4-pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) is a derivative of nicotinamide found physiologically in human body fluids that can be metabolized to mono-, di- or triphosphate derivatives (4PYMP, 4PYDP and 4PYTP respectively) and an analogue of NAD - the 1-β-D-ribonucleoside-4-pyridone-3-carboxamide adenine dinucleotide (4PYRAD) in human cells. The European Uremic Toxin Work Group (EUTox) has classified 4PYR as a uremic toxin that adversely affects endothelium. This study aimed to investigate the metabolism of 4PYR in murine brain microvascular endothelial cells (mBMECs). Incubation of mBMECs with 4PYR was carried out for 0, 24, 48 or 72 h. After incubation, a medium was removed and cellular concentrations of ATP, ADP, NAD, 4PYMP and 4PYRAD were analyzed using reversed-phase HPLC. 4PYR was metabolized by mBMECs to 4PYMP and 4PYRAD that reached concentrations of 2 ± 0.7 and 0.6 ± 0.2 nmol/mg protein (mean ± SEM), respectively, after 72 h incubation. However, unlike with endothelial cells studied so far this process has no effect on energy balance in the cell as indicated by maintained ATP/ADP ratio and adenine and nicotinamide intracellular pools. Further studies are required to explain whether the difference in 4PYR metabolism is related to differences between species or organs.
Keywords: 4-pyridone-3-carboxamide-1β-d-ribonucleoside (4PYR); endothelial cells; isolation method; mBMECs; primary culture.