Combined β-adrenergic and corticosteroid receptor activation regulates AMPA receptor function in hippocampal neurons

Shortly after stress, limbic neurons are exposed to high levels of noradrenaline and corticosterone. These hormones are necessary for optimal behavioural adaptation. Behavioural effects critically depend on noradrenaline acting via β-adrenergic receptors, but these effects are strongly modulated by corticosterone, indicating putative interactions between the two hormones. Since both noradrenaline and corticosterone are known to quickly affect properties of AMPA-type glutamate receptors (AMPAR), we here examined - in hippocampal neurons - three parameters which give insight in the functionality of AMPARs: phosphorylation, surface expression and spontaneous synaptic transmission. In homogenates of adult hippocampal slices, application of corticosterone (30 nM for 15 min) by itself did not affect phosphorylation of the AMPAR GluA1 subunit at S845 or S831. Co-application of the β-adrenergic receptor agonist isoproterenol (10 μM) largely increased S845 (but not S831) phosphorylation. Corticosterone also did not change GluA1 and GluA2 surface expression in hippocampal primary cultures. However, combined administration of corticosterone and 1 μM isoproterenol - which by itself was ineffective - enhanced surface expression. Interestingly, 10 μM isoproterenol alone enhanced GluA1 surface expression, but this was decreased by corticosterone. Finally, in hippocampal primary cultures, the inter-event interval of miniature excitatory postsynaptic currents (mEPSCs) was decreased by the combination of 1 μM isoproterenol and corticosterone (which were ineffective by themselves) while the same combination did not affect the amplitude. We conclude that AMPAR phosphorylation, surface expression and mEPSC inter-event interval respond most strongly to a combination of corticosterone and β-adrenergic receptors. These combined hormonal effects on glutamate transmission might contribute to their memory-enhancing effects.     

Current status of drug receptor nomenclature: receptor closure? The role of NC-IUPHAR

Pharmacology is at a crucial point, because we now have access to sequences, by homology, for almost all of the receptors in the human genome. The International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) has set up > 50 subcommittees to define the receptors, and their recommendations, when approved, are posted on a website freely available to all scientists. A major new effort is to functionally define relevant receptor polymorphisms. This initiative is open to all, and works only because of the freely given voluntary effort of scientists.     

Discovery of aminobenzyloxyarylamides as κ opioid receptor selective antagonists: application to preclinical development of a κ opioid receptor antagonist receptor occupancy tracer

Arylphenylpyrrolidinylmethylphenoxybenzamides were found to have high affinity and selectivity for κ opioid receptors. On the basis of receptor binding assays in Chinese hamster ovary (CHO) cells expressing cloned human opioid receptors, (S)-3-fluoro-4-(4-((2-(3-fluorophenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide (25) had a K(i) = 0.565 nM for κ opioid receptor binding while having a K(i) = 35.8 nM for μ opioid receptors and a K(i) = 211 nM for δ opioid receptor binding. Compound 25 was also a potent antagonist of κ opioid receptors when tested in vitro using a [(35)S]-guanosine 5'O-[3-thiotriphosphate] ([(35)S]GTP-γ-S) functional assay in CHO cells expressing cloned human opioid receptors. Compounds were also evaluated for potential use as receptor occupancy tracers. Tracer evaluation was done in vivo, using liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods, precluding the need for radiolabeling. (S)-3-Chloro-4-(4-((2-(pyridine-3-yl)pyrrolidin-1-yl)methyl)phenoxy)benzamide (18) was found to have favorable properties for a tracer for receptor occupancy, including good specific versus nonspecific binding and good brain uptake.     

Pyrazolo-triazolo-pyrimidine derivatives as adenosine receptor antagonists: a possible template for adenosine receptor subtypes?

Adenosine, a widely distributed modulator, regulates many physiological functions through specific cell membrane G-protein-coupled receptors classified as A(1), A(2A), A(2B) and A(3). An intense medicinal chemistry effort made over the last 20 years has led to a variety of selective adenosine receptor agonists and antagonists. In particular, the pyrazolo-triazolo-pyrimidine nucleus has been strongly investigated in the last years by our group. All the modifications performed and a tentative of structure-activity-relationship is reported. In fact, the combination of different substitutions at the N(7), N(8) and N(5) positions afford compounds which showed good affinity and selectivity for the different adenosine receptor subtypes. The data herein summarized, permit to speculate on the use of this nucleus as possible template for the adenosine receptor subtypes.     

