Abstract
Polarized distribution of chloride channels on the plasma membrane of epithelial cells is required for fluid transport across the epithelium of fluid-transporting organs. Ionotropic gamma-aminobutyric acid receptors are primary ligand-gated chloride channels that mediate inhibitory neurotransmission. Traditionally, these receptors are not considered to be contributors to fluid transport. Here, we report a novel function of gamma-aminobutyric acid receptors involving alveolar fluid homeostasis in adult lungs. We demonstrated the expression of functional ionotropic gamma-aminobutyric acid receptors on the apical plasma membrane of alveolar epithelial type II cells. gamma-Aminobutyric acid significantly increased chloride efflux in the isolated type II cells and inhibited apical to basolateral chloride transport on type II cell monolayers. Reduction of the gamma-aminobutyric acid receptor pi subunit using RNA interference abolished the gamma-aminobutyric acid-mediated chloride transport. In intact rat lungs, gamma-aminobutyric acid inhibited both basal and beta agonist-stimulated alveolar fluid clearance. Thus, we provide molecular and pharmacological evidence that ionotropic gamma-aminobutyric acid receptors contribute to fluid transport in the lung via luminal secretion of chloride. This finding may have the potential to develop clinical approaches for pulmonary diseases involving abnormal fluid dynamics.
Highlights
HL-083188, R01 HL-052146, and R01 HL-071628 and March of Dimes Grant 6FY05-76
We showed the expression of functional ionotropic GABA receptors on the apical plasma membrane of alveolar epithelial type II cells
We first carried out a systematic survey of the mRNA expression of 19 subunits of the ionotropic GABA receptors (␣1– 6, 1–3, ␥1–3, ␦, , ⑀, , and 1–3) in alveolar epithelial cells by using quantitative real-time PCR
Summary
Cell Isolation and Culture—Alveolar type II cells were isolated from adult rat lungs (ϳ200 g) as previously described and the purity was ϳ85–90% [12]. After overnight culture at 37 °C in a humidified 95% air and 5% CO2 incubator, non-adherent cells were removed and fresh medium was added only to the outside of the inserts. The cultures were continued for 3 more days and the resulting cells were used for immunostaining, RNA isolation, Western blot, and ClϪ transport study. Membrane Protein Biotinylation—To assess the polarity of the GABAA receptors on type II cell membranes, the biotinylation of cell membrane proteins was performed as previously described [17]. The supernatant containing the biotinylated proteins were analyzed by Western blot using anti-GABAA receptor subunit antibodies. To determine the silencing effects by siRNA, type II cells cultured on the inserts were double-labeled with goat anti-GABAA receptor subunit and anti-LB-180 antibodies, followed by incubation with Alexa 568-conjugated
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