Harris Ripps

Interphotoreceptor retinoid-binding protein (IRBP) - Molecular biology and physiological role in the visual cycle of rhodopsin

The regeneration of visual pigment in rod photoreceptors of the vertebrate retina requires an exchange of retinoids between the neural retina and the retinal pigment epithelium (RPE). It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid through the aqueous compartment (interphotoreceptor matrix) that separates the RPE and the photoreceptors. The first part of this review summarizes the cellular and molecular biology of IRBP. Work on the IRBP gene indicates that the protein contains a four-fold repeat structure that may be involved in binding multiple retinoid and fatty acid ligands. These repeats and other aspects of the gene structure indicate that the gene has had an active and complex evolutionary history. IRBP mRNA is detected only in retinal photoreceptors and in the pineal gland; expression is thus restricted to the two photosensitive tissues of vertebrate organisms. In the second part of this review, we consider the results obtained in experiments that have examined the activity of IRBP in the process of visual pigment regeneration. We also consider the results obtained on the bleaching and regeneration of rhodopsin in the acutely detached retina, as well as in experiments testing the ability of IRBP to protect its retinoid ligand from isomerization and oxidation. Taken together, the findings provide evidence that, in vivo, IRBP facilitates both the delivery of all-trans retinol to the RPE and the transfer of 11-cis retinal from the RPE to bleached rod photoreceptors, and thereby directly supports the regeneration of rhodopsin in the visual cycle.

Cloning and Expression of Two Related Connexins from the Perch Retina Define a Distinct Subgroup of the Connexin Family

We have cloned cDNAs for two closely related connexins (Cx), Cx35 and Cx34.7, from a perch retinal cDNA library. Sequencing of PCR products from genomic DNA revealed that both connexins have an intron 71 bp after the translation initiation site; in Cx35, the intron is 900 bp in length, whereas in Cx34.7 it is ∼20 kb. Southern blots of genomic DNA suggest that the two connexins represent independent single copy genes. In Northern blots, Cx35 and Cx34.7 transcripts were detected in retina and brain; Cx34.7 also showed a weak signal in smooth muscle (gut) RNA. Antibodies against Cx35 labeled a 30 kDa band on a Western blot of retinal membranes, and in histological sections, the pattern of antibody recognition was consistent with labeling of bipolar cells and unidentified processes in the inner plexiform and nerve fiber layers. When expressed in Xenopus oocytes, Cx35 and Cx34.7 formed homotypic gap junctions, but the junctional conductance between paired oocytes expressing Cx35 was 10-fold greater than that recorded for gap junctional channels formed by Cx34.7. The homotypic gap-junctional channels were closed in a voltage-dependent manner but with relatively weak voltage sensitivity. Heterotypic gap junctions formed by Cx35 and Cx34.7 displayed junctional conductances similar to those of Cx34.7 homotypic pairs and showed a slightly asymmetric current–voltage relationship; the side expressing Cx35 exhibited a higher sensitivity to transjunctional potentials. An analysis of the sequence and gene structure of the connexin family revealed that perch Cx35 and Cx34.7, skate Cx35, and mouse Cx36 constitute a novel γ subgroup.

Interphotoreceptor retinoid-binding protein: Role in delivery of retinol to the pigment epithelium

The ability of interphotoreceptor retinoid-binding protein (IRBP) to facilitate the incorporation of retinol into retinyl esters by the retinal pigment epithelium (RPE) was examined in toad (Bufo marinus) eyecup preparations devoid of neural retina (RPE-eyecup). Solutions containing purified bovine IRBP and all-trans[3H]retinol were introduced into the vitreal cavity of the RPE-eyecup. After incubation at 22 degrees C, [3H]retinyl ester was extracted from the RPE cells and isolated by high performance liquid chromatography. All-trans[3H]retinyl ester formed in the RPE increased with time of incubation (up to 2 hr) and with concentration of IRBP (up to 10 microM). The increase with IRBP concentration accompanied, and presumably resulted from, an increased transfer of [3H]retinol to the RPE-eyecup. With higher concentration of IRBP (20-30 microM), both the amount of [3H]retinyl ester formed (relative to the peak value at 10 microM IRBP) and the overall molar content of endogenous retinyl ester were reduced. On the other hand, bovine serum albumin at relatively high concentration (90 microM) was less effective than 3 microM IRBP in supporting the formation of [3H]retinyl ester, and it did not reduce the level of native retinyl ester in the RPE. Using 3 microM IRBP, levels of [3H]retinyl ester formed were comparable to or exceeded those obtained with phosphatidyl choline (0.9 mg ml-1) or serum retinol-binding protein (3 microM). The data are consistent with the hypothesized role of IRBP as a carrier of retinol between the retina and RPE in the operation of the visual cycle.

Ocular Lesions Produced by an Optical Maser (Laser)

Ocular lesions have been experimentally produced in rabbit by a pulsed optical maser (laser). The high-energy density delivered in a single 0.5 msec pulse was sufficient to cause instantaneous thermal injury to the pigmented retina and iris of the brown rabbit. Ophthalmoscopically, the retinal lesions resembled flash burns from an atomic fireball.

