Ilker Kanzik

Comparison of antioxidant activities of aminoguanidine, methylguanidine and guanidine by luminol-enhanced chemiluminescence

The objective of this study was to investigate the ability of aminoguanidine, methylguanidine and guanidine to inhibit free radicals or metabolites generated by either stimulated human leucocytes or cell‐free systems using luminol‐enhanced chemiluminescence (CL). Aminoguanidine (0.1 μM–10 mM), methylguanidine (10 μM–10 mM) and guanidine (10 μM–10 mM) produced concentration‐dependent inhibition (96±0.1%, n=7, 59±1.3%, n=6, and 62±3%, n=6, P<0.05 at 10 mM, respectively) in FMLP‐stimulated leucocytes CL. In cell‐free experiments, hydrogen peroxide (H2O2), hypochlorous acid (HOCl), hydroxyl radical and peroxynitrite‐induced CL responses were initiated by hydrogen peroxide (3.5 mM), NaOCl (50 μM), FeSO4 (40 nM) and peroxynitrite (20 nM), respectively. Aminoguanidine, methylguanidine and guanidine produced concentration‐dependent inhibition in H2O2‐(69±0.7%, n=7, 26±1%, n=6, and 15±0.5%, n=6, at 1 mM, respectively) and HOCl‐(84±0.3%, n=6, 50±1%, n=6, and 29±1%, n=7, at 1 mM, respectively) induced luminol CL. Peroxynitrite‐induced CL was markedly attenuated in a concentration‐dependent manner by aminoguanidine (99±0.1%, n=6, at 10 mM), methylguanidine (5±0.2%, n=6, at 10 mM) and guanidine (27±0.4%, n=7, at 10 mM). However, inhibition with aminoguanidine was found to be more marked than with methylguanidine and guanidine. Aminoguanidine (95±0.5%, n=6, at 1 mM) and methylguanidine (25±1%, n=6, at 1 mM), but not guanidine (2±1%, n=6, at 1 mM), significantly decreased ferrous iron‐induced CL. Collectively, these data suggest that aminoguanidine and a high concentration (0.1 mM) of methylguanidine have direct scavenging activities against H2O2, HOCl, hydroxyl radical and peroxynitrite. Guanidine, at a high concentration (0.1 mM), scavenges H2O2, HOCl and peroxynitrite, but not the hydroxyl radical. These direct scavenging properties may contribute to inhibitory effects of these compounds on human leucocyte CL.

Evidence for the involvement of peroxynitrite in ischaemic preconditioning in rat isolated hearts.

The aim of this study was to investigate the involvement of peroxynitrite, reactive metabolite originating from nitric oxide and superoxide, in preconditioning of the ischaemic myocardium in rat isolated hearts. Isolated hearts perfused with Krebs‐Henseleit solution were preconditioned either by 3 min of coronary artery occlusion (CAO) or by peroxynitrite administration at three different concentrations (0.1, 1, 10 μM) for 3 min, followed by 10 min reperfusion and 30 min of CAO. Peroxynitrite, at 1 μM concentration, decreased the incidence of VT from 100% (n=14) to 62% (n=13) and abolished the occurrence of VF (50% in the control group). N‐2‐mercaptopropionylglycine (MPG, 1 μM–10 mM) produced a concentration‐dependent inhibition of peroxynitrite signals in luminol chemiluminescence and 67±1% inhibition was observed at 100 μM (n=7). MPG (at 300 μM, n=7) added to the perfusate 10 min prior to ischaemic preconditioning or peroxynitrite infusion and maintained until CAO, significantly reversed the beneficial effects of the ischaemic and peroxynitrite‐treated groups. MPG administration in the peroxynitrite‐treated group increased the incidence of VT from 62% (n=13) to 100% (n=10) and total VF from 0% (n=0) to 67% (n=10). Similarly, MPG elevated the incidence of VT from 50% (n=10) to 100% (n=8) in the ischaemic preconditioned group. On its own, MPG did not affect the severity of cardiac arrhythmias. These results suggest that endogenously produced peroxynitrite plays a significant role in the antiarrhythmic effect of ischaemic preconditioning in the rat isolated hearts.

