G-protein Coupled Receptor Agonists Research Articles

The regulatory G protein signaling complex, Gβ5–R7, promotes glucose- and extracellular signal–stimulated insulin secretion

G protein-coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gβ5-R7, a protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit β 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M-stimulated glucose-stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5-/- islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K+-evoked membrane depolarization. These results indicate that Gβ5-R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gβ5-R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Retraction of "GPR40 Agonist GW9508 Ameliorates Oxidized LDL-Induced Endothelial Dysfunction via the AMPK/CREB/PGC-1α Pathway".

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction has been recognized as an important early event in atherosclerosis. G-protein-coupled receptor 40 (GPR40) is a cell surface receptor that is highly expressed in endothelial cells. The physiological function of GPR40 in endothelial cells remains to be elucidated. In this study, we found that ox-LDL stimulation reduced the expression of GPR40 in a dose-dependent manner from 50 to 150 μg/ml in human aortic endothelial cells (HAECs). Interestingly, we found that the activation of GPR40 by its agonist GW9508 ameliorated ox-LDL-induced reduced cell viability of HAECs. Furthermore, our results indicate that GW9508 treatment improved mitochondrial function by restoring ox-LDL-induced reduced mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) production. Mechanistically, we found that GW9508 mitigated ox-LDL-induced inactivation of adenosine 5'-monophosphate (AMP)-activated protein kinase α (AMPKα), however, the blockage of GPR40 by its antagonist GW1100 completely abolished the protective effect of GW9508 on AMPKα activation. Importantly, treatment with the AMPKα inhibitor compound C abolished the beneficial effects of GW9508 on NADPH oxidase activity, cellular reactive oxygen species (ROS) production, and superoxide dismutase (SOD) activity, suggesting that the cellular protective effects of GW9508 are mediated by AMPKα. Additionally, GW9508 also restored ox-LDL-induced dephosphorylation of cAMP-responsive element-binding protein (CREB), which was also abolished by compound C. Finally, we found that AMPKα and CREB participated in mediating the effects of GW9508 on the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial homeostasis. These findings suggest that GW9508 possesses an important protective effect in endothelial cells against ox-LDL-induced damages.

Effects of ICI 182,780, an ERα and ERβ antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells

ABSTRACTObjectiveTo investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability.MethodsCell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy.ResultsRegarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line.ConclusionThe effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors’ involvement in cellular processes of breast cancer resistance.

GPR55 Regulates Progression of Atherosclerosis by Monocyte Adhesion to Vascular Endothelium

G‐protein coupled receptor 55 (GPR55) is an orphan G‐protein coupled receptor, which is participate in the progression of atherosclerosis. however, the underlying mechanisms are unclear. Thus, this study investigated the molecular mechanisms responsible for monocyte adhesion to the vascular endothelium, which is a key early event in macrophage foam cell formation. In in vitro study, the pharmacological GPR55 agonist, O‐1602‐induced monocyte adhesion to the endothelial cells and adhesion molecule, Mac‐1 expression were significantly attenuated by pretreatment of GPR55 antagonist, CID 164002 in monocytes. In addition, in promoter activity analysis and chromatin immunoprecipitation assays to identify transcription factors involved in O‐1602‐induced Mac‐1 expression, both AP‐1 and NF‐κB played central roles to increase Mac‐1 transcription in O‐1602‐treated monocytes. Moreover, O‐1602‐induced activations of Gq/11 proteins, CaMKK, and AKT were found to be signaling components of the monocyte adhesion to the endothelial cells and Mac‐1 expression pathway. In in vivo study, a greater number of atherosclerotic plaques formed in ApoE(−/−) mice fed a high‐fat diet than in CID 164002 pretreated ApoE(−/−) mice, indicating a pivotal role for GPR55 in preventing vascular inflammation. Based on these results, GPR55‐mediated expression of Mac‐1 in monocytes plays a pivotal role on monocyte adhesion to vascular endothelium, leading to increased foam cell formation in the development of atherosclerosis.

