Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.     

Metabolism of adenosine and deoxyadenosine by human erythrocytes and CCRF-CEM leukemia cells

Human lymphocytes lacking adenosine deaminase die and T-cell leukemias are killed by deoxycoformycin (dCf), an inhibitor of adenosine deaminase, due to impaired metabolism of dAdo. The initial metabolism of exogenous adenosine (Ado) and deoxyadenosine (dAdo) has been compared in human erythrocytes and CCRF-CEM leukemia cells and the data obtained have been simulated using kinetic constants obtained in vitro for the enzymes involved. Cells were mixed with ³H-labelled Ado or dAdo, samples were taken at 3 sec intervals and progress curves for the ³H-labelled metabolites formed were determined by quantitative two-dimensional thin layer chromatography. Erythrocytes rapidly take up Ado and the predominant metabolite after 60 sec is hypoxanthine (Hyp), while for dAdo, deoxyinosine (dIno) predominates. By contrast, leukemia cells convert Ado predominantly to AMP, while dAdo is converted first to Hyp and then to AMP. The presence of dCf had little effect upon Ado metabolism but induced accumulation of dAdo. Erythrocytes rapidly degrade Ado and dAdo to Hyp, although the phosphorolysis of dIno is relatively slow. Human CCRF-CEM leukemia cells convert most of the Ado or dAdo to AMP after 60 sec. For dAdo, the sequence of reactions would be dAdo→dIno→Hyp→IMP→sAMP→AMP. dCf does not significantly affect the conversion of Ado→AMP, but dCf blocks AMP accumulation from dAdo, consistent with the reaction sequence shown above. A computer model has been developed for the metabolism of Ado and dAdo, but some of the kinetic constants determined in vitro for this model do not pertain to intact cells.     

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4

Adenosine is an important modulator of neuronal survival and differentiation in the CNS. Our previous work showed that nucleoside transporters (NTs) are present in cultures of chick retinal cells, but little is known about the mechanisms regulating adenosine transport in these cultures. Our aim in the present work was to study the participation of the adenosine metabolism as well as the ERK pathway on adenosine uptake in different types of retinal cultures (mixed and purified glial cultures). Kinetic analysis in both cultures revealed that the uptake reached equilibrium after 30 min and presented two components. Incubation of cultures with S-(p-nitrobenzyl)-6-thioinosine (NBTI) or dipyridamole, different inhibitors of equilibrative nucleoside transporters (ENTs), produced a significant and concentration-dependent uptake reduction in both cultures. However, while dipyridamole presented similar maximal inhibitory effects in both cultures (although in different concentrations), the inhibition by NBTI was smaller in glial cultures than in mixed cultures, suggesting the presence of different transporters. Moreover, pre-incubation of [³H]-adenosine with adenosine deaminase (ADA) or adenosine kinase (ADK) inhibition with iodotubercidin promoted significant uptake inhibition in both cultures, indicating that the uptake is predominantly for adenosine and not inosine, and that taken up adenosine is preferentially directed to the synthesis of adenine nucleotides. In both cultures, the MEK inhibitors PD98059 or UO126, but not the inactive analog U0124, induced a significant and concentration-dependent uptake decrease. We have not observed any change in adenosine metabolism induced by MEK inhibitors, suggesting that this pathway is mediating a direct effect on NTs. Our results show the expression of different NTs in retinal cells in culture and that the activity of these transporters can be regulated by the ERK pathway or metabolic enzymes such as ADK which are then potential targets for regulation of Ado levels in normal or pathological conditions.     

The Regional Concentrations of S-Adenosyl-l-Methionine, S-Adenosyl-l-Homocysteine, and Adenosine in Rat Brain

Abstract: The concentrations of S -adenosyl- l -methionine (SAM), S -adenosyl- l -homocysteine (SAH), and adenosine (Ado) were determined in whole brain and rat brain regions by HPLC. The whole brain contains, respectively, 22 nmol, 1 nmol, and 64 nmol of SAM, SAH, and Ado per g of wet tissue. Their distribution indicated that SAM and SAH levels are highest in brainstem, whereas the Ado level is highest in cortex. With aging the SAM concentrations decrease in whole brain, brainstem, and hypothalamus (–25%) and SAH levels increase by 90% in striatum and by 160% in cerebellum, while Ado levels are increased in all regions by 100–180%.     

