title: Activation of murine macrophage cell lines. Possible involvement of protein kinases in stimulation of superoxide production.
abstract: The mechanism by which phorbol myristate acetate (PMA) activates mononuclear phagocytes to produce O2- was investigated in two cloned murine macrophage-like cell lines. J774.16 cells produce O2- upon stimulation by PMA and aggregated IgG, but a variant clone derived from it, J774.C3C cells, do not, probably because of a functionally defective cytochrome b. 4-O-methyl 12-O-tetradecanoyl-phorbol-13-acetate (4-O-methyl TPA) did not stimulate O2- production in either clone. Studies on the ionic requirements for production of O2- indicated that the extracellular Ca2+ and Mg2+ concentrations did not affect O2- production of J774.16 cells. Furthermore, calcium flux studies with the use of 45Ca2+ indicated that PMA caused no detectable influx of Ca2+, produced a modest release of presumed loosely cell-associated 45Ca2+, but did not detectably change the rate of Ca2+ efflux. By using quin 2 acetoxymethyl ester, no evidence could be adduced that PMA caused intracellular Ca2+ redistribution. Because membrane potential changes are seen in other systems of activation, membrane potential changes in the macrophages stimulated by PMA were measured both by means of the fluorescence ionophore, dis-S-C3-(5), and by direct measurement by using a K+-electrode. Under conditions that stimulated O2- production, no membrane depolarization was observed. Under the conditions for stimulating O2- production, PMA also did not induce increased turnover of membrane phospholipids or arachidonic acid. Although J774.16 and J774.C3C produce prostaglandin D2 at resting state, PMA neither released arachidonic acid from membrane lipids, nor induced release of prostaglandins and thromboxane at least for 10 min after stimulation. Similarly, PMA failed to increase transmethylation of phospholipids or break down the methylated phospholipids. PMA and aggregated IgG, however, induced protein phosphorylation in both J774.16 and J774.C3C cells. Among these, mainly two sets of phosphorylated proteins with m.w. of 42,000 and pI 6.85 to 7.3 and m.w. of 58,000 and pI 8.15 to 9.3 are common in both stimuli. Biologically inactive 4-0-methyl TPA did not induce phosphorylation of these proteins. From the above results, we conclude that PMA induces O2- production in a macrophage line without detectable changes in membrane potential; Ca2+ flux, intracellular Ca2+ increase or redistribution, or increase of membrane lipid turnover. Because two stimuli that induce O2- production in J774.16 cells induced phosphorylation of common proteins, whereas a biologically inactive analog did not, protein kinases, possibly protein kinase C, may be involved in the activation for O2- production.
mesh_terms: D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D007473:Ion Channels / Q000378:metabolism; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000276:immunology*; D008563:Membrane Lipids / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D007473:Ion Channels; D008563:Membrane Lipids; D013481:Superoxides; D016718:Arachidonic Acid; D011494:Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium