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. 2007 Jan;150(2):220-6.
doi: 10.1038/sj.bjp.0706968. Epub 2006 Dec 18.

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea

A Dellabianca  1 , M Sacchi, L Anselmi, E De Amici, E Cervio, A Agazzi, S Tonini, C Sternini, M Tonini, S M Candura
Affiliations

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea

A Dellabianca et al. Br J Pharmacol. 2007 Jan.

Abstract

Background and purpose: Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non-adrenergic, non-cholinergic (NANC) relaxations in guinea-pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase-2 (HO-2), the CO-producing enzyme, in tracheal neurons.

Experimental approach: NANC responses to electrical field stimulation (EFS) at 3 and 10 Hz were evaluated in epithelium-free whole tracheal segments as intraluminal pressure changes. Drugs used were: L-nitroarginine methyl ester (L-NAME, 100 microM) to inhibit NO synthase (NOS), alpha-chymotrypsin (2 U ml(-1)) to inactivate VIP, zinc protoporphyrin-IX (ZnPP-IX, 10 microM) to inhibit HO-2, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor. For immunohistochemistry, tissues were exposed to antibodies to PGP 9.5, a general neuronal marker, HO-2 and NOS, and processed with an indirect immunofluorescence method.

Key results: alpha-Chymotrypsin did not affect NANC relaxations. ODQ inhibited NANC responses by about 60%, a value similar to that obtained by combining L-NAME and ZnPP-IX. The combination of ODQ, L-NAME and ZnPP-IX reduced the responses by 90%. Subpopulations of HO-2 positive neurons containing NOS were detected in tracheal sections.

Conclusions and implications: In the guinea-pig trachea, NANC inhibitory responses at 3 and 10 Hz use NO and CO as main transmitters. Their participation is revealed following inhibition of NOS, HO-2 and soluble guanylyl cyclase. The involvement of CO as a relaxing transmitter paves the way for novel therapeutic approaches in the treatment of airway obstruction.

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Figures

Figure 1
Figure 1
Representative tracing showing EFS-induced NANC relaxations evoked by frequencies of 3 and 10 Hz in the guinea-pig isolated whole trachea in the absence (a) and in the presence (b) of TTX.
Figure 2
Figure 2
EFS-induced NANC relaxations evoked by frequencies of 3 and 10 Hz in the guinea-pig isolated whole trachea were evaluated either as amplitude of the fast initial response (a) or as AUC (b) of the overall inhibitory response, including both the fast and the slow component. Note that the amplitude of the responses evoked by 10 Hz was significantly higher than that evoked by 3 Hz, whereas in terms of AUC the two responses were superimposable. Data represent mean and vertical lines show s.e.m. of 43 experiments. **P<0.01.
Figure 3
Figure 3
Effect of α-CT, ZnPP-IX and L-NAME on EFS-induced NANC relaxations evoked by frequencies of 3 and 10 Hz in the guinea-pig isolated whole trachea. α-CT (2 U ml−1) and ZnPP-IX (10 μM) were ineffective, whereas L-NAME (100 μM) significantly reduced both the amplitude (a) and the AUC (b) of the inhibitory responses. Data represent mean and vertical lines show s.e.m. of 5–8 experiments. *P<0.05, **P<0.01.
Figure 4
Figure 4
Effects of various pharmacological treatments with single drugs (ODQ and TTX), or with a combination of drugs (L-NAME+ZnPP-IX or L-NAME+ZnPP-IX+ODQ) on EFS-induced NANC relaxations evoked by frequencies of 3 and 10 Hz in the guinea-pig isolated whole trachea. For comparison purposes, the effect of L-NAME (as already presented in Figure 2) on both the amplitude (a) and the AUC (b) of the inhibitory responses has also been included. L-NAME+ZnPP-IX significantly reduced both the amplitude and the AUC of NANC responses either vs control relaxations or vs L-NAME alone (with the exception of the amplitude of relaxations elicited at 3 Hz). The effect of ODQ alone was similar to that of L-NAME+ZnPP-IX. The combination of L-NAME+ZnPP-IX+ODQ inhibited the responses in terms of amplitude and AUC to an extent similar to that of TTX (1 μM). Data represent mean and vertical lines show s.e.m. of 4–10 experiments. *P<0.05, **P<0.01.
Figure 5
Figure 5
Confocal images of neurons of the guinea-pig trachea showing HO-2 (green fluorescence), PGP 9.5 and NOS (red fluorescence) immunoreactivity in the cytoplasm of neuronal cell bodies in tracheal ganglion cells. HO-2/PGP 9.5 and HO-2/NOS show colocalization of HO-2 with PGP 9.5 or NOS immunoreactivity, respectively. Arrows point to examples of neurons that contain HO-2 and PGP 9.5 or NOS immunoreactivity. Calibration bar: 50 μm.
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