Cytidine metabolism in photoreceptor cells of the rat.

During brief (30-min) incubations, isolated rat retinas accumulated [3H]cytidine, converted it to cytidine triphosphate (CTP), and incorporated it into RNA and cytidine diphosphate-diacylglyceride (CDG), a phospholipid precursor of phosphatidylinositol (Pl). Labeled CTP, RNA, and CDG contents were found to be two- to three-fold higher in photoreceptor cells than in cells of the inner retina. Autoradiograms showed that, within photoreceptor cells, silver grains representing RNA were concentrated over the nuclei in dark and light, while silver grains representing CDG were concentrated over the inner segments only after incubation in dark. The formation of labeled CTP and the synthesis of RNA were enhanced in light, while labeled CDG levels became reduced in light concurrent with an increase in the incorporation of labeled inositol into Pl. The 3H-labeled CDG content, however, was increased two- to fourfold in light in the presence of actinomycin D, and autoradiograms show a heavy concentration of silver grains over the inner segments of photoreceptor cells. These findings establish a role for cytidine nucleotides in photoreceptor cell metabolism and in light-dependent increases in RNA and Pl synthesis. Furthermore, the observations indicate that a competition may exist in light for cytidine or CTP and suggest that availability of cytidine for CDG synthesis may have a regulatory role in Pl metabolism within the photoreceptor cells.

Light- and cytidine-dependent phosphatidylinositol synthesis in photoreceptor cells of the rat.

Incorporation of [3H]inositol into phosphatidylinositol (Pl) in isolated rat retinas is enhanced by light and by the addition of cytidine to the incubation media. In retinas preincubated with [3H]inositol in dark, [3H]inositol was chased into Pl in light by addition of unlabeled cytidine and was chased out of Pl in light by addition of unlabeled cytidine plus inositol. Autoradiograms of retinas show a heavy density of silver grains over photoreceptor cell inner segments (with chase-in) and a loss of labeling (with chase-out). Exogenous cytidine and inositol were shown to enhance not only the turnover of Pl within photoreceptor cells but the synthesis of Pl as well; in media supplemented with these precursors, approximately 50% of [14C]glycerol and 25% of [32Pi]incorporated into lipid in light were associated with Pl. These results suggest that availability of both cytidine and inositol may play a role in the light-dependent changes in Pl metabolism within photoreceptor cells.

Cyclic-nucleotide phosphodiesterase: an early defect in inherited retinal degeneration of C3H mice.

Cyclic nucleotides have been implicated in the differentiation and function of the vertebrate retina. In the normal retina of DBA mice, the specific activity of cyclic-nucleotide phosphodiesterase (PDE), with cyclic-AMP as the substrate (cAMP-PDE), increases eightfold between the 6th and 20th postnatal day. Kinetic analysis of retinae from newborn mice reveals a PDE with a single Michaelis constant (K(m)) value for cyclic-AMP (low K(m)-PDE). After the 6th postnatal day, a second PDE with a high K(m) for cyclic-AMP (high K(m)-PDE) can be demonstrated. The appearance and increasing activity of the high K(m)-PDE coincides with the differentiation and growth of photoreceptor outer segments. Additionally, the high K(m)-PDE is shown by microchemical techniques to be concentrated in the photoreceptor cell layer and the low K(m)-PDE within the inner layers of the normal retina. In C3H mice afflicted with an inherited degeneration of the photoreceptor layer, the postnatal increase in the specific activity of cAMP-PDE is substantially lower than in the normal retina. The postnatal increase in the specific activity of cAMP-PDE in two regions of the brain of C3H mice is the same as in the normal strain. A deficiency in high K(m)-PDE activity in the C3H retina is evident on the 7th postnatal day, when the activity of low K(m)-PDE, photoreceptor morphology, and rhodopsin content of these retina are essentially normal. In the adult C3H retina, the PDE activity with cyclic-GMP and cyclic-UMP as substrates is significantly below that of the normal retina. These data indicate that an alteration in cyclic-AMP metabolism occurs before photoreceptor cell degeneration in the retinae of C3H mice.

Retinal degeneration associated with taurine deficiency in the cat.

