Characterization of Inhibitor‐Sensitive and ‐Resistant Adenosine Transporters in Cultured Human Fetal Astrocytes

Abstract

The kinetic characteristics of [ 3H]adenosine uptake, the extent to which accumulated [3H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of KT and Vmnx, and the sensitivities with which nucleoside transport inhibitors blocked [3H]adenosine accumulations were determined in cultured human fetal astrocytes. KT and Vrnax values for accumulations of [3H]labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor e,ythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5'-iodotubercidin (ITU) were 6.2 1iM and 0.15 nmol/ mm/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components, respectively. In the presence of EHNA and ITU, where <4% of accumulated [ 3H]adenosinewas metabolized, transport per se was measured, and kinetic values for KT and V~axwere 179 1iM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [ 3H]adenosinewas rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of "concentrative" uptake in that the intracellular levels of [3H]-labeled purines (adenosine plus its metabolites) were 1.4-fold higher than in the medium. No apparent concentrative accumulations of [3H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [3H}adenosine transport; for the inhibitor-sensitive components the IC 50 values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC50 values were 2.5 1iM for NBI, 5.1 1j,M for dilazep, and 39.0 1jM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.

Nucleoside transporter expression and function in cultured mouse astrocytes

“…However, the lack of any tendency for the 3-s uptake of formycin B at low temperature to approach a plateau within the range of 1-100 lM may suggest a high K m value, which is consistent with published data for formycin B by the ENT1 and especially the ENT2 transporter (Burke et al, 1998). The presence of both ENTs is also consistent with previous uptake studies in human astrocytes by Gu et al (1996), but the distinct expression of mRNA for both ENT2 (ei) and ENT1 (es) is in contrast to C-6 glioma cells, where ENT2 accounts for >95% of the total equilibrative nucleoside transporter expression (Sinclair et al, 2001).…”

“…The presence of mRNA for each of the two ENTs is marked, consistent with the presence of diffusional uptakes for all four nucleosides, with the tendency toward a saturation of the uptake at high concentrations observed for the 3-s uptakes of adenosine and guanosine at low temperature, with the somewhat slower uptake of formycin B and of the initial phase of the thymidine uptake at 250 than at 1 lM and with the observation by Gu et al (1996) that part of the adenosine uptake in cultured human astrocytes is potently inhibited by NBTI. However, the lack of any tendency for the 3-s uptake of formycin B at low temperature to approach a plateau within the range of 1-100 lM may suggest a high K m value, which is consistent with published data for formycin B by the ENT1 and especially the ENT2 transporter (Burke et al, 1998).…”

Mechanisms of apoptosis induced by purine nucleosides in astrocytes

“…Therefore, by reducing the metabolism of adenosine released from the astrocytes, its extracellular concentration might be increased sufficiently to induce apoptosis. Cultured astrocytes metabolize extracellular adenosine to inosine by adenosine deaminase activity (ADA) (Ceballos et al, 1994;Gu et al, 1996). Therefore, we tested whether the ADA inhibitor, EHNA, would reveal a latent apoptotic effect of guanosine.…”

Purinergic Mechanisms in Epilepsy

“…In this regard, recent studies defining the conditions required to isolate and maintain adult human brain astrocytes in vitro will be important in this endeavour [35]. Of similar importance will be studies of the adenosine transporter in human astrocytes [36].…”