Inhibitory action of adenosine on synaptic transmission in the hippocampus of the guinea pig in vitro

Thin hippocampal slices were prepared from guinea pig brains. the postsynaptic field potential elicited in the pyramidal cell layer of CA3 region by mossy fiber stimulation was reversibly inhibited by application of adenosine to the perfusion medium. Among purine and pyrimidine derivatives, only adenosine and adenine nucleotides depressed the field potential with similar dose-response curves at concentrations of 10(-5).10(-3) M. In order to elucidate the mechanism of the inhibitory action of these agents, the effects of adenosine and adenine nucleotides on membrane events in pyramidal neurons were studied using intracellular recording techniques. Application of adenosine and adenine nucleotides hyperpolarized membrane potential and markedly depressed the EPSPs (excitatory postsynaptic potentials) elicited in the pyramidal cell by granular cell activation. However the spike generating mechanism of the neuron was not interfered with and membrane conductance was not increased by adenosine and adenine nucleotides. 4-Aminopyridine counteracted the inhibitory action of adenosine. These findings indicate that the mechanism of the inhibitory action of adenosine and adenine nucleotides is different from that of conventional inhibitory neurotransmitters such as gamma-aminobutyric acid and suggest a presynaptic action of adenosine and adenine nucleotides.     

Adenosine analogs and human platelets--II. Inhibition of ADP-induced aggregation by carbocyclic adenosine and imidazole-ring modified analogs. Significance of alterations in the nucleotide pools.

A number of adenosine analogs modified in the imidazole portion of the purine ring or in the carbohydrate moiety have been examined for their ability to inhibit the aggregation of human blood platelets induced by 10 μM ADP. Also, the effects of incorporation of adenosine analogs into the nucleotide pools, or of alteration of the natural adenine nucleotide levels, were studied. In accord with earlier findings, alterations in the ribose moiety of adenosine markedly diminished effectiveness in blocking ADP-induced aggregation. The C-nucleoside, formycin and its 1-methyl and 2-methyl derivatives, displayed little inhibitory activity and α-D-ribofuranosyl adenine and α-l-lyxofuranosyl adenine were without activity. Replacement of the 5'-hydroxyl group of the ribose by carboxyl, amino or S-methyl groups decreased capacity to inhibit aggregation. In contrast, carbocyclic adenosine (wherein an oxygen atom of the ribofuranosyl ring is replaced by a methylene group) retained full ability to inhibit ADP-induced aggregation. The effects of modifications in the imidazole portion of the purine ring of adenosine are complex. Tubercidin (7-deazaadenosine), 4-aminopyrazolo [3,4-d]pyrimidine ribonucleoside (4-APP-ribonucleoside) and 8-azaadenosine displayed negligible or weak inhibitory activity. However, analogs related to tubercidin, i.e. 6-aminotoyocamycin and sangivamycin (5-carboxamide tubercidin), as well as the 3-carboxamide derivative of 4-APP ribonucleoside, displayed inhibitory activity approaching that of adenosine. No relation was established between the incorporation of adenosine analogs into platelet nucleotide pools and their ability to inhibit ADP-induced aggregation. Analogs such as formycin, which in the presence of the adenosine deaminase inhibitor, deoxycoformycin, readily enter the platelet nucleotide metabolic pool with replacement of a substantial portion of the natural adenine nucleotides, cause only weak inhibitory effects, whereas analogs such as 6-aminotoyocamycin, and sangivamycin, which did not form analog nucleotides or formed only small quantities of the 5'-monphosphate nucleotides, display inhibitory activity comparable to that of adenosine. When platelets were incubated with 2-fluoroadenosine, a potent inhibitor of aggregation, large amounts of 2-fluoroadenosine, 5'-mono, di- and triphosphate nucleotides were formed with a coincident marked decrease in the ATP concentrations. When these platelets were washed free of extracellular 2-fluoroadenosine and resuspended in platelet-free plasma, normal aggregation was produced by ADP. However, incubation of this suspension for 10 min with 2-fluoroadenosine resulted in an almost complete inhibition of ADP-induced aggregation.     

