calcimycin and carbobenzoxyglycylphenylalanine-amide

Ribophorin I is a type I transmembrane glycoprotein specific to the rough endoplasmic reticulum. We have previously shown that, when expressed in transfected HeLa cells, a carboxyl-terminally truncated form of ribophorin I that contains most of the luminal domain (RI332) is, like the native protein, retained in the endoplasmic reticulum (ER). Brefeldin A (BFA) treatment of these HeLa cells leads to O-glycosylation of RI332 by glycosyltransferases that are redistributed from the Golgi apparatus to the ER (Ivessa, N. E., De Lemos-Chiarandini, C., Tsao, Y.-S., Takatsuki, A., Adesnik, M., Sabatini, D. D., and Kreibich, G. (1992) J. Cell Biol. 117, 949-958). Using the state of glycosylation of RI332 as a measure for the BFA-induced backflow of enzymes of the Golgi apparatus to the ER, we now demonstrate that the retrograde transport is inhibited when cells are treated with various agents that affect intracellular Ca2+ concentrations, such as the dipeptide benzyloxycarbonyl (Cbz)-Gly-Phe-amide, the Ca2+ ionophore A23187, and thapsigargin, an inhibitor of the Ca(2+)-transporting ATPase of the ER. These treatments prevent the BFA-induced O-glycosylation of RI332. Immunofluorescence localization of the Golgi markers, MG-160 and galactosyltransferase, shows that when BFA is applied in the presence of Ca2+ modulating agents, the markers remain confined to the Golgi apparatus and are not redistributed to the ER, as is the case when BFA alone is used. Cbz-Gly-Phe-amide does not, however, interfere with the BFA-induced release of beta-COP from the Golgi apparatus. We conclude that the maintenance of a Ca2+ gradient between the cytoplasm and the lumen of the ER and the Golgi apparatus is required for the BFA-induced retrograde transport from the Golgi apparatus to the ER to occur.

Topics: Biological Transport; Brefeldin A; Calcimycin; Calcium; Cell Compartmentation; Cyclopentanes; Dipeptides; Endoplasmic Reticulum; Glycosylation; Golgi Apparatus; HeLa Cells; Humans; Ionophores; Membrane Proteins; Metalloendopeptidases; Peptide Fragments; Protease Inhibitors; Protein Processing, Post-Translational

Inhibitors of metalloendopeptidases interfere with events involving Ca2(+)-dependent membrane fusion in a number of cell types. The divalent ion chelating agent 1,10-phenanthroline inhibited pancreatic amylase secretion stimulated by carbachol, cholecystokinin-octapeptide (CCK-8), or bombesin, but detailed studies indicated that this is unlikely to be a result of inhibition of metalloendopeptidase activity. The binding of [3H]N-methylscopolamine to pancreatic acini was reduced by 1,10-phenanthroline and this would explain the marked inhibition of carbachol-induced amylase secretion by the chelating agent. CCK-8-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate was reduced by 1,10-phenanthroline while the binding of CCK-8 to acini was not affected. This inhibition of hydrolysis would explain the inhibition of CCK-8- and bombesin-induced amylase secretion. The metalloendopeptidase substrate carbobenzoxyglycylphenylalanylamide did not affect bombesin-stimulated amylase secretion. Amylase secretion evoked by treating pancreatic acini with the ionophore A23187 or dibutyryl-cyclic AMP was not reduced by 1,10-phenanthroline, indicating a lack of involvement of metalloendopeptidases in the process of exocytosis in this cell type.

Topics: Amylases; Animals; Bombesin; Bucladesine; Calcimycin; Carbachol; Dipeptides; Metalloendopeptidases; Mice; N-Methylscopolamine; Pancreas; Phenanthrolines; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Receptors, Muscarinic; Scopolamine Derivatives; Sincalide