calcimycin and pancreastatin

To elucidate the regulatory pathway through which pancreastatin inhibits insulin secretion, RINm5F insulinoma cells were challenged with physiological and pharmacological probes known to stimulate insulin release through different mechanisms. Utilizing the electrophysiological technique of capacitance measurements as a correlate to exocytosis, pancreastatin was found to significantly diminish maximum capacitance changes evoked by glyceraldehyde, an effect which was attenuated in pertussis toxin-treated cells. In static incubations of this cell line, pancreastatin significantly inhibited insulin secretion stimulated by glyceraldehyde, carbachol and A23187, secretagogues known to directly elevate beta-cell cytosolic Ca2+. This peptide also inhibited insulin secretion stimulated by phorbol myristate acetate (PMA), but only at incubation times < or = 15 min. It was without effect on insulin secretion stimulated by mastoparan and longer incubations (30 min) with PMA, where the secretory mechanisms are not necessarily Ca(2+)-dependent. Additionally, pancreastatin had no effect on carbachol-generated inositol phosphate accumulation but inhibited simultaneously stimulated insulin secretion. All inhibitory effects of pancreastatin were pertussis toxin sensitive. These results suggest that pancreastatin inhibits insulin secretion in RINm5F cells through a G-protein regulated mechanism at a control point involved in the Ca(2+)-directed exocytotic machinery, a feature shared by other physiologic inhibitors of insulin secretion.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Carbachol; Chromogranin A; Exocytosis; Glyceraldehyde; GTP-Binding Proteins; Inositol Phosphates; Insulin; Insulin Secretion; Insulinoma; Intercellular Signaling Peptides and Proteins; Pancreatic Hormones; Pancreatic Neoplasms; Peptides; Pertussis Toxin; Phorbol Esters; Rats; Tumor Cells, Cultured; Virulence Factors, Bordetella; Wasp Venoms

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

We have investigated the effect of pancreastatin on cytosolic Ca2+ concentration in the insulin secreting cell line RINm5F. Changes in [Ca2+]i induced by pancreastatin were detected by Fluo-3 fluorescence using both flow cytometry and batch analysis measurements, and turned out to be from 90 to 315 nM equivalent to 80% of that caused by ATP, which increased [Ca2+]i from 90 nM to 400 nM. This effect of pancreastatin did not depend on extracellular calcium and was not mediated by alpha-adrenergic receptors since it was not prevented by the alpha-blocker yohimbine. It is concluded that pancreastatin has a role in the homeostasis of free cytosolic calcium in the insulin secreting cell line Rinm5F.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cell Line; Chromogranin A; Clonidine; Cytosol; Flow Cytometry; Insulin; Insulin Secretion; Pancreatic Hormones; Receptors, Adrenergic, alpha; Yohimbine

It is found that secretion of pancreastatin and somatostatin from QGP-1N cells is regulated through muscarinic receptor-mediated activation of phosphatidylinositide hydrolysis system. In this report, whether the cAMP pathway interacts with the phosphoinositide turnover system for the secretion of pancreastatin and somatostatin from QGP-1N cells through muscarinic receptors was studied. Stimulation of QGP-1N cells with carbachol increased intracellular cAMP levels. The carbachol-induced increase in cAMP levels was inhibited by atropine. Calcium ionophore (A23187) and phorbol 12-myristate 13-acetate increased cAMP synthesis. Dibutyryl cAMP, forskolin and theophylline stimulated secretion of pancreastatin and somatostatin. When either dibutyryl cAMP, forskolin or theophylline was added in culture medium with A23187, phorbol ester or carbachol, a synergistic effect was found on pancreastatin and somatostatin secretion. These results suggest that interaction between the phosphoinositide turnover system and the cAMP pathway occurs in QGP-1N cells through muscarinic receptor stimulation for the secretion of pancreastatin and somatostatin.

Topics: Adenoma, Islet Cell; Atropine; Bucladesine; Calcimycin; Carbachol; Cell Line; Chromogranin A; Colforsin; Cyclic AMP; Drug Synergism; Humans; Kinetics; Pancreatic Hormones; Pancreatic Neoplasms; Phosphatidylinositols; Somatostatin; Tetradecanoylphorbol Acetate; Theophylline; Tumor Cells, Cultured

Studies were made of pancreastatin (PST) secretion from a human PST-producing cell line (QGP-1N) in response to various secretagogues. Cells with immunoreactivity for PST were observed in monolayer cultures of QGP-1N cells. Carbachol stimulated PST secretion and the intracellular Ca2+ mobilization concentration dependently in the range of 10(-6)-10(-4) M. The PST secretion and Ca2+ mobilization induced by carbachol were inhibited by atropine. The calcium ionophore (A23187) stimulated PST secretion. However, cholecystokinin and gastrin-releasing peptide did not stimulate either PST secretion or Ca2+ mobilization. Secretin also did not stimulate PST secretion. The glucose concentration in the culture medium had no effect on PST secretion. These results suggest that PST secretion is mainly regulated by acetylcholine through a muscarinic receptor, and that an increase in intracellular Ca2+ plays an important role in stimulus-secretion coupling in QGP-1N cells.

Topics: Acetylcholine; Adenoma, Islet Cell; Atropine; Calcimycin; Calcium; Carbachol; Chromogranin A; Gastrin-Releasing Peptide; Humans; Pancreatic Hormones; Pancreatic Neoplasms; Parasympatholytics; Peptides; Piperidines; Pirenzepine; Receptors, Muscarinic; Sincalide; Tumor Cells, Cultured