Source Of Oxygen Free Radicals Research Articles

Measurement and characterization of superoxide generation from xanthine dehydrogenase: a redox-regulated pathway of radical generation in ischemic tissues.

The enzyme xanthine oxidoreductase (XOR) is an important source of oxygen free radicals and related postischemic injury. Xanthine dehydrogenase (XDH), the major form of XOR in tissues, can be converted to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. The conversion of XDH to XO has been assumed to be required for radical generation and tissue injury. It is also possible that XDH could generate significant quantities of superoxide, •O2–, for cellular signaling or injury; however, this possibility and its potential ramifications have not been previously considered. To unambiguously determine if XDH can be a significant source of •O2–, experiments were performed to measure and characterize •O2– generation using XDH from chicken liver that is locked in the dehydrogenase conformation. Electron paramagnetic resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide demonstrated that XDH in the presence of xanthine produces significant amounts of •O2–. NAD+ and NADH inhibited the generation of •O2– from XDH in a dose-dependent manner, with NAD+ exhibiting stronger inhibition than NADH at low physiological concentrations. Decreased amounts of NAD+ and NADH, which occur during and following tissue ischemia, enhanced the generation of •O2– from XDH in the presence of xanthine. It was observed that XDH-mediated oxygen radical generation markedly depressed Ca2+-ATPase activity of isolated sarcoplasmic reticulum vesicles from cardiac muscle, and this was modulated by NAD+ and NADH. Thus, XDH can be an important redox-regulated source of •O2– generation in ischemic tissue, and conversion to XO is not required to activate radical formation and subsequent tissue injury.

Human endothelial dysfunction: EDCFs

Human studies, conducted in the presence of clinical conditions characterized by endothelial dysfunction, evidenced that endothelial cells, in response to different agonists and physical stimuli, become a source of endothelium-derived contracting factors (EDCFs), mainly cyclooxygenase (COX)-derived prostanoids. Their production has been documented in several human diseases, mostly in essential hypertension and aging. The EDCF production was at first identified as responsible for impaired endothelium-dependent vasodilation in the forearm microcirculation of patients with essential hypertension. Subsequent studies demonstrated that COX-dependent EDCF products are also a characteristic of the aging process, and essential hypertension seems to only anticipate the phenomenon. Of note, in aging and hypertension, both indomethacin, a COX inhibitor, and vitamin C, an antioxidant, totally reverse the blunted vasodilation to acetylcholine by restoring NO availability, thus suggesting that EDCFs could be one of the major sources of oxygen free radicals. The presence of EDCFs was documented also in other clinical setting, such as coronary artery disease and estrogen deprivation. In conclusion, many human pathological conditions characterized by a decline in endothelial function are associated with a progressive decrease in NO bioavailability and increase in the production of EDCFs. The mechanisms that regulate the balance between NO and EDCFs and the processes transforming the endothelium from a protective organ to a source of vasoconstrictor, proaggregatory and promitogenic mediators, remain to be determined.

Superoxide Generation from Mitochondrial NADH Dehydrogenase Induces Self-inactivation with Specific Protein Radical Formation

Mitochondrial superoxide (O(2)(.)) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O(2)(.) production; its mechanism of O(2)(.) generation is not known. Therefore, the catalytic function of NDH in the mediation of O(2)(.) generation was investigated by EPR spin-trapping. In the presence of NADH, O(2)(.) generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O(2)(.) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O(2)(.) generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O(2)(.) generation. O(2)(.) production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O(2)(.)-dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200)-Ala-Gly-Ala-Tyr-Ile-Cys(206)-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)-Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.

Postresectional pulmonary oxidative stress in lung cancer patients. The role of one-lung ventilation

The authors conducted a prospective analysis in order to investigate through lipid peroxidation metabolites the generation of oxygen free radicals after one-lung ventilation (OLV). From 2001 to 2003, 212 patients were prospectively studied for lung reexpansion/reperfusion injury. They were classified in six groups. Group A, non-OLV lobectomy group; B, OLV pneumonectomy group; C-E, OLV lobectomy of 60, 90, and 120 min duration, respectively; F, normal subjects as baseline group. Preoperative, intraoperative and postoperative strict blood sampling protocol was followed. Malondialdehyde (MDA) plasma levels were measured. The recorded values were analyzed and statistically compared between groups and within each one. Comparison of groups C-E (OLV) to all other documented significant (P<0.001) increase of MDA levels during lung reexpansion and for the following 12h. The magnitude of oxidative stress was related to OLV duration (group E>D>C, all P<0.001). The removal of cancer-associated parenchyma led to MDA level decrease postoperatively (P<0.001) especially after pneumonectomy (A vs. B, P<0.001). (1) Lung reexpansion provoked severe oxidative stress. (2) The degree of the amount of generated oxygen free radicals was associated to the duration of OLV. (3) Patients with lung cancer had a higher production of oxygen free radicals than normal population. (4)Tumor resection removes a large oxidative burden from the organism. (5) Mechanical ventilation and surgical trauma are weak free radical generators. (6) Manipulated lung tissue is also a source of oxygen free radicals, not only intraoperatively but also for several hours later.

