N-(p-amylcinnamoyl) anthranilic acid attenuates remedial effects of memantine on memory deficits following intracerebroventricular streptozotocin administration in rats.

N-(p-amylcinnamoyl) anthranilic acid (ACA) is a blocker of transient receptor potential melastatin-2 (TRPM2) which is a non-selective, Ca2+-permeable and oxidative stress sensor cation channel. Intracerebroventricular (ICV) streptozotocin (STZ) induction successfully generates spatial memory deficits in rats. The purpose of this study was to investigate effects of ACA on a rat model of STZ-induced learning and memory deficits. A total of 60 Wistar rats randomly divided into six groups; (1) control, (2) sham-operated, (3) ICV-STZ administered, (4) ICV-STZ + memantine (5 mg/kg i.p.), (5) ICV-STZ + ACA (25 mg/kg i.p.) and (6) a combination therapy group, ICV-STZ + ACA (25 mg/kg) + memantine (5 mg/kg). Effects of the drugs on spatial memory deficits were appraised in Morris water maze (MWM) apparatus. Anxiety-like behavior of the rats were also assessed by using both the elevated plus maze (EPM) and open field maze (OFM) apparatuses. Western blot analysis of hippocampal tissues revealed TRPM2-L channel protein expression levels. Serum levels of tumor necrosis factor alpha (TNF-α) and malondialdehyde (MDA) were detected by enzyme-linked immunosorbent assay (ELISA) kits. Memantine treatment ameliorated the spatial memory deficits induced, as evidenced by the MWM tests. However, ACA treatment did not provide any improvement, instead positive effects of memantine were attenuated by ACA treatment. Western blot analysis in hippocampal tissues showed that TRPM2-L protein expression was markedly suppressed in ICV-STZ administered group. The ACA treatment reversed that suppression. Surprisingly, the memantine treatment resulted in overexpression of TRPM2-L, to a certain extent. Examination of the rats in EPM and OFM apparatuses, as a display of anxiety-like behavior, did not reveal any marked difference among groups. Serum levels of TNF-α and MDA also did not vary significantly among groups, as well. Conclusively, our findings showed for the first time that TRPM2-L protein expression was significantly suppressed in the ICV-STZ induced memory deficit model. Even though ACA reversed this suppression, no improvement in spatial memory was observed following ACA treatment.

Association between neutrophil/lymphocyte ratio with ductus arteriosus patency in preterm newborns.

OBJECTIVE: The aim of this study was to assess the influence of neutrophil to lymphocyte (N/L) ratio and platelet indices on patent ductus arteriosus (PDA) in preterm infants. BACKGROUND: PDA is a common problem with potentially serious associated morbidities in preterm neonates. METHODS: Premature infants with hemodynamically significant PDA (n = 47) and a control group without PDA (n = 50) who were hospitalized in the neonatal intensive care unit were retrospectively evaluated. The characteristics, perinatal factors, N/L ratio, platelet counts and other platelet indices of the infants in both groups during the first 3 days of life were recorded. RESULTS: Platelet counts were significantly lower in the patient group than in the control group (p = 0.0343). There was a marked positive correlation between body weight and N/L ratio in preterm infants with PDA (p = 0.0001). PDA was associated with low platelet count. CONCLUSION: Our results showed that N/L ratio is positively correllated with body weight in PDA group. These data suggest that platelet counts and N/L ratio might be useful predictors for the early diagnosis and evaluation of the clinical course of PDA in preterm infants (Tab. 2, Ref. 28).

Gene expressions of TRP channels in glioblastoma multiforme and relation with survival.

Glioblastoma multiforme (GBM) is one of the most lethal forms of cancer in humans, with a median survival of 10 to 12 months. Glioblastoma is highly malignant since the cells are supported by a great number of blood vessels. Although new treatments have been developed by increasing knowledge of molecular nature of the disease, surgical operation remains the standard of care. The TRP (transient receptor potential) superfamily consists of cation-selective channels that have roles in sensory physiology such as thermo- and osmosensation and in several complex diseases such as cancer, cardiovascular, and neuronal diseases. The aim of this study was to investigate the expression levels of TRP channel genes in patients with glioblastoma multiforme and to evaluate the relationship between TRP gene expressions and survival of the patients. Thirty-three patients diagnosed with glioblastoma were enrolled to the study. The expression levels of 21 TRP genes were quantified by using qRT-PCR with dynamic array 48 × 48 chip (BioMark HD System, Fluidigm, South San Francisco, CA, USA). TRPC1, TRPC6, TRPM2, TRPM3, TRPM7, TRPM8, TRPV1, and TRPV2 were found significantly higher in glioblastoma patients. Moreover, there was a significant relationship between the overexpression of TRP genes and the survival of the patients. These results demonstrate for the first time that TRP channels contribute to the progression and survival of the glioblastoma patients.

