Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil firm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium influx

Taxifolin has been reported to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1), a receptor-mediating firm adhesion with beta2 integrin (e.g., Mac-1) expressed on leukocytes. To evaluate whether taxifolin could modulate Mac-1-dependent firm adhesion by neutrophils, and the possible mechanism(s) underlying its anti-inflammatory action, its effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were studied. Pretreatment with taxifolin (1-100 microM) concentration-dependently diminished fMLP- or (PMA)-induced Mac-1-dependent firm adhesion and upexpression of surface Mac-1. Mobilisation of intracellular calcium and production of reactive oxygen species (ROS) signal the upexpression of Mac-1 and firm adhesion by neutrophils. Taxifolin impeded the calcium influx induced by fMLP (a receptor-mediated activator) or AlF(4)(-) (a G protein-mediated activator). Taxifolin also effectively inhibited the fMLP- or PMA-induced ROS production with 50% inhibitory concentration (IC(50)) less than 10microM, possibly through impairing the activation of NADPH oxidase, a major ROS-generating enzyme in neutrophils, by restricting the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC). In conclusion, we propose that impairment of ROS production by NADPH oxidase through interfering with p38 MAPK- and/or PKC-dependent signals, and antagonism of G protein-mediated calcium influx may account for the inhibition of Mac-1-dependent neutrophil firm adhesion that confers taxifolin the anti-inflammatory activity.     

Effective attenuation of acute lung injury in vivo and the formyl peptide-induced neutrophil activation in vitro by CYL-26z through the phosphoinositide 3-kinase gamma pathway

5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the polymorphonuclear leukocyte (PMNL) infiltration and protein leakage into the lungs in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as determined on the basis of PMNL and protein contents in bronchoalveolar lavage (BAL) fluid and myeloperoxidase (MPO) content in whole lung extracts. CYL-26z also attenuated the formyl-Met-Leu-Phe (fMLP)-induced neutrophil chemotaxis and respiratory burst in vitro (IC(50) 8.4+/-0.9microM and 2.0+/-0.6microM, respectively). CYL-26z had no effect on superoxide anion (O(2)(-)) generation during dihydroxyfumaric acid autoxidation or on the NADPH oxidase activity in two cell-free systems (the arachidonic acid-induced assembly of NADPH oxidase and the preassembled oxidase caused by phorbol ester treatment), whereas it inhibited NaF-induced respiratory burst. Inhibition of respiratory burst by CYL-26z was readily reversible by washing. Only slight, but significant, inhibition of extracellular signal regulated kinase (ERK) phosphorylation and p38 mitogen-activated protein kinase (MAPK) activation in response to fMLP by CYL-26z up to 30microM was obtained. CYL-26z effectively blocked the formation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) as determined by immunofluorescence microscopy and flow cytometry assays and the dual phosphorylation of protein kinase B (PKB/Akt) on S473 and T308 residues in fMLP-stimulated neutrophils. The membrane recruitment of p110gamma and Ras, the Ras activation, and the association between p110gamma and Ras were also attenuated by CYL-26z. These results indicate that the blockade of Ras activation by CYL-26z attenuated the downstream phosphoinositide 3-kinase (PI3K) gamma signaling, which is involved in chemoattractant-induced neutrophil chemotaxis and respiratory burst, and may have a beneficial anti-inflammatory effect on ALI.     

The effect of a brominated flame retardant, tetrabromobisphenol-A, on free radical formation in human neutrophil granulocytes: the involvement of the MAP kinase pathway and protein kinase C.

This study investigates the effects of one of the most frequently used brominated flame-retardants (BFR), tetrabromobisphenol-A (TBBPA), on formation of reactive oxygen species (ROS) and calcium levels in human neutrophil granulocytes. TBBPA enhanced ROS production in a concentration-depended manner (1-12 microM), measured as 2,7-dichlorofluorescein diacetate amplified (DCF) fluorescence. The results on ROS production by TBBPA was confirmed by lucigenin-amplified chemiluminescence. The TBBPA induced formation of ROS was due to activation of respiratory burst, as shown by the NADPH oxidase inhibitor DPI (10 microM). TBBPA induced activation of respiratory burst was also inhibited by the MEK 1/2 inhibitor U0126 (10 microM), the PKC inhibitor BIM (0.25 microM), and the tyrosine kinase inhibitor erbstatin-A (25 microM). We also found a small reduction in ROS formation in the absence of extracellular calcium and when verapamil was added. The phosphorylation of ERK 1/2 was confirmed by Western blotting. TBBPA also induced a concentration dependent increase in intracellular free calcium measured with Fura-2/AM. We suggest that exposure of human neutrophil granulocytes to the brominated flame retardant TBBPA leads to an activation of the NADPH oxidase primarily by an ERK 1/2 stimulated pathway. The data also show that PKC, calcium, and tyrosine kinases may be involved in the activation.     