From the Editors Perspective: Rational Approaches for Radical Entities

It is estimated that almost 400 million individuals suffer from nervous system disorders in the world. These disorders consist of both acute and chronic neurodegenerative diseases that include stroke, traumatic brain injury, presenile dementia, Alzheimer's disease, and Parkinson's disease. In addition, environmental toxin exposure also has become increasingly prominent as a precipitant of degenerative disease in the nervous system. Intimately coupled to each of these disorders is the generation of reactive oxygen species (ROS) in the brain. ROS consist of oxygen free radicals and other chemical entities that include superoxide free radicals, hydrogen peroxide, nitric oxide, and peroxynitrite. Under normal physiological conditions, these species are produced at low levels and are scavenged by endogenous antioxidant systems of the body that include superoxide dismutase, glutathione peroxidase, and catalase. Yet, oxidative stress in the brain occurs when the generation of ROS overrides the ability of the endogenous antioxidant system to remove excess oxygen free radicals. Oxidative stress is a formidable factor for injury in the nervous system, since it leads to the dysfunction of both neuronal and vascular cells as well as inciting inflammatory cellular demise. Oxidative stress leads to the peroxidation of cellular membrane lipids, cleavage of genomic DNA, and the oxidation of a variety of cellular proteins. The generation of ROS also can block complex enzymes in the electron transport chain of the mitochondria to halt mitochondrial respiration. During the release of free radicals in the brain, microglia also become activated to begin the process of phagocytosis for the removal of injured cells. As a result, microglia are believed to lead to cellular damage of neighboring neurons and vascular cells, partly through the generation of ROS products as well as through the production of cytokines. In consideration of the significant risks free radical generation can present to the nervous system, it is surprising to learn that the brain is highly susceptible to oxidative stress injury and has only limited capacity to avert cellular injury. A variety of observations support this premise. For example, the brain possesses the highest oxygen metabolic rate of any organ in the body, consuming twenty percent of the total amount of oxygen in the body and enhancing the possibility for the aberrant generation of free radicals. In addition, the brain is composed of significant amounts of unsaturated fats that can readily serve as a source of oxygen free radicals. Interestingly, given the increased risk factors for the generation of elevated levels of ROS in the brain, the brain also may suffer from an inadequate defense system against oxidative stress. Catalase activity in the brain, an endogenous antioxidant, has been reported to exist at levels markedly below other organs of the body, sometimes approaching catalase levels of ten percent in other organs such as the liver.

Octreotide ameliorates sepsis-induced pelvic inflammation in female rats by a neutrophil-dependent mechanism

Sepsis is a generalized inflammatory response, which involves organ systems remote from the locus of the initial infectious insult, accompanied by the release of cytokines and the subsequent formation of reactive oxygen and nitrogen species. The aim of this study was to investigate the possible protective effect of octreotide (OCT), a synthetic somatostatin analogue, against sepsis-induced oxidative damage in the uterine and ovarian tissues of rats. Sepsis was induced by caecal ligation and puncture method in female Wistar albino rats. Sepsis and sham operated (control) groups received either saline or OCT (50 μg/kg, i.p.; Novartis) immediately after the operation and at 12 h. Twenty-four hours after the surgery, rats were decapitated and serum TNF-α levels and tissue malondialdehyde (MDA) content, glutathione (GSH) levels and myeloperoxidase (MPO) activity were determined in the uterus and ovaries. Oxidant-induced tissue fibrosis was determined by tissue collagen contents, while the extent of tissue injuries was analyzed microscopically. Sepsis increased serum TNF-α levels and resulted in decreased GSH levels and increased MDA levels, MPO activity and collagen contents in both the uterus and the ovaries ( p < 0.05–0.001) indicating the presence of the oxidative damage, as also confirmed by histological analysis. On the other hand, OCT administration reversed these oxidant responses and reduced the severity of microscopic damage ( p < 0.001). In conclusion, OCT protects against sepsis-induced oxidative injury of the uterine and ovarian tissues by diminishing neutrophil infiltration, an important source of oxygen free radicals. Our results suggest that OCT may be of therapeutic value in ameliorating sepsis-associated pelvic inflammation.