Inhibition of antibody production by 2-chloroadenosine

The ability of 2-chloroadenosine (2Cl Ado) to modulate lymphocyte function was examined in culture and in vivo. Mitogenic stimulation of B cell DNA synthesis was antagonized by 2Cl Ado while adenosine produced both stimulations and inhibitions. In culture, 2Cl Ado was found to suppress antibody production to sheep erythrocytes (SRBC) regardless of whether the nucleoside was added at the initiation of culture or 48 hours after sensitization. Inhibiting adenosine deaminase (ADA) did not affect the response to 2Cl Ado, and 1-homocysteine thiolactone was found to potentiate the inhibition suggesting formation of S-adenosylhomocysteine. Similar responses were found with adenosine provided ADA was inhibited. When 2Cl Ado was administered to mice 3-4 days after SRBC, a concentration-dependent decrease in antibody producing cells was observed. These data suggest that nucleosides can inhibit antibody production by inhibiting transmethylation reactions. 2Cl Ado appears to be an effective immunosuppressant without concomitant cytotoxicity both in culture and in vivo.     

Isoprenylcysteine carboxyl methyltransferase activity modulates endothelial cell apoptosis

Extracellular ATP, adenosine (Ado), and adenosine plus homocysteine (Ado/HC) cause apoptosis of cultured pulmonary artery endothelial cells through the enhanced formation of intracellular S-adenosylhomocysteine and disruption of focal adhesion complexes. Because an increased intracellular ratio of S-adenosylhomocysteine/S-adenosylmethionine favors inhibition of methylation, we hypothesized that Ado/HC might act by inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT). We found that N-acetyl-S-geranylgeranyl-L-cysteine (AGGC) and N-acetyl-S-farnesyl-L-cysteine (AFC), which inhibit ICMT by competing with endogenous substrates for methylation, caused apoptosis. Transient overexpression of ICMT inhibited apoptosis caused by Ado/HC, UV light exposure, or tumor necrosis factor-alpha. Because the small GTPase, Ras, is a substrate for ICMT and may modulate apoptosis, we also hypothesized that inhibition of ICMT with Ado/HC or AGGC might cause endothelial apoptosis by altering Ras activation. We found that ICMT inhibition decreased Ras methylation and activity and the activation of the downstream signaling molecules Akt, ERK-1, and ERK-2. Furthermore, overexpression of wild-type or dominant active H-Ras blocked Ado/HC-induced apoptosis. These findings suggest that inhibition of ICMT causes endothelial cell apoptosis by attenuation of Ras GTPase methylation and activation and its downstream antiapoptotic signaling pathway.     

腺苷脱氨酶抑制巨噬细胞增殖迁移的作用研究

目的:探讨腺苷脱氨酶对鼠源巨噬细胞RAW264.7增殖、迁移、细胞周期、细胞凋亡的影响。方法:用不同浓度(0、0.25、1.25、2.5、5U/m L)的腺苷脱氨酶处理RAW264.7细胞后,用实时细胞分析系统检测细胞增殖能力,用流式细胞术检测腺苷脱氨酶对细胞凋亡和周期的影响,划痕修复实验检测RAW264.7细胞迁移能力。结果:与对照组相比,高浓度腺苷脱氨酶(2.5 U/m L、5 U/m L)处理可以显著抑制RAW264.7细胞的增殖能力,且抑制效果随腺苷脱氨酶浓度升高而增强(P<0.05)。流式细胞术检测结果显示,相较于对照组,高浓度腺苷脱氨酶(2.5 U/m L)处理可以诱导RAW264.7细胞凋亡,并导致细胞周期G2/M期阻滞(P<0.05)。此外,细胞划痕实验表明,高浓度腺苷脱氨酶(2.5 U/m L)处理可以显著抑制RAW264.7巨噬细胞的迁移能力(P<0.05)。结论:高浓度腺苷脱氨酶对巨噬细胞增殖和迁移具有抑制作用,并可诱导细胞凋亡和细胞周期阻滞。     

