Evidence that Adenine Nucleotides Modulate Nucleoside‐Transporter Function

Abstract

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 K_m 150 ± 45 μM, and V_max 414 ± 17 pmol · 10⁶ cells⁻¹· min⁻¹. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (K_i 3 nM) and dipyridamole (K_i 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 μM), 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 K_m value of 185 ± 11 μM and a V_max 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 V_max value but the K_m value was not significantly modified. Adenosine 5′‐[β,γ‐imino]triphosphate, 1,N⁶‐ethenoadenosine 5′‐triphosphate, and adenosine(5′)‐tetraphospho(5′)adenosine (Ap₄A) at 100 μM 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.