Structural requirements for long-chain (sphingoid) base inhibition of protein kinase C in vitro and for the cellular effects of these compounds

Sphingosine, sphinganine, and other long-chain (sphingoid) bases inhibit protein kinase C in vitro and block cellular responses to agonists that are thought to act via this enzyme. To gain further insight into the mechanism of this inhibition, a series of long-chain analogues differing in alkyl chain length (11-20 carbon atoms), stereochemistry, and headgroup were examined for (a) inhibition of protein kinase C activity in vitro, (b) the neutrophil respiratory burst in response to phorbol myristate acetate (PMA), (c) the PMA-induced differentiation of HL-60 cells, and (d) the growth of Chinese hamster ovary cells. In every instance, the effects were maximal with the 18-carbon homologues, which are the same length as the predominant naturally occurring long-chain base (sphingosine). The lower potency of the shorter chain homologues was partially due to decreased uptake by cells. Small differences were obtained with the four stereoisomers of sphingosine (i.e., D and L forms of erythro- and threo-sphingosine), with N-methyl derivatives of the different sphingosine homologues, and with simpler alkylamines (e.g., stearylamine). The potency of the different headgroup analogues may be affected by the degree of protonation at the assay pH. The pKa of sphingosine was measured to be 6.7; the pKa varied among the analogues. These findings establish that the major structural features required for inhibition of protein kinase C and cellular processes dependent on this enzyme are the presence of a free amino group and an aliphatic side chain and that other groups have more subtle effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the free sphingosine levels in human neutrophils by phorbol esters and other factors

Because free long-chain bases have been recently found to have potent pharmacological effects when added to neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623) and other cell types, the levels in human neutrophils were measured by high-performance liquid chromatography. Sphingosine was the major free long-chain base in freshly isolated cells and ranged from 13 to 101 pmol/10(7) cells for different donors (mean +/- S.E. of 50 +/- 5, n = 17). Upon incubation at 37 degrees C, there was a time-dependent increase in free sphingosine (57 +/- 8% in 1 h, n = 17), but no change was seen at 4 or 25 degrees C. The sphingosine was apparently derived from more complex sphingolipids because little (less than 1%) could be accounted for by new synthesis from [14C]serine. Greater increases in free sphingosine were obtained when neutrophils were incubated with serum, plasma, or serum lipoproteins (about 2-fold higher than for cells incubated alone). In contrast, agonists such as phorbol 12-myristate 13-acetate, A23187, arachidonic acid, low concentrations (10 nM) of N-formyl-methionyl-leucyl-phenylalanine, and opsonized zymosan either decreased the amount of free sphingosine or blunted the time-dependent increase. This may be due to enhanced removal of free sphingosine because phorbol 12-myristate 13-acetate-treated cells exhibited an increased conversion of exogenously added [3H]sphinganine to ceramides. Endogenous sphingosine was approximately one-tenth the level found in neutrophils when exogenous long-chain bases were added to inhibit protein kinase C. Hence, depending on the subcellular localization of the endogenous versus exogenous long-chain bases, the amounts of free sphingosine in neutrophils might be sufficient to affect the function of these cells.     

Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst

The roles of calmodulin and protein kinase C in the activation of the human neutrophil respiratory burst were characterized pharmacologically. The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not inhibit superoxide anion generation by neutrophils stimulated for 30 minutes with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). However, H-7 did depress superoxide production during the first 5 minutes following stimulation. In contrast, the specific calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and the dual calmodulin antagonist/protein kinase C inhibitor trifluoperazine (TFP) were potent inhibitors of the response throughout the 30 minute incubation. Stimulation of neutrophils with submaximal doses of FMLP or PMA failed to promote inhibition of the respiratory burst by H-7 or H-9, but did stimulate a respiratory burst response which was not inhibited by TFP or W-7. These results suggest that while protein kinase C may play a role in the initiation of the respiratory burst response, propagation of the response is dependent on calmodulin-dependent processes. The inability of TFP and W-7 to inhibit superoxide anion generation in response to submaximal stimulatory doses of FMLP or PMA suggests that calmodulin-independent processes may also be involved in activation of the respiratory burst.     