The distinct effect of carvedilol and metoprolol on the mitochondrial superoxide production under calcium overload of myocardium

Purpose: In heart failure or ischemia-reperfusion injury, intracellular calcium overload occurs in the myocardium. However, mitochondrial functions during calcium overload are not well known. Additionally failing myocardium have source of oxygen free radicals from mitochondrial electron transport complex 1. Recently, a beta-blocker is known to be useful for heart failure, and also suggested the difference between the drugs depending on the lipid solubility, and with or without antioxidant effect. Methods: Isolated mitochondria from SD rat hearts were loaded in the cuvette mounted by Clark oxygen sensor to measure the mitochondrial oxygen consumption. The amount of malondialdehyde (MDA) was measured as an index of superoxide production. Calcium concentration was varied at 10, 100 μM and carvedilol and metoprolol concentration was varied at 1, 10, 100 μM. Rotenone was used for injuring complex 1. Result: The oxygen consumption was significantly augmented by increasing the calcium concentration from 10 to 100 μM (p < 0.05). This increase of oxygen consumption was significantly suppressed by even 1 μM carvedilol (p < 0.05), but not metoprolol. The amount of MDA was augmented by rotenone (p < 0.01). This increase of MDA was suppressed by carvedilol dose dependently (p < 0.05), but not metoprolol. Conclusion: These data strongly suggest the distinct effect of carvedilol on superoxide production and oxygen consumption from metoprolol, which may be beneficial for the protection of cardiomyocyte from oxidant stress.

Transition metals and polyol pathway in the development of diabetic neuropathy in rats

The transition metal-catalyzed reaction is a major source of oxygen free radicals, which play an important role in vascular dysfunction leading to ischemia in diabetic tissues. The inhibition of polyol pathway hyperactivity has been reported to ameliorate neurovascular abnormalities in diabetic rats and has been proposed to improve the oxygen free radical scavenging capacity. The present study was conducted to compare the effect of a transition metal chelating agent, trientine (TRI), on diabetic neuropathy with that of an aldose reductase inhibitor, NZ-314 (NZ). Diabetic rats were divided into three groups: (1). untreated, (2). TRI-treated, and (3). NZ-treated. TRI (20 mg/kg) or NZ (100 mg/kg) was administered by gavage or chow containing NZ, respectively, for 8 weeks. Motor nerve conduction velocity (MNCV), coefficient of variation of the R - R interval on electrocardiogram (CVr-r), sciatic nerve blood flow (SNBF), platelet aggregation activities, and serum concentrations of malondialdehyde were measured. Untreated diabetic rats showed delayed MNCV, decreased CV(R-R), and reduced SNBF compared to normal rats. TRI or NZ completely prevented these deficits. Platelet hyperaggregation activities in diabetic rats were prevented by NZ, but not by TRI. Increased concentrations of malondialdehyde in diabetic rats were partially but significantly ameliorated by either TRI or NZ. These observations suggest that increased free radical formation through the transition metal-catalyzed reaction plays an important role in the development of diabetic neuropathy and that the preventive effect of an aldose reductase inhibitor on diabetic neuropathy may also be mediated by decreasing oxygen free radicals.

Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.

