TEGDMA modulates glutathione transferase P1 activity in gingival fibroblasts.

Dental resinous materials can contain large amounts (from 30 to 50%) of triethylene-glycol-dimethacrylate (TEGDMA). This compound leaches into aqueous media and is toxic to dental pulp, as well as to gingival fibroblasts in vitro. To elucidate the mechanism of TEGDMA toxicity, we investigated the effects on glutathione (GSH) level and glutathione transferase P1 (GSTP1) activity in cultured human gingival fibroblasts. TEGDMA cytotoxic concentrations (from 0.5 to 2 mM) induced a depletion of GSH without formation of oxidized GSH (GSSG). In fibroblasts expressing the wild-type GSTP1, TEGDMA both inhibited and potentiated GSTP1 activity at high (IC50 = 1.1 mM) and low concentrations, respectively. In contrast, cells expressing the GSTP1 *A/*B variant showed a weak inhibition of GST activity only, associated with greater sensitivity to drug toxicity. Biochemical analysis of GSTP1 inhibition revealed that TEGDMA is a non-competitive antagonist with respect to GSH and substrate. Thus, TEGDMA interference with GSH and GSTP1 activity may contribute to dental-resin-induced adverse effects.

Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst.

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.

Sodium stibogluconate (Pentostam) potentiates oxidant production in murine visceral leishmaniasis and in human blood.

Sodium stibogluconate (Sbb), a leishmanicidal drug, was studied for its in vivo effect on the formation of reactive oxygen species (ROS), assessed by chemiluminescence (CL) in the whole blood of mice infected with Leishmania infantum. Stimulation of ROS formation induced ex vivo by zymosan particles or the protein kinase C activator phorbol myristate acetate (PMA) was reduced by approximately 25% (P < 0.05) after infection of mice. Treatment of infected mice with Sbb (50 to 400 mg/kg of body weight) enhanced the blood CL induced by zymosan and PMA (47 to 96%, P < 0.01). The drug potentiation effect also occurred in uninfected mice. In vitro treatment of normal human blood with Sbb (1, 10, or 100 microg/ml) for 1 h primed the CL response to PMA (29 to 54%). The priming effect of Sbb was also observed on the production of superoxide by isolated polymorphonuclear leukocytes stimulated either by PMA and zymosan or by the chemoattractants N-formyl-Met-Leu-Phe and platelet-activating factor. These data provide the first evidence of priming of the phagocyte respiratory burst by Sbb. This novel property of Sbb may contribute to the drug's leishmanicidal effect.

Dental restorative biomaterials induce glutathione depletion in cultured human gingival fibroblast: protective effect of N-acetyl cysteine.

Eight biomaterials eluted from four different types of dental restorative biomaterials, that is, from glass-ionomer cement (GIC: Ketac-fil and Fuji II), resin-modified glass ionomer cement (RM-GIC: Fuji II LC and Photac-fil), composite (Z100 MP and Tetric-flow), and compomer (Compoglass F and F-2000), were studied for their cytotoxic properties in relation to glutathione (GSH) content in cultured human gingival fibroblasts. Z100 MP, Tetric-flow, and Compoglass F were less cytotoxic than the others, with a toxic concentration of 50% (TC 50) > 24% (of eluate), as determined by the MTT test. F-2000, Tetric-flow, and the other biomaterials were relatively more cytotoxic (TC 50 = 9-16%). With the exception of Z100 MP, all the biomaterials induced a depletion of cellular glutathione (GSH) that was variable depending upon the biomaterial eluates. The strongest GSH depletion was with F-2000, Fuji II, and Photac-fil. GSH depletion, with Compoglass and F-2000, was rapid-detectable after one h of cell treatment and complete within 3 h-whereas a longer period of incubation was required for the other biomaterials. Interestingly, the drug cytotoxic effects induced by all the biomaterials were prevented by cell treatment with the antioxidant N-acetylcysteine (NAC). This study provides evidence that the cytotoxic property of dental restorative biomaterials is associated with depletion of the glutathione level in gingival fibroblasts. While the molecular mechanisms of this phenomenon require further investigations, our data suggest that NAC may be useful in preventing the cellular damage induced by dental restorative biomaterials.

