BackgroundIn murine heart failure models and in humans with diabetic‐related heart hypertrophy, inhibition of phosphodiesterase 5 (PDE5) by sildenafil improves cardiac outcomes. However, the mechanism by which sildenafil improves cardiac function is unclear. We have observed a relationship between PDE5 and β2 adrenergic receptor (β2AR), which is characterized here as a novel mechanistic axis by which sildenafil improves symptoms of diabetic cardiomyopathy.Methods and ResultsWild‐type and β2AR knockout mice fed a high fat diet (HFD) were treated with sildenafil, and echocardiogram analysis was performed. Cardiomyocytes were isolated for excitation‐contraction (E‐C) coupling, fluorescence resonant energy transfer, and proximity ligation assays; while heart tissues were implemented for biochemical and histological analyses. PDE5 selectively associates with β2AR, but not β1 adrenergic receptor, and inhibition of PDE5 with sildenafil restores the impaired response to adrenergic stimulation in HFD mice and isolated ventriculomyocytes. Sildenafil enhances β adrenergic receptor (βAR)‐stimulated cGMP and cAMP signals in HFD myocytes. Consequently, inhibition of PDE5 leads to protein kinase G–, and to a lesser extent, calcium/calmodulin‐dependent kinase II–dependent improvements in adrenergically stimulated E‐C coupling. Deletion of β2AR abolishes sildenafil's effect. Although the PDE5‐β2AR association is not altered in HFD, phosphodiesterase 3 displays an increased association with the β2AR‐PDE5 complex in HFD myocytes.ConclusionsThis study elucidates mechanisms by which the β2AR‐PDE5 axis can be targeted for treating diabetic cardiomyopathy. Inhibition of PDE5 enhances β2AR stimulation of cGMP and cAMP signals, as well as protein kinase G–dependent E‐C coupling in HFD myocytes.
G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report neuronal excitability of β-blockers carvedilol and alprenolol at clinically relevant nanomolar concentrations. Carvedilol and alprenolol activate β2AR, which promote G protein signaling and cAMP/PKA activities without action of G protein receptor kinases (GRKs). The cAMP/PKA activities are restricted within the immediate vicinity of activated β2AR, leading to selectively enhance PKA-dependent phosphorylation and stimulation of endogenous L-type calcium channel (LTCC) but not AMPA receptor in rat hippocampal neurons. Moreover, we have engineered a mutant β2AR that lacks the catecholamine binding pocket. This mutant is preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant β2AR in mouse hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which β-blocker-dependent activation of GPCRs promotes spatially restricted cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.
GSL containing mixtures, regardless of the carbohydrate size, enhance the ordering of the surrounding lipids, resulting in a larger fraction of ordered phase of the monolayer and greater dimensions of the ordered domains. Reduction of the averaged area per molecule within the ordered domains was also observed but only in the cases where there was a size mismatch between the phospholipid headgroups and GSL components, suggesting that the condensation mechanism involves the relief of steric interactions between headgroups in mixtures.
G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report that some antagonists of β adrenergic receptors (βARs) such as β-blocker carvedilol and alprenolol activate β 2 AR at nanomolar concentrations, which promote G protein signaling and cAMP/PKA activity without action of G protein receptor kinases (GRKs). The cAMP/PKA signal is restricted within the local plasma membrane domain, leading to selectively enhance PKA-dependent augment of endogenous L-type calcium channel (LTCC) activity but not AMPA receptor in hippocampal neurons. Moreover, we have engineered a mutant β 2 AR that lacks serine 204 and 207 in the catecholamine binding pocket. This mutant can be preferentially activated by carvedilol but not the orthosteric agonist isoproterenol.Carvedilol activates the mutant β 2 AR in hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which β-blocker-dependent activation of GPCRs at low ligand concentrations promotes local cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.have recently identified spatially segregated subpopulations of β 2 AR undergoing exclusive phosphorylation by GRKs or PKA in a single cell, respectively. These findings indicate specific GPCR subpopulation-based signaling branches can co-exist in a single cell (12). GRK-mediated phosphorylation promotes pro-survival and cell growth signaling via β-arrestin-dependent mitogen-activated protein kinase (MAPK/ERK) pathways, prompting the search for biased ligands that preferentially activate β-arrestin pathways (13-18). On the other hand, our recent studies show that the cAMP/PKA-dependent phosphorylation of β 2 AR controls ion channel activity at the plasma membrane in primary hippocampal neurons (12). β-blockers are thought to reduce cAMP signaling because they either reduce basal activity of βARs or block agonist-induced receptor activation. While β-blockers are successful in clinical therapies of a broad range of diseases, their utility is limited by side effects in both the CNS and peripheral tissues (19,20). Indeed, studies have revealed that some β-blockers display partial agonism and can promote receptor-Gs coupling at high concentrations in vitro (21-23). Accordingly, some β-blockers display intrinsic properties mimicking sympathetic activation (sympathomimetic β-blockers) (24-26). The mechanism remains poorly understood because classic cAMP assay do not show even minimal cAMP signal induced by these β-blockers (24,25).In this study, we show that the β-blockers carvedilol and alprenolol can promote Gs protein coupling to β 2 AR and cAMP/PKA but not GRK activity at nanomolar concentrations. Thus these β-blockers are emerging as partial agonists rather than strict antagonists in mammalian cells. This cAMP/PKA signaling is spatially restricted, selectively promoting phosphorylation of β 2 AR and Ca V 1.2 by PKA which augments LTCC activity in primary hippocampal neurons. Furt...
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