Pathways of nucleotide metabolism in Schistosoma mansoni. IV. Incorporation of adenosine analogs in vitro

The incorporation in vitro of adenine or adenosine analogs into schistosome nucleotides is demonstrated. Tubercidin, 2-fluoroadenosine and 2-fluoroadenine were all shown to be converted into analog triphosphate nucleotides. Since tubercidin and 2-fluoroadenosine are not substrates for adenosine deaminase or purine nucleoside phosphorylase and are not susceptible to degradation to the free base level, it is assumed that they are converted to nucleotides by reaction with adenosine kinase. The incorporation of 2-fluoroadenine into the nucleotide pools indicates that it serves as a substrate for adenine phosphoribosyltransferase.Tubercidin, added to the culture medium, interferes with the maintenance of normal ATP levels. When the concentration of the analog greatly exceeded that of adenine or adenosine in the medium, virtual shutdown of adenosine triphosphate synthesis followed. It is suggested that stoichiometric competition for enzyme sites may determine the relative amounts of nucleotides formed.     

Mechanism of presynaptic inhibition of cholinergic transmission in guinea-pig ileum by adenine nucleotides

This study was designed to determine the mechanism of action of 2'-, 3'- and 5'-substituted adenine nucleotides on cholinergic transmission in guinea-pig ileum. Segments of ileum were continuously stimulated at 0.2 Hz and IC50 values of nucleotides for inhibition of twitch responses determined. All the nucleotides studied inhibited transmission, the effect being antagonised by 30 X 10(-6) M theophylline. The inhibitors of nucleoside transport, HNBTGR and dipyridamole, potentiated responses to all the nucleotides. Addition of adenosine deaminase reduced responses only to adenosine and 5'-AMP, while inhibition of adenosine deaminase with deoxycoformycin potentiated responses only to 5'-AMP and 5'-ADP. It was concluded that all the nucleotides studied inhibit cholinergic transmission through an action at P1-purinoceptors. However, it was not possible from this study to conclude whether these actions were direct or indirect following their hydrolysis to adenosine.     

Relaxations of isolated rabbit coronary artery by purine derivatives: A2-adenosine receptors

The pharmacological nature of purinoceptors in the rabbit coronary artery was investigated by comparing the extent of vasorelaxation induced by various purine derivatives, with or without amino group at C6 and with or without ribose at N9 in the purine structure. These derivatives were grouped according to structure--adenosine, inosine, adenine, and hypoxanthine analogs. The vasorelaxations produced by adenosine analogs were inhibited by aminophylline, while relaxations produced by the other three groups were unaffected. In the case of adenosine analogs, modification of amino group at C6 to secondary amine resulted in reduction of the potency of the vasorelaxing effect. The order of potency was as follows: adenosine greater than N6-(L)-phenylisopropyladenosine greater than N6-(D)-phenylisopropyladenosine greater than N6-cyclohexyl-adenosine greater than N6-(2,5-dioxo-3-pyrrolidinyl)-adenosine greater than kinetine riboside greater than N6-methyladenosine. On the other hand, the aminophylline-resistant relaxations of adenine analogs were augmented by modification of amino group at C6 to secondary or to tertiary amine. The order of potency was as follows: 6-cyclohexylaminopurine greater than 6-dimethylaminopurine greater than kinetine greater than 6-methylaminopurine adenine. Of adenosine, N6-cyclohexyladenosine, adenine, C6-cyclohexylaminopurine, and inosine, N6-cyclohexylaminopurine was the most potent phosphodiesterase inhibitor of crude enzyme prepared from rabbit aorta. From the order of potency of relaxations produced by adenosine analogs, adenosine receptors in the rabbit coronary artery seem to be of the A2-subtype. The relaxation induced by aminophylline-resistant adenine analogs may be due to activation of an unknown but specific site, since there is a structure-activity relationship in the relaxations produced by this group. Such relaxations may be linked to inhibition of phosphodiesterase in the rabbit coronary artery.     

