The signal transduction mechanism involved in kazinol B-stimulated superoxide anion generation in rat neutrophils

1. In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated.
2. Kazinol B concentration-dependently stimulated the superoxide anion (O₂[dot over 2]⁻) generation, with a lag but transient activation profile, in neutrophils but not in a cell-free system. The maximum response (13.2±1.4 nmol O₂[dot over 2]⁻ 10 min⁻¹ per 10⁶ cells) was observed at 10 μM kazinol B.
3. Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP) significantly enhanced the O₂[dot over 2]⁻ generation following the subsequent stimulation of cells with kazinol B.
4. Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B-induced O₂[dot over 2]⁻ generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response.
5. Kazinol B significantly stimulated [Ca²⁺]_i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP₂ and IP₃) formation with a 1 min lag time.
6. The membrane-associated PKC-α and PKC-θ but not PKC-ι were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-θ than PKC-α by kazinol B. Kazinol B partially inhibited the [³H]phorbol 12,13-dibutyrate ([³H]PDB) binding to the neutrophil cytosolic PKC.
7. Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B-induced O₂[dot over 2]⁻ generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein.
8. Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca²⁺]_i elevation in rat neutrophils.

     

Inhibition of formyl peptide-stimulated phospholipase D activation by Fal-002-2 via blockade of the Arf6, RhoA and protein kinase C signaling pathways in rat neutrophils

Three recently developed selective phospholipase D (PLD) inhibitors N-(2-(4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)-2-naphthamide (VU0155056), (S)-N-(1-(4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)propan-2-yl)-2-naphthamide (VU0155069), and N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]decan-8-yl)ethyl)quinoline-3-carboxamide (VU0285655-1) inhibited O₂ ^?? generation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel 2-phenyl-4-quinolone compound 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), which inhibited O₂ ^?? generation, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC₅₀ 16.0?±?5.0 μM). Fal-002-2 attenuated the interaction of PLD1 with ADP-ribosylation factor (Arf) 6, Ras homology (Rho) A and protein kinase C (PKC) isoforms (α, βI, and βII), and also inhibited the membrane recruitment of Arf6 and RhoA in fMLP-stimulated neutrophils, but not in GTPγS-stimulated cell-free system. The cellular levels of GTP-bound Arf6 and GTP-bound RhoA were reduced by Fal-002-2. Fal-002-2 also attenuated the membrane recruitment of Rho-associated protein kinase 1, phosphorylation of myosin light chain 2 at Thr18/Ser19 and PLD1 at Thr147, and the interaction of Arf6 with both arfaptin 1 and phosphatidylinositol 4-phosphate 5-kinase 1A. The association between RhoA and Vav, the interaction of Vav with both Lyn and Lck, the membrane recruitment of Vav, and the phosphorylation of Vav at Tyr174, but not Src family at Tyr416, were all attenuated by Fal-002-2 in fMLP-stimulated neutrophils. These results indicate that Fal-002-2 is not a direct PLD inhibitor, but the inhibition of fMLP-stimulated PLD activity by Fal-002-2, which partly accounts for its suppression of O₂ ^?? generation, is attributable to the blockade of both Arf6 and RhoA activation and attenuation of the interaction of Arf6, RhoA and PKC isoforms with PLD1 in rat neutrophils.     

Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils

1. The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated.
2. Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O₂^.−) generation with IC₅₀ values of 0.33±0.05 μg ml⁻¹ and 0.49±0.04 μg ml⁻¹, respectively.
3. Abruquinone A also inhibited O₂ consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O₂^.− in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation.
4. Abruquinone A inhibited both the transient elevation of [Ca²⁺]_i in the absence of [Ca²⁺]_o (IC₅₀ 7.8±0.2 μg ml⁻¹) and the generation of inositol trisphosphate (IP₃) (IC₅₀ 10.6±2.0 μg ml⁻¹) in response to fMLP.
5. Abruquinone A did not affect the enzyme activities of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA).
6. Abruquinone A had no effect on intracellular guanosine 3′ : 5′-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) levels.
7. The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/CB was inhibited by abruquinone A with IC₅₀ values of 2.2±0.6 μg ml⁻¹ and 2.5±0.3 μg ml⁻¹, respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73–78 kDa in activated neutrophils.
8. Abruquinone A inhibited both the O₂^.− generation in PMA-activated neutrophil particulate NADPH oxidase (IC₅₀ 0.6±0.1 μg ml⁻¹) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC₅₀ 1.5±0.2 μg ml⁻¹).
9. Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.

