Ethanol blocks adenosine uptake via inhibiting the nucleoside transport system in bronchial epithelial cells

Adenosine uptake into cells by nucleoside transporters plays a significant role in governing extracellular adenosine concentration. Extracellular adenosine is an important signaling molecule that modulates many cellular functions via 4 G-protein-coupled receptor subtypes (A(1), A(2A), A(2B), and A(3)). Previously, we demonstrated that adenosine is critical in maintaining airway homeostasis and airway repair and that airway host defenses are impaired by alcohol. Taken together, we hypothesized that ethanol impairs adenosine uptake via the nucleoside transport system.

To examine ethanol-induced alteration on adenosine transport, we used a human bronchial epithelial cell line (BEAS-2B). Cells were preincubated for 10 minutes in the presence and absence of varying concentrations of ethanol (EtOH). In addition, some cells were pretreated with S-(4-Nitrobenzyl)-6-thioinosine (100 microM: NBT), a potent adenosine uptake inhibitor. Uptake was then determined by addition of [(3)H]-adenosine at various time intervals.

Increasing EtOH concentrations resulted in increasing inhibition of adenosine uptake when measured at 1 minute. Cells pretreated with NBT effectively blocked adenosine uptake. In addition, short-term EtOH revealed increased extracellular adenosine concentration. Conversely, adenosine transport became desensitized in cells exposed to EtOH (100 mM) for 24 hours. To determine the mechanism of EtOH-induced desensitization of adenosine transport, cAMP activity was assessed in response to EtOH. Short-term EtOH exposure (10 minutes) had little or no effect on adenosine-mediated cAMP activation, whereas long-term EtOH exposure (24 hours) blocked adenosine-mediated cAMP activation. Western blot analysis of lysates from unstimulated BEAS-2B cells detected a single 55 kDa band indicating the presence of hENT1 and hENT2, respectively. Real-time RT-PCR of RNA from BEAS-2B revealed transcriptional expression of ENT1 and ENT2.

Collectively, these data reveal that acute exposure of cells to EtOH inhibits adenosine uptake via a nucleoside transporter, and chronic exposure of cells to EtOH desensitizes the adenosine transporter to these inhibitory effects of ethanol. Furthermore, our data suggest that inhibition of adenosine uptake by EtOH leads to an increased extracellular adenosine accumulation, influencing the effect of adenosine at the epithelial cell surface, which may alter airway homeostasis.