phosphorus-radioisotopes and 5-hydroxy-6-8-11-14-eicosatetraenoic-acid

Platelet-activating factor is a potent mediator of the inflammatory response. Studies of the actions of platelet-activating factor have centered mainly around neutrophils, monocytes, and platelets. In this report we begin to uncover the influence of platelet-activating factor on B lymphocytes. Employing the EBV-transformed human B cell line SKW6.4, we demonstrate that platelet-activating factor significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine, phosphatidylinositol, and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10(-9) to 10(-6) M. The inactive precursor, lyso-platelet-activating factor, at a concentration as high as 10(-7) M had no effect on any of the membrane phospholipids. We also show that platelet-activating factor from 10(-12) to 10(-6) M induced rapid and significant elevation in intracellular calcium levels, whereas lyso-platelet-activating factor was again ineffective. We further demonstrate the impact of platelet-activating factor binding to B cells by measuring platelet-activating factor induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production. Moreover, platelet-activating factor was capable of inducing transcription of the nuclear proto-oncogenes c-fos and c-jun. Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates platelet-activating factor induced arachidonic acid release perhaps acting at the level of phospholipase A2. In summary, platelet-activating factor is shown here to have a direct and profound effect on a pure B cell line.

Topics: Arachidonic Acid; Arachidonic Acids; B-Lymphocytes; Calcium; Cell Line, Transformed; DNA-Binding Proteins; Eicosanoids; Gene Expression Regulation; Herpesvirus 4, Human; Humans; Hydroxyeicosatetraenoic Acids; Membrane Lipids; Phospholipids; Phosphorus Radioisotopes; Platelet Activating Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Messenger; Transcription Factors; Up-Regulation

Neutrophils treated with optimal amounts of tumor promoters that activate protein kinase C (e.g. mezerein) release large quantities of superoxide (O2-) and exhibit an intense phosphorylation of two proteins with molecular masses of approximately 47 and 49 kDa. These cells can also be stimulated synergistically to release a comparable amount of O2-. This involves treatment with a suboptimal amount of a tumor promoter and an agent capable of elevating cellular Ca2+. Neutrophils treated in the former fashion exhibit a redistribution of the activity of protein kinase C from a soluble to a particulate fraction that is stable in the presence of Ca2+ chelators, whereas cells stimulated synergistically do not do so to an appreciable extent (Badwey, J. A., Robinson, J. M., Horn, W., Soberman, R. J., Karnovsky, M. J., and Karnovsky, M. L. (1988) J. Biol. Chem. 263, 2779-2786). In this paper, we report that neutrophils stimulated synergistically do exhibit a significant incorporation of 32P into the 47-kDa protein, but with little labeling of the 49-kDa species. This labeling of the 47-kDa protein was greater than the sum of those observed with each agent added separately but was less than that observed in cells stimulated with optimal amounts of tumor promoters alone. An inhibitor of protein kinase C (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) blocked O2- release and the phosphorylation of the 47-kDa protein under all conditions of stimulation mentioned, whereas an inhibitor of cyclic nucleotide-dependent kinases had no effect on these phenomena. Thus, labeling of the 47-kDa protein can occur in the absence of a "tight" translocation of protein kinase C to membrane and was always observed during synergy. The data support a role for protein kinase C and the 47-kDa phosphoprotein in the synergistic stimulation of neutrophils.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; Diterpenes; Drug Synergism; Guinea Pigs; Hydroxyeicosatetraenoic Acids; Isoquinolines; Molecular Weight; Neutrophils; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Piperazines; Sulfonamides; Superoxides; Terpenes