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1.
Sci Rep ; 10(1): 17395, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060647

RESUMO

Most G protein-coupled receptors (GPCRs) recruit ß-arrestins and internalize upon agonist stimulation. For the µ-opioid receptor (µ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for µ-OR recruitment of ß-arrestin and internalization. However, the contribution of GRK2 and GRK3 to ß-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in ß-arrestin2 recruitment and µ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced µ-OR internalization and ß-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of ß-arrestin2 recruitment to the plasma membrane upon µ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

2.
Biochim Biophys Acta Mol Cell Res ; 1867(12): 118849, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32916203

RESUMO

FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to ß-arrestin recruitment. ß-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of ß-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent ß-arrestin/AP2 interaction and ß-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/ß-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of ß-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in ß-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of ß-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of ß-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/ß-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.

3.
Proc Natl Acad Sci U S A ; 117(35): 21723-21730, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817560

RESUMO

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

4.
J Biol Chem ; 295(30): 10153-10167, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32439801

RESUMO

Agonist stimulation of G-protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called ß-arrestins (ßarrs). The GPCR-ßarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-ßarr complex formation can be used as a generic readout of GPCR and ßarr activation. Although several methods are currently available to monitor GPCR-ßarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated ßarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR-ßarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments and used them to monitor the localization and trafficking of ßarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of ßarr1, and the intrabodies co-localized with ßarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report ßarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for ßarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-ßarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

5.
Sci Rep ; 10(1): 8779, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471984

RESUMO

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different ß-adrenergic ligands to engage five distinct signalling pathways downstream of the ß1-adrenergic receptor (ß1AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the ßAR. These include coupling to Gz and G12 pathways. The signalling cascade linking the ß1AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both Gs and G12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.

6.
Cell Rep ; 31(7): 107660, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433969

RESUMO

In human cells, the expression of ∼1,000 genes is modulated throughout the cell cycle. Although some of these genes are controlled by specific transcriptional programs, very little is known about their post-transcriptional regulation. Here, we analyze the expression signature associated with all 687 RNA-binding proteins (RBPs) and identify 39 that significantly correlate with cell cycle mRNAs. We find that NF45 and NF90 play essential roles in mitosis, and transcriptome analysis reveals that they are necessary for the expression of a subset of mitotic mRNAs. Using proteomics, we identify protein clusters associated with the NF45-NF90 complex, including components of Staufen-mediated mRNA decay (SMD). We show that depletion of SMD components increases the binding of mitotic mRNAs to the NF45-NF90 complex and rescues cells from mitotic defects. Together, our results indicate that the NF45-NF90 complex plays essential roles in mitosis by competing with the SMD machinery for a common set of mRNAs.

8.
Cell Mol Life Sci ; 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32040695

RESUMO

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins ß-arrestins and G proteins. Depletion of ß-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that ß-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-ß-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK-ß-arrestin complex to the plasma membrane, ß-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient ß-arrestin and by a specific inhibitor of ß-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of ß-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.

9.
Neuropharmacology ; 166: 107718, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31351108

RESUMO

Binding and signaling kinetics have previously proven important in validation of biased agonism at GPCRs. Here we provide a comprehensive kinetic pharmacological comparison of clinically relevant µ-opioid receptor agonists, including the novel biased agonist oliceridine (TRV130) which is in clinical trial for pain management. We demonstrate that the bias profile observed for the selected agonists is not time-dependent and that agonists with dramatic differences in their binding kinetic properties can display the same degree of bias. Binding kinetics analyses demonstrate that buprenorphine has 18-fold higher receptor residence time than oliceridine. This is thus the largest pharmacodynamic difference between the clinically approved drug buprenorphine and the clinical candidate oliceridine, since their bias profiles are similar. Further, we provide the first pharmacological characterization of (S)-TRV130 demonstrating that it has a similar pharmacological profile as the (R)-form, oliceridine, but displays 90-fold lower potency than the (R)-form. This difference is driven by a significantly slower association rate. Finally, we show that the selected agonists are differentially affected by G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) expression. GRK2 and GRK5 overexpression greatly increased µ-opioid receptor internalization induced by morphine, but only had modest effects on buprenorphine and oliceridine-induced internalization. Overall, our data reveal that the clinically available drug buprenorphine displays a similar pharmacological bias profile in vitro compared to the clinical candidate drug oliceridine and that this bias is independent of binding kinetics suggesting a mechanism driven by receptor-conformations. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'.

