digitonin and 4-bromophenacyl-bromide

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.

Topics: Acetophenones; Amino Acid Sequence; Arachidonic Acids; Calcium; Cell Membrane; Cell Membrane Permeability; Digitonin; Diglycerides; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Molecular Sequence Data; Myelin Basic Protein; Neuroblastoma; Phospholipases A; Phospholipases A2; Phosphorus; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Quinacrine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

1. Previous studies have demonstrated that exocytosis in adrenal chromaffin cells appears to require zinc-dependent endoproteinase activity. 2. Chromaffin cells have metal-dependent endoproteinases in both the plasma membrane and the soluble fraction of homogenized cells. In order to further study critically the role of metalloproteinase in exocytosis, and prior to purification, we needed to determine which one of several adrenal metalloproteinases is implicated in exocytosis. 3. The studies described here demonstrate that the metal-dependent endoproteinases in these two subcellular fractions can be differentiated by selective inhibitors. In both intact and permeabilized cells, the plasma membrane metalloproteinase, but not the soluble proteinases, is inhibited by phosphoramidon. Phosphoramidon does not block exocytosis in either intact or permeabilized cells. 4. In addition, the plasma membrane metalloproteinase appears to have its catalytic site facing the outside of the cell. 5. Because of these observations the plasma membrane metalloproteinase does not appear to be required in exocytosis. Since soluble metalloproteinase activity is inhibited by proteinase inhibitors at concentrations which block exocytosis, a soluble, and not the plasma membrane, metalloproteinase appears to be required in exocytosis.

Topics: Acetophenones; Adrenal Medulla; Animals; Cattle; Cell Membrane; Cells, Cultured; Digitonin; Endopeptidases; Enzyme Inhibitors; Exocytosis; Glycopeptides; Metalloendopeptidases; Norepinephrine

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.

Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Catecholamines; Cattle; Chromaffin Granules; Chromaffin System; Digitonin; Ethylmaleimide; Exocytosis; Lipid Metabolism; Magnesium; Norepinephrine; Phorbols; Tetradecanoylphorbol Acetate