Effects of a selective Rho-kinase inhibitor Y-27632 on oxidative stress parameters in acute dichlorvos poisoning in rats.

This study examined the effects of Y-27632, a selective Rho-kinase inhibitor, on organophosphate-induced acute toxicity in rats. Rats were randomly divided into four groups as control (corn oil), dichlorvos (30 mg kg(-1) i.p.), 1 and 10 mg kg(-1) Y-27632 + dichlorvos groups. Cholinergic signs (fatigue, tremor, cyanosis, hyper-secretion, fasciculations) were observed in all the rats in the dichlorvos group and the mortality rate was 50%. No cholinergic findings and deaths were observed in the control and Y-27632 groups. Plasma cholinesterase activities were suppressed with dichlorvos and these reductions were attenuated with Y-27632 pretreatment. There was a marked increase in plasma malondialdehyde level in the dichlorvos group, but Y-27632 pretreatment abolished this elevation. Dichlorvos markedly depressed cardiac paraoxonase activity, but these changes were not markedly modified with Y-27632. Total antioxidant capacities, total oxidant status, oxidative stress index, total free sulfhydryl groups and catalase activities in plasma and cardiac tissues were not markedly different between the groups. No significant changes were observed with cardiac myeloperoxidase activities or plasma arylesterase and ceruloplasmin activities. In conclusion, our results suggest that Rho-kinase pathway is involved in organophosphate intoxication, and a decrease in cardiac paraoxonase activities may play a role in the pathogenesis of acute organophosphate poisoning in rats.

Effects of dexmedetomidine infusion in patients undergoing functional endoscopic sinus surgery under local anaesthesia.

BACKGROUND AND OBJECTIVES: Functional endoscopic sinus surgery can be performed under either local or general anaesthesia. The objective of this study was to investigate the haemodynamic effects of perioperatively administered dexmedetomidine, a new generation alpha-2-agonist, in patients for functional endoscopic sinus surgery. METHODS: Sixty-two patients who were planned to undergo functional endoscopic sinus surgery under local anaesthesia were included in the study. Following meperidine premedication, both groups were monitored in a standard manner with electrocardiogram, non-invasive blood pressure and percentages of peripheral saturation of oxygen. Saline intravenous infusion was started in the placebo group, and dexmedetomidine bolus intravenous infusion (an initial loading dose of 1 microg kg-1 given for a 10-min period followed by 0.7 microg kg-1 h-1) was administered to the treatment group. Maintenance dose infusion was stopped 15 min before the end of the surgical procedure. RESULTS: Systolic, diastolic and mean arterial pressures, and heart rate markedly decreased in the dexmedetomidine group. However, dexmedetomidine had no effect on serum nitric oxide levels, measured by a nitric oxide/ozone chemiluminescence method. No significant difference was found in oxygen saturation levels of the two groups. Postoperative nausea and vomiting rates were significantly lower in the dexmedetomidine group. No adverse effects were observed with this alpha-2-agonist. Dexmedetomidine provided appropriate levels of sedation. CONCLUSION: These results suggest that dexmedetomidine provides analgesia, adequate sedation and surgical comfort without adverse effects for patients undergoing functional endoscopic sinus surgery under local anaesthesia.

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.

Cardiotoxicity of digitalis glycosides: roles of autonomic pathways, autacoids and ion channels.

1 Cardiac glycosides have been used for centuries as therapeutic agents for the treatment of heart diseases. In patients with heart failure, digoxin and the other glycosides exert their positive inotropic effect by inhibiting Na(+)-K(+)-ATPase, thereby increasing intracellular sodium, which, in turn, inhibits the Na(+)/Ca(2+) exchanger and increases intracellular calcium levels. As the therapeutic index of digitalis is narrow, arrhythmias are common problems in clinical practice. The mechanisms and mediators of these arrhythmias, however, are not completely understood. 2 The involvement of the sympathetic and parasympathetic nervous system in digitalis cardiac toxicity is reviewed. 3 Receptors, channels, exchange systems or other cellular components involved in digitalis-induced cardiotoxicity are also reviewed. 4 Possible mediators of digitalis-induced cardiac toxicity are discussed. 5 Management of digitalis toxicity in patients is summarized. 6 The determination of the possible mediators of digitalis-induced cardiac toxicity will enhance our knowledge and lead to the development of new therapeutic strategies to treat these lethal arrhythmias.

Effects of whole-body hypoxic preconditioning on hypoxia/reoxygenation-induced intestinal injury in newborn rats.

PURPOSE: The precise cause of necrotizing enterocolitis (NEC) is elusive. Ischemia and reperfusion injury of the intestine has been considered to be a major contributing factor for NEC. Ischemic preconditioning is defined as one or more brief periods of ischemia with intermittent reperfusion that protects tissues against a sustained period of subsequent ischemia. Contribution of preconditioning to hypoxia/reoxygenation-induced intestinal injury in newborn rats has not been evaluated previously. METHODS: The study was carried out on 1-day-old Wistar albino rat pups. Whole-body hypoxia and reoxygenation (H/R) was achieved by 10 min hypoxia using 95 % N (2) + 5 % CO (2) followed by 10 min reoxygenation with 100 % oxygen. Whole body hypoxic preconditioning (HP) cycles were performed with 3 min hypoxia and 5 min reoxygenation. Thirty-three pups were randomly allocated into 4 groups. Group 1 served as untreated controls. The pups in group 2 were subjected to H/R only. In groups 3 and 4, 1 cycle and 3 cycles of HP were performed prior to H/R, respectively. Animals were killed at the end of the protocols. Intestine specimens were obtained to determine the histological changes, as well as to measure the tissue malondialdehyde (MDA) and nitric oxide (NO) levels, and xanthine oxidase (XO) and myeloperoxidase (MPO) activities. RESULTS: The microscopic lesions in H/R rat pups were virtually the same as those seen in neonatal NEC, with severe destruction of villi and crypts, in some cases extending to the muscularis. In both HP groups, the lesions were found to be milder. H/R resulted in a marked elevation in MDA and NO levels, and XO and MPO activities compared to the untreated controls. Both 1 cycle and 3 cycles of HP prior to H/R resulted in an obvious decrease in all biochemical parameters. Differences of the biochemical results between both HP groups were not statistically significant. CONCLUSION: This study revealed that whole-body hypoxic preconditioning is beneficial for hypoxia/reoxygenation-induced intestinal injury in newborn rats.

Association of Rho/Rho-kinase gene polymorphisms and expressions with obesity-related metabolic syndrome.

OBJECTIVE: The metabolic syndrome (MetS) is a common multicomponent condition including abdominal obesity, dyslipidemia, hypertension and hyperglycemia. The aim of this study was to investigate the associations of Rho GTPase and Rho-kinase (ROCK) gene polymorphisms and expressions with MetS in a Turkish population. PATIENTS AND METHODS: A total of 141 obese MetS patients and 163 healthy controls with similar age and sex were included to this study. Polymorphisms were analyzed in genomic DNA using a BioMark 96.96 dynamic array system. mRNA from blood samples was extracted, and real-time polymerase chain reaction was performed for gene expressions. RESULTS: We observed that genotype (CC, 18.1%; CA, 13.4%, and AA, 68.5%) and allele (C, 24.8%; A, 75.2%) frequencies for the rs35996865 polymorphism of the ROCK1 gene in patients were markedly different from controls (CC, 84.2%; CA, 2.9%, and AA, 12.9%; C, 85.6%; A, 14.4%, p < 0.0001). In the rs2230774 (Thr431Asn) polymorphism of the ROCK2 gene, there were increases in the CC genotype (16.5%) and C allele frequencies (20.4%) in MetS patients when compared with the control group (CC, 6.0%, p = 0.0009, and C, 6.7%, p < 0.0001). However, no associations with the other 18 polymorphisms studied were found. Although there were an increase in peripheral blood mRNA RhoH expressions, marked decreases in RhoC, RhoBTB1, RhoV, Rnd1, and CDC42 gene expressions were noted in MetS patients. CONCLUSIONS: This is the first study to provide evidence that ROCK gene polymorphisms and gene expressions of the Rho GTPase proteins may modify individual susceptibility to MetS in the Turkish population.

Role of the transient receptor potential (TRP) channel gene expressions and TRP melastatin (TRPM) channel gene polymorphisms in obesity-related metabolic syndrome.

