NAD Degradation Research Articles

Mechanisms of hypochlorite injury of target cells.

HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants.

A pathway linking NAD degradation and hypoxanthine formation associated with the tegument of the rat tapeworm Hymenolepis diminuta

A pathway linking NAD degradation and hypoxanthine formation associated with the tegument of the rat tapeworm <i>Hymenolepis diminuta</i>

Nicotinamide prolongs survival of primary cultured hepatocytes without involving loss of hepatocyte-specific functions

When hepatocytes isolated from adult rats were cultured in the presence of 10 mM nicotinamide, insulin- and epidermal growth factor-induced DNA synthesis and cell proliferation were found to be greatly stimulated, and the cells were able to be kept alive for more than one month. In the nicotinamide-treated hepatocytes, albumin and tryptophan 2,3-dioxygenase mRNAs were present at much higher levels than in the untreated control, and the inducibility of tryptophan oxygenase gene expression by dexamethasone and glucagon was also preserved. Without nicotinamide, primary cultured hepatocytes were viable for only 5-7 days and the hepatocyte-specific phenotypes were rapidly lost. The intracellular NAD level was maintained in the nicotinamide-treated hepatocytes at or above the level in intact liver but depleted in hepatocytes without nicotinamide. These results suggest that the maintenance of the intracellular NAD level is essential for the growth and functioning of hepatocytes and that nicotinamide can preserve the NAD level by blocking NAD degradation as well as by acting as a precursor for NAD synthesis.

Studies of the role of DNA fragmentation in selenium toxicity

The role of DNA damage in selenite cytotoxicity was studied in isolated hepatocyte model systems. An initial series of experiments, with hepatocytes in suspension, indicated that selenite-induced DNA fragmentation was oxygen dependent and could be inhibited by cyanide, HgCl 2 and CuDIPS. These findings were interpreted to imply that selenite-induced redox cycles were involved in this effect. In a second series of experiments, the effect of inhibitors of poly(ADP-ribose)polymerase (3-aminobenzamide and theophylline) and DNA alkylating agents on selenite-induced cellular lysis was studied. These experiments were performed with hepatocytes in primary culture and 20–30 μM selenite lysed the cultured cells after about 20 hr exposure. It was found that alkylators added 20 hr before selenite acted synergistically with selenite, and that inhibitors of poly(ADP-ribose)polymerase antagonized lysis. Further studies also indicated NAD degradation before lysis. These data indicate a modulating role for DNA damage in selenite cytotoxicity mediated by poly(ADP-ribose)polymerase. Taken together with previously published data on, for example, potentially lethal oxidation of NADPH (Anundi et al., Chem. Biol. Interact. 50, 277,1984) they also suggest that cell death resulted from interactions between several events that may deplete energy supplies. The results are compatible with a selective killing of DNA-damaged hepatocytes by low doses of selenite.

Pyridine nucleotide cycling and poly(ADP-ribose) synthesis in resting human lymphocytes.

NAD is a critical cofactor for the oxidation of fuel molecules. The exposure of human PBL to agents that cause DNA strand breaks to accumulate can deplete NAD pools by increasing NAD consumption for poly(ADP-ribose) formation. However, the pathways of NAD synthesis and degradation in viable PBL have not been carefully documented. The present experiments have used radioactive labeling techniques to trace the routes of NAD metabolism in resting PBL. The cells could generate NAD from either nicotinamide or nicotinic acid. PBL incubated with [14C]nicotinic acid excreted [14C]nicotinamide into the medium. Approximately 50% of a prelabeled [14C]NAD pool was metabolized during 6 to 8 hr in tissue culture. Basal NAD turnover was prolonged threefold to fourfold by 3-aminobenzamide (3-ABA), an inhibitor of poly(ADP-ribose) synthetase. Supplementation of the medium with 3-ABA also prevented the accelerated NAD degradation that ensued after exposure of PBL to deoxyadenosine plus deoxycoformycin at concentrations previously shown to cause DNA strand break accumulation. These results demonstrate that quiescent human PBL continually produce NAD and utilize the nucleotide for poly(ADP-ribose) synthesis.

Degradation of NAD by synaptosomes and its inhibition by nicotinamide mononucleotide: implications for the role of NAD as a synaptic modulator.

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.

Streptozotocin and alloxan induce DNA strand breaks and poly(ADP-ribose) synthetase in pancreatic islets.

Streptozotocin, which produces diabetes mellitus in experimental animals1–3, has been reported to reduce the level of NAD in pancreatic islets4,5 and to inhibit islet synthesis of proinsulin6. The decrease in NAD is due to increased NAD degradation mediated by islet nuclear poly(ADP–ribose) synthetase7,8. Evidence suggests that poly(ADP–ribose) synthetase is activated when DNA is fragmented9–17. Here we describe that both Streptozotocin and alloxan, which also produces experimental diabetes mellitus1,2, cause DNA strand breaks which stimulate nuclear poly(ADP–ribose) synthetase, thereby depleting intracellular NAD and inhibiting proinsulin synthesis in isolated pancreatic islets of rats.

Synthesis and degradation of NAD in guinea pig cardiac muscle: II. Studies about the different biosynthetic pathways and the corresponding intermediates.

