The Impact of Biology on Risk Assessment--workshop of the National Research Council''s Board on Radiation Effects Research. July 21-22, 1997, National Academy of Sciences, Washington, DC

Cholinesterases exhibit functions apart from their esterase activity. We have demonstrated an aryl acylamidase and a zinc stimulated metallocarboxypeptidase activity in human serum butyrylcholinesterase. To establish the presence of zinc binding sites in the enzyme we examined the effect of metal chelators on its catalytic activities. The metal chelators 1,10-phenanthroline and N,N,N',N'-tetrakis (2-pyridyl methyl)ethylene diamine (TPEN) inhibited all the three catalytic activities in the enzyme. However, EDTA inhibited the peptidase activity exclusively without affecting the cholinesterase and aryl acylamidase activities. The catalytic activities were recovered upon removal of the chelator by Sephadex G-25 chromatography. Pre-treatment of the enzyme with any one of the three chelators resulted in the binding of the enzyme to a zinc-Sepharose column or to 65Zn2+. Histidine modification of the enzyme pretreated with chelators resulted in abolition of 65Zn2+ binding and zinc-Sepharose binding. Whereas the binding studies demonstrated removal of a metal from a Zn2+ binding site, attempts to remove the metal responsible for catalytic activity were unsuccessful. Atomic absorption spectroscopy indicated approximately 2.5 mol of zinc per mol of enzyme before treatment with EDTA and 1 mol zinc per mol enzyme after EDTA treatment. The results indicate that there are at least two metal binding sites on butyrycholinesterase. The presence of two HXXE...H sequences in butyrylcholinesterase supports these findings. Our studies implicate a zinc dependent metallocarboxypeptidase activity in the non-cholinergic functions of butyrylcholinesterase.     

Fourth module of Xenopus interphotoreceptor retinoid-binding protein: activity in retinoid transfer between the retinal pigment epithelium and rod photoreceptors

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), an extracellular protein believed to support the exchange of retinoids between the neural retina and retinal pigment epithelium (RPE) in the vertebrate eye, exhibits a modular, i.e., repeat, structure. The present study was undertaken to determine whether an individual module of IRBP has activity in retinoid transfer between the RPE and rod photoreceptors. METHODS: The retinoid transfer activity of a recombinant protein corresponding to the fourth module of Xenopus laevis IRBP (X4IRBP) was examined in two ways. First, X4IRBP was tested for its ability to support the regeneration of porphyropsin in detached/reattached Xenopus retina/RPE-eyecups. Following illumination and removal of native IRBP, Xenopus eyecups supplemented with 42 microM X4IRBP or (as a control) Ringer's solution were incubated in darkness and then analyzed for regenerated porphyropsin. Second, toad (Bufo marinus) RPE-eyecup preparations were used to evaluate X4IRBP's ability to promote the release of 11-cis retinal from the RPE. RESULTS: The regeneration of porphyropsin in X4IRBP-supplemented Xenopus retina/RPE-eyecups (0.45 +/- 0.04 nmol; mean +/- SEM, n = 11) exceeded that in controls (0.13 +/- 0.02 nmol, n = 11). For promoting the release of 11-cis retinal from the toad RPE, 42 microM X4IRBP was more effective than equimolar bovine serum albumin although considerably less than that of 26 microM native bovine IRBP. CONCLUSIONS: The results indicate a low but significant activity of IRBP's fourth module in reactions relevant to retinoid exchange.     

Repair and adhesion mechanisms of the cryotherapy lesion in experimental retinal detachment

Cryotherapy to the pigment epithelium and retina induced a proliferation and metaplasia of pigment epithelial cells, Mueller cell hypertrophy, and proliferation of astrocytes. When cryotherapy was applied to the pigment epithelium and to the retina during retinal detachment surgery, a strong adhesion developed, characterized by the occurrence of true cell junctions between pigment epithelium and retinal cells. When only the pigment epithelium was treated, the adhesion appeared weak due to the absence of microvillous interdigitations normally present between pigment epithelium and retina.     

