Effects of gold-chloroquine complexes on respiratory burst of polymorphonuclear leukocytes

The effects of gold-chloroquine derivatives with the formula [Au(PR3)(CQ)]PF6 (where R = Ph (1), Et or Me) on the superoxide anion production by human neutrophils (PMNs, polymorphonuclear cells) were investigated. When these complexes (0.1-3 mumol/l) were added to PMNs prior to the activators formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), they inhibited isoluminol-horseradish peroxidase-dependent chemiluminescence (CLisol). The inhibition was a direct result of effects on PMNs since chemiluminescence in the cell-free system (horseradish peroxidase-hydrogen peroxidase-isoluminol) was not affected. The above mentioned concentrations of the complexes did not show in vitro toxicity on the cells. On the other hand, when 1 mumol/l of complex 1 was added to cells after stimulation, the chemiluminescence of PMNs stimulated by PMA was inhibited, but not the chemiluminescence stimulated by fMLP. The gold-chloroquine binding was essential for the referred activity as chemiluminescence was not influenced by the precursors chloroquine (CAS 54-05-7) and AuCl(PPh3). Furthermore, the extent of inhibition of chemiluminescence in PMNs activated by PMA did not increase with the duration of preincubation in presence of 1 mumol/l of 1. Extensive washing of cells after preincubation with 1 mumol/l of 1 reversed the aforementioned inhibition. All these results show that the gold-chloroquine binding can lead to compounds with specific properties that could make them useful in the treatment of some inflammatory diseases.     

Cimetidine modulates chemiluminescence and superoxide generation by neutrophils

Cimetidine, a known H2 blocker, markedly inhibited the generation of luminol-dependent chemiluminescence (LDCL) and the generation of Superoxide by human neutrophils (PMNs) stimulated by polycation-opsonized streptococci. Cimetidine also inhibited LDCL generation in peritoneal PMNs derived from mice pre-injected with this drug. The elucidation of the mechanisms of LDCL inhibition involved the employment of a variety of cimetidine analogues. The most effective inhibitory activity, besides cimetidine, was displayed by histamine, histidine, imidazole acetate, anserine and ergothionine. Imidazole, carnosine and homocarnosine had no inhibitory effect on oxygen radical generation. The possible mechanisms by which cimetidine and certain of its analogues affect the respiratory burst in leucocytes is discussed.     

Acetaminophen inhibits the human polymorphonuclear leukocyte function in vitro.

The aim of the study is to investigate the effect of Acetaminophen (Am) on the oxidative respiratory burst of isolated human polymorphonuclear leukocytes (PMNs). Acetaminophen inhibited the luminolchemiluminescence (CL) peak response of PMNs stimulated with phorbol myristate acetate (PMA) or opsonized zymosan in a concentration dependent manner. The inhibitory effect of Am on PMA-stimulated PMNs-CL response was partially reversible. The level of CL inhibition with Am plus the hydroxyl radical scavengers allopurinol, dimethyl sulfoxide (DMSO) or superoxide dismutase (SOD) is greater than that with Am alone. Generation of superoxide (O2-) by stimulated PMNs, as assayed by superoxide dismutase inhibitable reduction of Ferricytochrome c, was markedly inhibited by Am. Furthermore, the phagocytic activity of PMNs as tested for by the ingestion of opsonized dead yeast was significantly reduced in Am-treated cells. These results indicate clearly that Am causes significant inhibition of the human PMNs function in vitro.     

THE THERMAL POTENTIATION OF ACETAMINOPHEN-INHIBITED PMN OXIDATIVE METABOLISM IN VITRO

The effect of high temperatures (39, 41, and 43 °C) on acetaminophen (AM-) induced inhibition of the oxidative respiratory burst of polymorphonuclear leukocytes (PMNs) in vitro has been examined. Whole blood or isolated human PMNs were exposed to various temperatures in vitro in the presence or absence of AM for 0–90 min. Phagocyte membrane-bound NADPH oxidase was studied using the luminol chemiluminescence (CL) response and the superoxide dismutase inhibitable reduction of ferricytochrome C. The NADPH oxidase was stimulated by phorbol myristate acetate (PMA). The results showed that high temperatures (39–43 °C) potentiate the AM inhibitory effect on CL peak response of phagocytes in a temperature-dependent manner. Furthermore, the inhibition of superoxide (O⁻₂) production induced by AM was potentiated by incubating the cells at 39 or 43 °C at different time intervals. These studies suggest that high temperatures significantly potentiate the AM inhibitory effect on oxidative metabolism of PMNs in vitro. These actions of AM may influence the outcome in patients with infectious febrile conditions.     

