The matrix metalloproteinase inhibitor BB-1101 prevents experimental autoimmune uveoretinitis (EAU). (17/474)

EAU is characterized by breakdown of the blood-retinal barrier and extravasation of leucocytes into retinal tissue leading to destruction of photoreceptor cells. Matrix metalloproteinases (MMP) have been implicated in trafficking of cells into tissues, but their role in inflammatory eye disease is unclear. A synthetic MMP inhibitor, BB-1101, was administered subcutaneously, from either day 0 or day 7, to Lewis rats challenged with bovine S-antigen to induce EAU. When given up to day 14, BB-1101 reduced the incidence of disease and delayed the day of onset of clinical disease. When administered from day 7 until day 21, EAU was completely abrogated. A quantitative polymerase chain reaction (PCR) assay showed an increase of both matrilysin (MMP-7), neutrophil collagenase (MMP-8) and macrophage metalloproteinase (MMP-12) in retinas from EAU animals compared with naive controls. These enzymes are produced by activated leucocytes and act on components of the basement membrane. These results therefore implicate these MMP as integral to the development of pathology in EAU.  (+info)

Both PCE-1/RX and OTX/CRX interactions are necessary for photoreceptor-specific gene expression. (18/474)

RX, a homeodomain-containing protein essential for proper eye development (Mathers, P. H. Grinberg, A., Mahon, K. A., and Jamrich, M. (1997) Nature 387, 603-607), binds to the photoreceptor conserved element-1 (PCE-1/Ret 1) in the photoreceptor cell-specific arrestin promoter and stimulates gene expression. RX is found in many retinal cell types including photoreceptor cells. Another homeodomain-containing protein, CRX, which binds to the OTX element to stimulate promoter activity, is found exclusively in photoreceptor cells (Chen, S., Wang, Q. L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gillbert, D. J. Jenkins, N. A., and Zack, D. J. (1997) Neuron 19, 1017-1030; Furukawa, T., Morrow, E. M., and Cepko, C. L. (1997) Cell 91, 531-541). Binding assay and cell culture studies indicate that both PCE-1 and OTX elements and at least two different regulatory factors RX and CRX are necessary for high level, photoreceptor cell-restricted gene expression. Thus, photoreceptor specificity can be achieved by multiple promoter elements interacting with a combination of both photoreceptor-specific regulatory factors and factors present in closely related cell lineages.  (+info)

Mice lacking G-protein receptor kinase 1 have profoundly slowed recovery of cone-driven retinal responses. (19/474)

G-Protein receptor kinase 1 (GRK1) ("rhodopsin kinase") is necessary for the inactivation of photoactivated rhodopsin, the light receptor of the G-protein transduction cascade of rod photoreceptors. GRK1 has also been reported to be present in retinal cones in which its function is unknown. To examine the role of GRK1 in retinal cone signaling pathways, we measured in mice having null mutations of GRK1 (GRK1 -/-) cone-driven electroretinographic (ERG) responses, including an a-wave component identified as the field potential generated by suppression of the circulating current of the cone photoreceptors. Dark-adapted GRK1 -/- animals generated cone-driven ERGs having saturating amplitudes and sensitivities in both visible and UV spectral regions similar to those of wild-type (WT) mice. However, after exposure to a bright conditioning flash, the cone-driven ERGs of GRK1 -/- animals recovered 30-50 times more slowly than those of WT mice and similarly slower than the cone-driven ERGs of mice homozygously null for arrestin (Arrestin -/-), whose cone (but not rod) response recoveries were found to be as rapid as those of WT. Our observations argue that GRK1 is essential for normal deactivation of murine cone phototransduction and provide the first functional evidence for a major role of a specific GRK in the inactivation of vertebrate cone phototransduction.  (+info)

Kinetics of apoptotic cells in experimental autoimmune uveoretinitis. (20/474)

