leukotriene-b4 and 1-2-dioctanoylglycerol

Unopsonised zymosan particles bind to the CD11b/CD18 integrin on human neutrophils (PMN) and are phagocytosed. Binding stimulates the release of leukotriene (LT) B4. The present study examined the effect on this interaction of two agents that 'prime' PMN for augmented responses to a variety of agonists. The cell permeable diacyl glycerol, 1,2-dioctanoyl-glycerol (DiC8) and TNF alpha each increased CD11b/CD18 expression on PMN [maximal at 10-9 M TNF alpha or 10-8 M DiC8]. There was a decrease, however, in CD11b/CD18 expression above 10-8 M DiC8, which was not observed at high concentrations of TNF alpha. Pre-treatment with either DiC8 or TNF alpha dose-dependently augmented the zymosan-stimulated release of LTB4 from PMN. DiC8 and TNF alpha in combination, however, synergistically increased LTB4 release. In contrast, at concentrations above 10-8 M DiC8, whether in the presence or absence of TNF alpha, LTB4 release was inhibited and this was ameliorated by protein kinase C inhibitors. The response to neither TNF alpha nor DiC8 (below 10-8 M) was kinase inhibitor sensitive. Doses of DAG, which activate protein kinase C, inhibit CD11b/CD18-dependent responses by down-regulating receptor expression. In contrast, the mechanisms of TNF alpha and low dose DAG 'priming' are not clear but are independent of PKC activation. The synergy between these two priming agents, however, suggests independent, complementary signalling pathways that provide a novel, potentially important mechanism for the control of PMN CD11b/CD18 integrin-dependent activation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Diglycerides; Humans; Leukotriene B4; Macrophage-1 Antigen; Neutrophils; Phagocytosis; Protein Kinase C; Staurosporine; Tumor Necrosis Factor-alpha; Up-Regulation; Zymosan

Nucleotides have polymorphonuclear neutrophil (PMN)-stimulating actions resembling those of 5-hydroxyicosatetraenoate and its oxo analog, 5-oxoETE. Their effects on degranulation, however, are disputed even though this response may underlie their in vivo toxicity and is well-suited for comparing their mechanism of action with e.g., 5-oxoETE.. We measured the direct, synergistic, and cross-desensitizing actions of nine nucleotides and six other stimuli in degranulating unprimed and tumor necrosis factor (TNF)-alpha-primed human PMN.. Nucleotides weakly degranulated unprimed PMN but caused far larger responses in TNF-alpha-primed cells. Their actions, while differing from those of N-formyl-MET-LEU-PHE, platelet-activating factor, leukotriene B4, ionomycin, or dioctanoylglycerol, resembled those of 5-oxoETE. Nucleotides also enhanced PMN degranulation responses to the latter stimuli, particularly 5-oxoETE. Nucleotide degranulating and enhancing potencies were: UTP > or = ATP > or = ATP gamma S > ITP > ADP > 2-MeSATP, nonphosphohydrolyzable analogs lacked activity, and adenosine and AMP blocked PMN degranulation. Finally, nucleotides desensitized degranulation responses to each other but not to 5-oxoETE or other agonists, and 5-oxoETE desensitized to itself but not to nucleotides.. Nucleotides have intrinsic and synergistic degranulating actions that under appropriate conditions (i.e., in concert with TNF-alpha or 5-oxoETE) are exceedingly prominent. Recognition systems mediating their effects differ from those for various stimuli including 5-oxoETE. These systems likely involve a common "nucleotide" receptor, but studies do not exclude possibilities that other purinergic receptors contribute to their actions.

Topics: Adenine Nucleotides; Adenosine; Arachidonic Acids; Cytoplasmic Granules; Diglycerides; Drug Synergism; Humans; In Vitro Techniques; Ionomycin; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor; Ribonucleotides; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

