ENT-02

Adenosine plays a significant role in various physiological processes including cardioprotection. Nucleoside transporters modulate adenosine levels in the vicinity of adenosine receptors, which in turn modulate adenosine functional efficacy. In the current study, adenosine transport in the rat heart myoblast cell line H9c2 was characterized. Kinetic analysis of adenosine transport in H9c2 cells revealed a Km of 8.9+/-0.001 microM and a Vmax of 32.1+/-0.65 pmol/mg protein/min. Adenosine transport in H9c2 cells was Na+-independent. About 6% of the total adenosine uptake was sensitive to nitrobenzylmercaptopurine riboside (NBMPR); however, 94% was insensitive, suggesting that adenosine uptake by H9c2 cells was predominantly mediated by the equilibrative nucleoside transporter (ENT)-2 and only mildly by ENT-1. Results of RT-PCR demonstrated the presence of mRNA for ENT-1, ENT-2 and ENT-3. Upon culture in a cell differentiation medium containing fetal bovine serum (1%) and retinoic acid (10 nM), both the activity and mRNA expression of ENT-1 increased 3-fold, however, ENT-2 was unaffected. Pharmacological studies revealed that ENT-1 activity was stimulated by PKA and PKC-delta/epsilon, however, ENT-2 activity was unaffected. Taken together, the exceptionally high expression level of ENT-2 in H9c2 cells raises questions regarding the use of H9c2 cells as a model for physiological adenosine activity in the heart. Furthermore, this study may form the basis for further investigation into the effect of cell differentiation and protein kinases on the regulation of nucleoside transporters.