Sphinganine effects on chemoattractant-induced diacylglycerol generation, calcium fluxes, superoxide production, and on cell viability in the human neutrophil. Delivery of sphinganine with bovine serum albumin minimizes cytotoxicity without affecting inhibition of the respiratory burst

Long chain bases (sphinganine and sphingosine) are potent inhibitors of protein kinase C in an in vitro mixed micelle-reconstituted system (Hannun, Y. A., Loomis, C. R., Merrill, A. H. J., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609) and block activation of the superoxide-generating 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). In the present studies, we have investigated the effects of sphinganine on cellular levels of the second messengers related to phosphoinositide turnover: diacylglycerol and calcium. We find that sphinganine added from a stock solution containing equimolar or greater bovine serum albumin had no effect on either formyl-methionyl-leucyl-phenylalanine-stimulated calcium fluxes or diacylglycerol generation, at levels which completely blocked activation of superoxide generation. In addition, there was no effect of sphinganine on cell viability in this concentration range. These data indicate an inhibitory effect subsequent to the generation of second messengers and are consistent with protein kinase C as the locus of action. When sphinganine was added from a stock in dimethyl sulfoxide, significant cytotoxic effects (assayed by trypan blue exclusion, release of cellular lactate dehydrogenase, and leakage of Quin2) were seen at concentrations nearer those which inhibited the respiratory burst. Cytotoxicity was inversely proportional to cell concentration and was probably due to detergent micelle formation which occurs in the absence of albumin. These studies emphasize the importance of the method of delivery and the consideration of cytotoxic effects, but indicate that long-chain bases possess potent inhibitory properties which make them useful probes of signal transduction mechanisms.     

Temperature influence on stimulated PMN respiratory burst.

Body temperature can modulate the pathogenesis of infectious, metabolic and autoimmune diseases. This effect has been attributed to several hypothesized mechanisms. Body temperature could play an important role in influencing some cellular functions of human white blood cells. In this work we examined the temperature effect on the respiratory burst in human neutrophils. Human polymorphonuclear leucocytes (PMN) were obtained from heparinized venous blood by dextran sedimentation and erythrocyte lysis with NH4Cl (0.87%). Granulocytes were stimulated with opsonized zymosan (OZ), formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate (PMA), and monosodium urate (MSU) crystals at different temperatures (26, 37, 39, 40, 42 degrees C). The technique of luminol dependent chemiluminescence (CL) was used as indicator of oxygen free radicals (OFR) release by stimulated cells. OFR production from PMN stimulated with OZ, PMA, FMLP was higher at 37 degrees C than at 26, 39, 40, 42 degrees C (p < 0.001 OZ stimulated PMN at 40-42 degrees C; p < 0.05 PMA stimulated PMN at 42 degrees C. Significantly different from 37 degrees C value). OFR release from PMN stimulated with MSU crystals was significantly increased at 39 degrees C compared to 37 degrees C value (p < 0.001). This effect could not only be attributed to temperature influence on neutrophil activity. The specific polymorphonuclear leukocyte response to the microcrystals and the temperature influence on chemical and physical characteristics of the crystals may play an important role. We are now studying the temperature effect on activity of PMN exposed to others crystals.     

Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils

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.     

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.     

Sesquiterpene lactone from Wunderlichia crulsiana inhibits the respiratory burst of leukocytes triggered by distinct biochemical pathways

The sesquiterpene lactone tubiferin was chemically purified from the brazilian native plant Wunderlichia crulsiana and identified by NMR and GC/MS data. Its ability to inhibit the respiratory burst of peritoneal inflammatory polymorphonuclear leukocytes (PMN) stimulated upon addition of phorbol miristate acetate (PMA), opsonized zymosan (OZ), and N-formyl-methionyl-leucyl-phenylalanine (fMLP) was evaluated. The tubiferin inhibition was more pronounced when PMN were stimulated through the protein kinase C pathway (PMA) compared to the alternative complement pathway (OZ). The inhibition when PMN were triggered by a chemoattractant stimulus (fMLP) was similar to that achieved with OZ-stimulated phagocytes. Tubiferin showed dose-dependent effects on the PMN respiratory burst triggered by the three different substances, and also decreased substantially the carrageenan-induced mice paw edema.     

Zymosan but not phorbol myristate acetate induces an oxidative burst in rat bone marrow-derived macrophages

We found that rat bone marrow-derived macrophages responded to opsonized zymosan by releasing superoxide anion. However, these cells were defective in the response to the potent oxidative burst activator phorbol myristate acetate (PMA). This result was observed whatever the concentration of agonist used and with different concentrations of cells. Since it is strongly suspected that protein kinase C (PKC) is involved in the transductional pathway induced by PMA in numerous cell types, and particularly in phagocytes, we studied PKC and we observed that it was functional in rat bone marrow-derived macrophages, but only present at a low level. Thus, we suggest that our results are consistent with the possibility that zymosan-induced respiratory burst may be independent of PKC and that these cells may not possess the minimal level of PKC required for responding to PMA.     

Effect of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) on human neutrophil function: novel inhibition of neutrophil stimulus-response coupling?

Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.     

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.     

Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils.