The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen-related receptor α protein

BACKGROUND AND PURPOSE

AM251 is an inverse agonist of the cannabinoid 1 receptor (CB₁R) that can exert ‘off-target’ effects in vitro and in CB₁R knock-out mice. AM251 is also potent at modulating tumour cell growth, suggesting that growth factor-mediated oncogenic signalling could be regulated by AM251. Since dysregulation of the EGF receptor has been associated with carcinogenesis, we examined AM251 regulation of EGF receptor (EGFR) expression and function.

EXPERIMENTAL APPROACH

The various biological functions of AM251 were measured in CB₁R-negative human cancer cells. Pharmacological and genetic approaches were used to validate the data.

KEY RESULTS

The mRNA levels for EGFR and its associated ligands, including HB-EGF, were induced several fold in PANC-1 and HCT116 cells in response to AM251. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Exposure to XCT790, a synthetic inverse agonist of the orphan nuclear oestrogen-related receptor α (ERRα), also induced EGFR and HB-EGF expression to the same extent as AM251, whereas pretreatment with the ERRα-selective agonist, biochanin A, blunted AM251 actions. AM251 promoted the degradation of ERRα protein without loss of the corresponding mRNA. Knock-down of ERRα by siRNA-based approach led to constitutive induction of EGFR and HB-EGF levels, and eliminated the biological responses of AM251 and XCT790. Finally, AM251 displaced diethylstilbestrol prebound to the ligand-binding domain of ERRα.

CONCLUSIONS AND IMPLICATIONS

AM251 up-regulates EGFR expression and signalling via a novel non-CB₁R-mediated pathway involving destabilization of ERRα protein in selected cancer cell lines.     

Anti-nociception mediated by a κ opioid receptor agonist is blocked by a δ receptor agonist

BACKGROUND AND PURPOSE

The opioid receptor family comprises four structurally homologous but functionally distinct sub-groups, the μ (MOP), δ (DOP), κ (KOP) and nociceptin (NOP) receptors. As most opioid agonists are selective but not specific, a broad spectrum of behaviours due to activation of different opioid receptors is expected. In this study, we examine whether other opioid receptor systems influenced KOP-mediated antinociception.

EXPERIMENTAL APPROACH

We used a tail withdrawal assay in C57Bl/6 mice to assay the antinociceptive effect of systemically administered opioid agonists with varying selectivity at KOP receptors. Pharmacological and genetic approaches were used to analyse the interactions of the other opioid receptors in modulating KOP-mediated antinociception.

KEY RESULTS

Etorphine, a potent agonist at all four opioid receptors, was not anti-nociceptive in MOP knockout (KO) mice, although etorphine is an efficacious KOP receptor agonist and specific KOP receptor agonists remain analgesic in MOP KO mice. As KOP receptor agonists are aversive, we considered KOP-mediated antinociception might be a form of stress-induced analgesia that is blocked by the anxiolytic effects of DOP receptor agonists. In support of this hypothesis, pretreatment with the DOP antagonist, naltrindole (10 mg·kg⁻¹), unmasked etorphine (3 mg·kg⁻¹) antinociception in MOP KO mice. Further, in wild-type mice, KOP-mediated antinociception by systemic U50,488H (10 mg·kg⁻¹) was blocked by pretreatment with the DOP agonist SNC80 (5 mg·kg⁻¹) and diazepam (1 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

Systemic DOP receptor agonists blocked systemic KOP antinociception, and these results identify DOP receptor agonists as potential agents for reversing stress-driven addictive and depressive behaviours mediated through KOP receptor activation.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Protective effects of glycyrrhizin against β₂-adrenergic receptor agonist-induced receptor internalization and cell apoptosis

