calcimycin 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

Vasoactive intestinal peptide (VIP) primed the respiratory burst of human neutrophils induced by phorbol myristate acetate (PMA) and by the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP). The sigmoidal-shaped curve of the priming effect of VIP differs for both agonist since the Hill coefficient was close to three in the case of neutrophil activation by fMLP whereas the corresponding value for PMA was close to one. The priming effect of VIP was enhanced when neutrophils were stimulated by FMLP in the presence of sphinganine, a protein kinase C inhibitor, at concentrations which almost abolished the response to PMA. VIP failed to increase resting cytosolic free calcium and to modify the transient increase in [Ca2+]i induced by fMLP. The described results point out that the mechanism of the priming of neutrophils by VIP is also independent of calcium and protein kinase C. The absence of VIP receptors in plasma membrane of neutrophils suggests that a receptor-independent mechanism modulates the agonist-triggered signaling pathway. The priming of neutrophils by VIP can not be considered as a pharmacological effect, as may be deduced from the required VIP concentration; it should be rather considered that the enhancement of the formation of reactive oxygen metabolites by VIP may be interesting in the understanding of the neuroimmune axis.

Topics: Calcimycin; Humans; In Vitro Techniques; N-Formylmethionine Leucyl-Phenylalanine; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophil Activation; Neutrophils; Receptors, Vasoactive Intestinal Peptide; Respiratory Burst; Somatostatin; Sphingosine; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide

Alveolar macrophages (AM) differ from other macrophage (m phi) populations in their profile of eicosanoids synthesized from arachidonic acid (AA)3. Little information is available regarding possible differences in the regulation of AA metabolism among various m phi populations. In our study, we compared the ability of cultured resident rat AM and peritoneal m phi (PM) to release and metabolize AA in response to exogenous activators of protein kinase C (PKC). When stimulated with PMA, prelabeled PM released free [3H]AA in a dose-dependent manner over the concentration range 1 to 100 nM. As assessed by HPLC, PMA-stimulated PM metabolized AA to a variety of predominantly cyclooxygenase products. The dose-dependent synthesis of PGE2 by unlabeled PM stimulated with PMA was confirmed using RIA. The ability of PMA to trigger AA release and metabolism in PM was a function of its capacity to activate PKC, as indicated by the following: 1) an additional activator of PKC, oleoyl acetylglycerol, also triggered PM AA metabolism, whereas phorbol didecanoate, which lacks the ability to activate PKC, did not; 2) two structurally unrelated inhibitors of PKC activation (staurosporine and sphinganine) both abrogated PMA induced AA release in PM; and 3) pretreatment for 18 h with high dose PMA (used to deplete cellular PKC), but not phorbol didecanoate, rendered PM refractory to subsequent PMA stimulation of AA release. In contrast to PM, AM cultured in identical fashion failed to release or metabolize AA in response to either PMA or oleoyl acetylglycerol. PM and AM were also compared for their ability to release extracellular superoxide anion in response to PMA; once again, PM exhibited significantly greater release than did AM. Inasmuch as this unresponsiveness to activation of PKC distinguishes AM from other m phi populations, we conclude that it is a unique consequence of m phi differentiation in the lung. Moreover, because both AA metabolism and the respiratory burst are affected, this refractoriness appears to reflect a defect at some proximal level in PKC-mediated signaling.

Topics: Alkaloids; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Differentiation; Dinoprostone; Eicosanoids; Enzyme Activation; Female; Macrophages; Peritoneal Cavity; Phorbol Esters; Protein Kinase C; Pulmonary Alveoli; Rats; Rats, Inbred Strains; Sphingosine; Staurosporine; Superoxides; 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

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.

Topics: Calcimycin; Glucuronidase; Humans; In Vitro Techniques; Kinetics; Lactoferrin; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peroxidase; Protein Kinase C; Sphingosine; Tetradecanoylphorbol Acetate