Retinitis Pigmentosa: A Symposium on Terminology and Methods of Examination

This report represents a summary of opinions expressed at a meeting of specialists interested in retinitis pigmentosa (RP) and allied diseases, at which an attempt was made to define some minimum guidelines for ocular evaluation of these disorders. The term RP would be reserved for a group of hereditary disorders that diffusely involve photoreceptor and pigment epithelial function, and should not be used when a secondary cause is suspected. RP may be classified by genetic type (single cases without known affected relatives should be termed isolated or simplex), by the topography of retinal involvement, and by the severity of disease (to identify subtypes with mild or localized disease). Patients should have at least one comprehensive examination that conforms to basic standards, preferable early in the course of the disease. The visual field examination should use both a small and a large test light. Electroretinographic testing should (1) use a full-field stimulus, and (2) routinely document three independent responses (cone, rod, and mixed cone-rod). Patients should be identifiable for future study or therapeutic trials. They should be counseled about the disease and followed regularly. No specific therapy exists at present for most of these diseases, but optical and night vision aids are available. Sunglasses for outdoor use are recommended until more is known about whether long-term exposure to bright sunlight alters the course of these diseases.

FUNCTIONAL PROPERTIES OF INTERPHOTORECEPTOR RETINOID‐BINDING PROTEIN

Abstract– It has been hypothesized that interphotoreceptor retinoid‐binding protein (IRBP) functions as a two‐way carrier of retinoid between the retinal pigment epithelium (RPE) and rod photoreceptors in the vertebrate eye. This hypothesis has been tested in recent studies that have employed purified, initially ligand‐free, bovine IRBP and the “RPE‐eyecup” obtained from the toad (Bufo marinus) eye. The present experiments further characterize the IRBP/RPE‐eyecup system with respect to (i) the solubilization and protection of retinol by IRBP, and (ii) the time course of IRBP‐mediated release of 11‐cis retinal by the RPE. The data, together with previous findings in the IRBP/RPE‐eyecup preparation, support the view that 11‐cis retinal is the principal retinoid released by the RPE into IRBP‐supplemented aqueous medium, and that IRBP in vivo promotes the regeneration of rhodopsin by facilitating the exchange of retinoid between bleached rods and the RPE.

Antiretinal Antibodies in Serum of Patients with Age-related Macular Degeneration

Antibodies, immunoreactive with normal human retinal proteins, were detected by Western immunoblot analysis in the sera of 30 patients with age-related macular degeneration (AMD). Sera from 14 of these patients demonstrated positive binding predominantly to a doublet protein of molecular weight between 58 and 62 kD. The sera from the remaining 16 patients and from 12 control subjects reacted either weakly or not at all with the doublet protein. No correlation was found with any specific type of AMD. The serum antibodies also immunocrossreacted with the same proteins from isolated photoreceptor outer segments; this was confirmed by indirect immunofluorescence on intact retinas. The crossreactivity of the serum antibodies with a protein of Mr 58 to 62 kD, the lower band present in the bovine purified neurofilament-68 kD preparation, suggests strongly that this protein may be a component of the neuronal cytoskeleton. However, it is not clear whether these autoantibodies play a direct role in the etiology of AMD or represent a nonspecific response to retinal damage.

Cortical responses to stimulation of the human fovea

Abstract Functional properties of the human fovea were studied by means of the visual evoked response (VER), and the results compared with subjective measurements obtained for equivalent stimulus conditions. Employing a constant VER latency as the response criterion for sensitivity determinations, we found excellent agreement between the VER and subjective thresholds in studies of the area-intensity relation, Stiles-Crawford effect and spectral sensitivity. However, VER and subjective sensitivity measurements differed in one important respect. Whereas the subjective sensitivity of the parafovea exceeded that of the fovea, VER sensitivity decreased markedly with increasing distance from the fovea. Thus, several lines of evidence indicate that the VER provides a valid objective index of foveal function.

Functional characteristics of skate connexin35, a member of the γ subfamily of connexins expressed in the vertebrate retina

Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the γ subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage‐gated at transjunctional potentials ≥ 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the α or β subfamilies. Voltage‐activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 μm quinine resulted in a 1.8‐fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage‐activated currents of Cx46 hemichannels were also enhanced 1.6‐fold following quinine treatment, whereas Cx43‐injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells.

Hemichannel-Mediated and pH-Based Feedback from Horizontal Cells to Cones in the Vertebrate Retina

Background Recent studies designed to identify the mechanism by which retinal horizontal cells communicate with cones have implicated two processes. According to one account, horizontal cell hyperpolarization induces an increase in pH within the synaptic cleft that activates the calcium current (Ca2+-current) in cones, enhancing transmitter release. An alternative account suggests that horizontal cell hyperpolarization increases the Ca2+-current to promote transmitter release through a hemichannel-mediated ephaptic mechanism. Methodology/Principal Findings To distinguish between these mechanisms, we interfered with the pH regulating systems in the retina and studied the effects on the feedback responses of cones and horizontal cells. We found that the pH buffers HEPES and Tris partially inhibit feedback responses in cones and horizontal cells and lead to intracellular acidification of neurons. Application of 25 mM acetate, which does not change the extracellular pH buffer capacity, does lead to both intracellular acidification and inhibition of feedback. Because intracellular acidification is known to inhibit hemichannels, the key experiment used to test the pH hypothesis, i.e. increasing the extracellular pH buffer capacity, does not discriminate between a pH-based feedback system and a hemichannel-mediated feedback system. To test the pH hypothesis in a manner independent of artificial pH-buffer systems, we studied the effect of interfering with the endogenous pH buffer, the bicarbonate/carbonic anhydrase system. Inhibition of carbonic anhydrase allowed for large changes in pH in the synaptic cleft of bipolar cell terminals and cone terminals, but the predicted enhancement of the cone feedback responses, according to the pH-hypothesis, was not observed. These experiments thus failed to support a proton mediated feedback mechanism. The alternative hypothesis, the hemichannel-mediated ephaptic feedback mechanism, was therefore studied experimentally, and its feasibility was buttressed by means of a quantitative computer model of the cone/horizontal cell synapse. Conclusion We conclude that the data presented in this paper offers further support for physiologically relevant ephaptic interactions in the retina.