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The Journal of Physiology Nov 2016The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in nociceptive neurons and its activation causes ongoing pain and inflammation; TRPA1 is...
KEY POINTS
The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in nociceptive neurons and its activation causes ongoing pain and inflammation; TRPA1 is thought to play an important role in inflammation in the airways. TRPA1 is sensitised by repeated stimulation with chemical agonists in a calcium-free environment and this sensitisation is very long lasting following agonist removal. We show that agonist-induced sensitisation is independent of the agonist's binding site and is also independent of ion channel trafficking or of other typical signalling pathways. We find that sensitisation is intrinsic to the TRPA1 protein and is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials. Agonist-induced sensitisation may provide an explanation for sensitisation following long-term exposure to harmful irritants and pollutants, particularly in the airways.
ABSTRACT
The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) neurons and responds to a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here we show that in the absence of extracellular calcium the current passing through TRPA1 gradually increases (sensitises) during prolonged application of agonists. Activation by an agonist is essential, because activation of TRPA1 by membrane depolarisation did not cause sensitisation. Sensitisation is independent of the site of action of the agonist, because covalent and non-covalent agonists were equally effective, and is long lasting following agonist removal. Mutating N-terminal cysteines, the target of covalent agonists, did not affect sensitisation by the non-covalent agonist carvacrol, which activates by binding to a different site. Sensitisation is unaffected by agents blocking ion channel trafficking or by block of signalling pathways involving ATP, protein kinase A or the formation of lipid rafts, and does not require ion flux through the channel. Examination of the voltage dependence of TRPA1 activation shows that sensitisation is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials, and is therefore intrinsic to the TRPA1 channel. Sensitisation may play a role in exacerbating the pain caused by prolonged activation of TRPA1.
Topics: Animals; Calcium; Cells, Cultured; Cymenes; Female; Humans; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Monoterpenes; Neurons; Signal Transduction; Transient Receptor Potential Channels
PubMed: 27307078
DOI: 10.1113/JP272237 -
Scientific Reports Sep 2020Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivity and bias to activation of specific signalling pathways. The...
Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivity and bias to activation of specific signalling pathways. The operational model (OM) of pharmacological agonism is a useful means for achieving this goal. Allosteric ligands bind to receptors at sites that are distinct from those of endogenous agonists that interact with the orthosteric domain on the receptor. An allosteric modulator and an orthosteric agonist bind simultaneously to the receptor to form a ternary complex, where the allosteric modulator affects the binding affinity and operational efficacy of the agonist. Allosteric modulators are an intensively studied group of receptor ligands because of their selectivity and preservation of physiological space-time pattern of the signals they modulate. We analysed the operational model of allosterically-modulated agonism (OMAM) including modulation by allosteric agonists. Similar to OM, several parameters of OMAM are inter-dependent. We derived equations describing mutual relationships among parameters of the functional response and OMAM. We present a workflow for the robust fitting of OMAM to experimental data using derived equations.
Topics: Allosteric Regulation; Animals; Drug Synergism; Humans; Kinetics; Ligands; Protein Binding; Receptors, G-Protein-Coupled
PubMed: 32879329
DOI: 10.1038/s41598-020-71228-y -
Toxicology in Vitro : An International... Dec 2020Environmental exposures often occur in complex mixtures and at low concentrations. Generalized concentration addition (GCA) is a method used to estimate the joint effect...
Environmental exposures often occur in complex mixtures and at low concentrations. Generalized concentration addition (GCA) is a method used to estimate the joint effect of receptor ligands that vary in efficacy. GCA models have been successfully applied to mixtures of aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptor gamma (PPARγ) ligands, each of which can be modeled as a receptor with a single binding site. Here, we evaluated whether GCA could be applied to homodimer nuclear receptors, which have two binding sites, to predict the combined effect of full glucocorticoid receptor (GR) agonists with partial agonists. We measured transcriptional activation of GR using a cell-based bioassay. Individual concentration-response curves for dexamethasone (full agonist), prednisolone (full agonist), and medroxyprogesterone 17-acetate (partial agonist) were generated and applied in three additivity models, GCA, effect summation (ES), and relative potency factor (RPF), to generate response surfaces. GCA and RPF yielded adequate predictions of the experimental data for two full agonists. However, GCA fit experimental data significantly better than ES and RPF for all other binary mixtures. This work extends the application of GCA to homodimer nuclear receptors and improves prediction accuracy of mixture effects of GR agonists.
