| Summary: | 碩士 === 國立陽明大學 === 神經科學研究所 === 93 === Astrocytes are a group of important glial cells involving in the neuronal regeneration and degeneration. These cells are also known to be immunocompetent cells by possessing the ability to produce a wide variety of cytokines and chemokines. The high concentration of extracellular ATP (1 mM) activated P2X7 receptors have been demonstrated to affect such immune responses in various types of immune cells including astrocytes. In addition, researchers also revealed that activation of P2X7 receptor stimulated cellular injury and cell death, apoptosis or necrosis. RBA-2 type-2 astrocytes have been demonstrated to possess P2X7 receptors and activation of the receptor enhanced the expression of TGF-��1 cytokine and stress proteins, e.g. HSP60 and Cu/Zn SOD. Because P2X7 receptor-mediated morphological transformation and cellular injury may be important in immune responses of astrocytes, thus in the present study we sought to examine the mechanism involving in P2X7 receptor-mediated morphological transformation of RBA-2 astrocytes by confocal microscopy.
Our data revealed that activation of P2X7 receptor rapidly induced an rapidly alteration of cellular morphology of these cells. By double staining of cytoskeletal proteins, actin and GFAP, an initial change of process withdrawal was observed at 5 min. After treating the cells with 1 mM ATP or the P2X7 receptor selective agonist BzATP ( 100�n�嵱 ) for 30 min, the cells were nearly round up. Because PKC is an important regulatory molecule in P2X7 receptor-mediated signaling of RBA-2 astrocytes, we then examine the expression of PKC isozymes of these cells. Our results revealed that RBA-2 astrocytes express PKC-��, PKC-��, PKC-��, PKC-��, PKC-�� and PKC-��, PKC-��. In addition, a differential cellular distribution of various PKC isozymes was also observed. PKC inhibitors, chelerythrine chloride, GF109203X and Gö6976 all partially restored the P2X7 receptor-mediated morphological change indicating that the effect of PKC on morphological change may be mediated through an indirect process. Recently, a receptor tyrosine phosphatase, RPTP��, has been shown to associated with P2X7 receptor directly. We estimated the non-receptor protein tyrosine kinase in RBA-2 by RT-PCR. We revealed that two receptor protein tyrosine phosphatase, RPTP�� �nand RPTP��, two Src kinase family, Fyn and Lyn, two focal adhesion kinase family, FAK and Pyk2, were expressed in RBA-2. We then use the general tyrosine kinase inhibitor and Src kinase family inhibitor, genistein and PP2, to treat RBA-2 astrocytes. Our results revealed that genistein blocked the P2X7 receptor-mediated morphological transformation dose-dependently, but PP2 partially blocked the P2X7 receptor-mediated morphological transformation, suggesting that a tyrosine phosphorylation -dephosphorylation signaling cascade may affect the P2X7 receptor-mediated morphological transformation directly. Then we found that activated P2X7 receptor 5-30 mins dereased the FAK-Tyr397 autophosphorylation and changed the FAK-associated adaptor paxillin distribution. Collectively, a tyrosine kinase (eg. FAK or paxillin) may contribute to the PX7 receptor-mediated morphological transformation.
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