Effects of cyclic 3',5'-AMP and other adenine nucleotides on the melanophores of the lizard (Anolis carolinensis)

1. Cyclic 3',5'-AMP has been reported to darken skins of the frog, Rana pipiens. This suggests that cyclic 3',5'-AMP may mediate the action of MSH on amphibian chromatophores. Since MSH also darkens skins of the lizard, Anolis carolinensis, we investigated the effects of cyclic 3',5'-AMP and other nucleotides on Anolis melanophores to determine whether cyclic 3',5'-AMP may be the intracellular mediator of hormone action on melanophores of another vertebrate class.2. Cyclic 3',5'-AMP, itself, causes a rapid melanin granule aggregation within melanophores of Anolis. This response is, however, somewhat nonspecific in that both 5'-ATP and 5'-ADP also lighten the skins by aggregating the melanin granules. Another nucleotide, 5'-AMP, darkens the skins by dispersing melanin granules. Cyclic 2',3'-AMP does not darken or lighten Anolis skins.3. Dibutyryl cyclic 3',5'-AMP, which is considered to be better able to penetrate membranes and resist degradation by a specific phosphodiesterase, maximally darkens Anolis skins, as does MSH. This darkening by the potent dibutyryl cyclic 3',5'-AMP suggests that cyclic 3',5'-AMP may be the intracellular mediator of melanin granule dispersion within Anolis melanophores leading to skin darkening.4. Other evidence supporting the first-messenger-second-messenger hypothesis for melanophore regulation is discussed.5. The differences in responses of Anolis melanophores to adenine nucleotides may relate to the ability of these agents to penetrate melanophore membranes; thus, the nucleotides could exert their effects either intracellularly or extracellularly on the plasma membrane.     

Metabolic flux rates of adenosine in the heart

The quantitatively most important source of adenosine under well-oxygenated conditions is 5'-AMP hydrolyzed by cytosolic 5'-nucleotidase N-I. Hydrolysis of S-adenosylhomocysteine and extracellular dephosphorylation of 5'-AMP further contribute to total production. More than 90% of the total production occur intracellularly under well-oxygenated conditions. Besides cardiomyocytes, endothelial cells and smooth muscle contribute significantly to total cardiac adenosine production. Rapid enzymatic conversion of adenosine is provided by adenosine kinase and adenosine deaminase, keeping the cytosolic adenosine concentration in the nanomolar range. Due to the high intracellular rates of adenosine rephosphorylation and deamination the cytosolic is normally below the extracellular adenosine concentration, making the cytosol to a sink rather than a source of adenosine. It is for this reason that blockers of membrane transport enhance the plasma adenosine concentration. With increasing catabolism of adenine nucleotides the rate of intracellular adenosine production exceeds the rate of adenosine deamination and rephosphorylation. Thus, this condition will result in a concentration gradient from intra- to extracellular. Thence, membrane transport blockers would be expected to increase the intracellular adenosine concentration. A considerable insecurity on the importance of experimental data results from species differences of purine metabolism. Cardiac adenosine metabolism has recently been described in quantitative terms using mathematical model analysis. This analysis tool may prove useful in future when (1) clarifying the importance of various regulatory actions described for the different pathways of adenosine metabolism, (2) making quantitative comparisons of different experimental models possible and (3) deepening the insight from experimental data.     

Evidence for specific adenosine receptors at cholinergic nerve endings.

