Adenosine dialdehyde and neplanocin A: Potent inhibitors of S-adenosylhomocysteine hydrolase in neuroblastoma N2a cells

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase, E.C. 3.3.1.1) catalyzes the metabolism of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy) in mouse neuroblastoma N2a cells. AdoHcy hydrolase in N2a cells can be inhibited completely by adenosine dialdehyde (Ado dialdehyde) or neplanocin A. The inhibitory effects of Ado dialdehyde (2.5 μM) and neplanocin A (1 μM) on cellular AdoHcy hydrolase were time-dependent, with total enzyme inhibition occurring after 30 min and 15 min of incubation, respectively. The inhibition of AdoHcy hydrolase produced by Ado dialdehyde and neplanocin A persisted for up to 72 h of incubation, and was paralleled by a time-dependent increase in endogenous AdoHcy levels reaching a maximum 4-fold elevation after 8 h of incubation with Ado dialdehyde and an 11-fold increase in the neplanocin A-treated cells. This increase in AdoHcy levels produced a subsequent inhibition of S-adenosylmethionine (AdoMet)-dependent cellular methylations (e.g. protein carboxylmethylation (PCM), lipid methylation). In addition, neplanocin A was metabolically converted to the corresponding AdoMet analog, S-neplanocylmethionine (NepMet), in neuroblastoma N2a cells. NepMet reached maximum levels after 8 h of incubation of the cells with neplanocin A.     

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.     

The role of hyperhomocysteinemia in nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation.

Hyperhomocysteinemia (HHcy) is associated with impaired endothelial-dependent vasodilatation and increased risk of atherosclerosis and thrombosis. Here, we summarize some of our previous work on the effect of HHcy on pathways involved in endothelium-dependent vasodilatation, and present new data concerning the endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation. We showed that the 894 G>T single-nucleotide polymorphism in the human endothelial nitric oxide synthase gene (eNOS) increased the risk of recurrent venous thrombosis in individuals with elevated homocysteine levels, indicating that the pathophysiological mechanism in HHcy involves impaired NO-mediated vasodilatation. In addition, the EDHF-mediated vasodilatation of the renal artery was disturbed in diet-induced hyperhomocysteinemic rats. Interestingly, we demonstrated that pretreatment of rats with periodate-oxidized adenosine (Adox), which is an inhibitor of S-adenosylhomocysteine hydrolase, prevented the methionine-induced rise in plasma total Hcy (tHcy) levels but not the inhibition of the EDHF pathway. Furthermore, we demonstrated that S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) levels were increased in the kidneys of diet-induced HHcy rats, resulting in a decreased AdoMet:AdoHcy ratio. In addition, we demonstrated that mRNA expression of Connexin 40, which is one of the structural subunits of gap-junctions, was down-regulated in endothelial cells of HHcy rats, and correlated with elevated AdoHcy levels in kidney of these rats. These finding suggest a key role for AdoHcy in relation to decreased Cx40 mRNA expression and impaired EDHF-mediated vasodilatation of HHcy rats.     

Altered methionine metabolism occurs in all members of a set of diverse human tumor cell lines

Methionine dependence is a metabolic defect found thus far only in transformed and malignant cells. The defect is manifested as the inability of cells to grow in media in which methionine (Met) is replaced by its immediate precursor homocysteine (Hcy). We have termed this Met ^? Hcy ⁺ media. We demonstrate here that methionine-dependent cells derived from human tumors, compared to normal methionine-independent cells, have low levels of free Met, low levels of S-adenosylmethionine (AdoMet) and elevated levels of S-adenosylhomocysteine (AdoHcy) when incubated in Met ^? Hcy ⁺ medium. Methionine-independent human tumor cells also have very low levels of free Met compared to normal cells but generally have levels of AdoMet and AdoHcy comparable to normal cells in Met ^? Hcy⁺ medium. All tumor cell types incorporate amounts of Met into protein similar to normal methionine-pindependent human fibroblasts when incubated in Met ^? Hcy⁺ medium, thereby indicating apparently normal levels of Met synthesis in the tumor cells. The methionine-independent tumor cell lines in Met ^? Hcy⁺ medium seem able to regulate their AdoMet/AdoHcy ratios normally despite this defect in having very low levels of free Met. Thus, in a diverse set of human tumor cell lines, all are defective in at least one aspect of Met metabolism, giving rise to the possibility of a general metabolic defect in cancer.     

S-adenosylhomocysteine metabolism in different cell lines: effect of hypoxia and cell density.

