István Mucha

MODULATION OF ARACHIDONIC ACID METABOLISM BY PHENOLS : RELATION TO THEIR STRUCTURE AND ANTIOXIDANT/PROOXIDANT PROPERTIES

The effects of substituted catechols (3-methylcatechol, 4-methylcatechol, 4-nitrocatechol, and guaiacol) and trihydroxybenzenes (pyrogallol, propyl gallate, 1,2,4-trihydroxybenzene, and 1,3,5-trihydroxybenzene) on the synthesis of prostaglandin (PG)E2 and leukotriene (LT)B4 were tested in human A23187-stimulated polymorphonuclear leukocytes. The effects were related to their peroxyl-radical-scavenging (antioxidant), superoxide-scavenging (antioxidant), and superoxide-generating (prooxidant) properties. In general, compounds with hydroxyl groups in the ortho position increased PGE2/LTB4 ratio, and compounds with hydroxyl groups in the meta position decreased PGE2/LTB4 ratio. Catechols, which have hydroxyl groups in the ortho position, were the most potent peroxyl radical and superoxide anion scavengers. Trihydroxybenzenes (pyrogallol, 1,2,4-trihydroxybenzene, and 1,3,5-trihydroxybenzene) generated superoxide, whereas dihydroxybenzenes did not. Thus, the positions and number of hydroxyl groups seem to be the most important properties determining the action of phenolic compounds on PGE2/LTB4 ratio and their antioxidant/prooxidant activities.

Exercise enhances vasorelaxation in experimental obesity associated hypertension

OBJECTIVE Regular exercise is recommended for the non-pharmacological treatment of hypertension, but the mechanisms underlying the lowering of blood pressure remain controversial. Therefore, we studied the effects of 22-week-long training on blood pressure, arterial reactivity, and metabolic abnormalities in a model of genetic obesity and moderate hypertension. METHODS Obese and lean Zucker rats were subjected to treadmill exercise from 8 to 30 weeks of age. Blood pressures were measured by the tail-cuff method, and urine was collected in metabolic cages. At the end of the study, the samples for biochemical determinations were taken, and reactivity of isolated mesenteric and carotid arterial rings was examined in standard organ chambers. RESULTS The exercise prevented the elevation of blood pressure which was observed in non-exercised obese Zucker rats, and also reduced blood pressure in the lean rats. The relaxations of norepinephrine-preconstricted mesenteric and carotid arterial rings to acetylcholine and nitroprusside were clearly improved by exercise in the obese rats. In the lean rats exercise enhanced vasorelaxation to nitroprusside in the mesenteric and carotid rings, and to acetylcholine in the carotid preparations. The exercise-induced improvement of endothelium-mediated dilatation to acetylcholine was abolished by nitric oxide synthesis inhibition with NG nitro-L-arginine methyl ester, but not by cyclooxygenase inhibition with diclofenac or functional inhibition of endothelium-dependent hyperpolarization by precontractions with KCl. The urinary excretion of the systemic prostacyclin metabolite (2,3-dinor-6-ketoprostaglandin F1 alpha) was increased two-fold by exercise in the obese and lean rats, whereas that of the thromboxane A2 metabolite (11-dehydrothromboxane B2) remained unaffected. Treadmill training reduced blood glucose, cholesterol, and triglycerides, but did not affect the high levels of insulin in obese Zucker rats. CONCLUSIONS These results suggest that the antihypertensive effect of long-term exercise in experimental obesity related hypertension is associated with improved vasodilatation. This is expressed as enhanced relaxation via endogenous and exogenous nitric oxide, and increased endothelial prostacyclin production. The improved control of arterial tone after training could be attributed to the alleviation of hyperlipidemia and insulin resistance, whereas hyperinsulinaemia per se remained unaffected.

Modulation of arachidonic acid metabolism by phenols: Relation to positions of hydroxyl groups and peroxyl radical scavenging properties

We have shown earlier that catecholamines have opposite regulative effects on prostaglandin (PG)E2 and leukotriene (LT)B4 formation with a receptor-independent mechanism in human polymorphonuclear leukocytes (PMNs) and whole blood. To shed further light on the mechanisms involved and structure-action relationship, we tested the effects of phenols (catechol, hydroquinone, phenol, and resorcinol) on the synthesis of PGE2 and LTB4 in human A23187-stimulated PMNs. To study the mechanism of how phenols influence PGE2 and LTB4 synthesis, their peroxyl radical-scavenging properties were analyzed. In general, low concentrations of phenols stimulated (catechol > hydroquinone >> phenol) and high concentrations inhibited (resorcinol > catechol > hydroquinone > phenol) PGE2 formation. Resorcinol was different from the other phenols: It did not stimulate PGE2 synthesis at all, but it was effective inhibitor at high concentrations. Phenols had only an inhibitory effect on LTB4 formation (catechol = hydroquinone >> phenol > resorcinol). The order of both stochiometric factors and reactivities of phenols for scavenging peroxyl radicals was catechol > hydroquinone > resorcinol >> phenol. According to these results, phenols having hydroxyl groups in ortho- or paraposition have the greatest stimulative effect on PGE2 synthesis, the highest inhibitory action on LTB4 synthesis, and are good antioxidants. Resorcinol, having hydroxyl groups in the metaposition, behaves differently. It neither stimulates PGE2 nor inhibits LTB4 formation, but it is the most potent inhibitor of PGE2 formation. In spite of resorcinols two hydroxyl groups, it mimics as an antioxidant phenol more than catechol and hydroquinone.

