ABSTRACT
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.
Subject(s)
Neutrophils/metabolism , Reactive Oxygen Species/metabolism , Respiratory Burst/physiology , Adult , Humans , Male , Middle Aged , Young AdultABSTRACT
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.
Subject(s)
Adrenergic Agonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Carbazoles/pharmacology , Propanolamines/pharmacology , Receptors, Adrenergic/drug effects , Adrenergic alpha-Antagonists/pharmacology , Adrenergic beta-Antagonists/administration & dosage , Animals , Carbazoles/administration & dosage , Carvedilol , Cell Line , Dose-Response Relationship, Drug , Gene Expression Regulation, Enzymologic/drug effects , Lipopolysaccharides , Macrophages/drug effects , Macrophages/metabolism , Mice , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/drug effects , Nitric Oxide Synthase Type II/metabolism , Nitrites/metabolism , Propanolamines/administration & dosage , Receptors, Adrenergic/metabolism , Signal Transduction/drug effectsSubject(s)
Histamine H1 Antagonists/pharmacology , Mesentery , Pheniramine/pharmacology , Reperfusion Injury , Animals , Brompheniramine/pharmacology , Chlorpheniramine/pharmacology , Male , Mesentery/drug effects , Mesentery/pathology , Neutrophils/immunology , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Reperfusion Injury/pathology , Reperfusion Injury/prevention & controlSubject(s)
Histamine H1 Antagonists/metabolism , Ischemia , Mesentery , Reperfusion Injury , Animals , Benzothiepins/pharmacology , Loratadine/pharmacology , Male , Mesentery/blood supply , Mesentery/drug effects , Mesentery/pathology , Models, Biological , Rats , Rats, Wistar , Reperfusion Injury/prevention & controlABSTRACT
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.
Subject(s)
Carbolines/pharmacology , Neutrophils/drug effects , Neutrophils/physiology , Cell Degranulation/drug effects , Cell Degranulation/physiology , Dose-Response Relationship, Drug , Free Radicals/metabolism , Humans , Respiratory Burst/drug effects , Respiratory Burst/physiology , Solubility/drug effects , Superoxides/metabolismABSTRACT
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.
Subject(s)
Antioxidants/pharmacology , Blood Platelets/drug effects , Carbolines/pharmacology , Neutrophils/drug effects , Zymosan/pharmacology , Adult , Blood Platelets/metabolism , Humans , In Vitro Techniques , Male , Middle Aged , Neutrophils/metabolism , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Respiratory Burst/drug effectsABSTRACT
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.
Subject(s)
Antioxidants/pharmacology , Carbolines/pharmacology , Neutrophils/metabolism , Superoxides/blood , Free Radical Scavengers/pharmacology , Free Radicals/blood , Humans , In Vitro Techniques , Luminescent Measurements , Neutrophils/drug effects , Respiratory Burst/drug effects , Stimulation, Chemical , Tetradecanoylphorbol Acetate/antagonists & inhibitors , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
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.
Subject(s)
Acetic Acid , Benzothiepins/therapeutic use , Colitis/drug therapy , Histamine H1 Antagonists/therapeutic use , Animals , Colitis/chemically induced , Colitis/pathology , Colon/blood supply , Colon/enzymology , Colon/pathology , Histamine/physiology , Intestinal Mucosa/pathology , Male , Mast Cells/physiology , Organ Size , Peroxidase/metabolism , Rats , Rats, Wistar , gamma-Glutamyltransferase/metabolismABSTRACT
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.
Subject(s)
Antioxidants/pharmacology , Carbolines/pharmacology , Neutrophils/drug effects , Arachidonic Acid/antagonists & inhibitors , Arachidonic Acid/metabolism , Calcimycin/pharmacology , Glucuronidase/antagonists & inhibitors , Glucuronidase/metabolism , Humans , Ionophores/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/metabolism , Stimulation, Chemical , Superoxides/antagonists & inhibitors , Superoxides/metabolismABSTRACT
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.
Subject(s)
Arachidonic Acid/blood , Chloroquine/pharmacology , Neutrophils/drug effects , Thromboxane B2/blood , Cell Aggregation/drug effects , Humans , Neutrophils/metabolism , Thromboxane B2/biosynthesisSubject(s)
Antirheumatic Agents/pharmacology , Arachidonic Acid/metabolism , Chloroquine/pharmacology , Mast Cells/drug effects , Animals , Dose-Response Relationship, Drug , Male , Mast Cells/cytology , Mast Cells/metabolism , Membrane Proteins/metabolism , Phospholipids/metabolism , Prostaglandin D2/metabolism , Rats , Rats, Wistar , Thromboxane B2/metabolism , p-Methoxy-N-methylphenethylamine/pharmacologyABSTRACT
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.
