cAMP Inhibition Research Articles

Platelet inhibition by hypochlorous acid involves cAMP signalling.

Hypochlorous acid (HOCl), made by neutrophil-derived myeloperoxidase, has been suggested to inhibit platelets, however, the mechanisms involved have not been described. Here we confirm that HOCl exposure changes platelet morphology and inhibits platelet spreading and aggregation. HOCl effects could be reversed by glutathione suggesting a role for cysteine oxidation. Mass spectrometry-based proteomics of HOCl-exposed platelets revealed oxidised cysteine residues in 37 proteins including adenylate cyclase 6 and Rap1B. Adenylate cyclase is involved in the inhibitory cAMP pathway triggered by endothelium-derived prostacyclin and Rap1 is a small G protein required for integrin αIIbβ3 activation and platelet aggregation. We show that HOCl exposure stimulates cAMP production and phosphorylation of the cAMP-dependent protein kinase substrate VASP in platelets and transfected HEK293T cells. In addition, HOCl inhibited Rap1-GTP formation. These data suggest that HOCl inhibits platelets at least in part through the cAMP pathway and by regulating Rap1. Thus, this study provides new insights into HOCl mediated crosstalk between neutrophils and platelets.

Unique pharmacological properties of etrasimod among S1P receptor modulators.

Etrasimod (ADP334) is an oral, once-daily, selective sphingosine 1-phosphate (S1P)1,4,5 receptor modulator for the treatment of moderately to severely active ulcerative colitis and in development for the treatment of immune-mediated inflammatory diseases. Interaction between S1P and its five receptor subtypes (S1P1-S1P5) plays a role in several physiologic systems, including the cardiovascular and immune systems. Since differences in S1PR binding and downstream intracellular signaling could contribute to distinct profiles of drug efficacy and safety, we directly compared the S1P1-5 selectivity profile of etrasimod to three marketed S1PR modulators: fingolimod, ozanimod, and siponimod. Using both heterologous expression systems and human umbilical vein endothelial cells that spontaneously express S1P1, we profiled key S1P1 downstream signaling pathways and found that etrasimod had similar potency to the other tested S1PR modulators in promoting β-arrestin recruitment and S1P1 internalization. However, etrasimod was notably less potent than other S1PR modulators in assays measuring S1P1-mediated G protein activation (GTPγS binding and cAMP inhibition). Relatively lower potency of etrasimod in inducing G protein signaling corresponded to significantly diminished activation of human cardiac G protein-coupled inwardly rectifying potassium channels when compared to ozanimod. Together with pharmacokinetic properties, this hell profile of etrasimod may contribute to the positive benefit risk profile of etrasimod observed during the phase III ELEVATE UC 52 and ELEVATE UC 12 trials in patients with moderately to severely active ulcerative colitis.

New peripherally-restricted CB1 receptor antagonists, PMG-505–010 and −013 ameliorate obesity-associated NAFLD and fibrosis

The endocannabinoid system plays a crucial role in metabolic regulation, prompting the investigation of cannabinoid type 1 receptor (CB1R) antagonists for obesity and its complications like non-alcoholic fatty liver disease (NAFLD). Concerns over psychiatric side effects led to the development of peripheral CB1R antagonists that circumvent the blood-brain barrier (BBB). In this study, we synthesized PMG-505–010 and PMG-505–013 as peripherally restricted CB1 receptor antagonists by modifying rimonabant to minimize BBB penetration. Physicochemical analysis confirmed their reduced lipophilicity and increased polarity compared to rimonabant, indicating limited brain exposure. Molecular docking studies revealed similar binding modes to rimonabant at CB1R, characterized by robust hydrophobic interactions. Functionally, they acted as CB1R antagonists and inverse agonists, effectively reversing CP55,940-induced cAMP inhibition. In a murine model of obesity-related NAFLD, PMG-505–010 and −013 improved metabolic profiles, including fasting blood glucose levels and dyslipidemia. They also ameliorated hepatic injury, steatosis, and inflammation, evidenced by reduced liver enzymes, lipid peroxidation, hepatic lipid levels, and inflammatory cytokine levels. Notably, these compounds inhibited hepatic fibrosis by reducing extracellular matrix (ECM) deposition and altering fibrosis-related gene and protein expressions. In conclusion, PMG-505–010 and PMG-505–013 hold promise for treating obesity-related liver diseases, including NAFLD and fibrosis, through selective peripheral CB1R targeting, potentially avoiding CNS-related side effects seen with earlier CB1R antagonists.

