AUA UniversityMuscarinic M3- and M2- Receptor-Activated Signaling in the Bladder is Inversely Regulated by Caveolae.
Sources of Funding: Department of Veterans Affairs, Research Service BX001790
Introduction
Bladder smooth muscle (BSM) caveolae are cholesterol-enriched membrane microdomains that augment or attenuate detrusor functional responses by differentially regulating specific receptor-activated signaling pathways. However, BSM contractions induced by cholinergic activation in the rat are not altered by depletion of caveolae, unlike other smooth muscle systems in which muscarinic receptor signaling is evidently mediated by caveolae. Although this discrepancy may reflect the highly specific regulation imparted by caveolae among different tissues and species, a differential regulation of muscarinic M3 and M2 receptor subtypes in the bladder cannot be excluded. This study examined the functional and molecular relationship between caveolae and muscarinic acetylcholine receptor (mAChR) subtypes M3 and M2.
Methods
BSM tissue strips were prepared from Sprague Dawley rat bladders after removing the mucosa. Tissue was suspended in organ baths for isometric tension studies. Dose response curves to carbachol (CCh, 1nM-10μM) were generated at baseline, as well as in the presence of 4-DAMP (10nM) or AFDX (0.1μM) to inactive M3 or M2 receptors respectively. Responses to CCh were repeated after incubation with methyl-β-cyclodextrin (mβCD, 15mM), an agent that disrupts caveolae by depleting membrane cholesterol. Interaction between caveolin-1 (Cav-1, a protein required for caveolae biogenesis) and M3 or M2 mAChR subtype were investigated by co-immunoprecipitation.
Results
Compared to baseline responses, 4-DAMP decreased CCh-induced contractions at each dose. After mβCD treatment and in the presence of 4-DAMP, contractile responses to CCh were significantly enhanced. AFDX had little effect on CCh dose-response curves. However, the subsequent disruption of caveolae in the presence of AFDX attenuated significantly contractions induced by CCh. Immunoreactive bands corresponding to M3 and M2 mAChR subtypes were detected in Cav-1 immunoprecipitates.
Conclusions
The opposite effect of mβCD on CCh responses in the presence of M2 or M3 antagonists suggests that caveolae negatively regulate M2- and positively regulate M3-mediated signaling respectively, but this interaction is masked when only the aggregate effect of CCh is examined. Molecular interaction of Cav-1 and mAChRs is consistent with their localization within caveolae. Changes in the balance among caveolin-mAChR interactions, due to loss of caveolae or changes in mAChR subtype expression, may alter responses to cholinergic activation or the efficacy of anti-muscarinic agents in the bladder.
Funding
Department of Veterans Affairs, Research Service BX001790
Maryrose P. Sullivan