Contribution of mitogen-activated protein kinase to stimulation of phospholipase D by the chemotactic peptide fMet-Leu-Phe in human neutrophils.

Phospholipase D (PLD) plays an important role in signaling through phosphatidylcholine (PC) and in the production of superoxide (respiratory burst) by polymorphonuclear leukocytes (PMN) stimulated by the chemoattractant fMet-Leu-Phe (fMLP). However, the regulation of PLD activity by protein kinases is not fully understood. In the present study, we have used a mitogen-activated protein (MAP) kinase inhibitor (PD 98059) to investigate a possible connection between extracellular signal-regulated kinase (ERK) and PLD activity and respiratory burst. Using a range of concentrations (3-20 microM) which inhibit ERK activity, PD 98059 inhibited PLD activity induced by fMLP in cytochalasin B-primed PMN, as assessed by production-tritiated phosphatidylethanol (PEt), phosphatidic acid (PA), and hydrolysis of PC. However, the inhibition was partial (approximately 50%), while inhibition of PC hydrolysis was almost complete, suggesting a concomitant inhibition of PLA2 activity. In addition, PD 98059 reduced fMLP-induced respiratory burst by 50%, an effect which was correlated with PLD inhibition of PLD (r = 0.981, P < 0.01), and neither did PD 98059 inhibit the PLD activity and respiratory burst induced by PKC upon its direct activation by phorbol myristate acetate. These data provide the first evidence for implication of the ERK cascade in the stimulation of PLD through Gi signaling. They further indicate that PLD stimulation by fMLP receptors occurs through two pathways, dependent and independent on MAP kinase, the former pathway being linked to superoxide production.

Analysis of choline and phosphorylcholine content in human neutrophils stimulated by f-Met-Leu-Phe and phorbol myristate acetate: contribution of phospholipase D and C.

ABSTRACT. We analysed changes in choline (CHO) and phosphorylcholine (PCHO) content of stimulated human polymorphonuclear leukocytes (PMNs) by a chemiluminescence assay to further examine the relative contributions of phospholipase D (PLD) and PLC to phosphatidylcholine (PC) breakdown. PLD activation was also analysed by measuring tritiated phosphatidic acid (PA) and diglycerides (GDs) in PMNs labelled with tritiated alkyl-lyso PC. Stimulation of PMNs with formyl-methionyl-leucyl-phenylalanine fMLP; 0.1 microM induced a weak elevation of mass choline (+25% of basal level) that was strongly potentiated in PMNs primed with cytochalasin B (+350% relative to the control value of 657+/-53 pmol/10(7) cells). CHO production was rapid and transient, peaking within 1 min, and ran parallel to that of tritiated PA. Thereafter, the amount of tritiated PA declined strongly (40% of maximum by 3 min), whereas the elevated choline content induced by fMLP plateaued for at least 5 min. Phorbol myristate acetate (PMA) sustained the formation of CHO for as long as 20 min, which correlated with that of [3H]PA in a time- and concentration-dependent manner. PCHO content of resting PMN leukocytes (1560 +/- 56 pmol/10(7) cells) was not modified after stimulation of PMNs with fMLP or PMA for at least 10 min, which argues against breakdown of phosphatidylcholine by PLC. For longer treatment (10-20 min), fMLP stimulated a significant enhancement of PCHO level, which occurred concomitantly with a decrease in CHO level, suggesting that choline kinase rather than PLC may be activated. Unlike fMLP, PMA stimulated a fall in PCHO between 10 and 15 min after PMN stimulation, pointing to different regulatory mechanisms of PCHO level. These data indicate that DG formation from PC in PMNs is mediated by PLD but not by PLC and show that chemiluminescence measurement of choline is a reliable index of PLD activation.

Dermaseptin, a peptide antibiotic, stimulates microbicidal activities of polymorphonuclear leukocytes.