Subject(s)
Chloroquine/pharmacology , Mast Cells/drug effects , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , In Vitro Techniques , Male , Mast Cells/metabolism , Mast Cells/ultrastructure , Phospholipids/metabolism , Rats , Rats, Wistar , p-Methoxy-N-methylphenethylamine/pharmacologyABSTRACT
Lipophilic exaprolol and hydrophilic atenolol differ in their interaction with mast cell membranes. Exaprolol, as compared with atenolol, significantly decreased 32P incorporation into, but increased arachidonic acid liberation from, membrane phospholipids. Moreover, exaprolol significantly decreased phosphate incorporation in compound 48/80 and ConA-PS treated cells and decreased thromboxane formation in stimulated cells. On the other hand, atenolol decreased significantly only arachidonate liberation from stimulated mast cells. These results corroborate to some extent the effect of exaprolol and atenolol on histamine liberation which correlates with their membrane perturbing properties.
Subject(s)
Adrenergic beta-Antagonists/pharmacology , Atenolol/pharmacology , Cell Membrane/metabolism , Mast Cells/ultrastructure , Propanolamines/pharmacology , Animals , Arachidonic Acid , Arachidonic Acids/metabolism , Cell Membrane/drug effects , Concanavalin A/pharmacology , Male , Membrane Lipids/metabolism , Phosphates/metabolism , Phosphatidylserines/pharmacology , Phospholipids/metabolism , Phosphorus Radioisotopes/metabolism , Rats , Rats, Inbred Strains , Thromboxane B2/biosynthesis , p-Methoxy-N-methylphenethylamine/pharmacologyABSTRACT
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.
Subject(s)
Chloroquine/pharmacology , Histamine Release/drug effects , Mast Cells/physiology , Adrenergic beta-Antagonists , Animals , Calcimycin/pharmacology , Chloroquine/administration & dosage , Concanavalin A/pharmacology , Dose-Response Relationship, Drug , Male , Mast Cells/drug effects , Phosphatidylserines/pharmacology , Propanolamines/pharmacology , Rats , Rats, Inbred Strains , Thromboxane B2/biosynthesis , p-Methoxy-N-methylphenethylamine/pharmacologyABSTRACT
A release of histamine after the lipophilic betablockers exaprolol and propranolol correlates with their capability of displacing the bound membrane Ca2+ and increasing the disorder of phospholipidic membranes of the isolated mast cells. Electron microscopy confirmed intracellular displacement of histamine from granules of mast cells after exaprolol without marked structural changes on the plasmatic membrane. Hydrophilic and selective atenolol, which does not possess a histamine-liberating effect, decreases spontaneous transfer of the intracellular calcium, decreases the disorder of the mast-cell membranes, and together with exaprolol and propranolol inhibits, in dose-dependence way, the gain of extracellular histamine in cells. The inhibitory effect of EDTA, tetrodotoxine and suramine on histamine release after exaprolol explains the non-receptor mechanism of exaprolol effect, which confirms a possibility of induction of adverse effects of blockers of the beta-adrenergic receptor in the development of a bronchospasm.
Subject(s)
Adrenergic beta-Antagonists/toxicity , Propanolamines/toxicity , Propranolol/toxicity , Animals , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Cells, Cultured , Cytoplasm/drug effects , Cytoplasm/ultrastructure , Histamine Release/drug effects , Peritoneal Cavity/cytology , Pleura , Rats , Rats, Inbred StrainsABSTRACT
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.
Subject(s)
Adrenergic beta-Antagonists/pharmacology , Mast Cells/drug effects , Phospholipids/metabolism , Animals , Male , Mast Cells/metabolism , Metipranolol/pharmacology , Propanolamines/pharmacology , Propranolol/pharmacology , Rats , Rats, Inbred StrainsABSTRACT
The highly lipophilic drug exaprolol liberates histamine from isolated mast cells and decreases the uptake of extracellular histamine in a dose-dependent manner. Intracellular histamine depletion was confirmed by electron microscopy and was accompanied by calcium displacement from intracellular storage sites. The significant decrease in membrane fluidity due to exaprolol was temperature-dependent and was most probably a result of its high membrane affinity and intracellular penetration. Membrane perturbation by exaprolol may account for this nonreceptor interaction. This could contribute to the understanding of adverse reactions to beta-adrenoceptor blocking drugs.