10.
Nat Commun ; 10(1): 4075, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501422

RESUMO

Signaling diversity of G protein-coupled (GPCR) ligands provides novel opportunities to develop more effective, better-tolerated therapeutics. Taking advantage of these opportunities requires identifying which effectors should be specifically activated or avoided so as to promote desired clinical responses and avoid side effects. However, identifying signaling profiles that support desired clinical outcomes remains challenging. This study describes signaling diversity of mu opioid receptor (MOR) ligands in terms of logistic and operational parameters for ten different in vitro readouts. It then uses unsupervised clustering of curve parameters to: classify MOR ligands according to similarities in type and magnitude of response, associate resulting ligand categories with frequency of undesired events reported to the pharmacovigilance program of the Food and Drug Administration and associate signals to side effects. The ability of the classification method to associate specific in vitro signaling profiles to clinically relevant responses was corroborated using ß2-adrenergic receptor ligands.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Analgésicos Opioides/metabolismo , Animais , Análise por Conglomerados , Proteínas de Ligação ao GTP/metabolismo , Cobaias , Células HEK293 , Humanos , Ligantes , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides mu/metabolismo , beta-Arrestinas/metabolismo
11.
J Med Chem ; 62(16): 7400-7416, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31246024

RESUMO

In an effort to identify novel antithrombotics, we have investigated protease-activated receptor 4 (PAR4) antagonism by developing and evaluating a tool compound, UDM-001651, in a monkey thrombosis model. Beginning with a high-throughput screening hit, we identified an imidazothiadiazole-based PAR4 antagonist chemotype. Detailed structure-activity relationship studies enabled optimization to a potent, selective, and orally bioavailable PAR4 antagonist, UDM-001651. UDM-001651 was evaluated in a monkey thrombosis model and shown to have robust antithrombotic efficacy and no prolongation of kidney bleeding time. This combination of excellent efficacy and safety margin strongly validates PAR4 antagonism as a promising antithrombotic mechanism.

12.
Proc Natl Acad Sci U S A ; 116(26): 13006-13015, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31189595

RESUMO

Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln -/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln -/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln -/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr -/- mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.


Assuntos
Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/patologia , Apelina/metabolismo , Neprilisina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Angiotensina II/administração & dosagem , Animais , Aorta Abdominal/citologia , Aneurisma da Aorta Abdominal/tratamento farmacológico , Aneurisma da Aorta Abdominal/etiologia , Apelina/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/farmacologia , Fármacos Cardiovasculares/uso terapêutico , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Miócitos de Músculo Liso , Neprilisina/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Peptidil Dipeptidase A/metabolismo , Fenilefrina/administração & dosagem , Cultura Primária de Células , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/genética
13.
J Med Chem ; 62(10): 5111-5131, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31042379

RESUMO

Starting from the ß-adrenoceptor agonist isoprenaline and beta-blocker carvedilol, we designed and synthesized three different chemotypes of agonist/antagonist hybrids. Investigations of ligand-mediated receptor activation using bioluminescence resonance energy transfer biosensors revealed a predominant effect of the aromatic head group on the intrinsic activity of our ligands, as ligands with a carvedilol head group were devoid of agonistic activity. Ligands composed of a catechol head group and an antagonist-like oxypropylene spacer possess significant intrinsic activity for the activation of Gαs, while they only show weak or even no ß-arrestin-2 recruitment at both ß1- and ß2-AR. Molecular dynamics simulations suggest that the difference in G protein efficacy and ß-arrestin recruitment of the hybrid ( S)-22, the full agonist epinephrine, and the ß2-selective, G protein-biased partial agonist salmeterol depends on specific hydrogen bonding between Ser5.46 and Asn6.55, and the aromatic head group of the ligands.

14.
Int J Colorectal Dis ; 34(6): 1131-1140, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31044283

RESUMO

PURPOSE: Biofeedback therapy (BT) is a simple and effective technique for managing outlet constipation and fecal incontinence. Several clinical factors are known to predict BT response, but a 50% failure rate persists. Better selection of BT responsive patients is required. We aimed to determine whether the defecation disorder type per high-resolution manometry (HRM) was predictive of BT response. METHODS: We analyzed clinical, manometric, and ultrasound endoscopic data from patients who underwent BT in our department between January 2015 and January 2016. Patients were classified into four groups per the following defecation disorder classification criteria: rectal pressure > 40 mmHg and anal paradoxical contraction (type I); rectal pressure < 40 mmHg and anal paradoxical contraction (type II); rectal pressure > 40 mmHg and incomplete anal relaxation (type III); and rectal pressure < 40 mmHg and incomplete anal relaxation (type IV). An experienced single operator conducted ten weekly 20-min sessions. Efficacy was evaluated with the visual analog scale. RESULTS: Of 92 patients, 47 (50.5%) responded to BT. Type IV and type II defecation disorders were predictive of success (p = 0.03) (OR = 5.03 [1.02; 24.92]) and failure (p = 0.05) (OR = 0.41 [0.17; 0.99]), respectively. The KESS score severity before BT (p = 0.03) (OR = 0.9 [0.81; 0.99]) was also predictive of failure. CONCLUSION: The manometry types identified according to the defecation disorder classification criteria were predictive of BT response. Our data confirm the role of three-dimensional HRM in the therapeutic management of anorectal functional disorders.