BACKGROUND/AIMS: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. METHODS: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. RESULTS: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 +/- 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 +/- 1.1 and 21.1 +/- 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 +/- 0.7 and 7.1 +/- 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 +/- 0.2 nmol min(-1) mg(-1) protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. CONCLUSIONS: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.     

Tissue-specific effects of testosterone on S-adenosylmethionine formation and utilization in the mouse.

Exogenous administration of testosterone produced several metabolic tissue-specific changes in female mouse kidneys, but not in the liver. The hormone induced ornithine decarboxylase (ODC) activity, and also profoundly influenced metabolism of S-adenosylmethionine (AdoMet). Therefore, the activity of the AdoMet-synthesizing enzyme (AdoMet synthetase) and of cystathionine synthase, which commits homocysteine irreversibly to the transsulfuration pathway, were significantly increased. In contrast to the level of AdoMet in the liver the renal level of this metabolite was augmented, whereas the level of S-adenosylhomocysteine (AdoHcy) did not change. This resulted in an increase of the AdoMet/AdoHcy ratio. In testosterone-treated mice, pulse-labelled with [methyl-14C]methionine, the radioactivity recovered in the kidneys doubled, but in the liver remained the same. The rise in radioactivity recovered occurred mainly in TCA-soluble compounds and lipids, and to a smaller extent, in proteins and nucleic acids.     

Relationship between tissue levels of S-adenosylmethionine, S-adenylhomocysteine, and transmethylation reactions.

The concentrations of S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and various methyltransferases were determined in the cerebrum, cerebellum, and liver of rats during development and aging. The liver contained from 3 to 7 and from 10 to 15 nmol AdoHcy per gram in young and adult rats, respectively. The AdoMet concentration was 60 to 90 nmol/g liver from rats of the same age and sex. It did not vary significantly with age. In the brain the AdoMet concentration was 45 to 50 nmol/g at birth and decreased to 20 nmol/ g tissue with maturity of the organ. The level of AdoHcy in this organ was less than 1 nmol/g tissue throughout the life-span of the rat. Since the ratio of AdoMet to AdoHcy is relatively high, the rate of methylation of histones, DNA, or phosphatidylethanolamine in the liver or brain was not significantly influenced by AdoHcy. Under normal nutritional conditions, the tissue concentration of AdoMet is far above the Km values of histone and phosphatidylethanolamine methyltransferases. The levels of activity of these enzymes in liver and brain did not correlated with the cellular concentration of AdoHcy. Thi histone methyltransferase activity was elevated in rapidly proliferating tissues and declined markedly in the absence of histone biosynthesis. Phosphatidylethanolamine methyltransferase activity was elevated during development of the liver. The specific activity of the AdoHcy hydrolase remained relatively constant in the rat brain and liver. The activity of this enzyme was 10 times higher in liver than in brain, yet the concentration of AdoHcy was much lower in the latter organ. The tissue levels of this compound are evidently dependent on the rates of removal of homocysteine and adenosine. Adenosine deaminase was present in the liver and brain at relatively high concentrations, particularly during development.     

Förster resonance energy transfer measurements of cofactor‐dependent effects on protein arginine N‐methyltransferase homodimerization

Protein arginine N‐methyltransferase (PRMT) dimerization is required for methyl group transfer from the cofactor S‐adenosyl‐L ‐methionine (AdoMet) to arginine residues in protein substrates, forming S‐adenosyl‐L ‐homocysteine (AdoHcy) and methylarginine residues. In this study, we use Förster resonance energy transfer (FRET) to determine dissociation constant (K_D) values for dimerization of PRMT1 and PRMT6. By attaching monomeric Cerulean and Citrine fluorescent proteins to their N‐termini, fluorescent PRMTs are formed that exhibit similar enzyme kinetics to unconjugated PRMTs. These fluorescent proteins are used in FRET‐based binding studies in a multi‐well format. In the presence of AdoMet, fluorescent PRMT1 and PRMT6 exhibit 4‐ and 6‐fold lower dimerization K_D values, respectively, than in the presence of AdoHcy, suggesting that AdoMet promotes PRMT homodimerization in contrast to AdoHcy. We also find that the dimerization K_D values for PRMT1 in the presence of AdoMet or AdoHcy are, respectively, 6‐ and 10‐fold lower than the corresponding values for PRMT6. Considering that the affinity of PRMT6 for AdoHcy is 10‐fold higher than for AdoMet, PRMT6 function may be subject to cofactor‐dependent regulation in cells where the methylation potential (i.e., ratio of AdoMet to AdoHcy) is low. Since PRMT1 affinity for AdoMet and AdoHcy is similar, however, a low methylation potential may not affect PRMT1 function.     