Dermaseptin (DRs S1), a 34-amino acid residue cationic antimicrobial peptide was studied for its effects on the production of reactive oxygen species (respiratory burst) and exocytosis of polymorphonuclear leukocytes (PMN). Treatment of PMN with DRs S1 (10-100 nM) stimulated significant production of reactive oxygen species (approximately a 2-fold increase relative to control) and release of myeloperoxidase. In addition, low DRs S1 concentrations (1-10 nM) primed the stimulation of respiratory burst induced by zymosan particles. In contrast to the native peptide, a dermaseptin fragment without either the COOH-terminal (DRs 1-10) or NH2 terminal (DRs 16-34) portion was inactive. The DRs S1-induced respiratory burst was inhibited by a selective protein kinase C inhibitor, GF 109203X, and was associated with early signalling events such as a rapid and transient elevation of cytosolic-free calcium concentration and phospholipase D activity. These data provide the first evidence of stimulating and priming properties of a peptide antibiotic on microbicidal activities of neutrophils, suggesting a potential role of dermaseptin in modulating host-defense mechanisms.

Priming of phosphatidic acid production by staurosporine in f-Met-Leu-Phe-stimulated human neutrophils--correlation with respiratory burst.

Staurosporine, a microbial alkaloid known as a potent though non specific PKC inhibitor, enhances the production of superoxide anion (respiratory burst) of human polymorphonuclear leukocytes (PMN) stimulated by chemoattractants such as f-Met-Leu-Phe (fMLP). To gain insights into the mechanisms of this priming, we analysed staurosporine effects on formation of second messengers issued from phospholipase D (PLD), i.e., phosphatidic acid (PA) and its dephosphorylated form, diglycerides (DG). PA and DG were measured by two methods, in mass and after the labelling of PMN with a phosphatidylcholine precursor, [3H]-1-O-alkyl-2-lyso-3-phosphatidylcholine. Treatment of labelled PMN with low concentrations of staurosporine (12.5 and 50 nM) which prime respiratory burst had no significant effect on basal amounts of tritiated PA and DG, but potentiated fMLP-mediated formation of [3H]PA and phosphatidylethanol (PEt) pointing to a priming of PLD activity. PA mass in resting PMN increased (approximately 80 +/- 7%) in the presence of high drug concentrations only (250-500 nM), with no change in basal DAG mass. Low staurosporine concentrations (6.25-25 nM) markedly potentiated PA mass formation induced by fMLP and positive correlation (R = 0.95) was found between enhanced superoxide formation and generation of PA but not DG. Furthermore, cytochalasin B, which is known to prime PA production induced by fMLP, synergised the priming of respiratory burst by staurosporine, which further suggests a functional role of PA. In contrast to staurosporine, the more selective PKC inhibitor GF109203X neither stimulated PLD nor primed fMLP-induced PLD or respiratory burst. These data indicate that priming of fMLP-mediated PMN respiratory burst by staurosporine correlates with PA formation. This priming may be linked to alteration of early signalling events upstream of PLD rather than to feedback inhibition of PKC.

Redistribution of protein kinase C isoforms in human neutrophils stimulated by formyl peptides and phorbol myristate acetate.

The redistribution of protein kinase C (PKC) isoforms between the cytosolic and plasma membrane fractions of stimulated human polymorphonuclear leukocytes (PMN) was analysed by means of western blotting with antibodies against PKC beta I, beta II and Zeta. Treatment of PMN with 1 microM formyl-methionyl-leucyl-phenylalanine (fMLP) induced a rapid (5-10 sec) and sustained (at least 10 min) increase in the membrane association of PKC beta I, beta II, and the two immunoreactive proteins (76-81 kDa) recognized by the antibody directed against PKC zeta. Optimal translocation of PKC isoforms to the plasma membrane occurred in the presence of 10(-6) M fMLP and was not associated with a detectable fall in cytosolic PKC. In the absence of external calcium, the translocation of all PKC isoforms induced by fMLP was rapid (5 sec) but the membrane association of PKC was lost within one minute. Unlike fMLP, phorbol myristate acetate (PMA) induced a concentration-dependent translocation of the PKC isoforms, which persisted in the membrane in the absence of external calcium. These data provide the first evidence of redistribution of PKC isoforms by a chemoattractant. They further indicate that external calcium plays a crucial role in the persistence of the membrane association of PKC beta I, beta II and zeta induced by formyl peptides.

Contrasting effects of calyculin A and okadaic acid on the respiratory burst of human neutrophils.

