Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by differe nt types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nu cleotidase are located on the outer surface of human skin fibroblasts. Nucl eotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine . The absence of ADPribose and adenosine in the extracellular compartment c ould be due to further catabolism and/or uptake of these products. To clari fy the fate of the purine moiety of exogenous NAD, we investigated uptake o f the products of NAD hydrolysis using U-[C-14]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; A DP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptak e of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyz ing enzymes, whereas adenosine did not. Radioactive uptake by purine drasti cally dropped in fibroblasts incubated with C-14-NAD and dipyridamole, an i nhibitor of adenosine transport. Partial inhibition of [C-14]-NAD uptake ob served in fibroblasts depleted of ATP showed that the transport system requ ires ATP to some extent. All these findings suggest that adenosine is the p urine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with C-14-adenosine and analyzing nucleoside uptake an d intracellular metabolism under different experimental conditions. Fibrobl asts incubated with [C-14]-adenosine yield the same radioactive products as with [C-14]-NAD; the absence of inhibition of [C-14]-adenosine uptake by A DPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nuc leotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrola se is finally catabolised to adenosine. These results confirm that adenosin e is the NAD hydrolysis product incorporated by cells and further metaboliz ed to ATP, and that adenosine transport is partially ATP dependent. (C) 200 1 Wiley-Liss, Inc.