It has been reported that treatment with β? adrenergic receptor (β?AR) agonist bronchodilators may result in airway β?ARs internalization and cardiac muscle cells apoptosis. This could lead to the loss of pharmacological effect of β?AR agonists and increase adverse cardiovascular events in asthma patients receiving β?AR agonist therapy. Glycyrrhizin, the major bioactive component of licorice root extract, has been reported to exhibit protective effect on respiratory system. Here, we investigate the effects of glycyrrhizin against β?AR agonist salbutamol-induced receptor internalization and cell apoptosis. In our study, the live cell confocal imaging and fixed-cell enzyme-linked immunosorbent assay (ELISA) assay revealed that glycyrrhizin significantly inhibited salbutamol-induced surface β?AR internalization. The underlying mechanisms were then identified to be that glycyrrhizin could reduce the association of β?ARs with β-arrestins and clathrin heavy chain as well as the level of G protein-coupled receptor kinase (GRK) mediated phosphorylation of β?ARs. The inhibition of receptor internalization by glycyrrhizin further lead to stabilization of the β?AR mRNA and protein expression, thus amplified the transmembrane signaling via the β?ARs. We also proved that glycyrrhizin could profoundly attenuate salbutamol-induced early cellular apoptosis by regulating the expressions of B-cell lymphoma 2 (Bcl-2) family genes. Taken together, our results suggest that glycyrrhizin exhibits protective effects against β?AR agonist-induced receptor internalization and cell apoptosis. These findings might have practical implications for future strategies of combined application of glycyrrhizin with β?AR receptor agonists to improve the efficacy of bronchodilators in patients with asthma and chronic obstructive pulmonary disease (COPD).     

Thiophene bioisosteres of spirocyclic σ receptor ligands: relationships between substitution pattern and σ receptor affinity

On the basis of the 6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] framework, a series of more than 30 σ ligands with versatile substituents in 1-, 2'-, and 6'-position has been synthesized and pharmacologically evaluated in order to find novel structure-affinity relationships. It was found that a cyclohexylmethyl residue at the piperidine N-atom instead of a benzyl moiety led to increased σ(2) affinity and therefore to decreased σ(1)/σ(2) selectivity. Small substituents (e.g., OH, OCH(3), CN, CH(2)OH) in 6'-position adjacent to the O-atom were well tolerated by the σ(1) receptor. Removal of the substituent in 6'-position resulted in very potent but unselective σ ligands (13). A broad range of substituents with various lipophilic and H-bond forming properties was introduced in 2'-position adjacent to the S-atom without loss of σ(1) affinity. However, very polar and basic substituents in both 2'- and 6'-position decreased the σ(1) affinity considerably. It is postulated that the electron density of the thiophene moiety has a big impact on the σ(1) affinity.     

In vivo receptor occupancy assay of histamine H₃ receptor antagonist in rats using non-radiolabeled tracer

IntroductionRapid and reliable preclinical receptor occupancy measurement at the target organ in relevant species is critical in accelerating the drug hunting process. The aim of this study was to develop in vivo receptor occupancy assay for histamine H₃ receptors (H₃R) using the non-radiolabeled GSK189254 as a tracer and to correlate the occupancy–exposure relationship for H₃R antagonists in the rats.MethodsIn vivo tracer characterization studies like brain regional distribution, dose and time dependent uptake were carried out for GSK189254 in the male Wistar rats after intravenous administration. The tracer specificity was validated by pretreatment with H₃ antagonists like ciproxifan, thioperamide, and GSK334429. The brain regional tracer levels and H₃R antagonist concentrations in plasma and brain were quantified using liquid chromatography tandem mass spectrometry. Receptor occupancy was calculated using the ratio of total binding (striatum or frontal cortex) to the nonspecific binding (cerebellum) of the tracer in animals pretreated with H₃R antagonist.ResultsHigh degree of selective distribution of GSK189254 was found in striatum, frontal cortex, and low level in the cerebellum. Regional distribution of GSK189254 in the rat brain was consistent to that of H₃R distribution mapped using ³H or ¹¹C-GSK189254 in human, porcine, and rat. The calculated occupancy ED₅₀ values in the frontal cortex were 0.14, 1.58, and 0.14 mg/kg for ciproxifan, thioperamide, and GSK334429, respectively. The plasma EC₅₀ values (ng/mL) were found to be 2.33, 292.2, and 3.54 for ciproxifan, thioperamide and GSK334429, respectively.DiscussionResults from mass spectroscopy based approach to determine H₃R occupancy in rat brain is comparable with reported radiolabeled method by scintillation spectroscopy. In conclusion, non-radiolabeled GSK189254 was successfully employed as a tracer for assessing the H₃R occupancy in rats and it can be used as a preclinical tool for evaluation of novel H₃R ligands in the drug discovery.     