The involvement of serine/threonine protein-phosphatases in the production of superoxide (respiratory burst) by human neutrophils was investigated using calyculin A, a potent inhibitor of both protein phosphatases type 1 and 2A, and okadaic acid, which preferentially inhibits protein phosphatase type 2A. Treatment of neutrophils with calyculin A (25-75 nM) or okadaic acid (1-4 microM) had no stimulatory effect but potently enhanced total superoxide production induced by an optimal fMLP (N-formyl-methionyl-leucyl-phenylalanine) concentration (0.1 microM). The maximum increase plateaued with 50-75 nM calyculin A and 2-4 microM okadaic acid, reaching approximately 120 and 200% of control values, respectively. Unlike calyculin A, okadaic acid also primed the initial rate of superoxide production, suggesting that protein phosphatases may down-regulate both initiation and termination of respiratory burst. Optimal stimulation of the respiratory burst by PMA (160 nM) was inhibited by calyculin A and okadaic acid, with an IC50 of 60 nM and 2 microM, respectively, although both drugs caused protein hyperphosphorylation. The inhibition was partially prevented by a nonstimulatory concentration of A23187, indicating a role of calcium in the inhibitory effects of the drugs. Unlike the optimal respiratory burst, suboptimal respiratory burst induced by PMA (1-7 nM) was enhanced by calyculin A and okadaic acid. Unprimed and primed respiratory bursts were depressed by a selective antagonist of protein kinase C (GF 109203X), indicating positive regulation of these responses by protein kinase C. Thus, the use of calyculin A and okadaic acid distinguishes two regulatory processes of superoxide production.(ABSTRACT TRUNCATED AT 250 WORDS)

Okadaic acid, an inhibitor of type 1 and type 2A phosphatases, modulates the activation of phospholipase D in formyl peptide- and mastoparan-stimulated human neutrophils.

The activation of phospholipase D (PLD) induced by formyl peptides (fMLP), as evaluated by production of tritiated phosphatidylethanol (PEt) and phosphatidic acid (PA) in polymorphonuclear leukocytes (PMN), was markedly enhanced (50-125%) by low okadaic acid concentrations (0.25-0.5 microM) but inhibited by higher concentrations (2-3 microM), although the drug caused protein hyperphosphorylation. Both effects of okadaic acid were amplified when PLD activation was primed with cytochalasin B. Stimulation of PMN with mastoparan, a wasp venom toxin that activates Pertussis toxin(PTX)-sensitive G proteins, resulted in a weak calcium-dependent production of PEt which was respectively enhanced and inhibited by okadaic acid (1-2 microM) in unprimed and cytochalasin-primed PMN. The results show that low okadaic acid concentrations primed fMLP-mediated activation of PLD, in keeping with a down-regulatory role of protein phosphatases. The contrasting effects of okadaic acid in mastoparan-stimulated PMN further suggest that protein phosphatases may regulate the generation of second messengers through alteration of major signaling events at/or downstream of PTX-sensitive G proteins (Gi).

Immunochemical identification and translocation of protein kinase C zeta in human neutrophils.

Western blots of human polymorphonuclear leukocyte (PMN) extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. Two bands corresponding to PKC type zeta with apparent molecular masses of 81 kDa and 76 kDa were identified in the cytosolic fraction of resting cells, in addition to PKC types alpha and beta. PKC zeta was apparently abundant, like PKC beta, whereas PKC delta, -epsilon, and -gamma were not detectable. Following short stimulation (5 min) of PMN with phorbol-12-myristate-13-acetate (1 microgram/ml), physical translocation of PKC zeta from the cytosol to the plasma membrane fraction occurred, although this isoform does not bind phorbol esters. These data show that, in addition to the two calcium-dependent isoenzymes alpha and beta, human PMN express a calcium-independent isoenzyme zeta which translocates in stimulated cells, suggesting a role in the regulation of antibacterial activities.

Staurosporine up-regulates the expression of phorbol dibutyrate binding sites in human platelets.

