Adenosine transport by a variant of C1300 murine neuroblastoma cells deficient in adenosine kinase

The uptake of adenosine by an adenosine kinase deficient variant of C1300 murine neuroblastoma cells has been studied in the absence and in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent adenine deaminase inhibitor. Although 100 micro M inhibitor completely blocks the metabolism of adenosine under the conditions studied, the uptake of adenosine is concentrative, i.e., the intracellular adenosine concentration exceeds the extracellular concentration. This concentrative effect decreases as the concentration of adenosine increases and is hypothesized to be due to the binding of adenosine to an intracellular component. Despite this concentrative effect, we believe that the kinetics of uptake, as determined in experiments with short (10-20 s) uptake periods, reflect the kinetics of adenosine transport by a facilitated diffusion process. This nucleoside transport system appears to be nonspecific in that the transport of adenosine is competitively antagonized by thymidine. It does not appear to be necessary to inhibit adenosine deaminase in order to study transport in these cells as the Km for transport is not affected by the presence of erythro-9-(2-hydroxy-3-nonyl)adenine. However, erythro-9-(2-hydroxy-3-nonyl)adenine does depress the V for transport. This effect of the inhibitor is probably not due to the inhibition of adenosine deaminase as the transport of thymidine is similarly affected.     

Suppression of human lymphocyte DNA and protein synthesis in vitro by adenosine and eight modified adenine nucleosides in the presence or in the absence of adenosine deaminase inhibitors, 2′-deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA)

Adenosine and eight modified adenine nucleosides in the presence or in the absence of adenosine deaminase (ADA) inhibitors, 2′-deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl) adenine, were investigated with regard to their effects on phytohemagglutinin-stimulated human lymphocytes in vitro. Protein and DNA synthesis were inhibited depending upon the specific nucleoside, presence or absence of ADA inhibitor, and time of addition of nucleoside to cultures. A possible role for the modified adenine nucleosides in causing in vivo immunodeficiency is proposed.     

Interaction of adenosine deaminase with inhibitors. Chemical modification by diethyl pyrocarbonate

The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors— erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine—was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme–inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors—derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.     

Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency

From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.     

Purine enzyme inhibition fails to alter benzodiazepine receptor binding in brain

Binding of [³H]flunitrazepam to benzodiazepine receptors in brain from several species, including human, was measured in vitro in the presence and absence of purine-metabolizing enzyme inhibitors. Incubation with potent inhibitors of either adenosine deaminase (2′-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)-adenine) or guanine deaminase (5-amino-4-imidazole carboxamide) failed to alter [³H]flunitrazepam binding in homogenates of several different regions of human, rabbit, rat or guinea pig brain. These findings are in contrast to those of Norstrand et al. [Enzyme 29, 61–65 (1983)] who reported substantial alterations in [³H]flunitrazepam binding to human brain membranes in the presence of erythro-9-(2-hydroxy-3-nonyl)-adenine (increase) and 5-amino-4-imidazole carboxamide (decrease). In our studies, [³H]flunitrazepam binding was also unaltered in more anatomically intact brain sections following treatment with purine enzyme inhibitors. Furthermore, in vivo administration of erythro-9-(2-hydroxy-3-nonyl)-adenine to mice at a dose (200 mg/kg, i.p.) known to almost totally inhibit central adenosine deaminase activity also failed to alter brain [³H]flunitrazepam binding measured ex vivo, 30–120 min post injection.

While previous studies have shown that purines such as inosine interact with benzodiazepine receptors, our results raise some questions about the role of endogenous purines in regulating benzodiazepine receptors, at least in vitro and also acutely _vivo following purine enzyme inhibitor administration.     

An analysis of multiple mechanisms of adenosine toxicity in baby hamster kidney cells

Analysis of the response of baby hamster kidney cells to adenosine in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine has revealed two distinct mechanisms of toxicity. The first is apparent at low concentrations of adenosine (less than 5 microM) and is dependent upon the presence of a functional adenosine kinase. The initial toxicity is abolished by uridine, is unrelated to the inhibition of ribonucleotide reductase, and is accompanied by a decrease in the size of the pyrimidine nucleotide pool. Toxicity at higher concentrations of adenosine is adenosine kinase independent and is potentiated by homocysteine thiolactone. An elevation in the intracellular level of S-adenosylhomocysteine, which was observed following treatment with higher concentrations of adenosine (greater than 10 microM), is believed to mediate toxicity at these levels. Interestingly, BHK cells were resistant to intermediate levels of adenosine. The mechanism of resistance is currently unknown, but appears unrelated to a lack of inhibition of adenosine deaminase. It is proposed that substrate inhibition of adenosine kinase may be a determinant of this property.     

