sq-23377 and pancreastatin

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

The objective of these experiments was to investigate the influence of activation of three second messenger systems (protein kinase-C, adenylate cyclase-cAMP, and calcium mobilization) on the secretion of pancreastatin (PST) and chromogranin-A (CGA) by a human pancreatic carcinoid cell line (BON) in tissue culture. Stimulation of protein kinase-C by a phorbol ester (0.025-7.5 microM) caused a significant dose-related release of PST (186 +/- 22-4271 +/- 228% over controls). Treatment of BON cells with graded doses of 8-bromo-cAMP (0.14-3.0 mM) and isobutylmethylxanthine (IBMX; 0.01-1.0 mM) also stimulated a dose-related release of PST (107 +/- 22-284 +/- 28 and 16 +/- 12-1076 +/- 100% over controls, respectively). Incubation of BON cells with ionomycin (0.134-13.4 microM) increased the release of PST (102 +/- 15-554 +/- 21% over controls) in a dose-related manner. A combination of IBMX and ionomycin resulted in an additive effect, whereas treatment with a phorbol ester plus IBMX resulted in a synergistic effect on PST release. Pretreatment of BON cells with monensin, an agent that prevents processing of precursors to smaller peptides, significantly decreased PST, but not CGA, secretion in response to phorbol ester or ionomycin. These findings indicate that protein kinase-C, cAMP, and Ca2+ mobilization participate in CGA and PST secretion. Although the observation that secretions of PST and CGA in response to theophylline are quantitatively associated, the absence of a quantitative relationship in the release patterns of PST and CGA in response to phorbol ester and ionomycin do not support a simple precursor-product relationship between CGA and PST. The monensin experiments are consistent with the notion that PST is derived from CGA in BON cells.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Carcinoid Tumor; Cell Line; Chromatography, Gel; Chromogranin A; Chromogranins; Humans; Ionomycin; Kinetics; Monensin; Pancreatic Hormones; Pancreatic Neoplasms; Phorbol Esters; Radioimmunoassay; Second Messenger Systems; Theophylline