Tumor-promoting phorbol esters bind to and activate protein kinase C (PKC). Staurosporine, a potent PKC inhibitor, interferes with PKC catalytic activity without altering phorbol ester binding sites in cell-free systems. We found that, unlike cell-free systems, treatment of intact platelets with staurosporine enhances the expression of phorbol 12, 13-dibutyrate (PDBu) binding sites. Incubation of platelets at 37 degrees with staurosporine (25 nM to 1 microM and 2 nM tritiated PDBu ([3H]PDBu) increased the amount of [3H]PDBu specifically bound to intact platelets by approximately 10 to 200% of control values. This effect was rapid and plateaued after 10 min of cell treatment. Scatchard analysis of the data showed that staurosporine (500 nM) significantly increased the total binding capacity Bmax from 42.9 +/- 15.4 x 10(3) to 78 +/- 7.3 x 10(3) sites per platelet and reduced the apparent dissociation constant value Kd from 30.8 +/- 8.6 nM to 9.4 +/- 3.4 nM. Enhanced PDBu binding capacity and affinity were also observed with human mononuclear and polymorphonuclear leukocytes. Fractionation of staurosporine-treated platelets showed an increased binding capacity of the particulate fraction (102%) and decreased binding capacity of the soluble fraction (60%) compared to controls, with no change in the affinity of PDBu binding to these fractions. Chelation of internal calcium with BAPTA did not significantly attenuate the staurosporine-mediated rise in PBDu binding but prevented the platelet-activating factor-induced response, indicating that cytosolic calcium does not play an important role in these staurosporine effects. These results show that, in addition to interfering with PKC protein-phosphorylating activity, staurosporine enhances PDBu binding affinity and capacity in intact platelets. This latter effect appears to be due to translocation of soluble PDBu binding sites, presumably PKC units.

W-7, a calmodulin antagonist, primes the stimulation of human neutrophil respiratory burst by formyl peptides and platelet-activating factor.

Low concentrations of the calmodulin antagonist W-7 (1-10 microM) enhanced the respiratory burst (RB) of human polymorphonuclear leukocytes (PMN) stimulated by N-formyl-methionyl-leucyl-phenylalanine, whereas high drug concentrations (above 20 microM) depressed it. The maximal increase obtained with 5-10 microM W-7 affected both initial rate (50%) and total superoxide anion production (150%). W-7 also primed both parameters of the RB mediated by platelet-activating factor, although higher drug concentrations were required (15-50 microM). By contrast, W-7 depressed the RB induced by the calcium ionophore A23187 and by a protein kinase C activator, phorbol myristate acetate, with an IC50 of approximately 20 and 8 microM, respectively. These data show the enhancing effect of W-7 on chemoattractant-mediated RB and suggest that RB priming may involve calmodulin-dependent regulation of chemoattractant-mediated early signalling events.

Stimulation of the human neutrophil respiratory burst by formyl peptides is primed by a protein kinase inhibitor, staurosporine.

Stimulation of polymorphonuclear neutrophils (PMN) by phorbol esters or formyl peptides (fMLP) generates large quantities of superoxide anion, the so-called respiratory burst (RB), a phenomenon associated with intense phosphorylation of a 47-kD protein (p47 phox). Staurosporine, a potent protein kinase C (PKC) antagonist, inhibits both responses when PMN are stimulated by phorbol myristate acetate (PMA), suggesting a positive role of PKC. In this study, we reassessed these PMN responses in fMLP-stimulated cells and found that staurosporine had opposite effects depending on the duration of PMN treatment with staurosporine. Short PMN incubation (0.5 to 3 minutes) with 25 to 100 nmol/L staurosporine inhibited the fMLP-induced RB, whereas longer treatment (15 to 20 minutes) enhanced it by up to about 200% relative to controls. In contrast, the PMA-mediated RB was depressed by staurosporine in a time-dependent manner. A primed fMLP-induced RB was also observed after long (15 minutes) PMN treatment with 5 to 100 mumol/L H-7, whereas shorter treatment (5 minutes) resulted in a small decrease in RB. By contrast, the tyrosine kinase inhibitor genistein (2 to 80 mumol/L) depressed fMLP-induced RB whatever the duration of PMN treatment. Analysis of 32P-phosphorylated proteins in fMLP-stimulated cells showed that short PMN treatment (< 8 minutes) with staurosporine abolished the phosphorylation of the 47-kD protein, which was identified as p47 phox, whereas long treatment partially restored p47 phox phosphorylation up to approximately 50% of the control value. In PMA-stimulated PMN, phosphorylation was reduced in a time-dependent manner. Furthermore, the staurosporine-primed RB and the staurosporine-induced recovery of phosphorylation were inhibited by sphingosine but not by genistein. Thus, in addition to its known depressive effect, staurosporine markedly potentiated fMLP-stimulated RB as a function of the duration of PMN treatment. The restoration of p47 phox phosphorylation suggests that staurosporine may alter the interactions between different protein kinases, producing marked time-dependent changes in signalling pathways. These data emphasize the care that should be taken in interpreting data obtained using this kinase inhibitor that may, however, be helpful analyzing in signalling pathways.

