View Abstract

A6351 - Role of Alpha 4 Nicotinic Acetylcholine Receptors (a4 nAChR) in Regulation of Fibronectin Expression and Redox State in Lung Fibroblasts
Author Block: J. Ritzenthaler, W. H. Watson, Y. Zheng, T. J. Burke, E. Torres-González, G. Arteel, J. Roman; Medicine, University of Louisville, Louisville, KY, United States.
Rationale: Chronic ethanol abuse increases susceptibility to acute lung injury, but the mechanisms responsible for this effect remain incompletely elucidated. Previously, we demonstrated that ethanol stimulates the expression in murine lung fibroblasts of fibronectin, a matrix glycoprotein implicated in tissue disrepair. This effect appeared mediated by α4 nicotinic acetylcholine receptor (α4 nAChR) expressed on the surface of lung fibroblasts. Interestingly, lung fibroblasts cultured in media with an oxidized cysteine/cystine redox potential (Eh Cys/CySS) also produced fibronectin via stimulation of α4 nAChRs, thereby mimicking the effect of ethanol. Thus, we hypothesized that ethanol acts by inducing oxidative stress which, in turn, activates α4 nAChR-mediating signaling. Methods: To test the hypothesis, knockout C57BL/6 mice lacking α4 nAChRs (Transgenic Mouse Facility, University of California, Irvine, CA) and characterized. Primary lung fibroblasts harvested from knockout (KO) and wild type (WT) mice were used in experiments in vitro. NIH 3T3 fibroblasts in which wild type α4 nAChR was stably knocked down were used to express mutants of α4 nAChR. Fibronectin and CySS metabolizing genes were measured by real time-PCR. Intracellular and extracellular Cys, CySS, glutathione (GSH) and glutathione disulfide (GSSG) were measured by HPLC. Redox potentials (Eh) were calculated from the Nernst equation. Results: Both ethanol (60 mM) and oxidizing media (0 mV Eh Cys/CySS) stimulated fibronectin mRNA expression in WT lung fibroblasts, but not in α4 nAChR KO fibroblasts. In contrast, response to nicotine was not affected. Cells expressing mutants of α4 nAChR that lacked specific cysteine residues did not up-regulate fibronectin in response to ethanol or 0 mV redox media suggesting the involvement of redox-dependent events. Interestingly, lung fibroblasts lacking α4 nAChR also had defects in CySS metabolism and distribution as the expression of the CySS transporter Slc7a11 was low, extracellular CySS accumulated, intracellular GSH levels dropped, and lower amounts of Cys and GSH were exported in cultures of α4 nAChR KO fibroblasts. This was associated with oxidation of the extracellular Eh Cys/CySS of α4 nAChR KO fibroblasts, and total GSH concentrations in the conditioned media were lower. Conclusions: Our studies suggest that Cys residues on α4 nAChR mediate cellular responses to ethanol in primary mouse lung fibroblasts. Furthermore, these studies provide evidence for a feedback loop wherein activation of α4 nAChR by extracellular oxidation up-regulates intracellular production and export of Cys and GSH, providing a means to restore the redox state of α4 nAChR and limiting signaling.