Richard A. Bond

Experimental design and analysis and their reporting: new guidance for publication in BJP

This Editorial is part of a series. To view the other Editorials in this series, visit: http://onlinelibrary.wiley.com/doi/10.1111/bph.12956/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12954/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12955/abstract and http://onlinelibrary.wiley.com/doi/10.1111/bph.13112/abstract

Inverse agonism and the regulation of receptor number

Inverse agonists are ligands that preferentially stabilize inactive conformations of G protein-coupled receptors. In a range of systems, sustained treatment with inverse agonists can produce substantially greater upregulation of receptor levels than antagonists. The use of constitutively active mutant receptors can exaggerate this effect but may also allow agonists and antagonists to mimic the effect by preventing denaturation of the mutant receptor polypeptide. In this review Graeme Milligan and Richard Bond consider the basis for these effects and their therapeutic implications.

Agonist and antagonist characterization of a putative adrenoceptor with distinct pharmacological properties from the α- and β-subtypes

1 Experiments were done to characterize a putative adrenoceptor which functions to inhibit longitudinal muscle tension development in the guinea‐pig ileum. Several phenylethylamine based agonists were investigated: BRL 37344, (−)‐isoprenaline, (+)‐isoprenaline, noradrenaline, adrenaline, and fenoterol. Propranolol and nadolol were tested as antagonists. Agonist‐induced inhibition of the contractile response to histamine was measured under equilibrium conditions with α‐adrenoceptors and muscarinic cholinoceptors inhibited. 2 Inhibitory responses were obtained to (−)‐isoprenaline and BRL 37344 that were resistant to β‐adrenoceptor blockade with propranolol (5 μm) and nadolol (10μm). These resistant responses were antagonized by much higher concentrations of nadolol (30 to 1000 μm) yielding apparent pA2 values for nadolol of 4.31 with (−)‐isoprenaline as the agonist, and 4.68 with BRL 37344 as the agonist. Similar apparent pA2 values for nadolol at the putative adrenoceptor were obtained with noradrenaline (4.79), adrenaline (4.68), and fenoterol (4.38). 3 The order and relative potency of agonists at the putative adrenoceptor was: BRL 37344 (20) > (−)‐isoprenaline (8) > noradrenaline (1) > adrenaline (0.5) > fenoterol (0.35) > (H‐)‐isoprenaline (0.27). 4 The resistance to blockade by propranolol (5 μm), the low affinity of nadolol, and the order and relative potency of agonists, suggest the presence of an adrenoceptor with distinct pharmacological characteristics from currently defined α‐ and β‐adrenoceptors.

Enhanced Gi Signaling Selectively Negates β2-Adrenergic Receptor (AR)– but Not β1-AR–Mediated Positive Inotropic Effect in Myocytes From Failing Rat Hearts

Background—Myocardial contractile response to &bgr;1- and &bgr;2-adrenergic receptor (AR) stimulation is severely impaired in chronic heart failure, in which Gi signaling and the ratio of &bgr;2/&bgr;1 are often increased. Because &bgr;2-AR but not &bgr;1-AR couples to Gs and Gi with the Gi coupling negating the Gs-mediated contractile response, we determined whether the heart failure–associated augmentation of Gi signaling contributes differentially to the defects of these &bgr;-AR subtypes and, if so, whether inhibition of Gi or selective activation of &bgr;2-AR/Gs by ligands restores &bgr;2-AR contractile response in the failing heart. Methods and Results—Cardiomyocytes were isolated from 18- to 24-month-old failing spontaneously hypertensive (SHR) or age-matched Wistar-Kyoto (WKY) rat hearts. In SHR cardiomyocytes, either &bgr;-AR subtype–mediated inotropic effect was markedly diminished, whereas Gi proteins and the &bgr;2/&bgr;1 ratio were increased. Disruption of Gi signaling by pertussis toxin (PTX) enabled &bgr;2- but not &bgr;1-AR to induce a full positive inotropic response in SHR myocytes. Furthermore, screening of a panel of &bgr;2-AR ligands revealed that the contractile response mediated by most &bgr;2-AR agonists, including zinterol, salbutamol, and procaterol, was potentiated by PTX, indicating concurrent Gs and Gi activation. In contrast, fenoterol, another &bgr;2-AR agonist, induced a full positive inotropic effect in SHR myocytes even in the absence of PTX. Conclusions—We conclude that enhanced Gi signaling is selectively involved in the dysfunction of &bgr;2- but not &bgr;1-AR in failing SHR hearts and that disruption of Gi signaling by PTX or selective activation of &bgr;2-AR/Gs signaling by fenoterol restores the blunted &bgr;2-AR contractile response in the failing heart.

Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover

The p160 steroid receptor coactivators (SRCs) SRC-1, SRC-2 [nuclear receptor coactivator (NCOA)2], and SRC-3 [amplified in breast cancer 1 (AIB1)/NCOA3] are key pleiotropic “master regulators” of transcription factor activity necessary for cancer cell proliferation, survival, metabolism, and metastasis. SRC overexpression and overactivation occur in numerous human cancers and are associated with poor clinical outcomes and resistance to therapy. In prostate cancer (PC), the p160 SRCs play critical roles in androgen receptor transcriptional activity, cell proliferation, and resistance to androgen deprivation therapy. We recently demonstrated that the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger (POZ) domain protein (SPOP) interacts directly with SRC-3 and promotes its cullin 3-dependent ubiquitination and proteolysis in breast cancer, thus functioning as a potential tumor suppressor. Interestingly, somatic heterozygous missense mutations in the SPOP substrate-binding cleft recently were identified in up to 15% of human PCs (making SPOP the gene most commonly affected by nonsynonymous point mutations in PC), but their contribution to PC pathophysiology remains unknown. We now report that PC-associated SPOP mutants cannot interact with SRC-3 protein or promote its ubiquitination and degradation. Our data suggest that wild-type SPOP plays a critical tumor suppressor role in PC cells, promoting the turnover of SRC-3 protein and suppressing androgen receptor transcriptional activity. This tumor suppressor effect is abrogated by the PC-associated SPOP mutations. These studies provide a possible explanation for the role of SPOP mutations in PC, and highlight the potential of SRC-3 as a therapeutic target in PC.