Role of adenosine kinase in the control of Streptomyces differentiations: Loss of adenosine kinase suppresses sporulation and actinorhodin biosynthesis while inducing hyperproduction of undecylprodigiosin in Streptomyces lividans

Adenosine kinase (ADK) catalyses phosphorylation of adenosine (Ado) and generates adenosine monophosphate (AMP). ADK gene (adk(Sli), an ortholog of SCO2158) was disrupted in Streptomyces lividans by single crossover-mediated vector integration. The adk(Sli) disruption mutant (Deltaadk(Sli)) was devoid of sporulation and a plasmid copy of adk(Sli) restored sporulation ability in Deltaadk(Sli), thus indicating that loss of adk(Sli) abolishes sporulation in S. lividans. Ado supplementation strongly suppressed sporulation ability in S. lividans wild-type (wt), supporting that disruption of adk(Sli) resulted in Ado accumulation, which in turn suppressed sporulation. Cell-free experiments demonstrated that Deltaadk(Sli) lacked ADK activity and in vitro characterization confirms that adk(Sli) encodes ADK. The intracellular level of Ado was highly elevated while the AMP level was significantly reduced after loss of adk(Sli) while Deltaadk(Sli) displayed no significant derivation from wt in the levels of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Notably, Ado supplementation to wt lowered AMP content, albeit not to the level of Deltaadk(Sli), implying that the reduction of AMP level is partially forced by Ado accumulation in Deltaadk(Sli). In Deltaadk(Sli), actinorhodin (ACT) production was suppressed and undecylprodigiosin (RED) production was dramatically enhanced; however, Ado supplementation failed to exert this differential control. A promoter-probe assay verified repression of actII-orf4 and induction of redD in Deltaadk(Sli), substantiating that unknown metabolic shift(s) of ADK-deficiency evokes differential genetic control on secondary metabolism in S. lividans. The present study is the first report revealing the suppressive role of Ado in Streptomyces development and the differential regulatory function of ADK activity in Streptomyces secondary metabolism, although the underlying mechanism has yet to be elucidated.     

2-Chloroadenosine: a selective lethal effect to mouse macrophages and its mechanism

In our studies on the effects of purine compounds on immune responses in vitro, we found that 2-chloroadenosine (2-Cl Ado) exhibited a potent lethal effect on a viability of mouse adherent cells derived from the peritoneal cavity. The lethal effect was specific for adherent peritoneal cells (PC) (macrophages) and was prevented by exogenous addition of adenosine (Ado) or coformycin, a potent inhibitor of adenosine deaminase. A rapid decrease of intracellular ATP content (26% of control) in adherent PC was observed soon after 1 hr exposure to 2-Cl Ado (0.1 mM), and this decrease of ATP was comparable with that of monoiodoacetate (MIA, 0.1 mM)- or NaN3 (5 mM)-treated adherent PC. The ATP decrease by 2-Cl Ado was restored to 88 or 90% of control value by 1 hr addition of Ado or coformycin, respectively. Polymorphonuclear cells and lymphocytes to which 2-Cl Ado did not exhibit the lethal effect did not cause a significant ATP decrease of the cells. Therefore, the data suggested that the reason for the lethal effect on adherent PC treated with 2-Cl Ado could be attributed to a rapid decrease of ATP content at an early time. We assume that 2-Cl Ado competes with intracellular Ado in macrophages and then causes the adenosine starvation resulting in the ATP decrease.     