Direct detection of endogenous Gαi activity in cells with a sensitive conformational biosensor.

Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is not only a mechanism broadly used by eukaryotes to transduce signals across the plasma membrane, but also the target for a large fraction of clinical drugs. However, approaches typically used to assess this signaling mechanism by directly measuring G-protein activity, like optical biosensors, suffer from limitations. On one hand, many of these biosensors require expression of exogenous GPCRs and/or G-proteins, compromising readout fidelity. On the other hand, biosensors that measure endogenous signaling may still interfere with the signaling process under investigation or suffer from having a small dynamic range of detection, hindering broad applicability. Here, we developed an optical biosensor that detects the endogenous G-protein active species Gαi-GTP upon stimulation of endogenous GPCRs more robustly than current state-of-the-art sensors for the same purpose. Its design is based on the principle of bystander Bioluminescence Resonance Energy Transfer (BRET) and leverages the Gαi-binding protein named GINIP as a high affinity and specific detector module of the GTP-bound conformation of Gαi. We optimized this design to prevent interference with Gi-dependent signaling (cAMP inhibition) and to enable implementation in different experimental systems with endogenous GPCRs, including neurotransmitter receptors in primary astroglial cells or opioid receptors in cell lines, which revealed opioid neuropeptide-mediated activation profiles different from those observed with other biosensors involving exogenous GPCRs and G-proteins. Overall, we introduce a biosensor that directly and sensitively detects endogenous activation of G-proteins by GPCRs across different experimental settings without interfering with the subsequent propagation of signaling.

Baicalein isolated from Oroxylum indicum acts as a potent µ- and κ-opioid receptor antagonist agent via the reversal of agonist-mediated cAMP inhibition

The opioid receptors play a pivotal role in the treatment of several neuropsychiatric and neurological disorders. Oroxylum indicum (L.) Kurtz is a very important medicinal plant with several therapeutic applications. It is a main constituent of the Ayurvedic formulation ‘Dashmool’ used for multifaceted disorders by the Indians. However, the constituents of this plant in neurological conditions have not been well studied. Here, we performed activity-guided isolation of compounds for opioid receptor modulator activity. In the study, we found that the isolated compound baicalein (3) has shown the most potent and competitive antagonistic activity at 20 mg/kg dose in vivo experiments. The acute dose of 3 (20 mg/kg) and pan opioid receptor antagonist naloxone (20 mg/kg) block the morphine-induced antinociception and the paw withdrawal latency decreases up to 8.3 s and 9.6 s, respectively. The in silico studies also support our in vitro data that compound 3 binds with MOR and KOR.

Biased receptor signalling and intracellular trafficking profiles of structurally distinct formylpeptide receptor 2 agonists.

There is increasing interest in developing FPR2 agonists (compound 43, ACT-389949 and BMS-986235) as potential pro-resolving therapeutics, with ACT-389949 and BMS-986235 having entered phase I clinical development. FPR2 activation leads to diverse downstream outputs. ACT-389949 was observed to cause rapid tachyphylaxis, while BMS-986235 and compound 43 induced cardioprotective effects in preclinical models. We aim to characterise the differences in ligand-receptor engagement and downstream signalling and trafficking bias profile. Concentration-response curves to G protein dissociation, β-arrestin recruitment, receptor trafficking and second messenger signalling were generated using FPR2 ligands (BMS-986235, ACT-389949, compound 43 and WKYMVm), in HEK293A cells. Log(τ/KA) was obtained from the operational model for bias analysis using WKYMVm as a reference ligand. Docking of FPR2 ligands into the active FPR2 cryoEM structure (PDBID: 7T6S) was performed using ICM pro software. Bias analysis revealed that WKYMVm and ACT-389949 shared a very similar bias profile. In comparison, BMS-986235 and compound 43 displayed approximately 5- to 50-fold bias away from β-arrestin recruitment and trafficking pathways, while being 35- to 60-fold biased towards cAMP inhibition and pERK1/2. Molecular docking predicted key amino acid interactions at the FPR2 shared between WKYMVm and ACT-389949, but not with BMS-986235 and compound 43. In vitro characterisation demonstrated that WKYMVm and ACT-389949 differ from BMS-986235 and compound 43 in their signalling and protein coupling profile. This observation may be explained by differences in the ligand-receptor interactions. In vitro characterisation provided significant insights into identifying the desired bias profile for FPR2-based pharmacotherapy.

