title: Caveolae compartmentalise β2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte.
abstract: Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca(2+) current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser(16). Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled "Local Signaling in Myocytes".
authors: Institute of Membrane and Systems Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors: David A
authors: DA
authors: Macdougall
authors: Shailesh R
authors: SR
authors: Agarwal
authors: Elizabeth A
authors: EA
authors: Stopford
authors: Hongjin
authors: H
authors: Chu
authors: Jennifer A
authors: JA
authors: Collins
authors: Anna L
authors: AL
authors: Longster
authors: John
authors: J
authors: Colyer
authors: Robert D
authors: RD
authors: Harvey
authors: Sarah
authors: S
authors: Calaghan
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis*; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D015398:Signal Transduction; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D002867:Chromones; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D018343:Receptors, Adrenergic, beta-2; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium