Differential metabolism of organic nitrates by aldehyde dehydrogenase 1a1 and 2: substrate selectivity, enzyme inactivation, and active cysteine sites.

Organic nitrate vasodilators (ORN) exert their pharmacologic effects through the metabolic release of nitric oxide (NO). Mitochondrial aldehyde dehydrogenase (ALDH2) is the principal enzyme responsible for NO liberation from nitroglycerin (NTG), but lacks activity towards other ORN. Cytosolic aldehyde dehydrogenase (ALDH1a1) can produce NO from NTG, but its activity towards other ORN is unknown. Using purified enzymes, we showed that both isoforms could liberate NO from NTG, isosorbide dinitrate (ISDN), and nicrorandil, while only ALDH1a1 metabolized isosorbide-2-mononitrate and isosorbide-5-mononitrate (IS-5-MN). Following a 10-min incubation with purified enzyme, 0.1 mM NTG and 1 mM ISDN potently inactivated ALDH1a1 (to 21.9% ± 11.1% and 0.44% ± 1.04% of control activity, respectively) and ALDH2 (no activity remaining and 4.57% ± 7.92% of control activity, respectively), while 1 mM IS-5-MN exerted only modest inactivation of ALDH1a1 (reduced to 89% ± 4.3% of control). Cytosolic ALDH in hepatic homogenates incubated at the vascular EC(50) concentrations of ORN was inactivated by NTG (to 45.1% ± 8.1% of control activity) while mitochondrial ALDH was inactivated by NTG and nicorandil (to 68.2% ± 10.0% and 78.7% ± 19.8% of control, respectively). Via site-directed mutagenesis, the active sites of ORN metabolism of ALDH2 (Cys-319) and ALDH1a1 (Cys-303) were found to be identical to those responsible for their dehydrogenase activity. Cysteine-302 of ALDH1a1 and glutamate-504 of ALDH2 were found to modulate the rate of ORN metabolism. These studies provide further characterization of the substrate selectivity, inactivation, and active sites of ALDH2 and ALDH1a1 toward ORN.

Characterization of new medium-chain alcohol dehydrogenases adds resolution to duplications of the class I/III and the sub-class I genes.

Four additional variants of alcohol and aldehyde dehydrogenases have been purified and functionally characterized, and their primary structures have been determined. The results allow conclusions about the structural and evolutionary relationships within the large family of MDR alcohol dehydrogenases from characterizations of the pigeon (Columba livia) and dogfish (Scyliorhinus canicula) major liver alcohol dehydrogenases. The pigeon enzyme turns out to be of class I type and the dogfish enzyme of class III type. This result gives a third type of evidence, based on purifications and enzyme characterization in lower vertebrates, that the classical liver alcohol dehydrogenase originated by a gene duplication early in the evolution of vertebrates. It is discernable as the major liver form at about the level in-between cartilaginous and osseous fish. The results also show early divergence within the avian orders. Structures were determined by Edman degradations, making it appropriate to acknowledge the methodological contributions of Pehr Edman during the 65 years since his thesis at Karolinska Institutet, where also the present analyses were performed.

Suppression of heavy drinking and alcohol seeking by a selective ALDH-2 inhibitor.

BACKGROUND: Inherited human aldehyde dehydrogenase 2 (ALDH-2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH-2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH-2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH-2 inhibitor might affect other metabolic factors involved in regulating drinking. METHODS: Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co-crystal structure of ALDH-2 and daidzin. We tested the efficacy of a highly selective reversible ALDH-2 inhibitor, CVT-10216, in models of moderate and high alcohol drinking rats. We studied 2-bottle choice and deprivation-induced drinking paradigms in Fawn Hooded (FH) rats, operant self-administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue-induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm. RESULTS: CVT-10216 increases acetaldehyde after alcohol gavage and inhibits 2-bottle choice alcohol intake in heavy drinking rodents, including deprivation-induced drinking. Moreover, CVT-10216 also prevents operant self-administration and eliminates cue-induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT-10216 prevents alcohol-induced increases in NAc DA without changing basal levels. CVT-10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses. CONCLUSION: Our findings suggest that selective reversible ALDH-2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.

