Benzodiazepine diazepam regulates cell surface ß1-adrenergic receptor density in human monocytes.

Downregulation of cell surface ß-adrenergic receptors (ß-AR) is an important adaptive response that prevents deleterious effects of receptor overstimulation. Various factors including reactive oxygen species cause ß-AR downregulation. In this study, we evaluated the effects of ligands of the peripheral benzodiazepine receptor (PBR), a key protein in regulating oxidative stress, on surface density of endogenous ß1-and ß2-ARs in highly differentiated cells such as human monocytes, which express both ß-AR subtypes. ß-AR expression in human monocytes was evaluated by flow cytometry, qPCR and western blotting. Monocyte treatment with ß-AR agonist isoproterenol did not change surface ß1-AR density while downregulating surface ß2-AR density. This effect was antagonized by the ß-blocker propranolol. An opposite response was observed with benzodiazepine diazepam that led to a time-dependent reduction in ß1-AR density. In particular, while no significant downregulation was observed after 3 h of treatment, only 63% of ß1-ARs were still present on the cell surface after 48 h of treatment with diazepam at 1 µM. Treatment with the PBR antagonist PK11195, but not with propranolol, antagonized the effects of diazepam. No change in ß1-AR-mRNA or protein levels was observed at any time after diazepam treatment. We also found that diazepam did not affect Gs-protein or ß-arrestin-2 recruitment for both ß-ARs in engineered fibroblasts, further suggesting that diazepam activity on ß1-AR density is mediated by PBR. Finally, no sex-related differences were found. Collectively, these results indicate that monocyte ß1-ARs are resistant to catecholamine-mediated downregulation and suggest that PBR plays an important role in regulating ß1-AR density.

Novel signalling pathways in nephrogenic syndrome of inappropriate antidiuresis: functional implication of site-specific AQP2 phosphorylation.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare X-linked disease caused by gain-of-function mutations of arginine vasopressin receptor 2 (V2R). Patients with NSIAD are characterized by the inability to excrete a free water load and by inappropriately increased urinary osmolality despite very low levels of plasma vasopressin, resulting in euvolaemic hyponatraemia. To dissect the signalling downstream V2R constitutively active variants, Flp-In T-REx Madin-Darby canine kidney (FTM) cells, stably transfected with V2R mutants (R137L, R137C and F229V) and AQP2-wt or non-phosphorylatable AQP2-S269A/AQP2-S256A, were used as cellular models. All three activating V2R mutations presented constitutive plasma membrane expression of AQP2-wt and significantly higher basal water permeability. In addition, V2R-R137L/C showed significantly higher activity of Rho-associated kinase (ROCK), a serine/threonine kinase previously suggested to be involved in S269-AQP2 phosphorylation downstream of these V2R mutants. Interestingly, FTM cells expressing V2R-R137L/C mutants and AQP2-S269A showed a significant reduction in AQP2 membrane abundance and a significant reduction in ROCK activity, indicating the crucial importance of S269-AQP2 phosphorylation in the gain-of-function phenotype. Conversely, V2R-R137L/C mutants retained the gain-of-function phenotype when AQP2-S256A was co-expressed. In contrast, cells expressing the F229V mutant and the non-phosphorylatable AQP2-S256A had a significant reduction in AQP2 membrane abundance along with a significant reduction in basal osmotic water permeability, indicating a crucial role of Ser256 for this mutant. These data indicate that the constitutive AQP2 trafficking associated with the gain-of-function V2R-R137L/C mutants causing NSIAD is protein kinase A independent and requires an intact Ser269 in AQP2 under the control of ROCK phosphorylation. KEY POINTS: Nephrogenic syndrome of inappropriate antidiuresis is caused by two constitutively active variant phenotypes of AVPR2, one sensitive to vaptans (V2R-F229V) and the other vaptan resistant (V2R-R137C/L). In renal cells, all three activating arginine vasopressin receptor 2 (V2R) variants display constitutive AQP2 plasma membrane expression and high basal water permeability. In cells expressing V2R-R137L/C mutants, disruption of the AQP2-S269 phosphorylation site caused the loss of the gain-of-function phenotype, which, in contrast, was retained in V2R-F229V-expressing cells. Cells expressing the V2R-F229V mutant were instead sensitive to disruption of the AQP2-S256 phosphorylation site. The serine/threonine kinase Rho-associated kinase (ROCK) was found to be involved in AQP2-S269 phosphorylation downstream of the V2R-R137L/C mutants. These findings might have clinical relevance for patients with nephrogenic syndrome of inappropriate antidiuresis.