Estrogen,selective estrogen receptor modulators and now mechanism-specific ligands of the estrogen or androgen receptor?

Estrogen, the traditional treatment of both short- and long-term consequences of the menopause, has fallen into disfavor. Thus, selective estrogen receptor modulators (SERMs) have been used as an alternative approach to activate estrogen signaling pathways in a tissue-specific manner. In addition, recent findings indicate that some compounds might activate specific estrogen signaling pathways that are beneficial without affecting other pathways that might lead to the harmful side-effects associated with estrogen. These so-called mechanism-specific ligands of estrogen or androgen receptors hold considerable promise as novel approaches to treat (or at least ameliorate) the consequences of estrogen deficiency.     

Are CRF receptor antagonists potential antidepressants?

Corticotropin-releasing factor (CRF) is the major regulator of the hypothalamic-pituitary-adrenal (HPA) axis, and plays a key role in coordinating the endocrine, as well as autonomic and behavioral responses of an organism to stress. Direct CNS administration of CRF to laboratory animals produces an aggregate of effects that mimic the mammalian stress response. Impeding CRF function with CNS administration of a peptidergic CRF antagonist can block these manifestations of the stress response whether produced by exogenous CRF or occurring naturally in response to a stressor. A role for hypersecretion of CRF in the pathophysiology of depression is suggested by the finding that CNS administration of CRF mirrors many of the signs and symptoms utilized as diagnostic criteria for major depression. In addition, a large body of clinical evidence points to excess hypothalamic secretion of CRF and an accompanying HPA axis hyperactivity in patients with major depression. The recent development of selective, small molecule CRF(1) receptor antagonists, which block the effects of CRF both in vitro and in vivo, suggest that these compounds may be effective in the treatment of affective and anxiety disorders. Early evidence indicates that these agents possess anxiolytic and antidepressant activity in animal behavioral models. Copyright 2001 John Wiley & Sons, Ltd.     

β-Arrestin-mediated receptor trafficking and signal transduction

β-Arrestins function as endocytic adaptors and mediate trafficking of a variety of cell-surface receptors, including seven-transmembrane receptors (7TMRs). In the case of 7TMRs, β-arrestins carry out these tasks while simultaneously inhibiting upstream G-protein-dependent signaling and promoting alternate downstream signaling pathways. The mechanisms by which β-arrestins interact with a continuously expanding ensemble of protein partners and perform their multiple functions including trafficking and signaling are currently being uncovered. Molecular changes at the level of protein conformation as well as post-translational modifications of β-arrestins probably form the basis for their dynamic interactions during receptor trafficking and signaling. It is becoming increasingly evident that β-arrestins, originally discovered as 7TMR adaptor proteins, indeed have much broader and more versatile roles in maintaining cellular homeostasis. In this review paper, we assess the traditional and novel functions of β-arrestins and discuss the molecular attributes that might facilitate multiple interactions in regulating cell signaling and receptor trafficking.     

Investigational A₃ adenosine receptor targeting agents

INTRODUCTION: Adenosine is an endogenous nucleoside that accumulates in the extracellular space in response to metabolic stress and cell damage. Extracellular adenosine is a signaling molecule that signals by activating four GPCRs: the A(1), A(2A), A(2B) and A(3) receptors. Since the discovery of A(3) adenosine receptors, accumulating evidence has identified these receptors as potential targets for therapeutic intervention. AREAS COVERED: A(3) adenosine receptors are expressed on the surface of most immune cell types, including neutrophils, macrophages, dendritic cells, lymphocytes and mast cells. A(3) adenosine receptor activation on immune cells governs a broad array of immune cell functions, which include cytokine production, degranulation, chemotaxis, cytotoxicity, apoptosis and proliferation. In accordance with their multitudinous immunoregulatory actions, targeting A(3) adenosine receptors has been shown to impact the course of a wide spectrum of immune-related diseases, such as asthma, rheumatoid arthritis, cancer, ischemia and inflammatory disorders. EXPERT OPINION: Given the existence of both preclinical and early clinical data supporting the utility of A(3) adenosine receptor ligands in treating immune-related diseases, further development of A(3) adenosine receptor ligands is anticipated.     

