Metabolic fate of extracellular NAD in human skin fibroblasts (original) (raw)

2001, Journal of Cellular Biochemistry

Extracellular NAD is degraded to pyridine and purine metabolites by different 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 H-nucleotidase are located on the outer surface of human skin ®broblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5 H-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 could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[ 14 C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in ®broblasts incubated with 14 C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [ 14 C]-NAD uptake observed in ®broblasts depleted of ATP showed that the transport system requires ATP to some extent. All these ®ndings suggest that adenosine is the purine form taken up by cells, and this hypothesis was con®rmed incubating cultured ®broblasts with 14 C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [ 14 C]-adenosine yield the same radioactive products as with [ 14 C]-NAD; the absence of inhibition of [ 14 C]-adenosine uptake by ADPribose in the presence of a-b methyleneADP, an inhibitor of 5 H nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is ®nally catabolised to adenosine. These results con®rm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.