Immunohistochemical localizatio of protein components of catecholamine storage vesicles

Abstract

1. The distribution of specific proteins in sympathetic neurones has been examined by immunofluorescent histology using antibodies prepared against soluble protein components of the catecholamine storage vesicles of the adrenal medulla.2. Two antigen preparations were separated by ion exchange chromatography of the soluble proteins released on osmotic lysis of catecholamine storage vesicles which had been isolated by centrifugation from homogenates of sheep adrenal medulla. One fraction (AgDH) had high dopamine-beta-hydroxylase activity, while another (AgCB), consisting of the bulk of the protein, had some capacity to bind catecholamines. On disk gel electrophoresis the antigens ran as single bands with very different mobilities.3. Antisera (AsDH) and (AsCB) produced in rabbits to the two antigens were shown to react specifically with their antigens by immunodiffusion and electrophoresis in agarose.4. Indirect immunofluorescent staining of tissue sections was achieved by layering first the rabbit anti-sera, followed by goat anti-rabbit globulin serum which had been conjugated with fluorescein isothiocyanate.5. The adrenal medulla and the cell bodies of sympathetic ganglia showed the most intense green fluorescence with the immune rabbit sera, and hardly stained when pre-immune serum from the same animal was used. The reactivity of the antisera could be abolished by incubation with the corresponding antigen.6. The preterminal and terminal axons of sympathetic nerves also stained specifically but less intensely with both antisera. When the nerves were ligated for up to 24 hr, the portion immediately proximal to the constriction showed an enhanced reaction to the antisera.7. The results provide evidence that sympathetic neurones contain proteins immunologically identical to those involved in the synthesis and storage of noradrenaline in the adrenal medulla, and support the concept that granular vesicles are synthesized in the perikaryon of the neurone and are transported somatofugally in the axon.