Crosstalk between ß2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation.

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether ß-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of ß-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a ß-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a ß2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the ß2-AR stimulation. We conclude that the crosstalk between the ß2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.

Vasopressin receptor 2 mutations in the nephrogenic syndrome of inappropriate antidiuresis show different mechanisms of constitutive activation for G protein coupled receptors.

Vasopressin receptor 2 (V2R) mutations causing the nephrogenic syndrome of inappropriate antidiuresis (NSIAD) can generate two constitutively active receptor phenotypes. One type results from residue substitutions in several V2R domains and is sensitive to vaptan inverse agonists. The other is only caused by Arg 137 replacements and is vaptan resistant. We compared constitutive and agonist-driven interactions of the vaptan-sensitive F229V and vaptan-resistant R137C/L V2R mutations with ß-arrestin 1, ß-arrestin 2, and Gαs, using null fibroblasts reconstituted with individual versions of the ablated transduction protein genes. F229V displayed very high level of constitutive activation for Gs but not for ß-arrestins, and enhanced or normal responsiveness to agonists and inverse agonists. In contrast, R137C/L mutants exhibited maximal levels of constitutive activation for ßarrestin 2 and Gs, minimal levels for ß-arrestin 1, but a sharp decline of ligands sensitivity at all transducer interactions. The enhanced constitutive activity and reduced ligand sensitivity of R137 mutants on cAMP signaling persisted in cells lacking ß-arrestins, indicating that these are intrinsic molecular properties of the mutations, not the consequence of altered receptor trafficking. The results suggest that the two groups of NSIAD mutations represent two distinct molecular mechanisms of constitutive activation in GPCRs.

Intermittent ß-adrenergic blockade downregulates the gene expression of ß-myosin heavy chain in the mouse heart.

Expression of the ß-myosin heavy chain (ß-MHC), a major component of the cardiac contractile apparatus, is tightly regulated as even modest increases can be detrimental to heart under stress. In healthy hearts, continuous inhibition of ß-adrenergic tone upregulates ß-MHC expression. However, it is unknown whether the duration of the ß-adrenergic inhibition and ß-MHC expression are related. Here, we evaluated the effects of intermittent ß-blockade on cardiac ß-MHC expression. To this end, the ß-blocker propranolol, at the dose of 15mg/kg, was administered once a day in mice for 14 days. This dosing schedule caused daily drug-free periods of at least 6 h as evidenced by propranolol plasma concentrations and cardiac ß-adrenergic responsiveness. Under these conditions, ß-MHC expression decreased by about 75% compared to controls. This effect was abolished in mice lacking ß1- but not ß2-adrenergic receptors (ß-AR) indicating that ß-MHC expression is regulated in a ß1-AR-dependent manner. In ß1-AR knockout mice, the baseline ß-MHC expression was fourfold higher than in wild-type mice. Also, we evaluated the impact of intermittent ß-blockade on ß-MHC expression in mice with systolic dysfunction, in which an increased ß-MHC expression occurs. At 3 weeks after myocardial infarction, mice showed systolic dysfunction and upregulation of ß-MHC expression. Intermittent ß-blockade decreased ß-MHC expression while attenuating cardiac dysfunction. In vitro studies showed that propranolol does not affect ß-MHC expression on its own but antagonizes catecholamine effects on ß-MHC expression. In conclusion, a direct relationship occurs between the duration of the ß-adrenergic inhibition and ß-MHC expression through the ß1-AR.

ß-blockers Reverse Agonist-Induced ß2-AR Downregulation Regardless of Their Signaling Profile.

