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PubMed2004

NAD glycohydrolase activities and ADP-ribose uptake in erythrocytes from normal subjects and cancer patients.

Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [14C] ADP-ribose uptake values. When [adenosine-14C] NAD was used instead of free [14C] ADP-ribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-14C] NAD by Cibacron Blue. ADP-ribose uptake in erythrocytes appeared to be complex: upon incubation with free [14C] ADP-ribose, the radiolabel associated with erythrocytes was located in nearly equal parts in cytoplasm and plasma membrane. Part of [14C] ADP-ribose binding to the membrane was covalent, as indicated by its resistance to trichloroacetic acid-treatment. A preincubation with unlabeled ADP-ribose depressed subsequent erythrocyte NAD glycohydrolase activity and binding of [14C] ADP-ribose to erythrocyte membrane; but it failed to inhibit the transfer of labeled ADP-ribose to erythrocyte cytoplasm. On the other hand, incubation with [adenosine-14C] NAD did not result in a similar covalent binding of radiolabel to erythrocyte membrane. In line with this finding, a preincubation with unlabeled NAD was not inhibitory on subsequent NAD glycohydrolase reaction and ADP-ribose binding. ADP-ribose binding and NAD glycohydrolase activities were found also in solubilized erythrocyte membrane proteins and, after size fractionation, mainly in a protein fraction of around 45kDa-molecular weight.

ADP-ribosyl Cyclase↗
PubMed1982

Hydrolysis of nicotinamide-adenine dinucleotide by purified renal brush-border membranes. Mechanism of NAD+ inhibition of brush-border membrane phosphate-transport activity.

Purified rat renal brush-border membrane vesicles possess a heat-labile enzyme activity which hydrolyses NAD+. A reciprocal relationship exists between the disappearance of NAD+ and the appearance of adenosine; 2 mol of Pi are liberated from each mol of NAD+ incubated with brush-border membrane vesicles. Freezing and thawing brush-border membrane vesicles does not enhance the initial rate of NAD+ hydrolysis. Preincubation of brush-border membrane vesicles with NAD+ results in inhibition of Na+-dependent Pi-transport activity, whereas Na+-dependent glucose transport is not affected. EDTA, which prevents the release of Pi from NAD+ and which itself has no direct effect on brush-border membrane Pi transport, reverses the NAD+ inhibition of Na+-dependent Pi transport. These results suggest that it is the Pi liberated from NAD+ and not NAD+ itself that inhibits Na+-dependent Pi transport.

Animals↗
PubMed1996

Nitric oxide-dependent NAD linkage to glyceraldehyde-3-phosphate dehydrogenase: possible involvement of a cysteine thiyl radical intermediate.

Previous studies have demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes NAD(H) linkage to an active site thiol when it comes into contact with .NO-related oxidants. We found that a free-radical generator 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release either .NO or .NO-related species, was indeed able to induce the NAD(H) linkage to GAPDH. We performed spin-trapping studies with purified apo-GAPDH to identify a putative thiol intermediate produced by AAPH as well as by .NO-related oxidants. As .NO sources we used .NO gas and two .NO-donors, S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydno-nimine hydrochloride (SIN-1). Because SIN-1 produces .NO and a superoxide radical simultaneously, we also tested the effects of peroxynitrite. All the .NO-related oxidants were able to induce the linkage of NAD(H) to GAPDH and the formation of a protein free-radical identified as a thiyl radical (inhibited by N-ethylmaleimide). .NO gas and the .NO-donors required molecular oxygen to induce the formation of the GAPDH thiyl radical, suggesting the possible involvement of higher nitrogen oxides. Thiyl radical formation was decreased by the reconstitution of GAPDH with NAD+. Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging ability was decreased when its cysteine residues were alkylated or when it was reconstituted with NAD+. In addition, after treatment with AAPH, a thiyl radical of GAPDH was trapped at high enzyme concentrations. We suggest that the NAD(H) linkage to GAPDH is mediated by a thiyl radical intermediate not specific to .NO or .NO-related oxidants. The cysteine residue located at the active site of GAPDH (Cys-149) is oxidized by free radicals to a thiyl radical, which reacts with the neighbouring coenzyme to form Cys-NAD(H) linkages. Studies with the NAD+ molecule radio-labelled in the nicotinamide or adenine portion revealed that both portions of the NAD+ molecule are linked to GAPDH.