Adenosine-induced cell death: evidence for receptor-mediated signalling

Adenosine modulates the proliferation, survival and apoptosis of many different cell types, ranging from epithelial, endothelial and smooth muscle cells, to cells of the immune and neural lineages. In this review, we critically discuss the available in vitro and in vivo data which support a role for adenosine in both development-associated apoptosis, and in diseases characterized by either pathologically increased cell death (e.g., ischemia, trauma and aging-associated neurodegeneration) or abnormally reduced spontaneous apoptosis (e.g., cancer). Particular emphasis is given to the possible role of extracellular adenosine receptors, since these may represent novel and attractive molecular targets for the pharmacological modulation of apoptosis. In some instances, adenosine-induced cell death has been demonstrated to require entry of the nucleoside inside cells; however, in many other cases, activation of specific adenosine extracellular receptors has been demonstrated. Of the four G protein-coupled adenosine receptors so far identified, the A_2A and the A₃ receptors have been specifically implicated in modulation of cell death. For the A₃ receptor, results obtained by exposing both cardiomyocytes and brain astrocytes to graded concentrations of selective agonists suggest induction of both cell protection and cell death. Such opposite effects, which likely depend on the degree of receptor activation, may have important therapeutic implications in the pharmacological modulation of cardiac and brain ischemia. For the A_2A receptor, recent intriguing data suggest a specific role in immune cell death and immunosuppression, which may be relevant to both adenosine-deaminase-immunodeficiency syndrome (a pathology characterized by accumulation of adenosine to toxic levels) and in tumors where induction of apoptosis via activation of specific extracellular receptors may be desirable. Finally, preliminary data suggest that, in a similar way to the adenosine-deaminase-immunodeficiency syndrome, the abnormal accumulation of adenosine in degenerative muscular diseases may contribute to muscle cell death. Although the role of adenosine receptors in this effect still remains to be determined, these data suggest that adenosine-induced apoptosis may also represent a novel pathogenic pathway in muscular dystrophies.     

Adenosine induces apoptosis in human liver cancer cells through ROS production and mitochondrial dysfunction

Mitochondria are the most important sensor for apoptosis. Extracellular adenosine is well reported to induce apoptosis of tumor cells. Here we found that extracellular adenosine suppresses the cell growth by induction of apoptosis in BEL-7404 liver cancer cells, and identified a novel mechanism that extracellular adenosine triggers apoptosis by increasing Reactive Oxygen Species (ROS) production and mitochondrial membrane dysfunction in the cells. We observed that adenosine increases ROS production, activates c-Caspase-8 and -9 and Caspase effectors, c-Caspase-3 and c-PARP, induces accumulation of apoptosis regulator Bak, decreases Bcl-xL and Mcl-1, and causes the mitochondrial membrane dysfunction and the release of DIABLO, Cytochrome C, and AIF from mitochondria to cytoplasm in the cells; ROS inhibitor, NAC significantly reduces adenosine-induced ROS production; it also shows the same degree of blocking adenosine-induced loss of mitochondrial membrane potential (MMP) and apoptosis. Our study first observed that adenosine increases ROS production in tumor cells and identified the positive feedback loop for ROS-mediated mitochondrial membrane dysfunction which amplifies the death signals in the cells. Our findings indicated ROS production and mitochondrial dysfunction play a key role in adenosine-induced apoptosis of 7404 cells.     

John Montgomery's Legacy: Carbocyclic Adenosine Analogues as Sah Hydrolase Inhibitors with Broad-Spectrum Antiviral Activity

Ever since the S-adenosylhomocysteine (AdoHcy, SAH) hydrolase was recognized as a pharmacological target for antiviral agents (J. A. Montgomery et al., J. Med. Chem. 25:626–629, 1982), an increasing number of adenosine, acyclic adenosine, and carbocyclic adenosine analogues have been described as potent SAH hydrolase inhibitors endowed with broad-spectrum antiviral activity. The antiviral activity spectrum of the SAH hydrolase inhibitors include pox-, rhabdo-, filo-, arena-, paramyxo-, reo-, and retroviruses. Among the most potent SAH hydrolase inhibitors and antiviral agents rank carbocyclic 3-deazaadenosine (C-c³Ado), neplanocin A, 3-deazaneplanocin A, the 5′-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6′-R-alkyl (i.e., 6′-R-methyl) derivatives of neplanocin A. These compounds are particularly active against poxviruses (i.e., vaccinia virus), and rhabdoviruses (i.e., vesicular stomatitis virus). The in vivo efficacy of C-c³Ado and 3-deazaneplanocin A has been established in mouse models for vaccinia virus, vesicular stomatitis virus, and Ebola virus. SAH hydrolase inhibitors such as C-c³Ado and 3-deazaneplanocin A should in the first place be considered for therapeutic (or prophylactic) use against poxvirus infections, including smallpox, and hemorrhagic fever virus infections such as Ebola.     

Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N⁶-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25–1.0 nmol/g wet weight for renal tissue and 0.25–0.5 μM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1±2.6% and 102.5±4.0% for renal tissue and urine, respectively (mean±S.D., n=3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64±2.2, 0.67±0.18 and 46.2±1.9 nmol/g wet weight, respectively (mean±S.D., n=6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low μM range and are negatively correlated with urine flow-rate.     

Kinetic studies of adenosine kinase from L1210 cells: a model enzyme with a two-site ping-pong mechanism

Purified adenosine kinase from L1210 cells displayed substrate inhibition by high concentrations of adenosine (Ado), ATP, and MgCl2. When incubated with ATP and MgCl2, the enzyme was phosphorylated, and the phosphorylated kinase transferred phosphate to adenosine in the absence of ATP and MgCl2. Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a two-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among many possible pathways within this mechanism probably a random-bi ordered-bi route is the preferred sequence in which the two substrates, adenosine and MgATP, bind in a random order to form the ternary complex MgATP . E . Ado followed by the sequential dissociation of MgADP and AMP. Dissociation constants of various enzyme-substrate and enzyme-product complexes and the first-order rate constant of the rate-limiting step were estimated.     

Inhibition of adenosine kinase by phosphonate and bisphosphonate derivatives

The enzyme adenosine kinase (AK) plays a central role in regulating the intracellular and interstitial concentration of the purine nucleoside adenosine (Ado). In view of the beneficial effects of Ado in protecting tissues from ischemia and other stresses, there is much interest in developing AK inhibitors, which can regulate Ado concentration in a site- and event-specific manner. The catalytic activity of AK from different sources is dependent upon the presence of activators such as phosphate (Pi). In this work we describe several new phosphorylated compounds which either activate or inhibit AK. The compounds acetyl phosphate, carbamoyl phosphate, dihydroxyacetone phosphate and imidodiphosphate were found to stimulate AK activity in a dose-dependent manner comparable to that seen with Pi. In contrast, a number of phosphonate and bisphosphonate derivatives, which included clodronate and etidronate, were found to inhibit the activity of purified AK in the presence of Pi. These AK inhibitors (viz. clodronate, etidronate, phosphonoacetic acid, 2-carboxyethylphosphonic acid, N-(phosphonomethyl)-glycine and N-(phosphonomethyl)iminodiacetic acid), at concentrations at which they inhibited AK, were also shown to inhibit the uptake of ³H-adenosine and its incorporation into macromolecules in cultured mammalian cells, indicating that they were also inhibiting AK in intact cells. The drug concentrations at which these effects were observed showed limited toxicity to the cultured cells, indicating that these effects are not caused by cellular toxicity. These results indicate that the enzyme AK provides an additional cellular target for the clinically widely used bisphosphonates and related compounds, which could possibly be exploited for a new therapeutic application. Our structure–activity studies on different AK activators and inhibitors also indicate that all of the AK activating compounds have a higher partial positive charge (δ⁺) on the central phosphorous atom in comparison to the inhibitors. This information should prove helpful in the design and synthesis of more potent inhibitors of AK.     

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.     

[3H]Adenosine Transport in Synaptoneurosomes of Postmortem Human Brain

Abstract: Abstract: [³H]Adenosine transport was characterized in cerebral cortical synaptoneurosomes prepared from postmortem human brain using an inhibitor-stop/centrifugation method. The adenosine transport inhibitors dipyridamole and dilazep completely and rapidly blocked transmembrane fluxes of [³H]adenosine. For 5-s incubations, two kinetically distinguishable processes were identified, i.e., a high-affinity adenosine transport system with K_t and V_max values of 89 μM and 0.98 nmol/min/mg of protein, respectively, and a low-affinity adenosine transport system that did not appear to be saturable. For incubations with 1 μM [³H]adenosine as substrate, intrasynaptoneurosomal concentrations of [³H]adenosine were 0.26 μM at 5 s and 1 μM at 600 s. Metabolism of accumulated [³H]adenosine to adenine nucleotides was 15% for 5-s, 23% for 15-s, 34% for 30-s, 43% for 60-s, and 80% for 600-s incubations. The concentrations (μM) of total accumulated ³H-purines ([³H]-adenosine plus metabolites) at these times were 0.3, 0.5, 1.0, 1.3 and 5.6, respectively. These results indicate that in the presence of extensive metabolism, the intrasynaptoneurosomal accumulation of ³H-purines was higher than the initial concentration of 1 μM [³H]adenosine in the reaction medium. For 5-, 15-, 30-, 60-, and 600-s incubations in the presence of the adenosine deaminase inhibitor EHNA and the adenosine kinase inhibitor 5′-iodotubercidin, metabolism of the transported [³H]adenosine was 14, 14, 16, 14, and 38%, respectively. During these times, total ³H-purine accumulation was 0.3, 0.5, 0.5, 0.7, and 1.8 μM, respectively. Thus, the apparently “concentrative'’accumulation of ³H-purines can be prevented by inhibition of adenosine metabolism and, taken together, these results suggest that adenosine transport in at least synaptoneurosomes prepared from postmortem human brain is via a nonconcentrative and equilibrative system.     

