Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by the synthetic "C" nucleoside analogs,tiazofurin and selenazofurin. A new strategy for cancer chemotherapy.

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) are synthetic "C" nucleosides whose antineoplastic activity depends on their conversion to tiazofurin-adenine dinucleotide and selenazofurin-adenine dinucleotide which are analogs of NAD. The present study was conducted to determine whether these nucleoside analogs and their dinucleotide derivatives interfere with NAD metabolism and in particular with the NAD-dependent enzyme, poly(ADP-ribose) polymerase. Incubation of L1210 cells with 10 microM tiazofurin or selenazofurin resulted in inhibition of cell growth, reduction of cellular NAD content, and interference with NAD synthesis. Using [14C]nicotinamide to study the uptake of nicotinamide and its conversion to NAD, we showed that the analogs interfere with NAD synthesis, apparently by blocking formation of nicotinamide mononucleotide. The analogs also serve as weak inhibitors of poly(ADP-ribose) polymerase, which is an NAD-utilizing, chromatin-bound enzyme, whose function is required for normal DNA repair processes. Continuous incubation of L1210 cells in tiazofurin or selenazofurin resulted in progressive and synergistic potentiation of the cytotoxic effects of DNA-damaging agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea or N-methyl-N''-nitro-N-nitrosoguanidine. These studies provide a basis for designing chemotherapy combinations in which tiazofurin or selenazofurin are used to modulate NAD and poly(ADP-ribose) metabolism to synergistically potentiate the effects of DNA strand-disrupting agents.     

Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity to nondividing human lymphocytes.

Deoxyadenosine has been implicated as the toxic metabolite causing profound lymphopenia in immunodeficient children with a genetic deficiency of adenosine deaminase (ADA), and in adults treated with the potent ADA inhibitor deoxycoformycin. However, the biochemical basis for deoxyadenosine toxicity toward lymphocytes remains controversial. The present experiments have examined in detail the sequential metabolic changes induced in nondividing human peripheral blood lymphocytes by incubation with deoxyadenosine plus deoxycoformycin, or with 2-chlorodeoxyadenosine (CdA), an ADA resistant deoxyadenosine congener with anti-leukemic and immunosuppressive properties. The lymphotoxic effect of deoxyadenosine and CdA required their phosphorylation, and was inhibited by deoxycytidine. As early as 4 h after exposure to the deoxynucleosides, strand breaks in lymphocyte DNA began to accumulate, and RNA synthesis decreased. These changes were followed by a significant fall in intracellular NAD levels at 8 h, a drop in ATP pools at 24 h, and cell death by 48 h. Incubation of the lymphocytes with 5 mM nicotinamide, a NAD precursor and an inhibitor of poly(ADP-ribose) synthetase, prevented NAD depletion. The nicotinamide treatment also rendered the lymphocytes highly resistant to deoxyadenosine and CdA toxicity, without altering dATP formation or the accumulation of DNA strand breaks. The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide exerted a similar although less potent effect. These results suggest that NAD depletion, probably triggered by poly(ADP-ribose) formation, is the principle cause of death in normal resting human lymphocytes exposed to deoxyadenosine plus deoxycoformycin, or to CdA.     

A community-based comparison of trauma patient outcomes between d- and l-lactate fluids

Purpose

Ringer's lactate is used for patient resuscitation. Lactate naturally occurs in 2 stereoisometric forms, d- and l-lactate, that are added to fluid in equal amounts. Animal studies have demonstrated potentially deleterious effects of d-lactate on vital organs. Using an administrative database, we examined whether d- or l-lactate volume was associated with mortality in patients with trauma.

Basic procedures

The Trauma and Injury Severity Score could be calculated in 24?616 of 528?219 patients admitted in 2006 to 2009. Demographic characteristics, the use of blood products, mechanical ventilation, and mortality were compared among the following 3 groups of patients administered Ringer's lactate: group 1, fluids other than Ringer's lactate; group 2, fluids including Ringer's dl-lactate; and group 3, no d-lactate. The mean volume (in millimoles per day) of d- and l-lactate administered was calculated. Multivariate analyses were used to measure the impact of lactate volume on mortality, and mechanical ventilation started more than 48 hours after admission.

