Bile acids inhibit equilibrative adenosine transport to alter adenosine receptor signaling in cholestasis

High plasma bile acid (BA) levels in individuals with cholestasis affect adenosine (Ado) receptor (AdoR) signaling, but the underlying mechanisms are unclear. Here, we investigated BA interference with cellular Ado transport as a putative mechanism for altering extracellular Ado availability for AdoR signaling. Computational modeling and experimental studies revealed that equilibrative nucleoside transporter 2 (ENT2), but not ENT1, is capable of translocating BAs across the mammalian plasma membrane. ENT2-mediated BA transport has low affinity, is pH independent, and is partially sensitive to inhibition by nitrobenzylthioinosine (NBMPR). At cholestatic plasma concentrations of BAs, however, BAs interfere with Na⁺-independent, NBMPR-sensitive, ENTs without affecting Na⁺-driven, NBMPR-insensitive, concentrative nucleoside transporters. Interestingly, this BA interference with ENT transport was largely selective for Ado, with minimal to no impact on the transport of other purine or pyrimidine nucleosides. Xenopus oocyte-based studies demonstrated that BA inhibition of Ado transport is in the order ENT3≥ENT2>ENT1, which also corresponds to the intrinsic ability of individual ENTs to transport BAs. In silico analysis revealed that Ado and BA tend to occupy similar spaces within the ENT translocation pores and that the polar and hydrophilic pore-lining residues determine the interaction of ENTs with BAs. Furthermore, in vivo studies indicated that the accumulation of extraneously administered Ado decreases in the livers of cholestatic mice and that interference with Ado transport alters AdoR signaling. Together, these findings reveal novel ENT-dependent BA‒Ado interactions that may have implications for BA dysregulation of AdoR signaling in cholestatic liver diseases.