Mitochondrial nitrite reduction coupled to soluble guanylate cyclase activation: lack of evidence for a role in the bioactivation of nitroglycerin.

Reduction of nitrite to nitric oxide (NO) by components of the mitochondrial respiratory chain may link nitroglycerin biotransformation by mitochondrial aldehyde dehydrogenase (ALDH2) to activation of soluble guanylate cyclase (sGC). We used purified sGC as detector for NO-like bioactivity generated from nitrite and GTN by isolated heart and liver mitochondria. Exogenous NADH caused a pronounced increase in oxygen consumption that was completely inhibited by myxothiazol and cyanide. Oxygen depletion of cardiac mitochondria by NADH was accompanied by activation of sGC and cyanide-sensitive formation of NO. Mitochondrial biotransformation of nitroglycerin was sensitive to ALDH2 inhibitors and coupled to sGC activation but not affected by respiratory substrates or inhibitors. Our data suggest that cytochrome c oxidase catalyzes reduction of nitrite to NO at low O(2) tension but argue against the involvement of this pathway in mitochondrial bioactivation of nitroglycerin.

Structure of daidzin, a naturally occurring anti-alcohol-addiction agent, in complex with human mitochondrial aldehyde dehydrogenase.

The ALDH2*2 gene encoding the inactive variant form of mitochondrial aldehyde dehydrogenase (ALDH2) protects nearly all carriers of this gene from alcoholism. Inhibition of ALDH2 has hence become a possible strategy to treat alcoholism. The natural product 7-O-glucosyl-4'-hydroxyisoflavone (daidzin), isolated from the kudzu vine ( Peruraria lobata), is a specific inhibitor of ALDH2 and suppresses ethanol consumption. Daidzin is the active principle in a herbal remedy for "alcohol addiction" and provides a lead for the design of improved ALDH2. The structure of daidzin/ALDH2 in complex at 2.4 A resolution shows the isoflavone moiety of daidzin binding close to the aldehyde substrate-binding site in a hydrophobic cleft and the glucosyl function binding to a hydrophobic patch immediately outside the isoflavone-binding pocket. These observations provide an explanation for both the specificity and affinity of daidzin (IC50 =80 nM) and the affinity of analogues with different substituents at the glucosyl position.

Bioactivation of nitroglycerin by purified mitochondrial and cytosolic aldehyde dehydrogenases.

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC(50) values of 42 +/- 2.9 and 3.1 +/- 0.4 microm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

Eugenol and its structural analogs inhibit monoamine oxidase A and exhibit antidepressant-like activity.

Eugenol (1) is an active principle of Rhizoma acori graminei, a medicinal herb used in Asia for the treatment of symptoms reminiscent of Alzheimer's disease (AD). It has been shown to protect neuronal cells from the cytotoxic effect of amyloid beta peptides (Abetas) in cell cultures and exhibit antidepressant-like activity in mice. Results from this study show that eugenol inhibits monoamine oxidase A (MAOA) preferentially with a K(i)=26 microM. It also inhibits MAOB but at much higher concentrations (K(i)=211 microM). In both cases, inhibition is competitive with respect to the monoamine substrate. Survey of compounds structurally related to eugenol has identified a few that inhibit MAOs more potently. Structure activity relationship reveals structural features important for MAOA and MAOB inhibition. Molecular docking experiments were performed to help explain the SAR outcomes. Four of these compounds, two (1, 24) inhibiting MAOA selectively and the other two (19, 21) inhibiting neither MAOA nor MAOB, were tested for antidepressant-like activity using the forced swim test in mice. Results suggest a potential link between the antidepressant activity of eugenol and its MAOA inhibitory activity.

Contribution of aldehyde dehydrogenase to mitochondrial bioactivation of nitroglycerin: evidence for the activation of purified soluble guanylate cyclase through direct formation of nitric oxide.