Assuntos
Canal Anal/diagnóstico por imagem , Canal Anal/fisiopatologia , Biorretroalimentação Psicológica , Defecação/fisiologia , Imageamento Tridimensional , Manometria , Reto/diagnóstico por imagem , Reto/fisiopatologia , Endossonografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances
15.
Nat Chem Biol ; 15(5): 489-498, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30992568

RESUMO

Differentiating actions of short chain fatty acids (SCFAs) at free fatty acid receptor 2 (FFA2) from other free fatty acid-responsive receptors and from non-receptor-mediated effects has been challenging. Using a novel chemogenetic and knock-in strategy, whereby an engineered variant of FFA2 (FFA2-DREADD) that is unresponsive to natural SCFAs but is instead activated by sorbic acid replaced the wild-type receptor, we determined that activation of FFA2 in differentiated adipocytes and colonic crypt enteroendocrine cells of mouse accounts fully for SCFA-regulated lipolysis and release of the incretin glucagon-like peptide-1 (GLP-1), respectively. In vivo studies confirmed the specific role of FFA2 in GLP-1 release and also demonstrated a direct role for FFA2 in accelerating gut transit. Thereby, we establish the general principle that such a chemogenetic knock-in strategy can successfully define novel G-protein-coupled receptor (GPCR) biology and provide both target validation and establish therapeutic potential of a 'hard to target' GPCR.


Assuntos
Ácidos Graxos Voláteis/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Humanos , Camundongos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas-G/genética
16.
Nat Protoc ; 14(4): 1084-1107, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30911173

RESUMO

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein-protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Because the BRET donor does not require an external light source, it does not lead to phototoxicity or autofluorescence. It therefore represents an interesting alternative to fluorescence-based imaging such as FRET. However, the low signal output of BRET energy donors has limited the spatiotemporal resolution of BRET imaging. Here, we describe how recent improvements in detection devices and BRET probes can be used to markedly improve the resolution of BRET imaging, thus widening the field of BRET imaging applications. The protocol described herein involves three main stages. First, cell preparation and transfection require 3 d, including cell culture time. Second, image acquisition takes 10-120 min per sample, after an initial 60 min for microscope setup. Finally, image analysis typically takes 1-2 h. The choices of energy donor, acceptor, luminescent substrates, cameras and microscope setup, as well as acquisition modes to be used for different applications, are also discussed.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Medições Luminescentes/métodos , Imagem Óptica/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzenoacetamidas/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imidazóis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Pirazinas/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Renilla , Transfecção , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
17.
iScience ; 14: 47-57, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-30925410

RESUMO

G protein-coupled receptors are key signaling molecules and major targets for pharmaceuticals. The concept of ligand-dependent biased signaling raises the possibility of developing drugs with improved efficacy and safety profiles, yet translating this concept to native tissues remains a major challenge. Whether drug activity profiling in recombinant cell-based assays, traditionally used for drug discovery, has any relevance to physiology is unknown. Here we focused on the mu opioid receptor, the unrivalled target for pain treatment and also the key driver for the current opioid crisis. We selected a set of clinical and novel mu agonists, and profiled their activities in transfected cell assays using advanced biosensors and in native neurons from knock-in mice expressing traceable receptors endogenously. Our data identify Gi-biased agonists, including buprenorphine, and further show highly correlated drug activities in the two otherwise very distinct experimental systems, supporting in vivo translatability of biased signaling for mu opioid drugs.

