Gottfried C, Cechin S R, Gonzalez M A, Vaccaro T S, Rodnight R
Centro de Ciências da Saúde, Unisinos, Sao Leopoldo, Brazil.
Neuroscience. 2003;121(3):553-62. doi: 10.1016/s0306-4522(03)00557-8.
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.
在之前的研究中，我们发现，在聚-L-赖氨酸上培养的海马星形胶质细胞的多边形形态，在将培养基换成用HEPES缓冲的盐水后，会转变为星状形态，同时肌动球蛋白应激纤维消失[《脑研究》946 (2002)12]。相比之下，在含有Ca(2+)的碳酸氢盐缓冲盐水中，星形胶质细胞仍保持多边形，并继续表达应激纤维。有证据表明，HEPES缓冲盐水诱导的星状化与由于缺乏碳酸氢盐导致的细胞内酸化有关。在此，我们研究了用于制备星形胶质细胞培养物的基质的影响。在纤连蛋白基质上，HEPES盐水中会发生星状化，但如果存在Ca(2+)，在I型或IV型胶原基质上则不会。层粘连蛋白部分阻止了HEPES盐水中的星状化。此外，在聚赖氨酸上培养的星形胶质细胞中观察到的由HEPES盐水诱导的细胞内酸化，在胶原上培养的细胞中被消除，而在层粘连蛋白基质上则有所减弱。有两个观察结果表明整合素和粘着斑参与其中。(1) 用针对β1整合素亚基的阻断抗体处理胶原上的培养物，消除了对HEPES诱导的星状化的保护作用。(2) 与聚赖氨酸相比, 在胶原上培养的星形胶质细胞中，磷酸化酪氨酸蛋白、粘着斑蛋白纽蛋白和桩蛋白、β1整合素亚基的含量增加，并且用抗纽蛋白标记的粘着斑数量增多。与聚赖氨酸不同，在I型或IV型胶原上培养的星形胶质细胞在没有碳酸氢盐缓冲的情况下表达应激纤维且细胞内pH恒定，这一观察结果可能与以下事实有关：在完整大脑中，星形胶质细胞的突起（或终足）会与围绕脑毛细血管的内皮细胞基底膜中的IV型胶原和层粘连蛋白相遇并结合。也有可能星形胶质细胞从早期发育阶段就保留了这种能力，那时纤维基质蛋白就已存在。