Effect of 3-deazaadenosine on methionine metabolism in isolated perfused livers

The effect of the purine analog 3-deazaadenosine (dzAdo) on the metabolism of sulfur-containing compounds was examined in hepatocytes. The uptake of exogenous methionine by the liver was not affected by the addition of dzAdo to the perfusate, while the intracellular concentrations of S-adenosyl-L-methionine (AdoMet) and S-adenosyl-L-homocysteine (AdoHcy) continued to increase as long as exogenous methionine was available. In addition, large amounts of 3-deazaadenosyl-L-homocysteine (dzAdoHcy) accumulated in the cell. The specific radioactivity of the carbon chain of dzAdoHcy was the same as that of AdoMet and AdoHcy. Consequently, an equivalent amount of homocysteine (Hcy) must have been generated via hydrolysis of AdoHcy. Free Hcy could not be detected either in the tissue or perfusate when dzAdo was present, while Hcy was excreted into the perfusate by control livers. Consequently, the AdoHcy and DzAdoHcy that accumulate in the cell not only function as inhibitors of methylation reactions, but serve as a trap for Hcy. This could result in methionine starvation and hence, inhibition of protein synthesis.     

Tissue-specific relationship of S-adenosylhomocysteine with allele-specific H19/Igf2 methylation and imprinting in mice with hyperhomocysteinemia

DNA methylation is linked to homocysteine metabolism through the generation of S-adenosylmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy). The ratio of AdoMet/AdoHcy is often considered an indicator of tissue methylation capacity. The goal of this study is to determine the relationship of tissue AdoMet and AdoHcy concentrations to allele-specific methylation and expression of genomically imprinted H19/Igf2. Expression of H19/Igf2 is regulated by a differentially methylated domain (DMD), with H19 paternally imprinted and Igf2 maternally imprinted. F1 hybrid C57BL/6J x Castaneous/EiJ (Cast) mice with (+/−), and without (+/+), heterozygous disruption of cystathionine-β-synthase (Cbs) were fed a control diet or a diet (called HH) to induce hyperhomocysteinemia and changes in tissue AdoMet and AdoHcy. F1 Cast x Cbs+/− mice fed the HH diet had significantly higher plasma total homocysteine concentrations, higher liver AdoHcy, and lower AdoMet/AdoHcy ratios and this was accompanied by lower liver maternal H19 DMD allele methylation, lower liver Igf2 mRNA levels, and loss of Igf2 maternal imprinting. In contrast, we found no significant differences in AdoMet and AdoHcy in brain between the diet groups but F1 Cast x Cbs+/− mice fed the HH diet had higher maternal H19 DMD methylation and lower H19 mRNA levels in brain. A significant negative relationship between AdoHcy and maternal H19 DMD allele methylation was found in liver but not in brain. These findings suggest the relationship of AdoMet and AdoHcy to gene-specific DNA methylation is tissue-specific and that changes in DNA methylation can occur without changes in AdoMet and AdoHcy.     

Regulation of S-adenosylhomocysteine hydrolase in the halophilic cyanobacterium Aphanothece halophytica: a possible role in glycinebetaine biosynthesis

Aphanothece halophytica, a halophilic cyanobacterium capable of growing in saturated NaCl, accumulates high intracellular concentrations of glycinebetaine in response to increasing environmental NaCl. In this organism, intracellular levels of K⁺ rise dramatically with increasing external NaCl before an increase in glycinebetaine can be detected. Glycinebetaine synthesis requires three S-adenosylmethionine (AdoMet)-mediated transmethylations; each transmethylation reaction generates one molecule of the transmethylation inhibitor S-adenosylhomocysteine (AdoHcy). Thus, glycinebetaine synthesis should require continued removal of AdoHcy. In A. halophytica, catabolism of AdoHcy was shown to occur via the reversible reaction catalyzed by AdoHcy hydrolase (EC 3.3.1.1). Activity of AdoHcy hydrolase in the direction of synthesis of AdoHcy was inhibited by 0.4 M KCl in this organism. On the other hand, activity of AdoHcy hydrolase in the direction of AdoHcy hydrolysis was unaffected by 0.4 M KCl. Glycinebetaine increased synthesis of AdoHcy in the presence of 0.4 KCl, but had no effect on AdoHcy hydrolysis. Based upon these results, a mechanism is proposed for the regulation of glycinebetaine synthesis by K⁺ and glycinebetaine in A. halophytica. According to this mechanism, the regulatory response would be initiated by a K⁺-induced shift in the AdoMet/AdoHcy ratio.Abbreviations AdoMet S-adenosylmethionine - AdoHcy S-adenosyl homocysteine     