Human peripheral blood monocytes synthesize the potent lipid autacoid platelet-activating factor (PAF) following appropriate stimulation. We examined the role of protein kinase C (PKC) in regulating the synthesis of PAF by stimulated monocytes. 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol, which directly activate PKC, stimulated the synthesis of PAF. Sphingosine, a long-chain amine that inhibits PKC, blocked both the binding of phorbol esters to monocytes and the synthesis of PAF in response to PMA (half-maximal inhibition at 5 to 10 microM and complete inhibition at 10 to 30 microM sphingosine). Thus, the activation of PKC was necessary and sufficient for PAF synthesis in response to phorbol ester. Sphingosine also blocked PAF synthesis in response to the calcium ionophore A23187 and opsonized zymosan particles by specific inhibition of PKC. Two other PKC inhibitors, stearylamine and staurosporine, also blocked PAF synthesis following A23187 or opsonized zymosan stimulation. These experiments demonstrated that PKC activation was required for PAF synthesis in response to the calcium signal generated by A23187 or a receptor-mediated agonist, opsonized zymosan. The synthesis of PAF and leukotriene B4 were temporally coupled following cell stimulation. Further, production of these two lipid mediators, and the release of arachidonic acid, were inhibited in parallel by sphingosine. Thus, PKC regulate the synthesis of both PAF and leukotriene B4 at a common step, probably phospholipase A2.

Topics: Binding Sites; Calcimycin; Diglycerides; Enzyme Activation; Humans; Kinetics; Leukotriene B4; Monocytes; Opsonin Proteins; Phagocytosis; Platelet Activating Factor; Protein Kinase C; Sphingosine; Tetradecanoylphorbol Acetate; Zymosan

[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector e

Topics: Binding, Competitive; Caenorhabditis elegans Proteins; Calcimycin; Calcium; Carrier Proteins; Cell Membrane; Cytosol; Diglycerides; Humans; Ionomycin; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phorbol 12,13-Dibutyrate; Phospholipids; Protein Kinase C; Receptors, Drug; Sphingosine

The synthetic diacylglycerols (DG), sn-1,2-dihexanoylglycerol (diC6), sn-1,2-dioctanoylglycerol (diC8), and 1-oleoyl-2-acetylglycerol (OAG) stimulated the release of granule constituents from and superoxide anion (O2-) generation by human polymorphonuclear neutrophils (PMN). The DGs did not induce a rise in the cytosolic-free calcium concentration ([Ca2+]i), as monitored by the fluorescence of PMNs loaded with the fluorescent CA2+ indicator, Fura-2. DiC6, diC8, and OAG inhibited PMN degranulation elicited with the receptor-specific ligands, N-formyl-methionyl-leucyl-phenylalanine (FMLP), acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), and 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans eicosatetraenoic acid (LTB4) and the calcium ionophore, A23187. In contrast to their inhibitory effects on granule exocytosis, diC6, diC8 and OAG enhanced FMLP-, AGEPC-, LTB4 and A23187-stimulated O2- production. Activation of the respiratory burst with phorbol 12-myristate 13-acetate (PMA) was unaffected by the DGs. DiC8 inhibited the rise in [Ca2+]i elicited with FMLP, LTB4, and AGEPC; this effect, as well as the DG-mediated suppression of degranulation, could be reversed with the protein kinase C (PKC) inhibitor, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7). These data indicate that in addition to possessing the intrinsic capacity to activate PMNs, DG may function in a PKC-mediated autoregulatory mode to influence PMN activation in a response-specific manner by affecting certain components of receptor-coupled and receptor-independent signal transduction systems in a stimulus-specific manner.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Benzofurans; Calcium; Cytochalasin B; Cytoplasmic Granules; Diglycerides; Dose-Response Relationship, Drug; Exocytosis; Fura-2; Glycerides; Humans; Isoquinolines; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Piperazines; Platelet Activating Factor; Superoxides

Simultaneous measurements of the calcium rise, membrane potential change, and 90 degrees light scatter (shape change) responses exhibited by neutrophils upon activation, can be obtained with identical result as that obtained when independently performing each measurement. The putative intracellular mediator diacylglycerol depolarizes membrane potential and causes a decrease in light scatter. Leukotriene B4 causes a rise in calcium and a decrease in light scatter. The chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine, causes a depolarization of membrane potential, a calcium rise, and a decrease in light scatter. The fura 2 measurements of intracellular free calcium indicate that the calcium concentration of unstimulated cells is much lower than previously thought based on quin 2 studies.

Topics: Calcium; Diglycerides; Humans; In Vitro Techniques; Leukotriene B4; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Spectrometry, Fluorescence