Investigating selectivity and bias for G protein subtypes and β-arrestins by synthetic cannabinoid receptor agonists at the cannabinoid CB1 receptor

The cannabinoid CB1 receptor (CB1) is a G protein-coupled receptor (GPCR) with widespread expression in the central nervous system. This canonically G⍺i/o-coupled receptor mediates the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoid receptor agonists (SCRAs). Recreational use of SCRAs is associated with serious adverse health effects, making pharmacological research into these compounds a priority. Several studies have hypothesised that signalling bias may explain the different toxicological profiles between SCRAs and THC. Previous studies have focused on bias between G protein activation measured by cyclic adenosine monophosphate (cAMP) inhibition and β-arrestin translocation. In contrast, the current study characterises bias between G⍺ subtypes of the G⍺i/o family and β-arrestins; this method facilitates a more accurate assessment of ligand bias by assessing signals that have not undergone major amplification. We have characterised G protein dissociation and translocation of β-arrestin 1 and 2 using real-time BRET reporters. The responses produced by each SCRA across the G protein subtypes tested were consistent with the responses produced by the reference ligand AMB-FUBINACA. Ligand bias was probed by applying the operational analysis to determine biases within the G⍺i/o family, and between G protein subtypes and β-arrestins. Overall, these results confirm SCRAs to be balanced, high-efficacy ligands compared to the low efficacy ligand THC, with only one SCRA, 4CN-MPP-BUT7IACA, demonstrating statistically significant bias in one pathway comparison (towards β-arrestin 1 when compared with G⍺oA/oB). This suggests that the adverse effects caused by SCRAs are due to high potency and efficacy at CB1, rather than biased agonism.

The μ-opioid receptor-mediated Gi/o protein and β-arrestin2 signaling pathways both contribute to morphine-induced side effects

The μ-opioid receptor-biased agonist theory holds that Gio protein signaling mediates the analgesic effect of opioids and the related side effects via the β-arrestin2 signaling pathway. A series of μ-opioid-biased agonists have been developed in accordance with this theory, and the FDA has approved TRV130 (as a representative of biased agonists) for marketing. However, several reports have raised the issue of opioid side effects associated with the use of agonists. In this study, five permeable peptides were designed to emulate 11 S/T phosphorylation sites at the μ-opioid receptor (MOR) carboxyl-terminal. In vitro experiments were performed to detect the activation level of G proteins from the cAMP inhibition assay and the β-arrestin2 recruitment by the BRET assay. Designed peptides might effectively interfere with the activation of the Gio and β-arrestin2 pathways when combined with morphine. The resulting morphine-induced tolerance, respiratory inhibition, and constipation in mice showed that the β-arrestin2 pathway was responsible for morphine tolerance while the Gio signaling pathway was involved with respiratory depression and constipation and that these side effects were significantly related to phosphorylation sites S363 and T370. This study may provide new directions for the development of safer and more effective opioid analgesics, and the designed peptides may be an effective tool for exploring the mechanism by which μ-opioid receptors function, with the potential of reducing the side effects that are associated with clinical opioid treatment.