Efforts Toward GPR88 Deorphanization: Challenges and Alternative Approaches to Troublesome Cell‐Based Second Messenger Assays

G protein‐coupled receptors (GPCR) comprise a family of over 800 integral membrane proteins involved in signal transduction and serve as the primary cellular sensors for chemical stimuli. About 35% of current drugs act on GPCR targets. In addition, of the 300 agents in current clinical trials, greater than 20% target novel GPCRs for which there are no approved drugs (orphan GPCRs). Ligand discovery and deorphanization efforts seeking to assign function to understudied GPCRs are not trivial. Notoriously complex in nature, GPCR‐dependent signaling centers around receptor‐ligand interactions that serve to stabilize conformational access to intracellular signaling partners, G proteins. In GPCR signaling, receptors and heterotrimeric G proteins (made of the Gα and Gβγ subunits) work together to transmit signals via downstream effectors and distinct pathways. The measurement of GPCR mediated second messengers such as cyclic AMP (cAMP), intracellular calcium (Ca2+) mobilization, and ERK activation (MAP kinase) are commonly used as tools to detect ligand induced GPCR activation. However, experimental pathway elucidation is complicated by the tendencies for pathway crosstalk, receptor desensitization, and receptor‐G protein interaction promiscuity. Cell‐based assays that monitor second messenger responses are often used as screening tools for potential GPCR agonists and antagonists, however, commonly used cell‐lines such as HEK‐293, express many endogenous GPCRs that can complicate data analysis. Many classes of Gα subunits exist: Gαs and Gα i/o G‐proteins modulate cyclic AMP (cAMP) through stimulation or inhibition of adenylate cyclase while Gαq subunits activate phospholipase C and a subsequent rapid release of intracellular Ca2+. Specifically, the orphan receptor, GPR88, has been linked with psychiatric diseases including schizophrenia and bipolar disorder. Lacking a validated endogenous agonist, the GPR88 signaling pathway remains poorly understood, however, in vivo knockout experiments and in vitro preclinical studies with small molecule synthetic agonists indicate that GPR88 couples to Gαi/o G‐proteins to inhibit adenylate cyclase activity and reduce intracellular cAMP in cell‐based assays. In this work, we describe our challenges in the study of the GPR88 signaling pathway using second messenger cell‐based assays and explain our decision to move to a real‐time NanoLuc‐based GPCR/G protein complementation assay.Support or Funding InformationNIH grant 1 R15 MH109034

Regulator of G protein signaling complex, Gβ5‐R7, promotes glucose‐ and extracellular signal‐stimulated insulin secretion

Glucose‐stimulated insulin secretion (GSIS) is essential for maintaining energy homeostasis. G protein‐coupled receptors (GPCRs) are important modulators of this process by extracellular signals such as hormones, neurotransmitters and non‐glucose metabolites. We investigated the role of Gβ5‐R7, the protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling (RGS) of the R7 family. To analyze the effects of Gnb5 knockout on insulin secretion we used mouse insulinoma MIN6 cell line and pancreatic islets isolated from the Gnb5−/− mice. Consistent with previous work, Gnb5knockout diminished insulin secretion evoked by cholinergic agonist Oxo‐M. Here, we found that Gnb5 knockout also attenuated activity of other GPCR agonists ADP, AVP, GLP‐1, forskolin and, surprisingly, the response to high glucose. Our experiments also provided evidence that Gnb5 knockout eliminated the positive effect of cell adhesion on Oxo‐M‐stimulated GSIS, likely through adhesion GPCR GPR56. Total insulin content and the amount of insulin released in response to K+‐evoked membrane depolarization in Gnb5 −/− islets or MIN6 cells was normal. The pleiotropic effect on several GPCRs led us to the hypothesis that Gβ5‐R7 may play a non‐canonical role in insulin secretory pathway downstream of G protein signaling and glucose‐activated membrane depolarization. We found that Gnb5 knockout does not influence cortical actin depolymerization, but influences activity of protein kinase C, and thus far identified 14‐3‐3ɛ as one of the affected substrates. Together, these results allow us to propose that the Gβ5‐R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway, downstream of G protein signaling and actin depolymerization, but upstream of insulin granule release.Support or Funding InformationThis work was supported by National Institutes of Health Grants R01DK1054127(to V.S.), RO1‐DK073716, DK084236 (to E.B.M), EY11373 (to J.C.).