Phosphorylation of a 47 kDa protein in human neutrophils is induced by phorbol 12-myristate 13-acetate (PMA), opsonized latex beads, fMet-Leu-Phe, calcium ionophore A23187 and fluoride. All of these stimuli activate the specialized microbicidal respiratory burst of neutrophils, and in each case the kinetics of activation correspond with the kinetics of phosphorylation of the 47 kDa protein. Trifluoperazine (50 microM) and chlorpromazine (100 microM), inhibitors of calmodulin and protein kinase C, abolish the increase in oxygen consumption and selectively prevent phosphorylation of the 47 kDa protein after PMA stimulation. Treatment of neutrophils with pertussis toxin totally inhibits both superoxide production and phosphorylation of this protein in response to fMet-Leu-Phe, but not in response to PMA, indicating that a GTP-binding protein modulates the fMet-Leu-Phe receptor signal. Phosphorylation of the 47 kDa protein, a phenomenon absent from the neutrophils of subjects with autosomal recessive chronic granulomatous disease, which lack the respiratory burst, appears to be the common trigger for activation of the burst in normal neutrophils.     

The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation

The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) were examined for their ability to inhibit human neutrophil activation. At concentrations up to 100 micromolar, these compounds failed to inhibit either respiratory burst or the secretory response of neutrophils stimulated with particulate (serum-opsonized zymosan) or soluble (A23187, FMLP, PMA) stimuli. In contrast, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) inhibited both oxygen radical generation and lysosomal enzyme release in response to the same stimuli. These results suggest that calmodulin-dependent enzymes, rather than protein kinase C, may be essential for neutrophil activation.     

Inflammatory agonist stimulation and signal pathway of oxidative burst in neonatal chicken heterophils

Heterophils are the predominant polymorphonuclear leukocytes (PMNs) in poultry. The oxidative burst of activated heterophils, which generates reactive oxygen species (ROS), is one of the first line cellular defenses against invading microorganisms. In this report, the oxidative response of heterophils from neonatal chicks to in vitro stimulation by various inflammatory agonists was investigated using a fluorescence microplate assay. Both non-opsonized formalin-killed Salmonella enteritidis and Staphylococcus aureus were able to stimulate heterophil oxidative burst. The phorbol myristate acetate (PMA) was the most potent stimulant for the chicken heterophil oxidative response, whereas, the bacterial cell surface components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were less effective. Protein kinase C (PKC) is an essential signaling component regulating heterophil oxidative response to stimulation by PMA, LPS, LTA and S. enteritidis. However, inhibition of PKC did not affect the oxidative response to stimulation by S. aureus, suggesting differential signaling pathway responsible for the activation of oxidative burst by Gram-negative S. enteritidis and Gram-positive S. aureus. Inhibition of mitogen activated protein (MAP) kinase p38 and extracellular response kinase (ERK) by SB 203580 and PD 098059, respectively, did not inhibit activated oxidative burst.     

Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22(phox), the small subunit of the membrane-bound oxidase component flavocytochrome b(558), is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms (Regier, D. S., Waite, K. A., Wallin, R., and McPhail, L. C. (1999) J. Biol. Chem. 274, 36601-36608). Here we show that several neutrophil agonists (phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid production by PLD in stimulated cells. Phorbol myristate acetate-induced phosphorylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol. In contrast, ethanol reduced both activities when cells were stimulated by N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibitor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in parallel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylation of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphorylation are correlated and suggest two mechanisms (PLD-dependent and -independent) by which p22(phox) phosphorylation occurs.     

Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C

Preincubation of neutrophils with certain agonists may "prime" the cells to cause increased responses to a second stimulus ("primed stimulation"). We used two approaches to examine the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in priming and stimulation by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP): inhibition of protein kinase C by 1-(5-isoquinolinesulfonyl)-piperazine (C-I) and measurement of protein kinase C translocation induced by priming and stimulatory concentrations of OAG. C-I had little effect on stimulation or primed stimulation by fMLP, suggesting that fMLP invokes events independent of protein kinase C. C-I equally inhibited stimulation and primed stimulation by PMA. Direct stimulation by OAG was inhibited, but priming and primed stimulation by OAG was unaltered by C-I. OAG concentrations greater than or equal to 100 microM caused translocation of protein kinase C, in correlation with direct stimulation of the respiratory burst. Lower OAG concentrations (10-30 microM) primed to stimulation by fMLP and, conversely, stimulated neutrophils primed with fMLP, yet did not cause translocation of protein kinase C. The data are compatible with previous assumptions that PMA and OAG directly stimulate polymorphonuclear neutrophil leukocytes by translocation and activation of protein kinase C. However, priming and primed stimulation by OAG apparently invoke distinct transduction mechanisms other than protein kinase C translocation.     

Modulation of TNF-alpha-priming and stimulation-dependent superoxide generation in human neutrophils by protein kinase inhibitors.

