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Journal of Histochemistry and Cytochemistry, Vol. 45, 425-436, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Proteolytic Processing of Pro-opiomelanocortin Occurs in Acidifying Secretory Granules of AtT-20 Cells

Shigeyasu Tanakaa, Takao Yorab, Kazuhisa Nakayamac, Kinji Inoue1,a, and Kazumasa Kurosumia
a Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan
b Department of Medical Technology, Saitama Medical School Junior College, Moroyama, Japan
c Gene Experiment Center, University of Tsukuba, Ibaraki, Japan

Correspondence to: Shigeyasu Tanaka, Dept. of Cell Biology, Inst. for Molecular and Cellular Regulation, Gunma Univ., Showa-machi 3-39-15, Maebashi 371, Japan.

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. (J Histochem Cytochem 45:425-436, 1997)

Key Words: proteolytic processing, pro-opiomelanocortin, secretory granule, acidification, DAMP, bafilomycin A1, immunocytochemistry, prohormone convertase, AtT-20 cell


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