1 An electrophysiological study was made to determine if adenosine and adenine nucleotides affect cholinergic nerve endings to frog skeletal muscle through relatively non-specific nucleotide receptors or through specific adenosine receptors. 2 Non-hydrolysable derivatives of adenosine triphosphate failed to alter the mean number of acetylcholine (ACh) quanta released by the nerve impulse (m) or the miniature endplate potential frequency (m.e.p.p.f) but N6-methyladenosine and 2-chloroadenosine, two adenosine analogues with an unsubstituted ribose moiety (R-site agonists), produced marked reductions in m and m.e.p.p.f. 3 In contrast, 2'-deoxyadenosine, a derivative with an unsubstituted purine ring (P-site agonist), generally produced increases in m and m.e.p.p.f, which further increased after removing the drug. Other P-site agonists such as 5'-deoxyadenosine (in the presence of theophylline) and 9-beta-D-arabinofuranosyl adenine also increased m and m.e.p.p.f. 4 The results suggest that two types of adenosine receptors may be present at cholinergic nerve endings, one type (R-site) mediating depression and the other type (P-site) producing enhancement of ACh release.     

Regulation of adenosine kinase by adenosine analogs

The regulation of adenosine phosphorylation by adenosine analogs was studied using highly purified human placental adenosine kinase [ATP: adenosine 5'-phosphotransferase (EC 2.7.1.20)]. Our observations lead us to classify the analogs into three groups as follows: type I, 5'-N-ethylcarboxamidoadenosine and 5'-methylthioadenosine; type II, N6-cyclohexyladenosine, N6-L-phenylisopropyladenosine, and 2-chloroadenosine; and type III, 6-methylmercaptopurine riboside. Type I compounds are inhibitors of adenosine kinase at 0.5 microM adenosine with IC50 values of 25 microM for 5'-N-ethylcarboxamidoadenosine and 250 microM for 5'-methylthioadenosine. These compounds stimulate adenosine kinase at 5.0 microM adenosine up to a maximum of 30 to 50% above basal velocity. They are not substrates for adenosine kinase. Type II compounds are inhibitors of adenosine kinase at 0.5 microM adenosine with an IC50 of 220 microM for N6-cyclohexyladenosine and 200 microM for N6-L-phenylisopropyladenosine. These analogs also stimulate adenosine kinase at 5.0 microM adenosine. 2-Chloroadenosine, N6-cyclohexyladenosine, and N6-L-phenylisopropyladenosine are phosphorylated by adenosine kinase with apparent Km values of 1,330, and 205 microM, respectively. 6-Methylmercaptopurine riboside (type III) inhibited enzyme activity with an IC50 of 10 microM at 0.5 microM adenosine and 215 microM at 5 microM adenosine and is a substrate for adenosine kinase. These data are consistent with the following: (a) 2-chloroadenosine, N6-cyclohexyladenosine, and N6-L-phenylisopropyladenosine may not be good adenosine receptor agonists in vivo because they are phosphorylated into active derivatives by adenosine kinase; (b) 5'-N-ethylcarboxamidoadenosine and 5'-methylthioadenosine are superior candidates for adenosine receptor agonists in vivo because they are not phosphorylated; (c) 5'-N-ethylcarboxamidoadenosine, 5'-cyclohexyladenosine, N6-L-phenylisopropyladenosine, and 2-chloroadenosine may interact with adenosine kinase at two sites on the enzyme, a catalytic site and a regulatory site; and (d) 6-methylmercaptopurine riboside may interact with the enzyme at the catalytic site only.     

Differential effects of inhibitors of purine metabolism on two trichomonad species

