Detection of nitric oxide release induced by bradykinin in guinea pig trachea and main bronchi using a porphyrinic microsensor

Indirect evidence using nitric oxide (NO) synthase (NOS) inhibitors suggest s that in guinea-pig airways bradykinin releases bronchoprotective NO. In t his study, using a recently developed electrochemical method of NO measurem ent 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 mai n 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 mu M, with a maximum effect at 10 mu M Bradyk inin-induced NO release was higher in tracheal than in main bronchial segme nts. The selective bradykinin B-2 receptor antagonist D-Arg(0)-[Hyp(3), Thi (5), D-Tic(7), Oic(8)]bradykinin (1 mu M) inhibited NO release induced by a submaximum concentration of bradykinin (1 mu M). The ability of bradykinin to release NO was markedly reduced in epithelium-denuded segments, and abo lished in Ca2+-free conditions and after pretreatment with N-G-monomethyl-L -arginine (100 mu M), but not with N-G-monomethyl-D-arginine. Both cNOS iso forms 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 no t exclusively, derived from the epithelium. It also shows that both cNOS is oforms may be involved in bradykinin-evoked NO release.