RESUMO
In previous work we showed that the polygonal shape of hippocampal astrocytes cultured on poly-L-lysine changes to a stellate morphology with loss of actinomyosin stress fibers on exchanging the culture medium for saline buffered with HEPES [Brain Res 946 (2002)12]. By contrast, in bicarbonate-buffered saline containing Ca(2+) astrocytes remained polygonal and continued to express stress fibers. Evidence suggests that stellation induced by saline buffered with HEPES is related to intracellular acidification due to the absence of bicarbonate. Here we studied the influence of the matrix used in preparing astrocyte cultures. Stellation in HEPES-saline occurred on a matrix of fibronectin, but not on matrices of collagen I or IV provided Ca(2+) was present. Laminin partially prevented stellation in HEPES-saline. Further, the intracellular acidification induced by HEPES-saline observed in astrocytes cultured on polylysine was abolished in cells cultured on collagens and was attentuated on a matrix of laminin. Two observations suggested the involvement of integrins and focal adhesions. (1) Treatment of cultures on collagens with a blocking antibody to the beta1 integrin subunit abolished protection against HEPES-induced stellation. (2) Compared with polylysine, astrocytes cultured on collagens expressed increased contents of phosphotyrosine proteins, focal adhesion proteins vinculin and paxillin, the beta1 integrin subunit and increased numbers of focal adhesions labelled with anti-vinculin. The observation that astrocytes cultured on collagen I or IV, in contrast to polylysine, express stress fibers and a constant intracellular pH in the absence of buffering by bicarbonate may be related to the fact that in the intact brain astrocytic processes (or end-feet) encounter and bind to collagen IV and laminin in the basement membrane of the endothelial cells which surround the cerebral capillaries. It is also possible that astrocytes retain this capacity from early development when fibrous matrix proteins are present.