Cysteine↗
PubMed2000

NADP and NAD utilization in Haemophilus influenzae.

Exogenous NAD utilization or pyridine nucleotide cycle metabolism is used by many bacteria to maintain NAD turnover and to limit energy-dependent de novo NAD synthesis. The genus Haemophilus includes several important pathogenic bacterial species that require NAD as an essential growth factor. The molecular mechanisms of NAD uptake and processing are understood only in part for Haemophilus. In this report, we present data showing that the outer membrane lipoprotein e(P4), encoded by the hel gene, and an exported 5'-nucleotidase (HI0206), assigned as nadN, are necessary for NAD and NADP utilization. Lipoprotein e(P4) is characterized as an acid phosphatase that uses NADP as substrate. Its phosphatase activity is inhibited by compounds such as adenosine or NMN. The nadN gene product was characterized as an NAD-nucleotidase, responsible for the hydrolysis of NAD. H. influenzae hel and nadN mutants had defined growth deficiencies. For growth, the uptake and processing of the essential cofactors NADP and NAD required e(P4) and 5'-nucleotidase. In addition, adenosine was identified as a potent growth inhibitor of wild-type H. influenzae strains, when NADP was used as the sole source of nicotinamide-ribosyl.

Adenosine↗
PubMed1997

The oxidative half-reaction of xanthine dehydrogenase with NAD; reaction kinetics and steady-state mechanism.

The reaction between reduced xanthine dehydrogenase (XDH) from bovine milk and NAD has been studied in detail. An understanding of this reaction is necessary for a complete description of XDH turnover with its presumed natural electron acceptor and to address the preference of XDH for NAD over oxygen as a substrate. The reaction between pre-reduced XDH and NAD was studied by stopped-flow spectrophotometry. The reaction was found to involve two rounds of oxidation with 2 eq of NAD. The first round goes to completion, and the second round reaches a slightly disfavored equilibrium. Rapid binding of NAD with an apparent Kd of 25 +/- 2 microM is followed by NAD reduction at a rate constant of 130 +/- 13 s-1. NADH dissociation at a rate constant of 42 +/- 12 s-1 completes a round of oxidation. These steps have been successfully tested and modeled to repeat themselves in the second round of oxidation. The association rate constant for NAD binding was estimated to be much greater than any rate constant measured in the oxidation by molecular oxygen, thus explaining how NAD competes with oxygen for reducing equivalents. Rate constants for NAD reduction and NADH dissociation are respectively 21- and 7-fold greater than kcat, indicating that the reductive half-reaction of the enzyme by xanthine is mostly rate-limiting in xanthine/NAD turnover. A steady-state mechanism for XDH is discussed.

Animals↗
PubMed2002

NAD(P)H, a primary target of 1O2 in mitochondria of intact cells.

Direct reaction of NAD(P)H with oxidants like singlet oxygen ((1)O(2)) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2',4',5',7'-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of (1)O(2), which readily oxidized NAD(P)H to NAD(P)(+). In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating (1)O(2) within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5-10 s, Rho 123 for 60 s) led to a significant (29.6 +/- 8.2 and 30.2 +/- 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow increase. Prolonged photoactivation (> or =15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for (1)O(2) in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.

Animals↗
PubMed2004

Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition.

Extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1) by DNA damage is a major cause of caspase-independent cell death in ischemia and inflammation. Here we show that NAD(+) depletion and mitochondrial permeability transition (MPT) are sequential and necessary steps in PARP-1-mediated cell death. Cultured mouse astrocytes were treated with the cytotoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine or 3-morpholinosydnonimine to induce DNA damage and PARP-1 activation. The resulting cell death was preceded by NAD(+) depletion, mitochondrial membrane depolarization, and MPT. Sub-micromolar concentrations of cyclosporin A blocked MPT and cell death, suggesting that MPT is a necessary step linking PARP-1 activation to cell death. In astrocytes, extracellular NAD(+) can raise intracellular NAD(+) concentrations. To determine whether NAD(+) depletion is necessary for PARP-1-induced MPT, NAD(+) was restored to near-normal levels after PARP-1 activation. Restoration of NAD(+) enabled the recovery of mitochondrial membrane potential and blocked both MPT and cell death. Furthermore, both cyclosporin A and NAD(+) blocked translocation of the apoptosis-inducing factor from mitochondria to nuclei, a step previously shown necessary for PARP-1-induced cell death. These results suggest that NAD(+) depletion and MPT are necessary intermediary steps linking PARP-1 activation to AIF translocation and cell death.

Animals↗
PubMed2005

Structures of Escherichia coli NAD synthetase with substrates and products reveal mechanistic rearrangements.

Nicotinamide adenine dinucleotide synthetases (NADS) catalyze the amidation of nicotinic acid adenine dinucleotide (NAAD) to yield the enzyme cofactor nicotinamide adenine dinucleotide (NAD). Here we describe the crystal structures of the ammonia-dependent homodimeric NADS from Escherichia coli alone and in complex with natural substrates and with the reaction product NAD. The structures disclosed two NAAD/NAD binding sites at the dimer interface and an adenosine triphosphate (ATP) binding site within each subunit. Comparison with the Bacillus subtilis NADS showed pronounced chemical differences in the NAAD/NAD binding sites and less prominent differences in the ATP binding pockets. In addition, the E. coli NADS structures revealed unexpected dynamical rearrangements in the NAAD/NAD binding pocket upon NAAD-to-NAD conversion, which define a catalysis state and a substrate/product exchange state. The two states are adopted by concerted movement of the nicotinysyl moieties of NAAD and NAD, Phe-170, and residues 224-228, which may be triggered by differential coordination of a magnesium ion to NAAD and NAD. Phylogenetic structure comparisons suggest that the present results are relevant for designing species-specific antibiotics.

Adenosine Triphosphate↗
PubMed2005

Poly(ADP-ribose) polymerase-1-dependent cardiac myocyte cell death during heart failure is mediated by NAD+ depletion and reduced Sir2alpha deacetylase activity.

Robust activation of poly(ADP-ribose) polymerase-1 (PARP) by oxidative stress has been implicated as a major cause of caspase-independent myocyte cell death contributing to heart failure. Here, we show that depletion of myocyte NAD levels and the subsequent reduction of Sir2alpha deacetylase activity are the sequential steps contributing to PARP-mediated myocyte cell death. In both failing hearts and cultured cardiac myocytes, the increased activity of PARP was associated with depletion of cellular NAD levels and reduced Sir2alpha deacetylase activity. Myocyte cell death induced by PARP activation was prevented by repletion of cellular NAD levels either by adding NAD directly to the culture medium or by overexpressing NAD biosynthetic enzymes. The beneficial effect of NAD repletion was seen, however, only when Sir2alpha was intact. Knocking down Sir2alpha levels by small interfering RNA eliminated this benefit, indicating that Sir2alpha is a downstream target of NAD replenishment leading to cell protection. NAD repletion also prevented loss of the transcriptional regulatory activity of the Sir2alpha catalytic core domain resulting from PARP activation. We also show that PARP activation and the concomitant reduction of Sir2alpha activity in failing hearts regulate the post-translational acetylation of p53. These data demonstrate that, in stressed cardiac myocytes, depletion of cellular NAD levels forms a link between PARP activation and reduced Sir2alpha deacetylase activity, contributing to myocyte cell death during heart failure.

