The extracellular versus intracellular mechanisms of inhibition of TCR-triggered activation in thymocytes by adenosine under conditions of inhibited adenosine deaminase.
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The absence or low levels of adenosine deaminase (ADA) in humans result in severe combined immunodeficiency (SCID), which is characterized by hypoplastic thymus, T lymphocyte depletion and autoimmunity. Deficiency of ADA causes increased levels of both intracellular and extracellular adenosine, although only the intracellular lymphotoxicity of accumulated adenosine is considered in the pathogenesis of ADA SCID. It is shown that extracellular but not intracellular adenosine selectively inhibits TCR-triggered up-regulation of activation markers and apoptotic events in thymocytes under conditions of ADA deficiency. The effects of intracellular adenosine are dissociated from effects of extracellular adenosine in experiments using an adenosine transporter blocker. We found that prevention of toxicity of intracellular adenosine led to survival of TCR-cross-linked thymocytes in long-term (4 days) assays, but it was not sufficient for normal T cell differentiation under conditions of inhibited ADA. Surviving TCR-cross-linked thymocytes had a non-activated phenotype due to extracellular adenosine-mediated, TCR-antagonizing signaling. Taken together the data suggest that both intracellular toxicity and signaling by extracellular adenosine may contribute to pathogenesis of ADA SCID. Accordingly, extracellular adenosine may act on thymocytes, which survived intracellular toxicity of adenosine during ADA deficiency by counteracting TCR signaling. This, in turn, could lead to failure of positive and negative selection of thymocytes, and to additional elimination of thymocytes or autoimmunity of surviving T cells. (+info)
Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination.
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Template-independent nucleotide additions (N regions) generated at sites of V(D)J recombination by terminal deoxynucleotidyl transferase (TdT) increase the diversity of antigen receptors. Two inborn errors of purine metabolism, deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), result in defective lymphoid development and aberrant pools of 2'-deoxynucleotides that are substrates for TdT in lymphoid precursors. We have asked whether selective increases in dATP or dGTP pools result in altered N regions in an extrachromosomal substrate transfected into T-cell or pre-B-cell lines. Exposure of the transfected cells to 2'-deoxyadenosine and an ADA inhibitor increased the dATP pool and resulted in a marked increase in A-T insertions at recombination junctions, with an overall decreased frequency of V(D)J recombination. Sequence analysis of VH-DH-JH junctions from the IgM locus in B-cell lines from ADA-deficient patients demonstrated an increase in A-T insertions equivalent to that found in the transfected cells. In contrast, elevation of dGTP pools, as would occur in PNP deficiency, did not alter the already rich G-C content of N regions. We conclude that the frequency of V(D)J recombination and the composition of N-insertions are influenced by increases in dATP levels, potentially leading to alterations in antigen receptors and aberrant lymphoid development. Alterations in N-region insertions may contribute to the B-cell dysfunction associated with ADA deficiency. (+info)
Quantification of extracellular and intracellular adenosine production: understanding the transmembranous concentration gradient.
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BACKGROUND: Inhibitors of adenosine membrane transport cause vasodilation and enhance the plasma adenosine concentration. However, it is unclear why the plasma adenosine concentration rises rather than falls when membrane transport is inhibited. We tested the hypothesis that the cytosolic adenosine concentration exceeds the interstitial concentration under well-oxygenated conditions. METHODS AND RESULTS: In isolated, isovolumically working guinea pig hearts (n=50), the release rate of adenosine and accumulation of S-adenosylhomocysteine (after 20 minutes of 200 micromol/L homocysteine), a measure of the free cytosolic adenosine concentration, were determined in the absence and presence of specific and powerful blockers of adenosine membrane transport (nitrobenzylthioinosine 1 micromol/L), adenosine deaminase (erythro-9-hydroxy-nonyl-adenine 5 micromol/L), and adenosine kinase (iodotubericidine 10 micromol/L). Data analysis with a distributed multicompartment model revealed a total cardiac adenosine production rate of 2294 pmol. min-1. g-1, of which 8% was produced in the extracellular region. Because of a high rate of intracellular metabolism, however, 70.3% of extracellularly produced adenosine was taken up into cellular regions, an effect that was effectively eliminated by membrane transport block. The resulting approximately 2.8-fold increase of the interstitial adenosine concentration evoked near-maximal coronary dilation. CONCLUSIONS: We rejected the hypothesis that the cytosolic adenosine concentration exceeds the interstitial. Rather, there is significant extracellular production, and the parenchymal cell represents a sink, not a source, for adenosine under well-oxygenated conditions. (+info)
Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors.
