title: Effects of phorbol esters, diglyceride, and cholinergic agonists on the subcellular distribution of protein kinase C in intact or digitonin-permeabilized adrenal chromaffin cells.
abstract: Phorbol esters which activate protein kinase C increased the percentage of membrane-bound protein kinase C activity in bovine adrenal chromaffin cells from less than 10 to 20-50% within 30 min. Permeabilization of chromaffin cells with digitonin in the absence of Ca2+ and phorbol esters caused virtually 100% of the protein kinase C activity to leave the cells within 1 h, which is consistent with protein kinase C being soluble and cytosolic. However, if cells were incubated for 15-30 min with 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to permeabilization, 50-60% of the protein kinase C activity exited from the cells within 1 h of permeabilization. In cells not incubated with phorbol ester, permeabilization in the presence of 1-10 microM Ca2+ also decreased the rate at which protein kinase C exited from the cells. The slower release of protein kinase C caused by prior incubation of the cells with TPA or because of the presence of micromolar Ca2+ in permeabilized cells was associated with increased membrane-bound protein kinase C. The effects of TPA and permeabilization in the presence of micromolar Ca2+ were approximately additive. Active phorbol esters had different abilities to cause retention of protein kinase C in digitonin-treated cells. Dioctanoylglycerol, which activates protein kinase C in vitro and enhanced Ca2+-dependent secretion from permeabilized chromaffin cells similarly to TPA, also increased membrane-bound protein kinase C in intact cells, but had no effect on the retention of protein kinase C in permeabilized cells in the presence or absence of Ca2+. The different abilities of protein kinase C activators to cause retention of protein kinase C in subsequently permeabilized cells suggest differences in the reversibility of the binding. The mixed nicotinic-muscarinic agonist carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium, but not the muscarinic agonist muscarine, caused 3-10% of the total protein kinase C activity to become membrane-bound within 3 min in intact chromaffin cells. Thus, nicotinic stimulation of chromaffin cells may rapidly activate protein kinase C.
mesh_terms: D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology*; D004075:Diglycerides / Q000494:pharmacology*; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology*; D005989:Glycerides / Q000494:pharmacology*; D007700:Kinetics; D009116:Muscarine / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D004075:Diglycerides; D005989:Glycerides; D010879:Piperazines; D004246:Dimethylphenylpiperazinium Iodide; D009116:Muscarine; D002217:Carbachol; D011493:Protein Kinase C; D004072:Digitonin; D013755:Tetradecanoylphorbol Acetate