THE USE OF HYDROGEN PEROXIDE TO ENHANCE THE EFFICACY OF DOXORUBICIN HYDROCHLORIDE IN A MURINE BLADDER TUMOR CELL LINE

We determined whether the cytotoxicity of doxorubicin hydrochloride would be enhanced by adding hydrogen peroxide as a source of oxygen free radicals. Mouse bladder tumor cells (MBT-2) were grown in RPMI 1640 medium and treated with various concentrations of doxorubicin hydrochloride for 2 hours. Protein content was assayed as a measure of cell growth. A similar set of experiments was done with cells exposed to hydrogen peroxide only and combined doxorubicin and hydrogen peroxide. Protein content was again assayed as a measure of cell growth. Cells were also assayed for glutathione peroxidase and malonyl dialdehyde, a product of lipid peroxidation, to determine the mechanism of cell damage. Furthermore, MBT-2 cells were incubated with 100 M. alpha-tocopherol, a free radical scavenger, before exposure to hydrogen peroxide to determine whether the effects of hydrogen peroxide could be reversed. We observed a dose dependent inhibition of MBT-2 cell growth after exposure to doxorubicin hydrochloride. Exposure to doxorubicin and hydrogen peroxide resulted in greater cell growth inhibition than exposure to either agent alone. The effects of hydrogen peroxide on cell proliferation were reversed by pre-incubation with alpha-tocopherol. As a source of oxygen free radicals, hydrogen peroxide enhances the antiproliferative effect of doxorubicin hydrochloride on a mouse bladder tumor cell line. Thus, hydrogen peroxide may be a relatively inexpensive, nontoxic method of augmenting the cytotoxicity of doxorubicin hydrochloride. Further studies are warranted to determine whether these observations may have clinical application.

Mitochondrial electron transport complex I is the source of oxygen free radicals in the failing myocardium

Mitochondrial electron transport complex I is the source of oxygen free radicals in the failing myocardium

Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium.

Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5, 5'-dimethyl-1-pyrroline-N-oxide, in the presence of NADH and succinate as a substrate for NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (P<0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (274+/-13 versus 136+/-9 nmol. min(-1). mg(-1) protein, P<0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium.

Reoxygenation injury of human brain capillary endothelial cells.

1. Many studies have demonstrated that endothelial cells from several species can generate oxygen free radicals when subjected to anoxia and reoxygenation. However, due to the heterogeneity of the endothelium within different organs and species, the effects of superoxide dismutase (SOD), catalase, and allopurinol on reoxygenated cultured cells remain quite controversial. 2. This review outlines the possible sources of oxygen free radicals within brain endothelial cells. 3. We examine the aspects of the effects of SOD catalase and allopurinol on cultured human brain capillary endothelial cells upon reoxygenation. 4. Also, we introduce briefly a method of culturing human brain capillary endothelial cells and present our experimental results on the effects of SOD, catalase, and allopurinol in these cultured cells following anoxia and reoxygenation.

The role of alcohol in the oxidant antioxidant balance in heart.

The myocardium, like other tissues, has enzyme and non-enzyme systems to neutralize free radicals. The enzymes superoxide dismutase, catalase and glutathione peroxidase and glutathione reductase as well as the non-enzyme antioxidants vitamin E and ascorbic acid are the main antioxidants. Oxidants are produced by the mitochondria under normal conditions and by other sources under pathologic conditions. The quantity of antioxidants present in the myocardium is matched to the production of oxygen free radicals that may be produced under basal physiological conditions. However, the myocardium can be exposed to increased levels of oxygen free radicals under conditions in which myocardial metabolism, i.e. mitochondrial oxidative phosphorylation, is accelerated to match the adenosine triphosphate utilized to support the increased work load on the heart or can be exposed to oxygen free radicals under pathologic conditions such as ischemia and reperfusion, inflammation, and cardiotoxic drugs such as anti-cancer agents. Under such circumstances the normal heart has been shown to increase its antioxidant production and to be, with time, protected from further sources of oxygen free radicals. In particular, hearts previously exposed to a stimulus to produce greater antioxidant levels show less damage during ischemia reperfusion injury presumably because of neutralization of oxygen free radicals. This review will present several situations in which the myocardium increases its tolerance to ischemia reperfusion injury as a result of an initial oxidative stress.

Association of high plasma antioxidant capacity with new lesion formation in carotid atherosclerosis: a prospective study.

In atherosclerosis, both reductions and elevations in plasma levels of antioxidants have been reported. This study investigated total antioxidant capacity of plasma from subjects with atherosclerotic disease. The study population consisted of 48 men with or without carotid atherosclerosis. At baseline (1990) carotid arteries were evaluated by duplex sonography and plasma samples were obtained for testing antioxidant capacity by two different test systems. One assay system used neutrophils from healthy volunteers as a source of oxygen free radicals activating the non-fluorescent dichlorofluorescin diacetate in the presence of antioxidant containing plasma from study subjects. In the other test system, total plasma antioxidants were detected colorimetrically by using 2,2'-azino-di-(3-ethylbenzthiazoline sulphonate), metmyoglobin and superoxide in the presence of plasma. Carotid arteries were re-evaluated for the development of new plaques 5 years later (1995). Increased baseline total antioxidant capacity of plasma was significantly associated with the development of new atherosclerotic lesions during a period of 5 years. Endogenous antioxidant capacity of plasma is increased in patients with active atherosclerotic disease. As scavenging of oxygen free radicals is thought to protect from atherogenesis, elevated antioxidative capacity may represent an adaptive mechanism.