G-Protein-Coupled Receptors in CNS: A Potential Therapeutic Target for Intervention in Neurodegenerative Disorders and Associated Cognitive Deficits.

Neurodegenerative diseases are a large group of neurological disorders with diverse etiological and pathological phenomena. However, current therapeutics rely mostly on symptomatic relief while failing to target the underlying disease pathobiology. G-protein-coupled receptors (GPCRs) are one of the most frequently targeted receptors for developing novel therapeutics for central nervous system (CNS) disorders. Many currently available antipsychotic therapeutics also act as either antagonists or agonists of different GPCRs. Therefore, GPCR-based drug development is spreading widely to regulate neurodegeneration and associated cognitive deficits through the modulation of canonical and noncanonical signals. Here, GPCRs’ role in the pathophysiology of different neurodegenerative disease progressions and cognitive deficits has been highlighted, and an emphasis has been placed on the current pharmacological developments with GPCRs to provide an insight into a potential therapeutic target in the treatment of neurodegeneration.

Endocannabinoid hydrolysis inhibition unmasks that unsaturated fatty acids induce a robust biosynthesis of 2-arachidonoyl-glycerol and its congeners in human myeloid leukocytes.

The endocannabinoid (eCB) 2-arachidonoyl-gycerol (2-AG) modulates immune responses by activating cannabinoid receptors or through its multiple metabolites, notably eicosanoids. Thus, 2-AG hydrolysis inhibition might represent an interesting anti-inflammatory strategy that would simultaneously increase the levels of 2-AG and decrease those of eicosanoids. Accordingly, 2-AG hydrolysis inhibition increased 2-AG half-life in neutrophils. Under such setting, neutrophils, eosinophils, and monocytes synthesized large amounts of 2-AG and other monoacylglycerols (MAGs) in response to arachidonic acid (AA) and other unsaturated fatty acids (UFAs). Arachidonic acid and UFAs were ~1000-fold more potent than G protein-coupled receptor (GPCR) agonists. Triascin C and thimerosal, which, respectively, inhibit fatty acyl-CoA synthases and acyl-CoA transferases, prevented the UFA-induced MAG biosynthesis, implying glycerolipid remodeling. 2-AG and other MAG biosynthesis was preceded by that of the corresponding lysophosphatidic acid (LPA). However, we could not directly implicate LPA dephosphorylation in MAG biosynthesis. While GPCR agonists poorly induced 2-AG biosynthesis, they inhibited that induced by AA by 25%-50%, suggesting that 2-AG biosynthesis is decreased when leukocytes are surrounded by a pro-inflammatory entourage. Our data strongly indicate that human leukocytes use AA and UFAs to biosynthesize biologically significant concentrations of 2-AG and other MAGs and that hijacking the immune system with 2-AG hydrolysis inhibitors might diminish inflammation in humans.

FRET Reporter Assays for cAMP and Calcium in a 96-well Format Using Genetically Encoded Biosensors Expressed in Living Cells.

Stimulation of G protein-coupled receptors (GPCR) by hormones and neurotransmitters elicits cellular responses, many of which result from alterations in the concentrations of cytosolic cAMP and Ca2+. Here, we describe a microplate reader fluorescence resonance energy transfer (FRET) assay that uses the genetically encoded biosensors H188 and YC3.60 so that it is possible to monitor the kinetics with which alterations of [cAMP] or [Ca2+] occur in monolayers or suspensions of living cells exposed to GPCR agonists. This protocol uses HEK293 cell lines doubly transfected with a FRET biosensor and a recombinant GPCR of interest (e.g., glucagon receptors, CCK2 receptors, or NPY2R receptors). The protocol allows for rapid screening of small molecule GPCR agonists and antagonists, and it is also useful for discovery of synthetic mono-, dual-, and tri- agonist peptides with GPCR activating properties.

Allosteric modulation of metabotropic glutamate receptors in alcohol use disorder: Insights from preclinical investigations.

