Adrenoceptors: Receptors, Ligands and Their Clinical Uses, Molecular Pharmacology and Assays.

The nine G protein-coupled adrenoceptor subtypes are where the endogenous catecholamines adrenaline and noradrenaline interact with cells. Since they are important therapeutic targets, over a century of effort has been put into developing drugs that modify their activity. This chapter provides an outline of how we have arrived at current knowledge of the receptors, their physiological roles and the methods used to develop ligands. Initial studies in vivo and in vitro with isolated organs and tissues progressed to cell-based techniques and the use of cloned adrenoceptor subtypes together with high-throughput assays that allow close examination of receptors and their signalling pathways. The crystal structures of many of the adrenoceptor subtypes have now been determined opening up new possibilities for drug development.

Multipathway In Vitro Pharmacological Characterization of Specialized Proresolving G Protein-Coupled Receptors.

Specialized proresolving mediators (SPMs) and their cognate G protein-coupled receptors are implicated in autoimmune disorders, including chronic inflammation, rheumatoid arthritis, systemic scleroderma, and lupus erythematosus. To date, six G protein-coupled receptors (GPCRs) have been paired with numerous endogenous and synthetic ligands. However, the function and downstream signaling of these receptors remains unclear. To address this knowledge gap, we systematically expressed each receptor in a human embryonic kindney 293 (HEK293)-Flp-In-CD8a-FLAG cell system. Each receptor was pharmacologically characterized with both synthetic and putative endogenous ligands across different signaling assays, covering both G protein-dependent (Gs, Gi, and Gq) and independent mechanisms (ß-arrestin2 recruitment). Three orphan GPCRs previously identified as SPM receptors (GPR 18, GPR32 and GPR37) failed to express in HEK 293 cells. Although we were unsuccessful in identifying an endogenous ligand for formyl peptide receptor 2 (FPR2)/lipoxin A4 receptor (ALX), with only a modest response to N-formylmethionine-leucyl-phenylalanine (fMLP), we did reveal clear signaling bias away from extracelluar signal-related kinase (ERK) 1/2 phosphorylation for the clinically tested agonist N-(2-{[4-(1,1-difluoroethyl)-1,3-oxazol-2-yl]methyl}-2H-1,2,3-triazol-4-yl)-2-methyl-5-(3-methylphenyl)-1,3-oxazole-4-carboxamide (ACT-389949), adding further evidence for its poor efficacy in two phase I studies. We also identified neuroprotectin D1 as a new leukotriene B4 receptor 1 (BLT1) agonist, implying an alternative target for the neuroprotective effects of the ligand. We confirmed activity for resolvin E1 (RvE1) at BLT1 but failed to observe any response at the chemerin1 receptor. This study provides some much-needed clarity around published receptor-ligand pairings but indicates that the expression and function of these SPM GPCRs remains very much context-dependent. In addition, the identification of signaling bias at FPR2/ALX may assist in guiding design of new FPR2/ALX agonists for the treatment of autoimmune disorders. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to comprehensibly show how several natural mediators and synthetic ligands signal through three specialized proresolving mediator GPCRs using multiple ligands from different classes across four-six endpoint signaling assays. This study discovers new ligand pairings, refutes others, reveals poly-pharmacology, and identifies biased agonism in formyl peptide receptor 2/lipoxin A4 receptor pharmacology. This study highlights the potential of these receptors in treating specific autoimmune diseases, including rheumatoid arthritis, systemic scleroderma, and systemic lupus erythematosus.

G Protein-Coupled Receptors Targeting Insulin Resistance, Obesity, and Type 2 Diabetes Mellitus.

G protein-coupled receptors (GPCRs) continue to be important discovery targets for the treatment of type 2 diabetes mellitus (T2DM). Many GPCRs are directly involved in the development of insulin resistance and ß-cell dysfunction, and in the etiology of inflammation that can lead to obesity-induced T2DM. This review summarizes the current literature describing a number of well-validated GPCR targets, but also outlines several new and promising targets for drug discovery. We highlight the importance of understanding the role of these receptors in the disease pathology, and their basic pharmacology, which will pave the way to the development of novel pharmacological probes that will enable these targets to fulfill their promise for the treatment of these metabolic disorders.

AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts: Impact on the Therapeutic Targeting of Renal and Cardiac Fibrosis.

BACKGROUND: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT1R), which is expressed on myofibroblasts along with RXFP1 and AT2R, is unknown. METHODS: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo. RESULTS: The AT1R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT1Rs but that constitutive AT1R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT2R agonist (compound 21). CONCLUSIONS: These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.