18.
Br J Pharmacol ; 176(14): 2539-2558, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30809805

RESUMO

ß3 -Adrenoceptor agonists have proven useful in the treatment of overactive bladder syndrome, but it is not known whether their efficacy during chronic administration may be limited by receptor-induced desensitisation. Whereas the ß2 -adrenoceptor has phosphorylation sites that are important for desensitisation, the ß3 -adrenoceptor lacks these; therefore, it had been assumed that ß3 -adrenoceptors are largely resistant to agonist-induced desensitisation. While all direct comparative studies demonstrate that ß3 -adrenoceptors are less susceptible to desensitisation than ß2 -adrenoceptors, desensitisation of ß3 -adrenoceptors has been observed in many models and treatment settings. Chimeric ß2 - and ß3 -adrenoceptors have demonstrated that the C-terminal tail of the receptor plays an important role in the relative resistance to desensitisation but is not the only relevant factor. While the evidence from some models, such as transfected CHO cells, is inconsistent, it appears that desensitisation is observed more often after long-term (hours to days) than short-term (minutes to hours) agonist exposure. When it occurs, desensitisation of ß3 -adrenoceptors can involve multiple levels including down-regulation of its mRNA and the receptor protein and alterations in post-receptor signalling events. The relative contributions of these mechanistic factors apparently depend on the cell type under investigation. Which if any of these factors is applicable to the human urinary bladder remains to be determined. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

19.
Int Urogynecol J ; 30(12): 2049-2054, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30796476

RESUMO

INTRODUCTION AND HYPOTHESIS: Wearing high heels may not only produce deleterious effects on the musculoskeletal system and on the general posture, but also on the activity of pelvic floor muscles. However, no data are currently available concerning the link between fecal incontinence and wearing high heels. Our aim was to determine whether wearing high-heeled shoes could influence anal canal pressure values in patients suffering from fecal incontinence. METHODS: In this retrospective monocentric study, 338 female patients were included. Clinical data, including the Wexner score, and manometric data were recorded (using 3D high-resolution anorectal manometry). Wearing high heels was defined by the use of high-heeled shoes at least four times a week for 4 consecutive hours with at least 1 year of usage time and heels ≥3 cm. Two sub-groups were defined: "high heels" vs "no high heels." RESULTS: The two subgroups were comparable for clinical data, including Wexner score, except for a higher age, menopause and hormone replacement therapy, and urinary incontinence in the group with "high heels." No statistical difference was observed concerning the anal canal pressure. Using analysis by logistic regression, only age was related to a significantly lower resting pressure. CONCLUSIONS: In this retrospective cohort of women with fecal incontinence, no clinical or manometric differences were observed between women who wore high heels versus those who did not. Because there is limited knowledge on this potential link and because the pelvic tilt may vary according to age and the habit of walking with high heels, further studies are necessary.


Assuntos
Canal Anal/fisiopatologia , Incontinência Fecal/fisiopatologia , Pressão , Índice de Gravidade de Doença , Sapatos/efeitos adversos , Adulto , Feminino , Calcanhar , Humanos , Manometria , Pessoa de Meia-Idade , Diafragma da Pelve/fisiopatologia , Estudos Retrospectivos
20.
Int J Colorectal Dis ; 34(4): 719-729, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30706131

RESUMO

BACKGROUND: The aim of the study was to describe the results of 3D high-resolution anorectal manometry (3DHRAM) in a large cohort of patients with functional anorectal disorders. METHODS: In this single-center retrospective study, all consecutive patients referred for investigation of fecal incontinence (FI) or dyssynergic defecation (DD) underwent 3DHRAM. The parameters analyzed were usual manometric data, repartition of dyssynergic patterns, and the prevalence of a new "muscular subtype classification" underlying dyssynergia, anal sphincter defects, and pelvic floor disorders. RESULTS: Final analyses were performed in 1477 patients with a mean age 54 ± 16 years; 825 patients suffered from DD, and 652 patients suffered from FI. Among these patients, 86% met the diagnostic criteria for dyssynergia. Type II dyssynergia was the most frequently observed (56%) in women and men suffering from FI and in women with DD. Type I was the most frequently observed in men with DD (49%). Regarding the muscle type subgroups, combined puborectalis muscle involvement with an external anal sphincter profile was the most frequently observed. The global prevalence of rectal intussusception and excessive perineal descent were 12% and 21%, respectively. Type III dyssynergia was more frequently associated with pelvic floor disorders than were other types of dyssynergia (p < 0.001). CONCLUSION: This large cohort study provides reference values for 3DHRAM in patients with functional anorectal disorders. Further studies are necessary to assess the prevalence of pelvic floor disorders in healthy volunteers and to develop new scores and classifications including all of these new parameters.


Assuntos
Doenças do Ânus/diagnóstico por imagem , Doenças do Ânus/fisiopatologia , Imageamento Tridimensional , Manometria , Doenças Retais/diagnóstico por imagem , Doenças Retais/fisiopatologia , Doenças do Ânus/epidemiologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência
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