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 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.

Effects of histamine H1 antagonist dithiaden on acetic acid-induced colitis in rats.

To assess the possible involvement of mast cells and/or their mediators in inflammatory bowel diseases, the effect of the histamine H1 antagonist Dithiaden was studied on a model of acetic acid-induced colitis in rats. Dithiaden pretreatment by intracolonic administration was found to reduce the extent of acute inflammatory colonic injury. This was manifested by a decrease in the score of gross mucosal injury, by lowered colonic wet weight and by diminished myeloperoxidase activity reflecting reduced leukocyte infiltration. Vascular permeability and gamma-glutamyl transpeptidase activity, elevated by acetic acid exposure, were decreased after Dithiaden pretreatment. The results indicate that locally administered Dithiaden may protect the colonic mucosa against an acute inflammatory attack by interfering with the action of the major mast cell mediator histamine.

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.

Human polymorphonuclear leukocytes: effect of chloroquine on aggregation, arachidonic acid liberation and thromboxane B2 generation.

The effects of the antimalarial drug chloroquine (CQ) on arachidonic acid (AA) liberation from, thromboxane B2 (TXB2) formation in, and aggregation of isolated human polymorphonuclear (PMN) leukocytes stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) were investigated. CQ decreased aggregation of stimulated PMN leukocytes, however in contrast to AA liberation and TXB2 formation, lower concentrations were more effective than the highest one used. This effect may be associated with an increase in intracellular pH, reported to be induced by higher CQ concentrations, possibly counteracting the inhibition of aggregation, and/or eliminating negative feed back control of aggregation by lack of prostaglandins.

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 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.

[The effect of adrenergic beta receptor blockers on phospholipid metabolism in mast cells]. / Vplyv blokátorov betaadrenergického receptora na metabolizmus fosfolipidov v zírnych bunkách.

The aim of this study was to determine whether beta adrenergic receptor blocking drugs exaprolol, metipranolol and propranolol effect the metabolism of phospholipids in isolated rat mast cells. The phospholipids were labelled by 3H-arachidonic acid (3H-AA) and 32P. Exaprolol, metipranolol and propranolol significantly modulated 32P incorporation into phospholipids of resting and 48/80 stimulated cells. Atenolol had no effect. Studies with 3H-AA Labelled mast cells showed an enhanced liberation of arachidonate related radioactivity on exaprolol and propranolol treatment. The results indicated that 3H-activity was lost mainly from phosphatidylethanolamine. Atenolol and metipranolol significantly decreased the 48/80 stimulated 3H-AA release.

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.

Amine transport in isolated rat mast cells treated with betaadrenoceptor blocking drugs.

Histamine and serotonin uptake in isolated rat mast cells is decreased in the presence of betaadrenoceptor blocking (BAB) drugs. Such inhibition is dose-dependent and is evidently higher for the inhibition of serotonin uptake. The most potent drugs were the highly lipophilic compounds Kö 1124 and propranolol. No difference was found among the various BAB drugs in their ability to decrease histamine uptake. It is evident from the ratio between histamine and serotonin liberation on one side, to the uptake of both amines in mast cells on the other side, that with the exception of Kö 1124, all investigated BAB drugs possesses higher inhibition of extracellular amine uptake in comparison with histamine and serotonin liberation. Such an effect might be a result of a primary interaction of BAB drugs with mast cells at the plasma membrane.

On the interaction of beta-adrenoceptor-blocking drugs with isolated mast cells.

The interaction of beta-adrenoceptor blocking drugs (BAB drugs) with isolated mast cells resulted, according to the compound, in either a liberation of biogenic amines or an inhibition of stimulated amine release. The liberatory drugs exaprolol and Kö 1124 decreased the level of cAMP, stimulated the activity of cyclic nucleotide-phosphodiesterase, decreased the incorporation of orthophosphate into membrane phospholipids and rapidly displaced calcium from binding sites in mast cells. The inhibitory drugs alprenolol, metipranolol, oxprenolol, practolol and propranolol, possessing lower liposolubility, produced opposite effects. Drugs from both groups displaced histamine from binding sites in isolated mast cell granules. The interaction of BAB drugs with mast cells is a result of non-specific rather than specific receptor interactions. Inhibitory drugs interfere with mast cells at membrane sites while liberatory drugs penetrate the membrane, thus acting both at the level of membrane and intracellularly.

