RESUMO
Glial cell line-derived neurotrophic factor family receptor alpha-2 (GFR alpha-2) is a GPI-linked receptor that preferentially binds neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family. Three splice isoforms of GFR alpha-2 have been identified previously in mouse tissues, but the occurrence of splice isoforms in rats has not been described. The aim of this study was therefore to identify GFR alpha-2 splice isoforms in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and gene cloning. Three isoforms were identified and sequenced, and named GFR alpha-2(a), (b) and (c), according to the nomenclature used for the previously identified mouse isoforms. The GFR alpha-2(a) and (b) isoforms were identical to those previously described in mice. The GFR alpha-2(c) isoform was novel. Sequences for GFR alpha-2(b) and (c) were deposited in the GenBank database (accession numbers GI: 16797788 and 16797786, respectively). All three isoforms were expressed in the brain, kidney, and intestine of both postnatal and adult rats.
Assuntos
Processamento Alternativo/genética , Proteínas de Drosophila , Intestinos/inervação , Plexo Mientérico/crescimento & desenvolvimento , Plexo Mientérico/metabolismo , Proteínas Proto-Oncogênicas/isolamento & purificação , Receptores Proteína Tirosina Quinases/isolamento & purificação , Envelhecimento/genética , Animais , Animais Recém-Nascidos , Clonagem Molecular , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Masculino , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Oxidative stress is widely recognized to contribute to neuronal death during various pathological conditions and ageing. In the enteric nervous system (ENS), reactive oxygen species have been implicated in the mechanism of age-associated neuronal loss. The neurotrophic factors, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF), are important in the development of enteric neurons and continue to be expressed in the gut throughout life. It has therefore been suggested that they may have a neuroprotective role in the ENS. We investigated the potential of NT-3 and GDNF to prevent the death of enteric ganglion cells in dissociated cell culture after exposure to hydrogen peroxide (H(2)O(2)). H(2)O(2) treatment resulted in a dose-dependent death of enteric neurons and glial cells, as demonstrated by MTS assay, bis-benzimide and propidium iodide staining and immunolabelling. Cultures treated with NT-3 prior to exposure showed reduced cell death compared to untreated control or GDNF-treated cultures. GDNF treatment did not affect neuronal survival in H(2)O(2)-treated cultures. These results suggest that NT-3 is able to enhance the survival of enteric ganglion cells exposed to oxidative stress.
Assuntos
Sistema Nervoso Entérico/efeitos dos fármacos , Gânglios/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Peróxido de Hidrogênio/farmacologia , Neurônios/efeitos dos fármacos , Neurotrofina 3/farmacologia , Oxidantes/farmacologia , Animais , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corantes , Sistema Nervoso Entérico/citologia , Gânglios/citologia , Íleo/inervação , Plexo Mientérico/citologia , Plexo Mientérico/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Ageing is associated with gastrointestinal dysfunction, which can have a major impact on quality of life of the elderly. A number of changes in the innervation of the gut during ageing have been reported, including neuronal loss and degenerative changes. Evidence indicates that reactive oxygen species (ROS) are elevated in ageing enteric neurons, but that neurotrophic factors may reduce generation of neuronal ROS. Two such factors, glial cell line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) have also been found to protect enteric neurons against oxidative stress induced cell death of enteric ganglion cells in vitro. We have investigated the possible roles of neurotrophic factors further, by examining their expression in the gut during ageing, and by analysing their effects on antioxidant enzyme production in cultures of enteric ganglion cells. RESULTS: Analysis of the expression of GDNF and its receptors c-Ret and GFR α - 1 in rat gut by RT-PCR showed that expression continues throughout life and into ageing, in both ad libitum(AL) and calorically-restricted (CR) animals. Levels of expression of GDNF and GFR α - 1 were elevated in 24 month AL animals compared to 24 month CR animals, and to 24 CR and 6 month control animals respectively. The related factor Neurturin and its receptor GFR α - 2 were also expressed throughout life, the levels of the GFR - α-2(b) isoform were reduced in 24 m AL animals. Immunolabelling showed that c-Ret and GFR α - 1 proteins were expressed by myenteric neurons in ageing animals. GDNF, but not NT-3, was found to increase expression of Cu/Zn superoxide dismutase and catalase by cultured enteric ganglion cells. CONCLUSIONS: The neurotrophic factors GDNF and neurturin and their receptors continue to be expressed in the ageing gut. Changes in the levels of expression of GDNF , GFR α-1 and GFR α-2(b) isoform occurred in 24 m AL animals. GDNF, but not NT-3, increased the levels of antioxidant enzymes in cultured enteric ganglion cells, indicating a possible mechanism for the reported protective effect of GDNF against menadione-induced neuronal apoptosis in the ageing gut. Together these data suggest that GDNF family members may play a protective role in the gut throughout life, and support the suggestion that dysregulation of neurotrophic factor support could contribute to neuronal ageing in the gut.