bovine IPM comprises about 2% soluble macromolecules, with approximately 60% being
chondroitin sulfate. Hyaluronic acid constitutes about 14%, and low-molecular-weight
sialoglycansare also present.
•GAG synthesis studies suggest contributions from both photoreceptor inner segments and
the retinal pigment epithelium (RPE).
•Monoclonal antibodies reveal chondroitin sulfate's concentration in the basal inner
segment/outer segment zone, with immunogold cytochemistry indicating its presence on
the RPE apical surface.
•Proteins in bovine IPM, analyzed through SDS-PAGE, differ notably from adjacent retinal
tissues.

41. •Additional proteins, originating from adjacent compartments, are present.
•IRBP, accounting for nearly 70% oftotal IPM protein, is the primary protein
innontraumaticallyobtained samples.
•Few endogenous IPM proteins,like a 31-kDa glycoprotein, have been characterized.
•Lectin-binding glycoproteins and enzyme activities,such as cGMP phosphodiesterase and
lysosomal acid protease, are also detected in IPM.
•Lysosomal acidhydrolase release from the RPE suggests active secretion, possibly
regulated by mannose 6-phosphatereceptors.
•A 140-kDaglycoprotein, later identified as interphotoreceptor retinol-binding protein (IRBP),
is a major component.

42. Interphotoreceptor Retinol-Binding Protein
(IRBP)
•Light absorption by photoreceptors initiates visual processes in the retina. IRBP, identified
exclusively in the interphotoreceptor matrix (IPM), serves as a transport vehicle for retinol
between photoreceptors and the retinal pigment epithelium (RPE).
•IRBP's isolation from IPM and subsequent biochemical analysis revealed a glycoprotein with
an apparent Mrof about 250,000.
•It acts as a carrier for endogenous retinoids, facilitating their translocation during the visual
cycle.
•Although suggested as a buffer system, experiments indicate that IRBP may not directly
enhance retinol transfer through IPM.
•IRBP is synthesized mainly by photoreceptor cells, with its highest concentration on the RPE
apical surface.

43. •Carbohydrate analysis shows glycosylation, but its inhibition doesn't affect IRBP
secretion.IRBP is present in vitreous and aqueous humors, and the pineal gland.
•Chemical andphysicochemical properties of bovine and monkey IRBP are similar, with
thelatter termed aglycolipoproteindue to the presence of fatty acids.
•Molecularly, IRBP's gene structure, deduced amino acid sequences, and species
variationshave been studied.
•While a major function is retinoid binding, experiments suggest IRBP's involvement in
retinolesterification and rhodopsin regeneration.
•Despite being a significant IPM component,IRBP's actual role in transporting
boundretinoids across IPM remains debated.

44. •IRBP has relevance in experimental autoimmune uveitis (EAU), causing uveoretinitis upon
immunization. Human studies show lymphocyte reactions against IRBP, linking it to
autoimmune uveitis.
•Cyanogen bromide fragments of bovine IRBP induce EAU, suggesting multiple uveitogenic
sites.
•Synthetic peptides derived from IRBP induce uveitis, providing models for human
autoimmune disorders.
•IRBP concentrations in subretinal fluid (SRF) vary, with higher levels in shorter retinal
detachments.
•In contrast, SRF from retinopathy of prematurity patients lacks detectable IRBP, suggesting
impaired synthesis due to nonviable photoreceptors.

45. Retinal Pigment Epithelium
(RPE)
•Theretinalpigmentepithelium(RPE)isavitalpartofthevisualsystem,formingasinglelayer
betweenbruch'smembraneandphotoreceptors.
•Withapicalsurfacesfacingphotoreceptorsandbasalsurfaceslinkedtothechoroidvia
bruch'smembrane,RPEcreatestheouterblood-retinalbarrier.
•RPEcellsexhibitmorphologicalandbiochemicalpolarization,withspecificproteinslocalized
onapicalandbasalsurfaces.
•Notably,theRPEengagesinphagocytosis,retinoidstorage,andthesecretionof
componentsintotheinterphotoreceptormatrix.

