Conversion and storage of somatostatin are established before response to secretagogue stimuli in P19 neurons.

In mature neurons, neuropeptides are synthesized via limited proteolysis of propolypeptides by convertases. The bioactive peptides are then stored in secretory granules until they are released extracellularly upon the induction of a fusion between granules and the plasma membrane, in response to secretagogues. We used the mouse P19 embryonic carcinoma cells as a model to determine if the capacities to convert and store neuropeptides and to secrete them in a regulated fashion are established coordinately during neuronal differentiation. We have previously shown that both undifferentiated P19 cells and their neuronal derivatives express the largely distributed furin, PACE4 and PC5 convertases, whereas only neuronal derivatives express the neuroendocrine convertase PC2. In addition, undifferentiated cells displayed furin- rather than PC2-like converting capacities. The present work demonstrates that day 8 P19 neurons mainly convert prosomatostatin (proSS) to somatostatin-14 (SS-14) using HPLC and radioimmunoassay (RIA) analyses, indicating that P19 cells acquire PC2-like converting capacities as a consequence of neuronal differentiation. SS-14 was predominantly intracellular in neuronal cells which were shown to express several granins, markers of granules, by Western blotting. However, cell membrane depolarization with 50 mM K+, a general secretagogue stimulus, evoked the release of SS-14 by day 12, but not by day 8, P19 neurons. The results thus demonstrate that capacities to convert and store neuropeptides can be established before coupling of stimulus-secretion during neuronal differentiation.

Proteolytic profile of recombinant pro-opiomelanocortin in embryonal carcinoma P19 cells: conversion to beta-lipotropin and secretion are inhibited following incubation with canavanine.

A variety of proteins and peptides are produced through limited proteolysis of precursors at paired basic residues. This proteolytic bioactivation is carried out by subtilisin-like proteases, called convertases. The mRNAs of several convertases are expressed during prenatal life as well as in P19 embryonal carcinoma cells, which are a model of the totipotent cells of the embryo before and at the time of implantation. To determine whether converting activities accompany convertase mRNA expression in the early embryo, we transferred the gene of pro-opiomelanocortin (POMC) into P19 cells, by lipofection, and searched for the presence of mature peptides by high-performance liquid chromatography and radioimmunoassay techniques. In P19 cells, POMC, a precursor of several endocrine peptides, is mainly processed to beta-lipotropin rather than to beta-endorphin, both peptides having been identified by their immunoreactivity, polarity, and molecular size. These results indicate that converting capacities appear early in the embryo and that they are more similar to the activity of furin and of convertase PC1 than that of convertase PC2 in their cleavage selectivity of POMC sites. Efficiency of POMC processing can reach 50%, suggesting that convertases, with other proteases, can have an important role in ontogenesis. As for other peptide precursors in endocrine cells, the conversion of POMC in P19 cells was inhibited by the biosynthetic replacement of its arginine residues by the analog canavanine. However, the incorporation of canavanine into P19 cells also inhibited peptide secretion, suggesting that inhibition of conversion in these cells as well as in endocrine cells could indirectly result from the impairment of intracellular traffic and not only from a direct inhibition of the converting activity.