Main findings

Groups 2 and 3 consisted of 2?827 (11.5%) patients (88 hospitals) and 12?036 (48.9%) patients (145 hospitals), respectively. The use of mechanical ventilation best explained the variation in mortality. Greater d-lactate volume, but not fluid management category or l-lactate volume, was associated with mortality. l-Lactate decreased and d-lactate increased the use of mechanical ventilation more than 48 hours after admission.

Conclusions

Because early administration of d-lactate was associated with mortality and ventilation, physicians and policy makers should recognize the advantages of l-lactate and encourage research on the quality of d- and l-lactate in case mixes beyond trauma.     

Novel isoquinolinone-derived inhibitors of poly(ADP-ribose) polymerase-1: pharmacological characterization and neuroprotective effects in an in vitro model of cerebral ischemia

Excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme catalyzing the transfer of ADP-ribose units from NAD to acceptor proteins, induces cellular energy failure by NAD and ATP depletion and has been proposed to play a causative role in a number of pathological conditions, including ischemia/reperfusion injury. In this study, we used an in vitro enzyme activity assay to characterize a series of newly synthesized isoquinolinone derivatives as potential PARP-1 inhibitors. Several compounds displayed powerful inhibitory activity: thieno[2,3-c]isoquinolin-5-one (TIQ-A) displayed a submicromolar IC50 of 0.45 +/- 0.1 microM, whereas the 5-hydroxy and 5-methoxy TIQ-A derivatives had IC50 values of 0.39 +/- 0.19 and 0.21 +/- 0.12 microM, respectively. We then examined the neuroprotective effects of the newly characterized compounds in cultured mouse cortical cells exposed to 60 min of oxygen and glucose deprivation (OGD). When PARP-1 inhibitors were present in the incubation medium during OGD and the subsequent 24-h recovery period, they significantly attenuated neuronal injury. TIQ-A provided neuroprotection even when added to the culture 30 min after OGD and was able to reduce the early activation of PARP induced by OGD as detected by flow cytometry. When the IC50 values observed in the PARP-1 activity assay for selected compounds were compared with their IC50 values for the neuroprotective activity, a significant correlation (r = 0.93, P < 0.01) was observed. Our results suggest that TIQ-A and its derivatives are a new class of neuroprotectants that may be helpful in studies aimed at understanding the involvement of PARP-1 in physiology and pathology.     

A novel and potent poly(ADP-ribose) polymerase-1 inhibitor, FR247304 (5-chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone), attenuates neuronal damage in in vitro and in vivo models of cerebral ischemia

The activation of poly(ADP-ribose) polymerase-1 (PARP-1) after exposure to nitric oxide or oxygen-free radicals can lead to cell injury via severe, irreversible depletion of NAD. Genetic deletion or pharmacological inhibition of PARP-1 attenuates brain injury after focal ischemia and neurotoxicity in several neurodegenerative models in animals. FR247304 (5-chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone) is a novel PARP-1 inhibitor that has recently been identified through structure-based drug design. In an enzyme kinetic analysis, FR247304 exhibits potent and competitive inhibition of PARP-1 activity, with a K(i) value of 35 nM. Here, we show that prevention of PARP activation by FR247304 treatment protects against both reactive oxygen species-induced PC12 cell injury in vitro and ischemic brain injury in vivo. In cell death model, treatment with FR247304 (10(-8)-10(-5) M) significantly reduced NAD depletion by PARP-1 inhibition and attenuated cell death after hydrogen peroxide (100 microM) exposure. After 90 min of middle cerebral artery occlusion in rats, poly(ADP-ribosy)lation and NAD depletion were markedly increased in the cortex and striatum from 1 h after reperfusion. The increased poly(ADP-ribose) immunoreactivity and NAD depletion were attenuated by FR247304 (32 mg/kg i.p.) treatment, and FR247304 significantly decreased ischemic brain damage measured at 24 h after reperfusion. Whereas other PARP inhibitors such as 3-aminobenzamide and PJ34 [N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylactamide] showed similar neuroprotective actions, they were less potent in in vitro assays and less efficacious in an in vivo model compared with FR247304. These results indicate that the novel PARP-1 inhibitor FR247304 exerts its neuroprotective efficacy in in vitro and in vivo experimental models of cerebral ischemia via potent PARP-1 inhibition and also suggest that FR247304 or its derivatives could be attractive therapeutic candidates for stroke and neurodegenerative disease.     

