Department of Internal Medicine, Pulmonary, Critical Care, Sleep and Allergy Section, 985815 Nebraska Medical Center, Omaha, NE 68198-5815, USA.
Alcohol Clin Exp Res. 2009 May;33(5):791-8. doi: 10.1111/j.1530-0277.2009.00897.x. Epub 2009 Mar 6.
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.
核苷转运体将细胞内的腺苷摄取，对细胞外腺苷浓度的调控起着重要作用。细胞外腺苷是一种重要的信号分子，通过 4 种 G 蛋白偶联受体亚型（A1、A2A、A2B 和 A3）来调节多种细胞功能。先前，我们证明了腺苷在维持气道稳态和气道修复中具有重要作用，而酒精会损害气道宿主防御。综上所述，我们假设乙醇通过核苷转运系统损害腺苷摄取。
为了研究乙醇诱导的腺苷转运变化，我们使用了人支气管上皮细胞系（BEAS-2B）。细胞在用或不用不同浓度乙醇（EtOH）孵育 10 分钟前进行预孵育。此外，一些细胞用 S-（4-硝基苄基）-6-硫代次黄嘌呤（100 μM：NBT）预处理，这是一种有效的腺苷摄取抑制剂。然后通过在不同时间间隔添加[（3）H]-腺苷来测定摄取。
当在 1 分钟时测量时，增加 EtOH 浓度会导致对腺苷摄取的抑制作用增加。用 NBT 预处理的细胞可有效阻断腺苷摄取。此外，短期 EtOH 导致细胞外腺苷浓度增加。相反，暴露于 EtOH（100 mM）24 小时的细胞中，腺苷转运变得脱敏。为了确定 EtOH 诱导的腺苷转运脱敏的机制，我们评估了 cAMP 活性对 EtOH 的反应。短期 EtOH 暴露（10 分钟）对腺苷介导的 cAMP 激活几乎没有影响，而长期 EtOH 暴露（24 小时）阻断了腺苷介导的 cAMP 激活。来自未刺激的 BEAS-2B 细胞的裂解物的 Western blot 分析检测到分别表示 hENT1 和 hENT2 的单个 55 kDa 条带。来自 BEAS-2B 的 RNA 的实时 RT-PCR 显示 ENT1 和 ENT2 的转录表达。
总的来说，这些数据表明，细胞急性暴露于 EtOH 通过核苷转运体抑制腺苷摄取，而细胞慢性暴露于 EtOH 使腺苷转运体对乙醇的这些抑制作用脱敏。此外，我们的数据表明 EtOH 抑制腺苷摄取会导致细胞外腺苷积累增加，影响上皮细胞表面的腺苷作用，从而可能改变气道稳态。
