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DETANO and Nitrated Lipids Increase Chloride Secretion across Lung Airway Cells

Departments of ¹ Anesthesiology, ² Physiology and Biophysics, ³ Cell Biology, and ⁴ Pediatrics, ⁵ the Center for Free Radical Biology; and the ⁶ Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama

Correspondence and requests for reprints should be addressed to Sadis Matalon, Ph.D., Department of Anesthesiology, University of Alabama at Birmingham, 901 South 19th Street, BMR II Room 224, Birmingham, AL 35205-3703. E-mail: sadis{at}uab.edu

We investigated the cellular mechanisms by which nitric oxide (NO) increases chloride (Cl^–) secretion across lung epithelial cells in vitro and in vivo. Addition of (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETANONOate [DETANO];1–1,000 µM) into apical compartments of Ussing chambers containing Calu-3 cells increased short-circuit currents (I_sc) from 5.2 ± 0.8 to 15.0 ± 2.1 µA/cm² (X ± 1 SE; n = 7; P < 0.001). NO generated from two nitrated lipids (nitrolinoleic and nitrooleic acids; 1–10 µM) also increased I_sc by about 100%. Similar effects were noted across basolaterally, but not apically, permeabilized Calu-3 cells. None of these NO donors increased I_sc in Calu-3 cells pretreated with 10 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). Scavenging of NO either prevented or reversed the increase of I_sc. These data indicate that NO stimulation of soluble guanylyl cyclase was sufficient and necessary for the increase of I_sc via stimulation of the apical cystic fibrosis transmembrane regulator (CFTR). Both Calu-3 and alveolar type II (ATII) cells contained CFTR, as demonstrated by in vitro phosphorylation of immunoprecipitated CFTR by protein kinase (PK) A. PKGII (but not PKGI) phosphorylated CFTR immuniprecipitated from Calu-3 cells. Corresponding values in ATII cells were below the threshold of detection. Furthermore, DETANO, 8-Br-cGMP, or 8-(4-chlorophenylthio)-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.

Key Words: Calu-3 cells • cystic fibrosis transmembrane conductance regulator • nasal potential difference • protein kinase G type II • alveolar type II cells

CLINICAL RELEVANCE This research attempts to understand the basic mechanisms by which nitric oxide alters ion transport across lung epithelial cells. This may form the rational basis for the use of nitric oxide as a therapeutic tool in cystic fibrosis.