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Abstract
Adenosine has been reported to be transported by equilibrative nucleoside transporter 4 (ENT4), encoded by the SLC29A4 gene, in an acidic pH-dependent manner. This makes hENT4 of interest as a therapeutic target in acidic pathologies where adenosine is protective (e.g. vascular ischaemia). We examined the pH-sensitivity of nucleoside influx and efflux by hENT4 using a recombinant transfection model that lacks the confounding influences of other nucleoside transporters (PK15-NTD). We established that [3H]2-chloroadenosine, which is resistant to metabolism by adenosine deaminase, is a substrate for hENT4. Transport of [3H]2-chloroadenosine at a pH of 6.0 in PK15-NTD cells stably transfected with SLC29A4 was biphasic, with a low capacity (Vmax ~ 30 pmol/mg/min) high-affinity component (Km ~ 50 µM) apparent at low substrate concentrations, which shifted to a high capacity (Vmax ~ 500 pmol/mg/min) low affinity system (Km > 600 µM) displaying positive cooperativity at concentrations above 200 µM. Only the low affinity component was observed at a neutral pH of 7.5 (Km ~ 2 mM). Efflux of [3H]2-chloroadenosine from these cells was also enhanced by more than 4-fold at an acidic pH. Enhanced influx and efflux of nucleosides by hENT4 under acidic conditions supports its potential as a therapeutic target in pathologies such as ischaemia-reperfusion injury.
Highlights
Www.nature.com/scientificreports cardiomyocytes and vascular endothelial cells[1,3], and its activity is increased under acidic conditions, inhibition of hENT4 may selectively enhance the ability of adenosine to protect the acidic vasculature in ischaemia as well as prevent the loss of adenosine from the cells during early reperfusion
The uptake of 30 μM adenosine by the PK15-hENT4 cells was significantly greater at pH 6.0 versus pH 7.5, whereas no significant difference with pH was seen for adenosine uptake by the un-transfected PK15-NTD cells (Fig. 2a)
Our results show that equilibrative nucleoside transporter 4 (ENT4) mediates both the influx and efflux of 2-chloroadenosine, confirming that hENT4 is a bidirectional transporter for nucleosides like other members of the ENT family[35,36,37,38]
Summary
Www.nature.com/scientificreports cardiomyocytes and vascular endothelial cells[1,3], and its activity is increased under acidic conditions, inhibition of hENT4 may selectively enhance the ability of adenosine to protect the acidic vasculature in ischaemia as well as prevent the loss of adenosine from the cells during early reperfusion. While adenosine is the physiological substrate, it is rapidly metabolised giving it an exceptionally short half-life[19] This makes it difficult to differentiate between changes in intracellular metabolism and membrane transport kinetics as contributors to changes in the cellular accumulation of adenosine. This rapid intracellular conversion of adenosine to membrane impermeable adenine nucleotides makes adenosine unsuitable for assessing the efflux kinetics of hENT4 These prior studies were done using hENT4-transfected Xenopus oocytes[3] or MDCK cells[18]. NBMPR blocks hENT4, albeit at concentrations considerably higher than those required for inhibition of ENT118 To address these confounding issues, we assessed whether [3H]2-chloroadenosine, a poorly metabolized adenosine analogue[20], is a substrate of hENT4 when expressed in the PK15-NTD cell line (a nucleoside transporter deficient variant of the swine kidney tubular epithelial cell line PK15). Both the influx and efflux of 2-chloroadenosine by hENT4 are enhanced in an acidic environment
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