Topics: Biological Assay; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Ligands; Models, Theoretical; Receptors, Glucocorticoid
PubMed: 32858110
DOI: 10.1016/j.tiv.2020.104975 -
Frontiers in Immunology 2023Targeting costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily with agonistic antibodies is a promising approach in cancer immuno therapy. It...
INTRODUCTION
Targeting costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily with agonistic antibodies is a promising approach in cancer immuno therapy. It is known that their efficacy strongly depends on FcγR cross-linking.
METHODS
In this study, we made use of a Jurkat-based reporter platform to analyze the influence of individual FcγRs on the costimulatory activity of the 41BB agonists, Urelumab and Utomilumab, and the CD27 agonist, Varlilumab.
RESULTS
We found that Urelumab (IgG4) can activate 41BB-NFκB signaling without FcγR cross-linking, but the presence of the FcγRs (CD32A, CD32B, CD64) augments the agonistic activity of Urelumab. The human IgG2 antibody Utomilumab exerts agonistic function only when crosslinked via CD32A and CD32B. The human IgG1 antibody Varlilumab showed strong agonistic activity with all FcγRs tested. In addition, we analyzed the costimulatory effects of Urelumab, Utomilumab, and Varlilumab in primary human peripheral blood mononuclear cells (PBMCs). Interestingly, we observed a very weak capacity of Varlilumab to enhance cytokine production and proliferation of CD4 and CD8 T cells. In the presence of Varlilumab the percentage of annexin V positive T cells was increased, indicating that this antibody mediated FcγR-dependent cytotoxic effects.
CONCLUSION
Collectively, our data underscore the importance to perform studies in reductionist systems as well as in primary PBMC samples to get a comprehensive understanding of the activity of costimulation agonists.
Topics: Humans; Leukocytes, Mononuclear; Receptors, IgG; Immunoglobulin G
PubMed: 37575254
DOI: 10.3389/fimmu.2023.1208631 -
British Journal of Pharmacology Jul 2017The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein-coupled MT and...
BACKGROUND AND PURPOSE
The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein-coupled MT and MT receptors. In contrast to most other hormones targeting GPCRs, melatonin and its synthetic analogues are amphiphilic molecules easily penetrating into cells, but the existence of intracellular receptors is still unclear mainly due to a lack of appropriate tools.
EXPERIMENTAL APPROACH
We therefore designed and synthesized a series of hydrophilic melatonin receptor ligands coupled to the Cy3 cyanin fluorophore to reliably monitor its inability to penetrate cells. Two compounds, one lipophilic and one hydrophilic, were then functionally characterized in terms of their affinity for human and murine melatonin receptors expressed in HEK293 cells and their signalling efficacy.
KEY RESULTS
Among the different ligands, ICOA-13 showed the desired properties as it was cell-impermeant and bound to human and mouse MT and MT receptors. ICOA-13 showed differential activities on melatonin receptors ranging from partial to full agonistic properties for the G /cAMP and ERK pathway and β-arrestin 2 recruitment. Notably, ICOA-13 enabled us to discriminate between G /cAMP signalling of the MT receptor initiated at the cell surface and neuronal mitochondria.
CONCLUSIONS AND IMPLICATIONS
We report here the first cell-impermeant melatonin receptor agonist, ICOA-13, which allows us to discriminate between signalling events initiated at the cell surface and intracellular compartments. Detection of mitochondrial MT receptors may have an important impact on the development of novel melatonin receptor ligands relevant for neurodegenerative diseases, such as Huntington disease.
