Diphenyl ditelluride induces hypophosphorylation of intermediate filaments through modulation of DARPP-32-dependent pathways in cerebral cortex of young rats

摘要

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 Ca2+ channels (L-VDCC) blockers; 50 μM dantrolene, a ryanodine channel blocker, and the intracellular Ca2+ 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 Ca2+-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 Ca2+ levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.