Acetylcholine (ACh) and serotonin are bronchoconstrictors clinically relevant in airway diseases associated with airway hyperreactivity. Their receptors are incorporated in membrane signaling platforms termed caveolae. These are omega-shaped invaginations of the plasma membrane with a caveolar coat protein complex consisting of caveolins (Cav-1, -2 and -3) and cytoplasmic adapter proteins (cavin-1 to -4). There is evidence that Cav-3 might organize this caveolar coat complex in airway smooth muscle (ASM). To address this issue, we generated Cav-3-deficient mice to address the functional role of Cav-3 in ASM constriction using organ bath recording from extrapulmonary and videomicroscopy of precision cut-lung slices from intrapulmonary airways. Immunofluorescence, RT-PCR, real-time PCR, Western blotting and co-immunoprecipitation served to address the distribution and molecular composition of the caveolar coat complex in murine and human airways. Cav-3 was found in tracheal epithelium and ASM and was associated with Cav-1, cavin-1 and cavin-4. Cav-3 deficiency neither had impact on protein expression of caveolar coat complex members nor on EHD-2 expression, a caveolae abundancy marker. Thus, specific aspects of caveolar function rather than caveolar abundancy are dependent on the expression of Cav-3. The response of extrapulmonary airway to muscarine was not altered in Cav-3-/- mice, whereas a considerable increase in muscarinic bronchoconstriction was observed in Cav-3-/- intrapulmonary bronchi. This demonstrates an inhibitory regulatory role of Cav-3 in cholinergic bronchoconstriction. Cav-3 was previously found to interact with the muscarinic receptor subtype 2 (M2R) in bronchial SM, and in cardiac myocytes it couples M2R stimulation with endothelial NO synthase (eNOS) activation. Thus, the depletion of Cav-3 might cause an increased muscarine-induced bronchoconstriction through disruption of M2R-Cav-3-eNOS. Cav-3 deficiency fully abrogated serotonin-induced constriction of extrapulmonary airways while leaving intrapulmonary airways unaffected. Serotonergic airway constriction in murine extrapulmonary is considered to be indirectly mediated through ACh release from epithelial cells or nerve fibers. Since muscarinic constriction of extrapulmonary airways was unaffected in Cav-3-/- mice, the cholinergic component of this indirect effect shall not be responsible for the abrogated serotonergic effect. The selective expression of Cav-3 in epithelial cells present in tracheal but not in intrapulmonary bronchial epithelial cells might explain the differential effects of Cav-3 deficiency on serotonergic ASM constriction. Potentially, these data warrant consideration during pharmacological modulation of the cholinergic and serotonergic responses and provide an opportunity to modulate airway hyperreactivity.
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