Animals↗
PubMed2000

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity.

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe2+-H2O2 or Fe3+-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)+, only Fe2+-H2O2 also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe2+ iron (concentration range 3-180 microM) or H2O2 (concentration range 50-1000 microM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)+, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)+/ADP-ribose(P) ratio was about 4 at any Fe2+ or H2O2 concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)+/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)+ production of Fe2+-H2O2-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where "conventional" oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.

Adenosine Diphosphate Ribose↗
PubMed1995

Enzyme properties of Aplysia ADP-ribosyl cyclase: comparison with NAD glycohydrolase of CD38 antigen.

An ecto-enzyme of NAD glycohydrolase (NADase) induced by retinoic acid in HL-60 cells is attributed to the molecule of CD38 antigen [Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T. (1993) J. Biol. Chem. 268, 16895-16898]. CD38 antigen has an amino acid sequence homologous to Aplysia ADP-ribosyl cyclase which generates cyclic adenosine diphosphoribose (cADPR) and nicotinamide (NA) from beta-NAD+. On the basis of this sequence homology, we compared enzyme properties between CD38 NADase expressed as a fusion protein in Escherichia coli and ADP-ribosyl cyclase purified from the ovotestis of Aplysia kurodai. 1) beta-NAD+ analogs, nicotinamide 1, N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide, did not serve as good substrates for the ADP-ribosyl cyclase, suggesting that the intact adenine ring of beta-NAD+ was required for the cyclase-catalyzed reaction. On the other hand, CD38 NADase utilized the NAD analogs to form ADP-ribose and NA. 2) Kinetic analyses of the ADP-ribosyl cyclase reaction revealed that NA was first released from the substrate (beta-NAD+)-enzyme complex, followed by the release of another product, cADPR, which was capable of interacting with the free enzyme. 3) The enzyme reaction catalyzed by the ADP-ribosyl cyclase was fully reversible; beta-NAD+ could be formed from cADPR and NA with a velocity similar to that observed in the degradation of beta-NAD+. However, CD38 NADase did not catalyze the reverse reaction to form beta-NAD+ from ADP-ribopase and NA. 4) The CD38 NADase activity was, but the ADP-ribosyl cyclase activity was not, inhibited by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosyl Cyclase↗
PubMed1980

The effect of pH on the allosteric behaviour of ox-brain NAD+-dependent isocitrate dehydrogenase.

The sigmoid dependence of the initial rate of the reaction catalysed by NAD+-dependent isocitrate dehydrogenase on the concentration of isocitrate is greatly reduced as the pH is decreased. The apparent pK for this process is 7.3. At pH values of 6.0 and 6.5 the enzyme exhibits hyperbolic kinetics with respect to isocitrate at relatively high concentrations of NAD+, but the dependence becomes sigmoid at lower NAD+ concentrations. The allosteric activators ADP and citrate have only small effects on the activity of the enzyme at pH 6.5 but in the latter case the effects are increased by decreasing the concentration of NAD+. The dependence of initial velocity on the NAD+ concentration is hyperbolic at all pH values in the pH range 6--8.5. NADH is a competitive inhibitor of enzyme activity with respect to NAD+, and its presence induces a sigmoid dependence of initial velocity on isocitrate concentration at pH 6.5 and in the presence of high concentrations of NAD+. Kinetic studies at pH 6.5 indicate that the apparent maximum velocity of the reaction with respect to NAD+ is insensitive to changes in the isocitrate concentration and that with isocitrate as the substrate is relatively insensitive to changes in the NAD+ concentration under conditions where the behaviour appears to be hyperbolic. threo-Ds-Isocitrate is the true substrate for the enzyme and the corresponding Ls isomer is neither a substrate nor an inhibitor.

