4-Pyridone-3-carboxamide-1β-D-ribonucleoside metabolism in endothelial cells and its impact on cellular energetic balance.

4-Pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) is a naturally occurring compound related to nicotinamide that could be metabolized to mono-, di-, and triphosphates of 4PYR (4PYMP, 4PYDP, 4PYTP) and nicotinamide adenine dinucleotide (NAD) analogue (4PYRAD) in all types of cells. Previous studies demonstrated that formation of 4PYMP and 4PYTP was dependent on adenosine kinase activity. Pathway of 4PYRAD production is not yet identified, but most likely this process involves production of 4PYMP. This study aimed to evaluate influence of 4PYR on metabolism of endothelial cells and to test effect of nucleoside transport inhibitors. 4PYR was obtained by chemical synthesis. Endothelial cell line (HMEC-1) was incubated for 24 or 48 hours with 100 μM 4PYR. After incubation, cells were separated from medium and analyzed for concentrations of ATP, NAD, and 4PYR metabolites by using reversed-phase high performance liquid chromatography. We demonstrated progressive accumulation of 4PYR metabolites in endothelium that reached 33.2±0.8 nmol/mg protein for 4PYMP and 5.25±0.17 nmol/mg protein for 4PYRAD after 48-hour incubation with 4PYR. Dipyridamole protected from accumulation of 4PYR metabolites in endothelial cells. We conclude that endothelium is capable to convert 4PYR into intracellular metabolites and this causes disruption of cell energy balance. Nucleoside transport inhibition with dipyridamole could protect endothelium from this effect. This finding could be of clinical relevance in conditions associated with accumulation of 4PYR such as chronic renal disease.