RESUMO
The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.
Assuntos
Animais , Bovinos , Malha Trabecular , Transferrina/biossíntese , Transferrina , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/biossíntese , Análise de Sequência de RNA , Malha Trabecular/metabolismo , Transferrina/genéticaRESUMO
The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.
Assuntos
Malha Trabecular/metabolismo , Transferrina/biossíntese , Transferrina/metabolismo , Animais , Bovinos , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Transferrina/genéticaRESUMO
The contribution of the ciliary body to the origin of vitreous proteins was investigated in rabbits by incubating explants of this eye component under novel conditions. At the end of incubations for up to 21 h, the tissues were processed histologically and were shown to be in an excellent state of morphological preservation. When radioactive amino acids and fucose were added to the culture medium, protein and glycoprotein synthesis and secretion were detected using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) plus fluorography. The origin of these secretory products was traced by autoradiography to the ciliary epithelium. When samples of vitreous bodies - injected intravitreally with the same radioactive precursors - were run beside samples of the tissue culture media, comigration of at least 8 radioactively labelled bands including the one previously identified as transferrin was detected. This indicates that some vitreous proteins may be secreted by the ciliary body and that cultures of explants of ciliary body-iris are useful tools for studies on vitreous protein secretion.