Role of salvage and phosphorylation in the immunostimulatory activity of C8-substituted guanine ribonucleosides.
Lymphokine-like activity and selective stimulation of B cell growth is exerted by a group of synthetic ribonucleosides derivatized at C8 and exemplified by 8-bromoguanosine (8BrGuo), 8-mercaptoguanosine, and 7-methyl 8-oxoguanosine. However, relatively little is known about their molecular mechanism of action. Like naturally occurring nucleosides, 8BrGuo is taken up into lymphocytes by a process of facilitated diffusion. Naturally occurring nucleosides are then reclaimed by a well characterized salvage pathway, involving sequential phosphorolysis and phosphoribosylation. The studies reported in this communication demonstrate that, in contrast to naturally occurring nucleosides, 8BrGuo is not a substrate for salvage by purine nucleoside phosphorylase. The base that would be produced by putative phosphorolysis, 8-bromoguanine, is biologically inactive and is not a substrate for hypoxanthine-guanine phosphoribosyl-transferase. Accordingly, inhibitors of purine nucleoside phosphorylase-mediated salvage fail to inhibit nucleoside-induced immunostimulation selectively. Examination of the metabolism of 8BrGuo provides no direct evidence that 8BrGuo is phosphorylated by B lymphocytes. Direct enzymatic phosphorylation does not seem to be essential to the mechanism of action of the nucleoside insofar as competitive inhibition of deoxycytidine kinase (an enzyme that directly phosphorylates purines as well as pyrimidines) or of deoxyguanosine kinase fails to inhibit 8BrGuo stimulation selectively. Moreover, studies with synthetic nucleosides in which 3' and/or 5' hydroxyl groups were irreversibly blocked, precluding their phosphorylation, demonstrated that immunobiologic activity can occur in the absence of 3' and/or 5' phosphorylation. Finally, experiments with radiolabeled nucleosides provide no evidence to support the hypothesis that they are incorporated into cellular nucleic acid. These data, together with previous studies, suggest that it is the unmetabolized nucleoside that is active and, as such, is most likely to act in a regulatory capacity.
Goodman
M G
MG
Department of Immunology, Scripps Clinic, La Jolla, CA 92037.
D000276:Adjuvants, Immunologic / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D051379:Mice; D008808:Mice, Inbred CBA; D008934:Mitogens / Q000494:pharmacology; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis
D000276:Adjuvants, Immunologic; D008934:Mitogens; D011500:Protein Synthesis Inhibitors; D006151:Guanosine; C016276:8-bromocyclic GMP; C035169:8-bromoguanosine; D012313:RNA; D004247:DNA; D006152:Cyclic GMP