Pharmacology and toxicology of fibrates as hypolipidemic drugs mediated by nuclear receptor peroxisome proliferator—activated receptor

PPAR（peroxisome proliferator-activated receptor) is a family of nuclear receptor.In recent years,it has been focused for the discovery and development of new drugs which are mediated by PPARs.Fibrate hypolipidemic drugs are the specific and potent ligands to PPAR alpha and have been widely used for the treatment of hyperlipidemia.But these drugs induce hepatocarcinogenesis in rodent animals after the long-term administration.However,there are species differences on these phenomena which are not seen in mammals ioncluding human.To clarify the mechanism of carcinogenesis by these drugs in important for the evaluation of safety of these drugs in human.     

Functional efficacy of adenosine A₂A receptor agonists is positively correlated to their receptor residence time

BACKGROUND AND PURPOSE The adenosine A(2A) receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A(2A) receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A(2A) receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACH We set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A(2A) receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A(2A) receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTS A simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A(2A) receptor ligands at their receptor. A correlation was observed between the receptor residence time of A(2A) receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A(2A) receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONS This study indicates that the molecular basis of different agonist efficacies at the A(2A) receptor lies within their different residence times at this receptor.     

Solubilization of a mammalian β-adrenergic receptor

Summary Binding sites for iodohydroxybenzylpindolol with characteristics of a ₂-adrenergic receptor have been identified in a crude membrane fraction from guinea-pig lung. The binding sites could be solubilized by treatment of the membrane fraction with digitonin. Upon solubilization receptors retain their ₂-adrenergic type as indicated by the rank order of potencies of agonists in binding-inhibition experiments. The solubilized receptors demonstrate a marked increase in affinity for agonists compared with particulate receptors whereas antagonist affinity remains unchanged. Solubilized receptors are insensitive to divalent cations (Mg²⁺, Mn²⁺, Ca²⁺) which increase the potency of agonists for particulate receptors. The effects of Mg²⁺ can be reversed by Gpp(NH)p in particulate preparations; Gpp(NH)p is ineffective for the solubilized preparation. These experiments establish that -adrenergic receptors can be solubilized even from crude mammalian membrane preparations. They also show that the mammalian -adrenergic receptor in situ is under constraints with respect to agonist affinity and is modulated by divalent cations and guanyl nucleotides in the intact membrane.with technical assistance of L. Braun and C. KonradThis report is part of a dissertation to be presented by J. K. to the Fachbereich Humanmedizin, Justus Liebig-Universität Giessen, in partial fulfillment of the requirements for a Doctor of Medical Science degree     

CXCR2--the receptor to hit?

Emigration of leukocytes from the microcirculation into inflammatory tissues is one of the hallmarks of our immune system. However, excessive leukocyte recruitment can disturb the integrity of the organism and aggravate acute and chronic inflammatory diseases. Chemokines are chemoattractant peptides that are used as messengers to direct leukocytes to sites of inflammation. They mediate their effects through G-protein-coupled chemokine receptors on the surface of leukocytes and other, nonhematopoietic cells, where they induce a variety of cell responses including cell activation and transmigration. CXC chemokine receptor 2 (CXCR2) has been implicated in numerous inflammatory disorders. In many models of acute and chronic inflammatory diseases, blockade of CXCR2 substantially reduces leukocyte recruitment, tissue damage and mortality. The physiological importance of CXCR2 has led to the development of selective CXCR2 inhibitors that are now being tested in clinical trials. This review will summarize current knowledge about CXCR2 in inflammatory diseases and discuss its potential as a pharmaceutical target.