     

Effects of Diethyldithiocarbamate on Activating Mechanisms of Neutrophils

The effects of diethyldithiocarbamate on superoxide release by rat neurophils were investigated in a chemiluminescence study. Diethyldithiocarbamate augmented lucigenin-dependent chemiluminescence in a concentration-dependent manner and inhibited luminol-dependent chemiluminescence at concentrations of 0.1-1 μM. In contrast, after the addition of 0.1 mM diethyldithiocarbamate, the chemiluminescence was markedly enhanced. Diethyldithiocarbamate inhibited both the myeloperoxidase activity of neutrophils and the chemiluminescence generated in a cell-free horseradish peroxidase/H₂O₂ and H₂O₂/HOCl system. The increase in lucigenin-dependent chemiluminescence brought about by diethyldithiocarbamate was inhibited by H-7, ML-7, W-7, EGTA and pertussis toxin. These results suggest that diethyldithiocarbamate may stimulate O₂^? production by a guanosine 5′-triphosphate protein-mediated and Ca²⁺–dependent process and that the increase in O₂^? release by neutrophils may be dependent not only on the direct stimulation of the signal transduction pathway but also on the increase in O₂^? by reducing the effect of the hydroperoxide (H₂O₂)-myeloperoxidase system.     

Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O₂⁻ generation and cell migration but not degranulation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O₂⁻ generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC₅₀ 3.9 ± 1.2 μM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC) α, βI and βII isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca²⁺-dependent PKC in rat neutrophils.     

Modulation of immunoreactive protein kinase C-α and β isoforms and G proteins by acute and chronic treatments with morphine and other opiate drugs in rat brain

The abundance of protein kinase C-α and β isoforms (PKC-αβ), PKC-α messenger (m) RNA and guanine nucleotide-binding G protein subunits (Gα_i1/2, Gα_o and Gβ) were quantitated in the rat cerebral cortex after acute and chronic treatments with various opiate drugs. Acute (100mg/kg for 2h) and chronic (10 to 100mg/kg for 5 days) treatment with morphine decreased similarly the immunoreactivity of PKC-αβ (28% and 32%, respectively). Acute (2h) and chronic treatment (5 days) with other μ-agonists heroin (30mg/kg and 10 to 30mg/kg) and methadone (30mg/kg and 5 to 30mg/kg) also induced similar decreases of PKC-αβ (acute: 25% and 23%; chronic: 28% and 18%). After the chronic treatments, spontaneous (48h) or naloxone (2mg/kg)-precipitated opiate withdrawal (2h) resulted in up-regulation of PKC-αβ above control levels (30-38%), and in the case of morphine withdrawal in a concomitant marked increase in the expression of PKC-α mRNA levels (2.3-fold). Acute (2h) treatments with pentazocine (80mg/kg, mixed κ/δ-agonist and μ-antagonist), spiradoline (30mg/kg, selective κ-agonist) and [D-Pen², D-Pen⁵] enkephalin (14nmol i.c.v., selective δ-agonist) induced significant decreases of PKC-αβ (19–33%). Chronic (5 days) treatment with pentazocine (10 to 80mg/kg), but not spiradoline (2 to 30mg/kg), also induced a similar decrease of PKC-αβ (35%). In pentazocine- or spiradoline-dependent rats, naloxone (2mg/kg) did not induce up-regulation of brain PKC-αβ. Acute (10mg/kg for 2h) and chronic (2×10mg/kg for 5 and 14 days) treatment with naloxone did not alter PKC-αβ immunoreactivity. Chronic, but not acute, treatment with μ-agonists (morphine, heroin and methadone) increased the immunoreactivities of Gα_i1/2 (33-37%), Gα_o (25-41%) and Gβ (10-33%) protein subunits. In heroin- and methadone-dependent rats naloxone (2mg/kg)-precipitated withdrawal (2h) did not modify the up-regulation of these G proteins induced by chronic μ-opiate treatment. In marked contrast to μ-agonists, chronic treatment with high doses of pentazocine and spiradoline or acute treatment with [D-Pen², D-Pen⁵] enkephalin did not result in up-regulation of these G protein subunits. After chronic treatment with μ-agonists, significant negative correlations were found when the percentage changes in immunoreactivity of PKC-αβ were related to the percentage changes in immunoreactivity of Gα_i1/2 (r =-0.53, n = 29) and Gβ (r =-0.41, n = 24) in the same brains. PKC-αβ abundance did not correlate significantly with the density of Gα_o (r =-0.21, n = 28). Together the results indicate that the brain PKC-αβ system may play a major regulatory role in opiate tolerance and dependence. Moreover, the possible in vivo cross-communication between this regulatory enzyme and specific inhibitory G proteins may also be of relevance in the cellular and molecular processes of opiate addiction. Received: 15 July 1996 / Accepted: 22 November 1996     