Conformation of adenosine deaminase in complexes with inhibitors: application of selective quenching of fluorescence emission

The effect of inhibitors, 1-deazaadenosine (1-dAdo) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), on the conformation of adenosine deaminase was studied using the method of selective quenching of fluorescence emission by acrylamide, I- and Cs+. Both in free adenosine deaminase and in its complexes with the inhibitors, the wavelength maxima and half-width of the emission characterize the environment of fluorescing tryptophan residues in adenosine deaminase as weak polar with limited access to solvent. The formation of complexes with the ground state inhibitors used did not quench or change the main emission characteristics of tryptophan fluorescence in adenosine deaminase. Small blue shifts of emission maxima were observed upon quenching in all three samples. The Stern-Volmer parameters of tryptophan fluorescence quenching by acrylamide were not essentially influenced by complex formation of the enzyme with the inhibitors: in general, the folding of the enzyme molecule in the complexes is not perturbed. On the contrary, the emission quenching by charged heavy ions, I- and Cs+, in the complexes was hindered in comparison with free adenosine deaminase. In the complex with 1-deazaadenosine, the parameters for quenching by both ions evidence the essential worsening of their interaction with tryptophans. In the complex with erythro-9-(2-hydroxy-3-nonyl)adenine, along with the worse quenching by I-, complete prohibition of quenching by Cs+ was observed. These data indicate that the local environments of fluorescing tryptophan residues is substantially distorted compared with free adenosine deaminase, which leads to their screening from charged heavy ions.     

Endogenous Adenosine Modulation of 22Na Uptake by Rat Brain Synaptosomes

To evaluate if endogenous extracellular adenosine influences sodium channel activity in nerve terminals, we investigated how manipulations of extracellular adenosine levels influence ²²Na uptake by rat brain synaptosomes stimulated with veratridine (VT). To decrease extracellular adenosine levels, adenosine deaminase (ADA) that converts adenosine into an inactive metabolite was used. To increase extracellular adenosine levels, we used the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), as well as the inhibitor of adenosine transport, nitrobenzylthioinosine (NBTI). ADA (0.1–5U/ml) caused an excitatory effect on ²²Na uptake stimulated by veratridine, which was abolished in the presence of the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 25M). Both the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1–10M) and the adenosine deaminase inhibitor EHNA (10–25M) inhibited ²²Na uptake by rat brain synaptosomes. It is suggested that adenosine is tonically inhibiting sodium uptake by rat brain synaptosomes.     

Human malaria parasite adenosine deaminase. Characterization in host enzyme-deficient erythrocyte culture

Human malaria infected erythrocytes show a dramatic increase in adenosine deaminase activity in vitro. Using recently developed culture techniques, adenosine deaminase-deficient human erythrocytes were infected in vitro with the major human pathogen Plasmodium falciparum. Adenosine deaminase activity was undetectable in the uninfected host red cells, but increased by 2-fold over normal levels in these cells with an 8% parasitemia. The enzyme in these cells appeared unique in that its activity was markedly elevated over that of other parasite purine enzymes, was not cross-reactive with antibody against human erythrocyte adenosine deaminase, and though inhibited competitively by deoxycoformycin was relatively insensitive to erythro-9-(2-hydroxy-3-nonyl) adenine. The use of adenosine deaminase-deficient erythrocytes for the in vitro cultivation of Plasmodium provides a unique system for the study of parasite enzyme and allows further insight into the purine metabolism of the intraerythrocytic malaria parasite.     

Effects of exogenous adenosine deaminase and erythro-9-(2-hydroxy-3-nonyl) adenine on intracellular cyclic AMP levels in C1300 murine neuroblastoma in tissue culture

The cyclic AMP content of dense cultures of C1300 murine neuroblastoma cells (clone N2a) was elevated after incubation for short periods of time in minimal volumes of serum-free medium (SFM) containing Ro 20 1724, a potent nonxanthine phosphodiesterase inhibitor. This elevation was prevented by theophylline, an adenosine antagonist, and was retarded by dipyridamole or benzylthioinosine, inhibitors of nucleoside transport. Cyclic AMP was also elevated by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a potent adenosine deaminase inhibitor. This effect of EHNA was more pronounced in dense cultures, in small volumes of bathing medium, and was antagonized by dipyridamole. The addition of adenosine deaminase to growth medium or SFM lowered the cyclic AMP levels attained after the addition of Ro 20 1724. We conclude that N2a cells continually release adenosine into the growth or bathing medium $\text{via}$ the nucleoside transport system and that sufficient concentrations may be achieved to tonically stimulate adenylate cyclase and influence processes controlled by the cyclic AMP:cyclic AMP-dependent protein kinase system.     

