Using antibodies specific for pro-opiomelanocortin (POMC), amidated joining peptide (JP), and the prohormone convertase PC1, we showed immunocytochemically that PC1 in a corticotrophic tumor cell line, AtT-20, was co-localized either with POMC or with amidated JP in secretory granules, and also confirmed that POMC was cleaved mainly in secretory granules. Analysis using DAMP (3- [2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as the pH probe suggested a correlation between POMC processing and acidic pH in the secretory granules. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, completely inhibited POMC processing and caused constitutive secretion of the unprocessed precursor. By contrast, chloroquine, a weak base that is known to neutralize acidic organelles, was unable to inhibit POMC processing. Electron microscopic analysis revealed that, in AtT-20 cells treated with bafilomycin A1, the trans-Golgi cisternae were dilated and few secretory granules were present in the cytoplasm. These observations suggest that acidic pH provides a favorable environment for proteolytic processing of POMC by PC1 but is not required, and that integrity of the trans-Golgi network and sorting of POMC into secretory granules are important for POMC processing.
Immunohistochemical staining of somatostatin (SRIF) and a retrograde transport method with horseradish peroxidase (HRP) were simultaneously applied to the same section of the rat brain to identify the specific SRIF-containing neurons sending their fibers to the median eminence. After HRP injection into the median eminence, SRIF-positive neurons in the rostral parts of the periventricular nucleus were shown to be labeled with HRP, SRIF neurons in the other brain areas, such as the amygdala, the ventromedial nucleus, dorsomedial nucleus and arcuate nucleus, had no HRP-positive material. The present findings appear to demonstrate that SRIF neurons in the periventricular nucleus project directly to the median eminence.
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