Structure-function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins.

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity.

International Union of Basic and Clinical Pharmacology. XCV. Recent advances in the understanding of the pharmacology and biological roles of relaxin family peptide receptors 1-4, the receptors for relaxin family peptides.

Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1-4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gα(s), Gα(i), and Gα(o) proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gα(s)- and Gα(o)-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gα(i)/Gα(o) proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and ß-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1-4, the challenges facing the field, and current prospects for new therapeutics.

Isoform-Specific Biased Agonism of Histamine H3 Receptor Agonists.

The human histamine H3 receptor (hH3R) is subject to extensive gene splicing that gives rise to a large number of functional and nonfunctional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signaling, this has not been studied for the H3R; further, it is unknown whether splice variants of the same receptor engender the same or differential biased signaling. Herein, we profiled the pharmacology of histamine receptor agonists at the two most abundant hH3R splice variants (hH3R445 and hH3R365) across seven signaling endpoints. Both isoforms engender biased signaling, notably for 4-[3-(benzyloxy)propyl]-1H-imidazole (proxyfan) [e.g., strong bias toward phosphorylation of glycogen synthase kinase 3ß (GSK3ß) via the full-length receptor] and its congener 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether (iodoproxyfan), which are strongly consistent with the former's designation as a "protean" agonist. The 80 amino acid IL3 deleted isoform hH3R365 is more permissive in its signaling than hH3R445: 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate (imetit), proxyfan, and iodoproxyfan were all markedly biased away from calcium signaling, and principal component analysis of the full data set revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signaling between the two isoforms. Strikingly, hH3R365 was completely unable to stimulate GSK3ß phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signaling via isoforms of the same G protein-coupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants.

High throughput, quantitative analysis of human osteoclast differentiation and activity.

Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis. The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an 'osteoclast population' is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software. Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.

Orthosteric, Allosteric and Biased Signalling at the Relaxin-3 Receptor RXFP3.

Relaxin-3 is a neuropeptide that has roles in stress, memory and appetite regulation. The peptide acts on its cognate receptor RXFP3 to induce coupling to inhibitory G proteins to inhibit adenylyl cyclase and activate MAP-kinases such as ERK1/2, p38MAPK and JNK. Other relaxin family peptides can activate the receptor to produce alternative patterns of signalling and there is an allosteric modulator 135PAM1 that displays probe-selectivity. There are now a variety of selective peptide agonists and antagonists that will assist in the determination of the physiological roles of the relaxin-RXFP3 system and its potential as a drug target.

Label-Free Kinetics: Exploiting Functional Hemi-Equilibrium to Derive Rate Constants for Muscarinic Receptor Antagonists.

Drug receptor kinetics is as a key component in drug discovery, development, and efficacy; however, determining kinetic parameters has historically required direct radiolabeling or competition with a labeled tracer. Here we present a simple approach to determining the kinetics of competitive antagonists of G protein-coupled receptors by exploiting the phenomenon of hemi-equilibrium, the state of partial re-equilibration of agonist, antagonist, and receptor in some functional assays. Using functional [Ca(2+)]i-flux and extracellular kinases 1 and 2 phosphorylation assays that have short incubation times and therefore are prone to hemi-equilibrium "behaviors," we investigated a wide range of structurally and physicochemically distinct muscarinic acetylcholine receptor antagonists. Using a combined operational and hemi-equilibrium model of antagonism to both simulate and analyze data, we derived estimates of association and dissociation rates for the test set of antagonists, identifying both rapidly dissociating (4-DAMP, himbacine) and slowly dissociating (tiotropium, glycopyrrolate) ligands. The results demonstrate the importance of assay incubation time and the degree of receptor reserve in applying the analytical model. There was an excellent correlation between estimates of antagonist pK(B), k(on), and k(off) from functional assays and those determined by competition kinetics using whole-cell [(3)H]N-methylscopolamine binding, validating this approach as a rapid and simple method to functionally profile receptor kinetics of competitive antagonists in the absence of a labeled tracer.

Relaxin requires the angiotensin II type 2 receptor to abrogate renal interstitial fibrosis.