Lymphocyte dysfunction after DNA damage by toxic oxygen species. A model of immunodeficiency

The metabolic causes for immune impairment in patients with severe chronic inflammatory diseases have not been clearly defined. Recently, the overproduction of poly(ADP-ribose) in resting lymphocytes with unrepaired DNA strand breaks has been suggested to contribute to immune dysfunction in adenosine deaminase-deficient patients. Our experiments have determined to what extent DNA damage and poly(ADP-ribose) synthesis might also explain the impaired mitogen responsiveness of PBL exposed to toxic oxygen species. Treatment of normal resting human lymphocytes with xanthine oxidase and hypoxanthine dose-dependently induced DNA strand breaks and triggered the rapid synthesis of poly(ADP-ribose). Subsequently, NAD+ and ATP pools decreased precipitously. Lymphocytes exposed previously to the enzymatic oxidizing system did not synthesize DNA after stimulation with PHA. However, if the medium was supplemented with 3-aminobenzamide or nicotinamide, two compounds that inhibit poly(ADP-ribose) formation, cellular NAD+ and ATP pools were preserved, and the lymphocytes responded vigorously to a mitogenic challenge. Excessive poly(ADP-ribose) synthesis, provoked by DNA strand breakage, may represent a common pathway that connects the immunodeficiency syndromes associated with (a) exposure of lymphocytes to toxic oxygen species during chronic inflammatory states, (b) adenosine deaminase deficiency, and (c) certain DNA repair disorders.     

Some observations on serum lactate dehydrogenase (pyruvate leads to lactate) assays optimised at 37 degrees

One hundred and four sera were assayed for serum lactate dehydrogenase (l-lactate : NAD oxidoreductase, EC 1.1.1.27.) activity (pyruvate→ lactate) by three methods: the Henry assay used at 37°, and the McQueen and Eskalab assays which are claimed to be optimised at 37°. The McQueen assay gave significantly less lactate dehydrogenase activity, due to substrate inhibition, than the other two methods which appeared to be equivalent. An excess proportion of M-type lactate dehydrogenase subunits in samples was slightly overestimated by both the Eskalab and McQueen assays. It was concluded that an assay using pyruvate concentrations between 0.6 mM (Henry assay) and 1.5 Mm (Eskalab assay) would be more satisfactory.     

Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells

In this report, we show that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain activates the three major pathways of hyperglycemic damage found in aortic endothelial cells by inhibiting GAPDH activity. In bovine aortic endothelial cells, GAPDH antisense oligonucleotides activated each of the pathways of hyperglycemic vascular damage in cells cultured in 5 mM glucose to the same extent as that induced by culturing cells in 30 mM glucose. Hyperglycemia-induced GAPDH inhibition was found to be a consequence of poly(ADP-ribosyl)ation of GAPDH by poly(ADP-ribose) polymerase (PARP), which was activated by DNA strand breaks produced by mitochondrial superoxide overproduction. Both the hyperglycemia-induced decrease in activity of GAPDH and its poly(ADP-ribosyl)ation were prevented by overexpression of either uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD), which decrease hyperglycemia-induced superoxide. Overexpression of UCP-1 or MnSOD also prevented hyperglycemia-induced DNA strand breaks and activation of PARP. Hyperglycemia-induced activation of each of the pathways of vascular damage was abolished by blocking PARP activity with the competitive PARP inhibitors PJ34 or INO-1001. Elevated glucose increased poly(ADP-ribosyl)ation of GAPDH in WT aortae, but not in the aortae from PARP-1-deficient mice. Thus, inhibition of PARP blocks hyperglycemia-induced activation of multiple pathways of vascular damage.     

A coupled enzymatic method to measure blood lactate by amperometric monitoring of the rate of oxygen depletion with a Clark oxygen electrode.

