Caveolae Facilitate Muscarinic Receptor Mediated Intracellular Ca2+ Mobilization and Contraction in Airway Smooth Muscle

doi:10.1152/ajplung.00312.2007

Caveolae Facilitate Muscarinic Receptor Mediated Intracellular Ca²⁺ Mobilization and Contraction in Airway Smooth Muscle

Reinoud Gosens¹, Gerald L. Stelmack², Gordon Dueck³, Mark M. Mutawe², Martha A Hinton², Karol D. McNeill², Angela Paulson³, Shyamala Dakshinamurti⁴, William T Gerthoffer⁵, James A Thliveris⁶, Helmut Unruh⁷, Johan Zaagsma⁸, and Andrew John Halayko⁹^*

¹ Physiology, University of Manitoba, Winnipeg, Canada; Molecular Pharmacology, University of Groningen, Groningen, groningen, Netherlands; Internal Medicine, University of Manitoba, Winnipeg, Canada; Section of Respiratory Disease, University of Manitoba, Winnipeg, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Canada; CIHR National Training Program in Allergy and Asthma, University of Manitoba, Winnipeg, Canada
² Physiology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Canada
³ Physiology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Canada; CIHR National Training Program in Allergy and Asthma, University of Manitoba, Winnipeg, Canada
⁴ Pediatrics and Child Health, University of Manitoba, Winnipeg, Canada; Physiology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Canada
⁵ Pharmacology, University of Nevada, Reno, Nevada, United States
⁶ Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada
⁷ Section of Thoracic Surgery, University of Manitoba, Winnipeg, Canada
⁸ Molecular Pharmacology, University of Groningen, Groningen, groningen, Netherlands
⁹ Physiology, University of Manitoba, Winnipeg, Canada; Internal Medicine, University of Manitoba, Winnipeg, Canada; Section of Respiratory Disease, University of Manitoba, Winnipeg, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Canada; CIHR National Training Program in Allergy and Asthma, University of Manitoba, Winnipeg, Canada; Pediatrics and Child Health, University of Manitoba, Winnipeg, Canada

^* To whom correspondence should be addressed. E-mail: ahalayk{at}cc.umanitoba.ca.

Contractile responses of airway smooth muscle (ASM) determineairway resistance in health and disease. Caveolae microdomainsin the plasma membrane are marked by caveolin proteins, andare abundant in contractile smooth muscle associated with nanospacesinvolved in Ca²⁺ homeostasis. Via a scaffolding domain, caveolin-1modulates localization and activity of signaling proteins, includingtrimeric G-proteins. We investigated the role of caveolae incontraction and intracellular Ca²⁺ ([Ca²⁺]i) mobilization ofASM in response to the muscarinic receptor agonist, acetylcholine(ACh). Human and canine ASM tissues and cells predominantlyexpress caveolin-1. Muscarinic M₃ receptors (M₃R) and G_q/11co-fractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with ${beta}$ -cyclodextrin in canine tracheal stripsreduced sensitivity, but not maximum isometric force inducedby ACh. In fura-2 loaded canine and human ASM cells, exposureto methyl- ${beta}$ -cyclodextrin (m ${beta}$ CD) reduced sensitivity to, but notmaximum [Ca²⁺]i induced by ACh, but both parameters were reducedfor the partial muscarinic agonist, pilocarpine. Fluorescencemicroscopy revealed that m ${beta}$ CD disrupted the co-localization ofcaveolae-1 and M₃R, but H³-N-methyl-scapolamine receptor bindingassay revealed no effect on muscarinic receptor availabilityor affinity. To dissect the role of caveolin-1 in ACh induced[Ca²⁺]i flux, we disrupted its binding to signaling proteinsusing either a cell permeable caveolin-1 scaffolding domain(CSD) peptide mimetic or by siRNA knockdown. Similar to theeffects of m ${beta}$ CD, direct targeting of caveolin-1 reduced sensitivityto ACh but maximum [Ca²⁺]i mobilization was unaffected. Theseresults indicate caveolae and caveolin-1 facilitate [Ca²⁺]imobilization leading to ASM contraction induced by sub-maximalconcentrations of ACh.

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