PURPOSE: To investigate the role of apoptosis in immunopathogenic mechanisms of experimental autoimmune uveoretinitis (EAU), the kinetics of apoptotic cells and expression of Fas and Fas ligand (FasL) in the eye with EAU were studied. METHODS: Male inbred Lewis rats were immunized with S-antigen (40 microg/rat), and eyes were examined to detect apoptotic cells on days 1, 4, 8, and 10 post-immunization and days 0, 2, 4, 6, and 8 after the onset of EAU. The clinical and pathologic scores were used for estimating EAU. Apoptotic cells were analyzed by TdT-mediated dUTP nick-end labeling, electron microscopic and immunohistologic examinations, and agarose gel electrophoresis. The anti-rat Fas and anti-rat FasL antibodies were used to examine the expression of Fas and FasL. RESULTS: Apoptotic cells were detected in the infiltrating cells in the aqueous humor, the vitreous body, the iris-ciliary body, and the retina. Apoptotic cells were observed as early as the day of EAU onset and reached a peak on day 2 after the disease onset. Fas and FasL were expressed on the infiltrating cells in the aqueous humor and the vitreous. FasL was expressed on resident cells of the ciliary body. The kinetics of the expression of FasL corresponded with the kinetics of apoptotic cells. CONCLUSIONS: Fas-FasL-mediated apoptosis is considered to occur in the eye with EAU and plays a role in the immunopathogenic mechanisms to eliminate ocular infiltrating cells, thereby down-regulating the inflammatory processes.  (+info)

The retina of c-fos-/- mice: electrophysiologic, morphologic and biochemical aspects. (21/474)

PURPOSE: Mice without a functional c-Fos protein (c-fos-/- mice) do not exhibit light-induced apoptotic cell death of rods in contrast to their wild-type littermates (c-fos+/+ mice). To analyze the consequences of the absence of c-fos in the retina, we investigated whether the retinas of c-fos-/- mice have a reduced capacity to absorb and transduce light compared with c-fos+/+ mice. METHODS: Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ERGs) over more than 6 log units of intensity. Retinal morphology was studied by light- and electron microscopy. Arrestin and the heat shock protein 70 (Hsp70) were detected by Western blot analysis. The rhodopsin content and the kinetics of rhodopsin regeneration were determined in retinal extracts. RESULTS: Although the configuration of the ERGs was comparable in both groups of mice, c-fos-/- mice showed a marked variability in all quantitative ERG-measures with lower mean amplitudes, longer latencies, and a 0.9-log-unit lower b-wave sensitivity on average. Morphometry showed that c-fos-/- mice have 23% fewer rods on average, whereas the number of cones was comparable among c-fos+/+ and c-fos-/- mice. Arrestin levels appeared slightly reduced in c-fos-/- mice when compared with c-fos+/+ mice, whereas Hsp70 levels were comparable in both genotypes. The kinetics of rhodopsin regeneration were similar, but c-fos-/- mice had a 25% lower rhodopsin content on average. CONCLUSIONS: Compared with c-fos+/+ mice, retinal function in c-fos-/- mice is attenuated to a variable but marked degree, which may be, at least in part, related to the reduced number of rods and the reduced rhodopsin content. However, c-fos does not appear to be essential for the ability to absorb photons, nor for phototransduction or the function of second-order neurons. The resistance to light-induced apoptosis of photoreceptor cells in c-fos-/- mice may result from the acute deficit of c-fos in the apoptotic cascade rather than from developmental deficits affecting rod photoreceptor function.  (+info)

Arrestin binding to the M(2) muscarinic acetylcholine receptor is precluded by an inhibitory element in the third intracellular loop of the receptor. (22/474)