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1. The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X(1-4) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5'-adenylic deaminase, and purified using reverse-phase chromatography. 3. Against ATP-responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)>Ip4I (6.3)>Ip3I (>4.5). Ip5I (10-100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP effect. However, the Schild plot was non-linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non-surmountable antagonist. 4. Against ATP-responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6. 0)>Ip5I (5.6)>Ip3I (>4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. 5. Diinosine polyphosphates failed to inhibit ATP-responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1 - 4 receptors. 6. Thus, Ip5I acted as a potent and relatively-selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high-affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (100 nM) concentrations. (+info)
The anticonvulsant BW534U87 depresses epileptiform activity in rat hippocampal slices by an adenosine-dependent mechanism and through inhibition of voltage-gated Na+ channels.
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1. The cellular and molecular actions of BW534U87 were studied using intracellular and extracellular recordings from the CA1 region of rat hippocampal slices and whole-cell voltage-clamp recordings of recombinant human brain type IIA Na+ channels expressed in Chinese hamster ovary (CHO) cells. 2. Normal excitatory and inhibitory postsynaptic potentials evoked in hippocampal slices were unaffected by BW534U87 or the adenosine deaminase inhibitor EHNA. However, epileptiform activity was depressed by BW534U87 (50 micronM) and this inhibition was reversed by the adenosine receptor antagonist 8-phenyl theophylline (8-PT, 30 micronM). EHNA (10 micronM) mimicked the effects of BW534U87. Furthermore, BW534U87 enhanced the inhibitory effects of exogenous adenosine on evoked synaptic potentials. BW534U87 (50 micronM) also voltage- and use-dependently inhibited action potentials elicited by current injection, independent of the adenosine system, since it was not affected by 8-PT. 3. In CHO cells expressing the recombinant human brain Na+ channel, BW534U87 produced a concentration- and voltage-dependent inhibition of Na+ currents with a half-maximal inhibitory concentration of 10 micronM at a Vh of -60 mV. Use-dependent inhibition was evident at high-frequencies (20x20 ms pulse train at 10 Hz). 4 In conclusion, BW534U87 blocks hippocampal epileptiform activity by a dual mechanism. The first action is similar to that produced by EHNA and is dependent on endogenous adenosine probably by inhibition of adenosine deaminase. Secondly, BW534U87 directly inhibits voltage-gated Na+ channels in a voltage- and frequency-dependent manner. Both actions of BW534U87 are activity-dependent and may synergistically contribute to its overall anticonvulsant effects in animal models of epilepsy. (+info)
A normal level of adenosine deaminase activity in the red cell lysates of carriers and patients with severe combined immunodeficiency disease.
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The red cell lysates of two children with severe combined immunodeficiency disease (SCID) exhibited a virtually total absence of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) when standard volumes were assayed. Under these conditions the parents exhibited depressed specific activity except for one mother, whose lysate showed a normal value for activity. Upon storage of the lysate at 4 degrees, a significant amount of activity appeared in one of the SCID children, and the activity of the heterozygous carriers was stimulated. With the use of a sensitive spectrophotometric assay based on conversion of inosine to uric acid, it was shown that the specific enzymatic activity in each of the SCID patients increased progressively as the volume of lysate assayed was lowered. With the smallest amount of lysate this specific activity was in the normal range. Similarly, the specific activity of each of the parents' lysates increased to the level of normal (or, in one case, about twice normal) as smaller volumes were assayed. The activity in the SCID patient was inhibitable by 2-fluoroadenosine and N6-methyladenosine, known competitive inhibitors of human red cell adenosine deaminase. The lysate from the SCID patient was also shown to inhibit adenosine deaminase partially purified from a normal individual. The results are interpreted in terms of a genetically programmed production of an adenosine deaminase inhibitor in at least one variant of the severe combined immunodeficiency disease. (+info)
Age-related changes in A(1)-adenosine receptor-mediated bradycardia.
