Abstract
BackgroundThe production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to GABA. Based on studies suggesting a role of the airway epithelial GABAergic system in asthma-related mucus overproduction, we hypothesized that cigarette smoking, another disorder associated with increased mucus production, may modulate GABAergic system-related gene expression levels in the airway epithelium.MethodsWe assessed expression of the GABAergic system in human airway epithelium obtained using bronchoscopy to sample the epithelium and microarrays to evaluate gene expression. RT-PCR was used to confirm gene expression of GABAergic system gene in large and small airway epithelium from heathy nonsmokers and healthy smokers. The differences in the GABAergic system gene was further confirmed by TaqMan, immunohistochemistry and Western analysis.ResultsThe data demonstrate there is a complete GABAergic system expressed in the large and small human airway epithelium, including glutamate decarboxylase, GABA receptors, transporters and catabolism enzymes. Interestingly, of the entire GABAergic system, smoking modified only the expression of GAD67, with marked up-regulation of GAD67 gene expression in both large (4.1-fold increase, p < 0.01) and small airway epithelium of healthy smokers (6.3-fold increase, p < 0.01). At the protein level, Western analysis confirmed the increased expression of GAD67 in airway epithelium of healthy smokers compared to healthy nonsmokers (p < 0.05). There was a significant positive correlation between GAD67 and MUC5AC gene expression in both large and small airway epithelium (p < 0.01), implying a link between GAD67 and mucin overproduction in association with smoking.ConclusionsIn the context that GAD67 is the rate limiting enzyme in GABA synthesis, the correlation of GAD67 gene expression with MUC5AC expressions suggests that the up-regulation of airway epithelium expression of GAD67 may contribute to the increase in mucus production observed in association with cigarette smoking.Trial registrationNCT00224198; NCT00224185
Highlights
The production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to gamma-aminobutyric acid (GABA)
In the context that goblet cell hyperplasia and mucin overproduction is associated with cigarette smoking [10,11,12], we hypothesized that components of the GABAergic system may be altered in the airway epithelium of cigarette smokers
In the context that cigarette smoking is associated with mucus hypersecretion, in the present study we asked the question: Does smoking alter the gene expression pattern of GABAergic system genes in the respiratory epithelium? Assessment of our database of airway epithelial gene expression generated by microarrays showed that, while many of the GABAergic system genes are expressed in the human large and small airway epithelium, cigarette smoking is associated with changes in gene expression only of GAD67, a gene controlling the synthesis of GABA [2]
Summary
The production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to GABA. In the context that goblet cell hyperplasia and mucin overproduction is associated with cigarette smoking [10,11,12], we hypothesized that components of the GABAergic system may be altered in the airway epithelium of cigarette smokers. To assess this hypothesis, we examined our microarray database of large and small airway gene expression of healthy nonsmokers and healthy smokers to determine if the GABAergic system was expressed.
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