OBJECTIVE: Metabolic syndrome (MetS) is correlated with increased cardiovascular risk and characterized by several factors, including visceral obesity, hypertension, dyslipidemia, and insulin resistance. The etiology of MetS is complex, and can be influenced by genetic susceptibility. The aim of this study was to investigate a possible association of transient receptor potential (TRP) channels gene expressions and TRP melastatin (TRPM) gene polymorphisms with MetS in a Turkish population. PATIENTS AND METHODS: A total of 142 patients with obesity-related MetS and 166 healthy controls with similar age and sex were enrolled to this study. For polymorphism studies, genomic DNA from the participants was analyzed by a BioMark 96.96 dynamic array system (Fluidigm, South San Francisco, CA, USA). For gene expression studies, mRNA from blood samples was extracted, and real time polymerase chain reaction on the BioMark HD system was performed. RESULTS: There was an increase in A allele (64.6% in patients vs. 49.5% in controls) and decrease in G allele frequencies (35.4% in patients vs. 50.5% in control, p = 0.0019) of the TRPM5 gene rs4929982 (Arg578Gln) polymorphism. We also observed that the distribution of genotype and allele frequencies of the TRPM8 gene rs12472151 in MetS patients were significantly different from controls (p < 0.0001). Although there were marked decreases in TRPC1, TRPC3, TRPM2, TRPM5, TRPV4, TRPV5, TRPV6, MCOLN2 (TRPML2), and MCOLN3 (TRPML3) gene expressions, an augmentation was noted in TRPC6 gene expression. CONCLUSIONS: Genetic polymorphisms in TRPM5 and TRPM8 genes may modify individual susceptibility to MetS in the Turkish population. This study also revealed that there is a significant relationship between TRP channels gene expressions and MetS.

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

1. 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). 2. Aminoguanidine (0.1 microM-10 mM), methylguanidine (10 microM-10 mM) and guanidine (10 microM-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. 3. 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 microM), 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. 4. Collectively, these data suggest that aminoguanidine and a high concentration (> or = 0.1 mM) of methylguanidine have direct scavenging activities against H2O2, HOCl, hydroxyl radical and peroxynitrite. Guanidine, at a high concentration (> or = 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.

1. 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. 2. 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 microM) for 3 min, followed by 10 min reperfusion and 30 min of CAO. Peroxynitrite, at 1 microM 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). 3. N-2-mercaptopropionylglycine (MPG, 1 microM - 10 mM) produced a concentration-dependent inhibition of peroxynitrite signals in luminol chemiluminescence and 67+/-1% inhibition was observed at 100 microM (n=7). MPG (at 300 microM, 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. 4. These results suggest that endogenously produced peroxynitrite plays a significant role in the antiarrhythmic effect of ischaemic preconditioning in the 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.

Pharmacological evidence for the role of mediators in hypoxia-induced vasoconstriction in sheep isolated intrapulmonary artery rings.

The aim of this study was to determine the likely mediator(s) involved in the hypoxic-induced contraction in sheep pulmonary artery rings in vitro by studying the effects of selective receptor antagonists and enzyme inhibitors. Hypoxia caused a contraction in arteries under resting force and when precontracted with 5-hydroxytryptamine (5-HT). Flurbiprofen, a cyclooxygenase inhibitor, reduced the hypoxic contraction in 5-HT-precontracted rings but augmented the first part of the hypoxic contraction under baseline force. Inhibition of nitric oxide by haemolysate increased the hypoxic contraction under resting force. Superoxide dismutase and N-t-butyl-alpha-phenylnitrone (PBN), free radical scavenging agents, and trypsin, a proteolytic enzyme, did not produce any significant effect on hypoxia-induced constriction. Propranolol plus phentolamine, beta- and alpha-adrenoceptor antagonists respectively, did not produce any effect on hypoxic contraction under resting force, whereas these antagonists augmented hypoxic contraction in the presence of 5-HT. This combination of antagonists also caused a reduction of 5-HT contraction which was the result of alpha 2-adrenoceptor blockade. Verapamil, a calcium channel blocking drug, significantly reduced the 5-HT contraction, but did not reduce that caused by hypoxia either under resting force or in precontracted rings. These results suggest that hypoxic constriction in sheep isolated intrapulmonary artery is in part caused by reduced release of vasodilator prostanoids. This contraction does not involve voltage-operated calcium channels and is limited by release of endothelium-derived nitric oxide.

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.

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.

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.

Characterization of a method for the detection of drugs with free radical scavenging activity using porcine leukocytes.

