leukotriene-b4 and safingol

The sphingosine analog L-threo-dihydrosphingosine has been shown to inhibit protein kinase C (PKC) isoenzymes in mixed micelle and vesicle assays. This compound also inhibited the reactive oxygen intermediates (ROI) released from isolated neutrophils (IC50 approximately 2 microM) and phorbol ester-induced edema and neutrophil influx in the mouse ear model (ED50 approximately 11 mg/kg). Based on the anti-inflammatory activity of this compound, studies were done to determine its effect on arachidonate metabolism by the lipoxygenase pathway. Neutrophils were preincubated with test agents or vehicle for one minute and then incubated with 1 microM calcium ionophore A23187 for two minutes. Supernatants were assayed for LTB4 using a radioimmunoassay. The reference lipoxygenase inhibitor nordihydroguaiaretic acid exhibited 98.3% inhibition at 1 microM (n = 2) and prevented ROI production (IC50 approximately 6 microM). In contrast, the potent PKC inhibitor staurosporine was inactive against LTB4 in these studies (< 23% inhibition at 10 microM, n = 2), but inhibited ROI formation (IC50 approximately 3nM). L-threo-dihydrosphingosine inhibited LTB4 production 96.9 +/- 1.3%, at 10 microM (IC50 = 6 microM, n = 2). These data suggest that L-threo-dihydrosphingosine blocks the release of LTB4 from human neutrophils via a mechanism independent of PKC.

Topics: Animals; Calcimycin; Enzyme Inhibitors; Humans; In Vitro Techniques; Kinetics; Leukotriene B4; Masoprocol; Mice; Neutrophils; Protein Kinase C; Reactive Oxygen Species; Sphingosine; Staurosporine

[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

Three protein kinase C blockers (staurosporin, Cl, and sphinganine) acted temperature- and time-dependently on human neutrophils to lower the affinity and number of high affinity plasmalemma receptors available to leukotriene B4. The drugs did not alter the ligand's binding to isolated plasma membranes or reduce intact cell binding of platelet-activating factor. Thus, protein kinase C may regulate the expression of certain receptors in resting cells and blockers of this enzyme, by interfering with receptor expression, have secondary effects that complicate their use as pharmacological probes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Humans; In Vitro Techniques; Isoquinolines; Kinetics; Leukotriene B4; Neutrophils; Piperazines; Platelet Activating Factor; Protein Kinase C; Receptors, Immunologic; Receptors, Leukotriene B4; Sphingosine; Staurosporine; Tetradecanoylphorbol Acetate; Thermodynamics