A rapid, sensitive and reliable method to measure lactate in blood is described. The method is based on the enzymatic oxidation of lactate to pyruvate in the presence of nicotinamide adenine dinucleotide (NAD+) and lactate dehydrogenase (LDH). The reaction product, NADH, is then oxidized by molecular oxygen, carried in the buffered reagent medium, in the presence of horeseradish peroxidase and other cofactors. The maximum rate of oxygen depletion, which is directly proportional to the amount of lactate ion present in the sample, is amperometrically monitored by a membrane oxygen electrode. No sample pretreatment is required in the present procedure other than dilution, and a comparison study between the described method and a spectrophotometric method shows good correlation.     

Effect of the systemic fungicide carboxin on electron transport function in membranes of Micrococcus denitrificans

The systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) inhibited oxidation of succinate by membranes prepared from Micrococcus denitrificans, the K(i) being 16 muM. Oxycarboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4,4-dioxide), F831 (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4-oxide), and another succinate oxidase inhibitor, 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTB) were less effective inhibitors of succinate oxidation by membranes of M. denitrificans. Oxidation of other substrates (nicotinamide adenine dinucleotide, reduced form, d-lactate, l-lactate, malate, and d,l-alpha-hydroxybutyrate) was inhibited to a lesser degree by carboxin, and formate oxidation was entirely resistant. With all substrates tested, oxycarboxin, the dioxide analogue of carboxin, was less effective than carboxin. Carboxin also inhibited dichlorophenol indophenol (DCIP) reductase activities by these membranes in a manner both qualitatively and quantitatively similar to the inhibition of oxidation of the various substrates. The inhibition of DCIP reductase activities by TTB was qualitatively similar to carboxin, but TTB was a less effective inhibitor with all substrates tested. The inhibition of DCIP reductase by carboxin could be relieved by phenazine methosulfate with all substrates except d-lactate. Only slight inhibition of d-lactate-stimulated uptake of [(14)C]glycine by these membrane vesicles was seen with carboxin. Uptake of [(14)C]glycine could be stimulated to varying degrees with the other substrates tested, but in no case did carboxin cause significant inhibition. Membranes isolated from M. denitrificans are a useful system for investigating the mechanism of inhibition of electron transport function by carboxin, and the use of this system for evaluations of carboxin and its metabolites is suggested.     

Identification of a nicotinamide adenine dinucleotide glycohydrolase and an associated inhibitor in isoniazid-susceptible and -resistant Mycobacterium phlei.

Nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was demonstrated in the catalases fraction of Sephadex G-200-chromatographed sonic extracts of isoniazid (INH)-susceptible (Inhs) and -resistant (Inhr) Mycobacterium phlei. Since crude extracts had no demonstrable activity even after heating, active fractions of the NADase were purified chromatographically by removing the inhibitor with Sephadex G-200. Assays for oxidized nicotinamide adenine dinucleotide (NAD+) hydrolytic activity were done by following the disappearance of NAD+ by the methods of alcohol dehydrogenase or cyanide addition. The NADase activity was linear with respect to time as well as concentration of enzyme and was inhibited in the presence of 0.04 M NADP, benzoic acid hydrazide, or nicotinamide. Crude extracts or pooled concentrated Sephadex G-200 fractions eluting after the catalase inhibited NADase activity by at least 70%. Inhibitor activity was present in both the Inhs and Inhr strains of M. phlei. The activity of the partially purified inhibitors was reversible by INH or nicotinic acid hydrazide at levels between 10 and 100 mM. These findings indicate that an NADase inhibitor system which is sensitive to reversal by INH functions in both the Inhs and Inhr strains; however, unlike previous studies with other mycobacterial species, the enzyme is sensitive to inhibition by nicotinamide. Furthermore, the inhibitors are heat stable and sensitive to reversal by nicotinic acid hydrazide as well as INH.     

