title: Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle.
abstract: Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca(2+) homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca(2+) ([Ca(2+)](i)) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M(3) receptors (M(3)R) and Galpha(q/11) cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with beta-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-beta-cyclodextrin (mbetaCD) reduced sensitivity but not maximum [Ca(2+)](i) induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mbetaCD disrupted the colocalization of caveolae-1 and M(3)R, but [N-methyl-(3)H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca(2+)](i) flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mbetaCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca(2+)](i) mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca(2+)](i) mobilization leading to ASM contraction induced by submaximal concentrations of ACh.
authors: Department of Physiology, University of Manitoba, John Buhler Research Centre, Winnipeg, Manitoba, Canada.
authors: Reinoud
authors: R
authors: Gosens
authors: Gerald L
authors: GL
authors: Stelmack
authors: Gordon
authors: G
authors: Dueck
authors: Mark M
authors: MM
authors: Mutawe
authors: Martha
authors: M
authors: Hinton
authors: Karol D
authors: KD
authors: McNeill
authors: Angela
authors: A
authors: Paulson
authors: Shyamala
authors: S
authors: Dakshinamurti
authors: William T
authors: WT
authors: Gerthoffer
authors: James A
authors: JA
authors: Thliveris
authors: Helmut
authors: H
authors: Unruh
authors: Johan
authors: J
authors: Zaagsma
authors: Andrew J
authors: AJ
authors: Halayko
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D051242:Caveolin 1 / Q000737:chemistry; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D032342:Muscle Cells / Q000166:cytology / Q000187:drug effects; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D019832:N-Methylscopolamine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D012137:Respiratory System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014316:Tritium / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D051242:Caveolin 1; D034741:RNA, Small Interfering; D043587:Receptor, Muscarinic M3; D047392:beta-Cyclodextrins; D014316:Tritium; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C031215:betadex; D000109:Acetylcholine; D019832:N-Methylscopolamine