RESUMO
Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Receptores Opioides/imunologia , Animais , Anticorpos Anti-Idiotípicos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Linhagem Celular , Células Híbridas , Técnicas Imunoenzimáticas , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica , Testes de Precipitina , Coelhos , Ratos , Receptores Opioides deltaRESUMO
No opioid octapeptide Met-enkephalin-Arg-Gly-Leu was detected either in the brain or in the adrenal gland of the cat using a specific radioimmunoassay. Whereas it was possible to determine the Met-enkephalin and Leu-enkephalin contents. The Met-enkephalin versus Leu-enkephalin concentration ratio was around five in each area of the brain assayed. The presence of authentic Met-enkephalin and Leu-enkephalin was confirmed by high performance liquid chromatography analysis. All in all, these data seem to indicate that the cat proenkephalin is partly different from that previously analysed in mammalian species including humans, rats and cows.
Assuntos
Gatos/metabolismo , Encefalina Metionina/análogos & derivados , Sequência de Aminoácidos , Animais , Química Encefálica , Cromatografia Líquida de Alta Pressão , Encefalina Leucina/análise , Encefalina Metionina/análise , Dados de Sequência Molecular , RadioimunoensaioRESUMO
The time course changes in levels of mRNA encoding glutamic acid decarboxylase (GAD) and proenkephalin (PPE) was analyzed in the rat striatum following unilateral lesion of substantia nigra with 6-hydroxydopamine. The levels of both GAD and PPE mRNAs increased after the dopaminergic deafferentation, reaching concomitantly a maximal twofold increase on day 25. Thereafter, the mRNA levels declined; at 4 months, the amount of PPE mRNA remained slightly elevated whereas GAD mRNA had returned to the control value, suggesting the action of a compensatory mechanism. We also observed a rise of glial fibrillary acidic protein mRNA level which reflects a reactive astrocytosis. In contrast, alpha-tubulin mRNA level remained unchanged, indicating that no significant synaptogenesis occurs in this experimental situation. No obvious modification in mRNA levels was detected in the striatum contralateral to the lesion. These results highlight the role of the modulation of gene expression in adaptive processes to dopamine deficiency in striatal efferent pathways. Its relevance to the pathophysiology of Parkinson's disease is discussed.
Assuntos
Corpo Estriado/metabolismo , Dopamina/fisiologia , Encefalinas/genética , Glutamato Descarboxilase/genética , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Animais , Sondas de DNA , Denervação , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Hidroxidopaminas/farmacologia , Cinética , Masculino , Oxidopamina , Poli A/análise , RNA/análise , Ratos , Ratos Endogâmicos , Substância Negra/efeitos dos fármacos , Substância Negra/fisiologia , Tubulina (Proteína)/genéticaRESUMO
The possible influence of the corticostriatal (glutamatergic) pathway on denervation-induced striatal dopamine target cell supersensitivity has been investigated in the rat by measuring the changes in striatal acetylcholine levels induced by the dopamine agonist pergolide and the basal dopamine D2-receptor density after combined 6-hydroxydopamine-induced lesion of the substantia nigra and cortical ablation. Lesion of the nigrostriatal dopaminergic pathway alone enhanced the ability of pergolide (0.06-1 mg/kg i.p.) to increase acetylcholine levels and increased the maximal density of [3H]spiperone binding sites in the striatum. Similar changes in these biochemical parameters were observed after combined cortical ablation and nigral lesion. Cortical ablation by itself slightly diminished acetylcholine levels and reduced by 30% [3H]spiperone binding site density in the striatum. These results indicate that the corticostriatal tract does not influence striatal dopamine target cell supersensitivity caused by dopaminergic denervation.