腺苷预处理对大鼠心脏移植缺血再灌注损伤心肌的保护作用

目的探讨腺苷（adenosine,Ado）预处理在心脏移植缺血再灌注损伤的保护作用。方法健康雄性Sprague．Dawley（SD）大鼠40只,建立大鼠同系异位心脏移植模型。随机分为3组：假手术组、对照组、实验组（供体心在获取前15min给予Ado预处理后4℃改良St．Thoms液10mL。主动脉根部灌注停跳）。术后5h测定血清中丙二醛（MDA）、超氧化物歧化酶（SOD）、肌酸激酶同工酶（CK-MB）含量,电镜观测心肌细胞超微结构变化。结果实验组MDA、CK-MB明显减少（P〈0．01）,SOD明显增加（P〈0．01）,心肌超微结构改变明显减轻。实验组的移植心脏存活时间较对照组明显延长[（22．1±2．9）dw（12．0±1．8）d,P〈0．01）]。结论Ado预处理对心脏移植缺血再灌注损伤有保护作用,使受者的移植心脏存活期延长。     

Differential response of Drosophila cell lines to extracellular adenosine

Adenosine (Ado) is a crucial metabolite that affects a wide range of physiological processes. Key proteins regulating Ado signaling, transport and metabolism are conserved among vertebrates and invertebrates. It is well known that Ado influences proliferation of several vertebrate and invertebrate cells. Here we show that Ado negatively influences viability, changes morphology and mitochondrial polarity of the Drosophila imaginal disc cell line (Cl.8+) via a mechanism exclusively dependent on cellular Ado uptake. High transport of Ado is followed by phosphorylation and ATP production as a part of Ado salvation, which at higher concentrations may interfere with cellular homeostasis. In contrast, hematopoietic cell line Mbn2, which grows well in high Ado concentration, preferentially uses adenosine deaminase as a part of the purine catabolic pathway. Our results show that different types of Drosophila cell lines use different pathways for Ado conversion and suggest that such differences may be an important part of complex mechanisms maintaining energy homeostasis in the body.     

Simultaneous measurement of adenosine and hypoxanthine in human umbilical cord plasma using reversed-phase high-performance liquid chromatography with photodiode-array detection and on-line validation of peak purity

A new, robust and sensitive reversed-phase high-performance liquid chromatographic method was developed for concomitant measurement of plasma concentrations of the ATP catabolites adenosine and hypoxanthine in human umbilical cord blood. Deproteinized cord plasma was chromatographed on Hypersil C₁₈ columns, using UV photodiode-array detection, spectral analysis of peaks and on-line confirmation of peak purity. Elution with a gradient of acetonitrile–tetrahydrofuran in ammonium dihydrogen phosphate buffer pH 4.7, yielded sharp, well-resolved peaks of adenosine and hypoxanthine within 16 min. Peak areas were quantified from external calibration curves and converted to plasma concentrations via cord blood hematocrits. In seven deliveries, gestational ages 32–40 weeks, adenosine (range, 0.1–2.1 μM) was less than hypoxanthine (range, 1.6–18.5 μM) in the same cord plasma sample. Arteriovenous levels of each purine were similar, except in an abruptio placenta delivery.