Measurement of Inverse Agonism in β-Adrenoceptors

Taira, Carlos A.; Monczor, Federico; Höcht, Christián

doi:10.1016/b978-0-12-381296-4.00003-8

Cited by 5 publications

(3 citation statements)

References 111 publications

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“…; Taira et al. ), and indeed here, we show that each concentration dependently induces progressively positive‐deflected DMR responses (Fig. A and B).…”

Section: Resultsmentioning

confidence: 98%

Dynamic mass redistribution analysis of endogenous β‐adrenergic receptor signaling in neonatal rat cardiac fibroblasts

Carter

Grisanti

et al. 2014

Pharmacology Res & Perspec

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Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). The EPIC-BT by Corning was used to measure DMR responses in primary isolated NRCF treated with various βAR and EGFR ligands. Additional molecular assays for cAMP generation and receptor internalization responses were used to correlate the DMR findings with established βAR signaling pathways. Catecholamine stimulation of NRCF induced a concentration-dependent negative DMR deflection that was competitively blocked by βAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Subtype-selective βAR ligand profiling revealed a dominant role for β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. βAR-mediated cAMP generation profiles revealed similar kinetics to DMR responses, each of which were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, G protein-independent βAR signaling through epidermal growth factor receptor (EGFR) was assessed, revealing a smaller but significant contribution of this pathway to the DMR response to βAR stimulation. Measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein-dependent and –independent pathways.

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“…; Taira et al. ), and indeed here, we show that each concentration dependently induces progressively positive‐deflected DMR responses (Fig. A and B).…”

Section: Resultsmentioning

confidence: 98%

Dynamic mass redistribution analysis of endogenous β‐adrenergic receptor signaling in neonatal rat cardiac fibroblasts

Carter

Grisanti

et al. 2014

Pharmacology Res & Perspec

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“…We examined the effects of the inverse agonist timolol[21]. At concentrations up to 50 µM, timolol did not produce any significant change in the electrical signal from either β 2 -K -62-25 or β 2 -K -72-25 (Fig.…”

Section: Resultsmentioning

confidence: 99%

β2-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors

et al. 2011

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Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K+-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K+ channel Kir6.2 and muscarinic M2 or dopaminergic D2 receptors. Here, we extend the concept to the distinct, longer β2-adrenergic receptor which, unlike M2 and D2 receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of β2 constructs. We then demonstrate how engineering of both receptor and channel can produce β2-Kir6.2 ICCRs. Specifically, removal of 62–72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the β2 ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the β2 receptor.

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“…In contrast to the inverse agonist effects of bucindolol, the partial agonist xamoterol produced an expected positive inotropic effect and carvedilol was a neutral antagonist (no positive or negative inotropic effect) [49]. Inverse agonists decrease downstream signaling to a greater extent than antagonists by binding to their cognate receptor and stabilizing the inactive conformation, decreasing signal transduction below basal levels [61].…”

Section: Pharmacogenetic Modifiersmentioning

confidence: 99%

Bucindolol hydrochloride in atrial fibrillation and concomitant heart failure

Black‐Maier

Steinberg

Piccini

2015

Expert Review of Cardiovascular Therapy

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Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia and it increases the risk of thromboembolic stroke and death. AF is common in patients with heart failure and reduced ejection fraction (HFrEF), affecting between 30 and 40% of patients with HFrEF. AF increases the risk of death and hospitalization in patients with HFrEF. Only two antiarrhythmic drugs (amiodarone and dofetilide) are guideline-recommended in patients with AF and heart failure (HF). Meta-analyses of studies of major trials in HF suggest that patients with AF/HFrEF do not benefit from conventional β-blockers. Bucindolol has shown promise in the treatment of patients with AF/HFrEF. We will explore how the shared pathophysiology of AF/HF is targeted by the unique pharmacology of bucindolol and review the existing data for bucindolol in AF/HF. We will explore findings that support a pharmacogenetically modulated effect of bucindolol in patients with polymorphisms in β1-adrenergic receptor and provide an overview of ongoing studies.

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Measurement of Inverse Agonism in β-Adrenoceptors

Cited by 5 publications

References 111 publications

Dynamic mass redistribution analysis of endogenous β‐adrenergic receptor signaling in neonatal rat cardiac fibroblasts

Dynamic mass redistribution analysis of endogenous β‐adrenergic receptor signaling in neonatal rat cardiac fibroblasts

β2-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors

Bucindolol hydrochloride in atrial fibrillation and concomitant heart failure

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