Vascular relaxation to GTN (nitroglycerin) and other antianginal nitrovasodilators requires bioactivation of the drugs to NO or a related activator of sGC (soluble guanylate cyclase). Conversion of GTN into 1,2-GDN (1,2-glycerol dinitrate) and nitrite by mitochondrial ALDH2 (aldehyde dehydrogenase 2) may be an essential pathway of GTN bioactivation in blood vessels. In the present study, we characterized the profile of GTN biotransformation by purified human liver ALDH2 and rat liver mitochondria, and we used purified sGC as a sensitive detector of GTN bioactivity to examine whether ALDH2-catalysed nitrite formation is linked to sGC activation. In the presence of mitochondria, GTN activated sGC with an EC50 (half-maximally effective concentration) of 3.77+/-0.83 microM. The selective ALDH2 inhibitor, daidzin (0.1 mM), increased the EC50 of GTN to 7.47+/-0.93 microM. Lack of effect of the mitochondrial poisons, rotenone and myxothiazol, suggested that nitrite reduction by components of the respiratory chain is not essential to sGC activation. However, since co-incubation of sGC with purified ALDH2 led to significant stimulation of cGMP formation by GTN that was completely inhibited by 0.1 mM daidzin and NO scavengers, ALDH2 may convert GTN directly into NO or a related species. Studies with rat aortic rings suggested that ALDH2 contributes to GTN bioactivation and showed that maximal relaxation to GTN occurred at cGMP levels that were only 3.4% of the maximal levels obtained with NO. Comparison of sGC activation in the presence of mitochondria with cGMP accumulation in rat aorta revealed a slightly higher potency of GTN to activate sGC in vitro compared with blood vessels. Our results suggest that ALDH2 catalyses the mitochondrial bioactivation of GTN by the formation of a reactive NO-related intermediate that activates sGC. In addition, the previous conflicting notion of the existence of a high-affinity GTN-metabolizing pathway operating in intact blood vessels but not in tissue homogenates is explained.

Clozapine reduces alcohol drinking in Syrian golden hamsters.

Alcohol abuse contributes substantially to the overall morbidity of schizophrenia. While typical antipsychotic medications do not limit alcohol use in patients with schizophrenia, emerging data suggest that the atypical antipsychotic clozapine does. To further elucidate the effects of these antipsychotics on alcohol use, we initiated a study in alcohol-preferring rodents. Syrian golden hamsters were given free-choice, unlimited access to alcohol. Nine days of treatment (s.c. injection) with clozapine (2-4 mg/kg/day), but not haloperidol (0.2-0.4 mg/kg/day), reduced alcohol drinking. Clozapine reduced alcohol drinking by 88% (from 11.3+/-1.7 to 1.4+/-0.2 g/kg/day) while increasing both water and food intake. Alcohol drinking gradually (during 24 days) returned toward baseline in the clozapine-treated animals when vehicle was substituted for clozapine. Further increasing the doses of haloperidol (0.6-1.0 mg/kg/day) had no effect on alcohol drinking; moreover, very low doses of haloperidol (0.025-0.1 mg/kg/day) tested in separate groups of hamsters also had no effect on alcohol drinking. This study demonstrates that clozapine, but not haloperidol, can effectively and reversibly decrease alcohol consumption in alcohol-preferring hamsters. The results are compatible with the observations that clozapine, but not haloperidol, limits alcohol use in patients with schizophrenia. These data further suggest that clozapine may serve as a prototype for developing novel treatments for alcohol abuse.

Eugenol exhibits antidepressant-like activity in mice and induces expression of metallothionein-III in the hippocampus.

Here we show that eugenol has an antidepressant-like activity comparable to that of imipramine using a forced swim test and a tail suspension test in mice. Furthermore, we show that both eugenol and imipramine induce brain-derived neurotrophic factor (BDNF) in the hippocampus with and without induction of metallothionein-III (MT-III), respectively. It may be possible that MT-III expression is involved in the exhibition of antidepressant-like activity of eugenol, not of imipramine.

Synthesis of daidzin analogues as potential agents for alcohol abuse.