Insight into the mechanism of action of ORG27569 at the cannabinoid type one receptor utilising a unified mathematical model

Allosteric modulation of CB1 is therapeutically advantageous compared to orthosteric activation as it potentially offers reduced on-target adverse effects. ORG27569 is an allosteric modulator that increases orthosteric agonist binding to CB1 but decreases functional signalling. ORG27569 is characterised by a delay in disinhibition of agonist-induced cAMP inhibition (lag); however, the mechanism behind this kinetic lag is yet to be identified. We aimed to utilise a mathematical model to predict data and design in vitro experiments to elucidate mechanisms behind the unique signalling profile of ORG27569. The established kinetic ternary complex model includes the existence of a transitional state of CB1 bound to ORG27569 and CP55940 and was used to simulate kinetic cAMP data using NONMEM 7.4 and Matlab R2020b. These data were compared with empirical cAMP BRET data in HEK293 cells stably expressing hCB1. The pharmacometric model suggested that the kinetic lag in cAMP disinhibition by ORG27569 is caused by signal amplification in the cAMP assay and can be reduced by decreasing receptor number. This was confirmed experimentally, as reducing receptor number through agonist-induced internalisation resulted in a decreased kinetic lag by ORG27569. ORG27569 was found to have a similar interaction with CP55940 and the high efficacy agonist WIN55,212-2, and was suggested to have lower affinity for CB1 bound by the partial agonist THC compared to CP55940. Allosteric modulators have unique signalling profiles that are often difficult to interrogate exclusively in vitro. We have used a combined mathematical and in vitro approach to prove that ORG27569 causes a delay in disinhibition of agonist-induced cAMP inhibition due to large receptor reserve in this pathway. We also used the pharmacometric model to investigate the common phenomenon of probe dependence, to propose that ORG27569 binds with higher affinity to CB1 bound by high efficacy orthosteric agonists.

Macrophage β2-AR activation amplifies inflammation in wound healing by upregulating Trem1 via the cAMP/PKA/CREB pathway

BackgroundInflammation is an important part of the wound healing process. The stress hormone epinephrine has been demonstrated to modulate the inflammatory response via its interaction with β2-adrenergic receptor (β2-AR). However, the precise molecular mechanism through which β2-AR exerts its influence on inflammation during the wound healing process remains an unresolved question. MethodsTranscriptome datasets of wound and macrophages from the GEO database were reanalyzed using bioinformatics. The role of β2-AR in wound healing was explored by a mouse hind paw plantar wound model, and histological analyses were performed to assess wound healing. In vivo and in vitro assays were performed to elucidate the role of β2-AR on the inflammatory response. Triggering receptor expressed on myeloid cells 1 (Trem1) was knocked down with siRNA on RAW cells and western blot and qPCR assays were performed. ResultsTrem1 was upregulated within 24 h of wounding, and macrophage β2-AR activation also upregulated Trem1. In vivo experiments demonstrated that β2-AR agonists impaired wound healing, accompanied by upregulation of Trem1 and activation of cAMP/PKA/CREB pathway, as well as by a high level of pro-inflammatory cytokine production. In vitro experiments showed that macrophage β2-AR activation amplified LPS-induced inflammation, and knockdown of Trem1 reversed this effect. Using activator and inhibitor of cAMP, macrophage β2-AR activation was confirmed to upregulate Trem1 via the cAMP/PKA/CREB pathway. ConclusionOur study found that β2-AR agonists increase Trem1 expression in wounds, accompanied by amplification of the inflammatory response, impairing wound healing. β2-AR activation in RAW cells induces Trem1 upregulation via the cAMP/PKA/CREB pathway and amplifies LPS-induced inflammatory responses.

Ligand-directed biased agonism at human histamine H3 receptor isoforms across Gαi/o- and β-arrestin2-mediated pathways