The effect of putative protein kinase C inhibitors, K252a and staurosporine, on the human neutrophil respiratory burst activated by both receptor stimulation and post-receptor mechanisms.

1. Two compounds, reported to be potent inhibitors of protein kinase C (PKC), K252a and staurosporine, have been examined in order to gain further information as to the possible role played by PKC in the signal transduction sequence of the neutrophil respiratory burst as determined by superoxide (O2-) production. 2. A number of stimuli were used in the study, some acting at receptors i.e. fMet-Leu-Phe (fMLP), opsonized zymosan and heat-aggregated IgG (HAGG), one acting on a G-protein, fluoride, and two direct PKC activators, dioctanoylglycerol (diC8) and phorbol myristate acetate (PMA). 3. K252a and staurosporine inhibited the respiratory burst with all the stimuli but the order of agonist sensitivity was very different with the two inhibitors. 4. For K252a-induced inhibition of O2- release, the order of potency was fluoride greater than fMLP, HAGG greater than opsonized zymosan greater than PMA, DiC8. For staurosporine-induced inhibition of O2- release, the order of potency changed to fluoride greater than DiC8, PMA greater than HAGG, fMLP greater than opsonized zymosan. The significance of this unexpected difference in relative rank order of potency is discussed with reference to the reported mechanism of action of the two inhibitors and the events involved in the oxidative burst. 5. Staurosporine at low concentrations increased the fMLP-stimulated O2- response by 100%, the maximum effect occurring at 35 nM. 6. To the extent that the compounds used are specific inhibitors of PKC, these findings support a role for the enzyme PKC in stimulus-activation coupling in O2- generation with all the stimuli used in this study.     

Inhibition of protein kinase C mediated signal transduction by tamoxifen. Importance for antitumour activity

Recent studies have demonstrated tamoxifen inhibition of the enzyme protein kinase C (PKC) in vitro. The aim of this study was to investigate the effects of tamoxifen on PKC function in intact human cells. As PKC activates the neutrophil oxidase mechanism the neutrophil was chosen as an experimental model to assess PKC-tamoxifen interaction in these experiments. Neutrophils from healthy volunteers were separated by centrifugation through Ficoll Hypaque. Two separate parameters of oxidase activation; oxygen consumption and reactive oxygen metabolite production were monitored by a Clark electrode chamber and luminol dependent chemiluminescence respectively. Neutrophil chemiluminescence was markedly stimulated by 4 Phorbol-12 myristate-13 acetate (PMA). This stimulation was inhibited by tamoxifen; IC50 = 6.1 +/- 1.6 microM (means +/- S.E.M.) N = 6. Neutrophil oxygen consumption was similarly stimulated by PMA and inhibited by tamoxifen. The tamoxifen inhibition was not due to cell toxicity as assessment of cell integrity by the exclusion of trypan blue and measurement of intracellular concentrations of ATP showed no significant differences before and after treatment. Tamoxifen also inhibited neutrophil chemiluminescence which was stimulated by oleoyl acetyl glycerol and mezerein excluding interaction with PMA as an explanation of its inhibitory effect. These results are consistent with tamoxifen inhibition of PKC function in intact human cells. This may be central to its antitumour action.     

Clozapine prevents apoptosis and enhances receptor-dependent respiratory burst in human neutrophils