Immunohistochemical Localization of Xanthine Oxidase in Human Retina

Xanthine oxidase has been established as an important source of oxygen free radicals in ischemia-reperfusion injury. It has been localized in many different tissues such as heart and intestine, but it has not yet been localized in the eye. Xanthine oxidase was detected using immunohistochemistry on paraformaldehyde/glutaraldehyde fixed cryosections. Antibodies used included rabbit antibovine xanthine oxidase antibody and rabbit antihuman xanthine oxidase antibody. Xanthine oxidase was detected in the capillary endothelium cells of blood vessels in the retina of bovine and post mortem human eyes. Whole mount preparation of human retinas showed xanthine oxidase present throughout the small capillary network. Furthermore, whole mounts showed that xanthine oxidase was present in cones. This was confirmed by using mouse anticalbindin antibody for co-labelling. It is possible that xanthine oxidase can be a source of oxidative damage in the retina following ischemia-reperfusion injury.

Xanthine oxidase-derived superoxide causes reoxygenation injury of ischemic cerebral endothelial cells

Oxygen free radicals, generated by cerebral ischemia, have been widely implicated in the damage of vascular endothelium. Endothelial cells have been proposed as a significant source of oxygen free radicals. In the present study, we developed an anoxia–reoxygenation (AX/RO) model using pure cultures of cerebral endothelial cells (CECs) isolated from piglet cortex to measure CEC oxygen free radical production and determine its role in AX/RO-induced CEC injury. CEC injury, as measured by lactate dehydrogenase efflux into the culture medium, increased progressively with the duration of anoxic exposure, becoming significant after 10 h. Reoxygenation significantly increased CEC anoxic injury in a time-dependent manner. A 55% increase in oxygen free radical production, determined by fluorescence detection of dihydroethidium oxidation, was measured at the end of 4-h reoxygenation in CECs subjected to AX/RO conditions that killed 40% of the cells. Blockade of oxygen free radical production with superoxide dismutase (SOD; 250 and 1000 U/ml) or oxypurinol (50 and 200 μM), a potent xanthine oxidase inhibitor, reduced this injury by 32–36% and 30–39%, respectively. Results from our in vitro model indicate that CECs produce significant amounts of oxygen free radicals following ischemia, primarily from the xanthine oxidase pathway. These radicals ultimately have a cytotoxic effect on the very cells that produced them. Thus, reductions in oxygen free radical-mediated vascular injury may contribute to improvements in neurophysiologic outcome following treatment with oxygen free radical inhibitors and scavengers.

Protective effect of darodipine, a calcium antagonist, on rat cardiomyocytes against oxygen radical-mediated injury.

1. We used electrophysiological and electron spin resonance (e.s.r.) techniques to study the mechanism of the protective effect of darodipine on rat isolated cardiomyocytes exposed to an exogenous source of oxygen free radicals (OFR). 2. The L-type calcium current (I(Ca,L)), action potential and cell shortening were measured in patch-clamped cells in the whole-cell configuration. I(Ca,L) blockade by darodipine was concentration-dependent, peak current being reduced by 20% with 50 nM and by 58% with 100 nM darodipine. The lowest concentration of darodipine did not affect action potential or twitch profile. 3. Exposure to OFR-generating solution (5 mM dihydroxyfumarate, DHF) caused the appearance of electrophysiological alterations and/or spontaneous activity in 73% of cells (n=26) within 5 min; action potential duration (APD) was prolonged (195+/-16 ms vs 140+/-6 ms in the control) and maximum diastolic potential (MDP) was reduced (-59.5+/-2.6 mV vs -69.8+/-0.8 mV in the control) (P<0.05, n=25). 4. A 2 min pretreatment with 50 nM darodipine significantly reduced the incidence of these arrhythmogenic events following a 5 min exposure to OFR (36% of cells, n=14; P<0.05 vs nonpretreated cells). Pretreatment with darodipine also prevented APD prolongation caused by OFR (137+/-12 ms after DHF vs 117+/-6 ms before DHF n=14, not significant) but not the decrease of MDP (-63.4+/-2.5 mV after DHF vs -70.9+/-1.0 mV before DHF, P<0.05). 5. The e.s.r. spectra obtained from the DHF-DMPO solution in the absence of darodipine demonstrated the presence of two components corresponding to two DMPO adducts. The addition of darodipine (50-500 nM) led to a concentration-dependent decrease in intensity of the signals, the intensity of the DMPO-COO.- adduct being decreased more than that of the DMPO-OH. adduct. 6. Our results demonstrate that darodipine dose-depentently blocks I(Ca,L) in rat isolated cardiomyocytes. Furthermore it exerts protective effects against free-radical-induced electrophysiological alterations independently of its calcium antagonistic properties; this effect is possibly due to trapping of specific radical species.