Effect of stobadine on stimulated isolated mast cells.

Stobadine, an antiarrhythmic drug with antihistaminic properties, did not liberate histamine from mast cells in vitro. Compound 48/80-stimulated histamine liberation and degranulation was decreased in the presence of stobadine in a dose-dependent way. The spontaneous as well as stimulated calcium displacement in mast cells was significantly decreased by stobadine. Stobadine most probably possesses a membrane-stabilizing effect on isolated rat mast cells.

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.

Effect of chloroquine on mast cell membranes.

The effect of chloroquine (CQ) on phospholipid turnover and de novo synthesis in isolated rat mast cells (IRMC) was studied by determining the incorporation of 32P and 14C-glycerol into IRMC phospholipids. Incubation of mast cells with chloroquine increased 32P incorporation into PI and PS whilst it decreased 32P incorporation into PC, PE and PA. In mast cells pretreated with CQ and subsequently stimulated with compound 48/80, 32P incorporation into PI, PS and PA fractions was enhanced, while it was decreased into PC and PE, in comparison to 48/80 stimulated IRMC. 14C-glycerol incorporation into total IRMC phospholipids was not significantly changed by CQ and compound 48/80 treatment and neither was any dose-dependent effect of CQ on individual phospholipids detected. Our results indicate that chloroquine, similarly to other cationic amphiphilic drugs, may alter membrane PL turnover without changing de novo synthesis of phospholipids.

Beta-adrenoceptor blocking drugs and calcium transport in isolated rat mast cells.

The beta-adrenoceptor blocking (BAB) drugs exaprolol (EXA), metipranolol (MET) and propranolol (PRO) inhibited histamine liberation and degranulation from isolated rat mast cells stimulated with the calcium ionophore A23187. MET was the most and EXA the least active. Atenolol (ATE) had no effect. Inhibition by BAB drugs of secretion induced with A23187 was not accompanied by any change in 45Ca uptake. On the other hand, EXA, MET and PRO significantly decreased 45Ca uptake by mast cells stimulated with 48/80. The effect of BAB drugs on inhibition of A23187-induced secretion from isolated mast cells was dependent on the lipid solubility of the studied drugs.

Effect of beta-adrenoceptor blocking drugs on 32P incorporation into and arachidonic acid liberation from phospholipids in stimulated rat mast cells.

The lipophilic beta-adrenoceptor blocking (BAB) drugs metipranolol, propranolol and exaprolol significantly decreased 48/80- and A23187-induced 32P incorporation into rat mast cell phospholipids. Exaprolol was the most active, followed by propranolol and metipranolol. Atenolol and metipranolol significantly decreased the 48/80-stimulated, and metipranolol and exaprolol the A23187-stimulated 3H-arachidonic acid liberation from isolated mast cells.

Evidence for intracellular histamine liberation in isolated rat mast cells.

The beta-adrenoceptor blocking drug exaprolol liberated histamine from isolated rat mast cells in a dose- and time-dependent way. Histamine was liberated within seconds and was not followed by a parallel granule liberation. The inhibition of histamine liberation was induced with low temperature, low pH, high concentration of Ca2+, TTD, suramin and EDTA. Subcellular distribution of 3H-exaprolol demonstrated a quantitative relationship between histamine depletion against exaprolol uptake in isolated rat mast cell granules. A nonspecific mechanism of action in the effect of exaprolol on mast cells is discussed. It is proposed that the drug acts on mast cells due to the direct and indirect ion exchange mechanism resulted in disproportion between histamine and granule liberation.