The present study was undertaken to determine if the antipsychotic drug clozapine (CLZ) in the concentration range 2-50 microM can rescue polymorphonuclear cells (PMNs) from undergoing apoptosis. Our results indicate that 20 microM CLZ can rescue PMNs both from UVB-accelerated (28.0% vs. 45.9% for control without CLZ; P < 0.05) and from spontaneous (35.8% vs. 57.6%; P < 0.05) apoptosis whereas 50 microM CLZ could rescue PMNs from spontaneous (34.3% vs. 57.6%; P < 0.05) apoptosis only. Furthermore, since apoptosis has been reported to involve the impairment of PMN function, we evaluated the effects of CLZ on respiratory burst in UVB-irradiated and in unirradiated PMNs. When 20 or 50 microM CLZ-pretreated PMNs were aged in a culture during 4 h, the luminol-dependent chemiluminescence (CL) response was 3-fold (P < 0.01) and 2.5-fold (P < 0.05) increased, respectively, by subsequent exposure to serum opsonized zymosan (OZ). When 50 microM-pretreated PMNs were either UVB-irradiated or unirradiated, the CL response was 2.6-fold (P < 0.05) and 3.3-fold (P < 0.05) increased, respectively, after subsequent exposure to formyl-methionyl-leucyl-phenylalanine (fMLP). In contrast, the degree of enhancement was negligible upon subsequent exposure to ionomycin or phorbol myristate acetate (PMA). When incubation times were extended up to 22 h, the CL response induced by OZ in 20 microM CLZ-treated PMNs had a 4.9-fold increase (P < 0.001). This priming effect could be reverted when 20 microM CLZ-treated PMNs (aged 4 h in culture) were coincubated for 5 min with the protein tyrosine kinase inhibitor genistein as well as with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin. These findings suggest that CLZ primes respiratory burst and prevents PMN apoptosis as a consequence of tyrosine phosphorylation- and PI3-K activation-dependent signal transduction pathways.     

Inhibition of superoxide anion generation by YC-1 in rat neutrophils through cyclic GMP-dependent and -independent mechanisms

3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a soluble guanylyl cyclase (sGC) activator, inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O(2)*(-)) generation and O(2) consumption in rat neutrophils (IC(50) values of 12.7+/-3.1 and 17.7+/-6.9 microM, respectively). Inhibition of O(2)*(-) generation by YC-1 was partially reversed by the cyclic GMP-lowering agent 6-anilinoquinoline-5,8-quinone (LY83583) and by the Rp isomer of 8-(4-chlorophenylthio)guanosine-3',5'-monophosphorothioate (Rp-8-pCPT-cGMPS), a cyclic GMP-dependent protein kinase inhibitor. In cell-free systems, YC-1 failed to alter O(2)*(-) generation during dihydroxyfumaric acid autoxidation, phorbol 12-myristate 13-acetate (PMA)-activated neutrophil particulate NADPH oxidase preparation, and arachidonic acid-induced NADPH oxidase activation. YC-1 increased cellular cyclic GMP levels through the activation of sGC and the inhibition of cyclic GMP-hydrolyzing phosphodiesterase activity. The plateau phase, but not the initial spike, of fMLP-induced [Ca(2+)](i) changes was inhibited by YC-1 (IC(50) about 15 microM). fMLP- but not PMA-induced phospholipase D activation was inhibited by YC-1 (IC(50) about 28 microM). Membrane-associated ADP-ribosylation factor and Rho A in cell activation was also reduced by YC-1 at a similar concentration range. Neither cytosolic protein kinase C (PKC) activity nor PKC membrane translocation was altered by YC-1. YC-1 did not affect either fMLP-induced phosphatidylinositol 3-kinase activation or p38 mitogen-activated protein kinase phosphorylation, but slightly attenuated the phosphorylation of extracellular signal-regulated kinase. Collectively, these results indicate that the inhibition of the fMLP-induced respiratory burst by YC-1 is mediated by cyclic GMP-dependent and -independent signaling mechanisms.     

Human dopamine D(3) receptors mediate mitogen-activated protein kinase activation via a phosphatidylinositol 3-kinase and an atypical protein kinase C-dependent mechanism.