Topics: Animals; Carbocyanines; Cell Membrane Permeability; Cells, Cultured; Dose-Response Relationship, Drug; Drug Design; Ethylamines; HEK293 Cells; Humans; Indoles; Ligands; Mice; Molecular Structure; Pyrroles; Receptors, Melatonin; Structure-Activity Relationship
PubMed: 28493341
DOI: 10.1111/bph.13856 -
British Journal of Pharmacology Feb 2013The present-day concept of drug efficacy has changed completely from its original description as the property of agonists that causes tissue activation. The ability to... (Review)
Review
The present-day concept of drug efficacy has changed completely from its original description as the property of agonists that causes tissue activation. The ability to visualize the multiple behaviours of seven transmembrane receptors has shown that drugs can have many efficacies and also that the transduction of drug stimulus to various cellular stimulus-response cascades can be biased towards some but not all pathways. This latter effect leads to agonist 'functional selectivity', which can be favourable for the improvement of agonist therapeutics. However, in addition, biased agonist potency becomes cell type dependent with the loss of the monotonic behaviour of stimulus-response mechanisms, leading to potential problems in agonist quantification. This has an extremely important effect on the discovery process for new agonists since it now cannot be assumed that a given screening or lead optimization assay will correctly predict therapeutic behaviour. This review discusses these ideas and how new approaches to quantifying agonist effect may be used to circumvent the cell type dependence of agonism. This article, written by a corresponding member of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), reviews our current understanding of the interaction of ligands with seven transmembrane receptors. Further information on these pharmacological concepts is being incorporated into the IUPHAR/BPS database GuideToPharmacology.org.
Topics: Animals; Humans; Ligands; Molecular Dynamics Simulation; Phosphorylation; Receptors, Cell Surface; Signal Transduction; Treatment Outcome
PubMed: 22994528
DOI: 10.1111/j.1476-5381.2012.02223.x -
The Medical Journal of Malaysia Jun 2008The long agonistic protocol for controlled ovarian hyperstimulation (COH) is effective and used most often, thus is considered the gold standard. Therefore any new... (Comparative Study)
Comparative Study
The long agonistic protocol for controlled ovarian hyperstimulation (COH) is effective and used most often, thus is considered the gold standard. Therefore any new regimen has to be compared in its results with those obtained with the long protocol. This report compares the efficacy of GnRH agonist and antagonist in a retrospective study of IVF/ICSI carried out in a tertiary teaching hospital from 2003 to 2006. Only the first COH cycle followed by IVF-ICSI from 200 couples (agonist = 120 and antagonist = 80) were analysed. The end points studied included the number of oocytes recovered, number of mature (MII) oocytes, fertilization, cleavage, morphology based embryo quality, pregnancy rate, quantity and cost of gonadotrophin. The average age of female subjects was 35.1 +/- 4.7 years with 50% being 35 years and above. Major infertility factors were tubal blockage, male factor and endometriosis altogether comprising 68%. GnRH agonist and antagonist cycle parameters were comparable except lesser amount of gonadotrophin was used with lower resultant costs (both p < 0.0005) in antagonistic regime. Antagonist regime produce somewhat more good quality embryos (p = 0.065), an insignificant difference. A clinical pregnancy rate per embryo transfer of 16.3% in agonist and 20.6% in antagonist regime was achieved respectively. In conclusion, GnRH antagonist protocol produced a COH response, embryonic development and pregnancy rates on par to GnRH agonist regime. Moreover GnRH antagonist protocol required a shorter stimulation period plus fewer complications. Hence GnRH antagonist regime provided means for a friendlier, convenient and cost effective protocol for patients.