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The impact of age on functional sensitivity to A(1)-adenosine receptor activation was studied in Langendorff-perfused hearts from young (1-2 mo) and old (12-18 mo) male Wistar rats. Adenosine mediated bradycardia in young and old hearts, with sensitivity enhanced approximately 10-fold in old [negative logarithm of EC(50) (pEC(50)) = 4.56 +/- 0.11] versus young hearts (pEC(50) = 3.70 +/- 0. 09). Alternatively, the nonmetabolized A(1) agonists N(6)-cyclohexyladenosine and (R)-N(6)-phenylisopropyladenosine were equipotent in young (pEC(50) = 7.43 +/- 0.12 and 6.61 +/- 0.19, respectively) and old hearts (pEC(50) = 7.07 +/- 0.10 and 6.80 +/- 0. 11, respectively), suggesting a role for uptake and/or catabolism in age-related changes in adenosine sensitivity. In support of this suggestion, [(3)H]-adenosine uptake was approximately twofold greater in young than in old hearts (from 3-100 microM adenosine). However, although inhibition of adenosine deaminase and adenosine transport with 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride and 10 microM S-(4-nitrobenzyl)-6-thioinosine increased adenosine sensitivity three- to fourfold, it failed to abolish the sensitivity difference in old (pEC(50) = 4.95 +/- 0.08) versus young (pEC(50) = 4.29 +/- 0.13) hearts. Data indicate that 1) age increases functional A(1) receptor sensitivity to adenosine without altering the sensitivity of the A(1) receptor itself, and 2) age impairs adenosine transport and/or catabolism, but this does not explain differing functional sensitivity to adenosine. This increased functional sensitivity to adenosine may have physiological significance in the older heart. (+info)
Adenosine-mediated killing of cultured epithelial cancer cells.
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Because micromolar concentrations of adenosine (Ado) have been documented recently in the interstitial fluid of carcinomas growing in animals, we examined the effects of low concentrations of Ado on the growth of cultured human carcinoma cells. Ado alone had little effect upon cell growth. In the presence of one of a number of Ado deaminase (ADA) inhibitors, Ado led to significant growth inhibition of all cell lines tested. Similar effects were found when ATP, ADP, or AMP was substituted for Ado. Surprisingly, the ADA inhibitor coformycin (CF) had a much greater potentiating effect than did 2'-deoxycoformycin (DCF), although DCF is a more potent ADA inhibitor. The growth inhibition of the Ado/CF combination was not abrogated by pyrimidines or caffeine, a nonspecific Ado receptor blocker. Toxicity was prevented by the addition of the Ado transport inhibitor dipyridamole or the Ado kinase inhibitor 5'-amino 5'-deoxyadenosine. S-Adenosylhomocysteine hydrolase is not involved because neither homocysteine thiolactone nor an S-adenosylhomocysteine hydrolase inhibitor (adenosine dialdehyde) potentiated toxicity of the Ado/CF combination. Unexpectedly, substitution of 2'-deoxyadenosine (the toxic moiety in congenital ADA deficiency) for Ado, did not lead to equivalent toxicity. The Ado/CF combination inhibited DNA synthesis and brought about morphological changes consistent with apoptosis. Together, these findings indicate that the Ado-mediated killing proceeds via an intracellular route that requires the action of Ado kinase. The enhanced cofactor activity of CF may be attributable to its being a more potent inhibitor of AMP deaminase than is DCF. (+info)