The mitogen-activated protein kinase (MAPK) cascade is stimulated by both receptor tyrosine kinases and G protein-coupled receptors. We show that recombinant human dopamine D(3) receptors expressed in Chinese hamster ovary cells transiently activate MAPK via pertussis toxin-sensitive Gi and/or Go proteins. The involvement of D(3) receptors was confirmed by use of the D(3) agonists PD 128,907 and (+)-7-hydroxy-2-dipropylaminotetralin, which mimicked the response to dopamine (DA). Furthermore, haloperidol and the selective D(3) receptor antagonists S 14297 and GR 218,231 attenuated DA-induced MAPK activation; however, when tested alone, S 14297 weakly stimulated MAPK activity, suggesting partial agonist activity. The transduction mechanisms by which hD(3) receptors activate MAPK were explored with specific kinase inhibitors. Genistein and lavendustin A, inhibitors of tyrosine kinase activity, did not reduce DA-induced MAPK activation. In contrast, PD 98059, an inhibitor of MAPK kinase, and Ro 31-8220 and G? 6983, inhibitors of protein kinase C (PKC), blocked DA-induced MAPK activation. However, MAPK activation was insensitive to PKC down-regulation by phorbol esters, indicating the involvement of an "atypical" PKC. Furthermore, MAPK activation involved phosphatidylinositol 3-kinase inasmuch as its inhibition by LY 294002 and wortmannin reduced DA-induced MAPK activation. In conclusion, this study demonstrates that stimulation of hD(3) receptors activates MAPK. This action is mediated via an atypical isoform of PKC, possibly involving cross-talk with products of phosphatidylinositol 3-kinase activation.     

Daphnoretin-induced respiratory burst in rat neutrophils is,probably, mainly through protein kinase C activation

Daphnoretin, a dicoumarin isolated from Wikstroemia indica C.A. Mey. (Thymelaceae), induced superoxide anion (O₂⁻) formation in rat neutrophils in a concentration-dependent manner. Addition of staurosporine reduced daphnoretin-induced respiratory burst. Removal of extracellular free Ca²⁺ by EGTA did not affect the respiratory burst of neutrophils in response to daphnoretin. Prior exposure of neutrophils to phorbol 12-myristate 13-acetate (PMA) or daphnoretin reduced the O₂⁻ formation caused by a subsequent challenge with PMA and daphnoretin, but potentiated the response caused by a subsequent addition of formyl-Met-Leu-Phe (fMLP). Like PMA, daphnoretin did not increase the [Ca²⁺]_i during cell activation. In neutrophil suspension, daphnoretin increased the membrane associated protein kinase C activity. In the presence of Ca²⁺ and phosphatidylserine, daphnoretin also activated protein kinase C isolated from cytosolic fraction of resting neutrophils. Staurosporine inhibited the direct activation of protein kinase C caused by daphnoretin as well as by PMA. Daphnoretin reduced the [³H]Phorbol-12,13-dibutyrate ([³H]PDB) binding to the neutrophil cytosolic protein kinase C in a concentration-dependent manner with an IC₅₀ value of 1.77±0.37 μM. These results indicate that daphnoretin, like PMA, may direct activation of protein kinase C which in turn activated NADPH oxidase and elicited respiratory burst.     

Mechanism of N-formyl-methionyl-leucyl-phenylalanine- and platelet-activating factor-induced arachidonic acid release in guinea pig alveolar macrophages: involvement of a GTP-binding protein and role of protein kinase A and protein kinase C

Various pharmacological effectors were used to investigate the mechanism of arachidonic acid release by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) in guinea pig alveolar macrophages. The fMLP- and PAF-stimulated arachidonic acid release (i) was mimicked by sodium fluoride and inhibited by Bordetella pertussis toxin, suggesting the participation of a guanine nucleotide-binding protein; ii) was mimicked by A23187 but was insensitive to the calmodulin inhibitor R24571, making the involvement of a calmodulin-dependent pathway unlikely; and (iii) was mimicked by 12-O-tetra-decanoyl phorbol 13 acetate (TPA) and was, like the TPA-stimulated release, markedly decreased when protein kinase C (PKC) had been down-regulated by TPA (65% decrease) or inhibited by sphingosine, a diacylglycerol-competitive PKC inhibitor shown to completely abolish the enzyme activity from alveolar macrophages at 40 microM. Moreover, PAF and fMLP, under conditions where they stimulated arachidonic acid release, promoted an appreciable, albeit transient, translocation of PKC, suggesting a possible involvement of the enzyme in the agonist-stimulated process. However, staurosporine, another PKC inhibitor decreasing PKC activity from alveolar macrophages by 60% at 20 nM, failed to alter fMLP- and PAF-stimulated release. These data lead us to suggest that fMLP- and PAF-stimulated arachidonic acid release is mediated by mechanisms involving either a staurosporine-insensitive PKC isoform or a sphingosine-sensitive coupling between a pertussis toxin-sensitive guanine nucleotide-binding protein and phospholipase A2. Finally, the fMLP- and PAF-stimulated arachidonic acid release was inhibited by cholera toxin and was, like A23187-stimulated release, potentiated by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8), an exclusive protein kinase A inhibitor in alveolar macrophages, suggesting a negative regulation by protein kinase A.     

