Di Iorio Patrizia, Ballerini Patrizia, Traversa Ugo, Nicoletti Ferdinando, D'Alimonte Iolanda, Kleywegt Sonya, Werstiuk Eva S, Rathbone Michel P, Caciagli Francesco, Ciccarelli Renata
Department of Biomedical Sciences, School of Medicine, University of Chieti, Chieti, Italy.
Glia. 2004 May;46(4):356-68. doi: 10.1002/glia.20002.
Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase-3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (approximately 10-fold over basal apoptotic values) in 18-24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration-dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 x 10(-5) M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi-proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3-kinase (PI3K; LY294002, 30 microM) or the MAPK pathway (PD98059, 10 microM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase-3beta (GSK-3beta) and induced an upregulation of Bcl-2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine-induced apoptosis by activating multiple pathways, and these are mediated by a Gi-protein-coupled putative guanosine receptor.
鸟苷在中枢神经系统具有多种营养作用，包括刺激星形胶质细胞合成和释放神经营养因子，从而保护神经元免受兴奋性毒性死亡。因此，我们质疑鸟苷是否能保护星形胶质细胞免受星形孢菌素诱导的凋亡。我们通过吖啶橙染色、寡核苷酸或半胱天冬酶-3 ELISA 测定法，评估了培养的大鼠脑星形胶质细胞在暴露于 100 nM 星形孢菌素 3 小时后的凋亡情况。星形孢菌素能迅速促进凋亡，在将其施加于星形胶质细胞后的 18 - 24 小时达到最大效应（比基础凋亡值高约 10 倍）。从星形孢菌素处理前 1 小时开始，在培养基中添加鸟苷 4 小时，可呈浓度依赖性降低凋亡细胞的比例。鸟苷抑制作用的 IC50 值为 7.5×10⁻⁵ M。鸟苷的保护作用不受丙戊茶碱抑制核苷转运体、腺苷 A1 或 A2 受体的选择性拮抗剂（DPCPX 或 DMPX）或 P2X 和 P2Y 嘌呤受体的拮抗剂（苏拉明）的影响。相反，用百日咳毒素预处理星形胶质细胞，使 Gi 蛋白与其受体解偶联，消除了鸟苷的抗凋亡作用。用磷酸肌醇 3 -激酶（PI3K；LY294002，30 μM）或 MAPK 途径抑制剂（PD98059，10 μM）预处理星形胶质细胞，也会降低鸟苷的保护作用。添加鸟苷会导致 Akt/PKB 和糖原合酶激酶 - 3β（GSK - 3β）迅速磷酸化，并诱导 Bcl - 2 mRNA 和蛋白表达上调。这些数据表明，鸟苷通过激活多种途径保护星形胶质细胞免受星形孢菌素诱导的凋亡，且这些途径由 Gi 蛋白偶联的假定鸟苷受体介导。