Effect of the protein kinase C inhibitor GF 109 203X on elastase release and respiratory burst of human neutrophils

• 1.1. The effects of bisindolylmaleimide GF 109 203X, reported to be a potent and highly selective inhibitor of protein kinase C (PKC), have been investigated on some human neutrophil functions.
• 2.2. GF 109 203X prevented O₂⁻ production by NADPH-oxidase whatever the stimulus used for polymorphonuclear neutrophil (PMN) activation: directs PKC activators like phorbol myristate acetate (PMA) and dioctanoylglycerol, calcium ionophore (A23187), or receptor agonists like fMet-Leu-Phe (fMLP) and opsonized zymosan.
• 3.3. The effect of GF 109 203X was also examined on elastase exocytosis by neutrophils. PMA-mediated elastase release was prevented by GF 109 203X. However, GF 109 203X had no effect on exocytosis induced by A23187 and the effect of this compound on the fMLP response changed according to its concentration.
• 4.4. These data suggest that PKC might be essential for stimulus-mediated O₂⁻ production and also that PKC plays only a minor role in elastase secretion as compared to the role of the cytosolic calcium level.

     

Existence of a threshold for hydroxyl radical generation independent of hypoxia in rat striatum during carbon monoxide poisoning

We examined the role of hypoxia in the carbon monoxide (CO)-induced generation of the hydroxyl radical (^•OH) in the striatum, which could contribute to brain damage due to CO poisoning. Exposure of free-moving rats to 1,000 and 3,000 ppm CO or 8 and 5% O₂ for 40 min caused concentration-dependent hypoxic conditions in terms of carboxyhemoglobin (COHb), deoxyhemoglobin, oxyhemoglobin, and O₂ contents in arterial blood. The hypoxic conditions seemed comparable between 3,000 ppm CO and 5% O₂, although alterations of pH and partial O₂ pressure (PO₂) were complex and concentration independent. In the striatum, CO and low O₂ decreased tissue PO₂ levels in a concentration-dependent and concentration-independent manner, respectively, but levels at the end of exposure were comparable among all groups. This was also the case for the increase in striatal blood flow. Although the increases in extracellular glutamate (excitatory), taurine (inhibitory), and alanine (non-neurotransmitter), in the striatum in response to CO and low O₂ were complex, 3,000 ppm CO and 5% O₂ had comparable effects. Thus, 3,000 ppm CO and 5% O₂ seemed to induce comparable hypoxic conditions. Nevertheless, the former more strongly stimulated ^•OH generation in the striatum than the latter. In addition, in contrast to low O₂ which caused a concentration-dependent increase in ^•OH, 1,000 ppm CO had no effect. The findings suggest that striatal ^•OH generation due to CO poisoning may be independent of hypoxia per se and that a threshold might exist.     

