Abstract
Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.
Highlights
Protein Kinase C Activity Appears To Be Required for the Synthesis of Platelet-activatingFactor and LeukotrieneB4by Human Neutrophi~s*
The concentration of Ca2+required to activate thisactivity is lower in the presence of diacylglycerol [4], which is derived from the phospholipase C-catalyzed hydrolysis of phosphatidylinositol and perhaps phosphatidylcholine ( 5 ) .Tumor-promoting phorbol esters (e.g. PMA) activate protein kinase C activity in vitro by substituting for diacylglycerol, which allows activation at Ca2+concentrations equivalent to the intracellular free Ca2+levels in resting cells
The addition of inhibition by sphingosine occurred at 7 p ~ w,ith com- PMA to resting neutrophils results in the translocation of plete inhibition at 15p ~ P.MA by itself did not induce protein kinase C from the cytosol to intracellular membranes, the synthesisof PAF or LTB4, it did enhance activation of its kinase activity [6, 7],and the subsequent
Summary
The direct activation of protein kinase C, by the addition mobilize arachidonate is consistentwiththeinhibition of both PAF and LTB, accumulation. Palmitoylcarnitine and stearylaminewere employed since they inhibit protein kinase C activity in enzymatic assays (32, 3436) and responses attributed to protein kinase C in whole cells [32, 33, 37, 38] These twolong chainaminesalso inhibited the accumulation of PAF (Fig. 2) and LTB, (not shown) by A23187-stimulated neutrophils. Sought to determine if the failure to synthesize LTB, in the presence of protein kinase C inhibitors resulted from the inability to hydrolyze the appropriate precursor-containing phospholipids We examined this postulate by labeling neutrophils with [3H]arachidonic acid, washing them free of unincorporated label, and incubating them with BSA or asphingosine-BSA complex. The release of total radiolabel into themedium after stimulation with 10 p~ A23187 was quantitated as before. 0,control, PMA-treated W, sphingosine- and PMA-treated; A, control, PDD-treated; A, sphingosine- and PDDtreated
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