We recently reported the identification of hitherto unknown nucleotide that is present under physiological conditions in normal human erythrocytes: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PYTP). We reported massive accumulation of 4PYTP in the erythrocytes of patients with chronic renal failure together with 50-fold increase in plasma concentration of its nucleoside precursor: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR). Present report evaluates metabolism of this nucleotide and its precursor in erythrocytes, endothelium and cardiomyocytes and study its effect on cellular ATP concentration and cell function.

Human erythrocytes were isolated from peripheral blood, HMEC-1 cell line was used to study human endothelial metabolism. Cardiomyocytes were isolated from rat hearts using collagenase perfusion technique. Cells were incubated with chemically synthesized precursor of 4PYTP: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) followed by extraction and metabolic analysis with HPLC as well as cardiomyocyte action potential analysis by current clamp.

We demonstrated formation of 4PYTP in intact human erythrocytes during incubation with 4PYR. Concentration of 4PYTP increased in the erythrocytes from 16.1± 0.6 µM to 41.5±6.0, 74.9±9.2, 114.2±21.7 with 0.1, 0.3 and 1 mM 4PYR concentration, respectively after 6 h of incubation. We noted however, preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP. Concentration of 4PYMP increased in the erythrocytes from below 5 µM to 76.9±7.1, 254.7±13.9 and 674.3±34.3 µM after 6h incubation with 0.1, 0.3 and 1 mM 4PYR. 4PYMP progressively accumulated in the cultured endothelial cells during incubation with 4PYR up to 72 h. However, no 4PYTP formation was noted. Cardiomyocytes were also shown to accumulate 4PYMP but not 4PYTP. In all cell types formation of 4PYR derivatives was accompanied by decrease in cellular ATP concentration that in cardiomyocytes was accompanied by prolongation of action potential duration.

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