On the pharmacology of oxidative burst of human neutrophils.

The effect of three therapeutically used drugs and five polyphenolic compounds on the mechanism of oxidative burst was compared in whole blood and isolated neutrophils at cellular and molecular level. In 10 microM concentration, the compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin (N-f-5HT) > curcumin (CUR) > quercetin (QUER) > arbutin (ARB) > resveratrol (RES) > dithiaden (DIT) > carvedilol (CARV) > brompheniramine (BPA). The ratio between the percentage inhibition of extracellular versus intracellular chemiluminescence (CL) followed the rank order QUER > N-f-5HT > RES > CUR > DIT and is indicative of the positive effect of the compounds tested against oxidative burst of neutrophils, demonstrating suppression of reactive oxygen species extracellularly with minimal alteration of intracellular reactive oxygen species (ROS). Activation of protein kinase C was significantly decreased by DIT, CUR, QUER and N-f-5HT. CARV, DIT, QUER and ARB reduced activated neutrophil myeloperoxidase release more significantly compared with the effect on superoxide anion generation. All compounds tested increased the activity of caspase-3 in cell-free system. It is suggested that other regulatory mechanisms than protein kinase C might participate in the inhibition of neutrophil activation with the compounds tested. Different mechanisms are concerned in controlling the assembly of NADPH oxidase and the regulatory role of calcium ions is suggested. Compounds decreasing the amount of extracellular ROS generation, yet affecting but minimally intracellular ROS generation, are promising for further investigation in vivo.

Effect of plant polyphenols on ischemia-reperfusion injury of the isolated rat heart and vessels.

In the present study, we investigated the potential protective effect of selected natural substances in a rat model of heart and mesenteric ischemia-reperfusion (I/R). Experiments were performed on isolated Langendorff-perfused rat hearts, subjected to 30-min global ischemia, followed by 30-min reperfusion. Arbutin, curcumin, rosmarinic acid and extract of Mentha x villosa were applied in the concentration of 1 × 10⁻5 mol/l 10 min before the onset of ischemia and during reperfusion, through the perfusion medium. Mesenteric ischemia was induced by clamping the superior mesenteric artery (SMA) for 60 min, subsequent reperfusion lasted 30 min. Production of reactive oxygen species (ROS) by SMA ex vivo was determined by luminol-enhanced chemiluminiscence (CL). The effect of the substances was tested after their incubation with tissue. Curcumin and extract of Mentha x villosa were found to be the most effective in reducing reperfusion-induced dysrhythmias--ventricular tachycardia and fibrillation. This effect was accompanied by bradycardic effect. The mesenteric I/R induced an increase in CL in vascular tissue which was dampened by substances tested. All substances tested were found to have antioxidant properties, as demonstrated by a reduction in ROS production in mesenteric vessels. This effect was confirmed in curcumin and extract of Mentha x villosa which reduced reperfusion dyshythmias.

Effects of activated neutrophils on isolated rings of rat thoracic aorta.

Local inflammation and respiratory burst of polymorphonuclear leukocytes generate reactive oxygen species (ROS). The aim of our study was to analyze the effects of peritoneal neutrophils on changes of the muscle tension of isolated aorta and compare their effects with those of different ROS. While native neutrophils did not influence muscle tension, the N-formyl-methionyl-leucyl-phenylalanine activated ones evoked a biphasic response on the KCl-precontracted aorta. The effects of activated neutrophils were in both respects similar to those evoked by xanthine/xanthine oxidase (X/XO) and differed from the effects evoked by H(2)O(2) and Fe(2)SO(4)/H(2)O(2). Using H(2)O(2) we demonstrated that the effects of ROS were dependent on the KCl induced initial tension. To exclude the effect of extensive depolarization the action of different ROS was studied also on tissues precontracted by phenylephrine. Under such condition activated neutrophils caused a marked contraction similar to that evoked by X/XO. Their effects differed however, from those of H(2)O(2) and Fe(2)SO(4)/ascorbic acid. These findings and elimination of activated neutrophil-induced contractions as well as the chemiluminiscence by superoxide dismutase suggest that the primarily activated neutrophil-released ROS was superoxide, which can be transformed to peroxynitrite, and other ROS including H(2)O(2). Reduction of all followed-up contractions caused by nordihydroguaiaretic acid, indicate that 5-lipoxygenase metabolites unselectively reduce contractions. In contrast, selective inhibition of activated neutrophil-evoked contraction by indomethacin suggests that cyclooxygenase metabolites are involved mainly in their action on vascular smooth muscle.