The histamine H3 receptor (H3R) is a neurotransmitter receptor that is primarily found in the brain, where it controls the release and synthesis of histamine, as well as the release of other neurotransmitters (e.g. dopamine, serotonin). Notably, 20 H3R isoforms are differentially expressed in the human brain as a consequence of alternative gene splicing. The hH3R-445, -415, -365 and -329 isoforms contain the prototypical GPCR (7TM) structure, yet exhibit deletions in the third intracellular loop, a structural domain that is pivotal for G protein-coupling, signaling and regulation. To date, the physiological relevance underlying the individual and combinatorial function of hH3R isoforms remains poorly understood. Nevertheless, given their significant implication in physiological processes (e.g. cognition, homeostasis) and neurological disorders (e.g. Alzheimer’s and Parkinson’s disease, schizophrenia), widespread targeting of hH3R isoforms by drugs may lead to on-target side effects in brain regions that are unaffected by disease. To this end, isoform- and/or pathway-selective targeting of hH3R isoforms by biased agonists could be of therapeutic relevance for the development of region- and disease-specific drugs. Hence, we have evaluated ligand biased signaling at the hH3R-445, -415, -365 and -329 isoforms across various Gαi/o-mediated (i.e. [35S]GTPγS accumulation, cAMP inhibition, pERK1/2 activation, pAKT T308/S473 activation) and non Gαi/o-mediated (i.e. β-arrestin2 recruitment) endpoints that are relevant to neurological diseases. Our findings indicate that H3R agonists display significantly altered patterns in their degree of ligand bias, in a pathway- and isoform-dependent manner, underlining the significance to investigate GPCRs with multiple isoforms to improve development of selective drugs. Subject categoryNeuropharmacology.

Galacto-Oligosaccharides Alleviate LPS-Induced Immune Imbalance in Small Intestine through Regulating Gut Microbe Composition and Bile Acid Pool.

Recent evidence suggests that the protective effect of gut microbiota on intestinal inflammation can be achieved through a microbe-bile acids (BAs) mechanism. Galacto-oligosaccharides (GOS) are a kind of prebiotic that alter gut microbiota composition. To verify whether GOS has a protective effect on intestinal inflammation through a microbe-BAs mechanism, this research was performed in a lipopolysaccharide (LPS) porcine model with the presence or absence of GOS. GOS prevented LPS-induced production of pro-inflammatory cytokines, the decrease of bacterial bile salt hydrolase-containing bacteria abundance, and the decrease of chendoxycholic acid (CDCA) level in piglets. Additionally, CDCA decreased LPS-induced production of pro-inflammatory cytokines, induced the expression of the takeda G-protein receptor 5 (TGR5), and its downstream cyclic adenosine monophosphate (cAMP) production in lamina propria-derived CD11b+ cells. The cAMP inhibitor eliminated the protective effect of CDCA on lamina propria-derived CD11b+ cells. These results suggested that GOS reduced the production of pro-inflammatory cytokines and inhibited NF-κB activation via microbe-BA-dependent TGR5-cAMP signaling in LPS-challenged piglets.

Sphingosine-1-phosphate mediates FSH-induced cell viability but not steroidogenesis in bovine granulosa cells

Follicle-stimulating hormone (FSH) stimulates the proliferation, survival, and estradiol synthesis of granulosa cells by binding to their G protein-coupled receptors. Although FSH activates sphingosine kinase-1 (SPHK1) to induce sphingosine-1-phosphate (S1P) synthesis, which is required to mediate the proliferative and survival effect of this gonadotrophin, the mechanisms, and the role of S1P in estradiol synthesis have not been reported. This study aimed to evaluate the importance of FSH-induced S1P synthesis as a mediator of the effects of this gonadotrophin on granulosa cell viability and steroidogenesis and to determine if FSH-induced S1P synthesis depends on estradiol, cAMP, PKA, or PKC. To achieve these objectives, we tested the effects of FSH, a sphingosine kinase-1 inhibitor (SKI-178), estradiol and inhibitors of aromatase, cAMP, PKA, and PKC (Formestane, MDL-12330A, H-89 dihydrochloride hydrate and Calphostin C respectively), on granulosa cell viability, S1P and estradiol production, and the mRNA expression of CYP19A1 and STAR in four in vitro culture experiments. The addition of FSH (1 ng/mL) increased (P < 0.05) granulosa cells number and S1P concentration in the culture media. Conversely, the addition of SKI-178 (10 μM) reduced (P < 0.05) S1P concentration negating the effect of FSH on cell viability. Inhibition of PKC and PKA, but not cAMP, reduced (P < 0.05) S1P secretion of FSH treated granulosa cells. It is important to note that the reduction in S1P secretion was strong (49 %) with the use of the PKC inhibitor. The use of formestane (10 μg) did not modify (P > 0.05) S1P secretion in FSH-treated cells; however, the addition of 5 or 10 ng/mL of estradiol increased (P < 0.05) S1P secretion. Finally, FSH increased (P < 0.05) estradiol concentration in the culture media, but this effect was not blocked by the inhibition of S1P synthesis. Similarly, FSH, SKI-178 or their combination did not modify the mRNA expression of CYP19A1 and STAR. In conclusion, S1P synthesis is stimulated FSH in granulosa cells and mediated mainly by PKC. S1P in turn promotes the granulosa cell viability, however, this does not influence estradiol synthesis. Additionally, estradiol synthesis induced by FSH is not essential for S1P synthesis, however high estradiol concentration may stimulate S1P production by granulosa cells.