Daidzin, the active principle of an herbal remedy for 'alcohol addiction', has been shown to reduce alcohol consumption in all laboratory animals tested to date. Correlation studies using structural analogues of daidzin suggests that it acts by raising the monoamine oxidase (MAO)/mitochondrial aldehyde dehydrogenase (ALDH-2) activity ratio (J. Med. Chem. 2000, 43, 4169). Structure-activity relationship (SAR) studies on the 7-O-substituted analogues of daidzin have revealed structural features important for ALDH-2 and MAO inhibition (J. Med. Chem. 2001, 44, 3320). We here evaluated effects of substitutions at 2, 5, 6, 8, 3' and 4' positions of daidzin on its potencies for ALDH-2 and MAO inhibition. Results show that analogues with 4'-substituents that are small, polar and with hydrogen bonding capacities are most potent ALDH-2 inhibitors, whereas those that are non-polar and with electron withdrawing capacities are potent MAO inhibitors. Analogues with a 5-OH group are less potent ALDH-2 inhibitors but are more potent MAO inhibitors. All the 2-, 6-, 8- and 3'-substituted analogues tested so far do not inhibit ALDH-2 and/or have decreased potencies for MAO inhibition. This, together with the results obtained from previous studies, suggests that a potent antidipsotropic analogue would be a 4',7-disubstituted isoflavone. The 4'-substituent should be small, polar, and with hydrogen bonding capacities such as, -OH and -NH(2); whereas the 7-substituent should be a straight-chain alkyl with a terminal polar function such as -(CH(2))(n)-OH with 2< or =n < or =6, -(CH(2))(n)-COOH with 5< or =n < or =10, or -(CH(2))(n)-NH(2) with n > or =4.

Anti-dipsotropic isoflavones: the potential therapeutic agents for alcohol dependence.

Daidzin is the active principle of Radix puerariae (RP), an herbal remedy that has been used apparently safely and effectively for the treatment of "alcohol addiction" in China for more than a millennium. It has been shown to reduce alcohol consumption in all animal models tested to date. A link between daidzin's capacity to reduce alcohol consumption and its ability to increase liver mitochondrial monoamine oxidase (MAO): aldehyde dehydrogenase (ALDH-2) activity ratio has been established. Daidzin analogs that potently inhibit ALDH-2 but not MAO are the most anti-dipsotropic, whereas those that also inhibit MAO are not. On the basis of these findings, it was proposed that the liver mitochondrial MAO-ALDH-2 pathway is the primary site of action of daidzin and that a biogenic aldehyde derived from the action of MAO mediates its anti-dipsotropic action. Therefore, to design and synthesize more potent anti-dipsotropic analogs, structural features that would enhance ALDH-2 inhibition and/or decrease MAO inhibition needed to be evaluated. Structure-activity-relationship (SAR) studies have revealed that a sufficient set of criteria for a potent anti-dipsotropic analog is an isoflavone with a free 4'-OH function and a straight-chain alkyl at the 7 position that has a terminal polar function such as -OH, -COOH, or -NH2. The preferable chain lengths for the 7-O-omega-carboxy, 7-O-omega-hydroxy, and 7-O-omega-amino substituents are 5 < or = n < or = 10, 2 < or = n < or = 6, and n > or = 4, respectively. Analogs that meet these criteria have increased potency for ALDH-2 inhibition and/or decreased potency for MAO inhibition and are, therefore, likely to be potent anti-dipsotropic agents.

Rhizoma acori graminei and its active principles protect PC-12 cells from the toxic effect of amyloid-beta peptide.

The effects of water extracts of six medicinal herbs (Radix polygalae tenuifoliae, Radix salviae miltiorrhizae, Rhizoma acori graminei, Rhizoma pinelliae ternatae, Tuber curcumae and Scletrotium poriae cocos) on the cytotoxic action of Abeta(1-40) were tested with PC-12 cells. Only the extract of R. acori graminei (RAG) significantly decreased Abeta(1-40)-induced cell death. Further, eugenol and beta-asarone were isolated and identified as the major active principles. Both purified eugenol and beta-asarone protected PC-12 cells from the toxic effect of Abeta(1-40). Eugenol was active between 1 and 100 microM, and 10 microM eugenol gave approximately a 50% response. beta-Asarone was less potent and exhibited little, if any, activity at this concentration. Both eugenol and beta-asarone inhibited Ca(2+) intake by PC-12 cells: beta-asarone mainly inhibited basal Ca(2+) intake, whereas eugenol inhibited Abeta-induced Ca(2+) intake preferentially. These results suggest that eugenol may act by blocking Abeta-induced-Ca(2+) intake and provide a strong case for further pursuit of the therapeutic and prophylactic potentials of RAG and its active principles for the management of Alzheimer's disease.

