Rado Nosál

On the inhibitory effect of chloroquine on blood platelet aggregation

Chloroquine inhibited aggregation of rat blood platelets in a dose-dependent way. The inhibitory effect decreased, depending on the aggregation stimulus used, in the rank order of potency: ADP > thrombin > A23187. Effect of 1 millimolar chloroquine on A23187-stimulated aggregation was significantly decreased or completely abolished by addition of Ca2+ ions in the concentration of 0.1 or 1 mmol/l, respectively. For thrombin-stimulated aggregation the effect of chloroquine was partially decreased by corresponding isomolar calcium; platelet aggregation induced by ADP was not changed in the presence of extracellular Ca2+ ions. Addition of chloroquine during aggregation resulted in disaggregation of ADP-stimulated platelets and inhibition of thrombin- and A23187-induced aggregation.

The interaction of β-adrenoceptor blocking drugs with platelet aggregation, calcium displacement and fluidization of the membrane

beta-Adrenoceptor blocking drugs interfere with adenosine diphosphate-stimulated platelet aggregation. Alprenolol, exaprolol, Kö 1124 and propranolol inhibited the aggregation, metipranolol decreased the extent and rate of aggregation significantly. Atenolol potentiated the aggregation measured by amplitude significantly. The interaction of beta-adrenoceptor blocking drugs with aggregation correlated with the displacement of calcium ions from binding sites in isolated platelets and the fluidization of the whole platelets and isolated platelet membrane as measured with electron spin resonance of the spin probe. The most potent were highly liposoluble drugs alprenolol, exaprolol, metipranolol and propranolol which increased the calcium displacement and membrane fluidity, the least active was atenolol decreasing these phenomena. The inhibition by beta-adrenoceptor blocking drugs of stimulated platelet aggregation is rather a result of unspecific than specific receptor interaction.

Chloroquine inhibits stimulated platelets at the arachidonic acid pathway

Chloroquine inhibited arachidonic acid liberation from membrane phospholipids of thrombin- and A23187- stimulated platelets. In addition, it dose-dependently inhibited stimulated malondialdehyde formation and thromboxane B2 generation in the same platelets. The linear correlation between the inhibition of arachidonic acid liberation and malondialdehyde formation indicated that chloroquine inhibited activated phospholipase A2 in thrombin-stimulated platelets, similarly as it does in different cells and tissues. Yet, the nonlinear relationship between arachidonic acid liberation along with malondialdehyde formation and thromboxane generation as well as aggregation suggest that phospholipase A2 does not seem to be the only site of chloroquine action. Rather, it may affect platelets either at other levels of the arachidonic acid cascade too, or at some different stimulatory pathways, like intraplatelet calcium mobilisation, phosphoinositide cycle, calmodulin and protein kinase C activation.

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 of3H-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.

Cationic amphiphilic drugs and platelet phospholipase A2 (cPLA2)

The aim of the study was to verify and compare the effect of cationic amphiphilic drugs (CAD) from different pharmacological groups on activation of platelet phospholipase A2 (PLA2)--the essential enzyme of arachidonic pathway in blood platelets. Beta-adrenoceptor-blocking (BAB) drugs inhibited platelet aggregation in the rank order of potency: propranolol>alprenolol>metipranolol>atenolol. The higher the inhibition of arachidonic acid (AA) liberation by BAB drugs, the higher the inhibition of aggregation. Similarly did the H1-histamine antagonists bromadryl (BRO) and dithiaden (DIT) as well as the antimalarial chloroquine (CQ) show antiplatelet effect in vitro in the rank order of potency: DIT>BRO>CQ. Dose-dependent inhibition of aggregation was followed by the inhibition of AA liberation from membrane phospholipids of platelets stimulated either at the receptor site (thrombin) or by a stimulus bypassing membrane receptors (Ca2+ ionophore A23187). The rank order potency for inhibition of stimulated 3H-AA liberation from membrane phospholipids was: (a) for BAB drugs: propranolol>alprenolol>metipranolol, (b) for other drugs: DIT>BRO>CQ. The investigated drugs interference with stimulated liberation of AA showed nonspecific inhibition of platelet cytosolic PLA2 (cPLA2) by these drugs at intracellular level. The results revealed that besides the inhibition of cyclooxygenase pathway and receptors for adenosine diphosphate (ADP) and glycoproteins Gp IIbIIIa, the interaction of drugs with cPLA2 may represent a further site for antiplatelet action.