Phospholipase D-dependent and -independent p38MAPK activation pathways are required for superoxide production and chemotactic induction, respectively, in rat neutrophils stimulated by fMLP

Mitogen-activated protein kinase (MAPK)-mediated signal transduction pathways convert signals by extracellular stimulation into a variety of cellular functions. However, the roles of MAPKs in neutrophils are not well understood. To elucidate the temporal roles of p38MAPK during rat neutrophil activation stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), we examined the kinetics of this enzyme and the role of p38MAPK related to neutrophil functions (superoxide production and chemotaxis). SB203580, a potent and specific inhibitor of p38MAPK, significantly depressed both superoxide production and chemotaxis. Ethanol and 1-butanol, inhibitors of phospholipase D (PLD), suppressed p38MAPK activation in neutrophils under conditions (1 microM fMLP for 5 min) that stimulated superoxide production; and they significantly depressed superoxide production in rat neutrophils stimulated by fMLP. However, neither inhibitor had any effect on the activation of p38MAPK under the conditions (10 nM fMLP for 60 min) that gave optimal chemotaxis. These results indicate that multiple signaling pathways were involved in stimulating p38MAPK and that p38MAPK played different roles in regulating neutrophil function depending on the conditions for stimulation with fMLP. In addition, the activation of p38MAPK occurred dependent on or independent of PLD activation in neutrophils stimulated with fMLP.     

A commercial mixture of the brominated flame retardant pentabrominated diphenyl ether (DE-71) induces respiratory burst in human neutrophil granulocytes in vitro.

Polybrominated diphenyl ethers (PBDEs) are widely used brominated flame retardants (BFRs), which have become ubiquitous in the environment. This study investigates the effects of the pentabrominated diphenyl ether mixture, DE-71, on human neutrophil granulocytes in vitro. DE-71 enhanced production of reactive oxygen species (ROS) in a concentration-dependent manner measured as lucigenin-amplified chemiluminescence. Octabrominated diphenyl ether (OBDE), decabrominated diphenyl ether (DBDE), and the non-brominated diphenyl ether did not induce ROS formation at the concentrations tested. DPI (4 microM), an inhibitor of the NADPH oxidase completely inhibited DE-71 induced ROS formation, highlighting a role for NADPH oxidase activation. The protein kinase C inhibitor BIM (0.25 microM) and the selective chelator of intracellular calcium, BAPTA-AM (5 microM), also inhibited NADPH oxidase activation, indicating a calcium-dependent activation of PKC. ROS formation was also inhibited by the tyrosine kinase inhibitor tyrphostin (1 microM), the phospholipase C inhibitor ET-18-OCH3 (5 microM), and the phosphatidylinositol-3 kinase inhibitor LY294002 (25 microM). Alterations in intracellular calcium were measured using fura-2/AM, and a significant increase was measured after exposure to DE-71 both with and without extracellular calcium. The tetra brominated compound BDE-47 also enhanced ROS formation in a concentration dependent manner. The combination of DE-71 with the bacteria-derived N-formyl peptide fMLP and PCB153 induced an additive effect in the lucigenin assay. We suggest that tyrosine kinase mediated activation of PI3K could result in enhanced activation of calcium-dependent PKC by enhanced PLC activity, followed by intracellular calcium release leading to ROS formation in neutrophil granulocytes.     

Inhibitory effects of zafirlukast on respiratory bursts of human neutrophils

The effects of zafirlukast, a cysteinyl-leukotriene receptor antagonist, on the generation of the reactive oxygen species (ROS) released during respiratory bursts of human polymorphonuclear neutrophils (PMNs) is still unknown. The aim of this study was to investigate the ability of zafirlukast to interfere with the respiratory burst of PMNs. Respiratory burst responses of PMNs were investigated by luminol-amplified chemiluminescence (LACL) using particulate (Candida albicans and zymosan) and soluble stimulants [N-formyl-methionylleucyl-phenylalanine (fMLP) and phorbol 12 myristate 13 acetate (PMA)]. When incubated with PMNs for 10 min at concentrations ranging from 5 x 10(-9) M to 5 x 10(-6) M, zafirlukast did not significantly affect the respiratory bursts of PMNs induced by either the particulate or soluble stimuli. However, after incubation for 60 min, it did reduce the respiratory bursts of PMNs in a concentration-related fashion when the PMNs were stimulated with fMLP, and at a concentration of 5 x 10(-6) M when the stimulus was PMA. No significant effects were seen when the PMNs were challenged with particulate stimuli. Zafirlukast is able to interfere with the activation of the PMNs respiratory burst induced by soluble stimulants. The different behavior determined by different times of contact and different stimuli opens the way to interpretations concerning the antioxidant effect of zafirlukast.     

