AJP - Renal Physiology, Vol 267, Issue 5 758-F766, Copyright © 1994 by American Physiological Society
Regulation of nitrobenzylthioninosine-sensitive adenosine uptake by cultured kidney cells
J. Sayos, J. Blanco, F. Ciruela, E. I. Canela, J. Mallol, C. Lluis and R. Franco
Departament de Bioquimica i Fisiologia, Universitat de Barcelona, Spain.
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.