The elusive nature of intrinsic efficacy

In the discipline of pharmacology, drugs (ligands) are used as tools to elucidate the processes of biological systems. Because of this, pharmacologists strive to delineate all characteristics of drugs. Decades of research have resulted in the proposal that ligands possess two properties that are intrinsic to the ligand and are invariant of the system in which their effects are investigated. These properties are affinity (the capacity of a drug to bind to a receptor) and intrinsic efficacy (the capacity of a drug to activate or inactivate a receptor). Although affinity is a relatively easy parameter to measure with a variety of techniques, ways of quantifying intrinsic efficacy have remained elusive ever since its inception. Furthermore, recent evidence suggests that intrinsic efficacy might not be a single, ligand-dependent parameter but that agonists might have multiple intrinsic efficacies. William Clarke and Richard Bond discuss several reasons why the claim that intrinsic efficacy is a ligand-dependent parameter should be questioned, and the possible impact of these findings.

β2-Adrenoceptor signaling is required for the development of an asthma phenotype in a murine model

Chronic regular use of β2-adrenoceptor (β2-AR) agonists in asthma is associated with a loss of disease control and increased risk of death. Conversely, we have found that administration of β2-AR inverse agonists results in attenuation of the asthma phenotype in an allergen-driven murine model. Besides antagonizing agonist-induced signaling and reducing signaling by empty receptors, β-AR inverse agonists can also activate signaling by novel pathways. To determine the mechanism of the β-AR inverse agonists, we compared the asthma phenotype in β2-AR-null and wild-type mice. Antigen challenge of β2-AR-null mice produced results similar to what was observed with chronic β2-AR inverse agonist treatment, namely, reductions in mucous metaplasia, airway hyperresponsiveness (AHR), and inflammatory cells in the lungs. These results indicate that the effects of β2-AR inverse agonists are caused by inhibition of β2-AR signaling rather than by the induction of novel signaling pathways. Chronic administration of alprenolol, a β-blocker without inverse agonist properties, did not attenuate the asthma phenotype, suggesting that it is signaling by empty receptors, rather than agonist-induced β2-AR signaling, that supports the asthma phenotype. In conclusion, our results demonstrate that, in a murine model of asthma, β2-AR signaling is required for the full development of three cardinal features of asthma: mucous metaplasia, AHR, and the presence of inflammatory cells in the lungs.

The state of GPCR research in 2004

G-protein-coupled receptors (GPCRs) comprise the largest single class of cell-surface receptors, and have a history of being excellent therapeutic targets for a number of diseases. Yet several fundamental questions remain regarding their underlying biology, and many GPCRs that could be promising targets for drug discovery are still uncharacterized at the level of identified ligand or biological function. In an attempt to find some answers, we posed 20 questions of fundamental importance to the future development of the field to 20 of the worlds leading experts on GPCR research, and here we present their replies.

An inhibitory prejunctional 5-HT1-like receptor in the isolated perfused rat kidney. Apparent distinction from the 5-HT1A, 5-HT1B and 5-HT1C subtypes.

SummaryThe present study has identified a receptor for 5-hydroxytryptamine (5-HT) which functions to inhibit the stimulus-induced release of [3H] noradrenaline following sympathetic periarterial nerve stimulation to the isolated perfused rat kidney. In addition to 5-HT (IC30=4.5×10−8 mol/l), both 5-carboxamidotryptamine (IC30=8×10−9 mol/l) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl) indole (RU-24969, IC30=2.5×10−7 mol/l) acted as agonists whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was inactive. The inhibitory effect of 5-HT on the electrically-evoked release of tritium was antagonized in a concentration-dependent manner by methiothepin (IC50=4×10−9 mol/l), metergoline (IC50=4×10−8 mol/l) and methysergide (IC50=1.3×10−7 mol/l) but not by cyproheptadine, ketanserin, mesulergine, (−)-propranolol, (±)-pindolol, (±)-cyanopindolol, metoclopramide or phentolamine. It is concluded that the receptor to 5-HT conforms to general criteria defining 5-HT1-like receptors but at the present time the receptor site cannot be fitted to the designated 5-HT1A, 5-HT1B or 5-HT1C subtypes.

New perspectives regarding β2‐adrenoceptor ligands in the treatment of asthma

In the last two decades several significant changes have been proposed in the receptor theory that describes how ligands can interact with G protein‐coupled receptors (GPCRs). Here we briefly summarize the evolution of receptor theory and detail recent prominent advances. These include: (i) the existence of spontaneously active GPCRs that are capable of signalling even though they are unoccupied by any ligand; (ii) the discovery of ligands that can inactivate these spontaneously active receptors; (iii) the notion that a ligand may simultaneously activate more than one GPCR signalling pathway; and (iv) the notion that certain ligands may be able to preferentially direct receptor signalling to a specific pathway. Because the data supporting these receptor theory ideas are derived primarily from studies using artificial expression systems, the physiological relevance of these new paradigms remains in question. As a potential example of how these new perspectives in receptor theory relate to drug actions and clinical outcomes, we discuss their relevance to the recent controversy regarding the chronic use of β2‐adrenoceptor agonists in the treatment of asthma.