Fibrosis is a hallmark of chronic kidney disease, for which there is currently no effective cure. The hormone relaxin is emerging as an effective antifibrotic therapy; however, its mechanism of action is poorly understood. Recent studies have shown that relaxin disrupts the profibrotic actions of transforming growth factor-ß1 (TGF-ß1) by its cognate receptor, relaxin family peptide receptor 1 (RXFP1), extracellular signal-regulated kinase phosphorylation, and a neuronal nitric oxide synthase-dependent pathway to abrogate Smad2 phosphorylation. Since angiotensin II also inhibits TGF-ß1 activity through its AT2 receptor (AT2R), we investigated the extent to which relaxin interacts with the AT2R. The effects of the AT2R antagonist, PD123319, on relaxin activity were examined in primary rat kidney myofibroblasts, and in kidney tissue from relaxin-treated male wild-type and AT2R-knockout mice subjected to unilateral ureteric obstruction. Relaxin's antifibrotic actions were significantly blocked by PD123319 in vitro and in vivo, or when relaxin was administered to AT2R-knockout mice. While heterodimer complexes were formed between RXFP1 and AT2Rs independent of ligand binding, relaxin did not directly bind to AT2Rs but signaled through RXFP1-AT2R heterodimers to induce its antifibrotic actions. These findings highlight a hitherto unrecognized interaction that may be targeted to control fibrosis progression.

Role of G protein-coupled receptor kinases (GRKs) in ß2 -adrenoceptor-mediated glucose uptake.

Truncation of the C-terminal tail of the ß2 -AR, transfection of ßARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the ß2 -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant ß2 -ARs were generated and receptor affinity for [3 H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by ß2 -AR agonists, cAMP accumulation, GLUT4 translocation, [3 H]-2-deoxyglucose uptake, and ß2 -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between ß2 -AR and ß-arrestin2 or between ß2 -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to ß2 -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to ß2 -AR agonists occurred in CHO-GLUT4myc cells expressing ß2 -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type ß2 -AR. However, ß2 -ARs lacking phosphorylation sites failed to recruit ß-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the ß2 -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

The novel adrenergic agonist ATR-127 targets skeletal muscle and brown adipose tissue to tackle diabesity and steatohepatitis.

OBJECTIVE: Simultaneous activation of ß2- and ß3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of ß1-ARs - and thus inducing cardiovascular complications - are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel ß2-and ß3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models. METHODS: In the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective ß-AR agonist isoprenaline across various rodent ß-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5 mg/kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis. RESULTS: Exposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis. CONCLUSIONS: Our results demonstrate that ATR-127 is a highly effective, novel ß2- and ß3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies.

Interaction with caveolin-1 modulates G protein coupling of mouse ß3-adrenoceptor.

Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knock-out mice suggest that ß(3)-adrenoceptor (ß(3)-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the ß(3)-AR or solely with downstream signaling proteins. We have addressed this question by examining the impact of membrane rafts and caveolin-1 on the differential signaling of mouse ß(3a)- and ß(3b)-AR isoforms that diverge at the distal C terminus. Only the ß(3b)-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the ß(3a)-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the ß(3)-AR agonist CL316243 became PTX-sensitive, suggesting Gα(i/o) coupling. The ß(3a)-AR C terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant ß(3a)-ARs (F389A/Y392A/F398A or P384S/F389A) promoted PTX-sensitive cAMP responses, and in situ proximity assays demonstrated an association between caveolin-1 and the wild type ß(3a)-AR but not the mutant receptors. In membrane preparations, the ß(3b)-AR activated Gα(o) and mediated PTX-sensitive cAMP responses, whereas the ß(3a)-AR did not activate Gα(i/o) proteins. The endogenous ß(3a)-AR displayed Gα(i/o) coupling in brown adipocytes from caveolin-1 knock-out mice or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the ß(3a)-AR with caveolin inhibits coupling to Gα(i/o) proteins and suggest that signaling is modulated by a raft-enriched complex containing the ß(3a)-AR, caveolin-1, Gα(s), and adenylyl cyclase.

Quantification of functional selectivity at the human α(1A)-adrenoceptor.

Although G protein-coupled receptors are often categorized in terms of their primary coupling to a given type of Gα protein subunit, it is now well established that many show promiscuous coupling and activate multiple signaling pathways. Furthermore, some agonists selectively activate signaling pathways by promoting interaction between distinct receptor conformational states and particular Gα subunits or alternative signaling proteins. We have tested the capacity of agonists to stimulate Ca(2+) release, cAMP accumulation, and changes in extracellular acidification rate (ECAR) at the human α(1A)-adrenoceptor. Signaling bias factors were determined by novel application of an operational model of agonism and compared with the reference endogenous agonist norepinephrine; values significantly different from 1.0 indicated an agonist that promoted receptor conformations distinct from that favored by norepinephrine. Oxymetazoline was a full agonist for ECAR and a partial agonist for Ca(2+) release (bias factor 8.2) but failed to stimulate cAMP production. Phenylephrine showed substantial bias toward ECAR versus Ca(2+) release or cAMP accumulation (bias factors 21 and 33, respectively) but did not display bias between Ca(2+) and cAMP pathways. Cirazoline and N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide (A61603) displayed bias toward cAMP relative to Ca(2+) release (bias factors of 7.4 and 8.6). It is noteworthy that epinephrine, a second endogenous adrenoceptor agonist, did not display bias relative to norepinephrine. Our finding that phenylephrine displayed significant signaling bias, despite being highly similar in structure to epinephrine, indicates that subtle differences in agonist-receptor interaction can affect conformational changes in cytoplasmic domains and thereby modulate the repertoire of effector proteins that are activated.