Nicotinamide effects on the metabolism of human fibroblasts and keratinocytes assessed by quantitative,label-free fluorescence imaging

Alterations in metabolism are central to the aging process. Therefore, understanding the subcellular functional and structural changes associated with metabolic aging is critical. Current established methods for exploring cell metabolism either require the use of exogenous agents or are destructive to the tissue or cells. Two-photon excited fluorescence (TPEF) imaging has emerged as a method for monitoring subtle metabolic changes non-invasively. In this study, we use TPEF imaging to acquire high-resolution fluorescence images from two coenzymes, NAD(P)H (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), within human fibroblasts and keratinocytes in response to B3 (a nicotinamide precursor) supplementation and/or UV irradiation, without addition of exogenous labels. In addition, multi-parametric analysis methods are used to extract functional information of cellular metabolism, including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial organization. Our results demonstrate that such optical metabolic assessments can serve as sensitive, label-free, non-destructive reporters of known effects of B3 to maintain and in some cases even enhance the respiratory function of mitochondria, while lowering oxidative damage. Thus, TPEF imaging, supported by highly-quantitative analysis, can serve as a tool to understand aging-dependent metabolic changes as well as the effect of actives on human epidermal and dermal cells.     

Metabolic alterations in the human erythrocyte produced by increases in glucose concentration: The role of the polyol pathway

Human erythrocytes incubated in medium containing 50 mM glucose have increased intracellular sorbitol and fructose concentrations as compared with samples incubated with 5 mM glucose. Increased medium glucose concentration did not significantly alter total glucose consumption or lactate production. However, the intracellular lactate:pyruvate ratio rose, the concentrations of fructose diphosphate, and triose phosphates increased, and the 2,3-diphosphoglycerate concentration fell. [(14)C]O(2) production from glucose-1-(14)C also increased with increased medium glucose concentration. These changes are believed to reflect changes in the redox states of the diphosphopyridine nucleotide/reduced form of diphosphopyridine nucleotide (NAD/NADH) and nicotinamide-adenine dinucleotide phosphate/reduced form of nicotinamide-adenine dinucleotide phosphate (NADP/NADPH) couples resulting from increased activity of the polyol pathway. Addition of pyruvate to the incubation media prevented these changes. These studies illustrate that an increase in the red cell's normal substrate, glucose, can produce changes in red cell metabolism.     

ADP-ribosylation of membrane proteins: unveiling the secrets of a crucial regulatory mechanism in mammalian cells

Many bacterial toxins kill animal cells by adenosine diphosphate (ADP)-ribosylating intracellular target proteins. Mammalian cells express toxin-related cell surface ADP-ribosyltransferases (ARTs) that transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD) onto arginine residues of other membrane proteins. The association of these glycosylphosphatidylinositol (GPI)-anchored ectoenzymes with glycolipid rafts focuses them onto components of the signal transduction machinery. Exposing murine T cells to NAD, the ART substrate, induces a cascade of reactions that culminates in cell death by apoptosis. This mechanism, dubbed 'NAD-induced cell death' or NICD, is initiated when ART2 ADP-ribosylates the cytolytic P2X7 purinergic receptor, inducing formation of a cation channel, opening of a nonselective pore, shedding of CD62L from the cell surface, exposure of phosphatidylserine on the outer leaflet of the plasma membrane, breakdown of the mitochondrial membrane potential, and DNA-fragmentation. The ART substrate NAD is produced in large amounts inside the cell and can be released from damaged cells during inflammation and tissue injury. In the extracellular environment, the signaling function of NAD is terminated by NAD-degrading ectoenzymes such as CD38. We propose that ART2-catalyzed ADP-ribosylation of P2X7 represents the paradigm of a regulatory mechanism by which ART-expressing cells can sense and respond to the release of NAD from damaged cells.     

Role of nicotinamide adenine dinucleotide and adenosine triphosphate in glucocorticoid-induced cytotoxicity in susceptible lymphoid cells.

The possibility that corticosteroid cytotoxicity could be mediated by activation of poly(ADP-ribose) polymerase and consequent depletion of NAD and ATP was evaluated in steroid-sensitive S49.1 and steroid-resistant S49.143R mouse lymphoma cells and in lymphocytes from a patient with chronic lymphocytic leukemia. All cell types were shown to have the enzyme poly(ADP-ribose) polymerase and to increase activity in response to DNA strand breaks. Incubation of susceptible cells with 1 microM dexamethasone resulted in DNA strand breaks. Susceptible cells also showed a dose-dependent decrease in NAD and ATP that preceded loss of cell viability. These studies suggest that steroid-induced cytotoxicity in susceptible lymphocytes is due to the presence of DNA strand breaks that activate poly(ADP-ribose) polymerase to a sufficient degree to consume cellular pools of NAD with a consequent depletion of ATP and loss of cell viability.     