Hypoglycaemic effect of Momordica charantia extracts in normoglycaemic or cyproheptadine-induced hyperglycaemic mice

The hypoglycaemic effect of orally administered extracts of Momordica charantia L. fruits was examined in normoglycaemic or cyproheptadine-induced hyperglycaemic mice. The aqueous extract reduced the fasting glucose levels of hyperglycaemic or normoglycaemic mice. However, the ethanol extract did not affect the fasting or nonfasting glucose levels significantly in both groups of mice. There was no significant difference between the glucose-loaded and glucose-loaded plus aqueous extract given group. On the other hand, oral glucose-loading of the cyproheptadine-induced hyperglycaemic animals reduced the fasting glucose levels significantly. These results showed that aqueous extract of M. charantia fruits has a hypoglycaemic activity without improving the tolerance to glucose in cyproheptadine-induced diabetic mice.

Nitric-oxide production by human umbilical vessels in severe pre-eclampsia.

Objective: Pre-eclampsia is characterized by an increased vascular tone which might be related to an abnormal endothelial cell function. As representatives of the fetal circulation, we compared the nitric oxide (NO)-releasing capacity of human umbilical vessels from normal and pre-eclamptic pregnancies. Methods: Normal and pre-eclamptic umbilical vessels were mounted in parallel in an organ chamber with three perfusion lines superfusing the same detector tissue (rubbed rat aortic ring). In this cascade system the capacity of the umbilical vessels to release NO was measured under basal conditions and after stimulation with histamine, bradykinin or calcium ionophore A 23187. Results: Relaxations dependent on basal NO release were found to be significantly higher in pre-eclamptic vessels (especially in veins) than in normal vessels. Conversely, stimulaled NO release in response to histamine or bradykinin was significantly decreased in pre-eclamptic umbilical arteries, but not in veins, compared with normal vessels. However, there was no significant difference in the release of NO in response to A 23187 between normal and pre-eclamptic vessels. Conclusions: The NO-releasing and NO-producing capacity in the vessels from fetal circulation is not diminished in pre-eclampsia. However, in pre-eclamptic umbilical arteries the NO release in response to certain stimuli (histamine or bradykinin) is diminished, probably as a result of alterations in the receptor function.

Flow Injection Analysis for Monitoring Antioxidant Effects on Luminol Chemiluminescence of Reactive Oxygen Species

Abstract Knowledge of the antioxidant capacity of specific chemicals is essential to understanding the susceptibility to oxidative stress. Various assays have been developed for measuring the scavenging capacity of molecules. In the present study we used a continuous flow system for monitoring chemiluminescence (CL) reactions initiated by superoxide ( ) (derived from xanthine–xanthine oxidase reaction), hydrogen peroxide (H2O2), hypochlorite anion (−OCl) (derived from NaOCl), hydroxyl (·OH) (generated from O2–FeSO4-buffer), or peroxynitrite (ONOO−) (freshly synthesized). By adapting the flow injection analysis (FIA) method, inhibition of luminol-CL responses of these reactive oxygen species (ROS) by ascorbic acid (well-characterized, chain-breaking antioxidant) and other antioxidants were also investigated. Data showed that to monitor the antioxidant sensitivity of CL responses initiated by ROS is possible by using FIA method.

The beneficial effects of peroxynitrite on ischaemia–reperfusion arrhythmias in rat isolated hearts

The simultaneous production of nitric oxide (NO) and superoxide leads to the formation of a potent toxic metabolite peroxynitrite (ONOO(-)). However, ONOO(-) at low concentrations has been found to exert cardioprotective effects. The purpose of the present study was to investigate the effects of exogenous ONOO(-) on ischaemia-reperfusion arrhythmias. We studied the concentration-response effects of ONOO(-) (0.4, 4, 40 microM ml(-1) min(-1) for 20 min) in rat isolated hearts perfused with Krebs-Henseleit solution. The 0.4 microM concentration of ONOO(-) was selected for further experiments since it did not affect the sinus rhythm. In the hearts subjected to 10 min of ischaemia followed by 10 min of reperfusion during 0.4 microM ml(-1) min(-1) ONOO(-) infusion, the incidence of ventricular fibrillation was decreased significantly from 93% to 38% (n=8) and none of the hearts had an irreversible ventricular fibrillation. Urate, a ONOO(-) scavenger (at 1 mM, n=7), added to the perfusate 5 min prior to the coronary artery occlusion and maintained throughout the experimental period, did not significantly modify the beneficial effects of ONOO(-). Although L-N(G)-nitroarginine methylester (L-NAME) (100 microM, n=8) had no effect, superoxide dismutase (10 U ml(-1))+catalase (100 U ml(-1)) increased the number of ventricular ectopic beats from 91+/-32 to 286+/-83 (n=5) and augmented the incidence of irreversible ventricular fibrillation from 0% to 60%. There were no marked changes in the time of onset of the first arrhythmias in any group. These results suggest that ONOO(-) at a low concentration may exert beneficial effects on ischaemia-reperfusion-induced arrhythmias in rat isolated hearts.