SAT025 Characterization Of The Melatonin Type 1 Receptor / Cannabinoid Type 1 Receptor (MT1/CB1) Heteromers

Abstract Disclosure: Y. Cui: None. H. Cherdoud: None. G. Marsicano: None. L. Bellocchio: None. R. Jockers: None. O. Lahuna: None. Membrane receptors belonging to the G proteins coupled receptors (GPCRs), the largest family of proteins in the human genome with more than 800 members. GPCR functions were initially thought to take place at the plasma membrane upon binding of single monomeric receptor unit by their cognate ligand. This scheme was complexified after identification of cross-talks between GPCRs upon heteromerization and identification of functional GPCRs within intracellular organelles as nucleus or endosomes. Among a few others receptors, functional melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were found in neuronal mitochondria, suggesting a cross-talk between them. Here, we are identifying and characterizing MT1/CB1 heteromers as a new GPCR heteromer. Previously we showed that coexpression of the MT1 and CB1 receptors induced the constitutive formation of MT1/CB1 heteromers using coimmunoprecipitation experiments on transiently transfected HEK293 cells. Using simultaneous PLA (Proximity Ligation Assay) and immunofluorescence experiments, we described the existence of intracellular MT1/CB1 heteromers associated with the mitochondrial network. Since then, we have further characterized the properties of MT1/CB1 heteromers. Coimmunoprecipitation experiments performed on cerebellum from wild-type, MT1 or CB1 knock-out mice enabled us to identify MT1/CB1 heteromers in vivo. We established stable monoclonal HEK293 cell lines expressing both receptors. Transmission Electronic Microscopy (TEM) and PLA experiments showed the presence of MT1/CB1 complexes not only intracellularly but also at the cell surface. Using PLA experiments we identified a penetrating interfering peptide that specifically dissociate the MT1/CB1 complex and thus open the door to experiments targeting specifically the complex. We were able to follow the melatonin-dependent internalization and trafficking of the MT1/CB1 complex from the plasma membrane to the late endosomes using a combination of Nanoluciferase complementation experiments and bystander BRET (Bioluminescence Resonance Energy Transfer). Costimulation of HE293 cells expressing both receptors with melatonin and the selective CB1 agonist ACEA (Arachidonyl-2'-chloroethylamide), a derivative of the endocannabinoid AEA (Arachidonylethanolamide or anandamide) resulted in a potentiation of the melatonin-dependent cAMP inhibition. Use of the identified interfering peptide will enable us to identify actions on signaling pathways resulting from a direct cross-talk between MT1 or CB1 subunits. Presentation: Saturday, June 17, 2023

LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration

BackgroundG protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA1) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA1 as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA1 heteromer and characterized the unique molecular features and function of this heteromer.MethodsWe employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA1. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA1 heteromer, we performed various assays, including cAMP, BRET for G protein activation, β-arrestin recruitment, ligand binding, and transwell migration assays.ResultsWe observed that CXCR4 forms heteromers with LPA1 in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA1 with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gαi/o activation, and β-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA1-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gαi/o recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA1. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA1-selective antagonists enhanced CXCL12-induced Gαi/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA1-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA1 antagonists.ConclusionsThe presence and impact of CXCR4-LPA1 heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA1 inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA1 antagonists alone for such conditions.1EbjTZCQ7TNhXqMNgbxB5AVideo

