Enzymatic activities affecting exogenous nicotinamide adenine dinucleotide in human skin fibroblasts

Aleo, Maria Francesca; Sestini, Silvia; Pompucci, G; Preti, Augusto

doi:10.1002/(sici)1097-4652(199604)167:1<173::aid-jcp20>3.0.co;2-b

Cited by 6 publications

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References 42 publications

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“…In a previous study, we demonstrated that NAD‐glycohydrolase (cleaving NAD into nicotinamide and ADPR), nucleotide pyrophosphatase (cleaving NAD into NMN and AMP), and 5′‐nucleotidase are the enzymes involved in extracellular NAD catabolism in human skin fibroblasts. We also showed that the final products of the extracellular NAD metabolism are nicotinamide, NMN, hypoxanthine and adenine [Aleo et al, 1996]. In the present study, we investigated the metabolic fate of the extracellular NAD purine moiety, showing that adenosine is the form which permeates the cell membrane.…”

Section: Discussionmentioning

confidence: 66%

“…In a previous report [Aleo et al, 1996], 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.…”

mentioning

confidence: 51%

“…The main purine products of extracellular NAD hydrolysis in fibroblasts [Aleo et al, 1996], namely ADPribose and adenosine were then added to the incubation medium containing U‐[ 14 C]‐adenine‐NAD. Total radioactive cell uptake drastically decreased to 3.6 ± 0.9% in the presence of ADPribose and 5.0 ± 0.76% in the presence of adenosine (n = 5), while the relative proportion of intracellular labeled compounds did not vary with respect to control cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…HPLC analysis was performed as previously described [Aleo et al 1996] using 0.1 M K‐phosphate buffer containing 6 mM TBA at pH 5.5 and methanol as eluants.…”

Section: Methodsmentioning

confidence: 99%

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Metabolic fate of extracellular NAD in human skin fibroblasts

et al. 2000

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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.

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Section: Discussionmentioning

confidence: 66%

mentioning

confidence: 51%

Section: Resultsmentioning

confidence: 99%

“…HPLC analysis was performed as previously described [Aleo et al 1996] using 0.1 M K‐phosphate buffer containing 6 mM TBA at pH 5.5 and methanol as eluants.…”

Section: Methodsmentioning

confidence: 99%

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