The role of tyrosine kinase in hypoxic constriction of sheep pulmonary artery rings

Complex and incompletely understood mechanisms underline the vascular responses to hypoxia. Recent studies showed that the tyrosine kinase pathway is involved in vasoconstriction of vascular smooth muscle. Therefore, the aim of this study was to determine the tyrosine kinase pathway for the hypoxic contraction in large-diameter sheep pulmonary artery rings in vitro by studying the effects of selective inhibitors of tyrosine kinase and of a protein tyrosine kinase inhibitor. Lowering the pO2 from 96 to 5 mm Hg caused a contraction in arteries precontracted with 5-hydroxytryptamine (5-HT) but not under resting force. Preincubation of arteries with the tyrosine kinase inhibitors, genistein and tyrphostin, abolished the hypoxic contraction without affecting 5-HT contractions. Inhibition of tyrosine phosphatase activity by sodium orthovanadate increased the hypoxic vasoconstriction in 5-HT-precontracted arteries. These results suggest that the tyrosine kinase pathway is involved in hypoxic pulmonary vasoconstriction in sheep isolated pulmonary artery rings.

The comparison of vascular reactivities of arterial and venous grafts to vasodilators: Management of graft spasm

Graft spasm in the perioperative or postoperative period increases the risk of morbidity and mortality after coronary revascularization and hence necessitates urgent treatment. We have studied the effects of various vasodilators against noradrenaline- and endothelin-1-induced spasms in saphenous vein, internal mammary artery and gastroepiploic artery. In internal mammary and gastroepiploic arteries, the nitrovasodilators, sodium nitroprusside and glyceryl trinitrate, effectively reversed the spasms induced either with noradrenaline (for sodium nitroprusside; internal mammary artery: 101.07% +/- 1.63%; gastroepiploic artery: 94.10% +/- 2.07%) or endothelin-1 (for sodium nitroprusside; internal mammary artery: 97.67% +/- 4.94%; gastroepiploic artery: 90.69% +/- 2.61%). However, in saphenous vein contracted with endothelin-1, the responsiveness to nitrovasodilators was significantly blunted (for sodium nitroprusside: 52.33% +/- 5.19%) than that of rings contracted with noradrenaline (for sodium nitroprusside: 95.04% +/- 1.94%). Both arterial and venous grafts exhibited moderate beta-receptor function in response to isoproterenol. Isoproterenol was less effective in inhibiting the contractions of endothelin-1 in saphenous vein and gastroepiploic artery but not in internal mammary artery. On the other hand, nifedipine and papaverine were fully effective in reversing all the spasms in three of the graft materials. From these results, it can be deduced that saphenous vein is refractory against cyclic guanidine monophosphate (cGMP)-dependent and beta-receptor mediated relaxations when endothelin-1 was used as the spasmogenic agent. Internal mammary artery is the most responsive graft material to the vasodilators regardless of the nature of spasmogenic stimulus. Gastroepiploic artery exhibits functional similarity with internal mammary artery, with the exception of beta-receptor responsiveness.

Endothelium-dependent relaxing effect of histamine on the isolated guinea-pig main pulmonary artery strips

Contribution of endothelial cells (ECs) to the effects of histamine (HA) was investigated on the isolated guinea-pig main pulmonary artery (GPPA) strips precontracted with noradrenaline (NA). HA caused a dose-dependent relaxation at the concentrations ranged between 10−8 to 10−6M but produced a contraction when a relatively higher concentration (10−5M) was used in unrubbed strips. Cimetidine partially inhibited the relaxing effect of HA without altering its constrictive action. In rubbed strips, however, HA produced a dose-dependent contraction. The constrictive effect of HA in rubbed strips enhanced after addition of cimetidine to the incubation medium. HA elicited a concentration-dependent relaxation in both unrubbed and rubbed strips in the presence of mepyramine. Impromidine produced a relaxation in the strips with and without endothelium.These data was taken as an evidence indicating that HA caused a relaxation in the isolated GPPA strips, first causing the release of endothelium derived relaxing factor (EDRF) which is triggered by H1-receptors and secondly by the direct stimulation of H2-receptors.

Antinociceptive effect of some Amaryllidaceae plants in mice

The antinociceptive effects of ethanolic extracts of Pancratium maritimum L., Narcissus tazetta subspecies tazetta and Leucojum aestivum L. bulbs have been investigated in mice using the p‐benzoquinone‐induced abdominal constriction and hot‐plate tests.