Abstract P235: The Absence Of Inhibitory G Protein Regulation By Rgs2 Increases Basal Phosphodiesterase Activity To Decrease Forskolin-stimulated Camp Generation And Enhances Myogenic Response In Uterine Arteries Of Non-pregnant Mice

G protein regulation by regulators of G protein signaling (RGS) proteins is crucial to maintaining normal vascular tone for promoting uterine vascular bed perfusion. However, the specific regulatory mechanisms involved are incompletely understood. Previously, we showed that loss of regulation of Gi/o and Gq/11 by RGS2 contributes to enhanced myogenic tone and decreased uterine blood flow in non-pregnant mice. Similarly, RGS5, closely related to RGS2, was found to fine-tune G protein signaling and modulate GPCR-evoked vascular reactivity. RGS2 and RGS5 are co-expressed in the uterine vascular bed. However, it is unclear whether their regulatory activities are coordinated in fine-tuning G protein signaling to modulate vascular tone. Here, we tested whether the combined activity of RGS2 and 5 reduces myogenic tone by negatively regulating Gi/o signaling to promote cAMP-dependent uterine artery relaxation. We used commercially available kits to measure forskolin-stimulated cAMP generation and basal phosphodiesterase (PDE) activity in freshly isolated uterine arteries from non-pregnant wild type, Rgs5 KO and Rgs2 KO mice. Basal tone and myogenic response to increasing intraluminal pressure were assessed using ex vivo pressure myography. We found that the absence of RGS2 but not RGS5 decreased forskolin-stimulated cAMP generation. Conversely, basal PDE activity was augmented in both Rgs2 KO and Rgs5 KO arteries, though the increase was less robust in Rgs5 KO arteries. Basal tone and myogenic response were reduced in Rgs5 KO arteries compared to WT or Rgs2 KO arteries in the absence or presence of nitric oxide synthase blockade with L-NAME. Activating Gi/o with dopamine increased myogenic constriction in all genotypes but less robustly in Rgs5 KO arteries, while pre-incubation of the vessels with exogenous cAMP or PDE inhibition with IBMX normalized maximal myogenic response to similar levels in all groups. Collectively, these findings indicate that altered Gi/o regulation due to RGS2 absence enhances myogenic tone by promoting PDE-mediated inhibition of cAMP-dependent vasodilation. While both RGS2 and RGS5 negatively regulate this novel Gi/o-PDE-cAMP signaling pathway, RGS5 dampens the inhibitory efficacy of RGS2 towards Gi/o in uterine arteries.

A kinetic model for positive allosteric modulator (PAM)-antagonists for the type 1 cannabinoid receptor (CB1 ).

The type 1 cannabinoid receptor (CB1 ) is amongst the most abundant G-protein coupled receptors in brain. Allosteric ligands bind to a different site on receptors than the orthosteric ligand and can have effects that are both unique to the allosteric ligand and/or modulate orthosteric ligand activity. We aim to propose a unified mathematical model to describe the interaction effects of the allosteric ligand ORG27569 (ORG) and the orthosteric agonist CP55940 (CP) on CB1 . A ternary complex model was constructed which incorporated kinetic properties to describe the time-course of effects of ORG and CP reported in the literature: i) enhanced receptor binding of CP, ii) reduced internalisation and iii) time dependent modulation of cAMP. Underlying mechanisms of time-dependent modulation by ORG were evaluated by simulation. A hypothetical transitional state of CP-CB1 -ORG, which can internalise but cannot inhibit cAMP, was shown to be necessary and sufficient to describe the allosteric modulation by ORG, prior to receptors adopting an inactive conformation. The model indicated that the formation of this transitional CP-CB1 -ORG state and final inactive CP-CB1 -ORG state contributes to the enhanced CP binding. The inactive CP-CB1 -ORG cannot internalise or inhibit cAMP, leading to reduced internalisation and cessation of cAMP inhibition. In conclusion, a kinetic mathematical model for CB1 allosteric modulation was developed. However, a standard ternary complex model was not sufficient to capture the data and a hypothetical transitional state was required to describe the allosteric modulation properties of ORG.