Effect of bolesatine on phospholipid/calcium dependent protein kinase in vero cells and in rat thymus

 Bolesatine, a glycoprotein from Boletus satanas Lenz, has previously been shown to be mitogenic to rat and human lymphocytes at very low concentrations, whereas higher concentrations inhibit protein synthesis in vitro and in several in vivo systems. The mechanism whereby this mitogenic activity occurs was previously unknown. To elucidate this mechanism, the effects of bolesatine have been studied in a cell-free system, VERO cells in culture, and in rat thymus. Bolesatine was found to activate PKC, in vitro (cell free system), in VERO cells, and in vivo in rat thymus. In a cell-free system, bolesatine appears to be a direct effector of PKC. The activation is concentration dependent for 1 – 10 ng/ml. At the same time, VERO cells significantly proliferate when incubated with bolesatine (3, 5 and 10 ng/ml), since the DNA synthesis increases by 27, 48 and 59%, for respectively, 3, 5 and 10 ng/ml compared with control. Moreover, Bolesatine (5 and 10 ng/ml) induces InsP₃ release in a concentration-dependent manner (114 and 142%) as compared to control. In vivo, 24 h after oral administration of bolesatine to rats (20, 100 and 200 μg/kg), PKC activity is significantly increased in thymus. The most effective doses (100 and 200 μg/kg) give 590 – 620% increase in cytosolic PKC activity and 85 – 91% increase in total PKC activity as compared to control. This PKC activation by bolesatine in rat thymus is directly linked to the mitogenic activity observed in vivo. Bolesatine is thus capable of activating the PKC directly and/or indirectly (via InsP₃ release) during its mitogenic process. Received: 6 February 1995 / Accepted: 20 March 1995     

Zinc hydroxide induced respiratory burst in rat neutrophils

The effects of zinc hydroxide on the respiratory burst and phagocytosis by rat neutrophils were examined. Zinc hydroxide induced an increase in oxygen consumption and O₂⁻ production. Electronmicroscopy showed that neutrophils engulfed zinc hydroxide particles by phagocytosis. Pertussis toxin (0.25, 0.5, 1.0 μ/ml) and EGTA (1, 2, 5 mM) inhibited zinc hydroxide-induced O₂⁻ production in a dose-dependent manner. The inhibitors of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide inhibited zinc hydroxide-induced O₂⁻ production with IC₅₀ values ranging between 10 μM and 25 μM. The inhibitory study using an inhibitor of myosin light chain kinase, 1-(5-iodonapthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine, showed IC₅₀ values ranging from 5 μM to 10 μM. These findings indicate that zinc hydroxide induces respiratory burst and phagocytosis by rat neutrophils.     

Effects of NZ-107 on Airway Inflammation and Cell Activation in Guinea-pigs

Abstract— The effects of NZ-107 on some airway inflammation models and the generation of superoxide anion (O₂^?) were studied in guinea-pigs. Airway inflammation was caused by intra-tracheal injection of murine recombinant interleukin-5 (mrIL-5, 15 μg/animal), inhalation of platelet-activating factor (PAF, 0·003%) and intra-tracheal injection of leukotriene B₄ (LTB₄, 10 μg/animal). NZ-107 (4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone) at a dose of 50 mg kg^?1, intraperitoneally reduced mrIL-5- and PAF-induced eosinophilia. This compound at a dose of 25 and 50 mg kg^?1 also suppressed LTB₄-induced eosinophilia and neutrophilia in bronchoalveolar lavage fluid (BALF). On the other hand, prednisolone at a dose of 20 mg kg^?1, i.p., prevented the increased number of macrophages, eosinophils and neutrophils induced by mrIL-5, the increased number of eosinophils induced by PAF and the increased number of eosinophils and neutrophils induced by LTB₄ in BALF. Furthermore, both drugs reduced mrIL-5- or PAF-induced increase in the number of airway epithelial cells in BALF. The generation of O₂^? was measured by the method of cytochrome C reduction. NZ-107 (10–100 μg mL^?1) attenuated PAF- and FMLP-induced O₂^? production from macrophages and reduced PAF-induced O₂^? generation by eosinophils but had no effect on that from neutrophils. These results indicate that NZ-107 prevents the increased number of pulmonary eosinophils and airway epithelial cells and the activation of macrophages and eosinophils, suggesting that NZ-107 may be useful as a remedy for airway inflammatory diseases such as bronchial asthma.     

