Evidence that adenine nucleotides modulate nucleoside-transporter function. Characterization of uridine transport in chromaffin cells and plasma membrane vesicles.
Uridine transport was investigated in cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue. In intact cells, the kinetic parameters for uridine uptake were Km 150 +/- 45 microM, and Vmax 414 +/- 17 pmol . 10(6) cells-1 . min-1. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (Ki 3 nM) and dipyridamole (Ki 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC50 value for the adenosine transport inhibition by uridine was very high (240 microM), agreeing with the relative affinities of these nucleosides in the chromaffin cell nucleoside transport system, which was 150-fold higher for adenosine than for uridine. Uridine was significantly metabolized in chromaffin cells but not in plasma membrane vesicles. The affinity of uridine transport measured in these membrane vesicles was reproducible and similar to the affinity found for intact cells with a Km value of 185 +/- 11 microM and a Vmax value of 4.24 +/- 0.10 pmol . mg protein-1 . s-1. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the Vmax value but the Km value was not significantly modified. Adenosine 5'-[beta,gamma-imino]triphosphate, 1,N6-ethenoadenosine 5'-triphosphate, and adenosine(5')-tetraphospho(5')adenosine(Ap4A) at 100 microM were able to mimic the ATP effect. These results agree with a regulatory role of ATP, and the uridine transport on chromaffin plasma membrane vesicles is a good model for analyzing the nucleoside-transporter function and regulation.
Delicado
E G
EG
Department of Biochemistry, Universidad Complutense, Madrid, Spain.
Casillas
T
T
Sen
R P
RP
Miras-Portugal
M T
MT
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000313:Adrenal Medulla / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004176:Dipyridamole / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D033703:Nucleoside Transport Proteins; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism*
D000227:Adenine Nucleotides; D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D013868:Thioinosine; D004176:Dipyridamole; C001789:4-nitrobenzylthioinosine; D014529:Uridine