guanosine-triphosphate and 8-aminoguanosine

It has been reported that the immunosuppressant mizoribine (MZR) inhibits T cell proliferation by depleting intracellular guanine nucleotides via competitive inhibition of inosine 5'-monophosphate (IMP) dehydrogenase in the purine metabolism pathway. This study was performed to determine if the mechanism by which MZR suppresses the proliferation of mouse B cells and antibody production by these cells is dependent on the depletion of intracellular guanine nucleotides. Stimulation of purified splenic B cells of mice with lipopolysaccharide (LPS), a mitogen to B cells, increased both proliferation and antibody production. MZR suppressed both of these functions in a dose-dependent fashion. MZR also caused a decrease in the amount of intracellular guanosine 5'-triphosphate (GTP). When the cultures were grown on plates containing guanosine plus 8-aminoguanosine, the amount of intracellular GTP, which had been reduced by MZR, was restored. Furthermore, the repletion of GTP pools restored both proliferation and antibody production almost to their previous levels. These results suggest that MZR suppresses antibody production and proliferation of B cells by acting directly on B cells. Furthermore, it is suggested that the inhibitory effect of MZR on antibody production, as well as on T cell proliferation, is dependent on the decrease in intracellular guanine nucleotide pools of mouse B cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibody Formation; Antibody Specificity; B-Lymphocytes; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Guanosine; Guanosine Triphosphate; Immunosuppressive Agents; Lipopolysaccharides; Male; Mice; Mice, Inbred ICR; Ribonucleosides; Spleen

Purine nucleoside phosphorylase (NP; EC 2.4.2.1) deficiency is associated with selective T-cell dysfunction and normal B-cell immunity. In order to create an in vivo model of this immune deficiency, we administered 8-aminoguanosine to rats. This water-soluble nucleoside was rapidly converted by NP to the more potent inhibitor 8-aminoguanine, which has a Ki of 0.19 microM. The accumulation of inosine in plasma showed that administration of 8-aminoguanosine was effectively inhibiting NP activity. The administration of 8-aminoguanosine with deoxyguanosine produced increased levels of dGTP only in thymus cells, and increased levels of GTP in cells from thymus, spleen and lymph node and in red cells. This correlated with assays of deoxyguanosine kinase, which showed significantly higher activity in thymus cells than in cells from spleen and lymph node. The intraperitoneal injection of 8-aminoguanosine alone or with deoxyguanosine for 8 consecutive days caused significant decreases in the number of thymus cells (P less than 0.001) and in lymph node and spleen lymphocytes (P less than 0.01). These data showed that the administration of 8-aminoguanosine to rats provided an animal model of NP deficiency that will allow studies of the specific regulation of T-cell function.

Topics: Animals; Deoxyguanine Nucleotides; Deoxyguanosine; Disease Models, Animal; Erythrocytes; Female; Guanosine; Guanosine Triphosphate; Lymphoid Tissue; Male; Pentosyltransferases; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Purine-Nucleoside Phosphorylase; Rats; Rats, Inbred Lew

Topics: B-Lymphocytes; Cell Cycle; Cell Differentiation; Cells, Cultured; Deoxycytidine; Deoxyguanine Nucleotides; Deoxyguanosine; Guanosine; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; In Vitro Techniques; Lymphocyte Activation; Purine-Nucleoside Phosphorylase; T-Lymphocytes

Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8-aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'-deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4+ mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 microM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three- to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 microM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine. 8-Aminoguanosine appears to effectively inhibit extracellular PNP activity; thus, it prolongs the extracellular half-life of 2'-deoxyguanosine and guanosine, but does not completely inhibit intracellular PNP activity in these lymphoid cells. As a result, 2'-deoxyguanosine and guanosine are phosphorolyzed and actively salvaged within the cell, accounting for the accumulation of GTP. Partial inhibition of PNP activity in vivo, therefore, may lead to nonselective cellular toxicity by a mechanism independent of dGTP accumulation.

Topics: B-Lymphocytes; Cell Division; Deoxyguanosine; Drug Synergism; Guanine Nucleotides; Guanosine; Guanosine Triphosphate; Humans; T-Lymphocytes