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.     

Human neutrophils stimulated by cetyltrimethyl ammonium bromide generate luminol-amplified and non-amplified chemiluminescence but no superoxide production: A paradox

Human neutrophils (PMNs) stimulated by sub-toxic concentrations of cetyltrimethyl ammonium bromide (CETAB) (37 μmol/L) generated intense luminol-dependent chemiluminescence (LDCL) and moderate non-amplified chemiluminescence (CL), but, paradoxically, generation of superoxide (as assayed by cytochrome c reduction, lucigenin-dependent chemiluminescence, nitroblue tetrazolium reduction test (NBT), spin trapping or hydrogen peroxide (Thurman reaction) and also oxygen uptake, were not observed. LDCL generation, however, was dependent on the viability of the PMNs. On the other hand, CETAB failed to induce CL in PMNs obtained from two children with an X-linked chronic granulomatous disease of childhood. CETAB inhibited superoxide generation by PMNs stimulated by phorbol-12-myristate-13-acetate (PMA), histone or polyhistidine-opsonized streptococci. It also inhibited NBT reduction in PMNs stimulated by PMA or by cationized streptococci. Generation of LDCL by CETAB-stimulated PMNs was inhibited by azide, cyanide, thiourea, dimethylthiourea, histidine, cimetidine, chloroquine, nordihydroguaiaretic acid and bromophenacyl bromide and partially so, about 50%, by superoxide dismutase (SOD), by TEMPOL (a SOD mimetic) and H-7, a protein kinase c inhibitor, but not by catalase, desferrioxamine, taurine or methionine. PMNs stimulated by CETAB in the presence of azide generated a large peak of LDCL when treated with horseradish peroxidase (HRP), suggesting that hydrogen peroxide, perhaps of intracellular origin, was involved. Such enhanced HRP-stimulated light emission was inhibited by catalase and by desferrioxamine, suggesting that the HRP-catalysed reaction also depended on some source of trace metals. CETAB also markedly enhanced CL generated by a cell-free mixture of hydrogen peroxide and HRP, which was quenched to a large extent by catalase, dimethylthiourea or desferrioxamine, again suggesting that light emission might be linked with trace metals present in the salt solutions employed. It is postulated that CETAB-induced CL in human PMNs is the result of the interaction of hydrogen peroxide, presumably of intracellular source, a trace metal, and a peroxidase (myeloperoxidase). This phenomenon might be unrelated to the classical respiratory burst, which is always accompanied by oxygen consumption, and to the generation of a variety of oxygen-derived species linked with the activation of the NADPH oxidase present in the cell membrane.     

Effect of glycine on the release of reactive oxygen species in human neutrophils

The increase of extracellular glycine concentration prevents or mitigates a variety of pathological dysfunctional inflammatory responses. To eliminate the systemic effects of glycine as the reduction in the release of cytokines, this study was performed in isolated human neutrophils. The increase of the intracellular calcium concentration ([Ca2+](i)) and reactive oxygen species (ROS) release in cells incubated with glycine (0.1 to 10 mM) and stimulated with fMLP or PMA were compared with glycine-free controls. Glycine inhibited ROS production but increased [Ca2+](i) signal produced by fMLP. The inhibition of ROS production was observed even when glycine was added after the ROS release had reached maximal rate. The inhibitory effect was insensitive to strychnine and also obtained when PMA was used as stimulant. This study demonstrated that glycine impaired the activation of oxidative burst independently of glycine-gated chloride channel, presumably at the membrane level.     

The activation of gold complexes by cyanide produced by polymorphonuclear leukocytes--I. The effects of aurocyanide on the oxidative burst of polymorphonuclear leukocytes

It has been suggested that the antiarthritic gold complex, aurothiomalate (Autm), is activated by its conversion to aurocyanide by polymorphonuclear leukocytes (PMN) which generate cyanide from thiocyanate. In an examination of this hypothesis, a study has been conducted on the effects of aurocyanide on the oxidative burst of polymorphonuclear leukocytes (PMN) and monocytes activated by phorbol myristate acetate (PMA). Aurocyanide produced delayed inhibition of the oxidative burst as shown by its effect on both lucigenin and luminol-dependent chemiluminescence and on the production of superoxide. It was a more potent inhibitor of luminol-dependent chemiluminescence than free thiomalate and other by-products of the reaction between Autm and cyanide. Aurocyanide had a biphasic effect on the PMA-stimulated hexose monophosphate shunt of PMN, with enhancement at 0.1 microM and inhibition at 10 and 100 microM. The activity of aurocyanide was also compared with that of auranofin, an orally active gold complex, which inhibits a variety of functions of PMN and monocytes. At low concentrations, auranofin produced delayed inhibition of chemiluminescence in a similar fashion to aurocyanide but at high concentrations was an immediate inhibitor of the oxidative burst.     

