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.

Carvedilol and adrenergic agonists suppress the lipopolysaccharide-induced NO production in RAW 264.7 macrophages via the adrenergic receptors.

The interaction of adrenergic agonists and/or antagonists with the adrenergic receptors expressed on immunologically active cells including macrophages plays an important role in regulation of inflammatory responses. Our study investigated the effects of carvedilol, a unique vasodilating beta-adrenergic antagonist, and endogenous adrenergic agonists (adrenalin, noradrenalin, and dopamine) and/or antagonists (prazosin, atenolol) on lipopolysaccharide-stimulated nitric oxide (NO) production from murine macrophage cell line RAW 264.7. The production of NO was determined as the concentration of nitrites in cell supernatants (Griess reaction) and inducible nitric oxide synthase (iNOS) protein expression (Western blot analysis). Scavenging properties against NO were measured electrochemically. Carvedilol in a concentration range of 1, 5, 10 and 25 microM inhibited iNOS protein expression and decreased the nitrite concentration in cell supernatants. Adrenalin, noradrenalin or dopamine also inhibited the iNOS protein expression and the nitrite accumulation. Prazosine and atenolol prevented the effect of both carvedilol and adrenergic agonists on nitrite accumulation and iNOS expression in lipopolysaccharide-stimulated cells. These results, together with the absence of scavenging properties of carvedilol against NO, imply that both carvedilol and adrenergic agonists suppress the lipopolysaccharide-evoked NO production by macrophages through the activation and modulation of signaling pathways connected with adrenergic receptors.

New irradiation facility for biomedical applications at the RA-3 reactor thermal column.

A new irradiation facility has been developed in the RA-3 reactor in order to perform trials for the treatment of liver metastases using boron neutron capture therapy (BNCT). RA-3 is a production research reactor that works continuously five days a week. It had a thermal column with a small cross section access tunnel that was not accessible during operation. The objective of the work was to perform the necessary modifications to obtain a facility for irradiating a portion of the human liver. This irradiation facility must be operated without disrupting the normal reactor schedule and requires a highly thermalized neutron spectrum, a thermal flux of around 10(10) n cm(-2)s(-1) that is as isotropic and uniform as possible, as well as on-line instrumentation. The main modifications consist of enlarging the access tunnel inside the thermal column to the suitable dimensions, reducing the gamma dose rate at the irradiation position, and constructing properly shielded entrance gates enabled by logical control to safely irradiate and withdraw samples with the reactor at full power. Activation foils and a neutron shielded graphite ionization chamber were used for a preliminary in-air characterization of the irradiation site. The constructed facility is very practical and easy to use. Operational authorization was obtained from radioprotection personnel after confirming radiation levels did not significantly increase after the modification. A highly thermalized and homogenous irradiation field was obtained. Measurements in the empty cavity showed a thermal flux near 10(10) n cm(-2)s(-1), a cadmium ratio of 4100 for gold foils and a gamma dose rate of approximately 5 Gy h(-1).

Carvedilol--a beta-blocker with considerable antiaggregatory effect on human blood platelets.

BACKGROUND: Activated blood platelets play a key role in the genesis of many pathological states. Several studies have documented that beta-blockers can influence platelet aggregation. Carvedilol, a third generation non-selective agent with vasodilatory properties, is successfully used in pathological states accompanied with platelet hyperreactivity, however information on its antiplatelet activity is lacking. OBJECTIVES: The aim of this study was to analyse the in vitro effect of carvedilol on aggregation of human blood platelets, to compare this effect with the effect of propranolol and atenolol, and to determine whether its suggested antiaggregatory effect was accompanied with reduced thromboxane B2 formation. Moreover, some physico-chemical parameters of the drugs tested were calculated and compared. METHODS: Platelets were isolated by differential centrifugation and platelet aggregation was measured by the turbidimetric method. The amount of thromboxane B2 was measured by the radioimmunoassay method. Physico-chemical parameters of the drugs tested were calculated using the computer programme Hyperchem. RESULTS: Carvedilol and propranolol inhibited platelet aggregation in the rank order of stimuli: PMA > thrombin > A23187 > epinephrine. The reduction was accompanied by inhibition of thromboxane B2 formation. In comparison to propranolol, carvedilol was more effective, with the exception for aggregation stimulated with ADP. Atenolol did not affect any platelet function tested. From the drugs studied, the molecule of carvedilol was found to possess the highest partition coefficient, the highest index of molar refractivity, and the lowest dipole moment. CONCLUSION: Our study found carvedilol to be more potent than propranolol and atenolol in inhibiting platelet aggregation and thromboxane B2 production. This may be due to the different structure and more convenient physico-chemical parameters of the carvedilol molecule.

