A.Tuncay Demiryürek

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.

Ferrous iron-induced luminol chemiluminescence: a method for hydroxyl radical study.

We have investigated the chemiluminescence signal of the ferrous iron in the presence of the luminol and lucigenin. Ferrous, but not ferric, iron produced a transient signal in the presence of luminol, but not lucigenin. Ferrous iron-induced luminol chemiluminescence was significantly inhibited in a concentration-dependent manner by superoxide dismutase (SOD) and catalase. Specific hydroxyl radical scavengers, mannitol and dimethyl sulfoxide (DMSO), also markedly attenuated the ferrous iron-induced chemiluminescence. Additionally, antioxidants, urate, ascorbate, and methionine produced concentration-dependent significant inhibitions in this chemiluminescence. These results show that the hydroxyl radical generation is dependent on simultaneous formation of superoxide and hydrogen peroxide (H2O2). Ferrous iron does not generate a chemiluminescence signal in the presence of lucigenin suggesting that the formation of a hydroxyl radical is responsible for the luminol chemiluminescence. Thus, the present study has established a simple and inexpensive cell-free screening method for monitoring the scavenging effects of drugs on the hydroxyl radical.

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.

Superoxide in the pulmonary circulation

Superoxide formation in pulmonary tissue is modulated by cytokines, PO2, shear force, and disease states, and can be stimulated by drugs. Superoxide has diverse actions on pulmonary cells, including smooth muscle contraction, interaction with redox enzymes, cell proliferation, and gene transcription. In the lungs, there is an impressive array of specific defence mechanisms that destroy superoxide, especially superoxide dismutase (SOD) and metallothionein. Superoxide formation is increased in hyperoxia (e.g., oxygen therapy); however, superoxide-forming enzymes also can be up-regulated in hypoxia. Superoxide has been implicated in acute respiratory distress syndrome, lung ischaemia-reperfusion injury, and lung transplantation. Novel approaches to therapy have been explored, including SOD gene therapy and SOD targeting to the lung. In the future, new drugs interacting with superoxide may provide significant advances in the treatment of lung diseases.

Contribution of peroxynitrite to the beneficial effects of preconditioning on ischaemia-induced arrhythmias in rat isolated hearts.

We studied the effects of urate, a peroxynitrite scavenger, on ischaemia- and peroxynitrite-induced preconditioning in rat isolated hearts. Isolated hearts perfused with Krebs-Henseleit solution were preconditioned either by 3 min of coronary artery occlusion or by peroxynitrite administration (1 microM) for 3 min, followed by 10 min of reperfusion and 30 min of coronary artery occlusion. Both ischaemia and peroxynitrite produced a marked reduction in arrhythmias. Urate (1 mM) added to the perfusate 10 min prior to ischaemic preconditioning or peroxynitrite infusion and maintained until coronary artery occlusion, markedly reversed the beneficial effects in the ischaemic and peroxynitrite-treated groups. Urate administration in the peroxynitrite-treated group increased the incidence of ventricular tachycardia from 57% (n = 11) to 100% (n = 6) and total ventricular fibrillation from 0% (n=0) to 44% (n=4). Similarly, urate augmented the incidence of ventricular tachycardia from 47% (n=8) to 85% (n = 6) in the ischaemic preconditioning group. On its own, urate did not affect the severity of cardiac arrhythmias. Peroxynitrite infusion caused a marked increase in the effluent nitrate levels, from 0.05 +/- 0.1 microM (n = 5) to 0.4 +/- 0.2 microM (n = 6), and urate significantly decreased these levels to 0.08 +/- 0.03 microM (n = 9). These results suggest that peroxynitrite at low concentrations contributes to the beneficial effects of preconditioning on ischaemia-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.