Metabotropic glutamate (mGlu) receptors are family C G protein-coupled receptors (GPCRs) that modulate neuronal excitability and synaptic transmission throughout the nervous system. Owing to recent advances in development of subtype-selective allosteric modulators of mGlu receptors, individual members of the mGlu receptor family have been proposed as targets for treating a variety of neurological and psychiatric disorders, including substance use disorders. In this chapter, we highlight preclinical evidence that allosteric modulators of mGlu receptors could be useful for reducing alcohol consumption and preventing relapse in alcohol use disorder (AUD). We begin with an overview of the preclinical models that are used to study mGlu receptor involvement in alcohol-related behaviors. Alcohol exposure causes adaptations in both expression and function of various mGlu receptor subtypes, and pharmacotherapies aimed at reversing these adaptations have the potential to reduce alcohol consumption and seeking. Positive allosteric modulators (PAMs) of mGlu2 and negative allosteric modulators of mGlu5 show particular promise for reducing alcohol intake and/or preventing relapse. Finally, this chapter discusses important considerations for translating preclinical findings toward the development of clinically useful drugs, including the potential for PAMs to avoid tolerance issues that are frequently observed with repeated administration of GPCR agonists.

Recent Advances in the Drug Discovery and Development of Dualsteric/ Bitopic Activators of G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) represent the largest family of proteins targeted by drug design and discovery efforts. Of these efforts, the development of GPCR agonists is highly desirable, due to their therapeutic robust utility in treating diseases caused by deficient receptor signaling. One of the challenges in designing potent and selective GPCR agonists lies in the inability to achieve combined high binding affinity and subtype selectivity, due to the high homology between orthosteric sites among GPCR subtypes. To combat this difficulty, researchers have begun to explore the utility of targeting topographically distinct and less conserved binding sites, namely "allosteric" sites. Pursuing these sites offers the benefit of achieving high subtype selectivity, however, it also can result in a decreased binding affinity and potency as compared to orthosteric agonists. Therefore, bitopic ligands comprised of an orthosteric agonist and an allosteric modulator connected by a spacer and allowing binding with both the orthosteric and allosteric sites within one receptor, have been developed. It may combine the high subtype selectivity of an allosteric modulator with the high binding affinity of an orthosteric agonist and provides desired advantages over orthosteric agonists or allosteric modulators alone. Herein, we review the recent advances in the development of bitopic agonists/activators for various GPCR targets and their novel therapeutic potentials.

Is There a Role for GPCR Agonist Radiotracers in PET Neuroimaging?

Positron emission tomography (PET) is a molecular imaging modality that enables in vivo exploration of metabolic processes and especially the pharmacology of neuroreceptors. G protein-coupled receptors (GPCRs) play an important role in numerous pathophysiologic disorders of the central nervous system. Thus, they are targets of choice in PET imaging to bring proof concept of change in density in pathological conditions or in pharmacological challenge. At present, most radiotracers are antagonist ligands. In vitro data suggest that properties differ between GPCR agonists and antagonists: antagonists bind to receptors with a single affinity, whereas agonists are characterized by two different affinities: high affinity for receptors that undergo functional coupling to G-proteins, and low affinity for those that are not coupled. In this context, agonist radiotracers may be useful tools to give functional images of GPCRs in the brain, with high sensitivity to neurotransmitter release. Here, we review all existing PET radiotracers used from animals to humans and their role for understanding the ligand-receptor paradigm of GPCR in comparison with corresponding antagonist radiotracers.

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.

G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase Cα and ε in INS-1 cells.

ObjectiveThe mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells.MethodsUsing epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia.ResultsAt 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 μM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect.ConclusionGW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

The protein tyrosine phosphatase PTPN7 is a negative regulator of ERK activation and thromboxane generation in platelets

