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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 NG-monomethyl-l-arginine (100 μM), but not with NG-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.