Increase in S-adenosylhomocysteine concentration in interferon-treated HeLa cells and inhibition of methylation of vesicular stomatitis virus mRNA

A fraction of the viral mRNA synthesized in interferon-treated HeLa cells infected with vesicular stomatitis virus (VSV) lacks the 7-methyl group in the 5'-terminal guanosine of the cap; this mRNA is not associated with polyribosomes and does not bind to ribosomes in an assay for initiation of protein synthesis (de Ferra, F., and Baglioni, C. (1981) Virology 112, 426-435). To establish whether this defect in methylation is due to changes in the level of the methyl donor S-adenosylmethionine (AdoMet) and of its competitive inhibitor S-adenosylhomocysteine (AdoHcy), we measured the concentration of these compounds in HeLa cells treated with interferon. An increase in both AdoMet and AdoHcy was detected 3 to 6 h after addition of interferon. The level of these compounds increased gradually and in proportion to the interferon concentration used. With 125 reference units/ml of beta interferon, for example, the AdoHcy concentration increased more than 3-fold and that of AdoMet about 1.5-fold with a consequent change in the AdoHcy/AdoMet ratio. An increased AdoHcy/AdoMet ratio was also found in HeLa cells treated with pure alpha 2 interferon produced in Escherichia coli by recombinant DNA techniques. When the methylation of VSV mRNA was measured in assays carried out with permeabilized virions at the AdoHcy and AdoMet concentrations found in interferon-treated cells, a preferential inhibition of the viral (guanine-7-)methyltransferase activity was observed. Such an inhibition may account for the synthesis of VSV mRNA lacking the 7-methyl group of guanosine in the cap.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Regulation of S-adenosylmethionine levels in Saccharomyces cerevisiae

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine biosynthesis. Methionine can be activated by ATP to give rise to the universal methyl donor, S-adenosylmethionine (AdoMet). Previously, a chimeric MTHFR (Chimera-1) comprised of the yeast Met13p N-terminal catalytic domain and the Arabidopsis thaliana MTHFR (AtMTHFR-1) C-terminal regulatory domain was constructed (Roje, S., Chan, S. Y., Kaplan, F., Raymond, R. K., Horne, D. W., Appling, D. R., and Hanson, A. D. (2002) J. Biol. Chem. 277, 4056-4061). Engineered yeast (SCY4) expressing Chimera-1 accumulated more than 100-fold more AdoMet and 7-fold more methionine than the wild type. Surprisingly, SCY4 showed no appreciable growth defect. The ability of yeast to hyperaccumulate AdoMet was investigated by studying the intracellular compartmentation of AdoMet as well as the mode of hyperaccumulation. Previous studies have established that AdoMet is distributed between the cytosol and the vacuole. A strain expressing Chimera-1 and lacking either vacuoles (vps33 mutant) or vacuolar polyphosphate (vtc1 mutant) was not viable when grown under conditions that favored AdoMet hyperaccumulation. The hyperaccumulation of AdoMet was a robust phenomenon when these cells were grown in medium containing glycine and formate but did not occur when these supplements were replaced by serine. The basis of the nutrient-dependent AdoMet hyperaccumulation effect is discussed in relation to homocysteine biosynthesis and sulfur metabolism.     

Monoamine oxidase A from human placenta and monoamine oxidase B from bovine liver both have one FAD per subunit.

Choline and C1 metabolism pathways intersect at the formation of methionine from homocysteine. Hepatic S-adenosylmethionine (AdoMet) concentrations are decreased in animals ingesting diets deficient in choline, and it has been suggested that this occurs because the availability of methionine limits AdoMet synthesis. If the above hypothesis is correct, changes in hepatic AdoMet concentrations should relate in some consistent manner to changes in hepatic methionine concentrations. Rats were fed on a choline-deficient or control diet for 1-42 days. Hepatic choline concentrations in control animals were 105 nmol/g, and decreased to 50% of control after the first 7 days on the choline-deficient diet. Hepatic methionine concentrations decreased by less than 20%, with most of this decrease occurring between days 3 and 7 of choline deficiency. Hepatic AdoMet concentrations decreased by 25% during the first week, and continued to decrease (in total, by over 60%) during each subsequent week during which animals consumed a choline-deficient diet. Hepatic S-adenosylhomocysteine (AdoHcy) concentrations increased by 50% when animals consumed a choline-deficient diet. AdoHcy is formed when AdoMet is utilized as a methyl donor. In summary, choline deficiency can deplete hepatic stores of AdoMet under dietary conditions that only minimally decrease the availability of methionine within liver. Thus decreased availability of methionine may not have been the only mechanism whereby choline deficiency lowers hepatic AdoMet concentrations. We suggest that increased utilization of AdoMet might also have occurred.     

