Nucleoside transport in guinea pig myocytes: Comparison of the affinities and transport velocities for adenosine and 2-chloroadenosine

Abstract

The affinities of adenosine and 2-chloroadenosine for the nucleoside transport system of guinea pig myocytes were evaluated indirectly by studying the inhibition of the binding of [ 3H]nitro-benzylthioinosine and directly by measuring the influx of [ 3H]radiolabeled substrates. Maximal transport velocities of the two nucleosides were also obtained. [ 3H]Nitrobenzylthioinosine bound to a single class of high-affinity sites ( K D of 0.8 nM) which possessed a maximal binding capacity ( B max) of 870,000 sites/cell. Adenosine, 2-chloroadenosine or the nucleoside transport inhibitor, dipyridamole, competitively inhibited the site-specific binding of [ 3H]nitrobenzylthioinosine with K i values of 318 μM, 22 μM and 75 nM respectively. Both [ 3H]adenosine and [ 3H]2-chloroadenosine entered myocytes in a saturable and inhibitible manner. Observed transport kinetic constants ( K m and V max) were 146 μM and 24.2 pmoles/10 6 cells/sec, respectively, for adenosine and 36 μM and 11.7 pmoles/10 6 cells/sec, respectively for 2-chloroadenosine. Affinities of adenosine, 2-chloroadenosine, nitrobenzylthioinosine and dipyridamole for the nucleoside transport system derived from binding and influx methodologies were equivalent which confirms that [ 3H]nitrobenzylthioinosine binding sites are closely associated with the nucleoside transporter.