ABSTRACT
Previous studies have observed changes in the lacrimal gland and ocular surface related to diabetes mellitus and related it to insulin resistance or insufficiency and oxidative damage. The aim of this study was to evaluate whether insulin treatment inhibits those changes. Diabetes was induced in male Wistar rats with a single intravenous injection of streptozotocin and a subgroup was treated with insulin. After 5 and 10 weeks, the three groups (n = 5-10/group/experimental procedure) were compared for biochemical, functional, and histological parameters. After 5 weeks, changes in morphology and increased numbers of lipofucsin-like inclusions were observed in lacrimal glands of diabetic but not insulin-treated rats. After 5 weeks, malonaldehyde and total peroxidase activity were significantly higher in diabetic rats, but similar to control in insulin-treated diabetic rats (P = 0.03, P = 0.02, respectively). Our data indicate that diabetes induces histological alterations in lacrimal gland and suggests that hyperglycemia-related oxidative stress may participate in diabetic dry eye syndrome. Prevention by insulin replacement suggests direct hormone action and/or benefit by early sub optimal metabolic control.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Dry Eye Syndromes/chemically induced , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Lacrimal Apparatus/drug effects , Animals , Cornea/drug effects , Cornea/pathology , Dry Eye Syndromes/pathology , Injections, Intravenous , Lacrimal Apparatus/pathology , Male , Rats , Rats, WistarABSTRACT
PURPOSE: This paper deals with the capability of the ciliary epithelium to express neurolysin, involved in the inactivation of numerous neuropeptides. METHODS: Total RNAs from ciliary body (CB) were processed for RT-PCR, and the amplification products were sequenced. The whole-protein extracts of CBs were analyzed using the Western blot. The CBs were processed for neurolysin immunolocalization. RESULTS: The RT-PCR detected the presence of neurolysin mRNA in the ciliary body. The Western blot assays demonstrated immunochemical cross-reactivity with neurolysin. The immunoreactivity to neurolysin was observed in ciliary epithelium. CONCLUSIONS: These results indicate that the ciliary epithelium expresses neurolysin.
Subject(s)
Ciliary Body/metabolism , Metalloendopeptidases/metabolism , Animals , Blotting, Western , Cattle , Ciliary Body/cytology , Cross Reactions , Epithelial Cells/metabolism , Epithelium/metabolism , Immunochemistry , Male , Metalloendopeptidases/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Tissue DistributionABSTRACT
PURPOSE: To verify the capability of rabbit and rat ciliary body to synthesize and secrete ceruloplasmin. METHODS: Isolated ciliary body (CB) was cultured in the presence of [35S]-methionine, and the incubation medium was processed for immunoprecipitation. Total RNA from CB was processed for RT-PCR, and the amplification products were sequenced. Also, sections of CB were immunostained for the localization of ceruloplasmin. RESULTS: A labeled peptide, having a molecular weight of about 135 kDa, the expected size of ceruloplasmin, was immunopurified in the incubation media from both animal species. The RT-PCR and sequencing experiments detected the presence of ceruloplasmin mRNA in rat samples. Both layers of rabbit and rat ciliary epithelium (CE) exhibited ceruloplasmin reactivity after immunohistochemical processing. CONCLUSIONS: Taken altogether, these results indicate the CB, particularly its epithelium, as one of the possible sources of the ocular intrinsic ceruloplasmin.
Subject(s)
Ceruloplasmin/genetics , Ceruloplasmin/metabolism , Ciliary Body/enzymology , Animals , Immunoenzyme Techniques , Immunoprecipitation , Male , Organ Culture Techniques , RNA, Messenger/metabolism , Rabbits , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transferrin/geneticsABSTRACT
PURPOSE: To identify retinoids and retinoid processing proteins in the ocular ciliary epithelium (CE), and to compare in cultured ciliary epithelial cell lines promoter activities of the cellular retinaldehyde binding protein (CRALBP) and interphotoreceptor retinoid binding protein (IRBP). METHODS: Retinoid processing proteins were detected by RT-PCR, western analysis and immunocytochemistry. PCR products were verified by DNA sequence analysis. Retinoids were measured by normal phase HPLC and UV visible spectroscopy. Reporter product from CRALBP and IRBP promoter fragments was measured following transient transfection in bovine pigmented and nonpigmented CE cells. RESULTS: CRALBP, IRBP, cellular retinol binding protein (CRBP), 11-cis-retinol dehydrogenase (11-cis-RDH), lecithin:retinol acyltransferase (LRAT), and ATP binding cassette receptor (ABCR) were detected in human CE tissue by RT-PCR. Retinal pigment epithelium specific protein 65 kDa (RPE65) mRNA and protein were also detected. CRALBP and IRBP were detected by western analysis in tissue extracts from bovine CE and were localized to the PE and NPE cell layers, respectively, by immunocytochemistry. IRBP immunoreactivity was also detected in aqueous humor. Retinoids identified in the bovine CE include retinyl esters (7.4+/-3.5 pMol/mg of protein) and all-trans-retinol (14.9+/-1.1 pMol/mg of protein). Betacarotene was also tentatively identified. 11-cis-Retinoids were not detected. In CE cell cultures, the CRALBP p2.1-kb promoter construct exhibited reporter activity 15-30 fold above basal level, with 2 fold more activity in pigmented than nonpigmented CE cells. IRBP promoter constructs exhibited low level reporter activities in vitro in both CE cell layers. CONCLUSIONS: The ocular CE expresses genes encoding components of the rod visual cycle. The differential localization of CRALBP and IRBP along the bilayer of the CE suggests a potential role in retinoid transport and/or retinoid metabolism. However, the absence of 11-cis-retinoids suggests that the function of retinoid processing proteins in the CE differs from that of the retina.
Subject(s)
Carrier Proteins/genetics , Ciliary Body/metabolism , Eye Proteins/genetics , Pigment Epithelium of Eye/metabolism , Retinoids/metabolism , Retinol-Binding Proteins/genetics , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Acyltransferases/genetics , Acyltransferases/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Blotting, Western , Carrier Proteins/metabolism , Cattle , Cells, Cultured , Chromatography, High Pressure Liquid , Eye Proteins/metabolism , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation/physiology , Humans , Immunohistochemistry , Protein Transport , RNA, Messenger/metabolism , Rabbits , Retinoids/genetics , Retinol-Binding Proteins/metabolism , Retinol-Binding Proteins, Cellular , Reverse Transcriptase Polymerase Chain Reaction , cis-trans-IsomerasesABSTRACT
It has been shown that the vitreous contains several intrinsic glycoproteins whose origin remains to be clarified. Isolated ciliary epithelium (CE) was assayed to verify its role in the synthesis and secretion of transferrin for the vitreous body. It was cultured in the presence of [35S]methionine and the incubation medium was processed for immunoprecipitation. Total RNA from CE was processed for RT-PCR and the amplification products were sequenced. Also, whole preparations of isolated CE were processed for immunolocalization of transferrin. From the incubation assays, a labeled peptide of about 80 kDa was immunopurified that is the expected size of transferrin. The RT-PCR and sequencing experiments detected the presence of transferrin mRNA. Both layers of the CE exhibited transferrin reactivity, following immunohistochemical processing. Taken altogether, these results indicate the CE as one of the possible sources of vitreous intrinsic transferrin.