The effects of a range of free-radical scavenging drugs on luminol-enhanced chemiluminescence (CL) generated by porcine leukocytes, following activation by two nonreceptor-mediated stimulants, phorbol myristate acetate (PMA; a protein kinase activator) and ionomycin (a cation ionophore), and by xanthine plus xanthine oxidase (X-XO), have been examined. Superoxide dismutase (0.1 units/mL) and catalase (50 units/mL) inhibited X-XO, but they were ineffective in leukocyte suspensions except at concentrations 500 times and 20 times higher. Sodium azide (10(-5) to 10(-3) M) caused a marked inhibition in CL production in activated leukocytes, but not of X-XO CL. The antioxidants, glutathione (10(-3) M) and L-ascorbic acid (10(-3) M) were ineffective in activated leukocytes, but caused total inhibition of X-XO-induced CL. Mannitol (100 mM) had no effect on chemiluminescence in either system. Captopril (10(-3) M) produced an inhibition of CL in both systems and this inhibition was significantly modified by pH. Thus, the present study has established a standard screening procedure for the assessment of free-radical scavenging activity using activated porcine leukocytes and xanthine-xanthine oxidase.

Interaction of lidocaine with reactive oxygen and nitrogen species.

BACKGROUND AND OBJECTIVE: To investigate the ability of lidocaine to inhibit reactive oxygen and/or nitrogen species generation by either human leukocytes or cell-free systems via luminol- and lucigenin-enhanced chemiluminescence. METHODS: Venous blood was obtained from healthy volunteers and leukocytes were isolated, from which chemiluminescence was generated. Also, chemiluminescence, induced by H(2)O(2), HOCl, peroxynitrite or ferrous iron, was generated in cell-free systems. RESULTS: Lidocaine produced a concentration-dependent inhibition in chemiluminescence generated by leukocytes (92 +/- 1%, 1 mM). In cell-free experiments, lidocaine (1 mM) markedly inhibited chemiluminescence of xanthine-xanthine oxidase (24 +/- 3%), while it slightly suppressed hypochlorous acid-induced chemiluminescence (9 +/- 2%). Peroxynitrite-induced luminol- and lucigenin-enhanced chemiluminescence were also inhibited by lidocaine at 1 mM (19 +/- 3% and 48 +/- 8%, respectively). Lidocaine did not affect chemiluminescence generated by FeSO(4). However, lidocaine produced a biphasic effect on H(2)O(2)-induced chemiluminescence (37 +/- 5% inhibition at 0.01 mM and 61 +/- 17% activation at 1 mM). CONCLUSIONS: Lidocaine can elicit direct scavenging activity at high concentrations that might be important at or near the site of injection in local anaesthetic use.

Effects of prilocaine and articaine on human leucocytes and reactive oxygen species in vitro.

BACKGROUND: The aim of this study was to investigate the ability of local anaesthetics to inhibit reactive oxygen and nitrogen species generated by either stimulated human leucocytes or cell-free systems using luminol chemiluminescence (CL). METHODS: Free radical generation was stimulated in leucocyte assay by formyl-methionyl-leucyl-phenylalanine (FMLP, 2 microM). In cell-free experiments, hydrogen peroxide (H2O2) 3.5 mM, sodium hypochloride 5 microM, ferrous sulphate (FeSO4) 40 nM, peroxynitrite 50 nM and xanthine 0.1 mmol l(-1) plus xanthine oxidase 0.25 U ml(-1) were used to produce H2O2, hypochlorous acid (HOCl), hydroxyl radical, peroxynitrite and superoxide-induced CL, respectively. RESULTS: Prilocaine inhibited FMLP-induced CL in leucocytes (94+/-1%, at 1 mM), whereas articaine showed an activation (59+/-7%) at high concentration (1 mM) and inhibition (13+/-6%) at low concentration (0.1 mM). In cell-free experiments, prilocaine (22+/-6%, at 1 mM) and articaine (85+/-1%, at 1 mM) caused concentration-dependent inhibition in xanthine-xanthine oxidase-induced CL. Although articaine had no effect on H2O2-induced CL, prilocaine significantly attenuated the H2O2 signal (97+/-0.3%, at 1 mM). Prilocaine (99+/-0.04%, 1 mM) and articaine (70+/-6%, 1 mM) markedly inhibited HOCl-induced CL, whereas these drugs had no effect on FeSO4-induced CL. Articaine inhibited peroxynitrite CL (63+/-6%, 1 mM), but prilocaine did not produce any depression on this signal. CONCLUSION: Prilocaine interacted with superoxide, HOCl and H2O2, whereas articaine reacted with superoxide, HOCl, and peroxynitrite. The direct scavenging properties of these drugs might be involved in the inhibition observed in leucocyte assay and could provide experimental support for investigating the potential benefit of using these local anaesthetics in patients presenting pathologies associated with free radical reactions.