The biochemical basis for resistance to adenine arabinoside in a mutant of Toxoplasma gondii.

We have previously reported the isolation and preliminary characterization of a mutant of Toxoplasma gondii that was resistant to adenine arabinoside. Fiftyfold higher concentrations of adenine arabinoside were required to inhibit the growth of the resistant parasite in human fibroblast cultures. To determine the enzymic basis for resistance, we measured the kinases and deaminases that act on adenosine or deoxyadenosine. All of these enzymic activities were found in uninfected human fibroblast cells. The mutant and wild type parasite proved to have similar activities of adenosine deaminase, deoxyadenosine deaminase, and deoxyadenosine kinase. However, the adenine arabinoside resistant mutant had less than 0.1% of the adenosine kinase activity observed in the wild type T. gondii. The mutant parasite is presumably resistant because without adenosine kinase to phosphorylate adenine arabinoside it cannot carry out the first step in the conversion of the analogue to adenine arabinoside triphosphate, the active form. A mutant of 3T6 (mouse) cells previously selected for a loss of adenosine kinase also proved to be resistant to adenine arabinoside.     

Metabolism of O6-Propyl and N6-Propyl-carbovir in CEM Cells

Abstract

The metabolism of O⁶-propyl-carbovir and N⁶-propyl-carbovir, two selective inhibitors of HIV replication, has been evaluated in CEM cells. Both compounds were phosphorylated in intact cells to carbovir-5′-triphosphate. The metabolism of these two agents was inhibited by deoxycoformycin and mycophenolic acid, but not erythro-9-(2-hydroxy-3-nonyl)adenine. No evidence of the 5′-triphosphate of either compound was detected in CEM cells.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

The effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2'-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20-40% and 60-80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF. The majority of thymocytes present at Day 9 and at Day 14 in inhibitor-treated rats had the characteristics of subcapsular cortical thymocytes which are probably the most ancestral of the thymocytes. Thus, an induced ADA deficiency blocked the proliferation and differentiation of subcapsular cortical thymocytes which are the precursors of cortical and medullary thymocytes.     

Inhibition of phosphoinositide metabolism in human polymorphonuclear leukocytes by S-adenosylhomocysteine

Transmembrane signaling by chemoattractants in leukocytes appears to require activation of phosphoinositide metabolism with subsequent generation of the second messenger substances, inositol(1,4,5)trisphosphate and diacylglycerol. In addition, previous studies have shown that conditions which lead to an intracellular increase in S-adenosylhomocysteine (AdoHcy), a by-product and competitive inhibitor of S-adenosylmethionine-mediated methylation reactions, inhibit all chemoattractant-mediated functions of leukocytes, suggesting that AdoHcy also interferes with chemoattractant transmembrane signaling. In the present study, we determined whether AdoHcy altered the metabolism of phosphoinositides in human polymorphonuclear leukocytes. Treatment of 32P-labeled polymorphonuclear leukocytes with the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, plus exogenous adenosine and L-homocysteine thiolactone, conditions which cause an increase in AdoHcy, produced as much as a 37% decrease in the amount of [32P]phosphatidylinositol 4-monophosphate associated with the cells. The formation of inositol bisphosphate was inhibited by as much as 45% by erythro-9-(2-hydroxy-3-nonyl)adenine, adenosine, and L-homocysteine thiolactone suggesting decreased availability of phosphatidylinositol 4-monophosphate. In support of this, AdoHcy, in concentrations ranging from 0.01 to 0.1 mM, inhibited the transfer of gamma-32P from gamma-[32P] ATP to phosphatidylinositol (PtdIns). The inhibition of PtdIns kinase was competitive with an apparent Ki for AdoHcy of 43 microM. Increased intracellular AdoHcy reduced chemoattractant-mediated increases in inositol(1,4,5)trisphosphate formation suggesting abrogation of transmembrane signaling. These findings for the first time demonstrate that AdoHcy is a competitive inhibitor of PtdIns kinase and thus a regulator of the phosphoinositide pathway.     