α2-Adrenoceptors activate noradrenaline-mediated glycogen turnover in chick astrocytes.

In the brain, glycogen is primarily stored in astrocytes where it is regulated by several hormones/neurotransmitters, including noradrenaline that controls glycogen breakdown (in the short term) and synthesis. Here, we have examined the adrenoceptor (AR) subtype that mediates the glycogenic effect of noradrenaline in chick primary astrocytes by the measurement of glycogen turnover (total (14) C incorporation of glucose into glycogen) following noradrenergic activation. Noradrenaline and insulin increased glycogen turnover in a concentration-dependent manner. The effect of noradrenaline was mimicked by stimulation of α(2) -ARs (and to a lesser degree by ß(3) -ARs), but not by stimulation of α(1) -, ß(1) -, or ß(2) -ARs, and occurred only in astrocytes and not neurons. In chick astrocytes, studies using RT-PCR and radioligand binding showed that α(2A) - and α(2C) -AR mRNA and protein were present. α(2) -AR- or insulin-mediated glycogen turnover was inhibited by phosphatidylinositol-3 kinase inhibitors, and both insulin and clonidine caused phosphorylation of Akt and glycogen synthase kinase-3 in chick astrocytes. α(2) -AR but not insulin-mediated glycogen turnover was inhibited by pertussis toxin pre-treatment indicating involvement of Gi/o proteins. These results show that the increase in glycogen turnover caused by noradrenaline is because of activation of α(2) -ARs that increase glycogen turnover in astrocytes utilizing a Gi/o-PI3K pathway.

Modulation of the glucagon-like peptide-1 receptor signaling by naturally occurring and synthetic flavonoids.

The glucagon-like peptide 1 receptor (GLP-1R) is a promising target for the treatment of type II diabetes mellitus because of its role in metabolic homeostasis. In recent years, difficulties with peptide therapies have driven the search for small-molecule compounds to modulate the activity of this receptor. We recently identified quercetin, a naturally occurring flavonoid, as a probe-dependent, pathway-selective allosteric modulator of GLP-1R-mediated signaling. Using Chinese hamster ovary cells expressing the human GLP-1R, we have now extended this work to identify the structural requirements of flavonoids to modify GLP-1R binding and signaling (cAMP formation and intracellular Ca(2+) mobilization) of each of the GLP-1R endogenous agonists, as well as the clinically used exogenous peptide mimetic exendin-4. This study identified a chemical series of hydroxyl flavonols with the ability to selectively augment calcium (Ca(2+)) signaling in a peptide agonist-specific manner, with effects only on truncated GLP-1 peptides [GLP-1(7-36)NH(2) and GLP-1(7-37)] and exendin-4, but not on oxyntomodulin or full-length GLP-1 peptides [GLP-1(1-36)NH(2) and GLP-1(1-37)]. In addition, the 3-hydroxyl group on the flavone backbone (i.e., a flavonol) was essential for this activity, however insufficient on its own, to produce the allosteric effects. In contrast to hydroxyl flavonols, catechin had no effect on peptide-mediated Ca(2+) signaling but negatively modulated peptide-mediated cAMP formation in a probe-dependent manner. These data represent a detailed examination of the action of different flavonoids on peptide agonists at the GLP-1R and may aid in the development of future small molecule compounds targeted at this receptor.

Pharmacological Insights Into Safety and Efficacy Determinants for the Development of Adenosine Receptor Biased Agonists in the Treatment of Heart Failure.

