R. Nosál

The combined luminol/isoluminol chemiluminescence method for differentiating between extracellular and intracellular oxidant production by neutrophils

Abstract To address the question why isoluminol, but not luminol, failed to detect oxidants produced intracellularly, differences between these luminophores were investigated with respect to physicochemical parameters and the character of chemiluminescence signal. Our results showed the isoluminol molecule to be more polar, more hydrophilic and possessing lower ability to form intramolecular bonds than the luminol molecule. Therefore, isoluminol: (i) only slightly pervaded biological membranes; (ii) depended essentially on extracellular peroxidase; (iii) did not produce chemiluminescence in the presence of extracellular scavengers; and (iv) it could be considered a specific detector of extracellular radicals. On the other hand, the physicochemical parameters of luminol and partial resistance of its chemiluminescence to the effect of extracellular inhibitors proved the lipo/hydrophilic character of this luminophore and thus its ability to interact with radicals both outside and inside of cells. The luminol chemiluminescence measured in the presence of extracellular scavengers and the isoluminol chemiluminescence were used with the intention to differentiate the effects of two antihistamine drugs on intra- and extracellular radical formation. In activated human neutrophils, brompheniramine inhibited the extracellular and potentiated the intracellular part of chemiluminescence signal, whereas a reducing effect of loratadine was observed in both compartments.

Reactive oxygen metabolite production is inhibited by histamine and H1-antagonist dithiaden in human PMN leukocytes.

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 w mol/l) and by the H 1 -antagonist dithiaden (1-100 w mol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemilumiescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H 1 -antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A 2 ) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.

An inhibitor of thrombin-stimulated blood platelet aggregation from the salivary glands of the hard tick Amblyomma variegatum (Acari: Ixodidae)

The tropical bont tick. Amblyomma variegation can cause intense skin irritation and inflammation and bites that often develop into septic wounds or abscess in their host. Crude salivary gland extract (SGE) of partially engorged A. variegatum females as well as SGE protein fractions purified by three-step reverse phase HPLC procedure were tested for their antiaggregatory effect on isolated human blood platelets stimulated with thrombin and compared with the effect of recombinant hirudin. At concentrations 10−3 and 5 × 10−3 μg protein/ml the following rank order of antiplatelet activity was detected: AV 16/3 (inhibitor purified from AV-III, third purification) > SGE > AV-II (fraction from first purification) > AV-III (fraction from first purification) > hirudin. The effect of all fractions tested was dose-dependent. For fraction AV 16/3, the inhibitory effect was 49 and 61% for 10−3 and 5 × 10−3 μg protein/ml, respectively. The results suggest that protein fractions from A. variegatum SGE possess an antithrombin effect on human blood platelets with hirudin-like activity.

Effect of chloroquine on isolated mast cells

Chloroquine liberated a relatively low amount of histamine from isolated rat mast cells. In a dose-dependent way, this drug inhibited histamine liberation from mast cells stimulated with compound 48/80, A23187, concanavalin A plus phosphatidylserine (Con A+PS) and abolished histamine liberation induced by exaprolol. The degranulation was decreased in cells stimulated with 48/80, Con A+PS and exaprolol. Chloroquine significantly inhibited the formation of thromboxane B2 in mast cells stimulated with 48/80, Con A+PS and A23187. We assume that chloroquine interferes with mast cells at a plasmic membrane site as well as intracellularly.

The effect of beta-adrenergic blocking drugs and inhibitors of phosphodiesterase on histamine release from isolated mast cells

Differences in the histamine liberation from isolated rat mast cells after beta-adrenergic blocking drugs were demonstrated. In equimolar concentrations histamine release was induced by Kö 1124, Kö 1500, Kö 1560, Kö 1561 and propranolol. Alprenolol, oxprenolol, propranolol, and trimepranol significantly decreased the histamine release induced by compound 48/80. The release of granules from cells was inhibited quantitatively more than the release of histamine. This enabled us to surmise the selective effect of beta-adrenergic blocking drugs on cell membranes of mast cells. The possible mechanisms of release reaction are discussed.

The effect of trimepranol® on thrombocyte function and histamine release in the rat

Trimepranol® a beta-adrenergic blocking drug released serotonin from rat isolated and plasma-rich thrombocytes in vitro. The release was time- and dose-dependent. With the same concentrations this drug inhibited the histamine release from isolated rat mast cells. Aggregation of isolated or plasma-rich thrombocytes induced by ADP was inhibited by Trimepranol®. The inhibition was dose, time dependent and reversed by calcium ions.

Antiplatelet activity of carvedilol in comparison to propranolol

The non-selective vasodilating g -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 w mol/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 w mol/l. In platelets stimulated with thrombin, carvedilol (10 w mol/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 w mol/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 f - and g -adrenergic receptors.

The Natural Stilbenoid Piceatannol Decreases Activity and Accelerates Apoptosis of Human Neutrophils: Involvement of Protein Kinase C

Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role) were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular) membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC)—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.

Role of histamine receptors in the effects of histamine on the production of reactive oxygen species by whole blood phagocytes

AIMS The diverse physiological functions of histamine are mediated through distinct histamine receptors. In this study we investigated the role of H2R and H4R in the effects of histamine on the production of reactive oxygen species by phagocytes in whole blood. MAIN METHODS Changes in reactive oxygen species (ROS) production by whole blood phagocytes after treatment with histamine, H4R agonists (4-methylhistamine, VUF8430), H2R agonist (dimaprit) and their combinations with H4R antagonist (JNJ10191584) and H2R antagonist (ranitidine) were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all compounds were measured using several methods (TRAP, ORAC, and luminol-HRP-H2O2 based CL). KEY FINDINGS Histamine, 4-methylhistamine, VUF8430 and dimaprit inhibited the spontaneous and OZP-activated whole blood CL in a dose-dependent manner. On the other hand, only VUF8430 was able to inhibit PMA-activated whole blood CL. Ranitidine, but not JNJ10191584, completely reduced the effects of histamine, 4-methylhistamine and dimaprit. The direct scavenging ability of tested compounds was negligible. SIGNIFICANCE Our results demonstrate that the inhibitory effects of histamine on ROS production in whole blood phagocytes were caused by H2R. Our results also suggest that H4R agonists in concentrations higher than 10(-6)M may also influence ROS production via binding to H2R.

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

Abstract.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.