Poly(adp-ribose) synthetase inhibition reduces bacterial translocation in rats after endotoxin challenge

We investigated whether 3-aminobenzamide (3-AB), a poly(ADP-ribose) synthetase (PARS) inhibitor, reduces bacterial translocation (BT) after intraperitoneal endotoxin administration. Wistar rats were randomized to receive intraperitoneal saline (control, n = 6); endotoxin (n = 8); 3-AB (n = 6); and 3-AB plus endotoxin (n = 8). Six hours later, to evaluate the endotoxin-related intestinal injury and BT, tissue and blood samples were collected. Administration of intraperitoneal endotoxin caused severe intestinal injury and BT to mesenteric lymph nodes. PARS inhibition with 3-AB completely prevented endotoxin-induced BT. No colony-forming bacteria was isolated from the samples obtained from 3-AB-pretreated animals under endotoxin challenge. Treatment with 3-AB significantly reduced the endotoxin-induced intestinal mucosal injury. The inhibition of PARS by its blocker 3-aminobenzamide during endotoxemia prevents bacterial translocation and intestinal injury in rats. PARS activation may provide a novel therapeutic approach in reducing gut barrier failure seen in endotoxemia.     

Activation of poly(ADP-ribose) polymerase contributes to development of doxorubicin-induced heart failure

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) by oxidant-mediated DNA damage is an important pathway of cell dysfunction and tissue injury in conditions associated with oxidative stress. Increased oxidative stress is a major factor implicated in the cardiotoxicity of doxorubicin (DOX), a widely used antitumor anthracycline antibiotic. Thus, we hypothesized that the activation of PARP may contribute to the DOX-induced cardiotoxicity. Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the phenanthridinone PARP inhibitor PJ34, we now demonstrate the role of PARP in the development of cardiac dysfunction induced by DOX. PARP-1+/+ and PARP-1-/- mice received a single injection of DOX (25 mg/kg i.p). Five days after DOX administration, left ventricular performance was significantly depressed in PARP-1+/+ mice, but only to a smaller extent in PARP-1-/- ones. Similar experiments were conducted in BALB/c mice treated with PJ34 or vehicle. Treatment with a PJ34 significantly improved cardiac dysfunction and increased the survival of the animals. In addition PJ34 significantly reduced the DOX-induced increase in the serum lactate dehydrogenase and creatine kinase activities but not metalloproteinase activation in the heart. Thus, PARP activation contributes to the cardiotoxicity of DOX. PARP inhibitors may exert protective effects against the development of severe cardiac complications associated with the DOX treatment.     

Role of tight junction derangement in the endothelial dysfunction elicited by exogenous and endogenous peroxynitrite and poly(ADP-ribose) synthetase

DNA single-strand breakage and activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) triggers an energy consuming, inefficient repair cycle, which contributes to peroxynitrite-induced cellular injury. Here, we investigated whether peroxynitrite and PARS activation are involved in tight junctions (tight junction) derangement in the endothelial dysfunction in cells exposed to peroxynitrite and in vascular rings of animals subjected to zymosan non-septic shock. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, as measured by the mitochondrial-dependent conversion of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan. Moreover, peroxynitrite caused activation of PARS. Inhibition of PARS by 3-aminobenzamide (3-AB; 1 mM) reduced the peroxynitrite-induced suppression of mitochondrial respiration in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. Peroxynitrite incubation also caused a significant derangement of zonula occludens (ZO)-1, which was significantly affected by pharmacological inhibition of PARS. 3-AB ameliorated the development of this peroxynitrite-induced endothelial dysfunction. In vascular rings obtained from the zymosan-treated rats, there was a marked suppression of the endothelium-dependent relaxation ex vivo, which was reduced by in vivo 3-AB treatment. A significant derangement of ZO-1 was observed in vascular rings from zymosan-treated rats. Tight junction alteration was significantly reduced by in vivo 3-AB treatment. Thus, activation of PARS by exogenous and endogenous peroxynitrite may be involved in the tight junction derangement associated with endothelial dysfunction. Inhibition of PARS may be a novel pharmacological approach to preserve endothelial tight junction function in shock and inflammation.