Development of the interphotoreceptor matrix in Xenopus laevis

Xenopus laevis interphotoreceptor matrix (IPM) contains a relatively aqueous insoluble wheat germ agglutinin (WGA)-binding component containing unidentified sialoglycoconjugates (Wood et al [1984] J. Comp. Neurol. 228:299-307). The appearance of WGA-binding macromolecules in the IPM was assessed during late embryonic stages (32-45) and in retinal rudiment cultures, using lectin cytochemistry and Western blotting techniques. Metabolic labeling of the neural retina versus retinal pigment epithelium (RPE)-choroid of juvenile Xenopus with 35S-MET was also evaluated in vivo and in vitro. Lectin cytochemistry of eyes from developmental stages 32-42 demonstrated distinct WGA-ferritin-binding sites on the developing outer segment membranes and in the IPM compartment. At stages 44-46 extensive WGA-binding domains were present as an extracellular network with other randomly scattered domains near the retinal pigment epithelium. Retinal rudiments from stage 32-33 were isolated and allowed to differentiate in hanging drop culture (Hollyfield and Witkowsky [1974] J. Exp. Zool. 189:357-377) with or without an investing pigment epithelium. Cultures developing with RPE exhibited an elaborate IPM with an anastomosing meshwork of WGA-ferritin binding sites. In the absence of RPE only limited amounts of binding restricted to the immediate vicinity of the developing photoreceptor outer segment membranes was observed. When Western blots were probed with WGA-HRP, stage 32-45 retinas demonstrated a major WGA-binding band of 126 kD. Similar amounts of WGA-binding macromolecules were synthesized in preparations cultured in the presence or absence of the investing RPE. During development the major WGA-binding component is a 126-kD protein. Equivalent synthesis of this protein in the presence and absence of RPE suggests that the PE is not required for synthesis of this 126-kD component. These results suggest that the retina is the primary site of synthesis of the WGA-binding components of the Xenopus IPM, whereas the PE plays a principal role in their assembly and organization.     

The optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.     

Light-induced apoptosis: differential timing in the retina and pigment epithelium

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.     

Cellular and molecular regulation of serotonin N-acetyltransferase activity in chicken retinal photoreceptors

Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and large changes in the activity of this enzyme appear to regulate the rhythm in melatonin synthesis. Recent advances have made it possible to study the mRNA encoding chicken AA-NAT, which has only been detected in the retina and pineal gland. Within the retina, AA-NAT mRNA is expressed primarily in photoreceptors. The levels of chicken retinal AA-NAT mRNA and activity exhibit 24-hour rhythms with peaks at night. These rhythms appear to reflect circadian clock control of AA-NAT mRNA abundance and independent effects of light and darkness on both mRNA levels and enzyme activity. The effects of darkness and light may occur through alterations in cAMP-dependent protein phosphorylation, which increases AA-NAT activity in photoreceptor cell cultures. The cAMP-dependent increase of AA-NAT enzyme activity reflects, at least in part, increased mRNA levels and inhibition of enzyme inactivation by a posttranslational mechanism. This review discusses a hypothetical model for the cellular and molecular regulation of AA-NAT activity by circadian oscillators and light in chicken retinal photoreceptor cells.     

Irregular retinal and RPE damage after pressure-induced ischemia in the rabbit

PURPOSE: Pressure-induced ocular ischemia is a frequent model for the investigation of the mechanisms and therapy of retinal ischemic damage. It is important to know whether the tissue damage in such experiments is uniform or irregular. METHODS: We reviewed histologic features of Dutch rabbit eyes after 60-80 min of pressure-induced ischemia. The eyes were enucleated 4 hr, 1 day, or 1 wk after circulation was restored, at which times the electroretinogram b-wave was moderately reduced. RESULTS: Light microscopy showed an irregular distribution of damage involving all retinal layers and retinal pigment epithelium. Some regions of damage (or preservation) were several millimeters wide; others were as small as a few cell widths. Correlation with electroretinogram reduction in individual eyes was difficult. CONCLUSIONS: These results show that pressure-induced ischemic damage in the rabbit, sufficient to reduce the electroretinogram, has a patchy and irregular effect on retina and retinal pigment epithelium. Erroneous judgments may be made about ischemic damage, or therapeutic intervention, if only small or selected regions of retina are examined histologically.     

