Recruitment of ß-arrestin 1 and 2 to the ß2-adrenoceptor: analysis of 65 ligands.

UNLABELLED: Beyond canonical signaling via Gαs and cAMP, the concept of functional selectivity at ß2-adrenoceptors (ß2ARs) describes the ability of adrenergic drugs to stabilize ligand-specific receptor conformations to initiate further signaling cascades comprising additional G-protein classes or ß-arrestins (ßarr). A set of 65 adrenergic ligands including 40 agonists and 25 antagonists in either racemic or enantiopure forms was used for ßarr recruitment experiments based on a split-luciferase assay in a cellular system expressing ß2AR. Many agonists showed only (weak) partial agonism regarding ßarr recruitment. Potencies and/or efficacies increased depending on the number of chirality centers in (R) configuration; no (S)-configured distomer was more effective at inducing ßarr recruitment other than the eutomer. ßarr2 was recruited more effectively than ßarr1. The analysis of antagonists revealed no significant effects on ßarr recruitment. Several agonists showed preference for activation of Gαs GTPase relative to ßarr recruitment, and no ßarr-biased ligand was identified. IN CONCLUSION: 1) agonists show strong bias for Gαs activation relative to ßarr recruitment; 2) agonists recruit ßarr1 and ßarr2 with subtle differences; and 3) there is no evidence for ßarr recruitment by antagonists.

Apoptotic and anti-proliferative effect of guanosine and guanosine derivatives in HuT-78 T lymphoma cells.

The effects of 100 µM of 3',5'-cGMP, cAMP, cCMP, and cUMP as well as of the corresponding membrane-permeant acetoxymethyl esters on anti-CD3-antibody (OKT3)-induced IL-2 production of HuT-78 cutaneous T cell lymphoma (Sézary lymphoma) cells were analyzed. Only 3',5'-cGMP significantly reduced IL-2 production. Flow cytometric analysis of apoptotic (propidium iodide/annexin V staining) and anti-proliferative (CFSE staining) effects revealed that 3',5'-cGMP concentrations > 50 µM strongly inhibited proliferation and promoted apoptosis of HuT-78 cells (cultured in the presence of αCD3 antibody). Similar effects were observed for the positional isomer 2',3'-cGMP and for 2',-GMP, 3'-GMP, 5'-GMP, and guanosine. By contrast, guanosine and guanosine-derived nucleotides had no cytotoxic effect on peripheral blood mononuclear cells (PBMCs) or acute lymphocytic leukemia (ALL) xenograft cells. The anti-proliferative and apoptotic effects of guanosine and guanosine-derived compounds on HuT-78 cells were completely eliminated by the nucleoside transport inhibitor NBMPR (S-(4-Nitrobenzyl)-6-thioinosine). By contrast, the ecto-phosphodiesterase inhibitor DPSPX (1,3-dipropyl-8-sulfophenylxanthine) and the CD73 ecto-5'-nucleotidase inhibitor AMP-CP (adenosine 5'-(α,ß-methylene)diphosphate) were not protective. We hypothesize that HuT-78 cells metabolize guanosine-derived nucleotides to guanosine by yet unknown mechanisms. Guanosine then enters the cells by an NBMPR-sensitive nucleoside transporter and exerts cytotoxic effects. This transporter may be ENT1 because NBMPR counteracted guanosine cytotoxicity in HuT-78 cells with nanomolar efficacy (IC50 of 25-30 nM). Future studies should further clarify the mechanism of the observed effects and address the question, whether guanosine or guanosine-derived nucleotides may serve as adjuvants in the therapy of cancers that express appropriate nucleoside transporters and are sensitive to established nucleoside-derived cytostatic drugs.

cUMP hydrolysis by PDE3A.

As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a K M value of ~143 µM and a V max value of ~42 µmol/min/mg. PDE3A hydrolyzed cAMP with a K M value of ~0.7 µM and a V max of ~1.2 µmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (K i = 57 nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions.

Interaction of fenoterol stereoisomers with ß2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the ß2-adrenoceptor (ß2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using ß2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor.

The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

Structure-bias relationships for fenoterol stereoisomers in six molecular and cellular assays at the ß2-adrenoceptor.

Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic efficacy and fewer adverse effects. Dual coupling of the ß2-adrenoceptor (ß2AR) triggers a signal transduction via Gsα and Giα proteins. Here, we examined 12 fenoterol stereoisomers in six molecular and cellular assays. Using ß2AR-Gsα and ß2AR-Giα fusion proteins, (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol were identified as biased ligands with preference for Gs. G protein-independent signaling via ß-arrestin-2 was disfavored by these ligands. Isolated human neutrophils constituted an ex vivo model of ß2AR signaling and demonstrated functional selectivity through the dissociation of cAMP accumulation and the inhibition of formyl peptide-stimulated production of reactive oxygen species. Ligand bias was calculated using an operational model of agonism and revealed that the fenoterol scaffold constitutes a promising lead structure for the development of Gs-biased ß2AR agonists.

Dissociations in the effects of ß2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

In neutrophils, activation of the ß2-adrenergic receptor (ß2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various ß2AR ligands on adenosine-3',5'-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2(•-)) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2(•-) formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. ß2AR agonists were generally more potent in inhibiting fMLP-induced O2(•-) production than in stimulating cAMP accumulation. (-)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2(•-) production. Moreover, (-)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2(•-) assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2(•-) production after ß2AR-stimulation although cAMP-increasing substances inhibited O2(•-) production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the ß2AR inhibits O2(•-) production in a cAMP-independent manner.