Solid-phase extraction of urinary 11-dehydrothromboxane B2 for reliable determination with radioimmunoassay

In this paper we elaborate a one-step procedure for the selective extraction of urinary 11-dehydrothromboxane B2 on octylsilyl silica cartridges for reliable determination with radioimmunoassay. The immunoreactivity profile of nonselectively extracted urine after HPLC separation showed that as much as 70% of the total 11-dehydrothromboxane B2 immunoreactivity comigrates with polar interfering material. Its amount could be considerably decreased using acetonitrile:water (18:82, v/v) as wash solvent before elution of 11-dehydrothromboxane B2 from the cartridge. Alternatively, very high immunoreactive purity was achieved without the preceding wash step by selective elution of the analyte with dichloromethane:hexane (70:30). After both optimized steps in the extraction procedure were combined, immunoreactivity was found only in HPLC fractions corresponding to the retention volume of authentic 11-dehydrothromboxane B2. The homogeneity of this immunoreactivity was confirmed by two-step HPLC separation. A significant correlation of values was observed between samples measured after extraction and those measured after subsequent HPLC purification. A high correlation was also found with concentrations determined by radioimmunoassay using four different antisera. The values of 24 h excretion of 11-dehydrothromboxane B2 in 10 male volunteers (595 +/- 114 ng/g creatinine, mean +/- SD) as well as the inhibitory effect of acetylsalicylic acid (80 +/- 13%) closely correspond with those reported in the literature. This selective extraction procedure provides a high validity in radioimmunoassay without requiring any further purification step.

Nicotine stereoisomers and cotinine stimulate prostaglandin E2 but inhibit thromboxane B2 and leukotriene E4 synthesis in whole blood

The effects of (-)-nicotine (0.0005-500 microM), (+)-nicotine (0.0005-50 microM) and (-)-cotinine (0.0005-500 microM) on arachidonic acid metabolism were investigated in Ca2+ ionophore A23187 (calcimycin)-stimulated human whole blood in vitro. (-)-Nicotine and (-)-cotinine stimulated prostaglandin E2 but inhibited thromboxane B2 synthesis, as has been observed previously in A23187-stimulated polymorphonuclear leukocytes and platelet-rich plasma [Saareks, V., Riutta, A., Mucha, I., Alanko, J., Vapaatalo, H., 1993. Nicotine and cotinine modulate eicosanoid production in human leukocytes and platelet rich plasma. Eur. J. Pharmacol., 248, 345-349.]. (+)-Nicotine also stimulated prostaglandin E2 but inhibited thromboxane B2 synthesis. High concentrations of (-)-nicotine and (-)-cotinine and even nanomolar concentrations of (+)-nicotine inhibited leukotriene E4 synthesis. These results indicate that (-)-nicotine and (-)-cotinine stimulate cyclooxygenase but inhibit thromboxane synthase and 5-lipoxygenase in whole blood in vitro. (+)-Nicotine is capable of affecting in the same direction as well.

Nicotine and cotinine modulate eicosanoid production in human leukocytes and platelet rich plasma

We investigated the effects of nicotine and cotinine (0.5 nM-0.5 mM) on prostaglandin E2 and leukotriene B4 production in human polymorphonuclear leukocytes and on thromboxane B2 formation in human platelet-rich plasma, stimulated by calcium ionophore A23187. Nicotine and cotinine dose-dependently increased prostaglandin E2 synthesis in polymorphonuclear leukocytes from 25% (0.5 nM) up to nearly four-fold (0.5 mM). In concentrations found in the plasma of smokers, nicotine and cotinine increased prostaglandin E2 production by 33% (50 nM) and 50% (500 nM), respectively. Nicotine and cotinine equipotentially reduced both leukotriene B4 production in polymorphonuclear leukocytes and thromboxane B2 production in platelet rich plasma, the inhibition increasing from 20% (0.5 nM) to 60% (0.5 mM). The stimulation of prostaglandin E2 and inhibition of leukotriene B4 and thromboxane B2 production by nicotine and cotinine may due to the pyridine moiety that these compounds have in common.

Prostaglandin and thromboxane synthesizing activity in isolated murine hepatocytes and nonparenchymal liver cells.

