Angiotensin II-mediated endothelial dysfunction: role of poly(ADP-ribose) polymerase activation

Angiotensin II (AII) contributes to the pathogenesis of many cardiovascular disorders. Oxidant-mediated activation of poly(adenosine diphosphate-ribose) polymerase (PARP) plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovascular diseases. We have investigated whether activation of the nuclear enzyme PARP contributes to the development of AII-induced endothelial dysfunction. AII in cultured endothelial cells induced DNA single-strand breakage and dose-dependently activated PARP, which was inhibited by the AII subtype 1 receptor antagonist, losartan; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin; and the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester. Infusion of sub-pressor doses of AII to rats for 7 to 14 d induced the development of endothelial dysfunction ex vivo. The PARP inhibitors PJ34 or INO-1001 prevented the development of the endothelial dysfunction and restored normal endothelial function. Similarly, PARP-deficient mice infused with AII for 7 d were found resistant to the AII-induced development of endothelial dysfunction, as opposed to the wild-type controls. In spontaneously hypertensive rats there was marked PARP activation in the aorta, heart, and kidney. The endothelial dysfunction, the cardiovascular alterations and the activation of PARP were prevented by the angiotensin-converting enzyme inhibitor enalapril. We conclude that AII, via AII receptor subtype 1 activation and reactive oxygen and nitrogen species generation, triggers DNA breakage, which activates PARP in the vascular endothelium, leading to the development of endothelial dysfunction in hypertension.     

Regulatory mechanisms of poly(ADP-ribose) polymerase

Here, we describe the latest developments on the mechanistic characterization of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], a DNA-dependent enzyme that catalyzes the synthesis of protein-bound ADP-ribose polymers in eucaryotic chromatin. A detailed kinetic analysis of the automodification reaction of PARP in the presence of nicked dsDNA indicates that protein-poly(ADP-ribosyl)ation probably occurs via a sequential mechanism since enzyme-bound ADP-ribose chains are not reaction intermediates. The multiple enzymatic activities catalyzed by PARP (initiation, elongation, branching and self-modification) are the subject of a very complex regulatory mechanism that may involve allosterism. For instance, while the NAD+ concentration determines the average ADP-ribose polymer size (polymerization reaction), the frequency of DNA strand breaks determines the total number of ADP-ribose chains synthesized (initiation reaction). A general discussion of some of the mechanisms that regulate these multiple catalytic activities of PARP is presented below.     

Cytological detection of poly (ADP-ribose) polymerase

An attempt was made to demonstrate poly (ADP-ribose) polymerase cytologically. In vitro incorporation from the nucleotide, [³H]NAD was detected in frozen sections of onion embryo and meristematic tissue by autoradiography. In meristematic tissue, there was a correlation between the number of cells displaying intensein vitro incorporation from [³H]NAD and cytological DNA polymerase activity. Performed enzymes effecting a distinct incorporation from [³H]NAd were localized in the nuclei of all tissues of the ungerminated seed except the endosperm. Evidence for poly (ADP-ribose) polymerase has been obtained for the first time from higher plant cells and localized cytologically.     

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.     

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A⁺) mRNA detection by hybridization to [³H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000–120000 range. The Mr 120000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.Abbreviations ADP-ribose adenosine (5) diphospho(5)--D ribose - poly(ADP-ribose) polymer of ADP-ribose - mRNP messenger ribonucleoprotein particles - PMSF phenylmethylsulfonyl fluoride - LDS lithium dodecyl sulfate - TCA trichloroacetic acid     

The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity

Recent work has demonstrated that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron-transport chain triggers several pathways of injury [(protein kinase C (PKC), hexosamine and polyol pathway fluxes, advanced glycation end product formation (AGE)] involved in the pathogenesis of diabetic complications by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Increased oxidative and nitrosative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP). PARP activation, on one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport and ATP formation. On the other hand, PARP activation results in inhibition of GAPDH by poly-ADP-ribosylation. These processes result in acute endothelial dysfunction in diabetic blood vessels, which importantly contributes to the development of various diabetic complications. Accordingly, hyperglycemia-induced activation of PKC and AGE formation are prevented by inhibition of PARP activity. Furthermore, inhibition of PARP protects against diabetic cardiovascular dysfunction in rodent models of cardiomyopathy, nephropathy, neuropathy, and retinopathy. PARP activation is also present in microvasculature of human diabetic subjects. The present review focuses on the role of PARP in diabetic complications and emphasizes the therapeutic potential of PARP inhibition in the prevention or reversal of diabetic complications.     

