INITIAL RATE KINETICS AND EVIDENCE FOR DUALITY OF MEDIATED TRANSPORT OF ADENOSINE, RELATED PURINE NUCLEOSIDES, AND NUCLEOSIDE ANALOGS IN L1210 CELLS

Abstract

In studies using a rapid kinetic technique, evidence was derived for multiplicity of systems mediating [3H]adenosine transport in [murine leukemia] L1210 cells. A variety of approaches were used in discriminating between transport and kinase-mediated phosphorylation. Under these conditions, 2 systems mediating influx were delineated which exhibited high-affinity [Km = 13.9 .+-. 2 (SE) .mu.M] or low-affinity [Km = 199 .+-. 27 .mu.M] for [3H]-adenosine. Both systems exhibited high capacities, but that associated with the low-affinity system .**GRAPHIC**. = 263 .+-. 43 nmol/s per g, dry wt) was 2- to 3-fold greater than that for the high-affinity system .**GRAPHIC**. = 99.6 .+-. 12 nmol s per g, dry wt). The relative difference in affinity of these 2 systems during influx was also reflected in the values for influx Ki obtained with other nucleosides and nucleoside analogs. Influx of [3H]-adenosine by each mediated system was inhibited by 6-(2-hydroxy-5-nitrobenzyl)thioguanosine, a specific transport inhibitor, and by 9-.beta.-D-arabinofuranosylpurine-6(1H)thione which is not phosphorylated in L1210 cells, in ATP L1210 cells, in cells (L1210/ara-C/MMPR) [resistant to both 1-.beta.-D-arabinofuranosylcytosine and 5-methyl-6-thioinosine] which have substantially reduced ability for [3H]adenosine phosphorylation, and in the presence of 2''-deoxycoformycin, a potent inhibitor of adenosine deaminase. The same multiplicity in mediated influx of [3H]adenosine was shown at 0.degree. when transport became rate limiting to total uptake. The high-affinity system mediating [3H]adenosine influx was also elucidated in L1210 cell plasma membrane vesicles in the presence or absence of 2-deoxycoformycin. Almost all of the natural nucleosides examined competed less effectively with [3H]adenosine for influx by the high-affinity system than by the low-affinity system. These results are discussed with respect to possible pharmacological implications.