A protein kinase inhibitor, staurosporine, enhances the expression of phorbol dibutyrate binding sites in human polymorphonuclear leucocytes.

Staurosporine, a potent protein kinase C (PKC) inhibitor, was studied for its effects on the binding of phorbol 12,13-dibutyrate (PDBu) to human polymorphonuclear leucocytes (PMNs). Treatment of PMNs with staurosporine concentrations in the range 50 nM-2 microM at 37 degrees C (but not at 4 degrees C) and with 1 nM [3H]PDBu at both temperatures enhanced specific PDBu binding to PMNs by approx. 10-600% relative to control values. The potentiation was rapid (detectable within 1 min) and reached a plateau after 10 min of cell treatment. Scatchard analysis of the binding showed that staurosporine increased the total number of PDBu-binding sites (Bmax) from (178 +/- 9) x 10(3) (control) to (324 +/- 15) x 10(3) sites per PMN and lowered the apparent dissociation constant (Kd) from 9.6 +/- 0.8 (control) to 3.3 +/- 0.3 nM. In Ca(2+)-depleted cells, staurosporine induced similar changes in Kd values, whereas the Bmax. increased by 60%. Treatment of PMNs with 500 nM staurosporine enhanced PDBu binding in the particulate fraction by 120 +/- 7% and decreased PDBu binding in the soluble fraction by 69 +/- 4%, whereas PKC histone-phosphorylating activity of both fractions was almost completely inhibited. Incubation of staurosporine-pretreated particulate fractions with soluble fractions enriched the particulate fraction in PDBu-binding sites at the expense of the soluble fraction, without altering the binding affinity of PDBu for either fraction. Stimulation of PMNs with chemotactic N-formyl peptides induced a transient increase in PDBu binding. This effect was potentiated by approx. 52% by staurosporine. These results show that, in addition to its interference with PKC protein-phosphorylating activity, staurosporine enhances both PDBu-binding capacity and affinity to PMNs, through a mechanism involving Ca(2+)-dependent and -independent processes. Alterations of PDBu binding to soluble and particulate fractions suggest that the enhanced binding capacity in intact PMNs may be due to translocation of PDBu receptors, presumably PKC units. This phenomenon may affect PKC-dependent cellular responses mediated by physiological stimulation.

Staurosporine stimulates phospholipase D activation in human polymorphonuclear leukocytes.

Treatment of 1-O-[3H]alkyl-2-acyl-phosphatidylcholine-prelabeled human polymorphonuclear leukocytes (PMNs) with staurosporine (50 nM to 1 microM) induced a time- and concentration-dependent generation of tritiated phosphatidic acid (PA), reaching approximately 225% of the control value at 15-20 min. In the presence of ethanol, staurosporine induced a production of phosphatidylethanol (PEt) reaching, 250% of control values, and partial inhibition of PA production, consistent with PLD activation. The amount of ether-linked acylglycerol (EAG) was weakly enhanced (29%) after 5 min of PMN treatment; longer treatment resulted in no significant EAG production, suggesting a possible late inhibition of PA hydrolase activity. Staurosporine concentrations that induced an elevation in PA completely depressed protein kinase C (PKC) activity in both soluble and particulate cell fractions, suggesting that PLD activation may occur independently from PKC activation. PLD may thus represent a potential cellular target for staurosporine action.

