Nitric oxide inhibits heterologous CFTR expression in polarized epithelialcells

Nitric oxide (.NO) has been implicated in a wide range of autocrine and par acrine signaling mechanisms. Herein, we assessed the role of exogenous NO i n the modulation of heterologous gene expression in polarized kidney epithe lial cells (LLC-PK1) that were stably transduced with a cDNA encoding human wildtype cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC-PK1-WT CFTR). Exposure of these cells to 125 mu M DETA NONOate at 37 degrees C for 24 h (a chemical .NO donor) diminished Zn2+-induced and uninduced CFTR pro tein levels by 43.3 +/- 5.1 and 34.4 +/-. 17.1% from their corresponding co ntrol values, respectively. These changes did not occur if red blood cells, effective scavengers of .NO, were added to the medium. Exposure to .NO did not alter lactate dehydrogenase release in the medium or the extent of apo ptosis. Coculturing LLC-PK1-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible .NO synthase cDNA gene also inhi bited CFTR protein expression in a manner that was antagonized by 1 mM N-G- monomethyl-L-arginine in the medium. Pretreatment of LLC-PK1-WTCFTR with OD Q, an inhibitor of guanylyl cyclase, did not affect the ability of .NO to i nhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effec t on heterologous CFTR expression. These data indicate that .NO impairs the heterologous expression of CFTR in epithelial cells at the protein level v ia cG-MP-independent mechanisms.