Desensitization of G protein-coupled receptors (GPCRs) involves the binding of members of the family of arrestins to the receptors. In the model system involving the visual GPCR rhodopsin, activation and phosphorylation of rhodopsin is thought to convert arrestin from a low to high affinity binding state. Phosphorylation of the M(2) muscarinic acetylcholine receptor (mAChR) has been shown to be required for binding of arrestins 2 and 3 in vitro and for arrestin-enhanced internalization in intact cells (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). For the M(2) mAChR, arrestin binding requires phosphorylation at multiple serine and threonine residues at amino acids 307-311 in the third intracellular (i3) loop. Here, we have investigated the molecular basis for the requirement of receptor phosphorylation for arrestin binding. Constructs of arrestin 2 that can bind to other GPCRs in a phosphorylation-independent manner were unable to interact with a mutant M(2) mAChR in which the Ser/Thr residues at 307-311 were mutated to alanines. However, although phosphorylation-deficient mutants of the M(2) mAChR that lacked 50-157 amino acids from the i3 loop were unable to undergo agonist-dependent internalization when expressed alone in tsA201 cells, co-expression of arrestin 2 or 3 restored agonist-dependent internalization. Furthermore, a deletion of only 15 amino acids (amino acids 304-319) was sufficient to allow for phosphorylation-independent arrestin-receptor interaction. These results indicate that phosphorylation at residues 307-311 does not appear to be required to activate arrestin into a high affinity binding state. Instead, phosphorylation at residues 307-311 appears to facilitate the removal of an inhibitory constraint that precludes receptor-arrestin association in the absence of receptor phosphorylation.  (+info)

Purification and characterization of bovine cone arrestin (cArr). (23/474)

To elucidate the quenching mechanism of phototransduction in vertebrate cone photoreceptors, a cDNA clone encoding cone specific arrestin (cArr) was isolated from a bovine retinal cDNA library using a human cArr cDNA probe. Affinity-purified anti-peptide antibody specific to cArr was prepared. Immunohistochemical staining displayed specific labeling of cArr in cone photoreceptors and immunoblotting identified a 46 kDa protein band. We purified cArr from bovine retinas by sequential column chromatography using DEAE-cellulose, gel filtration and mono Q columns. Binding studies revealed no binding of cArr to rhodopsin regardless of whether it was bleached and/or phosphorylated. cArr also failed to bind to heparin-Sepharose under conditions which rod arrestin (rArr) bound to the column. The present data suggest that cArr may play a role in the quenching of phototransduction in cone photoreceptors and that its activity therein is different to that of rArr.  (+info)

LEDGF: survival of embryonic chick retinal photoreceptor cells. (24/474)

PURPOSE: Lens epithelium-derived growth factor (LEDGF) is a novel adhesive, survival, and growth factor for lens epithelial cells, keratinocytes, fibroblasts, and cos7 cells. In the presence of LEDGF, these cells acquire resistance to environmental stresses, and in the absence of LEDGF they die. The effects of LEDGF on survival of embryonic chick retinal photoreceptor cells under serum starvation and heat stress were studied. METHODS: The expression pattern of LEDGF in embryonic chick retinal photoreceptor cells was investigated with protein blot analysis and immunohistochemistry using antibodies (Abs) to LEDGF. Retinal cells were cultured in serum-free medium for up to 6 days in the presence of varying amounts of LEDGF at 37 degrees or 41 degrees C. The photoreceptor cells were immunostained with Abs to arrestin and counted to evaluate the photoreceptor cell viability. Heat shock proteins in the cultured cells were quantified by protein blot analysis with Ab probes and semiquantitative reverse transcription-polymerase chain reaction analysis. RESULTS: LEDGF was found predominantly in the nucleus of neuroretinal cells, including photoreceptor cells. In the presence of LEDGF, photoreceptor cells manifested increased resistance to serum starvation and thermal stress and survived for a longer period. The levels of heat shock protein 90 were elevated in those cells. Most retinal cells died in the absence of LEDGF. CONCLUSIONS: LEDGF enhanced survival of retinal photoreceptor cells under serum starvation and heat stress. Thus, LEDGF has a potency to enhance survival of neuronal cell types against environmental stresses, and it may be applicable as a therapeutic agent for those cells.  (+info)