Cell-type- and developmental-stage-specific metabolism and storage of retinoids by embryonic chick retinal cells in culture

Biological functions of retinoids in the vertebrate retina include the role of 11-cis retinaldehyde as visual pigment chromophore, and possible effects of retinoic acid in histogenesis and cell survival. Qualitative and quantitative regulation of retinoid availability for these complex processes could involve several cell types, including retinal pigment epithelium, Müller glia and retinal photoreceptors and non-photoreceptor neurons; their relative contributions, however, have not been fully elucidated. Using purified cultures, we have carried out a study of cell-type-specific metabolism and storage of retinoids in chick embryo retinal photoreceptors and other neuronal cells, as compared to those of retinal glia. Retinal glia were found to synthesize both retinoic acid and retinyl esters, and to hydrolyse the latter; they also displayed retinol dehydrogenase activities. Cultured neurons and photoreceptors also synthesized and hydrolysed retinyl esters; their capacity for retinaldehyde synthesis from a retinol or retinyl ester substrate suggested the presence of retinol dehydrogenase activity. Retinoic acid was not synthesized in differentiated neuronal cultures, although some synthesis was detectable at early culture stages when the cells were still morphologically undifferentiated. These findings indicate that cell-type-specific metabolic activities are expressed during retinal cell differentiation in vitro, and that embryonic retinal photoreceptors and nonphotoreceptor neurons are active participants in the metabolism and storage of retinoids.     

Circadian clock functions localized in xenopus retinal photoreceptors

A circadian oscillator that regulates visual function is located somewhere within the vertebrate eye. To determine whether circadian rhythmicity is generated by retinal photoreceptors, we isolated and cultured photoreceptor layers from Xenopus retina. On average, 94% of the viable cells in these preparations were rod or cone photoreceptors. Photoreceptor layers produced melatonin rhythmically, with an average period of 24.3 hr, in constant darkness. The phase of the melatonin rhythm was reset by in vitro exposure of the photoreceptor layers to cycles of either light or quinpirole, a D2 dopamine receptor agonist. These data indicate that other parts of the eye are not necessary for generation or entrainment of retinal circadian melatonin rhythms and suggest that rod and/or cone photoreceptors are circadian clock cells.     

Oxygen dependency of retinal adhesion

PURPOSE: Normal retina is firmly attached to the retinal pigment epithelium, but the force of this adhesion drops precipitously within the first 2-3 min after enucleation. The purpose was to study metabolic factors that might be relevant to this postmortem failure of adhesion. METHODS: Dutch rabbit retina was manually peeled from the retinal pigment epithelium on strips of enucleated eyecup within a 37 degrees C bath. Retinal adhesiveness was measured by observing the amount of retinal pigment epithelium that remained adherent to the retina. RESULTS: Autologous whole blood in place of salt solution retarded the decrease in adhesiveness. A solution of hemoglobin alone was similarly effective, whereas methemoglobin solution failed to help the persistence of retinal adhesion. Bubbling oxygen into the salt solution and circulating it to avoid oxygen depletion at the tissue boundary also proved effective at sustaining retinal adhesiveness. Eyes made ischemic in vivo for 5 min or longer, by elevating intraocular pressure, showed virtually no retinal adhesion when enucleated immediately thereafter. However, eyes made ischemic for 10 min, but allowed to regain circulation for 5 min before enucleation, showed a return of retinal adhesiveness to 80% of normal. CONCLUSIONS: Oxidative metabolism is critical to the maintenance of retinal adhesiveness, and the effects of oxygen deprivation on adhesion are reversible within a certain time period.     

A long-term course of experimentally produced choroidal neovascularization in the rat

We studied morphologically a long-term course of experimental choroidal neovascularization (ChNV) induced by krypton laser photocoagulation in the rat retina. Fifty-two weeks after photocoagulation, ChNV was enveloped completely by the retinal pigment epithelium. Vascular endothelial cells of ChNV were thin, with many fenestrations and wide lumen. The ChNV maintained the morphological characteristics of mature leaky capillaries similar to choriocapillaris. The lumen of the neovascularizations tended to be compressed by massive collagen fibers produced by the retinal pigmented epithelium. We found that experimental ChNV in the rat retina retains the characteristics of leaky capillaries for a long time unlike that in the monkey ChNV.     

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.     

