Purpose: Recently, Tsukamoto et al. have demonstrated that COA-Cl (6-amino-2-chloro-9-[trans-trans-2,3-bis(hydroxymethyl)cyclobutyl]purine) synthesized as a novel adenosine analogue elicits potent angiogenic and neuroprotective effects, which mimics vascular endothelial growth factor (VEGF) as well as nerve growth factor (NGF). Thus, COA-Cl is one candidate of promising preventive and therapeutic medicines after ischemic stroke. However, it has yet to be elucidated how COA-Cl penetrates across the membrane. The purpose of the present study was to elucidate whether a concentrative nucleoside transporter (CNT) can recognize and transport COA-Cl, which may determine the pharmacokinetic profile of COA-Cl.
Methods: The human CNT2 (SLC28A2) and CNT3 (SLC28A3) were expressed heterologously in Xenopus oocytes by capped cRNA injections. Oocytes were injected with 50 ng CNT cRNA in a 50 nl volume and incubated for 3 – 6 days. The oocytes were used for transport studies 3 - 6 days after cRNA injection. The nucleoside transport activity via CNTs was determined by the uptake of [3H]-adenosine or [3H]-uridine by the oocytes and by the substrate-induced current due to the transport via CNTs by electrophysiological studies with two-electrode voltage clamp technique.
Results: We determined the uptake of uridine and adenosine by hCNT2 and hCNT3 heterologously expressed in Xenopus oocytes, of which both can transport purine and pyrimidine nucleosides. In cRNA injected oocytes, the uptakes of 1 μM uridine and adenosine by cRNA injected oocytes (hCNT2 and hCNT3) were enhanced more than 50-fold, compared to control, indicating that hCNTs were expressed robustly in Xenopus oocytes. These enhanced uptakes showed Na+-dependency as the uptake was reduced substantially when measured in the absence of Na+. The transport activities of adenosine and uridine via hCNT2 and hCNT3 were inhibited potently by 1 mM various nucleosides. By contrast, the uptake activities via hCNT3 were inhibited only by 1 mM COA-Cl, whereas the uptake activities via hCNT2 remained unchanged irrespective of 10 mM COA-Cl, suggesting that the only hCNT3 might recognize and transport COA-Cl. The IC50 value for uridine transport activity via hCNTs by COA-Cl was estimated to be approximately 5 mM. Furthermore, we assessed the ability of hCNTs to transport COA-Cl by monitoring an inward current due to its transport via CNTs in the oocyte, because CNTs are electrogenic Na+/nucleoside cotransporters. Superfusion of hCNTs-expressing oocytes with 200 μM various nucleosides elicited substantially inwards currents via both CNTs. One mM COA-Cl also induced substantially inward currents in hCNT3-expressing oocytes, which were abolished completely by Na+ removal in the perfusate. Such currents were not detectable in water-injected oocytes. By contrast, hCNT2-expressing oocytes did not show any inward current induced by COA-Cl. In the light of these findings, hCNT3 could exhibit a relatively broad substrate recognition and transport COA-Cl in a concentrative manner.
Conclusion: We here demonstrated that hCNT3 can transport a nucleoside analogue, COA-Cl in a Na+-coupled electrogenic manner. The ability of this transporter to exhibit a relative broad recognition raises the possibility that it can be useful to deliver COA-Cl to a tissue. Since hCNT3 is expressed not only in the intestinal tract and kidney, but also in various tissues such as the lung, pancreas and skin, hCNT3 might have the potential to govern the bioavailability, pharmacokinetics, and pharmacodynamics of COA-Cl, which might be useful therapeutically in the prevention and/or treatment of ischemia, arteriosclerosis and multiple sclerosis.
Masayuki Masuda– Toho University, Funabashi, Chiba
Kazuaki Sugio– Toho University, Funabashi, Chiba
Shotaro Sasaki– Lecturer, Toho University, Funabashi, Chiba
Kazumi Shimono– Professor, Sojo University, Kumamoto, Kumamoto
Ikuko Tsukamoto– Professor, Kagawa University, Kagawa