The synergistic effect of adenosine A2A receptors agonist, type IV phosphodiestease inhibitor and ATP-sensitive K channels activation on free radicals production and aggregation of human polymorphoneuclear leukocytes.

The adenosine A2A receptor agonist CGS21680 (50, 100 and 200 microg/ml), the phosphodiserease type IV (PDE IV) inhibitor Rolipram (50, 100 and 200 microg/ml) and, ATP-sensitive K+ channels activator Cromakalim (30 and 40 microg/ml), when added separately, inhibit oxygen free radicals production from isolated human polymorphoneuclear leukocytes (PMNLs), stimulated with phorbol myristate acetate (PMA), in a dose dependent manner. When both CGS21680 and Rolipram were combined, in vitro, the inhibitory effect on PMNLs free radicals production was synergistic. On the other hand, when both the ATP-sensitive K+ channels opener (KATP) Cromakalim and the type IV PDE inhibitor Rolipram were combined, produced negative synergism (the inhibitory effect of both drugs disappeared). Furthermore, CGS21680, Rolipram, Cromakalim and Forskolin produced no significant inhibitory effect on PMNLs aggregation when added separately. But when various combinations of the above drugs were used, produced significant inhibition of aggregation. Only CGS21680 exhibited a scavenging effect on free radicals production. From the above results, combination of adenosine A2A agonists and type IV PDE inhibitors could serve as potentially novel anti-inflammatory drugs. Furthermore, ATP-sensitive K+ channels activators should be considered for further investigation as anti-inflammatory drug.     

Effects of diarylpentanoid analogues of curcumin on chemiluminescence and chemotactic activities of phagocytes

Objectives A series of 43 curcumin diarylpentanoid analogues were synthesized and evaluated for their inhibitory effects on the chemiluminescence and chemotactic activity of phagocytes in vitro. Methods The effects of the compounds on the respiratory burst of human whole blood and isolated human polymorphonuclear leukocytes (PMNs) were evaluated using a luminol‐based chemiluminescence assay and their effect on chemotactic migration of PMNs was investigated using the Boyden chamber technique. Key findings Compounds 6 , 17 , 25 and 30 exhibited significant inhibitory activity on the oxidative burst of PMNs. The presence of methoxy groups at positions 2 and 5, and methoxylation and fluorination at positions 4 and 2 of both phenyl rings, respectively, may contribute significantly to their reactive oxygen species inhibition activity. Compounds 7 , 17 , 18 , 24 and 32 showed strong inhibition of the chemotaxis migration of PMNs. Chlorination at various positions of both phenyl rings of cyclohexanone diarylpentanoid resulted in compounds with potent inhibitory effects on PMN migration. Conclusions The results suggest that some of these diarylpentanoid analogues are able to modulate the innate immune response of phagocytes at different steps, emphasizing their potential as a source of new immunomodulatory agents.     

Auranofin inhibits the activation pathways of polymorphonuclear leukocytes at multiple sites.

In order to characterize the mechanism by which the anti-rheumatic gold complex auranofin (AF) affects the functions of resting and activated polymorphonuclear leukocytes (PMN) the following studies were performed: (1) The effect of AF on the major processes involved in the respiratory burst of PMN: glucose transport and phosphorylation; hexose monophosphate (HMP) shunt activity in intact cells and in a cell-free system; superoxide production by particulate fractions and intact PMN measured as lucigenin-dependent chemiluminescence. (2) A comparison of the effects of AF added to the PMN before, at the time of, or subsequent to the stimulants [N-formyl-methionyl-leucyl phenylalanine (FMLP), concanavalin A (ConA), calcium ionophore (A23187) and phorbol myristate acetate (PMA)]. (3) The effect of AF on PMN activated by two stimulates (PMA, ConA) added sequentially. AF (0.1-10 microM) caused a dose-dependent inhibition of lucigenin-dependent chemiluminescence regardless of the activator (FMLP, ConA, A23187, PMA) when AF was added before the activator. In contrast, when AF was added to PMN after stimulation, it inhibited only the chemiluminescence of PMN stimulated by PMA. Furthermore, the chemiluminescence was largely unaffected by AF in sequentially activated PMN. The relative sensitivity to AF of the various processes studied indicates that blockade of the activation signal appears to be responsible for inhibition of the respiratory burst of PMN.     