Mechanisms of poly(ADP-ribose) polymerase catalysis; mono-ADP-ribosylation of poly(ADP-ribose) polymerase at nanomolar concentrations of NAD

Calf thymus and rat liver poly(ADP-ribose) polymerase enzymes, and the polymerase present in extracts of rat liver nuclei synthesize unstable mono-ADP-ribose protein adducts at 100 nM or lower NAD concentrations. The isolated enzyme-mono-ADP-ribose adduct hydrolyses to ADP-ribose and enzyme protein at pH values slightly above 7.0 indicating a continuous release of ADP-ribose from NAD through this enzyme-bound intermediate under physiological conditions. NH2OH at pH 7.0 hydrolyses the mono-ADP-ribose enzyme adduct. Desamino NAD and some other homologs at nanomolar concentrations act as 'forward' activators of the initiating mono-ADP-ribosylation reaction. These NAD analogs at micromolar concentrations do not affect polymer formation that takes place at micromolar NAD concentrations. Benzamides at nanomolar concentrations also activate mono-ADP-ribosylation of the enzyme, but at higher concentrations inhibit elongation at micromolar NAD as substrate. In nuclei, the enzyme molecule extensively auto-ADP-ribosylates itself, whereas histones are trans-ADP-ribosylated to a much lower extent. The unstable mono-ADP-ribose enzyme adduct represents an initiator intermediate in poly ADP-ribosylation.     

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health.     

Lipid peroxidation, protein thiol oxidation and DNA damage in hydrogen peroxide-induced injury to endothelial cells: role of activation of poly(ADP-ribose)polymerase

These experiments are a continuation of work investigating the mechanism of oxidant-induced damage to cultured bovine pulmonary artery endothelial cells (BPEC). Earlier experiments implicated DNA strand breakage and activation of poly(ADP-ribose)polymerase as critical steps in cell injury. In the current report, a better defined model of oxidant stress was used to investigate DNA damage, lipid peroxidation and protein thiol oxidation in BPEC following oxidant stress. The dose and time response of LDH release following exposure to H2O2 were established. H2O2 was metabolized rapidly by BPEC (t1/2 = 20 min). Hydrogen peroxide-induced increases in thiobarbituric acid (TBA) reactive material were prevented by pretreatment with the lipophilic antioxidant diphenylphenylinediamine (DPPD). However, DPPD did not decrease LDH release. Conversely, pretreatment with 5 mM 3-aminobenzamide (3AB), a competitive inhibitor of poly(ADP-ribose)polymerase, prevented LDH release from BPEC following H2O2 treatment. Dithiothreitol (DTT), a sulfhydryl reducing agent, also prevented LDH release. The effects of 3AB and DTT on H2O2-induced changes in DNA strand breaks and NAD+ and ATP levels were investigated as well as the effect of H2O2 on soluble and protein-bound thiols. As DPPD inhibited peroxidation without preventing LDH release, lipid peroxidation does not appear to play a role in the loss of BPEC viability in response to oxidant stress. As protein thiol oxidation was not caused by H2O2, it does not appear to play a causative role in cytotoxicity, although DTT may protect via maintenance of soluble thiols. H2O2 induces DNA strand breaks, which activate poly(ADP-ribose)polymerase, leading to depletion of cellular NAD+ and ATP and loss in cell viability. This supports earlier studies implicating the activation of poly(ADP-ribose)polymerase in oxidant injury to cultured endothelial cells.     

Detection of poly(ADP-ribose) polymerase and its reaction product poly(ADP-ribose) by immunocytochemistry

Summary Poly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD⁺ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.     

Purines inhibit poly(ADP-ribose) polymerase activation and modulate oxidant-induced cell death.

