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, 150 2 45 pM, and V,,, 414 2 17 pmol . lo6 cells-' . min-I. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (K, 3 nM) and dipyridamole (K, 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC,* value for the adenosine transport inhibition by uridine was very high (240 pM), 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,,, value of 185 % 11 pM and a V,, value of 4.2420.10 pmol . mg protein-' . s-I. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the V,,, value but the K, value was not significantly modified. Adenosine 5'-[p,y-imino]triphosphate, 1 ,W-ethenoadenosine 5'-triphosphate, and adenosine(5')-tetraphospho(5')adenosine (Ap,A) at 100 pM 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.A wide variety of mammalian cells have been reported to possess nucleoside transport systems [l]. The presence of high-affinity nucleoside transport mediated mainly by a facilitated-diffusion mechanism has been reported in neural cells and synaptosomal preparations. This system recognizes adenosine as the preferred endogenous substrate with K , values in the range 1-20 pM for this nucleoside [2-51. In neurochromaffin cells, which are a homogeneous neural cell population, the nucleoside transport mechanism has a high affinity for adenosine (K, 1 pM) [6]. This transport system was not dependent on a Na' gradient and was completely inhibited by low concentrations of nitrobenzylthioinosine (NPhCH,SIno; K, 0.01 nM), a specific inhibitor for the facilitated-diffusion systems [7, 81. The chromaffin cell model has proved to be very useful in studies of adenosine transport regulation. In recent studies, it was reported that activation of protein kinase A and protein Correspondence to M. Teresa Miras-Portugal,

Molecular biology and regulation of nucleoside and nucleobase transporter proteins in eukaryotes and prokaryotes

“…Rat fibroblasts producing a transforming protein tyrosine kinase exhibited in- creased levels of es and ei activities compared with wildtype fibroblasts (Meckling-Gill and Cass 1992). Protein kinase C activation in chromaffin cells resulted in decreased adenosine transport by reducing transporter abundance, as measured by quantitating the number of NBMPR-binding proteins in preparations of plasma membrane vesicles (Delicado et al 1994). Protein kinase A and protein phosphatases, which may be regulated by protein kinase C, have been implicated in the ethanol sensitivity of adenosine transport in neuroblastoma × glioma hybrid cells (Coe et al 1996a(Coe et al , 1996b.…”

Adenosine: An immune modulator of inflammatory bowel diseases

“…The role of the activation of purinergic receptors on adenosine uptake was also studied in vascular endothelial cells and chromaffin cells. ATP has been shown to up-regulate adenosine transport and protein expression in vascular endothelial cells and chromaffin cells [78][79][80] . However, the effect of ATP on adenosine transport in normal and inflamed intestine remains to be elucidated.…”

Nucleoside transporter and nucleotide vesicular transporter: Two examples of mnemonic regulation

“…In addition, transport studies of plasma membrane vesicle preparations of chromaffin cells that were resealed in the presence of ATP showed a significant increase in uridine transport and a parallel increase in [ 3 H]NBTI binding [Casillas et al, 1993a]. These findings suggested for the first time the existence of an intracellular ATP binding site on the nucleoside transporter [Delicado et al, 1994]. More recent studies have shown that besides allosteric modulation by intracellular effectors, such as ATP, the adenosine transporter in chromaffin cells is regulated by extracellular levels of its own substrate, adenosine.…”