Metabolic fate of extracellular NAD in human skin fibroblasts

Abstract

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'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide 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 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 fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts 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 alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.

High Sensitivity of CD4+CD25+ Regulatory T Cells to Extracellular Metabolites Nicotinamide Adenine Dinucleotide and ATP: A Role for P2X7 Receptors

“…A concentration of 1 M NAD, i.e., 1/1000 that inside cells and only ϳ10 times that in the extracellular fluid (22)(23)(24)(25), induces almost instantaneous death. Effects of ATP are concordant, but not as impressive, because concentrations of 300 M, i.e., 1/10th those inside cells, are required to induce effects comparable to those of NAD.…”

P2X7 Receptor-Dependent and -Independent T Cell Death Is Induced by Nicotinamide Adenine Dinucleotide

“…The results above confirm the observation (11) that T cells substantially hydrolyze NAD to ADPR, and that upon prolonged incubation ADPR exerts effects similar but not identical to those induced by NAD. It should also be noted that ADPR is metabolized to adenosine (25), which could act on T cells via action on purinergic receptors. Indeed effects of adenosine on T cells via action on A2 A receptors are well documented in the literature (for review see Ref.…”

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

“…This activity cleaves NAD ϩ to NMN and AMP (40). The nucleotide pyrophosphatase inhibitor pyridoxyl phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (41) prevented restoration of the mitochondrial NAD ϩ pool and cell survival when NAD ϩ was used as extracellular precursor (Fig.…”