Adenine/Ribose Supply Increases Adenosine Production and Protects ATP Pool in Adenosine Kinase-inhibited Cardiac Cells

The objective of the present study was to establish the optimal combination of inhibitors of adenosine metabolism and nucleotide precursors resulting in long-term increase in endogenous adenosine concentration without adverse metabolic consequences in non-ischemic cardiomyocytes and endothelial cells. Cardiomyocytes and endothelial cells were isolated after collagenase digestion of the rat heart. Freshly isolated cardiac myocytes or cultured endothelial cells were incubated for up to 8 h with no inhibitors or substrates or with various combinations of adenosine deaminase inhibitor: 5μ erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), adenosine kinase inhibitors: 10μm5′-iodotubercidin (ITu) or 10μ 5′-aminoadenosine (AA) and nucleotide precursors: 100μ adenine, 2.5 m ribose and 5 m inorganic phosphate. Nucleotide, nucleoside and base concentrations were evaluated at the end of the incubation by HPLC in cardiomyocyte or endothelial cells extracts and in incubation media. Adenosine content in cardiomyocyte suspension was enhanced after 3 h incubation in the presence of ITu+EHNA as compared to EHNA alone (2.8±0.2v0.9±0.2 nmol/mg protein, respectively). ATP decreased from an initial value of 22.7±0.7 nmol/mg protein to 18.9±0.7 in the presence of ITu+EHNA, while ATP was maintained at 21.8±0.7 nmol/mg protein with EHNA. With adenine+ITu+EHNA, the changes were similar to those observed with ITu+EHNA. However, with ribose+adenine+ITu+EHNA, ATP increased to 25.8±1.2 nmol/mg protein and adenosine concentration was elevated to 3.9±0.3 nmol/mg protein. Similar results were observed if AA was used instead of ITu to inhibit adenosine kinase. All the changes were maintained after 8 h of incubation. Adenosine content was increased in endothelial cells incubated with ITu+EHNA to 3.1±0.4 nmol/mg protein as compared to 1.1±0.2 nmol/mg protein with EHNA alone after 3 h, while ATP decreased (18.1±1.1v22.0±1.4 nmol/mg protein with EHNA+ITu or EHNA, respectively). In the presence of adenine+ITu+EHNA, adenosine content increased after 3 h to 6.5±0.9 nmol/mg protein while ATP was elevated to 26.1±0.8 nmol/mg protein. Additional presence of ribose was without effect. No changes in adenylate energy charge were observed in cardiomyocytes or endothelium under any conditions studied. Inhibition of adenosine kinase and adenosine deaminase caused a decrease in ATP together with increased adenosine content both in endothelial cells and cardiomyocytes. However, the addition of adenine (endothelial cells) or adenine with ribose (cardiomyocytes) together with inhibitors of adenosine metabolism protected cells from ATP depletion and further increased adenosine concentration.