Tritrichomonas foetus and Trichomonas vaginalis are both incapable of de novo purine nucleotide synthesis. Previous studies indicated that T. foetus relies mainly on the salvage of hypoxanthine and subsequent conversion of IMP to AMP and GMP, whereas T. vaginalis depends on direct conversions of exogenous adenosine to AMP and guanosine to GMP without much interconversion between the two nucleotides. These two different types of purine salvage suggest the possibility of differential sensitivities between the two species of trichomonad flagellates toward different purine antimetabolites. Mycophenolic acid, hadacidin, 8-azaguanine, and formycin B inhibited the growth of T. foetus but had no effect on T. vaginalis. Mycophenolic acid acted by blocking conversion of IMP to GMP, hadacidin inhibited conversion of IMP to AMP, and 8-azaguanine was incorporated into the T. foetus nucleotide pool, likely via hypoxanthine phosphoribosyl transferase. Formycin B was converted to 5'-monophosphate in T. foetus and inhibited the conversion of IMP to AMP. Its precise mechanism of action on T. foetus remains, however, to be elucidated. Alanosine, whose ribonucleotide derivative is a potent inhibitor of adenylosuccinate synthetase, had no effect on the growth or hypoxanthine incorporation in T. foetus, which may be due to the lack of conversion of alanosine to the ribonucleotide because of the absence of de novo purine nucleotide synthesis in parasites. Four adenosine analogs, adenine arabinoside, tubercidin, sangivamycin, and toyocamycin, were found inhibitory to the growth of T. vaginalis but showed little effect on T. foetus growth. Further investigations suggested that these four compounds acted on T. vaginalis by blocking incorporation of adenosine into the adenine nucleotide pool.     

Pathways of nucleotide metabolism in Schistosoma mansoni--VII. Inhibition of adenine and guanine nucleotide synthesis by purine analogs in intact worms.

Twelve analogs of adenosine or its related purines have been tested as single agents or in combinations as possible antischistosomal compounds. Measurements were made of the effect of these drugs on the anabolism and catabolism of [8-¹⁴C]adenosine by Schistosoma mansoni in vitro. Based on the degree of inhibition of biosynthesis of adenine and guanine nucleotides by intact worms. the best currently available purine analogs are 7-deaza-adenosine (tubercidin) and N⁶-phenyladenosine. These drugs reduced the total synthesis of nueleotides to 30 and 25 per cent, respectively, of controls. Blockade of the catabolic pathway (adenosine deaminase) by coformycin resulted in significantly increased synthesis of adenine nucleotides rather than the expected decrease. Thus, adenosine kinase must play a more prominent role in nucleotide synthesis than had been previously estimated. The implications of these findings in the development of new anti-schistosomal drugs are discussed.     

Papaverine enhances the effect of adenosine in guinea-pig atria

Summary Papaverine, while enhancing the force of contraction of guinea-pig atria, remarkably and dose-dependently enhanced the negative inotropic response of the atria to adenosine. It also enhanced the actions of ATP and other adenine nucleotides, but not those of 2-chloroadenosine and ACh. At similar concentrations, papaverine inhibited the uptake of adenosine by the atrial tissue during incubation with adenosine. Adenosine in the medium was degraded to inactive inosine during incubation with the atrial tissue, and papaverine reduced its degradation.The enhancing effect of papaverine on the action of adenosine on guinea-pig atria was like those of dipyridamole, 6-(2-hydroxy-5-nitrobenzyl)thioguanosine and cinepazide. The effect seemed to be due mainly to inhibition of adenosine uptake into the tissue. Inhibition of adenosine degradation may also have contributed to the action of papaverine, but this action was probably much less important than inhibition of adenosine uptake.     

5'-deoxy-5'-methylthioadenosine phosphorylase--V. Acycloadenosine derivatives as inhibitors of the enzyme