Prostanoid synthesis from 3H-arachidonic acid was compared in isolated parenchymal and nonparenchymal murine liver cells. The cells incorporated arachidonic acid into phospholipids but no prostanoid synthesis could be measured during 30 min incubation. Conditions necessary for prostanoid synthesis were different in parenchymal and nonparenchymal cells and the products were also different. Prostanoid synthesis could be induced by in vitro partial hepatectomy: parenchymal cells synthesized thromboxane A2 whereas nonparenchymal cells produced prostaglandin E2 and F2 alpha. Prostaglandin E2 and F2 alpha synthesis could be provoked also by homogenization of the nonparenchymal cells prepared from normal liver, while the homogenates of parenchymal cells prepared from normal liver did not synthesize thromboxane. Imidazole and indomethacin inhibited the production of thromboxane and prostaglandins, respectively. Our results suggest that the various cell types of the liver respond by the synthesis of different and specific prostanoids after the same injury.

Smoking cessation and nicotine substitution modulate eicosanoid synthesis ex vivo in man

The effects of smoking cessation with and without nicotine substitution on prostaglandin E2, leukotriene B4, leukotriene E4, and thromboxane B2 synthesis ex vivo in man were investigated. Blood samples were obtained from 20 healthy non-smoking controls and from 30 healthy smoking volunteers before and 3, 7 and 14 days after smoking cessation. Half of the smokers used nicotine chewing gum as a substitution therapy. Urinary cotinine and trans-3′-hydroxycotinine as well as thiocyanate excretions were used as indicators for the use of nicotine chewing gum and smoking, respectively. Prostaglandin E2, leukotriene E4, and thromboxane B2 were measured from whole blood after calcium ionophore A23187 stimulation by direct radioimmunoassay and leukotriene B4 by RP-HPLC.Prostaglandin E2 and thromboxane B2 syntheses were about three times and leukotriene B4 and E4 syntheses four times higher in smokers than in controls. Three days after smoking cessation without nicotine substitution, levels were lowered significantly to about 70%, 80%, 45% and 60% of the initial values; and after 14 days to 55%, 80%, 45% and 50%, respectively. In the nicotine substitution group no significant decreases were seen during the two-week follow-up.The increased level of eicosanoid synthesis detected in smokers in this ex vivo study may contribute to the harmful cardiovascular effects of smoking. Long-term nicotine substitution might diminish the beneficial effects of smoking cessation due to the possible stimulatory effects of nicotine and cotinine on eicosanoid synthesis even in vivo.

Catecholamines decrease leukotriene B4 and increase thromboxane B2 synthesis in A23187-stimulated human whole blood

Catecholamines (adrenaline, dopamine, isoprenaline, noradrenaline) and caffeic acid (catecholic compound without adrenergic receptor activity) decreased leukotriene (LT)B4 synthesis in A23187-stimulated human whole blood. Salbutamol, a non-catecholic beta 2-adrenergic agonist, did not influence LTB4 synthesis. Catecholamines stimulated thromboxane (TX)B2 synthesis with a concomitant inhibition of LTB4 synthesis; caffeic acid and salbutamol did not stimulate TXB2 synthesis. These results, obtained in A23187-stimulated whole blood, which also takes into account the complex interaction between different cell types, are similar to our previous results with polymorphonuclear leukocytes. Catecholamines show an opposite effect on lipoxygenase and cyclooxygenase pathways, which may give rise to a marked change in LT/TX ratio in physiological or pathological conditions where sufficient concentrations of catecholamines are present.

Prostaglandin E2 adn gestational hypotension in rabbits

Arterial levels of 13,14-dihydro-15-keto-PGE2 (PGE2-M), a stable metabolite of prostaglandin E2 (PGE2) were compared between unanesthetized pregnant (n = 12) and nonpregnant (n = 8) rabbits with the aim of elucidating the role PGE2 in the development of physiological hypotension associated with pregnancy. On the 20th and 22nd days of the 30 day gestation period the mean arterial concentrations of PGE2-M were about 10-times higher (p less than 0.05) and largely variable as compared to that of nonpregnant rabbits. Mean arterial pressure was not lower on either the 20th (69 +/- 4 mmHg, mean +/- SD) or the 22nd (70 +/- 3 mmHg) days of gestation (dg) than in nonpregnant rabbits (69 +/- 4 and 73 +/- 6 mmHg, respectively). On the 23rd dg hypotension was invariably present (61 +/- 5 mmHg vs 72 +/- 4 in nonpregnants, p less than 0.001), but arterial levels of PGE2-M (31.0 +/- 31.6 ng/ml) did not overcome those measured on earlier, normotensive days of gestation. Hypotension was also evident in a subgroup of pregnant rabbits (n = 4) with low PGE2-M concentrations in the nonpregnant range (3.2 +/- 1.5 ng/ml vs 1.9 +/- 1.2 in nonpregnant rabbits, ns). Since the arterial level of PGE2-M proved to correlate (p less than 0.001) with both the uteroplacental venous and renal venous PGE2 concentrations, we suggest that a key role of uteroplacental and renal PGE2 played in the development of gestational hypotension is not probable in rabbits.