Antioxidant properties of dipyridamole as assessed by chemiluminescence

The ability of dipyridamole (DIP) to scavenge oxygen metabolites generated by either activated human neutrophils (PMNs) or cell-free systems using luminol(s)- and lucigenin-enhanced chemiluminescence was investigated. In the presence of DIP (15-50 microM) a dose-dependent inhibition period was seen in phorbol myristate acetate (PMA)-stimulated PMNs as assayed by isoluminol-enhanced chemiluminescence (ILCL) with horseradish peroxidase (HRP). Although such a lag period was not observed in the absence of HRP, 50 microM DIP inhibited extracellular ILCL by more than 50%. Intracellular luminol-enhanced chemiluminescence (LCL) as assayed in either PMA- or in ionomycin-activated PMNs was not affected by dipyridamole (15-50 microM). In cell-free systems, DIP produced concentration-dependent inhibition in H2O2-(45% at 50 microM), OH- (40%, at 0.1 microM) and HOCl-(20% at 10 microM). Both absorbance and fluorescence scans revealed that DIP is able to react with equimolar quantities of either H202 or HOCl. These results suggest that DIP scavenges reactive oxygen species (ROS) presumably secreted by activated human PMNs in the following decreasing order: *OH > HOCl > H2O2 > O2-.     

Reciprocal effects between opioid peptides and human polymorphonuclear leukocytes--I. Chemical modifications of Leu-enkephalin by phorbol myristate acetate-stimulated polymorphonuclear leukocytes.

When L-tyrosyl-glycyl-L-phenylalanyl-L-leucine (Leu-enkephalin) is exposed to the activated oxygen species produced by phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMNs), hydroxylation of the phenylalanyl residue in position 4 of the peptide occurs, producing hydroxy-phenylalanyl derivatives which are identified by HPLC analysis and mass spectrometry. Attack of hydroxyl radicals generated by the Cu (II)/ascorbate system upon Leu-enkephalin also produces isomeric o-, m- and p-hydroxy-phenylalanyl derivatives. When PMNs are incubated with a synthetic peptide, L-tyrosyl-glycyl-glycyl-L-tyrosyl-L-leucine used as a model of hydroxylated Leu-enkephalin, their chemiluminescence response to PMA activation is higher than that of PMNs incubated with Leu-enkephalin.     

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.     

Alteration by flutamide of neutrophil response to stimulation: Implications for tissue injury

When activated, inflammatory cells such as polymorphonuclear leukocytes (PMNs) can damage isolated hepatocytes in vitro. These studies were performed to determine if flutamide activates PMNs. Flutamide (Eulexin) is an orally active, nonsteroidal antiandrogen that can cause liver injury associated with inflammation. Activation of PMNs was assessed from the production of superoxide anion and the release of myeloperoxidase in the presence or absence of flutamide and phorbol myristate acetate (PMA) or f-methionylleucyl phenylalanine (fmlp). In addition, hepatocytes were cocultured with PMNs stimulated with PMA or fmlp in the presence or absence of flutamide, and cytotoxicity to hepatocytes was evaluated from the release of alanine aminotransferase into the medium. Flutamide alone did not stimulate the generation of superoxide anion by PMNs but potentiated its production in response to PMA. At lower concentrations of flutamide (i.e. 25 μM), there was a tendency toward increased release of myeloperoxidase, whereas at higher concentrations (i.e. 75–100 μM) flutamide inhibited degranulation in response to fmlp. In coculture with hepatocytes, PMNs exposed to either flutamide, fmlp, or PMA alone caused a significant increase in release of alanine aminotransferase. Hepatocellular toxicity caused by PMNs incubated with flutamide and PMA was additive and was not affected by the addition of superoxide dismutase and catalase. Flutamide had no significant effect on fmlp-induced injury in cocultures. These data indicate that flutamide alters the activation of PMNs by subsequent stimuli in vitro. In addition, in the presence of flutamide, minor PMN-mediated injury to isolated hepatocytes was observed.     

Suppressive effect of zearalenone, an estrogenic mycotoxin, on bovine neutrophil chemiluminescence