Topics: Adult; Female; Fertilization in Vitro; Gonadotropin-Releasing Hormone; Humans; Male; Middle Aged; Ovulation Induction; Pregnancy; Retrospective Studies; Sperm Injections, Intracytoplasmic
PubMed: 18942295
DOI: No ID Found -
Oncology Letters Nov 2020Cluster of differentiation 40 (CD40) mediates many immune activities. Preclinical studies have shown that activation of CD40 can evoke massive antineoplastic effects in... (Review)
Review
Cluster of differentiation 40 (CD40) mediates many immune activities. Preclinical studies have shown that activation of CD40 can evoke massive antineoplastic effects in several tumour models , providing a rationale for using CD40 agonists in cancer immunotherapy. To date, several potential agonistic antibodies that target CD40 have been investigated in clinical trials. Early clinical trials have shown that the adverse events associated with agonists of CD40 thus far have been largely transient and clinically controllable, including storms of cytokine release, hepatotoxicity and thromboembolic events. An antitumour effect of targeting CD40 for monotherapy or combination therapy has been observed in some tumours. However, these antitumour effects have been moderate. The present review aimed to provide updated details of the clinical results of these agonists, and offer information to further investigate the strategies of combining CD40 activation with chemotherapy, radiotherapy, targeted therapy and immunomodulators. Furthermore, biomarkers should be identified for monitoring and predicting responses and informing resistance mechanisms.
PubMed: 32934743
DOI: 10.3892/ol.2020.12037 -
Diabetes, Obesity & Metabolism Aug 2018We performed acute and chronic studies in healthy and diet-induced obese animals using mouse-specific or monkey-specific dual GLP-1R/GCGR agonists to investigate their... (Comparative Study)
Comparative Study
Running on mixed fuel-dual agonistic approach of GLP-1 and GCG receptors leads to beneficial impact on body weight and blood glucose control: A comparative study between mice and non-human primates.
AIM
We performed acute and chronic studies in healthy and diet-induced obese animals using mouse-specific or monkey-specific dual GLP-1R/GCGR agonists to investigate their effects on food intake, body weight, blood glucose control and insulin secretion. The selective GLP-1R agonist liraglutide was used as comparator.
METHODS
The mouse-specific dual agonist and liraglutide were tested in lean wild type, GLP-1R knockout and diet-induced obese mice at different doses. A chronic study was performed in DIO mice to investigate the effect on body weight, food consumption and total energy expenditure (TEE) in obese and diabetic monkeys with a focus on body weight and energy intake.
RESULTS
The mouse-specific dual agonist and liraglutide similarly affected glycaemic control. A higher loss in body weight was measured in dual agonist-treated obese mice. The dual agonist significantly enhanced plasma glucose excursion in overnight fed GLP-1R mice, probably reflecting a potent GCGR agonist activity. It increased TEE and enhanced fat and carbohydrate oxidation, while liraglutide produced no effect on TEE. In obese and diabetic monkeys, treatment with the monkey-specific dual agonist reduced total energy intake to 60%-70% of baseline TEI during chronic treatment. A decrease in body weight and significant improvement in glucose tolerance was observed.
CONCLUSIONS
In DIO mice and non-human primates, dual agonists elicited robust glycaemic control, similar to the marketed GLP-1R agonist, while eliciting greater effects on body weight. Results from DIO mice suggest that the increase in TEE is caused not only by increased fat oxidation but also by an increase in carbohydrate oxidation.
Topics: Animals; Animals, Outbred Strains; Appetite Depressants; Body Weight; Diabetes Mellitus, Type 2; Diet, High-Fat; Dose-Response Relationship, Drug; Drug Therapy, Combination; Energy Intake; Energy Metabolism; Female; Glucagon-Like Peptide-1 Receptor; Hyperglycemia; Hypoglycemic Agents; Insulin Secretion; Macaca fascicularis; Male; Mice, Inbred C57BL; Mice, Knockout; Obesity; Random Allocation; Receptors, Glucagon
PubMed: 29938884
DOI: 10.1111/dom.13212