Adenosine Diphosphate↗
PubMed1980

Metabolism of NAD and N1-methylnicotinamide in growing and growth-arrested cells.

Nicotinamide is metabolized primarily into NAD and N1-methylnicotinamide in cultured cells of normal rat kidney. The metabolic pathways for the nicotinamide metabolites are independently regulated and are influenced by the growth stage of the cells. N1-Methylnicotinamide levels are 1.5--2-fold elevated in cells growth-arrested by treatment with histidinol, thymidine, or picolinic acid, or by serum starvation. This increase is due to a more rapid rate of synthesis rather than decrease in excretion. The rates of both synthesis and degradation of NAD are increased in serum-starved cells so that the NAD concentration is the same as it is in growing cells. NAD and N1- methylnicotinamide levels are not significantly increased when the intracellular nicotinamide concentration is increased 20-fold by addition of excess nicotinamide to the culture medium, demonstrating that the size of the nicotinamide pool does not limit synthesis of these compounds. In medium containing normal amounts of nicotinamide, the apparent first-order rate constant for the decay of NAD, radioactively labeled in the nicotinamide moiety, is about 4 h-1. Labeled N1-methylnicotinamide is not metabolized, but rather is excreted into the medium with a first-order rate constant of 3.9 h-1. The rate of loss of label from NAD, but not from N1-methylnicotinamide, is increased about twofold by addition of excess nicotinamide to the culture medium. This could be explained by a dilution of a labeled nicotinamide pool which is formed during NAD degradation and which is recycled into NAD but not into N1-methylnicotinamide. The results demonstrate a rapid turnover of NAD at the bond joining nicotinamide and ADP-ribose, in agreement with previous studies. In addition, the results show that nicotinamide is metabolized into N1-methylnicotinamide with what appears to be a carefully regulated synthetic mechanism. The existence of significant amounts of N1-methylnicotinamide in cultured cells raises the question of the physiological importance of this compound.

Animals↗
PubMed2006

Zinc neurotoxicity is dependent on intracellular NAD levels and the sirtuin pathway.

Zinc neurotoxicity has been demonstrated in ischemic, seizure, hypoglycemic, and trauma-induced neuronal death where Zn(2+) is thought to be synaptically released and taken up in neighbouring neurons, reaching toxic concentrations. We previously demonstrated that toxicity of extracellular Zn(2+) depended on entry, elevation in intracellular free Zn(2+) ([Zn(2+)](i)), a reduction in NAD(+) and ATP levels, and dysfunction of glycolysis and cellular metabolism. We suggested that PARP-1 activation alone can not explain this loss of neuronal NAD(+). NAD(+) was recently demonstrated to permeate neurons and glia, and we have now shown that exogenous NAD(+) can reduce Zn(2+) neurotoxicity, and 3-acetylpyridine, which generates inactive NAD(+), potentiated Zn(2+) neurotoxicity. Sirtinol and 2-hydroxynaphthaldehyde, inhibitors of the sirtuin pathway (SIRT proteins are NAD(+)-catabolic protein deacetylases), attenuated both acute and chronic Zn(2+) neurotoxicity. Resveratrol and fisetin (sirtuin activators) potentiated NAD(+) loss and Zn(2+) neurotoxicities. Furthermore, neuronal cultures derived from the Wld(s) mouse, which overexpress the NAD(+) synthetic enzyme nicotinamide mononucleotide adenyl transferase (NMNAT-1), had reduced sensitivity to Zn(2+) neurotoxicity. Finally, nicotinamide was demonstrated to attenuate CA1 neuronal death after 10 min of global ischemia in rat even if administered 1 h after the insult. Together with previous data, these results further implicate NAD(+) levels in Zn(2+) neurotoxicity.

Aldehydes↗
PubMed1987

Negative modulation of Escherichia coli NAD kinase by NADPH and NADH.

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2+ of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyme was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme. These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2+ and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.

