Caveolae facilitate muscarinic receptor-mediated intracellular Ca²⁺mobilization and contraction in airway smooth muscle

Abstract

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 2ϩ 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 2ϩ ([Ca 2ϩ ]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 M3 receptors (M3R) and G␣q/11 cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with ␤-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-␤-cyclodextrin (m␤CD) reduced sensitivity but not maximum [Ca 2ϩ ]i induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that m␤CD disrupted the colocalization of caveolae-1 and M 3R, 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 2ϩ ]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 m␤CD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca 2ϩ ]i mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca 2ϩ ]i mobilization leading to ASM contraction induced by submaximal concentrations of ACh.

β-Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca2+ release

“…Indeed, in -dystroglycan-silenced myocytes, there was a significant reduction in the sensitivity to MCh (EC50 Control 50.3±14 nM versus EC50 shRNAi 220±24 nM; P<0.01). By contrast, and consistent with previous studies in which caveolin-1 expression was silenced (Gosens et al, 2007b), peak [Ca 2+ ] i induced with maximum concentrations of MCh were unaffected. Collectively, these data demonstrate that the requirement of -dystroglycan for caveolae integrity is a crucial determinant of the spatial profile of proximal signaling effectors, which has important consequences on the initiation of contraction by physiologically relevant agonists.…”

“…We and others have shown in smooth muscle cells that depletion of cholesterol or silencing of caveolin-1 expression disrupts the organization of membrane caveolae and alters functional responses, as measured by contractile agonist-induced intracellular Ca 2+ release and force generation (Gosens et al, 2007b;Prakash et al, 2007). Our new data are consistent with these observations and those showing a role for the actin cytoskeleton in modulating Ca 2+ responses in several other cell types (Calaghan et al, 2004;Rosado et al, 2000;Sabala et al, 2002), because induced changes in ultrastructure and the protein profile of caveolae were associated with a reduction both sensitivity to MCh and in maximum peak [Ca 2+ ] i .…”

“…Our new data are consistent with these observations and those showing a role for the actin cytoskeleton in modulating Ca 2+ responses in several other cell types (Calaghan et al, 2004;Rosado et al, 2000;Sabala et al, 2002), because induced changes in ultrastructure and the protein profile of caveolae were associated with a reduction both sensitivity to MCh and in maximum peak [Ca 2+ ] i . Trimeric G-proteins are sequestered to caveolae through the interaction of -subunits with caveolin-1, and the signaling machinery required to mobilize Ca 2+ within smooth muscle cells is organized in caveolae (Darby et al, 2000;de Weerd and LeebLundberg, 1997;Gosens et al, 2007b;Kifor et al, 1998;Li et al, 1995). Although PLC1 is concentrated at the cell membrane, it is also present throughout the cell, whereas G q is more-or-less membrane specific, where it stably associates with PLC1 to allow for rapid transmission of intracellular signals via GPCRs such as the M3 muscarinic receptor (Dowal et al, 2006).…”

“…Collectively, these results support a role for the actin cytoskeleton, through its tethering to a DGC-caveolin-1 complex, in the establishment and maintenance of discrete caveolae microdomains in smooth muscle cells. release in the smooth muscle and other cell systems (Daniel et al, 2009;Darby et al, 2000;El-Yazbi et al, 2008;Gosens et al, 2007b;Prakash et al, 2007;Sengupta et al, 2008;Zhu et al, 2008). We assessed whether this functional role is linked to the unique ordered distribution of caveolae in contractile smooth muscle cells.…”

“…Biochemical fractionation has revealed that caveolae are enriched in Ca 2+ -handling and Ca 2+ -binding proteins, and trimeric G-proteins are sequestered to these microdomains (Darby et al, 2000;de Weerd and Leeb-Lundberg, 1997;Gosens et al, 2007a;Li et al, 1995). Caveolae are functionally important, because they are needed to facilitate contraction and Ca 2+ mobilization mediated by some G-protein-coupled receptors (GPCRs) in airway smooth muscle (Gosens et al, 2007b;Prakash et al, 2007). These observations suggest that the spatial organization of caveolae and the repertoire of proteins that localize to them are fundamental determinants of the contractile response of smooth muscle.…”