Regulation of nitrobenzylthioninosine-sensitive adenosine uptake by cultured kidney cells.
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.
Departament de Bioquímica i Fisiologia, Universitat de Barcelona, Spain.
J
J
Sayós
J
J
Blanco
F
F
Ciruela
E I
EI
Canela
J
J
Mallol
C
C
Lluis
R
R
Franco
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D007668:Kidney; D007672:Kidney Cortex / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D013552:Swine; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014316:Tritium
D000345:Affinity Labels; D014316:Tritium; D003571:Cytochalasin B; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D005947:Glucose; D000241:Adenosine