Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8.
A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology; D009994:Osmolar Concentration; D010704:Phorbols / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013481:Superoxides / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004075:Diglycerides; D010704:Phorbols; D010718:Phosphatidylserines; D013481:Superoxides; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C013965:diolein