S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase variations during differentiation of Dictyostelium discoideum

We have analyzed the level of substrate (AdoMet) and products (AdoHcy) of transmethylations throughout the developmental cycle of the primitive eukaryote Dictyostelium discoideum. The ratio AdoMet/AdoHcy varied dramatically during differentiation. The intracellular level of AdoHcy decreased sharply after the beginning of starvation reaching a value of 18% of that in vegative cells within 4 h. In contrast, there was a two-fold transient increase in AdoMet at the time of aggregation. However, these changes were not related to changes in AdoHcy hydrolase since constant levels of both the protein and the activity were found until 16 h of differentiation. In particular, there was no indication of an in vivo inactivation of the enzyme by cAMP at the time of aggregation. These results are discussed with respect to the previously postulated role of AdoHcy hydrolase in the regulation of the AdoMet/AdoHcy ratio in eukaryotic cells.     

The transmethylation pathway as a source for adenosine in the isolated guinea-pig heart.

In order to quantify adenosine production from the transmethylation pathway [S-adenosylmethionine (AdoMet)----S-adenosylhomocysteine (AdoHcy) in equilibrium adenosine + L-homocysteine] in the isolated guinea-pig heart under basal conditions (normoxic perfusion with 95% O2) and during elevated adenosine production (hypoxic perfusion with 30% O2), two methods were used. (1) Hearts were perfused with normoxic medium containing [2,5,8-3H]adenosine (5 microM) and L-homocysteine thiolactone (0.1 mM), which brings about net AdoHcy synthesis via reversal of the AdoHcy hydrolase reaction and labels the intracellular pool of AdoHcy. From the decrease in AdoHcy pool size and specific radioactivity of AdoHcy in the post-labelling period, the rate of transmethylation, which is equivalent to the rate of adenosine production, was calculated to be 0.98 nmol/min per g. Adenosine release from the hearts was 40-50 pmol/min per g. (2) Hearts were perfused with hypoxic medium containing [35S]homocysteine (50 microM). Owing to the hypoxia-induced increase in adenosine production, this procedure also results in expansion and labelling of the AdoHcy pool. From the dilution of the specific radioactivity of AdoHcy relative to that of [35S]homocysteine, the rate of AdoHcy synthesis from AdoMet (transmethylation) was calculated to be 1.12 nmol/min per g. It is concluded that in the oxygenated heart the transmethylation pathway is quantitatively an important intracellular source of adenosine, which exceeds the rate of adenosine wash-out by the coronary system by about 15-fold. Most of the adenosine formed by this pathway is re-incorporated into the ATP pool, most likely by adenosine kinase. The transmethylation pathway is essentially O2-independent, and the known hypoxia-induced production of adenosine must be derived from an increase in 5'-AMP hydrolysis.     

Inhibitory effect of Epimedium extract on S-adenosyl-L-homocysteine hydrolase and biomethylation

In the present paper, the inhibitory effect of Epimedium extract on the activity of S-adenosyl-L-homocysteine (AdoHcy) Hydrolase was studied. The results showed that Epimedium extract inhibited the activity of recombinant human AdoHcy hydrolase in a dose-dependent manner. This inhibitory effect was also observed in hepatic cell line 7701 and hepatoma HepG2, however, the effect in 7701 cells was more potent than in HepG2 cells. The extract could significantly reduce AdoMet/AdoHcy ratio in 7701 cells in a dose-dependent manner, suggesting reduced biomethylation level in 7701 cells. In contrast, it resulted in elevated AdoMet/AdoHcy ratio in the HepG2 cells. The result of MALDI-MS assay indicated that epimedin A and ikarisoside F from the extract could bind to AdoHcy hydrolase. The present data suggested that Epimedium extract could inhibit the activity of AdoHcy hydrolase, thus regulating the cellular biomethylation as well as reducing cellular Hcy level. These results will provide new clues to the mechanisms of Epimedium in curing of cardiovascular disease and regulating tumor cell growth.