RESUMO
Tobacco smoking and aging are among the few factors linked to age-related macular degeneration (AMD), a major cause of blindness in the elderly. Recent studies indicate that cadmium (Cd), an environmental toxic trace metal, is approximately four-fold higher in the retinas of smokers compared to non-smokers. In this study, we determined the effects of age and gender on Cd accumulation in human retinal tissues, specifically the neural retina, retinal pigment epithelium (RPE), and choroid. Cadmium levels in cultured RPE cells or retinal tissues isolated from frozen donor eyes were measured using inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrophotometry (GF-AAS). Cadmium uptake in cultured human RPE cells (ARPE-19) was also assessed using GF-AAS. Toxic effects of cadmium were determined from cell loss (measured as a decrease in cell density) and lactate dehydrogenase release (an indicator of membrane disruption). In "young" eyes (< 55 years) Cd was highest in the retinal pigment epithelium and lowest in the neural retina. Cd was higher in all tissues in aged eyes (>or=55 years) and was significantly higher in the neural retina and RPE in older females. Cultured RPE cells exposed to Cd showed altered cell morphology, decreased cell survival, elevated ROS levels and concentration-dependent disruption of membrane integrity. We conclude that cadmium is accumulated differently in the neural retinal and RPE of older men and women. The deleterious effects of Cd on RPE cells indicate that this environmental toxin is a potentially important factor in age-related retinal disease.
Assuntos
Envelhecimento/metabolismo , Cádmio/análise , Retina/química , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cádmio/farmacocinética , Cádmio/toxicidade , Morte Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Criança , Pré-Escolar , Canais de Cloreto/efeitos dos fármacos , Corioide/química , Relação Dose-Resposta a Droga , Feminino , Humanos , Lactente , L-Lactato Desidrogenase/metabolismo , Masculino , Espectrometria de Massas/métodos , Potenciais da Membrana/efeitos dos fármacos , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Técnicas de Patch-Clamp , Epitélio Pigmentado Ocular/química , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , Retina/efeitos dos fármacos , Fatores Sexuais , Espectrofotometria Atômica/métodosRESUMO
Retinal pigment epithelium (RPE) possesses regulated chloride channels that are crucial for transepithelial fluid and ion transport. At present, little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H(2)O(2). The inhibitory effects of H(2)O(2) were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.
Assuntos
Antioxidantes/farmacologia , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Oxidantes/farmacologia , Epitélio Pigmentado Ocular/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Idoso , Canais de Cloro CLC-2 , Canais de Cloreto/genética , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Glutationa/farmacologia , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Epitélio Pigmentado Ocular/citologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Venenos de Escorpião/farmacologiaRESUMO
Genetic mutations of the Cl(-) channel ClC-5 cause Dent's disease in humans. We recently cloned an amphibian ortholog of Xenopus ClC-5 (xClC-5) from the A6 cell line. We now compare the properties and regulation of ClC-5 currents expressed in mammalian (COS-7) cells and Xenopus oocytes. Whole cell currents in COS-7 cells transfected with xClC-5 cDNA had strong outward rectification, Cl(-) > I(-) anion sensitivity, and were inhibited at low pH, similar to previous results in oocytes. In oocytes, antisense xClC-5 cRNA injection had no effect on endogenous membrane currents or the heterologous expression of human ClC-5. Activators of cAMP and protein kinase C inhibitors had no significant effects on ClC-5 currents expressed in either COS-7 cells or oocytes, whereas H-89, a cAMP-dependent protein kinase (PKA) inhibitor, and hydrogen peroxide decreased the currents. We conclude that the basic properties of ClC-5 currents were independent of the host cell type used for expression. In addition, ClC-5 channels may be modulated by PKA and reactive oxygen species.