ACTH/cAMP-Mediated Skin Pigmentation Caused By 5-Fluorouracil Administration.

Anticancer drugs exhibit many side effects, including skin pigmentation, which often lowers patient quality of life. However, the mechanism of pigmentation caused by anticancer drugs remains unknown. The purpose of this study was to elucidate the mechanism of anticancer drug-induced skin pigmentation using 5-fluorouracil (5-FU), a widely used anticancer drug. Specific pathogen-free, 9-week-old Hos:HRM-2 male mice were intraperitoneally administered 5-FU daily for 8 weeks. Skin pigmentation was observed at the end of the study. Mice treated with 5-FU were also administered inhibitors of cAMP, α-melanocyte-stimulating hormone (α-MSH), and adrenocorticotropic hormone (ACTH) for analysis. Administration of oxidative stress, nuclear factor-kappa B (NF-κB), cAMP, and ACTH inhibitors reduced pigmentation in 5-FU-treated mice. These results indicate that the oxidative stress/NF-κB/ACTH/cAMP/tyrosinase pathway plays an important role in pigmentation in 5-FU-treated mice.

Pistacia vera Extract Potentiates the Effect of Melatonin on Human Melatonin MT1 and MT2 Receptors with Functional Selectivity.

Melatonin is a tryptophan derivative synthesized in plants and animals. In humans, melatonin acts on melatonin MT1 and MT2 receptors belonging to the G protein-coupled receptor (GPCR) family. Synthetic melatonin receptor agonists are prescribed for insomnia and depressive and circadian-related disorders. Here, we tested 25 commercial plant extracts, reported to have beneficial properties in sleep disorders and anxiety, using cellular assays (2─[125I]iodomelatonin binding, cAMP inhibition, ERK1/2 activation and β-arrestin2 recruitment) in mock-transfected and HEK293 cells expressing MT1 or MT2. Various melatonin receptor-dependent and -independent effects were observed. Extract 18 (Ex18) from Pistacia vera dried fruits stood out with very potent effects in melatonin receptor expressing cells. The high content of endogenous melatonin in Ex18 (5.28 ± 0.46 mg/g extract) is consistent with this observation. Ex18 contains an additional active principle that potentiates the effect of melatonin on Gi protein-dependent pathways but not on β-arrestin2 recruitment. Further active principles potentiating exogenous melatonin were detected in several extracts. In conclusion, we identified plant extracts with various effects in GPCR-based binding and signalling assays and identified high melatonin levels and a melatonin-potentiating activity in Pistacia vera dried fruit extracts that might be of therapeutic potential.

Suppression of cAMP/PKA/CREB signaling ameliorates retinal injury in diabetic retinopathy.

The blood-retinal barrier (BRB), homeostasis, neuronal integrity, and metabolic processes are all directly influenced by Müller cells, the most important retinal glial cells. We isolated primary Müller cells from Sprague-Dawley (SD) neonatal rats and treated them with glucose at varying doses. CCK-8 was used to quantify cellular viability, and a TUNEL assay was performed to detect cell apoptosis. ELISA, immunofluorescence, and western blotting were used to assess cAMP/PKA/CREB signaling, Kir4.1, AQP4, GFAP, and VEGF levels, respectively. H&E staining was used to examine histopathological alterations in diabetic retinopathy (DR)-affected retinal tissue in rats. As glucose concentration increases, gliosis of Müller cells became apparent, as evidenced by a decline in cell activity, an increase in apoptosis, downregulation of Kir4.1 level, and overexpression of GFAP, AQP4, and VEGF. Treatments with low, intermediate, and high glucose levels led to aberrant activation of cAMP/PKA/CREB signaling. Interestingly, blocking cAMP and PKA reduced high glucose-induced Müller cell damage and gliosis by a significant amount. Further in vivo results suggested that cAMP or PKA inhibition significantly improved edema, bleeding, and retinal disorders. Our findings showed that high glucose exacerbated Müller cell damage and gliosis via a mechanism involving cAMP/PKA/CREB signaling.