Characterization of bidirectional adenosine transporters in rat brain synaptosomes and clonal DDT|1 MF-2 smooth muscle cells using [3H]L-adenosine

Abstract

Membrane-bound nucleoside transport processes and their inhibitors regulate intra- and extra-cellular levels of endogenous D-adenosine and the actions of adenosine are mediated through adenosine receptors. Our research involved (1) developing a method to measure adenosine transport and release in animal cells and CNS tissue, (2) characterizing the transport processes involved, and (3) determining the effects of transport inhibitors on these processes. Using rat brain synaptosomal preparations we showed that ($\sp3$H) L-adenosine, the metabolically stable enantiomer of ($\sp3$H) D-adenosine, was released in a concentration- and time-dependent, and temperature-sensitive manner that was linear with synaptosomal protein content. Release of ($\sp3$H) L-adenosine was inhibited by the nucleoside transport inhibitors dipyridamole, nitrobenzylthioinosine (NBI) and dilazep, as well as unlabelled L-adenosine, D-adenosine and uridine, suggesting that release occurred at least in part by NBI-sensitive equilibrative nucleoside transporters. To determine further which subtype of equilibrative nucleoside transporter was involved, we conducted similar studies using cells, DDT$\sb1$ MF-2 cells, that contain only NBI-sensitive (es) nucleoside transporters. In DDT$\sb1$ MF-2 cells, accumulation of ($\sp3$H) D-adenosine was more rapid than that of ($\sp3$H) L-adenosine. ($\sp3$H) D-Adenosine uptake was inhibited by unlabelled L-adenosine to a greater degree than ($\sp3$H) L-adenosine uptake was inhibited by unlabelled D-adenosine. Nitrobenzylthioinosine inhibited uptake of ($\sp3$H) D- and ($\sp3$H) L-adenosine. ($\sp3$H) L-Adenosine release was temperature- and time-dependent, and was inhibited by NBI. Our studies support the use of ($\sp3$H) L-adenosine as a useful probe for the characterization adenosine transport and release, and show that release occurs in part by nucleoside transporters that are sensitive to inhibition by nanomolar concentrations of NBI. Such studies may help identify therapeutically useful adenosine transport inhibitors.