Various adenosine acyclonucleoside derivatives were tested as inhibitors of 5'-deoxy-5'-methylthioadenosine (MeSAdo) phosphorylase, an enzyme involved in the salvage of adenine and methionine from MeSAdo. The 2-halogenated derivatives of acyloadenosine [9-(2-hydroxyethoxy-methyl)adenine], including the chloro-, bromo- and iodo-congeners, all inhibited murine Sarcoma 180 (S180) MeSAdo phosphorylase, with Ki values in the range of 10(-6) to 10(-5) M. Halogenated derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)adenine, which more closely resemble the natural substrate, were substantially more potent inhibitors of the enzyme, with Ki values in the range of 2-7 x 10(-7) M. 5'-Methylthio and 5'-halogenated analogs of 2'-deoxy-1',2'-seco-adenosine were weak inhibitors, with Ki values of 10(-4) M or greater. 9-[(1-Hydroxy-3-iodo-2-proxy)methyl]adenine. (HIPA), the derivative with the lowest Ki values among these analogs, was a competitive inhibitor of S180 MeSAdo phosphorylase. In preliminary studies, HIPA inhibited MeSAdo phosphorylase in intact HL-60 human promyelocytic leukemia cells, as it limited the incorporation of [8-14C]MeSAdo into cellular adenine nucleotide pools. In addition, 9-(phosphonoalkyl)adenines, representing potential multisubstrate inhibitors of MeSADo phosphorylase, were synthesized. Of these the heptyl derivative was the most potent inhibitor, with a Ki of 1.5 x 10(-5) M at low (3.5 mM) phosphate concentrations. The inhibitory effects of these analogs could be ablated at high phosphate concentrations (50 mM), suggesting that they interact with the phosphate binding site on the enzyme. Some of these novel MeSAdo phosphorylase inhibitors may have a role in cancer chemotherapy as potentiators of agents that block purine de novo synthesis, e.g. antifolates and 6-methylmercaptopurine ribonucleoside.     

Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides.

1. In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A(2a)-adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2. Endogenous adenosine tonically inhibited synaptic transmission, since 0.5-2 u ml-1 of adenosine deaminase increased both the population spike amplitude (30 +/- 4%) and field excitatory post-synaptic potential (f.e.p.s.p.) slope (27 +/- 4%) recorded from hippocampal slices and the evoked [3H]-acetylcholine ([3H]-ACh) release from the motor nerve terminals (25 +/- 2%). 3. alpha, beta-Methylene adenosine diphosphate (AOPCP) in concentrations (100-200 microM) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 +/- 5% and f.e.p.s.p. slope by 32 +/- 3% in hippocampal slices and [3H]-ACh release from motor nerve terminals by 27 +/- 3%. 4. Addition of exogenous 5'-nucleotidase (5 u ml-1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43-57%, whereas the P2 antagonist, suramin (100 microM), did not modify the effect of AOPCP. 5. In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5-2 u ml-1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3-dipropyl-8-cyclopentylxanthine (2.5-5 nM), but was abolished by the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (10 microM). 6. These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CAI pyramid synapses and in phrenic motor nerve endings.     

Inhibition of lymphocyte-mediated cytolysis by adenosine analogs. Biochemical studies concerning mechanism of action.

A number of adenosine (Ado) analogs have been found to inhibit lymphocyte-mediated cytolysis (LMC) in vitro at μM concentrations. Those analogs which are substrates for adenosine deaminase were more inhibitory to LMC in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of the deaminase. The inhibitory activity of most of these analogs (the exceptions being 2′-deoxyadenosine, 9-β-d-arabinofuranosyladenine and 7-deazaadenosine) was markedly enchanced by an inhibitor (Ro 20-1724) of cyclic AMP phosphodiesterase. With the exceptions of 2-fluoroadenosine, 7-deazaadenosine and formycin A, the inhibition of LMC caused by the Ado analogs was fully reversible upon removal of the analogs from the medium. In general, the Ado analogs did not cause a reduction in the pool sizes of endogenous ribonucleoside 5′-triphosphates in the lymphocytes. Some inhibitory and non-inhibitory analogs were metabolized to their corresponding 5′-triphosphates. Lymphocytes pretreated with a reversible inhibitor of LMC, 2-aminoadenosine, retained most of the resultant 2-amino-ATP during subsequent incubation in analog-free medium. Most of the Ado analogs which were inhibitory to LMC caused a substantial elevation of lymphocyte cyclic AMP; the magnitude of this elevation was enhanced by Ro 20-1724. Collectively, these results suggest that Ado and many of its structural analogs inhibit LMC by reason of their ability to stimulate the formation and consequent build-up of cyclic AMP in the cytotoxic lymphocytes. This stimulation of adenylate cyclase appears to result from the binding of an appropriate nucleoside to an adenosine receptor located on the membrane of the lymphocytes.     

