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The Ordered Secretion of Bioactive Peptides: Oldest or Newest First?

Department of Neuroscience, Johns Hopkins University School of Medicine Baltimore, Maryland 21205

Address requests for reprints to: Dr. Richard E. Mains, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205.

Abstract

The order of secretion of newly synthesized and older bioactive peptides was investigated using primary rat intermediate pituitary melanotropes, which synthesize, store, and secrete peptides derived from pro-ACTH/endorphin (PAE; also POMC). PAE-derived peptides produced by the cells were biosynthetically labeled by incubating the cells with radioactive amino acids at various times preceding the period during which secretion was examined; secreted and cellular peptides were characterized and quantitated by immunoprecipitation, using affinitypurified antibodies to selected regions of PAE, followed by polyacrylamide gel electrophoretic analysis. Release in the absence of secretagogues (basal or constitutive release) was compared to release in the presence of maximally effective levels of 8-bromo-cAMP and BaCI₂ (stimulated or regulated release). Both cell types showed short-lived preferential basal release of newly synthesized and not fully mature peptides (<2–3 h old). Conversely, the cells showed preferential stimulated secretion of older peptides. A process of maturation occurred, taking 2–4 h, after which the secretion of newly synthesized and older peptides in response to secretagogues was nearly indistinguishable for the smallest product peptides. The data support a model of gradual processing of peptides from precursors into smaller products and maturation from molecules only available for basal release into peptides available for stimulated secretion as well as for basal release. Basal secretion was found to include mature peptides as well as intermediates and precursor molecules. The data do not support the existence of any preferential regulated secretion of newly synthesized peptides.

FOOTNOTES

This work was supported by grants from the NIDA (DA-00266 and DA-00097) and the Medical Research Council of Canada.

^* Current address: Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec, Canada H3G 1A4.

Received for publication February 6, 1991. Revision received March 15, 1991. Accepted for publication March 26, 1991.

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