A degeneration of the retinal photoreceptor cells develops in cats when casein is the source of dietary protein. Amino acid profiles indicate that the degeneration is associated with a selective decrease in plasma and retinal taurine concentrations. A sulfur amino acid deficit in the casein diet combined with specific amino acid requirements of the cat appear related to this unique expression of taurine deficiency.

Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes in GCL.

Detailed analyses of neuronal and astrocyte cell numbers in the ganglion cell layer (GCL) of whole-mounted peripheral retinas from 16 Alzheimer's disease (AD) and 11 control eyes (11 and 9 cases, respectively) demonstrate extensive neuronal loss throughout the entire retina in AD as compared to control eyes. The observed neuronal loss is most pronounced in the superior and inferior quadrants, ranging between 40 and 49% throughout the midperipheral regions, and reaching 50-59% in the far peripheral inferior retina, while the overall neuronal loss throughout the entire retina amounts to 36.4% (p < 0.004). Although the 16% increase in astrocyte numbers is not significant, the ratio of astrocytes to neurons is significantly higher (82%; p < 0.0008) in AD as compared to normal retina (0.238 +/- 0.070 vs. 0.131 +/- 0.042). These results are strengthened by the close agreement (within +/- 15% of respective means) found between fellow eyes. Analysis of glial fibrillary acidic protein immunoreactivity (GFAP-ir) in sections of retinas from an additional 12 AD and 19 control cases show increased GFAP-ir with more extensive labeling of astrocytes in the GCL as well as increased labeling of Müller cell end-feet and radial processes in AD as compared to control retinas. The extensive loss of neurons documented in these retinas, accompanied by an increased astrocyte/neuron ratio, provides further support for the substantial involvement of the retina in AD.

Protein phosphorylation in cultured rat RPE. Effects of protein kinase C activation.

Incubation of confluent cultures of rat retinal pigment epithelium (RPE) with 32P-orthophosphate resulted in the incorporation of 32P into proteins, RNA and the nucleoside phosphates ADP, GDP, ATP and GTP. RPE cultures incubated with phorbol-12-myristate-13-acetate (PMA), a known activator of protein kinase C, did not significantly change the incorporation of 32P into total protein, RNA or the nucleoside phosphates ADP, GDP, ATP and GTP. However, PMA exposure specifically increased phosphorylation of five proteins with molecular weights of 80 kilodaltons (K), 56K, 35K, 33K, and 29K having isoelectric points between 4.3 and 6.5. PMA treated cultures also showed dephosphorylation of two proteins having molecular weights of about 33K. The observed increase in 80K phosphorylation suggests that protein kinase C is present and activated by PMA in the RPE.

Reductions in taurine secondary to photoreceptor loss in Irish setters with rod-cone dysplasia.

These studies show that onset of photoreceptor cell degeneration preceded the loss of taurine in retinas of Irish setters with rod-cone dysplasia. The numbers of photoreceptor cell nuclei were within the normal adult range in affected setters at 10 through 26 days of age but declined rapidly between 26 and 45 days and more gradually thereafter; their numbers became reduced to 50% of normal at 45 days and then to 12-20% and 3-10% of normal at 192 and 346 days, respectively. Taurine concentrations increased within the photoreceptor cell layer during normal development to peak values (50 mM) at a time (45 days of age) corresponding to the development of adult photoreceptor function. In the affected setters, taurine levels increased as in the normal until 26 days of age and then remained at that value until 40-50% of the photoreceptor cells had degenerated. Thereafter, taurine levels declined gradually throughout the period of photoreceptor cell degeneration and were reduced to 30-40% of the normal adult level at the time (346 days) when the thickness of the outer nuclear layer was reduced to less than one complete row of nuclei. These observations agree with findings in retinal degeneration (rd) mice and RCS rats and indicate that in all three of these animal models of hereditary retinal degenerations, reductions in retinal taurine levels occur secondary to the loss of photoreceptor cells.

Opsin phosphorylation in retinas from autopsy eyes with retinitis pigmentosa.

