Biomedical subjects
Publications and source records attributed to V Jancinová.
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Carvedilol inhibits luminol-enhanced chemiluminescence of reactive oxygen metabolites in vitro. In this study it was found that, in the cell-free system, carvedilol dose-dependently decreased chemiluminescence in the following ranking order of radicals: hydroxyl radical > hydrogen peroxide > superoxide radical. The inhibition of myeloperoxidase was significant with carvedilol concentrations of 10 and 100 micromol/l and manifested in the concentration-dependent shift of chemiluminescence peaks to the right. In whole blood, carvedilol in concentrations of 10 and 100 micromol/l significantly inhibited chemiluminescence induced by both receptor-bypassing stimuli (A23187, PMA) and receptor-operating stimuli (fMLP, OpZ). Carvedilol dose-dependently inhibited chemiluminescence of isolated human polymorphonuclear leucocytes in the ranking order of stimuli: A23187 > OpZ > fMLP. In the presence of blood platelets, carvedilol did not substantially change chemiluminescence induced by fMLP and OpZ, while it was much more effective on chemiluminescence stimulated with calcium ionophore A23187. This could be the result of the supportive effect of serotonin liberated from platelets by A23187.
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The non-selective vasodilating beta-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 micromol/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 micromol/l. In platelets stimulated with thrombin, carvedilol (10 micromol/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 micromol/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 alpha- and beta-adrenergic receptors.
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.
Chloroquine inhibited human platelet aggregation in vitro both at receptor- and nonreceptor-operated stimuli. The inhibition was dose-dependent, recorded on isolated platelets as well as in platelet-rich plasma, and followed the rank order of stimuli: adrenaline (second phase)>phorbol 12-myristate 13 acetate>adenosine diphosphate>adrenaline (first phase)>thrombin>calcium ionophore A23187. In thrombin-activated platelets, chloroquine decreased in a dose-dependent manner phospholipase A(2)-induced arachidonic acid liberation from membrane phospholipids, malondialdehyde formation (a marker of membrane phospholipid peroxidation), and thromboxane generation, considered the most potent autoaggregatory agent. Chloroquine only slightly altered the arachidonic acid cascade of platelets stimulated with A23187 and phorbol 12-myristate 13 acetate. Histamine formation and liberation induced with thrombin and A23187 were not affected by chloroquine. On the other hand, thrombin-stimulated serotonin secretion was significantly decreased with chloroquine in the concentration of 10 micromol/L. This indicated that chloroquine might interfere with stimulated secretion from platelets. The results suggest that chloroquine inhibited activated platelets: first, intracellularly; second, in a close relationship to the intraplatelet Ca(2+) mobile pool; and third, most probably at the site of platelet phospholipase A(2) activation.
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.
Using simultaneous recording of aggregation and chemiluminescence, responses of human polymorphonuclear leukocytes, blood platelets and their mixture were investigated after stimulation by specific as well as non-specific stimuli for each cell. In our experimental settings, aggregation of platelets and PMN leukocytes was increased in the following order of stimuli: PMA<A23187<thrombin and FMLP<A23187<PMA, respectively. FMLP selective for PMN leukocytes did not activate platelet aggregation, and, on the other hand, aggregation of PMN leukocytes was not induced by thrombin. The presence of PMN leukocytes decreased aggregatory responses of platelets to all the stimuli applied. Luminol amplified chemiluminescence of PMN leukocytes was increased in the order: thrombin<FMLP<A23187<PMA, but platelets alone did not show detectable chemiluminescence response to any stimulation. Platelets significantly decreased chemiluminescence of PMN leukocytes and their inhibitory effect did not depend on the type of stimulation. These observations suggest that under defined experimental conditions human PMN leukocytes and platelets might decrease mutually their dominant responses in vitro. The inhibitory effect was dependent either on the selectivity of stimuli, or on cell to cell contact before stimulus addition.
The antiarrhythmic and cardioprotective drug stobadine, possessing antioxidant and neuroprotective properties, was studied as to its in vitro effect on aggregation of human blood platelets. Pretreatment of platelets with stobadine for 30 s inhibited stimulated platelet aggregation in a dose-dependent way. Depending on the aggregation stimulus used, the minimal effective concentrations of the drug were 1 micromol/l (adrenaline), 200 micromol/l (ADP), and 1,000 micromol/l (PMA). Aggregation induced with thrombin or Ca2+-ionophore A23187 was not changed in the presence of stobadine even in the concentration of 1,000 micromol/l. Addition of stobadine 30 s after adrenaline was also effective and terminated aggregation (100 and 1,000 micromol/l) or prolonged onset of its second phase (10 micromol/l). The presented experiments showed stobadine as a potent inhibitor of adrenaline-induced aggregation, indicating its involvement in the observed antithrombotic and cytoprotective activity.
On the basis of values corresponding to concentrations exhibiting 50% inhibition of platelet aggregation induced with different stimuli and 50% inhibition of arachidonic acid liberation and thromboxane generation, we compared the antiplatelet effect of two cationic amphiphilic drugs--chloroquine and dithiaden. Compared to chloroquine, dithiaden was much more effective in inhibiting platelet aggregation, A-23187 induced arachidonic acid liberation and thromboxane generation. Chloroquine, on the other hand, was more effective in the inhibition of thrombin-induced arachidonic acid liberation.
Stimulation of human blood platelets with Ca2+-ionophore A23187 at the concentrations of 0.5, 1, 2 and 3 micromol/ litre increased the total platelet histamine content from 25 ng (resting platelets) to 35, 40, 42 and 47 ng/10(8) platelets, respectively. The maximum rise was 90% , while in the presence of thrombin (0.01-1 NIH U/ml) or phorbol 12-myristate 13-acetate (PMA) (10-100 nmol/l) histamine content rose maximally by 40%. Ionophore induced a histamine increase in the same concentration range as that needed to induce platelet aggregation and liberation of [3H]-arachidonic acid. Decrease of temperature from 37 to 0 degrees C or inhibitors of platelet functions, such as acetylsalicylic acid (1 mmol/litre) or [H1]-histamine receptor antagonist Dithiaden (0.1 mmol/litre), 1 inhibited histamine liberation from A23187- stimulated platelets, but did not affect the total rise in histamine content. The presented results indicate that, similarly as with other stimuli, Ca2+-ionophore A23187 activation of human blood platelets is accompanied by histamine synthesis.
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The effects of bromadryl, dithiaden, chloroquine and propranolol on thrombin-stimulated rat platelet aggregation (measured turbidimetrically) and thromboxane B2 generation (detected by an RIA method) were compared with four selected physico-chemical parameters of these drugs. Platelet aggregation was inhibited in the rank order of potency: bromadryl > dithiaden > propranolol > chloroquine, which corresponded with the decrease in the net charge of the terminal methyl-(or ethyl-) groups in the side chain and with the increase of the dipole moment of drug molecules. On the other hand, the rank order of potency in which the drugs tested inhibited thromboxane B2 formation (chloroquine > dithiaden > bromadryl > propranolol) correlated well with the decline in molar refractivity of the drugs. No relationship was found between inhibitory effects of drugs and their partition coefficients. The results presented indicate that inhibition of platelet functions might consist of several types of drug-cell interactions, depend on the structure and physico-chemical properties of the drugs and cannot be estimated simply on the basis of partition coefficients.
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.