Concentrations of adenosine and its metabolites in the rat retina/choroid during reperfusion after ischemia

PURPOSE. Little is known about the nature of biochemical disturbances during reperfusion after retinal ischemia. Previous studies have suggested that adenosine is responsible for regulation of retinal blood flow soon after ischemia has ended. Therefore, in this study we measured concentrations of adenosine and its metabolites in the rat retina/choroid after brief (10 min) or prolonged (60 min) periods of ischemia, and the functional consequences of inhibiting adenosine metabolism. METHODS. Ischemia was produced in anesthetized rats by ligation of the central retinal artery. The eyes were frozen in situ and purine nucleoside concentration was determined by high performance liquid chromatography. The functional effects of pre-ischemic inhibition of xanthine dehydrogenase/xanthine oxidase were assessed by measurement of the electroretinogram before, during, and up to 7 days following 60 min ischemia. RESULTS. Changes in the concentrations of adenosine and its metabolites were significant early in the reperfusion period, and were greater in magnitude and occurred earlier in prolonged, compared to brief, ischemic periods. Concentrations of adenosine, inosine, and hypoxanthine remained elevated for 30 min following the end of 60 min ischemia, and xanthine concentration was significantly elevated until 60 min after the end of either 10 or 60 min of ischemia. The onset of its peak value after ischemia was delayed in comparison to that of adenosine. Ischemia-evoked increases in xanthine concentration were attenuated by inhibition of adenosine deaminase or xanthine oxidase/xanthine dehydrogenase. Pre-ischemic inhibition of xanthine oxidase/xanthine dehydrogenase by oxypurinol (40 or 80 mg/kg intraperitoneally [IP]) resulted in a significant improvement in recovery of the a and b waves of the electro-retinogram in comparison to a saline-treated control group. CONCLUSIONS. These results indicate that adenosine is a major component of the biochemical changes that occur after retinal ischemia. Long-lasting increases in xanthine concentration during reperfusion after ischemia could be a source of oxygen free radicals that may contribute to delayed injury of the retina; attempts to decrease xanthine concentration would ideally be initiated within one hour after the end of ischemia.

Association of cytotoxin production and neutrophil activation by strains of Helicobacter pylori isolated from patients with peptic ulceration and chronic gastritis.

Helicobacter pylori is associated with neutrophil infiltration within the gastroduodenal mucosa. Neutrophil activation provides a major source of oxygen free radicals, which have been implicated in the pathogenesis of peptic ulceration. To investigate if cytotoxin producing strains of H pylori are associated with the generation of oxidative burst in polymorphonuclear neutrophils (PMNs). 76 patients undergoing endoscopy of whom 45 had peptic ulcer and 31 chronic gastritis only were studied. Strains of H pylori were cultured in Brucella broth. After 48 hours, bacteria were harvested by centrifugation and a bacterial suspension prepared as a stimulus for PMN oxidative burst using chemiluminescence. PMNs were prepared from health blood donors. To test the ability of strains to produce cytotoxin, culture supernatants of each were concentrated by polyethylene glycol and tested on cultured Vero cells for intracellular vacuolation. 30 of 45 (66.7%) peptic ulcer patients induced cell vacuolation versus nine of 31 (29%) strains from patients with chronic gastritis only (p < 0.01). Cytotoxin positive strains of H pylori regardless of the presence or absence of peptic ulcer displayed an increased induction of respiratory burst in PMNs compared with toxin negative strains from patients with chronic gastritis only (p < 0.05). Among the toxin negative strains, those from patients with peptic ulcer did not show a significant increase of the oxidative burst than those from patients without peptic ulcer (NS). Toxinogenicity of strains of H pylori seems to be correlated with neutrophil respiratory burst and peptic ulceration. The ability of some strains of H pylori to produce cytotoxin and to induce the oxidative burst in neutrophils may be important in the pathogenesis of peptic ulcer disease.