Inhibition of chemiluminescence by carvedilol in the cell-free system, whole human blood and blood cells.

Carvedilol inhibits luminol-enhanced chemiluminescence of reactive oxygen metabolites in vitro. In this study it was found that, in the cell-free system, carvedilol dose-dependently decreased chemiluminescence in the following ranking order of radicals: hydroxyl radical > hydrogen peroxide > superoxide radical. The inhibition of myeloperoxidase was significant with carvedilol concentrations of 10 and 100 micromol/l and manifested in the concentration-dependent shift of chemiluminescence peaks to the right. In whole blood, carvedilol in concentrations of 10 and 100 micromol/l significantly inhibited chemiluminescence induced by both receptor-bypassing stimuli (A23187, PMA) and receptor-operating stimuli (fMLP, OpZ). Carvedilol dose-dependently inhibited chemiluminescence of isolated human polymorphonuclear leucocytes in the ranking order of stimuli: A23187 > OpZ > fMLP. In the presence of blood platelets, carvedilol did not substantially change chemiluminescence induced by fMLP and OpZ, while it was much more effective on chemiluminescence stimulated with calcium ionophore A23187. This could be the result of the supportive effect of serotonin liberated from platelets by A23187.

Blood platelets decrease concentration of reactive oxygen species produced by polymorphonuclear leukocytes.

BACKGROUND: Reactive oxygen species produced by polymorphonuclear leukocytes (PMNL) participate substantially in vascular injury induced by ischaemia and reperfusion. Blood platelets, accumulated simultaneously with PMNL may modulate this process. OBJECTIVE: To compare effects of resting and completely stimulated platelets on PMNL-derived oxidants. METHODS: Autologous human platelets and PMNL were co-incubated in the physiological cell ratio 50:1, and the formation of reactive oxygen species was detected by luminol- and isoluminol-enhanced chemiluminescence methods. To compare effects of platelets at different degrees of their activation, FMLP (selective PMNL stimulus) and Ca2+-ionophore A23187 (activates both PMNL and platelets) were used as chemiluminescence stimuli. The liberation of serotonin from platelets was estimated fluorometrically. RESULTS: Both stimulated and non-stimulated platelets inhibited PMNL chemiluminescence. However, while the decreasing effect of resting platelets disappeared at increased extracellular peroxidase concentration, the inhibition of chemiluminescence by activated platelets became even more pronounced after the addition of peroxidase and was accompanied by liberation of serotonin. The concentrations of serotonin released from platelets were sufficiently high to inhibit PMNL chemiluminescence. CONCLUSION: The obtained data indicate that by interference of platelets with peroxidase liberation from PMNL and the scavenging effect of platelet serotonin, resting and stimulated platelets might be respectively operative in inhibiting chemiluminescence. Since the presence of blood platelets in the proximity of PMNL effectively decreased the concentration of reactive oxygen species, platelets may represent a unique protective mechanism, active only in case of emergency and selectively at sites exposed to toxic effects of reactive oxygen species. (Tab. 1, Fig. 4, Ref. 42.).

Effect of H1-antagonist Dithiaden on human PMN-leukocyte aggregation and chemiluminescence is stimulus-dependent.

