Wing Ming Keung

Kudzu root: An ancient chinese source of modern antidipsotropic agents

Kudzu (Pueraria lobata) is one of the earliest medicinal plants used in traditional Chinese medicine. It has many profound pharmacological actions including antidipsotropic (antialcohol abuse) activity. Although both the roots and flowers of kudzu, Radix and Flos puerariae, respectively, have been used to treat alcohol abuse safely and effectively in China for more than a millennium, their true efficacy, active constituents, sites and mechanisms of action have never been critically examined. Recently, we have demonstrated that a crude extract of Radix puerariae suppresses the free-choice ethanol intake of ethanol-preferring golden Syrian hamsters and have identified two of its isoflavones, daidzin and daidzein, that account for this effect. Since then, we and other investigators have confirmed these findings in rats that were either trained or genetically bred to prefer and consume large amounts of ethanol. This article summarizes recent progress on the pharmacological and biochemical studies of the antidipsotropic isoflavones isolated from Radix puerariae.

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.

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.

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 EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm 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.

Rhizoma acori graminei and its active principles protect PC-12 cells from the toxic effect of amyloid-β 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 Alzheimers disease.

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.

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.

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.

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.

Identification of human alcohol dehydrogenase isozymes by disc polyacrylamide gel electrophoresis in 7 m urea

Polyacrylamide gel electrophoresis in the presence of 7 M urea provides a simple, reproducible method for the identification of cathodic alcohol dehydrogenase (ADH) isozymes. Treatment of native ADH dimers with 7 M urea and 1 mM dithiothreitol results in a complete dissociation of the 40,000 Mr subunits. Electrophoresis of urea-dissociated ADH isozymes yields a single protein band for homodimers and two bands of equal intensity for heterodimers. The ADH subunits pi, alpha, gamma 2, gamma 1, and beta exhibit electrophoretic mobilities of 0.71, 0.79, 0.88, 0.95, and 1.0, respectively. Thus, the identity of any cathodic ADH isozyme can be determined from the electrophoretic mobilities of its component subunits.