INFLUENCE OF CARVEDILOL ON SUPEROXIDE GENERATION AND ENZYME RELEASE FROM STIMULATED HUMAN NEUTROPHILS

Activation of neutrophils induces generation of reactive oxygen species and release of granule enzymes, which not only participate in the bactericidal mechanisms of these cells, but also in possible tissue damage. We studied the effect of carvedilol (CARV) [0.1-100μmol/l], an antihypertensive and cardiovascular drug with antioxidative properties, on superoxide generation (SO) and myeloperoxidase (MPO) release from isolated human neutrophils stimulated with fMLP, a specific receptor activator, or with PMA, a receptor bypassing stimulus. Unstimulated cells showed neither SO formation nor MPO release after preincubation with drug. CARV decreased fMLP and PMA stimulated MPO release and SO generation dose dependently. The inhibitory effect of CARV may attributed to non-specific action since its effect was not influenced by the type of stimulation. It might inhibit SO generation as well as MPO release either by membrane-operating stimulus (fMLP) or membrane bypassing activator (PMA).

Neutrophils represent the most important source of free radicals and actively contribute to damage of the vessel wall in cardiac and cerebrovascular diseases 6 .Activation of neutrophils induces generation of reactive oxygen species and release of granule enzymes, which not only participate in the bactericidal mechanisms of these cells, but also in possible tissue damage 7 .Dandona et al. 8 described the inhibitory effect of CARV on SO anion release from activated neutrophils ex vivo.CARV was also found to inhibit luminol-enhanced chemiluminescence of reactive oxygen species in blood cells in vitro 9 .
In this work we studied the effect of CARV on the first member of the chain of oxygen species -superoxide anion (SO) and on myeloperoxidase (MPO) release by human neutrophils stimulated with N-formyl-methionyl-leucylphenylalanine (fMLP), a specific receptor activator, or with phorbol-12-myristate-13-acetate (PMA), a receptor bypassing stimulus.
The stock solution of carvedilol tartrate was diluted freshly before each experiment with deionised water to the desired concentrations.

Enzyme determination
For myeloperoxidase (MPO) determination, neutrophils were preincubated with cytochalasin B (5 µg/ml) at room temperature for 5 min.Subsequently the suspension of neutrophils (2 × 10 6 cells/ml PBS) was incubated with carvedilol (0.1-100 µmol/l) for 5 min at 37 o C and then stimulated with fMLP (0.1µmol/l) for 3 min or with PMA (1 µmol/l ) for 15 min at 37 o C. Controls for direct interaction of carvedilol and MPO were included.The activity of MPO was assayed in the supernatant after centrifugation 2000 × g for 10 min at 4 o C by determining the oxidation of o-dianisidine in the presence of hydrogen peroxide in a Hewlett Packard 8452 A spectrophotometer at 463 nm 12 .

Statistical analysis
All values are given as means ± SEM.Statistical significance of differences between means was established by Student's t-test and p values below 0.05 were considered statistically significant.