Pathways of nucleotide metabolism in schistosoma mansoni. V. Adenosine cleavage enzyme and effects of purine analogues on adenosine metabolism in vitro

Schistosoma mansoni extracts have been found to possess an active anabolic pathway for nucleotide biosynthesis in which adenosine is cleaved to adenine followed by conversion of adenine to AMP via adenine phosphoribosyltransferase. A significant fraction of labeled adenosine was found to enter the nucleotide pool by this pathway; howeverm, most of the nucleoside was converted to nucleotides by a pathway which employs adenosine deaminase, purine nucleoside phosphorylase and hypoxanthine phosphoribosyltransferase enzymes. Formycin A has been found to be a potent blocker of adenosine cleavage when tested in worm extracts. Arabinosyl-6-mercaptopurine and 6-thioguanosine are inhibitors of worm adenosine deaminase, and formycin B and 6-thioguanosine were found to inhibit the purine nucleoside phosphorylase of this parasite. Combinations of arabinosyl-6-mercaptopurine with either formycin A or formycin B result in substantial blockage of adenosine utilization for nucleotide synthesis. These studies thus suggest that adenosine analogues in combination might be useful in vivo for the chemotherapy of schistosomiasis.     

C(2′)-substituted purine nucleoside analogs: Interactions with adenosine deaminase and purine nucleoside phosphorylase and formation of analog nucleotides

Four C(2′)-substituted 2′-deoxyadenosines were examined as substrates for human erythrocytic adenosine deaminase and for formation of intracellular nucleotide analogs in human erythrocytes, lymphocytes and murine Sarcoma 180 cells: 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)adenine, 9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine, 9-(2′-azido-2′-deoxy-β-D-ribofuranosyl)adenine (2′-N₃-riboA) and 9-(2′-azido-2′-deoxy-β-D-arabinofuranosyl)adenine. All four adenosine analogs were substrates of human erythrocytic adenosine deaminase, but the corresponding inosine analogs (synthesized by the adenosine deaminase reaction) were highly resistant to cleavage by human erythrocytic purine nucleoside phosphorylase. Only 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)hypoxanthine underwent very slow phosphorolysis, and no inhibition of inosine phosphorolysis was detected when a 30 μM concentration of any studied inosine analog was added to a reaction mixture containing 30 μM inosine (the K_m concentration). Kinetic parameters were determined for the deamination of the adenosine analogs. The greatest affinity for adenosine deaminase was found with 2′-N₃-riboA (K_i=2μM), but the reaction velocity was highest with the F-substituted analogs. All four adenosine analogs formed triphosphate nucleotides after incubation with human erythrocytes, murine Sarcoma 180 cells, or human lymphocytes (tested only with the F analogs) in the presence of deoxycoformycin.     

Stimulation of P1-purinoceptors by ATP depends partly on its conversion to AMP and adenosine and partly on direct action

The effects of degradative enzymes and enzyme inhibitors were examined on the inhibitory actions of adenosine, AMP and ATP on atrial muscle and on the cholinergic responses of the ileum to transmural stimulation of the guinea-pig, in order to determine whether ATP responses are mediated by its breakdown products, AMP and adenosine. In both the atria and the ileum, adenosine deaminase reduced responses to ATP, although when combined with 5'-nucleotidase it had no further effect. In the atrium, the 5'-nucleotidase inhibitor, alpha,beta-methylene ADP (APCP), had no effect on its own, but prevented the potentiating effect of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) on responses to ATP. In the ileum, EHNA had no effect, but APCP potentiated responses to ATP. The enzyme 5'-AMP deaminase was shown to have a non-specific inhibitory effect on purine responses in both preparations. It is concluded for both preparations, that (1) the inhibitory responses to ATP are partly mediated by AMP and adenosine following the ectoenzymatic breakdown of ATP, and partly mediated by ATP itself, and (2) that AMP as well as adenosine can act directly on P1-purinoceptors. It is suggested that of the two breakdown products of ATP, AMP and adenosine, a larger proportion of the response is mediated by AMP in the ileum, whereas adenosine is the major mediator in the atrium.     

