Metabolic fate of extracellular NAD in human skin fibroblasts

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

Anti-oxidant potential and gap junction-mediated intercellular communication as early biological markers of mercuric chloride toxicity in MDCK cell line.

In this study, the early nephrotoxic potential of mercuric chloride (HgCl(2)) has been evaluated in vitro, by exposing a renal-derived cell system, the tubular epithelial Madin-Darby canine kidney (MDCK) cell line, to the presence of increasing HgCl(2) concentrations (0.1-100 microM) for different periods of time (from 4 to 72 h). As possible biological markers of the tubular-specific toxicity of HgCl(2) in exposed-MDCK cultures we analysed: (i) critical biochemical parameters related to oxidative stress conditions and (ii) gap-junctional function (GJIC). HgCl(2) cytotoxicity was evaluated by cell-density assay. The biochemical analysis of the pro-oxidant properties of the mercuric ion (Hg(2+)) was performed by evaluating the effect of the metal salt on the antioxidant status of the MDCK cells. The cell glutathione (GSH) content and the activity of glutathione peroxidase (Gpx) and catalase (Cat), two enzymes engaged in the H(2)O(2) degradation, were quantified. HgCl(2) influence on MDCK GJIC was analysed by the microinjection/dye-transfer assay. HgCl(2)-induced morphological changes in MDCK cells were also taken into account. Our results, proving that subcytotoxic (0.1-10 microM) HgCl(2) concentrations affect either the antioxidant defences of MDCK cells or their GJIC, indicate these critical functions as suitable biological targets of early mercury-induced tubular cell injury

Enzymatic activities affecting exogenous nicotinamide adenine dinucleotide in human skin fibroblasts

The fate of nicotinamide adenine dinucleotide (NAD), AMP, and ADP-ribose supplied to intact human skin fibroblasts was monitored, and the concentrations of intra- and extracellular pyridine and purine compounds were determined by HPLC analysis. Two enzymatic activities affecting extracellular NAD were detected on the plasma membrane, one hydrolyzing the pyrophosphoric bond and yielding nicotinamide mononucleotide (nucleotide pyrophosphatase) and the other cleaving the glycoside link and releasing nicotinamide (NAD-glycohydrolase). No AMP or ADP-ribose was found in the extracellular medium of cells incubated with NAD, the former being completely catabolized to hypoxanthine and the latter degraded to adenine and hypoxanthine

Nephrotoxic metals and stress response: a comparative in vitro study

accettato come contributo oral