Signal response transduction in rabbit neutrophil leucocytes. The effects of exogenous phospholipase A2 suggest two pathways exist

Rabbit neutrophils stimulated by chemotactic peptide (fMLP) or phorbol ester (PMA) respond with a metabolic burst which can be assayed by following luminol-enhanced chemiluminescence. Depending upon the agonist used, exogenous bee-venom phospholipase A2 (PLA2) will enhance or inhibit the response. Neutrophil activation by fMLP is enhanced by PLA2 or by the addition of arachidonic acid, but unaffected by lysophosphatide. The cellular response to PMA is markedly inhibited by PLA2 or by lysophosphatide, though not completely abrogated, but is enhanced by arachidonic acid. The lysophosphatide inhibition overrides the arachidonic acid potentiation of the PMA-induced response. Neither PLA2 nor arachidonic acid alone will activate the cells; it seems that agonist is essential. We interpret these results to mean that at least two signal-response transduction systems are involved in agonist-induced metabolic activation of rabbit neutrophil leucocytes.     

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.     

Staurosporine inhibits protein kinase C and prevents phorbol ester-mediated leukotriene D4 receptor desensitization in RBL-1 cells

The aim of the present study was to investigate the effects of staurosporine on phorbol-myristate acetate (PMA)-induced activation of protein kinase C (PKC) and the desensitization of leukotriene D4 (LTD4)-stimulated Ca2+ mobilization in rat basophilic leukemia (RBL-1) cells. Staurosporine, one of the most potent PKC inhibitors known to date, markedly inhibited partially purified PKC from RBL-1 cells with an IC50 of 3 nM. Exposure of RBL-1 cells to PMA resulted in inhibition of LTD4-stimulated Ca2+ mobilization. However, prior treatment of the cells with staurosporine completely prevented PMA-induced desensitization of LTD4-stimulated Ca2+ mobilization. This reversal of Ca2+ desensitization by staurosporine was dose dependent with an IC50 of 0.1 microM. Treatment of RBL-1 cells with PMA resulted in translocation and activation of PKC from the cytosol to the membrane fraction. Pretreatment of RBL-1 cells with staurosporine inhibited the PMA-induced activation of PKC in the membrane fraction. The inhibition of PKC activity by staurosporine was time and dose dependent with an IC50 of 0.9 microM. These results show that PMA-induced heterologous desensitization is mediated by PKC and staurosporine prevented this process by directly inhibiting PKC in intact RBL-1 cells.     

Activation of phosphoinositide 3-kinase, protein kinase C, and extracellular signal-regulated kinase is required for oridonin-enhanced phagocytosis of apoptotic bodies in human macrophage-like U937 cells

Our previous study showed that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic cells by macrophage-like U937 cells through tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta release. In this study, we further investigated signaling events involved in oridonin-augmented phagocytosis. Phagocytic stimulation was significantly suppressed by inhibitors, including a phosphoinositide 3-kinases (PI3K) inhibitor (wortmannin), a protein kinase C (PKC) inhibitor (stauroporine), and a phospholipase C (PLC) inhibitor (U73122). Exposure of U937 cells to oridonin caused an increase in PKC activity time- dependently, which was prevented by pretreatment with inhibitors of PI3K and PLC. Simultaneously, the activation of protein kinase B (PKB/Akt) and the increased expression of PLCgamma2 were also blocked by wortmannin. In addition, an extracellular signal-regulated kinase (ERK) MAPK inhibitor, PD98059, suppressed oridonin-augmented phagocytosis, whereas the p38 MAPK inhibitor (SB203580) and c-Jun N-terminal kinase (JNK) MAPK inhibitor (SP98059) had no inhibitory effect. Furthermore, pretreatment of U937 cells with anti-TNFalpha and anti-IL-1beta antibodies blocked oridonin-induced phagocytic stimulation as well as phosphorylation of ERK, but did not block the activation of PKC, indicating that these signaling events are triggered by oridonin, whereas secreted TNFalpha or IL-1beta only activate the ERK-dependent pathway. Taken together, oridonin is suggested to enhance phagocytosis of apoptotic bodies by activating PI3K, PKC, and ERK-dependent pathways.     