OBJECTIVE AND DESIGN: Contradictory data published on histamine-PMN leukocyte interactions stimulated us to study to the role of histamine and H1-antagonist Dithiaden in generation of reactive oxygen species (ROS) and aggregation of human neutrophils. METHODS AND MATERIALS: Whole blood or isolated PMN-leukocytes were exposed in a dose-dependent way to histamine or H1-antagonist Dithiaden and subsequently stimulated. Whole blood was stimulated with opsonised zymosan (OZ). Isolated cells were stimulated with membrane stimuli (OZ, N-formyl-methionyl-leucyl-phenylalanine--fMLP), or membrane bypassing stimuli (Ca2+-ionophore A23187, phorbol-myristate-acetate--PMA). The luminol-enhanced chemiluminescence (CL) was measured separately (whole blood) in a luminometer or simultaneously with neutrophil aggregation in a whole blood lumiaggregometer. RESULTS: Depending on the concentration used, Dithiaden" was 1.5- to 25.0-times more effective in inhibiting activated CL of whole blood than histamine. In isolated neutrophils both histamine and Dithiaden inhibited OZ- and A23187-stimulated CL dose-dependently, with potentiation observed after stimulation with PMA and fMLP. Histamine did not alter aggregation with any of the stimuli tested. Dithiaden inhibited A23187-, OZ- and PMA-stimulated PMN-leukocytes but potentiated fMLP-induced aggregation of isolated neutrophils. Simultaneous application of Dithiaden and histamine abolished the effect of Dithiaden on fMLP-stimulated CL. CONCLUSIONS: Dithiaden, depending on the stimuli applied, inhibited human neutrophils, both isolated or in whole blood, more markedly than histamine. The inhibition of aggregation and CL was dose- and stimulus-dependent. Histamine administered simultaneously abolished the effect of Dithiaden on fMLP-stimulated PMN-leukocytes. It seems likely that the interaction of Dithiaden with neutrophils operated both at an extra- and intracellular level.

Cooperation of chloroquine and blood platelets in inhibition of polymorphonuclear leukocyte chemiluminescence.

Effect of activated blood platelets and chloroquine on concentration of reactive oxygen species produced by polymorphonuclear leukocytes (PMNL) stimulated with Ca(2+)-ionophore A23187 was investigated. Oxygen metabolites localized outside PMNL were visualized by isoluminol enhanced chemiluminescence, whereas chemiluminescence, enhanced with luminol and measured in the presence of the extracellular scavengers superoxide dismutase and catalase, was used for the detection of radicals originated intracellularly. Significant reduction of chemiluminescence was observed in the presence of platelets (added to PMNL in the physiological cell ratio 50:1) and of chloroquine (10 and 100 micromol/L). Although chloroquine decreased effectively both the extra- as well as the intracellular part of the chemiluminescence signal, the activity of platelets occurred largely outside PMNL. Serotonin liberated from platelets by A23187 appeared to be involved in inhibition of chemiluminescence; its concentrations achieved in platelet supernatants were found to be sufficient for elimination of PMNL-derived oxygen metabolites. The presented results indicated that chloroquine and blood platelets cooperate in inhibition of chemiluminescence because their common effect was found to be much more extensive than reduction induced by these inhibitors separately. Therefore, for accurate prediction of drug effect in the whole organism, the use of multicellular test systems seems to be pertinent.

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence.

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.

Human blood platelets, PMN leukocytes and their interactions in vitro. Responses to selective and non-selective stimuli.

Using simultaneous recording of aggregation and chemiluminescence, responses of human polymorphonuclear leukocytes, blood platelets and their mixture were investigated after stimulation by specific as well as non-specific stimuli for each cell. In our experimental settings, aggregation of platelets and PMN leukocytes was increased in the following order of stimuli: PMA

Effect of stobadine on oxygen free radical generation in stimulated human polymorphonuclear leukocytes.

The generation both superoxide and a mixture of reactive oxygen species was recorded in a suspension of human polymorphonuclear leukocytes stimulated with phorbol myristate acetate. While stobadine dose-dependently decreased chemiluminescence, only its highest concentration used reduced significantly superoxide generation. The results suggest that stobadine is a more effective scavenger of free radicals rather than a quencher of superoxide anion.

Effect of stobadine on human stimulated polymorphonuclear leukocytes.

The effect of stobadine (0.1-100 microM) on human polymorphonuclear (PMN) leukocytes stimulated with N-formyl-methionyl-leucyl-phenylalanine, a specific receptor activator, or with the calcium ionophore, A-23187 (receptor bypassing stimulus) was investigated with respect to: i) superoxide generation, ii) beta-glucuronidase release and iii) 3[H]-arachidonic acid liberation. Stobadine was found to exert an inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine but not on A-23187-stimulated PMN leukocytes. The effect was more intensive on superoxide generation and beta-glucuronidase release than on 3[H]-arachidonic acid liberation. These results indicate that the inhibitory effect of stobadine is most probably via a mechanism dependent on signal transduction across the plasma membrane. This effect may occur through inhibition of arachidonate signal transduction through a regulatory G-protein.