The cGMP Modulator,LY83583 Alters Oxygen Metabolites Differently in Cultured Endothelial Cells and Isolated Neutrophilic Granulocytes

Abstract: The present study was designed to assess the effect of LY83583 on H₂O₂/O₂^- production from endothelial cells and neturophils, as determined by chemiluminiscence generation in vitro. We found that LY83583 increased H₂O₂/O₂^-production from endothelial cells, but inhibited the H₂O₂/O₂^- production from phorbol myristate acetate-stimulated neutrophils. Furthermore, LY83583 consumed NADPH under certain conditions. Since neutrophils generate superoxide anion radicals via an NADPH-oxidase, we suggest that the reduction of chemiluminiscence, seen after addition of LY83583 to phorbol myristate acetate-stimulated neutrophils, is due to increased consumption of NADPH. In endothelial cells, NADPH is required as a co-factor in the generation of nitric oxide, which may interact with superoxide anion. A consumption in NAPDH would therefore be expected to decrease the production of nitric oxide and increase H₂O₂/O₂^- generation. The consumption of NADPH in endothelial cells could also cause reduced scavenger functions of the glutathion system, resulting in a further increase in H₂O₂ release.     

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.     

The hederagenin saponin SMG-1 is a natural FMLP receptor inhibitor that suppresses human neutrophil activation

The pericarp of Sapindus mukorossi Gaertn is traditionally used as an expectorant in Japan, China, and Taiwan. Activated neutrophils produce high concentrations of the superoxide anion (O₂⁻) and elastase known to be involved in airway mucus hypersecretion. In the present study, the anti-inflammatory functions of hederagenin 3-O-(3,4-O-di-acetyl-α-l-arabinopyranoside)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (SMG-1), a saponin isolated from S. mukorossi, and its underlying mechanisms were investigated in human neutrophils. SMG-1 potently and concentration-dependently inhibited O₂⁻ generation and elastase release in N-Formyl-Met-Leu-Phe (FMLP)-activated human neutrophils. Furthermore, SMG-1 reduced membrane-associated p47^phox expression in FMLP-induced intact neutrophils, but did not alter subcellular NADPH oxidase activity in reconstituted systems. SMG-1 attenuated FMLP-induced increase of cytosolic calcium concentration and phosphorylation of p38 MAPK, ERK, JNK, and AKT. However, SMG-1 displayed no effect on cellular cAMP levels and activity of adenylate cyclase and phosphodiesterase. Significantly, receptor-binding analysis showed that SMG-1 inhibited FMLP binding to its receptor in a concentration-dependent manner. In contrast, neither phorbol myristate acetate-induced O₂⁻ generation and MAPKs activation nor thapsigargin-caused calcium mobilization was altered by SMG-1. Taken together, our results demonstrate that SMG-1 is a natural inhibitor of the FMLP receptor, which may have the potential to be developed into a useful new therapeutic agent for treating neutrophilic inflammatory diseases.     

白屈菜红碱逆转不同浓度葡萄糖培养的乳鼠心肌细胞肥大及其相关机制的探讨

 研究白屈菜红碱对不同浓度葡萄糖培养的乳鼠心肌细胞肥大的作用,并进一步探讨其相关的作用机制。通过建立乳鼠心肌细胞培养模型,随机分成对照组（5 mmol·L^-1）、高糖组（10、15、20及25.5 mmol·L^-1）、高糖（25.5 mmol·L^-1）+ 不同浓度白屈菜红碱（1及8 μmol·L^-1）组、对照（5 mmol·L^-1）+ 不同浓度白屈菜红碱 (1及8 μmol·L^-1) 组,分别测定各组心肌细胞直径和蛋白质含量,并应用Western blotting检测心肌细胞蛋白激酶C ( protein kinase C,PKC )-α、PKC-β₂的表达及其磷酸化水平。结果表明,高糖培养组心肌细胞直径、蛋白质含量以及PKC-α、PKC-β₂的表达和磷酸化水平均高于对照组,并呈现浓度依赖性; 而分别在对照组和高糖组 ( 25.5 mmol·L^-1）加入不同浓度的白屈菜红碱后,心肌细胞直径、蛋白质含量、PKC-α、PKC-β₂的磷酸化水平以及PKC-α的总表达水平均降低,并且这种效应随着白屈菜红碱浓度的增高而增强; 但在对照+白屈菜红碱 ( 1 μmol·L^-1) 组, PKC-β₂的总表达水平并没有出现显著性降低。本研究表明白屈菜红碱可逆转葡萄糖诱导的乳鼠心肌细胞肥大, 对高糖环境中的心肌细胞具有保护作用,其机制可能与PKC途径有关。     