Structure-activity relationship of ligands of human plasma adenosine deaminase2

Diethylaminoethyl-cellulose chromatography was used to separate the two isoenzymes of adenosine deaminase (EC3.5.4.4), adenosine deaminase1 (ADA1) and adenosine deaminase2 (ADA2), in human plasma. One hundred and fifteen purine base, nucleoside, and nucleotide analogs were tested as inhibitors of this partially purified preparation of ADA2. Coformycin and 2'-deoxycoformycin were by far the most potent inhibitors of this isoenzyme (apparent Ki values 20 and 19 nM, respectively). ADA2 was also inhibited by nebularine (apparent Ki 1.5 mM) but was resistant to the potent ADA1 inhibitor (+)-erythro-9(2-S-hydroxy-3-R-nonyl)adenine. alpha-D-Adenosine also inhibited ADA2, as did several halogenated purine and adenine base analogs. Structural requirements for the binding of purine analogs to ADA2 are presented which provide a general basis for the design of specific inhibitors of ADA2. Such inhibitors may be useful in studies designed to provide an understanding of the physiological role of ADA2 both in the normal state and in diseases such as human immunodeficiency virus-1 infection where levels in plasma are increased markedly.     

5'-deoxy-5'-methylthioadenosine phosphorylase--IV. Biological activity of 2-fluoroadenine-substituted 5'-deoxy-5'-methylthioadenosine analogs

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAPase) phosphorolyzes 5'-deoxy-5'-methylthioadenosine (MTA) generated during polyamine biosynthesis to adenine and 5-methylthioribose-1-phosphate. Two doubly-substituted, 2-fluoroadenine-containing analogs of MTA, 5'-deoxy-2-fluoroadenosine (5'-dFAdo) and 5'-deoxy-5'-iodo-2-fluoroadenosine (5'-IFAdo), were synthesized and studied as substrates of MTAPase: their reaction with this enzyme resulted in the liberation of the cytotoxic base, 2-fluoroadenine, as well as potentially cytotoxic analogs of 5-methylribose-1-phosphate. The activities of these MTA analogs were compared to that of the singly-substituted analog, 5'-deoxy-5'-methylthio-2-fluoroadenosine (5'-MTFAdo). The cytotoxic action of these MTA analogs depended primarily on their conversion to 2-fluoroadenine-containing nucleotides, as a cell line that contains both MTAPase and adenine phosphoribosyltransferase (APRT) activity (HL-60 human promyelocytic leukemia) readily converted these MTA analogs to 2-fluoroadenine-containing nucleotides (especially 2-fluoroadenosine triphosphate) and was highly sensitive to the growth-inhibitory effects of all three compounds (IC50 values in the 10(-8) M range), whereas cell lines lacking MTAPase (CCRF-CEM human T-cell leukemia) or APRT (HL-60/aprt1 cells) did not form analog nucleotides and were relatively insensitive to these compounds (IC50 values in the 10(-5) M range). The doubly-substituted analogs were not more growth inhibitory than 5'-MTFAdo in wild type HL-60 cells as the potent effects of 2-fluoroadenine may mask the activity of the 5-methylthioribose-1-phosphate analogs generated in the reaction of these compounds with MTAPase. 5'-dFAdo and 5'-IFAdo also were irreversible inhibitors of S-adenosylhomocysteine hydrolase, which may explain in part the weak but observable growth inhibitory action of these compounds against MTAPase-deficient cell lines.