Altered ß-adrenergic receptor (ß-AR) density has been reported in cells, animals, and humans receiving ß-blocker treatment. In some cases, ß-AR density is upregulated, but in others, it is unaffected or even reduced. Collectively, these results would imply that changes in ß-AR density and ß-blockade are not related. However, it has still not been clarified whether the effects of ß-blockers on receptor density are related to their ability to activate different ß-AR signaling pathways. To this aim, five clinically relevant ß-blockers endowed with inverse, partial or biased agonism at the ß2-AR were evaluated for their effects on ß2-AR density in both human embryonic kidney 293 (HEK293) cells expressing exogenous FLAG-tagged human ß2-ARs and human lymphocytes expressing endogenous ß2-ARs. Cell surface ß2-AR density was measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Treatment with propranolol, carvedilol, pindolol, sotalol, or timolol did not induce any significant change in surface ß2-AR density in both HEK293 cells and human lymphocytes. On the contrary, treatment with the ß-AR agonist isoproterenol reduced the number of cell surface ß2-ARs in the tested cell types without affecting ß2-AR-mRNA levels. Isoproterenol-induced effects on receptor density were completely antagonized by ß-blocker treatment. In conclusion, the agonistic activity of ß-blockers does not exert an important effect on short-term regulation of ß2-AR density.

Gain-of-function mutations of the V2 vasopressin receptor in nephrogenic syndrome of inappropriate antidiuresis (NSIAD): a cell-based assay to assess constitutive water reabsorption.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently identified chromosome X-linked disease associated with gain-of-function mutations of the V2 vasopressin receptor (V2R), a G-protein-coupled receptor. It is characterized by inability to excrete a free water load, hyponatremia, and undetectable vasopressin-circulating levels. Hyponatremia can be quite severe in affected male children. To gain a deeper insight into the functional properties of the V2R active mutants and how they might translate into the pathological outcome of NSIAD, in this study, we have expressed the wild-type V2R and three constitutively active V2R mutants associated with NSIAD (R137L, R137C, and the F229V) in MCD4 cells, a cell line derived from renal mouse collecting duct, stably expressing the vasopressin-sensitive water channel aquaporin-2 (AQP2). Our findings indicate that in cells expressing each active mutant, AQP2 was constitutively localized to the apical plasma membrane in the absence of vasopressin stimulation. In line with these observations, under basal conditions, osmotic water permeability in cells expressing the constitutively active mutants was significantly higher compared to that of cells expressing the wild-type V2R. Our findings demonstrate a direct link between activating mutations of the V2R and the perturbation of water balance in NSIAD. In addition, this study provides a useful cell-based assay system to assess the functional consequences of newly discovered activating mutations of the V2R on water permeability in kidney cells and to screen the effect of drugs on the mutated receptors.

Systematic errors in detecting biased agonism: Analysis of current methods and development of a new model-free approach.

Discovering biased agonists requires a method that can reliably distinguish the bias in signalling due to unbalanced activation of diverse transduction proteins from that of differential amplification inherent to the system being studied, which invariably results from the non-linear nature of biological signalling networks and their measurement. We have systematically compared the performance of seven methods of bias diagnostics, all of which are based on the analysis of concentration-response curves of ligands according to classical receptor theory. We computed bias factors for a number of ß-adrenergic agonists by comparing BRET assays of receptor-transducer interactions with Gs, Gi and arrestin. Using the same ligands, we also compared responses at signalling steps originated from the same receptor-transducer interaction, among which no biased efficacy is theoretically possible. In either case, we found a high level of false positive results and a general lack of correlation among methods. Altogether this analysis shows that all tested methods, including some of the most widely used in the literature, fail to distinguish true ligand bias from "system bias" with confidence. We also propose two novel semi quantitative methods of bias diagnostics that appear to be more robust and reliable than currently available strategies.

Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo).

INTRODUCTION: Opioid receptors are currently classified as Mu (µ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ. METHODS: We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats. RESULTS: DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats. CONCLUSION: Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties.

Ligands raise the constraint that limits constitutive activation in G protein-coupled opioid receptors.

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and µ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

Morphine-like opiates selectively antagonize receptor-arrestin interactions.

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of mu and delta receptors with G protein or beta-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gbeta(1). In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was G alpha-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at delta and mu receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (delta) or partial agonists (mu) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.