Effects of Calcium Blockers on the Cytosolic Calcium,H2O2 Production and Elastase Release in Human Neutrophils

Abstract: Activated neutrophils are assumed to be one plausible cause of tissue injury in the ischaemic and reperfused myocardium. We studied the inhibitory effects of the calcium antagonists felodipine, nimodipine and verapamil on human neutrophil activation in order to elucidate the mechanisms underlying their myocardioprotective effects and to determine whether calcium antagonists with different chemical structures vary in their effect on neutrophil activation. Neutrophils were stimulated with formyl-Met-Leu-Phe (0.1 μM) or by phorbol myristate acetate (0.16 μM), and the rise in cytosolic calcium and the H₂O₂ production were determined. For felodipine, the inhibitory effect on granulocyte elastase release was also studied. The calcium antagonists reduced formyl-Met-Leu-Phe and phorbol myristate acetate-induced neutrophil activation in a concentration-dependent manner, the order of potency being: felodipine >nimodipine >verapamil. For felodipine, the IC₅₀ (concentration causing 50% reduction) values were 3×10^?6 and 2×10^?6 M for the formyl-Met-Leu-Phe-induced cytosolic calcium increase and H₂O₂ production, respectively. The IC₅₀ -value for the phorbol myristate acetate-induced cytosolic calcium increase was 6×10^?6 and for H₂O₂ production 4×10^?6 M. For formyl-Met-Leu-Phe-induced granulocyte elastase release, the IC₅₀-value was 5×10^?6 M. The inhibitory effect of felodipine on the phorbol myristate acetate-induced granulocyte elastase release did not exceed 50%. Nimodipine was a less potent inhibitor than felodipine for both formyl-Met-Leu-Phe- and phorbol myristate acetate-induced cell activities. Verapamil was even less potent than the other two agents. The present study demonstrates that felodipine potentially suppresses neutrophil activation at micromolar concentrations. However, this observation should not be directly extrapolated to explain the tissue protection by the compounds without evidence of profound local accumulation.     

Effects of Zinc on Production of Active Oxygen Species by Rat Neutrophils

Abstract: The effects of zinc on the production of active oxygen species were investigated in rat neutrophils by chemilumi-nescence and spectrophotometric assays. The luminol-dependent chemiluminescence in unstimulated neutrophils showed a single peak. Zinc at concentrations lower than 0.1 mM augmented the intensity of chemiluminescence and showed a bimodal pattern, the first peak of which was inhibited by superoxide dismutase and catalase, while the second peak disappeared in the presence of catalase, but was unaffected by superoxide dismutase. At the same concentrations of zinc, O₂^? and H₂O₂ production increased, but secretion and activity of myeloperoxidase were not affected. Zinc at 0.1 mM enhanced the second peak of luminol-dependent chemiluminescence, and concomitantly O₂^? and H₂O₂ production of neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine. Homogenized neutrophils showed a bimodal pattern on induction by zinc, the second peak of which was inhibited slightly by catalase and completely by sodium azide, but was not inhibited by superoxide dismutase. Zinc-induced O₂^? production was inhibited by pertussis toxin, but was not significantly inhibited by a protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), or a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). These results suggest that zinc can augment luminol-dependent chemiluminescence by increasing O₂^? production through the classical signal transduction pathway, and by increasing H₂O₂ not via O₂^?.     