46. Methods for Isolation and Culture
•To overcome past limitations in studying retinal pigment epithelium (RPE) biochemistry,
various isolation and culture methods are employed.
oBovine RPE cells can be isolated by removing the anterior segment and neural retina,
though this may cause membrane damage.
oAlternatively, trypsinization methods minimize cell injury.
oCultured RPE cells form viable monolayers, preserving morphology and functionality.
However, they exhibit drawbacks, such as difficulty maintaining pigmentation and
reduced retinoid metabolism. Despite these limitations, both freshly prepared RPE cells
and cultured preparations are widely used for studying RPE biochemistry.

47. ChemicalComposition
•The chemical composition of bovine retinal pigment epithelium (RPE) cells reveals
significant differences in protein profiles, lipid classes, and fatty acid composition
compared to neural retina.
oProteins:
oActin is a major component of the RPE cytoskeleton, along with some othernumerous
proteins, with some variability among donors. Plasma membrane proteins in RPE are
glycoproteins with varying molecular weights.
oLipids:
oBovine RPE has a total lipid content of 3%, with phospholipids comprising about half.
While the total content phospholipid is similar to neural retina, the fatty acid profiles,
especially in phospholipids, show striking differences.
oRPE phospholipids contain higher proportions of saturated fatty acids, arachidonic
acid (20:4), and lower levels of polyunsaturates(22:6) compared to neural retina.
oPlasma membrane-enriched fractions of RPE have been isolated, indicating
differences in cholesterol content and fatty acid composition.
oLipid inclusion bodies in RPE contain triglycerides with a unique fatty acid composition,
suggesting selective clearance and recycling of certain fatty acids.

48. Metabolism
of RPE

49. •Carbohydrate and Energy Metabolism:
•The RPE, rich in mitochondria, exhibits active oxidative pathways for glucose
metabolism.
•While direct biochemical studies on glucose metabolism in RPE cells are limited,
early findings suggest glycolysis and tricarboxylic acid oxidation.
•Energy for phagocytosis primarily comes from glucose oxidation via the
tricarboxylic acid cycle.

50. •Vitamin A and Cellular Retinoid-BindingProteins:
•The RPE, a key player in vitamin A handling for the visual cycle, stores vitamin A in
concentrations rivaling theliver.
•Receptors for serum retinol-binding protein mediate retinol uptake, leading to
esterification and storage inthe RPE.
•Cytosolic retinol-binding protein (CRBP) and cytosolic retinal-binding protein
(CRALBP) are crucial in retinoidtransport and metabolism.
•The balance of retinyl esters in different species, influenced by factors like
ageand light adaptation,emphasizes the intricate regulation of vitamin A in the
RPE.

51. •Production of ExtracellularMatrix :
•The polarized RPE secretes specialized extracellular matrices (ECMs) on its apical
and basal surfaces.
•Bruch's membrane, the basal ECM,consists of proteoglycans, type IV collagen,
laminin, and fibronectin.
•Studies on feline and human RPE reveal polarized proteoglycansecretion, with
distinctions in size distribution and composition.
•Human RPE cells also produce collagen types I-III, fibronectin, and laminin.

52. •Transport of Ions, Solutes, and Fluid :
•The RPE regulates subretinal fluidvolume and the ionic environmentaround
photoreceptors throughactive solute transport to thechoroid.
•Ouabain-sensitive Na+, K+-ATPase activity is present in frog andhuman RPE,
facilitating Na+, taurine,and amino acid transport.
•Zinc uptake inhuman RPE and ascorbateaccumulation in feline RPE
involvefacilitated diffusion and energy-dependent processes, respectively.
•Fluid transport is linked to ion andsolute transport, primarily driven byHCO3-.
•cAMP elevation reducesfluid absorption, implicating asecretory flux of Na+ and
Cl-.

53. •Adrenergic Receptors, Adenylate Cyclase, and PhosphoinositideTurnover :
•RPE responds to hormones and neurotransmitters through adenylate cyclase,
producing cAMP.
•Catecholamines stimulate cAMP production via β-adrenergic receptors. cAMP
modulation affects fluid transport and phagocytosis.
•α1-adrenergic receptors, acting independently of adenylatecyclase, couple to
phosphoinositide turnover, influencing Ca2+ mobilization and protein kinase C
activation.
•RPE may generate superoxide anion inresponse to phagocyticchallenges.

