Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways

Abstract

Cholinergic bronchoconstriction is mediated by M(2) and M(3) muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-β-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R(-/-) and M3R(-/-) mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.

Fig. 1.

Protein and mRNA detection of caveolins Cav-1 and Cav-3 in murine bronchial smooth muscle cells (smc). A and C: double-labeling immunofluorescence of murine bronchi, confocal laser scanning microscopy (CLSM). A and C′: Cav-1 immunoreactivity is located in the plasma membrane of bronchial smc. B and C: bronchial epithelial cells (ec) and smc display labeling for Cav-3. Smooth muscle cells also exhibit plasma membrane staining for Cav-3. A′ and B′: muscarinic receptor subtype M2R immunoreactivity is found predominantly in the plasma membrane of bronchial smc. Additionally, an unspecific labeling was detected in the cilia of the ciliated cells. A″, B″, and C″: merged images. Colocalization of the labeling results in yellow color. Arrowheads, endothelium; alv, alveolar region; lu, airway lumen. D and E: caveolin mRNA detection in laser-assisted microdissected murine airway smc (D) with subsequent RT-PCR (E) in tracheal (1) and bronchial (4 and 5) smc but not in control samples including oil (2), bronchial luminal regions proximate to the dissected smc (3), and without template (6).

Fig. 2.

Validation of antibody specificity. A and B: the anti-M2R antibody labels airway smooth muscle cells (ASMC) in lung sections from wild-type (A) but not from M2R-deficient (B) mice. C: the anti-Cav-1 antibody labels ASMC and cells of the alveolar region in wild-type mice. D: this labeling is not present in Cav-1-deficient tissue. Smooth muscle cell staining with anti-Cav-3 antibody in a lung section from an FVB mouse (E) and a Cav-1-deficient mouse (F) is shown. Inset in E: preabsorption with the corresponding peptide abolished the anti-Cav-3 antibody labeling. Arrows: smooth muscle cells. Bar = 50 μm. G and H: Western blotting-based validation of Cav-1 and Cav-3 antibody specificities. Bands for Cav-1α detected in wild-type mice are not present in Cav-1-deficient mice (G). H: bands corresponding to the molecular mass of Cav-3 can be preabsorbed with the corresponding Cav-3 antigen. Controls for the specificity of the secondary reagents: Ø primary antibody.

Fig. 3.

Detection of Cav-1 and Cav-3 in human bronchus. A–E: double-labeling immunohistochemistry, CLSM. A: cross-section of a human bronchus stained for Cav-3 (yellow) and α-smooth muscle actin (α-sma; green). Omitting the primary anti-Cav antibody (B) and preabsorption of Cav-3 antibody with a corresponding peptide (C) resulted in exclusive α-sma labeling in the smooth muscle. Cav-1 (D), Cav-3 (E), and α-sma immunoreactivities (D′ and E′) are present in human bronchial smc and epithelium, whereas α-sma immunoreactivity is restricted to smc (D′ and E′). To distinguish the specific labeling for Cav-1 (orange; D) and Cav-3 (orange; E) from autofluorescence (af) of elastic fibers, these fibers were visualized in blue using a short wavelength filter (D′ and E′). Images were merged, resulting in elastic fibers appearing in pink color (A–E, D″, and E″). Arrows, smooth muscle cells; arrowheads, basement membrane. F: expression of Cav-1 and Cav-3 mRNA detected in homogenates from human lungs (n = 5) by RT-PCR using different primer pairs (Table 1) performed in duplicates. Hypoxanthine phosphoribosyltransferase (HPRT) was used as housekeeping gene to control for the RT-PCR efficacy. H₂O, without DNA template.

Fig. 4.

Detection of close spatial association of M2R/Cav-3 and Cav-1/Cav-3 by double-labeling indirect immunofluorescence and CLSM-fluorescence resonance energy transfer (FRET) analysis in airway smooth muscle of murine bronchi in situ. Changes in donor fluorescence (ΔIF) in the membrane area of bronchial smc in experimental compared with control group are shown. For M2R/Cav-3 and Cav-1/Cav-3, ΔIF is higher in experimental groups than in controls. ***P ≤ 0.001, *P ≤ 0.01; n = number of regions of interest/animals. Box plots: percentiles 0, 25, median, 75, 100; extreme values (○), outlier (☐).

Fig. 5.

Transmission electron microscopy images of the airway wall of wild-type mice derived from precision-cut lung slices (PCLS) included in videomorphometric experiments. A: ciliated cells (cc), basal cells (ba), and secretory cells (sec) of lamina epithelialis mucosae, lamina propria with elastin (double arrowheads), and underlying tunica muscularis with bronchial smc. Bar = 1 μm. B: vehicle-treated murine ASMC containing areas with caveolae (arrows) in the plasma membrane and other subsurface vesicles (arrowheads). C: cell surface region of an equivalent bronchial smc after caveolae disruption by methyl-β-cyclodextrin (MCD). Black arrows point to plasma membrane without caveolae. nucl, Nucleus.

Fig. 6.

Bronchoconstrictor responses of M3R^−/− and M3R^+/+ mice (A) of M2R^−/− and M2R^+/+ mice (B) to stimulation with muscarine (1 μM), KCl (60 mM), and 5-HT (1 μM) after vehicle (—) or MCD treatment (- - -). Data represent luminal area with prestimulus value set as 100%. A: caveolae disruption reduces the response to muscarine in bronchi from M3R^+/+ mice and fully abrogated in M3R^−/− mice. No differences in response to KCl occur after MCD or vehicle treatment in either mouse strain. 5-HT-induced responses are fully abrogated by MCD in both strains. B: in M2R^−/− and M2R^+/+ mice, the muscarine-induced constriction is not significantly different from M2R^+/+ mice and is reduced to less than 10% after MCD treatment. No differences in response to KCl occurred after MCD or vehicle treatment in either mouse strain. Responses to 5-HT were less distinct in M2R^+/+ compared with M2R^−/− mice but were reduced after caveolae disruption in both strains. Data are presented as means ± SE; n = number of bronchi/animals.

Fig. 7.

A–C: real-time RT-PCR. cDNA were derived from lung homogenate (A; n = 5), tracheal muscle (B; n = 4), and urinary bladder (C; n = 4) from M3R^+/+ and M3R^−/− mice each. Changes in cycle threshold (ΔCT) values were calculated for M2R and M3R using β-microglobulin (β-MG) as reference gene and PCR products of M2R, M3R, and β-MG separated on agarose gels. Images in A–C originate from the same gel, each, after excluding irrelevant samples. Box plots: percentiles 0, 25, median, 75, 100; extreme values (○), outlier (☐); n = number of animals; n.s., not significant.