Herbal remedies for alcoholism: promises and possible pitfalls.

This review summarizes the findings of the effects on alcohol intake in alcohol-preferring rats of extracts or purified compounds from two of the most promising herbs: kudzu (Pueraria lobata) and St. John's Wort (Hypericum perforatum). It is a summary of a symposium presented at the 2002 RSA meeting in San Francisco. The meeting organizers/co-chairs were David Overstreet and Wing-Ming Keung. The presentations were (1) Introduction to the symposium, by David Y. W. Lee and David H. Overstreet; (2) Effects of daidzin on alcohol intake-search for mechanisms of action, by Wing-Ming Keung; (3) Long-term suppressive effects of puerarin on alcohol drinking in rats, by David Overstreet and David Y. W. Lee; (4) St. John's Wort extract reduces alcohol intake in FH and P rats, by Amir Rezvani and David Overstreet; and (5) extracts reduce alcohol intake in Marchigian Sardinian alcohol-preferring rats, by Maurizio Massi.

Anti-amyloid beta activity of metallothionein-III is different from its neuronal growth inhibitory activity: structure-activity studies.

Recent studies have shown that metallothionein-III (MT-III), but not MT-I or -II, antagonizes both the neurotrophic and neurotoxic effects of amyloid beta peptides (Abetas). Further, its anti-Abeta-toxicity effect was attributed to the fact that it inhibits the formation of fibrillar Abeta. MT-III alone also affects neuron survival in culture-promoting at low but inhibiting at high concentrations. To characterize these biological activities of MT-III in relation to its neuronal growth inhibitory activity discovered by Uchida et al. [Neuron 7 (1991) 337-347], we here studied effects of the P7S/P9A double mutant, and the N- and C-terminal domains of MT-III on primary cultures of rat embryonic cortical neurons in the presence and absence of Abeta. Results show that (i). only the wild-type MT-III inhibited the formation of SDS-resistant Abeta aggregates and protected cortical neurons from the toxic effect of Abeta, and (ii). both the wild type and the N-terminal domain of MT-III promote neuron survival at low concentrations but inhibited it at high concentrations. On the basis of these findings, we conclude that the anti-Abeta activity of MT-III is different from its neuronal growth inhibitory activity and suggest that the increased trophic activity of AD brain extracts could be attributed to its low MT-III content.

S-nitrosothiols react preferentially with zinc thiolate clusters of metallothionein III through transnitrosation.

Metallothionein (MT) is a two-domain protein with zinc thiolate clusters that bind and release zinc depending on the redox states of the sulfur ligands. Since S-nitrosylation of cysteine is considered a prototypic cellular redox signaling mechanism, we here investigate the reactions of S-nitrosothiols with different isoforms of MT. MT-III is significantly more reactive than MT-I/II toward S-nitrosothiols, whereas the reactivity of all three isoforms toward reactive oxygen species is comparable. A cellular system, in which all three MTs are similarly effective in protecting rat embryonic cortical neurons in primary culture against hydrogen peroxide but where MT-III has a much more pronounced effect of protecting against S-nitrosothiols, confirms this finding. MT-III is the only isoform with consensus acid-base sequence motifs for S-nitrosylation in both domains. Studies with synthetic and zinc-reconstituted domain peptides demonstrate that S-nitrosothiols indeed release zinc from both the alpha- and the beta-domain of MT-III. S-Nitrosylation occurs via transnitrosation, a mechanism that differs fundamentally from that of previous studies of reactions of MT with NO*. Our data demonstrate that zinc thiolate bonds are targets of S-nitrosothiol signaling and further indicate that MT-III is biologically specific in converting NO signals to zinc signals. This could bear importantly on the physiological action of MT-III, whose biological activity as a neuronal growth inhibitory factor is unique, and for brain diseases that have been related to oxidative or nitrosative stress.