Effects of iodotubercidin on adenosine kinase activity and nucleoside transport in DDT ₁ MF-2 smooth muscle cells

Iodotubercidin is an adenosine kinase inhibitor that through its ability to increase levels of endogenous adenosine can enhance adenosine's receptor- mediated effects. We investigated whether iodotubercidin can inhibit [ ³H]adenosine accumulation by inhibiting transport processes in addition to inhibition of intracellular trapping of labeled adenine nucleotides. Under conditions in which extensive metabolism of intracellular adenosine was present, [ ³H]adenosine accumulation by DDT ₁ MF-2 cells was almost completely inhibited by iodotubercidin and the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine or by the nucleoside transport inhibitor nitrobenzyl-thioinosine. By using similar conditions, [ ³H]adenosine accumulation was significantly greater in Na ⁺ buffer than in buffer containing N-methyl-D-glucamine in place of Na ⁺; however, this effect may be explained by an observed 40% inhibition of adenosine kinase activity by N-methyl-D-glucamine. By using uptake intervals of 14 sec to represent the transport component of uptake, iodotubercidin decreased the affinity for adenosine, by about 3-fold, but had no effect on maximum velocity of transport. That these effects of iodotubercidin were due to direct interactions with nucleoside transporters was supported by findings that iodotubercidin inhibited [ ³H]nitrobenzylthioinosine binding to nucleoside transporters with a K(i) value of 4 μM and inhibited [ ³H]uridine and [ ³H]formycin B uptake with IC ₅₀ values of 7 and 15 μM, respectively. These data suggest that iodotubercidin, at pharmacologically relevant concentrations, inhibits nucleoside transport independently of its well characterized inhibition of adenosine kinase and that N-methyl-D-glucamine must be used with caution in experiments to determine the possible presence of Na ⁺ gradient-dependent concentrative nucleoside transporters.