A novel pineal night-specific ATPase encoded by the Wilson disease gene

We have identified a pineal night-specific ATPase (PINA), a novel splice variant of the ATP7B gene disrupted in Wilson disease (WD). PINA expression exhibits a dramatic diurnal rhythm in both pineal gland and retina with 100-fold greater expression at night than at day. PINA is expressed in pinealocytes and a subset of photoreceptors in adult rats and is transiently expressed in the retinal pigment epithelium and the ciliary body during retinal development. Nocturnal pineal expression of PINA is under the control of a suprachiasmatic nucleus clock mediated by superior cervical ganglion innervation of the pineal. In vitro, PINA expression in pineal cells can be stimulated by agents activating the cAMP signal transduction pathway. PINA is able to restore copper transport activity in Saccharomyces cerevisiae deficient in the homologous copper-transporting ATPase CCC2, suggesting that this protein may function as a copper transporter in rat pinealocytes. These studies suggest a potential role of rhythmic copper metabolism in pineal and/or retina circadian function.     

Sensitivities of ocular tissues to acute pressure-induced ischemia

Intraocular pressure was artificially elevated for eight hours in eight owl monkeys. The first permanent effect (produced at a perfusion pressure of plus 15 mm Hg) was partial necrosis of iris stroma and ciliary processes, associated with microscopic lesions in the photoreceptors and retina pigment epithelium around the disc and in the retinal periphery. At a slightly higher pressure, visual nerve fibers in the retina and optic nerve and their ganglion cells were affected. Simultaneously, the outer retinal layers showed damage to the pigment epithelium, photoreceptors, and other nuclear layers. At even higher pressures, nearly all the other intraocular tissues were affected except for Müller cells, astroglia in the optic nerve head, epithelium of the pars plana, and the pigment cells of the choroid. The possibility is raised of a nonischemic pressure-induced mechanism for destruction of disc astrocytes in human chronic glaucoma.     

Effects of intravenous iodoacetate and iodate on pH outside rod photoreceptors in the cat retina

PURPOSE: Effects of intravenous iodoacetate (a glycolysis inhibitor) and iodate (a metabolism inhibitor selective to retinal pigment epithelium) on light-evoked alkalinizations and hypoxia-induced acidifications were studied in the dark-adapted cat retina, in vivo, to learn about pH regulation. METHODS: pH was recorded in the extracellular space surrounding rod photoreceptors with double-barrelled H(+)-selective microelectrodes. RESULTS: Intravenous infusion of 5 mg/kg iodoacetate-induced alkalinizations in the outer nuclear layer and suppressed both light-evoked and hypoxia-induced pH responses immediately. Iodate injection (30 mg/kg) produced acidifications in the subretinal space and affected light-evoked alkalinizations gradually but not hypoxia-induced acidifications. CONCLUSIONS: These results suggest that rods glycolysis plays an important role in both light-evoked and hypoxia-induced pH responses. And the retinal pigment epithelium may have little concern with light-evoked alkalinizations except that it plays an important role in regenerating the rhodopsin to be needed for the light responses of photoreceptors. Furthermore, the finding of the intravenous-iodoacetate-induced alkalinization in the outer nuclear layer supports that acid production by rods in the dark is originated from glycolysis to support the dark current. The iodate-induced acidification in the subretinal space indicators that the retinal pigment epithelium might actively transport acids from the subretinal space to the choroid.     

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells1

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.     

The localization of cholinesterase in the retina of the fetal and newborn guinea pig

Retinae of guinea pigs from the fortieth day of gestation to one day postnatally were processed for the localization of cholinesterases in the electron microscope according to the method of Lewis and Shute ('66). Selective inhibition served to distinguish acetylcholinesterase from non-specific cholinesterase activity. Acetylcholinesterase activity was found initially in small amounts in some regions of the outer plexiform layer at the fortieth day of gestation. At later stages it increased in distribution being observed at some photoreceptor terminals and in non-synaptic regions of the layer. Activity was less intense initially in the inner plexiform layer but increased rapidly so that by birth it encompassed a majority of processes. Perikarya of horizontal and some amacrine and ganglion cells possessed acetylcholinesterase activity in their nuclear envelope and rough endoplasmic reticulum. The possible role of the enzyme in inhibitory circuits of the fetal retina is discussed.     

Decrease of hepatic triglyceride lipase levels and increase of cholesteryl ester transfer protein levels in patients with primary biliary cirrhosis: relationship to abnormalities in high-density lipoprotein

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.