Differences among betaadrenoceptor blocking drugs in modifying platelet aggregation and arachidonic acid liberation under thrombin stimulation

The dose-dependent inhibition of thrombin stimulated platelet aggregation due to beta-adrenoceptor blocking drugs followed the rank order of potency: propranolol greater than alprenolol greater than metipranolol and correlated with arachidonic acid (3H-AA) liberation. Atenolol which slightly potentiated stimulated aggregation increased also the liberation of 3H-AA from membrane phospholipids of isolated platelets. Stimulation of platelets resulted in decreased 3H-AA incorporation into phosphatidylcholine, phosphatidylinositol and phosphatidic acid and increased incorporation into phosphatidylethanolamine and phosphatidylserine. Alprenolol, metipranolol and propranolol enhanced the incorporation of 3H-AA into phosphatidylcholine of stimulated platelets.

Cooperation of chloroquine and blood platelets in inhibition of polymorphonuclear leukocyte chemiluminescence.

Effect of activated blood platelets and chloroquine on concentration of reactive oxygen species produced by polymorphonuclear leukocytes (PMNL) stimulated with Ca(2+)-ionophore A23187 was investigated. Oxygen metabolites localized outside PMNL were visualized by isoluminol enhanced chemiluminescence, whereas chemiluminescence, enhanced with luminol and measured in the presence of the extracellular scavengers superoxide dismutase and catalase, was used for the detection of radicals originated intracellularly. Significant reduction of chemiluminescence was observed in the presence of platelets (added to PMNL in the physiological cell ratio 50:1) and of chloroquine (10 and 100 micromol/L). Although chloroquine decreased effectively both the extra- as well as the intracellular part of the chemiluminescence signal, the activity of platelets occurred largely outside PMNL. Serotonin liberated from platelets by A23187 appeared to be involved in inhibition of chemiluminescence; its concentrations achieved in platelet supernatants were found to be sufficient for elimination of PMNL-derived oxygen metabolites. The presented results indicated that chloroquine and blood platelets cooperate in inhibition of chemiluminescence because their common effect was found to be much more extensive than reduction induced by these inhibitors separately. Therefore, for accurate prediction of drug effect in the whole organism, the use of multicellular test systems seems to be pertinent.

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 the membrane and intracellularly.

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.

Naturally appearing N-feruloylserotonin isomers suppress oxidative burst of human neutrophils at the protein kinase C level.

N-feruloylserotonin (N-f-5HT) isomers, isolated from seeds of Leuzea carthamoides (Wild) DC, inhibited dose-dependent oxidative burst in human whole blood and isolated neutrophils in vitro, which were measured by luminol- and/or isoluminol-enhanced chemiluminescence in the following rank order of stimuli: PMA > OpZ > calcium ionophore A23187. In isolated neutrophils that were stimulated with PMA, N-f-5HT isomers were effective against extracellular and intracellular reactive oxygen species. Liberation of ATP, analysis of apoptosis, and recombinant caspase-3 activity revealed that N-f-5HT isomers, used in concentrations up to 100 μM, did not alter the viability and integrity of isolated neutrophils. Western blot analysis documented that in concentrations of 10 and 100 μM, N-f-5HT isomers significantly decreased PMA-induced phosphorylation of PKC α/β II. The results suggest that N-f-5HT isomers are an effective, naturally occurring substance with a potent pharmacological effect on the oxidative burst of human neutrophils. It should be further investigated for its pharmacological activity against oxidative stress in ischemia-reperfusion, inflammation and other pathological conditions.