Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells

Abstract

CNT2 (concentrative nucleoside cotransporter) is a plasma membrane high-affinity Na + -coupled adenosine transporter, also localized in intracellular structures. This transporter protein may play additional roles other than nucleoside salvage, since it has recently been shown to be under purinergic control via K ATP channels, by a mechanism that does not seem to involve changes in its subcellular localization. In an attempt to identify the agents that promote CNT2 trafficking, bile acids were found to increase CNT2-related transport activity in a K ATP channel-independent manner in both Fao hepatoma and rat liver parenchymal cells. A maximum effect was recorded after treatment with hydrophilic anions such as TCA (taurocholate). However, this effect did not involve changes in the amount of CNT2 protein, it was instead associated with a subcellular redistribution of CNT2, resulting in an accumulation of the transporter at the plasma membrane. This was deduced from subcellular fractionation studies, biotinylation of plasma membrane proteins and subsequent CNT2 detection in streptavidin precipitates and in vivo confocal microscopic analysis of the distribution of a YFP (yellow fluorescent protein)-CNT2 construct. The induction of CNT2 translocation, triggered by TCA, was inhibited by wortmannin, dibutyryl-AMPc, PD98059 and colchicine, thus suggesting the involvement of the PI3K/ERK (phosphoinositide 3-kinase/extracellular-signal related kinase) pathway in microtubule-dependent activation of recombinant CNT2. These are novel effects of bile-acid physiology and provide the first evidence for short-term regulation of CNT2 translocation into and from the plasma membrane.

Ribavirin Uptake into Human Hepatocyte HHL5 Cells Is Enhanced by Interferon-α via up-Regulation of the Human Concentrative Nucleoside Transporter (hCNT2)

“…Short-term activation of hCNT2 after IFNα addition would be consistent with this cytokine binding to its receptor, IFNR, inducing a signaling cascade that results in ERK 1/2 phosphorylation and Rho activation, thereby promoting movement of hCNT2-containing vesicles toward the cell surface and thus requiring intact cytoskeletal structures. These considerations support the idea of a nontranscriptional and rapid effect of IFNα, as previously reported. ,− CNT2 is abundant in epithelial barriers, where its activity and localization are tightly regulated. In particular, bile acids have been shown to promote CNT2 trafficking to the plasma membrane in rat hepatocyte-derived cells, thus anticipating an additional strategy for the improvement of hCNT2-mediated drug bioavailability.…”

“…16,28−30 CNT2 is abundant in epithelial barriers, 5 where its activity and localization are tightly regulated. In particular, bile acids have been shown to promote CNT2 trafficking to the plasma membrane in rat hepatocyte-derived cells, 28 thus anticipating an additional strategy for the improvement of hCNT2-mediated drug bioavailability. CNT2 is also known to be under purinergic control, its activity being up-regulated in an energy-dependent manner in minutes in rat hepatocyte-derived cells via the activation of adenosine A1 receptors.…”

Structural determinants for rCNT2 sorting to the plasma membrane of polarized and non-polarized cells

“…A link of CNT2 with intracellular energy metabolism had been anticipated previously by demonstrating energy-dependent purinergic regulation of CNT2 [34] and CNT2-mediated adenosine intracellular signalling via AMPK [12]. Evidence for regulated trafficking of CNT2 has also been provided [11]. Although the N-terminal tail of the transporter might be relevant for many of its regulatory properties, this has not been mapped previously as a first step to understand constitutive sorting of CNT2 in nonpolarized and polarized epithelia Within the first 80 amino acids of the rCNT2 sequence, which comprise the intracellular N terminal tail, there are two highly conserved regions, N 21 PGLELME 28 and S 46 LKD.…”

“…In this regard, it has been described that bile acids can increase plasma membrane rCNT2 activity by promoting the transporter translocation from intracellular compartments to the plasma membrane [11]. CNT2 has also been described as a key player in energy metabolism, necessary for the activation of AMPK (AMP-activated protein kinase) by extracellular adenosine [12].…”

A guide to plasma membrane solute carrier proteins

“…SLC28A2 has a varied subcellular distribution, localizing to both the cell membrane and intracellular vesicles in certain cell lines. This localization has been shown to be regulated by bile acids (BA), with extracellular BA triggering translocation to the cell membrane [251].…”