In continuation of the previous report the possible pathways for the incorporation of the precursors nicotinamide or nicotinic acid into NAD of isolated guinea pig atria have been analyzed by combined methods of paper- and column chromatography. After incubation with a high concentration of 20 mM14C-nicotinamide a deamidation was proved by finding high activities of14C-nicotinic acid in cardiac tissue. An important intermediate,14C-NaAD, could be also detected in a ratio of 1.35∶1 related to the measured14C-NAD. Therefore for the new NAD synthesis thePreiss-Handler pathway is operating. No significant activity was found in NMN and NADP. From a total increase of 24.4 nMol NAD/8 h · 100 mg atrial tissue (+74%) 7.1 nMol can be attributed to a new synthesis of14C-NAD and 17.3 nMol NAD to the inhibition of the degradation. It is assumed that nicotinamide inhibits the NAD degrading glycohydrolase in high concentrations. After incubation with 20 mM14C-nicotinic acid only a very low14C-incorporation into NAD was observed with thePreiss-Handler pathway. The total NAD level does not change during an incubation time of 24 h. With a concentration of 10 μM14C-nicotinamide no deamidation to14C-nicotinic acid could be measured in atrial tissue. However, significant activities as14C-NMN,14C-NAD and14C-NADP were observed. Therefore theDietrich pathway is operating here. With 10 μM14C-nicotinic acid a higher incorporation rate into NAD could be measured than was observed with 10 μM14C-nicotinamide during the same incubation period. The high radioactivity found in NaAD demonstrated that the synthesis proceeds here via thePreiss-Handler cycle. Under these conditions, however,14C-NAD can be degraded easily by the NAD glycohydrolase to14C-nicotinamide and ADPR.14C-nicotinamide was found in atrial tissue as well as in the nutrient solution and can be reincorporated into NAD via NMN in theDietrich pathway, since significant radioactivity was observed in this intermediate. The analysis of the investigated precursors and intermediates of NAD synthesis and degradation could be established only by the combined methods of paper- and column chromatography, because14C-NaAD can be separated from14C-NAD only with column chromatography and14C-nicotinic acid from14C-NAD only with paper chromatography.

NAD glycohydrolase activity in hearts with acute experimental infarction

Myocardial ischemia was produced in dogs by occluding the descending coronary artery. NAD decreased in the ischemic tissue taken 2 h after the arterial ligature, and an equivalent amount of nicotinamide was detected instead. A further breakdown occurred when fragments of ischemic and nonischemic tissue were incubated at 37 degrees C. In contrast, NAD concentration remained unchanged for as long as 60 min in incubated fragments from normal heart. When normal tissue was homogenized, an immediate hydrolysis of NAD was observed with the formation of stoichiometric amounts of nicotinamide. An excess of nicotinamide completely inhibited the NAD degradation. The NAD glycohydrolase activity assayed in vitro was similar in normal, ischemic, and nonischemic cardiac homogenates. The conclusions are that the NAD loss in the ischemic heart is due to the tissue NAD glycohydrolase activity and that the cell disorganization provoked by the occlusion of the coronary artery seems to facilitate the reaction between the substrate and the enzyme.

The Action of Streptozotocin on Mouse Leukaemia Cells

Streptozotocin, the glucose derivative of N-methyl-N-nitrosourea, is, in addition to being carcinogenic, a powerful diabetogen. When administered to mice, it rapidly lowers the amount of islet-cell NAD and causes considerable 8-cell toxicity (Schein ef a/., 1973 ; Gunnarsson et al., 1974). Administration of nicotinamide before streptozotocin, or up to 2h afterwards, completely inhibits the fall in NAD and prevents the diabetes. The diabetogenic action of streptozotocin is thus thought to be through the lowering of the amount of 8-cell NAD. In experimental systems, streptozotocin is a potent anti-cancer agent (Bhuyan, 1970), but it has found limited clinical use. Simultaneous administration of nicotinamide potentiates the survival of tumour-bearing animals, acting probably through a modulating effect on streptozotocin toxicity (Schein et al., 1967). However, nicotinamide can potentiate the carcinogenic action of the drug (Rakieten et al., 1971). There is a large literature associating changes in NAD concentrations with changes in the growth state of cells. An increasing amount of evidence associates such changes with the chromosomal enzyme, poly(ADP-ribose) polymerase, which degrades NAD. 5-Methylnicotinamide, which inhibits NAD degradation, but is not a precursor for its synthesis, will block the diabetogenic action of streptozotocin in mouse islets (Hinz et al., 1973). Whish et al. (1975) showed that administration of streptozotocin to Physarum polycephalum causes a marked decrease in NAD, which correlates with a twofold

Metabolism of labeled nicotinamide coenzyme in different organs of mice and rats.

SummaryReplacement of radioactive NAD in different organs of rats and mice was found to be most rapid in kidney, liver, and intestine and slowest in brain, muscle, and testis. Experiments with nicotinamide as precursor indicate that nicotinamide derived from NAD degradation is extensively reutilized in the latter organs. Injection of labeled nicotinamide together with a large amount of cold nicotinic acid suggests that this reutilization in brain and muscle probably does not involve deamidation of nicotinamide to nicotinic acid.

Studies on Metabolic Pathway of NAD in Yeast Part I

The enzyme system of NAD degradation was extracted from autolysate of Saccharomyces oviformis. The enzymes were partially separated by ammonium sulfate fractionation and DEAE-cellulose column chromatography, and then the metabolic pathway of NAD in yeast was presented, in which four enzymes were contained. It has been found that, among them, the 5'nucleotidase has more affinity for AMP and the nucleosidase has strict affinity for nicotinamide riboside. In the degradation of NAD in the yeast, nucleotide pyrophosphatase was main enzyme, but NADase, nucleotide pyrophosphorylase and adenosine deaminase seemed not to play an important role.