Stimulation of human polymorphonuclear leukocytes potentiates the uptake of diclofenac and the inhibition of chemotaxis.

Diclofenac sodium, a non-steroidal anti-inflammatory drug, has been shown to impair the stimulation of human polymorphonuclear leukocytes (PMNs) by chemoattractants. To gain insight into the mechanism of action of this agent, we investigated the uptake of diclofenac by resting and activated PMNs and the effect of the drug on PMN locomotion. During incubation of resting PMNs at 37 degrees in the presence of 78 microM (25 micrograms/mL) diclofenac, drug uptake reached a plateau in less than 2 min. The resulting cellular to extracellular diclofenac concentration ratio (C/E) was 1.01 +/- 0.13 (mean +/- SD). Stimulation of PMNs at 37 degrees but not at 4 degrees with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), induced a rise in diclofenac uptake, which was dependent on incubation time and diclofenac and stimulus concentrations. Maximal C/E was 1.83 +/- 0.18 and 4.40 +/- 0.60 (mean +/- SD) for PMNs stimulated with 10 microM fMLP and 0.16 microM PMA, respectively. The diclofenac associated with PMNs was predominantly present in the soluble fraction of disrupted cells. Interestingly, PMNs which were pretreated with diclofenac and stimulated with fMLP, exhibited impaired random and directional locomotion induced by activated serum, as compared to controls, i.e. PMNs treated with diclofenac alone or fMLP alone. Thus, stimulation of PMNs enhances diclofenac uptake and potentiates the drug impairment of chemotactic activity. These findings could explain, in part, the observed anti-inflammatory properties of this compound.

Priming effect of fibronectin on respiratory burst of human neutrophils induced by formyl peptides and platelet-activating factor.

Fibronectin (FN), a glycoprotein present in the plasma and the extracellular matrix, has been shown to enhance adherence-related functions of polymorphonuclear leukocytes (PMNs). In this study we investigated the effects of FN on the activation of human PMNs in suspension by soluble stimuli, as determined by the generation of superoxide radicals (respiratory burst). FN (up to 100 micrograms/ml) did not directly stimulate the PMN respiratory burst assessed using a sensitive assay, luminol-dependent chemiluminescence (CL). Low FN concentrations (Up to 25 micrograms/ml) caused a dose-dependent enhancement of the CL induced by two chemoattractants. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet-activating factor (Paf), and also by phorbol myristate acetate (PMA), a known protein kinase C activator. Higher FN concentrations were less effective. The potentiation involved both initial rate and total CL responses and was more active on extracellular than intracellular generation of oxygen radicals. FN potentiation persisted after cell washing and was abolished by treatment of FN with trypsin. Measurement of the respiratory burst using the cytochrome c reduction assay confirmed that FN enhanced both the initial rate and total amount of superoxide anion generated by FMLP-stimulated PMNs. These data indicate that FN facilitates the respiratory burst of chemoattractant-stimulated PMNs and suggest that FN can prepare PMNs in suspension for amplified biological functions induced by soluble inflammatory stimuli.

Staurosporine, a protein kinase inhibitor, up-regulates the stimulation of human neutrophil respiratory burst by N-formyl peptides and platelet activating factor.

Staurosporine (STAR), a potent protein kinase C (PKC) antagonist, was found to modulate the chemoattractant-induced respiratory burst of human polymorphonuclear leukocytes (PMNs) according to drug concentration. Low STAR concentrations from 10 to 200 nM potentiated the N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet activating factor (Paf)-induced respiratory burst, affecting both the initial rate and the total amount of superoxide anion generated. The maximal increase occurred in the presence of 100 nM STAR and optimal fMLP concentration and reached 60-100% of control values. Above 250 nM, STAR inhibited the respiratory burst with an IC50 of 360 and 320 nM for fMLP and Paf, respectively. The respiratory burst induced by PKC activators such as phorbol myristate acetate or phorbol 12, 13 dibutyrate was inhibited effectively by STAR, with a low IC50 (25 nM) for both stimuli. Thus, the use of low STAR concentrations points to two possible roles of PKC in the regulation of NADPH oxidase activity, i.e. a positive regulation in phorbol ester-treated cells and a negative regulation in chemoattractant-stimulated PMNs.