RESUMO
The functional role of AT(2) receptors is unclear and it activates unconventional signaling pathways, which in general do not involve a classical activation of a G-protein. In the present study, we aimed to investigate the transduction mechanism of AT(2) Ang II receptors in PND15 rat hindbrain membrane preparations, which represents a physiological developmental condition. To determine whether Ang II AT(2) receptors induced association to SHP-1 in rat hindbrain, co-immunoprecipitation assays were performed. Stimulation of Ang II AT(2) receptors induced both a transient tyr-phosphorylation and activation of SHP-1. The possible participation of c-Src in Ang II-mediated SHP-1 activation, we demonstrated by recruitment of c-Src in immunocomplexes obtained with anti AT(2) or anti-SHP-1 antibodies. The association of SHP-1 to c-Src was inhibited by PD123319 and the c-Src inhibitor PP2. Similarly, SHP-1 activity determined in AT(2)-immunocomplexes was inhibited by PD123319 and the c-Src inhibitor PP2. Following stimulation with Ang II, AT(2) receptors recruit c-Src, which was responsible for SHP-1 tyr-phosphorylation and activation. Since AT(2) receptors are involved in neuron migration, we tested the presence of FAK in immunocomplexes. Surprisingly, AT(2)-immunocomplexes contained mainly the 85kDa fragment of FAK. Besides, p125FAK associated to SHP-1. In summary, we demonstrated the presence of an active signal transduction mechanism in PND15 rat hindbrain, a developmental stage critical for cerebellar development. In this model, we showed a complex containing AT(2)/SHP-1/c-Src/p85FAK, suggesting a potential role of Ang II AT(2) receptors in cerebellar development and neuronal differentiation.
Assuntos
Angiotensina II/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor Tipo 2 de Angiotensina/fisiologia , Rombencéfalo/citologia , Rombencéfalo/metabolismo , Animais , Animais Recém-Nascidos , Proteína Tirosina Quinase CSK , Movimento Celular/fisiologia , Cerebelo/citologia , Cerebelo/enzimologia , Cerebelo/metabolismo , Quinase 1 de Adesão Focal/química , Substâncias Macromoleculares/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Ratos Wistar , Receptor Tipo 2 de Angiotensina/agonistas , Rombencéfalo/enzimologia , Transdução de Sinais/fisiologia , Quinases da Família srcRESUMO
Endo-oligopeptidase A (EOPA, formerly EC 3.4.22.19), a thiol-activated oligopeptidase, is able to degrade both bradykinin and neurotensin, and also to convert enkephalin-containing peptides into enkephalins. The expression of this enzyme was studied in the rat brain by in situ hybridization using non-radiotopic probes. The distribution of EOPA transcripts included many regions of the rat central nervous system, with higher expression in some regions, such as the hippocampus, cerebellum, and basal nucleus of Meynert. The marked EOPA expression in these areas could be anticipated, since they are rich in neuropeptides that are known to be EOPA substrates in vitro. The data characterize a widespread occurrence of EOPA in the rat brain and reinforce the suggestion of a critical role for EOPA in peptide processing.