Protein tyrosine phosphatase nonreceptor type 7 (PTPN7), also called hematopoietic protein tyrosine phosphatase, controls extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase in T lymphocytes. Because ERK1/2 plays an important role in regulating thromboxane A2 (TXA2) generation in platelets, we investigated the function of PTPN7 in these cells. Using immunoblot analysis, we detected PTPN7 in both human and mouse platelets but not in PTPN7-null mice. PTPN7 KO mouse platelets exhibited increased platelet functional responses, including aggregation, dense granule secretion, and TXA2 generation, compared with platelets from WT littermates, upon stimulation with both G protein-coupled receptor (GPCR) and glycoprotein VI (GPVI) agonists. Using the GPCR agonist AYPGKF in the presence of the COX inhibitor indomethacin, we found that PTPN7 KO mouse platelets aggregated and secreted to the same extent as WT platelets, suggesting that elevated TXA2 is responsible for the potentiation of platelet functional responses in PTPN7-KO platelets. Phosphorylation of ERK1/2 was also elevated in PTPN7 KO platelets. Stimulation of platelets with the GPVI agonist collagen-related peptide along with the COX inhibitor indomethacin did not result in phosphorylation of ERK1/2, indicating that GPVI-mediated ERK phosphorylation occurs through TXA2 Although bleeding times did not significantly differ between PTPN7-null and WT mice, time to death was significantly faster in PTPN7-null mice than in WT mice in a pulmonary thromboembolism model. We conclude that PTPN7 regulates platelet functional responses downstream of GPCR agonists, but not GPVI agonists, through inhibition of ERK activation and thromboxane generation.

Common umbilical cord insertion in a monochorionic, monoamniotic twin pregnancy - a case report.

Ischemia and reperfusion remain inseparable elements of numerous medical procedures such as by-pass surgery, organ transplantation or other cardiology and intervention radiology. The contraction of the smooth muscle of the vessel is considered to be one of the basic components leading to impaired perfusion, an increase in the oxygen deficit of the endothelium of the vessel, and subsequently also to tissues vascularized by the vessel.Main aim of this study was to evaluate the effect of ischemia and reperfusion on vascular smooth muscle cells stimulated pharmacologically with mastoparan-7 (direct G-protein activator) in comparison to stimulation of G-protein coupled receptor agonist - phenylephrine, and direct calcium channel activator - Bay K8644.Experiments were performed on isolated and perfused tail artery of Wistar rats. Contraction force in our model was measured by increased level of perfusion pressure with a constant flow.Concentration-response curves obtained for phenylephrine, mastoparan-7 and Bay K8644 presented a sigmoidal relation. Ischemia induced hyporreactivity of vessels, whereas during reperfusion the significant time related hyperreactivity for phenylephrine and mastoparan-7 only but not for Bay K8644. These reactions were secondary to the modulation of calcium influx from intra- and extracellular calcium stores.Results of our experiments suggest that mastoparan-7 significantly induces contraction of vascular smooth muscle cells not only for controls but in the presence of ischemia and reperfusion too. Potential therapeutic applications of the observed reactions are important. They may include regenerative processes within the nervous system, studies on the improvement of blood flow within the microcirculation, or antimicrobial activity. Modulation of the G protein-phospholipase C response may also be an interesting point of action of future drugs modifying the response to stimulation during ischemia in particular, such activities could take place during the transport of organs for transplantation.

Dynamin 2 is required for GPVI signaling and platelet hemostatic function in mice.

Receptor-mediated endocytosis, which contributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration, requires endocytic vesicle release by the large GTPase dynamin 2. Here, the role of dynamin 2 was investigated in platelet hemostatic function using both pharmacological and genetic approaches. Dnm2fl/fl Pf4-Cre (Dnm2Plt−/−) mice specifically lacking dynamin 2 within the platelet lineage developed severe thrombocytopenia and bleeding diathesis and Dnm2Plt−/− platelets adhered poorly to collagen under arterial shear rates. Signaling via the collagen receptor GPVI was impaired in platelets treated with the dynamin GTPase inhibitor dynasore, as evidenced by poor protein tyrosine phosphorylation, including that of the proximal tyrosine kinase Lyn on its activating tyrosine 396 residue. Platelet stimulation via GPVI resulted in a slight decrease in GPVI, which was maintained by dynasore treatment. Dynasore-treated platelets had attenuated function when stimulated via GPVI, as evidenced by reduced GPIbα downregulation, α-granule release, integrin αIIbβ3 activation, and spreading onto immobilized fibrinogen. By contrast, responses to the G-protein coupled receptor agonist thrombin were minimally affected by dynasore treatment. GPVI expression was severely reduced in Dnm2Plt−/− platelets, which were dysfunctional in response to stimulation via GPVI, and to a lesser extent to thrombin. Dnm2Plt−/− platelets lacked fibrinogen in their α-granules, but retained von Willebrand factor. Taken together, the data show that dynamin 2 plays a proximal role in signaling via the collagen receptor GPVI and is required for fibrinogen uptake and normal platelet hemostatic function.