The effects of zearalenone (ZEA), an estrogenic mycotoxin produced by Fusarium fungi, on bovine neutrophils were investigated in vitro using chemiluninescence, a bactericidal parameter. ZEA suppressed luminol-dependent, phorbol 12-myristate 13-acetate (PMA)-elicited chemiluminescence in a dose-dependent manner at concentrations of 10(-4) M and 10(-5) M. No significant suppression was observed at concentrations lower than 10(-6) M. The possible mode ofaction of 10(-4) M ZEA on the cell activity was investigated with special reference to intracellular Ca2+ ([Ca2+]i) release and estrogen receptors. The 10(-4) M ZEA treatment significantly impaired [Ca2+]i release. When pretreated with a low dose (10(-6) M) of PMA, the cells resisted the ZEA-induced chemiluminescence suppression. However, pretreatment of the cells with the estrogen receptor blockers Tamoxifen and ICl 182,780 (both at 10(-6) M) did not annul the suppressive ZEA action. Considering that PMA is an activator of protein kinase C (PKC), a signal transducing enzyme, and in association with a rise in [Ca2+]i causes cytosolic PKC to shift to the plasma membrane where the activated PKC triggers a varied array of cellular responses, the pharmacological dose of ZEA might have suppressed chemiluminescence by hindering the release of [Ca2+]i and the PKC shift. The results of pretreatment with estrogen receptor blockers, however, did not support the suggestion that the ZEA treatment affected the cells via estrogen receptor pathways.     

Oxidative metabolism of peripheral blood neutrophils in experimental acute hypercapnia in the mechanically ventilated rabbit

BACKGROUND AND AIM: The aim of this study was to investigate the effect of acute hypercapnia due to the mechanical hypoventilation on the oxidative metabolism of peripheral blood neutrophils in the rabbit. METHODS: The study was performed on 24 Chinchilla rabbits, randomized into normo- and hypercapnia groups (P(a)CO(2) between 9 and 11 kPa over 180 min). At the baseline point and after 180 min of mechanical ventilation, a neutrophil count, luminol-dependent chemiluminescence of the neutrophils stimulated with opsonized zymosan (OZ) or PMA (phorbol myristate acetate), and the hydrogen peroxide production of the cells upon the PMA stimulation were measured. Serum cortisol levels were additionally determined. RESULTS: In both studied groups, a significant neutrophilia after 180 min of mechanical ventilation was observed (P<.05). Neither chemiluminescence nor hydrogen peroxide production of peripheral neutrophils was changed (P>.05). CONCLUSIONS: Hypercapnia lasting for 180 min did not affect the oxidative metabolism of circulating neutrophils but a rise in a neutrophil count was observed in the mechanically ventilated rabbit.     

Effect of some psychotropic drugs on the activated macrophage-induced luminol-dependent chemiluminescence

The effect of some psychotropic drugs on the activity of macrophages to produce superoxide radicals during phagocytosis was tested. Three-cyclic antidepressants, imipramine and amitriptyline, and the thioxanthene neuroleptic, chlorprothixene, were studied. The superoxide production was measured by luminol-dependent chemiluminescence. The drugs were investigated in the concentration range of 10(-7)-10(-4) mol/l. It was seen that all tested drugs caused a concentration-dependent decrease of the luminol-dependent chemiluminescence. The inhibitory effect of imipramine and amitriptyline on the macrophage superoxide production was moderate, while the effect of chlorprothixene was significantly stronger (a decrease more than 100 times that of macrophage chemiluminescence). Essentially, the luminol-dependent chemiluminescence reflects the level of superoxide radicals in the system. Therefore, the effect of drugs may be due to the possible activity for scavenging superoxide. In additional experiments with different systems of generations of O2- and different methods of registration, this possibility was discarded. Therefore, the effect of the drugs on the luminol-dependent chemiluminescence seems to be due to drug-induced decrease of the ability of activated macrophages to produce superoxide radicals.     

Effect of mammalian lignans on fMLP-induced oxidative bursts in human polymorphonuclear leucocytes.

We examined the effects of mammalian lignans, enterolactone, prestegane B and 2,3-dibenzylbutane-1,4-diol (DBB) on superoxide production and luminol-dependent chemiluminescence (LCL) response in human polymorphonuclear leucocytes (PMNs). The three lignans had no direct effect on the responses of human PMNs. DBB and prestegane B enhanced the superoxide production and LCL response induced by formylmethionyl-leucyl-phenylalanine (fMLP), but enterolactone inhibited fMLP-induced effects. The effects of DBB were stronger than those of prestegane B and the effects of DBB were inhibited by bromophenacyl bromide, mepacrine, N-(6-aminophenyl)-5-chloro-1-naphthalene, sulphonamide and trifluoroperazine, but not by gossypol, nordihydroguaretic acid, indomethacin, staurosporine, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride or (R,S)-2-methoxy-3-(octadecyl-carbamoyloxy)-propyl-2-(2-thiazoli o)-ethylphosphate. These results suggest that DBB primes the responses of human PMNs, and the priming effect is caused by the activation of phospholipase A2--and Ca(2+)-calmodulin-pathways, but not by the activation of lipoxygenase, cyclo-oxygenase and protein kinase C or by the release of platelet activating factor.     

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.