Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers.

Geetha Samak,Avtar S Meena,Radhakrishna Rao,Roshan G Rao,Pradeep K Shukla,Ruchika Gangwar,Damodaran Narayanan,Jonathan H Jaggar,Bhargavi Manda

doi:10.1038/srep38899

Geetha Samak, Avtar S Meena + Show 7 more

Open Access

https://doi.org/10.1038/srep38899

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Abstract

Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca2+-free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or CaV1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

Highlights

Epithelial tight junctions confer the intestinal mucosal barrier function in the intestine
We investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity and barrier function in Caco-2 cell monolayers, and delineated the role of calcium influx and mitochondrial oxidative stress in the mechanism involved in the synergistic effect of ethanol and acetaldehyde
Confocal immunofluorescence microscopy showed that ethanol produced no obvious effect on junctional distribution of occludin and ZO-1, but it caused a dramatic redistribution of occludin and ZO-1 from the intercellular junctions into intracellular compartment in ADH1B-transfected cell monolayers (Fig. 1D)

Summary

Introduction

Epithelial tight junctions confer the intestinal mucosal barrier function in the intestine. Previous studies showed that acetaldehyde is more effective than ethanol in tight junction disruption in Caco-2 cell monolayer[1]. To determine the role of oxidative stress in ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction we evaluated the effect of antioxidants.

Results

Conclusion