neuromedin-n and Carcinoid-Tumor

Brief phorbol ester treatment of BON cells results in a persistent release and cellular depletion of immunoreactive chromogranin A (CGA-IR) and neurotensin (NT-IR) cell contents. The purpose of the present study was to characterize the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on the secretion, biosynthesis, and steady-state messenger RNA (mRNA) levels of chromogranin A (CGA) and of a coresident peptide, neurotensin, by a novel human pancreatic carcinoid cell line, called BON. Acute TPA treatment (100 nM, 1 h) of BON cells resulted in 20- and 40-fold elevations in release of CGA-IR and NT-IR, respectively; and a 70-90% depletion of CGA-IR and NT-IR cell contents. TPA treatment also increased the biosynthetic rate of CGA-IR. Steady-state mRNA levels of CGA and NT/N (neurotensin/neuromedin N) were unchanged. Cell contents of CGA-IR and NT-IR were not replenished for a period of up to 6 days; secretion of CGA-IR and NT-IR persisted. In addition, BON cells failed to release CGA in response to stimulation by ionomycin and A23187 several days after acute TPA treatment. Our data indicate that the lack of replenishment of cell contents of CGA-IR and NT-IR is not due to decreases in steady-state CGA-IR and NT-IR mRNA levels, nor is it due to a decrease in biosynthesis of CGA-IR, but it is the result of a loss in the ability of TPA-treated BON cells to store and secrete CGA-IR and NT-IR in a regulated manner. These effects of TPA are mediated through the PKC pathway.

Topics: Analysis of Variance; Blotting, Northern; Calcimycin; Carcinoid Tumor; Cell Division; Cell Line; Chromogranin A; Chromogranins; Dose-Response Relationship, Drug; Gene Expression; Humans; Ionomycin; Kinetics; Methionine; Neurotensin; Pancreatic Hormones; Pancreatic Neoplasms; Peptide Fragments; RNA, Messenger; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

Antisera towards the bioactive peptides, neurotensin (NT, 13 residues) and neuromedin N (NMN, 6 residues), as well as towards three regions of their 147-residue canine precursor were used to identify and to quantitate precursor-derived peptides in extracts of human BON cells. This cell-line, which was obtained from a human pancreatic carcinoid tumor, constitutively expresses NT/NMN mRNA and secretes NT. Quantitation of seven precursor-derived peptides led us to conclude that BON cells display the intestinal pattern of NT/NMN precursor processing, which is primarily characterized by the production of a large molecular (125 amino acid) form of NMN. Four large molecular components, identified by immunochemical analyses and Western blotting, displayed physico-chemical properties which, for the most part, were consistent with the structures predicted from the partially-known human mRNA sequence. However, as shown previously for these peptides in canine gut, the empirically determined M(r) and pI values were slightly higher than those predicted solely from the amino acid content, perhaps due to the presence of additional substituents. These results suggest that BON cells may provide a good in vitro model in which to study the regulation of intestinal NT/NMN precursor processing and the nature of the enzyme(s) involved.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Carcinoid Tumor; Chromatography, High Pressure Liquid; Dogs; Gene Expression; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Molecular Sequence Data; Neurotensin; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Radioimmunoassay; RNA, Messenger; Tumor Cells, Cultured