RESUMO
Glial cells prevail in number and in diversity of cellular phenotypes in the nervous system. They have also gained prominence due to their multiple physiological and pathophysiological roles. Our current knowledge of the asymmetry and heterogeneity of the plasma membrane demands an in depth analysis of the diverse array of membrane microdomains postulated to exist in the context of glial cells. This review focuses and analyzes the studies reported to date on the detection of caveolae membrane rafts and the caveolin family members in glial cell model systems, the conditions leading to changes in their level of expression, and their functional and clinical significance. Outstanding in this work emerge the ubiquitous expression of caveolins, including the typically regarded 'muscle-specific' cav3, in diverse glial cell model systems, their participation in reactive astrogliosis, cancer, and their key relevance to calcium signaling. The knowledge obtained to date demands incorporation of the caveolins and caveolae membrane rafts in our current models on the role of glial cells in heath and neurological disease.
Assuntos
Caveolinas/metabolismo , Membrana Celular/metabolismo , Neuroglia/citologia , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Modelos BiológicosRESUMO
Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Regulação para Baixo/genética , Ácido Glutâmico/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Bário/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Ácido Glutâmico/farmacocinética , Homeostase/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/genética , Interferência de RNA/fisiologia , RatosRESUMO
Caveolae, a specialized form of lipid rafts, are cholesterol- and sphingolipid-rich membrane microdomains implicated in potocytosis, endocytosis, transcytosis, and as platforms for signal transduction. One of the major constituents of caveolae are three highly homologous caveolin isoforms (caveolin-1, caveolin-2, and caveolin-3). The present study expands the analysis of caveolin isoform expression in C6 glioma cells. Three complementary approaches were used to assess their differential expression during the dibutyryl-cyclic AMP-induced differentiation of C6 cells into an astrocyte-like phenotype. Immunoblotting, conventional RT-PCR, and real-time RT-PCR analysis established the expression of the caveolin-3 isoform in C6 cells, in addition to caveolin-1 and caveolin-2. Similar to the other isoforms, caveolin-3 was associated with light-density, detergent-insoluble caveolae membrane fractions obtained using sucrose-density gradient centrifugation. The three caveolin isoforms display different temporal patterns of mRNA/protein expression during the differentiation of C6 cells. Western blot and real-time RT-PCR analysis demonstrate that caveolin-1 and caveolin-2 are up-regulated during the late stages of the differentiation of C6 cells. Meanwhile, caveolin-3 is gradually down-regulated during the differentiation process. Indirect immunofluorescence analysis via laser-scanning confocal microscopy reveals that the three caveolin isoforms display similar subcellular distribution patterns. In addition, co-localization of caveolin-1/caveolin-2 and caveolin-1/caveolin-3 was detected in both C6 glioma phenotypes. The findings reveal a differential temporal pattern of caveolin gene expression during phenotypic differentiation of C6 glioma cells, with potential implications to developmental and degenerative events in the brain.