54. •Drug-MetabolizingEnzymes :
•RPE contains cytochrome P450 enzymes, part of microsomal drug-metabolizing
systems.
•Theseenzymes, inducible by aromatic hydrocarbons,detoxify substances
entering the RPEfrom the uveal circulation.
•Studiesreveal RPE microsomes capable of metabolizing drugs and
chemicals,emphasizingtheir potential protectiverole.

55. •Melanin:
•RPE melanin,synthesized in utero,undergoes gradualmaturation andshows age-
relatedchanges.
•Melaningranules,mainly apical, varyin size andshape.Tyrosinase, key
inmelaninbiosynthesis,is present inembryonic andadult RPE.
•Melaninconcentrations decrease withage, withregionalvariationsand the
emergenceofcomplex granules,suggesting lysosomalinvolvement
andpotentialreorganization.

56. Phagocytosis
and
Maintenance
ofPhotoreceptor
Cells
•In the retina, the retinal pigment epithelium
(RPE) plays a crucial role in
maintainingphotoreceptor cells through the
process of phagocytosis.
•This involves the shedding of rodouter
segments (ROS), their subsequent
phagocytosis by the RPE, and the
synchronizedshedding mechanism that
keeps the length of photoreceptor cells
relatively constant.

57. Binding andUptake Mechanisms inPhagocytosis :
•The phagocytic processinvolves binding anduptake mechanisms, withstudies
suggesting bothspecific andnonspecificcapabilities of RPE cells.
•While the precise biologicalmechanisms initiatingshedding are notfullyunderstood,
once ROSpackets are released, theymust rapidly bind to theRPE's apical
surfacebeforeingestion.
•Recentinvestigations indicate thatROS are preferredsubstrates over otherparticle
types,andrhodopsin may not be theprimary ligand for ROSbinding.
Factors InfluencingPhagocytosis:
•Several factors influencephagocytosis, including therole of cytoskeletalcomponents like
actinfilaments andthemodulation of intracellularcAMP levels.
•Studiessuggest that cAMP may actas a secondmessengercontrolling the rate
ofphagocytosis.
•Additionally,attention has been focusedon identifyingpotentialreceptors for ROS on
theRPE cell surface, includingglycoproteins, mannose 6-phosphate receptors,andthe
role of cyclicnucleotides

58. PhagosomeDegradation andLipofuscin Formation:
•Once phagocytosed, ROS undergo efficientdegradation through
thephagolysosomalsystem.
•Lysosomal acid hydrolases,including cathepsin D, playa vital role in breakingdown
macromolecularsubstances.
•The passageexplores the specificactivities of acid hydrolasesand their involvement inthe
degradation of ROSmembranes.
•The process of lipofuscinformation is also discussed,emphasizing the age-related
accumulation ofauto-fluorescentlipofuscinbodies in the RPE.
•Thecomposition of lipofuscinfluorophoresrelationship with vitamin Ametabolites.
Additionally,the passage touches upondrusen, extracellulardeposits beneath the
RPEassociated with aging andage-related maculardegeneration, and
theirheterogeneouscomposition.

59. Metabolismofthe
Microvasculature
•Theretinalcapillariesarecharacterizedbytwocelltypes:endothelialcells
andpericytes.
•Thesecellsformabarrierwithtightjunctions,maintainingtheinnerblood-
retinalbarrier.
•Thelossofpericytesisahallmarkofdiabeticretinopathy.

60. Glucose Transport, Insulin, and Aldose Reductase:
•Glucose transport in retinal microvasculature involves facilitated diffusion, with insulin-
independent uptake.
•High glucose levels affect the transport of physiological substances, such as inositol and
ascorbate.
•Studies suggest a link between sugar alcohols from the polyol pathway and interference
with inositol transport.
•Specific insulin receptors exist, influencing glucose uptake.
•Aldose reductase, present in pericytes, converts glucose to sorbitol and may play a role in
diabetic retinopathy.
Production of Extracellular Matrix:
•Heparan sulfate, a crucial component of basement membranes, is found in retinal micro-
vessels.
•Bovine pericytes and endothelial cells show variations in glycosaminoglycan synthesis.
•Collagen composition primarily consists of type IV collagen, but cultured cells produce
mainly type I collagen.
•These alterations in collagen expression require further investigation.