Manganese promotes increased formation of hydrogen peroxide by activated human macrophages and neutrophils in vitro

Although pro-inflammatory mechanisms have been implicated in the pathogenesis of manganese (Mn²⁺)-related neurological and respiratory disorders, relatively little is known about the potential of this metal to interact pro-oxidatively with human phagocytes. The primary objective of the current study was to investigate the effects of Mn²⁺ as MnCl₂ (0.5–100 µM) on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide (H₂O₂), and hypohalous acids by isolated human blood neutrophils and monocyte-derived macrophages following activation of these cells with the chemotactic tripeptide, FMLP (1 µM), or the phorbol ester, PMA (25?ng/mL). Generation of ROS was measured using the combination of oxygen consumption, lucigenin/luminol-enhanced chemiluminescence, spectrofluorimetric detection of oxidation of 2,7-dichlorodihydrofluorescein, radiometric assessment of myeloperoxidase (MPO)-mediated protein iodination, release of MPO by ELISA, and spectrophotometric measurement of nitrite formation. Treatment of activated neutrophils with either FMLP or PMA resulted in significantly decreased reactivity of superoxide in the setting of increased formation of H₂O₂ and MPO-mediated iodination, with no detectable effects on either oxygen consumption or MPO release. Similar effects of the metal with respect to superoxide reactivity and H₂O₂ formation were observed with activated macrophages, while generation of NO was unaffected. Taken together with the findings of experiments using cell-free ROS-generating systems, these observations are compatible with a mechanism whereby Mn²⁺, by acting as a superoxide dismutase mimetic, increases the formation of H₂O₂ by activated phagocytes. If operative in vivo, this mechanism may contribute to the toxicity of Mn²⁺.     

Investigation of the inhibitory effect of broussochalcone A on respiratory burst in neutrophils

Broussochalcone A, a prenylated chalcone isolated from Broussonetia papyrifera (L.) VENT. (Moraceae), inhibited O₂ consumption in formylmethionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC₅₀ values of 70.3±4.9 and 63.9±7.1 μM, respectively. Broussochalcone A did not affect the fMLP-induced increase of cellular inositol trisphosphate (IP₃) and [Ca²⁺]_i. However, the enzyme activity of neutrophil cytosolic protein kinase C was effectively suppressed by broussochalcone A. Broussochalcone A had no effect on either [³H]phorbol 12,13-dibutyrate ([³H]PDB) binding to neutrophil cytosolic protein kinase C or on PMA-induced membrane translocation of protein kinase C-β in neutrophils. Broussochalcone A suppressed the enzyme activity of trypsin-treated rat brain protein kinase C in a concentration-dependent manner. In PMA-activated neutrophil particulate NADPH oxidase, broussochalcone A attenuated superoxide anion radical (O₂⁻) generation with an IC₅₀ value of 61.8±5.4 μM. These results show that the inhibitory effect of broussochalcone A on respiratory burst in neutrophils is not mediated by the reduction of phospholipase C activity, but is mediated partly by the suppression of protein kinase C activity through interference with the catalytic region and by the attenuation of O₂⁻ generation from the NADPH oxidase complex.     

Inhibition of phospholipase A2 activities and some inflammatory responses by the marine product ircinin

The marine product ircinin has been tested for its effects on secretory and cytosolic phospholipase A₂ (PLA₂) activities in vitro as well as for inhibition of cellular functions in human neutrophils and inflammatory responses in mice. Ircinin inhibited Naja naja venom, human synovial recombinant, bee venom and zymosan-injected rat air pouch PLA₂ with IC₅₀ values in the M range, similar to those of the known inhibitor scalaradial. On the other hand, ircinin was less active on cytosolic PLA₂ from human monocytes and decreased potently the release of LTB₄ in human neutrophils. This marine product affected weakly human neutrophil functions like superoxide generation and degranulation. In the zymosaninjected rat air pouch ircinin inhibited in vivo the activity of PLA₂ present in exudates and reduced dose-dependently myeloperoxidase levels, whereas cell migration was inhibited only at the highest dose tested. This compound exerted a potent anti-oedematous effect after topical application in the mouse ear oedema test. Ircinin is a new inhibitor of PLA₂ activity and our results suggest a potential role for this marine product as an inhibitor of inflammatory processes.