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.).

Stobadine-modulated human neutrophil functional responsiveness: effect on particular and soluble stimulation.

The effect of stobadine on degranulation (myeloperoxidase release) and on oxidative burst, measured as superoxide anion production, was investigated in human neutrophils activated with receptor-specific (fMLP, opsonized zymosan) and nonreceptor stimuli (PMA, A 23187). Wortmannin, a specific inhibitor of 1-phosphatidylinositol 3-kinase, significantly inhibited fMLP-stimulated generation only. This effect was pronounced by stobadine. Stobadine dose-dependently decreased superoxide generation and myeloperoxidase release after receptor-specific stimuli, with the highest effect on fMLP stimulation of superoxide generation and on opsonized zymosan stimulation of myeloperoxidase release.

Effect of stobadine on opsonized zymosan stimulated generation of reactive oxygen species in human blood cells.

To predict more precisely the effect of stobadine, a pyridoindole antioxidant agent, in the whole organism, we studied its effect on opsonized zymosan-stimulated free radical generation in whole blood, on superoxide generation in the mixture of PMNL : platelets (1:50), as well as on superoxide generation and myeloperoxidase release in isolated PMNL. Without stimulation, stobadine had no effect on reactive oxygen species (ROS) generation and myeloperoxidase release. Stobadine in a concentration of 10 or 100 micromol/l significantly decreased luminol-enhanced chemiluminescence in opsonized zymosan-stimulated whole blood. In concentrations of 10 and 100 micromol/l, it reduced myeloperoxidase release from isolated neutrophils. Stobadine significantly decreased superoxide generation in isolated neutrophils in 100 micromol/l concentration. Its effect was much less pronounced in the mixture of neutrophils and platelets in the ratio close to physiological conditions (1:50). Our results suggest that stobadine might exert a beneficial effect in diseases or states where superfluous ROS generation could be deleterious.

Antiplatelet activity of carvedilol in comparison to propranolol.

The non-selective vasodilating beta-blocker carvedilol was found to inhibit platelet aggregation as well as thromboxane B(2) formation more effectively than propranolol. The antiaggregatory activity of carvedilol decreased, depending on the stimulus used, in the following rank order of potency (in parentheses the respective mean inhibitory concentrations of carvedilol and propranolol are given in micromol/l): PMA (19 and 34) > thrombin (55 and 77) > Ca(2+)-ionophore A23187 (58 and 81) > epinephrine (86 and 118). However, aggregation of platelets activated with ADP was not affected by carvedilol in concentrations up to 100 micromol/l. In platelets stimulated with thrombin, carvedilol (10 micromol/l) reduced thromboxane B(2) formation by 64%, whereas propranolol was ineffective at this concentration. Moreover, A23187-induced formation of thromboxane B(2), not affected by propranolol, was completely blocked by 100 micromol/l carvedilol. In comparison to propranolol, the molecule of carvedilol is more lipophilic and possesses lower dipole moment and higher molar refractivity, thus penetrating into platelet membranes readily and in large quantities. The antiplatelet effect was assumed to result from interactions of carvedilol with membrane macromolecules (phospholipids, ion channels, enzymes, etc.) rather than from blockade of alpha- and beta-adrenergic receptors.