Opsin phosphorylation in light was detected in three retinas from autopsy eyes with retinitis pigmentosa (RP) including one with sex-linked RP and two with autosomal recessive RP, that were studied at postmortem intervals of 1-4 hr. In these retinas from RP eyes, opsin phosphorylation in light was reduced compared with that in normal human retinas, a finding that is compatible with reduced amounts of opsin due to extensive loss of photoreceptor cells. ATP and GTP levels, although reduced below normal, also were easily detectable, supporting the idea that the reduction in opsin phosphorylation was due to loss of photoreceptor cells and not to a reduced capacity for energy metabolism. These findings in these RP retinas contrast with those in rd mice and Irish setters with rod-cone dysplasia in which a failure of opsin phosphorylation has been detected prior to onset of photoreceptor cell degeneration.

Protein phosphorylation in retinal pigment epithelium of Long-Evans and Royal College of Surgeons rats.

Cultures of retinal pigment epithelium (RPE) from normal Long-Evans (LE) and dystrophic Royal College of Surgeons (RCS) rats were incubated with 32P-orthophosphate and then phagocytically challenged with isolated rod outer segments (ROS) or polystyrene latex spheres (PSL). The 32P incorporation into individual proteins was quantified by image analysis of two-dimensional gel autoradiograms, and changes in phosphorylation were identified by comparison with unchallenged control cultures. Phosphorylation changes that were similar in response to either ROS or PSL were classified as nonspecific and omitted from further analysis; those associated solely with ROS exposure were classified as ROS specific and compared between the two strains. None of the 30 ROS-specific changes in protein phosphorylation identified in normal LE RPE were the same as in RCS RPE. However, unique ROS-specific changes in phosphorylation were observed in 13 RCS RPE proteins. Three RCS proteins showed ROS-specific decreases; ten showed ROS-specific increases. Six of these ten RCS proteins with increased phosphorylation showed ROS-specific decreases in LE RPE. No other correspondence in ROS-specific changes was found among the remaining LE or RCS RPE proteins, but several RCS proteins were phosphorylated at abnormal levels under control conditions. Even though ROS-induced changes in phosphorylation were aberrant in RCS RPE, their presence indicated that a ROS-specific transmembrane signal was generated after interaction with ROS. The abnormal increases and decreases observed in ROS-specific phosphorylation in the RCS suggested that the defect in ROS phagocytosis was associated with the misregulation or malfunction of both protein kinases and phosphatases.

Postmortem metabolic capacity of photoreceptor cells in human and rat retinas.

Retinas of postmortem human donor eyes retained the high-affinity mechanism for uptake of 3H-taurine, the capacity to synthesize rhodopsin from 14C-amino acids, and the ability to incorporate inorganic 32P-phosphate into rhodopsin with exposure to light for 4 to 4 1/2 hr. These processes declined at a rate of about 16% to 19% per hour between 2 and 4 1/2 hr after death. Parallel studies with rats maintained after death at room temperature (22 degrees C) showed that all three processes declined linearly at rates of 8% to 12% per hour. In rats maintained after death at body temperature (37 degrees C) or on ice (4 degrees C), the rates of decline in rhodopsin synthesis were respectively 20% to 22% and 7% to 9% per hour, whereas the rates of decline of rhodopsin phosphorylation at these temperatures were, respectively, 20% to 22% and 3% to 5% per hour. Retinas from rats maintained after death in light or dark at room temperature showed no differences in their capacity to synthesize or phosphorylate rhodopsin from la 7% to 9% per hour, whereas the rates of decline of rhodopsin phosphorylation at these temperatures were, respectively, 20% to 22% and 3% to 5% per hour. Retinas from rats maintained after death in light or dark at room temperature showed no differences in their capacity to synthesize or phosphorylate rhodopsin from la 7% to 9% per hour, whereas the rates of decline of rhodopsin phosphorylation at these temperatures were, respectively, 20% to 22% and 3% to 5% per hour. Retinas from rats maintained after death in light or dark at room temperature showed no differences in their capacity to synthesize or phosphorylate rhodopsin from labeled precursors. These findings in human and rat eyes show that photoreceptor cells can perform energy-requiring processes for several hours after death.

Melanin concentration in normal human retinal pigment epithelium. Regional variation and age-related reduction.