Enhancement of biotinidase activity in mouse serum by inhibitors of methylation

Summary DL-ethionine increases the activity of liver biotinidase, an enzyme which hydrolyzes biotinylesters and biotinylpeptides. Chronic DL-ethionine feeding increases transiently the activity of biotinidase in mouse and rat liver, after which it remains elevated in the serum. In the present work we show that both isomers of DL-ethionine are equally good enhancers of the liver biotinidase, while, 3-ethylthiopropionate, the toxic metabolite of DL-ethionine, has no effect on the biotinidase activity of either liver or serum. We have also employed two different combinations of inhibitors of the hydrolytic pathway of SAH, a transmethylation product and potent inhibitor of methylation. It was found that these inhibitors (EHNA and Ara-A, 2-deoxycoformycin and adenosine) increase the activity of serum biotinidase as was the case with ethionine. Because SAH does not ethylate biomolecules, these changes in biotinidase activity, which can not be preveneted by adenine, biotin or lecithin are most probably related to the inhibition of methylation.Abbreviations Ara-A 9--D-arabinofuranosyladenine - EHNA erythro-9-(2-hydroxy-3-nonyl)adenine - SAE S-adenosylethionine - SAH S-adenosylhomocysteine - SAM S-adenosylmethionine     

Adenosine deaminase from bovine brain: purification and partial characterization.

Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.     

Deoxyadenosine toxicity in an adenosine deaminase-inhibited human CCRF-CEM T-lymphoblastoid cell line causes cell swelling

The human T-lymphoblastoid cell line CCRF-CEM, pre-treated with 2′-deoxycoformycin, was used as a model for adenosine deaminase deficiency to investigate how 2′-deoxyadenosine exerts its cytotoxic effects. Incubation of these cells with 1 μM or 5 μM deoxyadenosine for 24 and 48 h caused an increase of up to 50% in their modal cell volume as measured by a Coulter Size Distribution Analyzer and this increase in cell volume was accompanied by an increase in their fragility and deformability. The swelling of cells was concomitant with the phosphorylation of deoxyadenosine and its intracellular accumulation as dATP. There was no evidence of osmotic imbalance or of inhibition of the Na⁺/K⁺-dependent ATPase activity as the intracellular concentrations (and the intracellular: extracellular ratios)_of Na⁺, K⁺ and Ca²⁺ were essentially unchanged. Cytochalasin B (20 μM) also caused lymphoblasts to swell over a 6-h period and its effect on cell size was similar to that of either 1 μM or 5 μM deoxyadenosine over 24 or 48 h. Longer time-courses of incubation with cytochalasin B caused severe toxicity leading to the death and lysis of a significant proportion of the cells. Other drugs, such as colchicine, vincristine and vinblastine that are known to affect various components of the cytoskeleton also caused swelling of cells in a concentration- and time-dependent manner but there was no evidence that these effects were additive or synergistic with those of deoxyadenosine. Inhibition of DNA synthesis, either directly by aphidicolin or indirectly by hydroxyurea, was less cytotoxic than the effect caused by deoxyadenosine. We conclude that one of the toxic effects resulting from the excessive phosphorylation of deoxyadenosine and its accumulation as dATP in human T-lymphoblasts is not dependent on inhibition of DNA synthesis but may be caused by the disruption of the cytoskeleton in these cells.     

Effects of adenosine analogs on rat cerebral cortical neurons

Adenosine and the adenosine 5'-phosphates (5'-AMP, 5'-ADP and 5'-ATP) depress the spontaneous firing of cerebral cortical neurons. In this study adenosine analogs, adenosine transport blockers and adenosine deaminase inhibitors have been used to gain further insight into the nature of the adenosine receptor and the likely routes of metabolism of extracellularly released adenosine. The firing rate of cortical neurons, including identified corticospinal neurons, was depressed by 2-substituted derivatives of adenosine. 2-Halogenated derivatives of adenosine were potent depressors while 2-aminoadenosine and 2-hydroxyadenosine (crotonoside) were slightly less potent than adenosine. The α,β-methylene isosteres of 5'-ADP and 5'-ATP were almost devoid of agonist activity while the β,γ-methylene analog was an active agonist. This suggests that ADP and ATP must be converted to AMP or possibly adenosine before they can activate the adenosine receptor. 2'-, 3'- and 5'- deoxyadenosine depressed spontaneous firing without antagonizing the effect of adenosine. Adenosine deaminase inhibitors, deoxycoformycin and $\text{erythro}$-9-(2-hydroxy-3-nonyl) adenine had potent, long lasting depressant actions on the spontaneous firing of cortical neurons and concurrently potentiated the actions of adenosine or 5'-AMP. Inhibitors of adenosine transport, papaverine and 2-hydroxy-5-nitrobenzylthioguanosine, prolonged the duration of action of adenosine and 5'-AMP. Intracellular recordings show that 5'-AMP hyperpolarizes cerebral cortical neurons and suppresses spontaneous and evoked excitatory postsynaptic potentials, in the absense of any pronounced alterations in membrane resistance.