Purines such as adenosine, inosine, and hypoxanthine are known to have potent antiinflammatory effects. These effects generally are believed to be mediated by cell surface adenosine receptors. Here we provide evidence that purines protect against oxidant-induced cell injury by inhibiting the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Upon binding to broken DNA, PARP cleaves NAD+ into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins such as histones and PARP itself. Overactivation of PARP depletes cellular NAD+ and ATP stores and causes necrotic cell death. We have identified some purines (hypoxanthine, inosine, and adenosine) as potential endogenous PARP inhibitors. We have found that purines (hypoxanthine > inosine > adenosine) dose-dependently inhibited PARP activation in peroxynitrite-treated macrophages and also inhibited the activity of the purified PARP enzyme. Consistently with their PARP inhibitory effects, the purines also protected interferon gamma + endotoxin (IFN/LPS) -stimulated RAW macrophages from the inhibition of mitochondrial respiration and inhibited nitrite production from IFN/LPS-stimulated macrophages. We have selected hypoxanthine as the most potent cytoprotective agent and PARP inhibitor among the three purine compounds, and investigated the mechanism of its cytoprotective effect. We have found that hypoxanthine protects thymocytes from death induced by the cytotoxic oxidant peroxynitrite. In line with the PARP inhibitory effect of purines, hypoxanthine has prevented necrotic cell death while increasing caspase activity and DNA fragmentation. As previously shown with other PARP inhibitors, hypoxanthine acted proximal to mitochondrial alterations as hypoxanthine inhibited the peroxynitrite-induced mitochondrial depolarization and secondary superoxide production. Our data imply that purines may serve as endogenous PARP inhibitors. We propose that, by affecting PARP activation, purines may modulate the pattern of cell death during shock, inflammation, and reperfusion injury.     

Structure and function of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (PARP) participates in the intricate network of systems developed by the eukaryotic cell to cope with the numerous environmental and endogenous genetoxic agents. Cloning of the PARP gene has allowed the development of genetic and molecular approaches to elucidate the structure and the function of this abundant and highly conserved enzyme. This article summarizes our present knowledge in this field.     

Novel alkoxybenzamide inhibitors of poly(ADP-ribose) polymerase

We have previously described poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors based on a substituted benzyl-phthalazinone scaffold. As an alternative chemical template, a novel series of alkoxybenzamides were developed with restricted conformation through intramolecular hydrogen bond formation; the compounds exhibit low nM enzyme and cellular activity as PARP-1 inhibitors.     

Analysis of the activation of poly(ADP-ribose) polymerase by various types of DNA.

In this work, we examined the activation of poly(ADP-ribose) polymerase by various types of DNA. We have found that the best activator for poly(ADP-ribose) polymerase is an octanucleotide. However, the use of a defined activated DNA is also suitable.     

Identification of minimal size requirements of DNA for activation of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase requires DNA as an essential enzyme activator. Using enzyme purified from lamb thymus and double-stranded deoxynucleotide oligomers of defined length, we conducted studies to identify the smallest size DNA fragment capable of successfully activating poly(ADP-ribose) polymerase. These studies revealed that a double-stranded hexadeoxynucleotide activated the enzyme 30% as effectively as highly polymerized calf thymus DNA and a double-stranded octadeoxynucleotide activated the enzyme even more effectively than calf thymus DNA. When histone H1 was also included in the reaction system, the enzyme could be activated by even smaller DNA fragments. Thus, in the presence of histone H1, a double-stranded tetradeoxynucleotide activated the enzyme 25% as effectively as calf thymus DNA, and a double-stranded hexadeoxynucleotide was equally as effective as calf thymus DNA. The time courses for activation and the stabilities of the products were identical when the enzyme was activated by a double-stranded hexadeoxynucleotide or by calf thymus DNA. Double-stranded oligodeoxynucleotides containing dephosphorylated termini were more effective activators than those containing 3'-phosphorylated termini which in turn were more effective than those containing 5'-phosphorylated termini.     

The role of terminal DNA groups in the activation of (ADP-ribose)polymerase]

Some peculiarities of activation of (ADP-ribose) polymerase by DNA fragments were studied. DNA fragments were produced by the digestion of calf thymus DNA by micrococcal nuclease and with a subsequent enzymatic modification of their end groups by nuclease S1, polynucleotide kinase of phage T4 and alkaline phosphatase. The dependence of the activating effect of DNA on the chemical structure of its end groups was established. It was shown that the terminal phosphate groups are involved in the formation of a catalytically active complex of (ADP-ribose) polymerase with DNA.     

Acetaldehyde activation of poly(ADP-ribose)polymerase in hepatocytes of mice treated in vivo

The activities of poly(ADP-ribose) polymerase and of DNA polymerases alpha and beta and the level of cytochrome P450 were determined in mouse parenchymal liver cells 5 h after treatment with 0.1, 0.3, 1.0, and 3.0 mumole of acetaldehyde. Injection with 1.0 and 3.0 mumole of acetaldehyde induced an increase in poly(ADP-ribose) polymerase activity and in the P450 level, but had no effect on DNA polymerases. The stimulation of poly(ADP-ribose) polymerase activity can be used as an index of induced DNA damage. The possibility of using this experimental approach with other cells derived from mice treated in vivo with different xenobiotics is discussed.