Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, Porto Alegre, RS, 90035-003, Brazil.
Arch Toxicol. 2012 Feb;86(2):217-30. doi: 10.1007/s00204-011-0746-6. Epub 2011 Aug 24.
We studied the effect of different concentrations of diphenyl ditelluride (PhTe)(2) on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and neurofilament (NF) subunits from cerebral cortex and hippocampus of rats during development. (PhTe)(2)-induced hypophosphorylation of GFAP and NF subunits only in cerebral cortex of 9- and 15-day-old animals but not in hippocampus. Hypophosphorylation was dependent on ionotropic glutamate receptors, as demonstrated by the specific inhibitors 10 μM DL-AP5 and 50 μM MK801, 100 μM CNQX and 100 μM DNQX. Also, 10 μM verapamil and 10 μM nifedipine, two L-voltage-dependent Ca(2+) channels (L-VDCC) blockers; 50 μM dantrolene, a ryanodine channel blocker, and the intracellular Ca(2+) chelator Bapta-AM (50 μM) totally prevented this effect. Results obtained with 0.2 μM calyculin A (PP1 and PP2A inhibitor), 1 μM Fostriecin a potent protein phosphatase 2A (PP2A) inhibitor, 100 μM FK-506 or 100 μM cyclosporine A, specific protein phosphatase 2B inhibitors, pointed to PP1 as the protein phosphatase directly involved in the hypophosphorylating effect of (PhTe)(2). Finally, we examined the activity of DARPP-32, an important endogenous Ca(2+)-mediated inhibitor of PP1 activity. Western blot assay using anti-DARPP-32, anti-pThr34DARPP-32, and anti-pThr75DARPP-32 antibodies showed a decreased phosphorylation level of the inhibitor at Thr34, compatible with inactivation of protein kinase A (PKA) by pThr75 DARPP-32. Decreased cAMP and catalytic subunit of PKA support that (PhTe)(2) acted on neuron and astrocyte cytoskeletal proteins through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells. Moreover, in the presence of Bapta, the level of the PKA catalytic subunit was not decreased by (PhTe)(2), suggesting that intracellular Ca(2+) levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.
我们研究了不同浓度的二苯并二碲（PhTe）（2）对发育过程中大鼠大脑皮层和海马区神经丝（NF）亚基和胶质纤维酸性蛋白（GFAP）磷酸化的体外影响。PhTe）（2）诱导的 9 至 15 日龄动物大脑皮层 GFAP 和 NF 亚基的低磷酸化，但海马区没有。低磷酸化依赖于离子型谷氨酸受体，这一点可以通过特定抑制剂 10 μM DL-AP5 和 50 μM MK801、100 μM CNQX 和 100 μM DNQX 来证明。此外，10 μM 维拉帕米和 10 μM 硝苯地平，两种 L-电压依赖性钙（Ca2+）通道（L-VDCC）阻断剂；50 μM 丹曲洛林，一种ryanodine 通道阻断剂，以及细胞内 Ca2+螯合剂 Bapta-AM（50 μM）完全阻止了这种作用。用 0.2 μM 钙调素 A（PP1 和 PP2A 抑制剂）、1 μM Fostriecin（一种有效的蛋白磷酸酶 2A（PP2A）抑制剂）、100 μM FK-506 或 100 μM 环孢菌素 A（特异性蛋白磷酸酶 2B 抑制剂）获得的结果表明，PP1 是直接参与（PhTe）（2）低磷酸化作用的蛋白磷酸酶。最后，我们检查了 DARPP-32 的活性，DARPP-32 是一种重要的内源性 Ca2+介导的 PP1 活性抑制剂。用抗 DARPP-32、抗 pThr34 DARPP-32 和抗 pThr75 DARPP-32 抗体进行 Western blot 分析表明，抑制剂在 Thr34 处的磷酸化水平降低，与 pThr75 DARPP-32 对蛋白激酶 A（PKA）的失活一致。cAMP 和 PKA 催化亚基的减少支持（PhTe）（2）通过 PKA 介导的 DARPP-32 失活作用于神经元和星形胶质细胞细胞骨架蛋白，促进 IF 蛋白的释放和这些神经细胞的低磷酸化。此外，在 Bapta 存在的情况下，（PhTe）（2）没有降低 PKA 催化亚基的水平，这表明细胞内 Ca2+水平可能是这种神经毒性物质引发的信号通路的上游，并针对细胞骨架。