Melanin concentrations were analyzed in the pigment epithelium of 61 postmortem normal human eyes from donors 14-97 yr of age. The content of soluble melanin in the pigment epithelium declined with age from the highest values of 95 micrograms/mg in the 14-50 yr age group to the lowest values of 22 micrograms/mg dry weight in the over 70 yr age group. In 16 of these eyes, regional measurements revealed that melanin concentrations were lowest in the macula-perimacular area (a region 5-8 mm in diameter centered on the fovea), were higher in the mid-periphery, and were highest in the far periphery. In the macular region, melanin concentrations were similarly low at all ages, whereas, in the mid- and far-peripheral regions, melanin concentrations decreased with age. These studies demonstrate age-related reductions and regional variations in melanin concentrations in the pigment epithelium of postmortem normal human eyes, and establish a baseline for future comparison with donor eyes from patients with hereditary retinal degenerations.

Deficiency in light-dependent opsin phosphorylation in Irish setters with rod-cone dysplasia.

A deficiency in light-dependent opsin phosphorylation and a slight reduction in opsin synthesis were observed during photoreceptor cell development (22-26 days) preceding photoreceptor cell loss in Irish setters with rod-cone dysplasia. In addition to opsin, two other phosphoprotein bands were found associated with the photoreceptor cell layer; synthesis and phosphorylation of one of these (band 3; 44-48 Kd) appeared reduced, while synthesis and phosphorylation of the other (band 1; 29-31 Kd) was within the normal range in 25-day-old affected setters. The deficiency in light-dependent opsin phosphorylation in affected setters was not due to a deficiency in opsin kinase, since soluble proteins from affected or normal outer segments catalyzed equally well opsin phosphorylation in partially kinase-depleted outer segment membranes from normal, while both kinase preparations failed to promote light-dependent opsin phosphorylation in those from affected setters. A deficiency in light-dependent opsin phosphorylation was also observed in rd/rd mice at all ages studied. In contrast, in Royal College of Surgeons (RCS) rats, light-dependent opsin phosphorylation was within the normal range prior to photoreceptor loss, and became nondetectable only after 50% or more of the photoreceptors had degenerated.

Identification of proteins in retinas and IPM from eyes with retinitis pigmentosa.

Opsin, the alpha-subunit of transducin, S-antigen, interphotoreceptor retinoid-binding protein (IRBP) and cathepsin D were assessed in autopsy eyes from patients with retinitis pigmentosa (RP) and normal autopsy eyes. Immunochemical methods were used to determine the presence of these proteins on Western blots of retinal homogenates from five RP donors and on blots of interphotoreceptor matrix (IPM) preparations from six other RP eyes. The amounts of immunoreactive opsin, S-antigen, alpha-transducin, and IRBP appeared below normal in retinas from RP eyes. All six IPM samples from patients with advanced RP had reduced amounts of S-antigen and no detectable IRBP or transducin. Cathepsin D (an RPE protein) was present in IPM or RP eyes in amounts comparable to that in IPMs from normal eyes. Small amounts of cathepsin D were also detected in retinas from both normal and RP eyes. These studies show that proteins specific to the photoreceptor-pigment epithelium complex in normal eyes can be detected in autopsy eyes from patients with RP and suggest that the observed reductions in photoreceptor-specific proteins occur as a consequence of photoreceptor loss.

Plasma amino-acids in hereditary retinal disease. Ornithine, lysine, and taurine.

Plasma free amino-acids were measured in 41 patients with hereditary chorio-retinal degenerations including 26 with retinitis pigmentosa and five with gyrate atrophy of the choroid, six relatives of patients with gyrate atrophy, and 13 normal subjects. Patients with gyrate atrophy had very increased levels of ornithine and slightly decreased mean lysine values. Most relatives had slightly increased ornithine. Taurine, known to be deficient in the plasma of casein-fed cats with photoreceptor degeneration, was normal in all patients. Amino-acid precursors and metabolites of ornithine and taurine were also normal in the plasma. Although the association of high ornithine and gyrate atrophy appears constant, high levels of ornithine alone do not necessarily lead to this degeneration; one patient with known hyperammonaemia, homocitrullinuria and a tenfold increase in plasma ornithine was found to have a normal fundus appearance and normal electroretinogram.

Effects of IBMX on the ERG of the isolated perfused cat eye.