Adenine Nucleotides↗
PubMed1986

5-Methylnicotinamide inhibits renal brush-border phosphate transport with no change in NAD.

Administration of nicotinamide to rats produces a dose-dependent increase in nicotinamide adenine dinucleotide (NAD) content in the renal cortex and specific inhibition of sodium gradient-dependent phosphate transport across the renal brush-border membrane (BBM). Nicotinamide acts both as a precursor for NAD synthesis and as an inhibitor of NAD hydrolysis. To determine if the latter effect contributes to the change in phosphate transport, a nicotinamide analogue was used in the present study. 5-Methylnicotinamide (5MN) cannot support NAD synthesis, but it is an equipotent inhibitor of NAD-hydrolyzing enzymes. The characteristics of the specific inhibitory effect of nicotinamide on sodium gradient-dependent phosphate transport were reproduced by an equimolar dose (4 mmol/kg body wt) of 5MN. Unlike nicotinamide, the action of 5MN did not involve a change in renal cortical NAD content. These data indicate that inhibition of NAD hydrolyzing enzymes may be associated with inhibition of BBM phosphate transport, and this effect occurs independently of changes in NAD levels. These conclusions are supported by the finding that BBM phosphate transport was not inhibited by administration of nicotinic acid, an analogue that does not inhibit NAD hydrolysis.

Animals↗
PubMed2006

NAD metabolism and sirtuins: metabolic regulation of protein deacetylation in stress and toxicity.

Sirtuins are recently discovered NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins, thereby regulating the biological function of their targets. Sirtuins have been shown to increase organism and tissue survival in diverse organisms, ranging from yeast to mammals. Evidence indicates that NAD(+) metabolism and sirtuins contribute to mechanisms that influence cell survival under conditions of stress and toxicity. For example, recent work has shown that sirtuins and increased NAD(+) biosynthesis provide protection against neuron axonal degeneration initiated by genotoxicity or trauma. In light of their protective effects, sirtuins and NAD(+) metabolism could represent therapeutic targets for treatment of acute and chronic neurodegenerative conditions. Our work has focused on elucidating the enzymatic functions of sirtuins and quantifying perturbations of cellular NAD(+) metabolism. We have developed mass spectrometry methods to quantitate cellular NAD(+) and nicotinamide. These methods allow the quantitation of changes in the amounts of these metabolites in cells caused by chemical and genetic interventions. Characterization of the biochemical properties of sirtuins and investigations of NAD(+) metabolism are likely to provide new insights into mechanisms by which NAD(+) metabolism regulates sirtuin activities in cells. To develop new strategies to improve cell stress resistance, we have initiated proof of concept studies on pharmacological approaches that target sirtuins and NAD(+) metabolism, with the goal of enhancing cell protection against genotoxicity.

Acetylation↗
PubMed2007

Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia.

Excessive poly(ADP-ribose) polymerase-1 (PARP-1) activation plays a significant role in ischemic brain damage. Increasing evidence has supported the hypothesis that PARP-1 induces cell death by depleting intracellular NAD+. Based on our in vitro finding that NAD+ treatment can abolish PARP-1-mediated cell death, we hypothesized that NAD+ administration may decrease ischemic brain injury. In this study, we used a rat model of transient focal ischemia to test this hypothesis. We observed that intranasal NAD+ delivery significantly increased NAD+ contents in the brains. Intranasal delivery with 10 mg/kg NAD+ at 2 hours after ischemic onset profoundly decreased infarct formation when assessed either at 24 or 72 hours after ischemia. The NAD+ administration also significantly attenuated ischemia-induced neurological deficits. In contrast, intranasal administration with 10 mg/kg nicotinamide did not decrease ischemic brain damage. These results provide the first in vivo evidence that NAD+ metabolism is a new target for treating brain ischemia, and that NAD+ administration may be a novel strategy for decreasing brain damage in cerebral ischemia and possibly other PARP-1-associated neurological diseases.

Administration, Intranasal↗