Published ahead of print on February 28, 2008, doi:10.1165/rcmb.2008-0005OC Am. J. Respir. Cell Mol. Biol., Volume 39, Number 2, August 2008, 150-162 A more recent version of this article appeared on August 1, 2008
Submitted on January 3, 2008 Detanonoate and Nitrated Lipids Increase Chloride Secretion Across Lung Airway CellsLan Chen1,1 Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL, United States, 2 Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL, United States; Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, USA; Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, USA, 3 Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA, 4 Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA, 5 Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA * To whom correspondence should be addressed. E-mail: sadis{at}uab.edu.
We investigated the mechanisms by which nitric oxide (NO) increases chloride (Cl-) secretion across lung epithelial cells in vitro and in vivo. Addition of DETANONOate (1-1000 µM), into apical compartments of Ussing chambers containing Calu-3 cells, increased short circuit currents (Isc) from 5.2±0.8 to 15.0 ±2.1 (µA/cm2; X ± 1 S.E; n=7; p<0.001). NO generated from two nitrated lipids [nitrolinoleic (LNO2) and nitrooleic (ONO2) acids; 1-10 µM] also increased Isc by about 100%. Similar effects were noted across basolaterally but not apically permeabilized Calu-3 cells. None of these NO donors increased Isc in Calu-3 cells pre-treated with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM an inhibitor of soluble guanylyl cyclase (sGC)). Scavenging of NO either prevented or reversed the increase of Isc. These data indicates that NO stimulation of sGC was sufficient and necessary for the increase of Isc via stimulation of the apical CFTR. Both Calu-3 and ATII cells contained CFTR as demonstrated by in vitro phosphorylation of immunoprecipitated CFTR by protein kinase A; PKA). PKGII (but not PKGI) phosphorylated CFTR immuniprecipitated from Calu-3 cells. Corresponding values in ATII cells were below the threshold of detection. Furthermore, DETANO, Br-cGMP or CPT-cGMP (up to 2 mM each) did not increase Cl- secretion across amiloride-treated ATII cells in vitro. Measurements of nasal potential differences in anesthetized mice showed that perfusion of the nares with DETANO activated glybenclamide-sensitive Cl- secretion. These findings suggest that small concentrations of NO donors may prove beneficial in stimulating Cl- secretion across airway cells without promoting alveolar edema.
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