Anti-Apoptotic Effect of G-Protein-Coupled Receptor 40 Activation on Tumor Necrosis Factor-α-Induced Injury of Rat Proximal Tubular Cells

G-protein-coupled receptor 40 (GPR40) has an anti-apoptotic effect in pancreatic β-cells. However, its role in renal tubular cell apoptosis remains unclear. To explore the role of GPR40 in renal tubular apoptosis, a two-week unilateral ureteral obstruction (UUO) mouse model was used. The protein expression of GPR40 was decreased, while the Bax/Bcl-2 protein expression ratio, the expression of tumor necrosis factor (TNF)-α mRNA, and angiotensin II type 1 receptor (AT1R) protein were increased in mice with UUO. In vitro, pretreatment of rat proximal tubular (NRK52E) cells with GW9508, a GPR40 agonist, attenuated the decreased cell viability, increased the Bax/Bcl-2 protein expression ratio, increased protein expression of cleaved caspase-3 and activated the nuclear translocation of nuclear factor-κB (NF-κB) p65 subunit induced by TNF-α treatment. TNF-α treatment significantly increased the expression of AT1R protein and the generation of reactive oxygen species (ROS), whereas GW9508 treatment markedly reversed these effects. Pretreatment with GW1100, a GPR40 antagonist, or silencing of GPR40 in NRK52E cells promoted the increased expression of the cleaved caspase-3 protein by TNF-α treatment. Our results demonstrate that decreased expression of GPR40 is associated with apoptosis via TNF-α and AT1R in the ureteral obstructed kidney. The activation of GPR40 attenuates TNF-α-induced apoptosis by inhibiting AT1R expression and ROS generation through regulation of the NF-κB signaling pathway.

Lodoxamide Attenuates Hepatic Fibrosis in Mice: Involvement of GPR35

A previous pharmacogenomic analysis identified cromolyn, an anti-allergic drug, as an effective anti-fibrotic agent that acts on hepatocytes and stellate cells. Furthermore, cromolyn was shown to be a G protein-coupled receptor 35 (GPR35) agonist. However, it has not been studied whether anti-fibrotic effects are mediated by GPR35. Therefore, in this study, the role of GPR35 in hepatic fibrosis was investigated through the use of lodoxamide, another anti-allergic drug and a potent GPR35 agonist. Long-term treatment with carbon tetrachloride induced hepatic fibrosis, which was inhibited by treatment with lodoxamide. Furthermore, CID2745687, a specific GPR35 antagonist, reversed lodoxamide-mediated anti-fibrotic effects. In addition, lodoxamide treatment showed significant effects on the mRNA expression of collagen Iα1, collagen Iα2, and TGF-β1 in the extracellular matrix. However, a transforming growth factor α (TGF-α) shedding assay revealed lodoxamide not to be a potent agonist of mouse GPR35 in vitro. Therefore, these results showed anti-fibrotic effects of lodoxamide in mice and raise concerns how lodoxamide protects against liver fibrosis in vivo and whether GPR35 is involved in the action.

Quercetin-3-oleoyl derivatives as new GPR40 agonists: Molecular docking studies and functional evaluation.

The G-protein-coupled receptor 40 (GPR40) is an attractive molecular target for the treatment of type 2 diabetes mellitus. Previously, based on the natural oleic acid substrate, an exogenous ligand for this receptor, named AV1, was synthesized. In this context, here we validated the activity of AV1 as a full agonist, while the corresponding catechol analogue, named AV2, was investigated for the first time. The ligand-protein interaction between this new molecule and the receptor was highlighted in the lower portion of the GPR40 groove that generally accommodates DC260126. The functional assays performed have demonstrated that AV2 is a suitable GPR40 partial agonist, showing a therapeutic potential and representing a useful tool in the management of type 2 diabetes.