RESULTS AND DISCUSSION
Superoxide (SO) radical generated during oxidative burst by neutrophil NADPH oxidase, a precursor of other reactive oxygen species used for host defense against bacterial and fungal pathogens, but they also inflict damage on adjacent tissue, and are thought to be of pathogenic significance in a large number of diseases 13 .Proteolytic enzymes released from activated neutrophils in the process of degranulation have been recognised as an important factor contributing to neutrophil-mediated tissue injury.
CARV is an antihypertensive agent with several mechanisms of antiarrhythmic and antiischaemic activities, underlying its cardio-and cerebroprotective 14,15 as well as antioxidant properties.The antioxidant action may be relevant for preserving myocardial viability, particularly during ischaemia and reperfusion, conditions linked to the occurrence of oxidative stress [1][2][3]16 . Ischemia and reperfusion are potent stimuli for eliciting cardiomyocyte apoptosis, with neutrophils involved in the development of myocardial injury.CARV (at a dose range 1-10 nmol/l) significantly prevented activated neutrophil-induced cardiomyocyte apoptosis on cultured primary neonatal rat cardiomyocytes, possibly due to its antioxidant properties 17 .CARV inhibited oxidative stress in neutrophils and mononuclear cells in patients with hypertension 18 and exerted a marked scavenger effect on free radical generation-induced red blood cell membrane damage 19 .
CARV inhibits luminol-enhanced chemiluminiscence of reactive oxygen metabolites in vitro.Nosáľ et al. 9 found that in the cell-free system CARV dose-dependently decreased chemiluminiscence in the following range of order of radicals: hydroxyl radicals > hydrogen peroxide > superoxide radical.CARV also dose-dependently inhibited chemiluminiscence of isolated human neutrophils in the ranging order of stimuli: A 23187 > OpZ > fMLP.Dandona et al. 8 demonstrated that CARV in patients who were given this drug significantly inhibited SO generation by neutrophils and mononuclear cells via chemiluminiscence with fMLP stimulation.(PMA) lead to the conclusion that the inhibitory effect may be attributed to its non-specific action.Inhibition of SO generation by CARV in stimulated human neutrophils is not only able to reduce the toxicity caused by SO itself, but also, even more importantly, to prevent the formation of higher reactive and toxic hydroxyl radical, and thus to protect against further damage induced by the oxygen-radical chain reactions.
Åsbrink et al. 20 observed that CARV dose dependently modulated generation of SO ions by NADPH oxidase when induced by fMLP or PMA.
In the present work, we studied the effect of CARV on SO generation and MPO release by human neutrophils stimulated with fMLP, a specific receptor activator, or with PMA, a receptor bypassing stimulus.In every experiment we used parallel incubation of unstimulated controls.These cells showed neither SO formation nor enzyme release after preincubation with CARV.Table 1 illustrates the effect of CARV (0.1, 1, 10 and 100 µmol/l) on fMLP activated SO generation in isolated human neutrophils.CARV inhibited dose-dependently SO generation.In the concentration 100 µmol/l, CARV significantly inhibited SO generation to 84.08 ± 2.99 %.
CARV suppressed superoxide generation of human neutrophils stimulated with PMA, as determined by MCLA-dependent chemiluminiscence 21 .The ability of CARV to prevent cell damage and death of activated neutrophils may be due to its property to scavenge SO ions and to inhibit the production of SO radicals by neutrophils.In a concentration-dependent manner.CARV was demonstrated to inhibit the production and generation of SO ions by neutrophils, based on direct measurement of SO release from PMA activated human neutrophil, or its generation during autooxidation of dihydroxyfumaric acid 2,3 .
In our experimental conditions, we observed that the effect of CARV (0.1, 1, 10 and 100 µmol/l) on PMA activated SO generation in isolated human neutrophils was dose-dependent (Table 2).CARV in the concentration 100 µmol/l significantly decreased SO generation to 70.21 ± 4.45 %.
The haeme-containing enzyme myeloperoxidase (MPO) is a major constituent of the azurophilic cytoplasmic granules of neutrophils, which is gradually released either intracellulary into the formed phagocytes or extracellulary upon stimulation of cells.Together with the membraneous NADPH oxidase, MPO is involved in the formation of reactive oxygen species and oxidation of biological material.CARV inhibited MPO activity of the cellfree system in a concentration-dependent manner 9 .CARV treatment reduced MPO levels at both the area-at-risk and the necrotic zone in the ischaemic myocardium 22 .
Our results showing that CARV inhibited SO generation as well as MPO release both by membrane-operating stimulus (fMLP) and by or membrane bypassing activator Influence of carvedilol on superoxide generation and enzyme release from stimulated human neutrophils

Table 1 .
Effect of carvedilol on fMLP (0.1µmol/l, 3 min/ 37 o C) stimulated superoxide generation and myeloperoxidase release in human neutrophils