Transformation products of extracellular NAD+ in the rat liver: kinetics of formation and metabolic action

Abstract

The perfused rat liver responds in several ways to NAD(+) infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption and gluconeogenesis are some of the effects that were observed. Extracellular NAD(+) is also extensively transformed in the liver. The purpose of the present work was to determine the main products of extracellular NAD(+) transformation under various conditions and to investigate the possible contribution of these products for the metabolic effects of the parent compound. The experiments were done with the isolated perfused rat liver. The NAD(+) transformation was monitored by HPLC. Confirming previous findings, the single-pass transformation of 100 microM NAD(+) ranged between 75% at 1.5 min after starting infusion to 95% at 8 min. The most important products of single-pass NAD(+) transformation appearing in the outflowing perfusate were nicotinamide, ADP-ribose, uric acid, and inosine. The relative proportions of these products presented some variations with the time after initiation of NAD(+) infusion and the perfusion conditions, but ADP-ribose was always more abundant than uric acid and inosine. Cyclic ADP-ribose (cADP-ribose) as well as adenosine were not detected in the outflowing perfusate. The metabolic effects of ADP-ribose were essentially those already described for NAD(+). These effects were sensitive to suramin (P2(XY) purinergic receptor antagonist) and insensitive to 3,7-dimethyl-1-(2-propargyl)-xanthine (A2 purinergic receptor antagonist). Inosine, a known purinergic A3 agonist, was also active on metabolism, but uric acid and nicotinamide were inactive. It was concluded that the metabolic and hemodynamic effects of extracellular NAD(+) are caused mainly by interactions with purinergic receptors with a highly significant participation of its main transformation product ADP-ribose.

Transformation and action of extracellular NAD+ in perfused rat and mouse livers

“…AMP and inosine comprised very small fractions of the transformation products, but the contributions of ADP-ribose and uric acid were highly significant ( Figure 5). This is again partly similar to what was found in livers from the Wistar strain, but in those rats, ADP-ribose was considerably more abundant than uric acid, especially at the end of the infusion period [6] .…”

“…An extraction of 97% corresponds to a transformation rate of 0.31 µmol·min -1 ·g -1 . A similar, but considerably higher, recovery peak was also found in livers from rats of the Wistar strain, and it coincided with the time of maximal increase in perfusion pressure caused by NAD + [6] . Nicotinamide tended to approach 90% of the infused NAD + at the end of the infusion period ( Figure 5).…”

“…No transformation product with a retention time longer than 20 min was found, an observation that excludes free adenosine, which had a retention time of 23.64±0.07 min. Furthermore, cyclic-ADPribose (cADPR) and β-nicotinic acid adenine dinucleotide phosphate (β-NAADP) were expected to appear with retention times between 7 and 8 min [6] . This region, however, was practically empty in all chromatograms obtained in the present work (Figures 1 and 2).…”

“…Recent investigations have shown that the main products in the effluent perfusate in the liver of Wistar rats after portal infusion of NAD + are ADP-ribose, uric acid and inosine [6] . Extracellular nucleotides such as NAD + are considered to be ideally suited as extracellular signal transmitters because they can be rapidly mobilized from intracellular stores and the signal is rapidly terminated by degradation by nucleotide catabolizing enzymes.…”

“…These effects are Ca 2+ -dependent and seem to result from an interaction between parenchymal and non-parenchymal cells via eicosanoid production. Furthermore, recent evidence indicates that these metabolic effects of NAD + in the liver are partly due to its transformaTransformation and action of extracellular NAD + in perfused rat and mouse livers tion products, such as ADP-ribose, and that these effects are mediated by purinergic receptors [6] . Unlike the rat liver, little is known about the action and transformation of NAD + in the mouse liver.…”