61. Adrenergic Receptors and PhosphoinositideTurnover :
•Retinal vessels contain adrenergic receptors, although devoid of autonomic innervation.
•The receptors may respond to circulatinghormones and neurotransmitters.
•Phosphoinositide turnover may regulate pericyte proliferation through second messengers
like IP3 andDG.
•Glucose concentrations affect inositol transport, potentially impacting pericyteviability.
Regulation ofMicrovesselCaliber :
•Pericytes, considered contractile elements, show characteristics of smooth muscle cells.
•Actin, a major contractile protein, activatesskeletal muscle myosin Mg2+-ATPase.
Eicosanoid production in pericytes suggests a role in vasodilation.
•Loss of pericytes in diabeticretinopathy may disruptmicro-vesselregulation, impacting
caliber and stability.
•Further research is needed to fully understand thesecomplex mechanisms.

62. Glia (Muller cells)
•Mullercells,theprimaryglialcellsinthevertebrateretina,providemechanical
supportandcontributetotheb-waveintheelectroretinogram.
•Recentstudiesrevealtheirunexpectedroleinneuralretinametabolism.
•Intensestainingforaldosereductaseinmullercellssuggestsalinktoimpaired
polyolmetabolism,potentiallyleadingtoosmoticfluxesandstructural
changes.
•Aldosereductaseinhibitors,suchastolrestat,effectivelypreventthickeningof
theinnerlimitingmembraneinducedbylong-termgalactosefeedinginrats.

63. Retinoid-Binding Proteins :
•Three retinoid-binding proteins (CRBP, CRALBP, CRABP) in neural retina are unexpectedly
localized in Muller cells, contrary to the assumption of photoreceptor localization.
•The significance and specific role of these proteins in retinoid metabolism within the retina
warrant further exploration.
Glial Fibrillary Acidic Protein (GFAP) :
•GFAP, a 47-to 50-kDa cytoskeletal protein, is typically found in astrocytes but shows a
remarkable increase in Muller cells in response to injury or photoreceptor degeneration.
•This heightened immunoreactivity indicates Muller cells' early response to various metabolic
insults in photoreceptor cells.

64. •Insulin Synthesis (Insulin-Specific mRNA):
•Immunoreactive insulin-like activity is present in glial cells of the mouse and human
retina,particularly in Muller cells.
•Studies using a tritiated insulin eDNA probe and in situ hybridization reveal that Muller
cellscontain mRNA for de novo insulin synthesis.
•The function of locally produced insulin in Muller cells is yet to be fully understood,
withpotential roles as a neurotransmitter, neuromodulator, or regulator of glucose
metabolismand glycogen turnover.

65. Neurotransmitters
•Neurotransmittersandneuromodulatorsarevitalforneuralcommunication.
•Neurotransmitterselicitrapidresponses,whileneuromodulatorshavelonger
effects.
•Theretinafeatures15+suchsubstances,categorizedintoaminoacids,
biogenicamines,andneuropeptides.
•Identificationinvolvesdetectability,synthesismechanisms,releaseupon
stimulation,postsynapticresponses,andefficientterminationmechanisms,
oftenenzymaticinactivationorhigh-affinityreuptake.

66. Amino Acids :
•Aspartate and glutamate, abundant in the retina, act as neurotransmitters in
photoreceptor cells, showing species variation.
•GABA, a crucial inhibitory transmitter, is present in three neuron classes, and glycine, mainly
in amacrine cells, meets neurotransmitter criteria.
Biogenic Amines :
•Acetylcholine is a major transmitter in the retina, with cholinergic cells in amacrine layers.
•Dopamine serves as a neurotransmitter and neuromodulator, influencing cAMP levels in
horizontal cells. Serotonin, present in various retinas, has uncertain neurotransmitter status.
•Epinephrine and norepinephrine in rat retinas may be neurotransmitters.
Neuropeptides :
•Over 30 neuropeptides, identified through immunohistochemistry, reside in amacrine cells,
with colocalization observed.
•Functions, often slow-acting and long-term, remain unclear, warranting further
investigation into their roles in retinal physiology.

