CYCLIC ADENOSINE MONOPHOSPHATE-MEDIATED RELEASE OF NITRIC-OXIDE FROM CANINE CULTURED TRACHEAL EPITHELIUM

Nitric oxide (NO) may play a part in pulmonary vascular regulation and bronchomotor control and has been detected in exhaled air. We report the release of NO from airway epithelial cells and its regulation by c yclic adenosine monophosphate (cAMP). To directly measure NO release, a highly specific amperometric sensor for NO made of Pt/Ir alloy coate d with a three-layered membrane consisting of KCl, NO-selective resin, and normal silicon resin was developed. Immersion of this sensor in t he medium containing canine cultured tracheal epithelium detected base line levels of NO at 9.6 +/- 1.6 nM (mean +/- SE), which was reduced b y N-G-nitro-L-arginine methylester (L-NAME) but not by D-NAME. This in hibition was reversed by L-arginine. Addition of isoproterenol, 3-isob utyl-1-methylxanthine, a nd forskolin caused a rapid increase in NO, a n effect that was not altered by Ca2+-free medium in the presence of t he intracellular Ca2+ chelator BAPTA-AM and the calmodulin antagonist W-7. Bradykinin, ionomycin, and ATP were without effect on NO release. The forskolin-induced NO release was accompanied by intracellular acc umulation of cAMP and Ca2+. In contrast, bradykinin increased intracel lular Ca2+ but not cAMP levels. Cytochemistry of cultured tracheal epi thelium showed a positive staining with NADPH diaphorase activity. The se results suggest that airway epithelial cells spontaneously release NO and that the release may be stimulated specifically through a cAMP- dependent mechanism.