Human peripheral blood polymorphonuclear leukocytes (HPPMN) from healthy individuals are not primed and, hence, weak stimulation-dependent responses are induced by certain stimuli which bind to membrane receptors. When HPPMN were exposed to recombinant human tumor necrosis factor alpha (rHuTNF-alpha) or recombinant human granulocyte colony stimulating factor (rG-CSF), they underwent priming and the rate of superoxide anion (O.-2) generation was increased by subsequent exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). However, the degree of enhancement was very small upon exposure to phorbol myristate acetate (PMA) or dioctanoyl glycerol (DOG). The oxygen burst induced by FMLP or OZ was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), which are inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) and staurosporine, which are inhibitors of protein kinase C (PKC). Without priming, however, O.-2 generation from HPPMN by high concentrations of FMLP was not inhibited strongly by genistein or ST638. On the contrary, the oxygen burst induced by PMA or DOG was stimulated by genistein or ST638 and was inhibited by H-7 or staurosporine. Furthermore, O.-2 generation by guinea pig peritoneal neutrophils, which are already primed in vivo, was induced markedly by FMLP by a mechanism which was stimulated by a low concentration of genistein or ST638. Thus, FMLP-mediated O.-2-generation of HPPMN is coupled with rHuTNF-alpha- or rG-CSF-priming and is inhibited by TK inhibitors, whereas PMA- or DOG-induced O.-2 generation is not coupled with TNF-alpha or G-CSF-priming and is inhibited by PKC inhibitors. These results suggest that both PKC and TK play critical roles in the regulatory mechanism of priming and NADPH-oxidase activation in neutrophils.     

Calcium ionophore potentiates chemotactic peptide and platelet activating factor in stimulating thromboxane B2 and leukotriene B4 biosynthesis in human neutrophils

Formyl-Met-Leu-Phe (FMLP) and platelet activating factor (PAF) stimulated the synthesis of thromboxane B2 (TXB2) and leukotriene B4 (LTB4) to a small degree in human neutrophils. Calcium ionophore A-23187 enhanced synergistically both FMLP and PAF induced eicosanoid synthesis, whereas phorbol ester PMA attenuated PAF but not FMLP stimulated arachidonate metabolism. These results suggest that calcium mobilization may be a rate limiting step in FMLP and PAF induced synthesis of TXB2 and LTB4 and that protein kinase C activation may play a negative regulatory role in PAF stimulated eicosanoid synthesis.     

Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme

Protein kinase C may be important in leukocyte function, because it is activated by phorbol myristate acetate (PMA), a potent stimulus of the respiratory burst in neutrophils. The localization of protein kinase C was compared in unstimulated and PMA-stimulated human neutrophils. Protein kinase C was primarily cytosolic in unstimulated cells but became associated with the particulate fraction after treatment of cells with PMA. The particulate-associated kinase activity did not require added calcium and lipids, but when extracted by Triton X-100 (greater than or equal to 0.2%), calcium and phospholipid dependence could be demonstrated. The EC50 of PMA for stimulating kinase redistribution and activation of NADPH oxidase, the respiratory burst enzyme, were similar (30 to 40 nM). Redistribution of protein kinase C occurred rapidly (no lag) and preceded NADPH oxidase activation (30 sec lag). These results suggest that redistribution of protein kinase C is linked to activation of the respiratory burst in human neutrophils.     

Diacylglycerol generation and phosphoinositide turnover in human neutrophils: effects of particulate versus soluble stimuli

Serum-treated, or "opsonized" zymosan (OZ), a particulate material which can be phagocytized by polymorphonuclear leukocytes, activates the superoxide-generating respiratory burst in these cells. The use of dual wavelength spectroscopy in the present studies has allowed accurate continuous monitoring of superoxide generation (cytochrome c reduction) upon cellular activation by this turbid material; activation occurs after a short lag period (about 20 s) which is similar to the lag seen after activation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Unlike the fMLP response which terminates after about 90 s, superoxide generation in response to OZ continues beyond 10 min, and is similar in this regard to the response seen with the protein kinase C activator phorbol myristate acetate (PMA). OZ and fMLP, but not PMA, also activate receptor-linked phospholipase C mechanisms as judged by the appearance of inositol trisphosphate (IP3) (as well as other inositol phosphates) and diacylglycerol (DAG), with the latter measured by a mass assay. The appearance of these potential mediators corresponded to the loss of phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphate (PIP2). The magnitude of DAG and inositol sugar generation as well as the breakdown of PIP2 was considerably greater using OZ than with fMLP. In addition, while fMLP resulted in a transient increase in IP3 and DAG, OZ resulted in a sustained elevation of these molecules. With both agonists, the onset and duration of generation of putative mediators corresponded to the period of generation of O2-, consistent with a role for DAG and/or IP3 in the activation of the respiratory burst.