Suppression of Adenosine-Activated Chloride Transport by Ethanol in Airway Epithelia

Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM) for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (ISC) in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A2B adenosine receptor (A2BAR), largely abolished the adenosine-stimulated chloride transport, suggesting that A2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections

Adenosine-stimulated short circuit current is chloride conductance.

<p><b>A</b>) I_SC of Calu-3 epithelia was measured by using the asymmetric chloride buffers. After stimulated with 100 µM of apical adenosine, the cells gave rise to I_SC of ∼75 µA/cm². The I_SC was blocked by ∼58% by basolateral addition of bumetanide (100 µM), but not by acetazolamide (20 µM) or DNDS (100 µM). <b>B</b>) I_SC of Calu-3 epithelia was measured with either asymmetric chloride buffers or symmetric chloride buffers. The adenosine-stimulated I_SC with no chloride-gradient buffers was ∼92% lower than that with chloride-gradient buffers. Significance of the difference was determined by Student's t-test (<i>p</i><0.01, n = 5).</p

Effect of ethanol pre-exposure on adenosine-induced epithelial ion transport.

<p>Calu-3 cells were cultured at an air-liquid interface on membrane filters and basolaterally exposed to 0, 25, 50 and 100 mM of ethanol for 24 hours. These Calu-3 epithelia were placed in an Ussing chamber with asymmetrical buffers of chloride (apical 10.4 mM and basolateral 139.8 mM). Following voltage clamp, stable short circuit baselines were attained in 15 to 20 min. I_SC was measured after blocking Na⁺ channels with 100 µM of apical amiloride and stimulated with 100 µM of apical adenosine. Alterations in ion transport are expressed as difference in I_SC from their baseline. Ethanol pre-exposure decreased 100 µM adenosine mediated epithelial ion transport in a dose-dependent manner. Asterisks indicate significant differences between groups by One-way ANOVA test (<i>p</i><0.05, n = 5 for each condition).</p

Adenosine-stimulated transepithelial I_SC was mediated by CFTR channel.

<p>Calu-3 epithelia were stimulated with 100 µM of apical adenosine, resulting in a significant increase in I_SC above baseline. Such an adenosine-induced I_SC was mostly absent in CFBE41o cells in which CFTR channel was dysfunctional or was significantly inhibited with 25 µM of CFTR inhibitor CFTR_inh172. Student's t-test was performed to determine the statistic significance (<i>p</i><0.05, n = 5).</p

Adenosine stimulates chloride conductance through the A_2B adenosine receptor (A_2BAR).

<p>The air-liquid interface cultures of Calu-3 cells were stimulated with 100 µM of apical adenosine, followed by apical addition of 50 µM of alloxazine, a specific inhibitor for A_2BAR. The adenosine-stimulated I_SC was decreased by ∼75% (n = 6).</p

Phosphodiesterase inhibitors restore the ethanol suppression of adenosine-induced transepithelial chloride conductance.

<p>The air-liquid interface cultures of Calu-3 cells were exposed to 0 and 100 mM of ethanol for 24 hours. Adenosine-stimulated I_SC was measured, showing that ethanol-exposed Calu-3 epithelia had significantly lower adenosine-stimulated I_SC than the no ethanol control. However, phosphodiesterase inhibitors, IBMX (100 µM) or papaverine (50 µM), fully restored the ethanol-suppressed adenosine-stimulated I_SC. Asterisks indicate statistically significant differences between groups (p<0.05, n = 4 for each group).</p

Effect of ethanol exposure on cellular cAMP level and adenosine analog antagonizes ethanol suppression of adenosine-induced chloride secretion.

<p><b>A</b>) Calu-3 epithelia, exposed to 0 and 100 mM of ethanol for 24 hours, were apically stimulated with 100 µM of adenosine for 10 min. Cells were lysed and cAMP levels were estimated by immunoassay. Ethanol pre-exposure significantly decreased the adenosine-induced cellular cAMP level to ∼64% of the no ethanol control (<i>p</i><0.05, n = 5). <b>B</b>) In Calu-3 epithelia, ethanol pre-exposure decreased the adenosine-stimulated I_SC. Such an inhibitory effect of ethanol on adenosine-induced I_SC was antagonized by Sp-cAMPS, a phoshodiesterase-resistant cAMP analog (<i>p</i><0.05, n = 6).</p