Am. J. Respir. Cell Mol. Biol., Volume 22, Number 1, January, 2000 97-104

Detection of Nitric Oxide Release Induced by Bradykinin in Guinea Pig Trachea and Main Bronchi Using a Porphyrinic Microsensor

Fabio L. M. Ricciardolo, Luciana Vergnani, Silke Wiegand, Franco Ricci, Nadia Manzoli, Axel Fischer, Silvia Amadesi, Renato Fellin, and Pierangelo Geppetti

Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands; Department of Clinical and Experimental Medicine, University of Ferrara, Ferrara, Italy; and Institute for Anatomy and Cell Biology, Justus-Liebig University, Giessen, Germany

Indirect evidence using nitric oxide (NO) synthase (NOS) inhibitors suggests that in guinea-pig airways bradykinin releases bronchoprotective NO. In this study, using a recently developed electrochemical method of NO measurement based on a porphyrinic microsensor, we investigated whether bradykinin releases NO from guinea-pig airways and whether the epithelium is the main source of NO. Further, the Ca²⁺-dependence of bradykinin-induced NO release was assessed stimulating airway preparations with bradykinin in Ca²⁺-free conditions. We also studied the immunohistochemical distribution of the Ca²⁺- dependent constitutive isoforms of NOS (constitutive NOS [cNOS]: neuronal and endothelial [ecNOS]) in our preparations. The porphyrinic microsensor was placed in the bathing fluid onto the mucosal surface of tracheal or main bronchial segments. Addition of bradykinin vehicle (0.9% saline) did not cause any detectable change of the baseline signal. Addition of bradykinin caused an upward shift of the baseline that reached a maximum within 1 to 2 s. The amplitude of the response to bradykinin was concentration-dependent between the range 1 nM to 10 µM, with a maximum effect at 10 µM. Bradykinin-induced NO release was higher in tracheal than in main bronchial segments. The selective bradykinin B₂ receptor antagonist D-Arg⁰-[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]bradykinin (1 µM) inhibited NO release induced by a submaximum concentration of bradykinin (1 µM). The ability of bradykinin to release NO was markedly reduced in epithelium-denuded segments, and abolished in Ca²⁺-free conditions and after pretreatment with N^G-monomethyl-L-arginine (100 µM), but not with N^G-monomethyl-D-arginine. Both cNOS isoforms were present in trachea and main bronchi, ecNOS being the predominant isoform in the epithelium. The study shows that bradykinin via B₂ receptor activation caused a rapid and Ca²⁺-dependent release of NO, mainly, but not exclusively, derived from the epithelium. It also shows that both cNOS isoforms may be involved in bradykinin-evoked NO release.

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