YC-1 increases cyclo-oxygenase-2 expression through protein kinase G- and p44/42 mitogen-activated protein kinase-dependent pathways in A549 cells

YC-1, an activator of soluble guanylate cyclase (sGC), has been shown to increase the intracellular cGMP concentration. This study was designed to investigate the signaling pathway involved in the YC-1-induced COX-2 expression in A549 cells. YC-1 caused a concentration- and time-dependent increase in COX activity and COX-2 expression in A549 cells. Pretreatment of the cells with the sGC inhibitor (ODQ), the protein kinase G (PKG) inhibitor (KT-5823), and the PKC inhibitors (Go 6976 and GF10923X), attenuated the YC-1-induced increase in COX activity and COX-2 expression. Exposure of A549 cells to YC-1 caused an increase in PKC activity; this effect was inhibited by ODQ, KT-5823 or Go 6976. Western blot analyses showed that PKC-alpha, -iota, -lambda, -zeta and -mu isoforms were detected in A549 cells. Treatment of A549 cells with YC-1 or PMA caused a translocation of PKC-alpha, but not other isoforms, from the cytosol to the membrane fraction. Long-term (24 h) treatment of A549 cells with PMA down-regulated the PKC-alpha. The MEK inhibitor, PD 98059 (10 - 50 microM), concentration-dependently attenuated the YC-1-induced increases in COX activity and COX-2 expression. Treatment of A549 cells with YC-1 caused an activation of p44/42 MAPK; this effect was inhibited by KT-5823, Go 6976, long-term (24 h) PMA treatment or PD98059, but not the p38 MAPK inhibitor, SB 203580. These results indicate that in human pulmonary epithelial cells, YC-1 might activate PKG through an upstream sGC/cGMP pathway to elicit PKC-alpha activation, which in turn, initiates p44/42 MAPK activation, and finally induces COX-2 expression.     

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts

Bradykinin (1 μM 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 μM) to cells preincubated with [³H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5–10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg⁹-bradykinin (1 μM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE₂) in MC3T3-EI cells. This stimulatory effect was seen after 15–20 min incubation. The Ca²⁺ ionophore A23187 at and above 1 μM induced a rapid (within seconds) burst of PGE₂ formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE₂ formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. In conclusion: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.     

Cellular mechanisms of inhibition of superoxide anion generation in rat neutrophils by the synthetic isoquinoline DMDI.

This study was undertaken to assess the cellular localization of the inhibitory effect of a chemically synthetic isoquinoline compound 1-(3',4'-dimethoxybenzyl)-6,7-dichloroisoquinoline (DMDI) on the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils. The DMDI concentration dependently inhibited the superoxide anion (O(2)(*-)) generation and O(2) consumption (IC(50) 12.2+/-4.9 and 15.2+/-8.4 microM, respectively) of neutrophils. DMDI did not scavenge the O(2)(*-) generated during the autoxidation of dihydroxyfumaric acid in a cell-free system. DMDI did not elevate cellular cyclic AMP levels. Inhibition of O(2)(*-) generation by DMDI in neutrophils was not reversed by a cyclic AMP-dependent protein kinase inhibitor, (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5720). The DMDI concentration dependently inhibited the late plateau phase but not the initial spike of fMLP-induced [Ca(2+)](i) changes in the presence of extracellular Ca(2+). However, DMDI had no effect on the fMLP-induced [Ca(2+)](i) changes in the absence of extracellular Ca(2+). In addition, DMDI did not affect the fMLP-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activation. DMDI produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol in the presence of ethanol from fMLP-stimulated neutrophils (IC(50) 13.3+/-4.0 and 9.4+/-4.3 microM, respectively). On the basis of the immunoblot analysis of the phosphorylation of the mitogen-activated protein (MAP) kinase, DMDI attenuated the fMLP-stimulated MAP kinase phosphorylation in a similar concentration range. Collectively, these results indicate that the inhibition of the respiratory burst by DMDI in rat neutrophils is mediated through the blockade of phospholipase D and MAP kinase signaling pathways.