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The effect of nitrobenzylthioinosine (NBTI) on [3H]adenosine uptake and the characterization of the [3H]NBTI binding in cell (primary cultures and LLC-PK1 cell line) plasma membrane and brush-border membrane (BBM) vesicles from pig renal cortices and LLC-PK1 cells was analyzed. [3H]adenosine uptake was strongly inhibited by NBTI in nonconfluent cells, whereas it was totally insensitive to the reagent in BBM. The concentration dependence of [3H]adenosine uptake in BBM was linear, suggesting simple diffusion. In both cell membranes and BBM high-affinity [3H]NBTI binding was observed. [3H]NBTI binding as well as NBTI-sensitive [3H]adenosine uptake was strongly reduced when cells grew to confluence. Both reduction effects were reproduced by treatment of nonconfluent cells with chlorophenyl adenosine 3',5'-cyclic monophosphate (cAMP), which indicates that the transporter is regulated by a cAMP-dependent protein kinase. To confirm this hypothesis, the binding of [3H]NBTI was analyzed in pig kidney BBM obtained in the presence of orthovanadate and alkaline phosphatase. With respect to control membranes, BBM obtained in the presence of orthovanadate showed a lower maximum number of binding sites (Bmax), whereas those obtained in the presence of alkaline phosphatase showed a slight increase in Bmax for [3H]NBTI binding. Taken together, these results suggest that the reduction in both [3H]NBTI-binding capacity and NBTI-sensitive [3H]adenosine uptake takes place by a mechanism that involves phosphorylation of the transporter molecule or of a protein that interacts with it.
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