Effect of Stobadine on Leukocyte Free Radical Generation in Streptozotocin-Diabetic Rats: Comparison with Vitamin E

We investigated a possible alteration in the ability of leukocytes to produce reactive oxygen species by stobadine, a pyridoindole antioxidant, in streptozotocin-diabetic rats. The production of free radicals from whole blood was assessed by luminol-enhanced chemiluminescence after stimulation by phorbol myristate acetate. The effects of vitamin E treatment were also evaluated and compared with the effects of combined treatment with stobadine. Diabetes was induced by streptozotocin (55 mg/kg i.p.). Some of diabetic rats and their age-matched controls were treated orally with a low dose of stobadine (24.7 mg/kg/day), vitamin E (400–500 IU/kg/day), or stobadine plus vitamin E for 10 weeks. Stobadine and vitamin E separately produced, to a similar degree, a reduction in diabetes-induced hyperglycemia. The phorbol myristate acetate stimulated chemiluminescence signal was markedly depressed in both moderate and severe diabetic rats. Stobadine treatment prevented this depression of the chemiluminescence response. Vitamin E treatment also eliminated the depression of the chemiluminescence signal in diabetic rats, and the combination with stobadine did not produce further improvement in leukocyte function. These results suggest that stobadine treatment alone is able to produce beneficial effects on leukocyte function and to maintain leukocyte free radical release during diabetes.

Involvement of tyrosine kinase pathway in acute hypoxic vasoconstriction in sheep isolated pulmonary vein.

Tyrosine kinase pathway has been shown to be involved in the effects of hypoxia in pulmonary arteries, but its role in pulmonary vein is not known. The aims of this study were to determine the effect of hypoxia in sheep isolated pulmonary veins and to identify the role of tyrosine kinase pathway in hypoxic response. Genistein and tyrphostin were used as selective tyrosine kinase inhibitors, and sodium orthovanadate was administered for tyrosine kinase activation. Hypoxia (95% N(2) to 5% CO(2)) caused a vasoconstriction either under resting tone or in U46619-precontracted pulmonary veins. Genistein and tyrphostin inhibited hypoxia-induced vasoconstriction both under resting tone and in precontracted veins, while sodium orthovanadate increased these hypoxic contractions. Our findings suggest that tyrosine kinase pathway is involved in hypoxic pulmonary vasoconstriction in sheep isolated pulmonary vein rings.

Nuclear factor-κB inhibitors abolish hypoxic vasoconstriction in sheep-isolated pulmonary arteries

The aim of this study was to determine the role of nuclear factor-kappaB (NF-kappaB) in hypoxic constriction of isolated pulmonary arteries. Rings were suspended in an organ bath filled with Krebs-Henseleit solution and isometric contractions were recorded continuously. Hypoxia (%95 N(2)-%5 CO(2)) had no marked effect on resting force in artery rings. However, hypoxia caused further contractions in serotonin-precontracted arteries. Hypoxia-induced vasoconstrictions were abolished by preincubation with NF-kappaB inhibitors, pyrrolidine dithiocarbamate (100 microM) or pyrithione (10 microM). These results suggest that reactive oxygen species and/or NF-kappaB activation may be involved in the hypoxia-induced vasoconstriction in sheep-isolated pulmonary arteries.

Involvement of tyrosine kinase in peroxynitrite-induced preconditioning in rat isolated heart.

We have investigated the role of tyrosine kinase in the antiarrhythmic effects of peroxynitrite preconditioning in rat isolated heart by using a tyrosine phosphatase inhibitor, sodium orthovanadate, and tyrosine kinase inhibitors, genistein and tyrphostin. Rat hearts were preconditioned by peroxynitrite administration at 1 microM for 3 min, which was followed by 10-min washout and 30 min of ischemia. None of the hearts had ventricular fibrillation in the peroxynitrite preconditioning group (from 64%, n=11, to 0%, n=11). Neither sodium orthovanadate (10 microM) nor genistein (50 microM) or tyrphostin (100 microM) alone showed any effects on arrhythmias. Peroxynitrite preserved its beneficial effects on arrhythmias (to 0% ventricular fibrillation, n=7) during sodium orthovanadate infusion (for 23 min) prior to 30 min of an ischemic period. On the other hand, genistein or tyrphostin treatment significantly reversed the protective effects of the peroxynitrite preconditioning (to 71% ventricular fibrillation, n=14, genistein and, to 75% ventricular fibrillation, n=8, tyrphostin). These results suggest that the tyrosine kinase pathway plays a significant role in peroxynitrite-induced preconditioning in rat isolated heart.