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Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse

Kummer W, Wiegand S, Akinci S, Wessler I, Schinkel AH, Wess J, Koepsell H, Haberberger RV, Lips KS - Respir. Res. (2006)

Bottom Line: Corticosterone (1 microM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice.This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi.These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.

Affiliation: Institute for Anatomy and Cell Biology, Justus-Liebig-University, 35385 Giessen, Germany. wolfgang.kummer@anatomie.med.uni-giessen.de

ABSTRACT

Background: It has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs.

Methods: We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry.

Results: Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 +/- 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 +/- 3.6 %, n = 11). In wild-type mice, 5-HT (1 microM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 microM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 microM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle.

Conclusion: The doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.

Immunohistochemical localization of OCTs in murine bronchi. OCT1-immunolabelling is localized to the apical membrane of ciliated epithelial cells in wild-type FVB mice (arrows in A). The specificity of this labelling is indicated by its absence after preabsorption of the antiserum with its corresponding antigenic peptide (B) and the lack of labelling in OCT1/2-/- mice (C). Neither of the two OCT2-antibodies used in this study showed specific labelling of mouse bronchi (D, E). The spotty labelling of epithelial cells observed with the OCT2-antibody raised against the human sequence (E) was also observed in OCT1/2-/- mice (F), indicating that this signal is non-specific. Specific OCT3-immunolabelling, documented by its absence in the preabsorption control (inset in G), is observed primarily on the bronchial smooth muscle (sm) and, less intensely, on epithelial cells (G). OCT3-localization in smooth muscle cells is confirmed by double-labelling immunofluorescence with OCT3-antibody and a monoclonal antibody against α-smooth muscle actin (SMA) (G') yielding the yellow signal in the merged image (G'). Bar represents 10 μm in A-F and 20 μm in G-G".
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Figure 5: Immunohistochemical localization of OCTs in murine bronchi. OCT1-immunolabelling is localized to the apical membrane of ciliated epithelial cells in wild-type FVB mice (arrows in A). The specificity of this labelling is indicated by its absence after preabsorption of the antiserum with its corresponding antigenic peptide (B) and the lack of labelling in OCT1/2-/- mice (C). Neither of the two OCT2-antibodies used in this study showed specific labelling of mouse bronchi (D, E). The spotty labelling of epithelial cells observed with the OCT2-antibody raised against the human sequence (E) was also observed in OCT1/2-/- mice (F), indicating that this signal is non-specific. Specific OCT3-immunolabelling, documented by its absence in the preabsorption control (inset in G), is observed primarily on the bronchial smooth muscle (sm) and, less intensely, on epithelial cells (G). OCT3-localization in smooth muscle cells is confirmed by double-labelling immunofluorescence with OCT3-antibody and a monoclonal antibody against α-smooth muscle actin (SMA) (G') yielding the yellow signal in the merged image (G'). Bar represents 10 μm in A-F and 20 μm in G-G".

Mentions: Immunohistochemistry revealed OCT1-immunoreactivity in the apical membrane of ciliated cells (Fig. 5A). This labelling was OCT1-specific since it was absent when the antiserum was preabsorbed with the corresponding antigenic peptide and when tissue from OCT1/2-/- mice was used for immunohistochemistry (Fig. 5B, C). No specific OCT2-immunolabelling was observed in the bronchial wall (Fig. 5D–F). Specific OCT3-immunoreactivity was most intense in the bronchial smooth muscle and weaker on epithelial cells (Fig. 5G–G").

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Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse

Kummer W, Wiegand S, Akinci S, Wessler I, Schinkel AH, Wess J, Koepsell H, Haberberger RV, Lips KS - Respir. Res. (2006)

Immunohistochemical localization of OCTs in murine bronchi. OCT1-immunolabelling is localized to the apical membrane of ciliated epithelial cells in wild-type FVB mice (arrows in A). The specificity of this labelling is indicated by its absence after preabsorption of the antiserum with its corresponding antigenic peptide (B) and the lack of labelling in OCT1/2-/- mice (C). Neither of the two OCT2-antibodies used in this study showed specific labelling of mouse bronchi (D, E). The spotty labelling of epithelial cells observed with the OCT2-antibody raised against the human sequence (E) was also observed in OCT1/2-/- mice (F), indicating that this signal is non-specific. Specific OCT3-immunolabelling, documented by its absence in the preabsorption control (inset in G), is observed primarily on the bronchial smooth muscle (sm) and, less intensely, on epithelial cells (G). OCT3-localization in smooth muscle cells is confirmed by double-labelling immunofluorescence with OCT3-antibody and a monoclonal antibody against α-smooth muscle actin (SMA) (G') yielding the yellow signal in the merged image (G'). Bar represents 10 μm in A-F and 20 μm in G-G".
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Figure 5: Immunohistochemical localization of OCTs in murine bronchi. OCT1-immunolabelling is localized to the apical membrane of ciliated epithelial cells in wild-type FVB mice (arrows in A). The specificity of this labelling is indicated by its absence after preabsorption of the antiserum with its corresponding antigenic peptide (B) and the lack of labelling in OCT1/2-/- mice (C). Neither of the two OCT2-antibodies used in this study showed specific labelling of mouse bronchi (D, E). The spotty labelling of epithelial cells observed with the OCT2-antibody raised against the human sequence (E) was also observed in OCT1/2-/- mice (F), indicating that this signal is non-specific. Specific OCT3-immunolabelling, documented by its absence in the preabsorption control (inset in G), is observed primarily on the bronchial smooth muscle (sm) and, less intensely, on epithelial cells (G). OCT3-localization in smooth muscle cells is confirmed by double-labelling immunofluorescence with OCT3-antibody and a monoclonal antibody against α-smooth muscle actin (SMA) (G') yielding the yellow signal in the merged image (G'). Bar represents 10 μm in A-F and 20 μm in G-G".
Mentions: Immunohistochemistry revealed OCT1-immunoreactivity in the apical membrane of ciliated cells (Fig. 5A). This labelling was OCT1-specific since it was absent when the antiserum was preabsorbed with the corresponding antigenic peptide and when tissue from OCT1/2-/- mice was used for immunohistochemistry (Fig. 5B, C). No specific OCT2-immunolabelling was observed in the bronchial wall (Fig. 5D–F). Specific OCT3-immunoreactivity was most intense in the bronchial smooth muscle and weaker on epithelial cells (Fig. 5G–G").

Bottom Line: Corticosterone (1 microM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice.This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi.These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.

Affiliation: Institute for Anatomy and Cell Biology, Justus-Liebig-University, 35385 Giessen, Germany. wolfgang.kummer@anatomie.med.uni-giessen.de

ABSTRACT

Background:

Background: It has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs.

Methods: We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry.

Results: Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 +/- 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 +/- 3.6 %, n = 11). In wild-type mice, 5-HT (1 microM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 microM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 microM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle.

Conclusion: The doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.

View Similar Images In: Results  - Collection
View Article: Pubmed Central - HTML -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=1468398&rFormat=json&query=null&req=5