Full-field electroretinograms (ERGs) were recorded from isolated cat eyes perfused through the ophthalmociliary artery with isobutylmethylxanthine (IBMX), an inhibitor of cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase. Low doses of IBMX (0.1-0.3 mM) produced decreased rod ERG amplitudes at low stimulus luminances and increased rod ERG amplitudes at high stimulus luminances. A high dose of IBMX (1.0 mM) initially produced the same effect as the low doses and then led to decreased rod ERG amplitudes at all stimulus luminances. Perfusion with IBMX also resulted in elevations in the semi-saturation luminance (sigma), delayed rod a-wave latencies, delayed rod a-wave and b-wave implicit times, and reduced rod a-wave slopes. Eyes perfused with IBMX (1.0 mM) were also found to have elevated levels of retinal cyclic GMP. These effects of IBMX on the rod ERG are considered in the context of previously described ERGs in selected cases of human retinal degeneration.

Phagocytic challenge induces changes in phosphorylation of retinal pigment epithelium proteins.

Changes in protein phosphorylation induced by phagocytic challenge were identified in cultured rat retinal pigment epithelium (RPE) following exposure to isolated rat rod outer segments (ROS) or to polystyrene latex microspheres (PSL). RPE phosphoproteins were characterized based on molecular weight and isoelectric point and 32P incorporation into phosphoproteins was quantified by digitized image analysis of two-dimensional gel autoradiograms. Changes in the phosphorylation of RPE proteins were determined by comparing 32P gel data from phagocytically challenged cultures with control cultures. ROS-specific changes were defined as those occurring only in response to ROS while nonspecific changes were those associated with either ROS or PSL phagocytosis. A parallel study was conducted to identify those proteins which also show increased phosphorylation following protein kinase C (PKC) activation by phorbol-12-myristate-13-acetate. ROS-specific increases in the phosphorylation of 2 RPE proteins were found, 1 of which also showed an increase with PKC activation. Nonspecific increases included the phosphorylation of 11 RPE proteins, 10 of which were also phosphorylated with PKC activation. ROS-specific decreases were observed in 12 RPE phosphoproteins while 3 proteins showed nonspecific decreases in their phosphorylation. These findings demonstrate that phagocytic challenge of the RPE with either specific or nonspecific particles is linked to the activation of phosphatases and kinases and that activation of PKC may play a role in phagocytosis of both particle types. The identification of two distinct groups of changes in phosphorylation supports the hypothesis that different pathways exist for phagocytosis of ROS-specific and nonspecific particles by the RPE.

Light enhances the turnover of phosphatidylinositol in rat retinas.

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light-dependent increase in [3H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [3H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light-enhanced incorporation of [3H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [3H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer. PI turnover involved 2%/min of the total PI content in dark and 6-8%/min in light. In contrast to what has been reported for stimulus-enhanced turnover of PI in some tissues, this light-enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (22Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of 22Na in light but not in dark.

Phosphatidylinositol synthesis and phosphorylation are enhanced by light in rat retinas.

Incorporation of labeled precursors (glycerol, glucose, or 32Pi) into phosphatidylinositol (PI) was 2-3-fold higher in rat retinas incubated in light compared to those in dark. During brief (30 min) incubations with labeled glycerol, there was a selective increase in the radioactivity associated with PI and phosphatidic acid, whereas, upon longer (60 min) incubations, synthesis of other lipids was also enhanced in light. Accumulation of the precursors was similar in light or dark throughout 60 min of incubation. Phosphorylation of PI to triphosphoinositide (TPI) was also enhanced in light, and, in both light and dark, up to 40-50% of the total 32Pi incorporated was associated with TPI. A model is proposed for PI metabolism based on two pathways: 1) a PI cycle representing synthesis and turnover of PI, and 2) phosphorylation of PI to TPI and possible hydrolysis of TPI. Light stimulation was shown to enhance the synthesis of PI within the photoreceptor cell layer and truncated rods and to increase the phosphorylation of PI to TPI within the inner retina and synaptosomes. Parallel studies with retinas from Royal College of Surgeons rats with advanced photoreceptor cell degeneration and intact inner retina showed that loss of the photoreceptor cells did not affect the capacity for phosphorylation of PI to TPI, but was associated with reduced capacity for PI synthesis. These results provide evidence that light stimulation affects different pathways of PI metabolism within different cells of the retina.