67. Visualexcitation

68. Initiation of Phototransduction in Vertebrate Rods:
•Photon absorption by rhodopsin in outer segment.
•Rapid isomerization of 11-cis retinal to all-trans retinal.
•Formation of intermediates like bathorhodopsinand metarhodopsinII (R*).
Chemical Messenger in Vertebrate Phototransduction:
•cGMP, not Ca2+, established as the transmitter.
•Light-induced cGMP hydrolysis closes cGMP-gated channels.
•Resulting hyperpolarization decreases transmitter release.
Recovery of Dark State in Vertebrate Rods:
•Guanylate cyclase stimulated by lowered Ca2+ levels.
•Resynthesis of cGMP essential for restoring dark current.

69. Role of G Protein (Transducin) in Vertebrate Rods:
•Transducinactivated by R* after photon capture.
•Activation of cGMP phosphodiesterase (PDE) bytransducin.
•Signal amplification through PDE hydrolysis of cGMP.
Invertebrate Phototransduction Differences:
•Utilization of inositol 1,4,5-trisphosphate (IP3), not cGMP.
•Light-induced breakdown of PIP2 increases IP3 levels.
•G proteins in invertebrate phototransduction coupled with phospholipase C activation.

70. HumanDisorders

71. Retinitis Pigmentosa (RP) :
•Generic term for heterogeneous inherited disorders.
•Initial symptoms: night blindness, followed by peripheral visual field loss.
•Rod photoreceptors primarily affected; cone cells survive longer.
•Variable onset and degeneration rate among individuals.
•Autosomal dominant, autosomal recessive, or X-linked inheritance.
•RP considered a family of disorders with diverse clinical signs.
•Biochemical studies involve postmortem analysis and blood samples.
•Altered proteoglycans, IRBP absence, and cGMP variations observed.

72. Gyrate Atrophy :
•Rare autosomal recessive disorder affecting choroid and retina.
•Clinical signs: myopia, night blindness, and later, cataracts.
•Linked to deficiency in ornithine-8-aminotransferase (OAT).
•OAT catalyzes ornithine metabolism; deficiency leads to hyperornithinemia.
•Clinical heterogeneity observed; vitamin B6 therapy and arginine-restricted diets explored.
•OAT gene mutations identified, contributing to clinical and biochemical variability.
•Potential therapeutic avenues explored with human OAT gene expression.

73. Retinoblastoma:
•Biochemical Studies on Cell Line Y-79
•Introduction:
•Childhood tumor, hereditary or nonhereditary, on chromosome 13q 14.
•Y-79 cell line established for thorough investigation, neuroectodermal,
undifferentiated.
•Biochemical studies often use Y-79 suspension cultures.
•Retinoid-Binding Proteins:
•Receptors for retinol and retinoic acid detected in Y-79 cell extracts.
•Varied binding in retinoblastomas; low CRABP levels; IRBP synthesized and secreted.
•IRBP presence correlates with tumor differentiation; butyrate stimulates IRBP synthesis.

74. •Insulin and IGF-1 Receptors:
•Insulin and IGF-I receptors found in Y-79 cells; down-regulation after prolonged insulin
exposure.
•Two-site binding for insulin; one-site, one-affinity system for IGF-1.
•Insulin and IGF-I enhance glycine uptake in Y-79 cells, implications in neuronal
characteristics.
•Cyclic-AMP-Dependent Protein Kinases:
•Y-79 cells contain both type I and type II cAMP-dependent protein kinases.
•Imbalance in RI subunit synthesis speculated to contribute to unregulated cell growth.
•Attached (Monolayer) Y-79 Cell Cultures:
•Conditions for monolayer growth manipulated for partial differentiation.
•Butyrate induces IRBP synthesis; laminin promotes attachment and morphological
changes.
•Agents like butyrate and retinoic acid alter gene expression in Y-79 cell attachment
cultures.

75. THANKYOU!!!