adenosine-kinase and beta-glycerophosphoric-acid

Previous results demonstrated that the adenosine that accumulates in human fat cell suspensions is derived from extracellular sources (Kather, H. (1988) J. Biol. Chem. 263, 8803-8809). To get insight into the mechanisms responsible for the lack of adenosine release, extracellular adenine nucleotide catabolism was minimized by 10 mmol/liter beta-glycerophosphate and 10 mumol/liter alpha,beta-methyleneadenosine 5'-diphosphate. Intracellular adenine nucleotide catabolism resulted in a release of inosine and hypoxanthine under these conditions that was increased markedly by isoproterenol. Experiments with inhibitors of adenosine deaminase and adenosine kinase indicated that the production of inosine and hypoxanthine proceeded via AMP deamination. Consistently, IMP levels were increased transiently in the presence of isoproterenol. In addition, the cells possessed a nucleotide phosphomonoesterase that was resistant to the inhibitory actions of ATP and alpha,beta-methyleneadenosine 5'-diphosphate and showed preference for IMP over AMP. Adenosine (approximately 1 nmol/10(6) cells/h) was also produced inside the cells. However, adenosine production was unrelated to ATP turnover via adenylate cyclase, and any adenosine formed was immediately reconverted to adenine nucleotides in the absence and presence of isoproterenol. It was concluded that adenosine is not released by intact human adipocytes, because the alternative routes of intracellular AMP catabolism are compartmentalized (at least in functional terms), and adenosine kinase is not saturated with substrate in the absence and presence of isoproterenol.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Kinase; Adenosine Monophosphate; Adenosine Triphosphate; Adipose Tissue; Glycerophosphates; Humans; Hypoxanthine; Hypoxanthines; Inosine; Inosine Monophosphate; Isoproterenol; Lipolysis; Pentostatin; Purines; Tubercidin

The mechanisms responsible for the large increases of intracellular ATP levels seen after isolated rabbit proximal tubules are treated with exogenous adenine nucleotides were studied. Exogenous ATP was rapidly degraded via adenosine as far as hypoxanthine. Degradation of AMP to adenosine was substantially inhibited by beta-glycerol phosphate. In studies of the ability of individual exogenous purines to increase intracellular ATP levels, single large doses of adenosine were less effective than equimolar doses of exogenous ATP but were substantially more effective than exogenous inosine or hypoxanthine. Exogenous guanine derived compounds increased only cell GTP. Incremental delivery of smaller doses of adenosine to maintain medium levels greater than 5 microM or inhibition of adenosine deaminase with erythro-9-[3-(2-hydroxynonyl)]adenine or 2'-deoxycoformicin enhanced the nucleoside's effectiveness. However, the initial increase of cell ATP was still greater after treatment with exogenous ATP than after adenosine and, in the presence of adenosine deaminase inhibition, larger increases of cell ATP were produced by 50 microM adenosine than by 250 microM adenosine. These observations are most consistent with substrate inhibition of adenosine kinase by adenosine. Furthermore, the adenosine kinase inhibitor, 5-iodotubercidin, prevented the increases of cell ATP resulting from exogenous adenosine or exogenous ATP. These studies demonstrate how the differential uptake and utilization characteristics of nucleosides and bases can fully account for the increases of intracellular nucleotides produced in isolated tubules by exogenous purines.

Topics: Adenine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Adenosine Triphosphate; Animals; Biomechanical Phenomena; Extracellular Space; Female; Glycerophosphates; In Vitro Techniques; Kidney Tubules; Nucleosides; Nucleotides; Purines; Rabbits