Adenosine A1 receptors (A1R) are a potential target for cardiac injury treatment due to their cardioprotective/antihypertrophic actions, but drug development has been hampered by on-target side effects such as bradycardia and altered renal hemodynamics. Biased agonism has emerged as an attractive mechanism for A1R-mediated cardioprotection that is haemodynamically safe. Here we investigate the pre-clinical pharmacology, efficacy and side-effect profile of the A1R agonist neladenoson, shown to be safe but ineffective in phase IIb trials for the treatment of heart failure. We compare this agent with the well-characterized, pan-adenosine receptor (AR) agonist NECA, capadenoson, and the A1R biased agonist VCP746, previously shown to be safe and cardioprotective in pre-clinical models of heart failure. We show that like VCP746, neladenoson is biased away from Ca2+ influx relative to NECA and the cAMP pathway at the A1R, a profile predictive of a lack of adenosine-like side effects. Additionally, neladenoson was also biased away from the MAPK pathway at the A1R. In contrast to VCP746, which displays more 'adenosine-like' signaling at the A2BR, neladenoson was a highly selective A1R agonist, with biased, weak agonism at the A2BR. Together these results show that unwanted hemodynamic effects of A1R agonists can be avoided by compounds biased away from Ca2+ influx relative to cAMP, relative to NECA. The failure of neladenoson to reach primary endpoints in clinical trials suggests that A1R-mediated cAMP inhibition may be a poor indicator of effectiveness in chronic heart failure. This study provides additional information that can aid future screening and/or design of improved AR agonists that are safe and efficacious in treating heart failure in patients.

GPR55 regulates the responsiveness to, but does not dimerise with, α1A-adrenoceptors.

Emerging evidence suggests that G protein coupled receptor 55 (GPR55) may influence adrenoceptor function/activity in the cardiovascular system. Whether this reflects direct interaction (dimerization) between receptors or signalling crosstalk has not been investigated. This study explored the interaction between GPR55 and the alpha 1A-adrenoceptor (α1A-AR) in the cardiovascular system and the potential to influence function/signalling activities. GPR55 and α1A-AR mediated changes in both cardiac and vascular function was assessed in male wild-type (WT) and GPR55 homozygous knockout (GPR55-/-) mice by pressure volume loop analysis and isolated vessel myography, respectively. Dimerization of GPR55 with the α1A-AR was examined in transfected Chinese hamster ovary-K1 (CHO-K1) cells via Bioluminescence Resonance Energy Transfer (BRET). GPR55 and α1A-AR mediated signalling (extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation) was investigated in neonatal rat ventricular cardiomyocytes using AlphaScreen proximity assays. GPR55-/- mice exhibited both enhanced pressor and inotropic responses to A61603 (α1A-AR agonist), while in isolated vessels, A61603 induced vasoconstriction was attenuated by a GPR55-dependent mechanism. Conversely, GPR55-mediated vasorelaxation was not altered by pharmacological blockade of α1A-ARs with tamsulosin. While cellular studies demonstrated that GPR55 and α1A-AR failed to dimerize, pharmacological blockade of GPR55 altered α1A-AR mediated signalling and reduced ERK1/2 phosphorylation. Taken together, this study provides evidence that GPR55 and α1A-AR do not dimerize to form heteromers, but do interact at the signalling level to modulate the function of α1A-AR in the cardiovascular system.

Deletion of GPR21 improves glucose homeostasis and inhibits the CCL2-CCR2 axis by divergent mechanisms.

INTRODUCTION: A potential role for the orphan G protein-coupled receptor, GPR21, in linking immune cell infiltration into tissues and obesity-induced insulin resistance has been proposed, although limited studies in mice are complicated by non-selective deletion of Gpr21. RESEARCH DESIGN AND METHODS: We hypothesized that a Gpr21-selective knockout mouse model, coupled with type 2 diabetes patient samples, would clarify these issues and enable clear assessment of GPR21 as a potential therapeutic target. RESULTS: High-fat feeding studies in Gpr21-/- mice revealed improved glucose tolerance and modest changes in inflammatory gene expression. Gpr21-/- monocytes and intraperitoneal macrophages had selectively impaired chemotactic responses to monocyte chemoattractant protein (MCP)-1, despite unaltered expression of Ccr2. Further genotypic analysis revealed that chemotactic impairment was due to dysregulated monocyte polarization. Patient samples revealed elevated GPR21 expression in peripheral blood mononuclear cells in type 2 diabetes, which was correlated with both %HbA1c and fasting plasma glucose levels. CONCLUSIONS: Collectively, human and mouse data suggest that GPR21 influences both glucose homeostasis and MCP-1/CCL2-CCR2-driven monocyte migration. However, a Gpr21-/- bone marrow transplantation and high-fat feeding study in mice revealed no effect on glucose homeostasis, suggesting that there is no (or limited) overlap in the mechanism involved for monocyte-driven inflammation and glucose homeostasis.