Date of Award


Degree Type


Degree Name

Doctor of Philosophy


In rabbit and guinea pig cortical synaptosomes, the nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole were used to test the hypotheses that the nitrobenzylthioinosine-sensitive and -resistant ({dollar}\sp3{dollar}H) dipyridamole binding sites are associated with the es and ei nucleoside transporters, respectively. In addition, the hypothesis that the es and ei nucleoside transporters in rabbit cortical synaptosomes differ in their selectivity for compounds besides nitrobenzylthioinosine was tested, Nitrobenzylthioinosine-sensitive ({dollar}\sp3{dollar}H) dipyridamole binding and ({dollar}\sp3{dollar}H) nitrobenzylthioinosine binding involved the same site on the es transporter. The relative proportions and inhibitor sensitivities of nitrobenzylthioinosine-resistant ({dollar}\sp3{dollar}H) dipyridamole binding could not be correlated with nitrobenzylthioinosine-resistant nucleoside transport. This, and other data, suggested that the nitrobenzylthioinosine-resistant ({dollar}\sp3{dollar}H) dipyridamole binding site(s) involved membrane components distinct from those associated with functional, ei nucleoside transporters. In addition, none of the substrates examined in the rabbit were selective for one transporter subtype over the other.;R75231 is a newly developed mioflazine derivative which has extremely tight binding characteristics in vitro and in vivo. The hypothesis that R75231 bound to the es nucleoside transporter in an irreversible manner was tested. In rabbit synaptosomes, R75231 was shown to bind extremely tightly and to be a "mixed" inhibitor of ({dollar}\sp3{dollar}H) nitrobenzylthioinosine binding. Binding of ({dollar}\sp3{dollar}H) R75231 to human erythrocyte ghost membranes was reversible, but the rate of dissociation depended upon the displacer used. R75231 and mioflazine slowed the rate of dissociation of ({dollar}\sp3{dollar}H) R75231, and caused an initial increase of site-bound ({dollar}\sp3{dollar}H) R75231. These and other results, indicate that R75231 binding to the nucleoside transporter is a reversible, complex reaction involving multiple interacting sites exhibiting positive cooperativity.;The hypothesis that the nucleoside transporter characteristics changed upon the differentiation of LA-N-2 cells was tested. Undifferentiated cells accumulated ({dollar}\sp3{dollar}H) formycin B by the es nucleoside transport system exclusively. Cell differentiation, induced by growth in serum-free medium, increased the initial rate of ({dollar}\sp3{dollar}H) formycin B transport 25%. However, there was no concomitant change in the number of ({dollar}\sp3{dollar}H) NBMPR binding sites. The enhanced uptake in the differentiated cells appeared to be due to an increased expression of ei nucleoside transporters.



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