Fluorescent Agonist Research Articles

Design and synthesis of first environment-sensitive coumarin fluorescent agonists for MrgX2

Mas related G-protein-coupled receptor member X2 (MrgX2) has been identified as the crucial receptor in drug induced pseudo-allergic reactions and allergic diseases. In this research, the first type of fluorescent agonists (ZX1, ZX2 and ZX3) for MrgX2 were developed by conjugating environment-sensitive fluorophore coumarin to MrgX2 selective agonists (R)-ZINC-3573. Their environment-sensitive property was confirmed by the dramatically increase of fluorescent intensity after binding to the hydrophobic ligand binding domain MrgX2, which help to overcome the high background signal. Based on these characteristics, they can be used for selective visualization of MrgX2 in living cells even with their own background interference. Among these fluorescent agonists, compound ZX2 possessed splendid spectroscopic properties, outstanding pharmacological activities (EC50 = 0.93 μM, KD = 1.97 μM). And a competitive binding assay was established with ZX2 to analysis the binding affinity of MrgX2 agonists, which shown high coherence with the results of cell membrane chromatography. To our knowledge, these probes are the first fluorescent ligands of MrgX2 with agonistic activity and environment-sensitive property, which is expected to use for the development of MrgX2 molecular pharmacology and serve as a convenient high-throughput screening tool for the drug candidates targeting MrgX2.

Photoinduced Electron Transfer-Based Fluorescent Agonists for α1-Adrenergic Receptors Imaging.

The novel fluorescent agonists were discovered herein for α1-adrenergic receptors (α1-ARs) based on photoinduced electron transfer (PeT) off-on switch by conjugating the fluorophore 7-(diethylamino)coumarin-3-carboxylic acid with phenylephrine. After careful evaluation, these probes exhibited efficient binding affinity with α1-ARs and could be applied to selectively imaging α1-ARs or successfully tracing the dynamic process of α1-AR internalization in living cells. Meanwhile, a bioluminescence resonance energy transfer binding assay with these new probes has been well-established and applied. Therefore, these PeT-based on-off agonists may serve as powerful tools for the α1-AR-associated study during drug discovery.

Efficient G protein coupling is not required for agonist-mediated internalization and membrane reorganization of the adenosine A3 receptor.

Organization of G protein‐coupled receptors at the plasma membrane has been the focus of much recent attention. Advanced microscopy techniques have shown that these receptors can be localized to discrete microdomains and reorganization upon ligand activation is crucial in orchestrating their signaling. Here, we have compared the membrane organization and downstream signaling of a mutant (R108A, R3.50A) of the adenosine A3 receptor (A3AR) to that of the wild‐type receptor. Fluorescence Correlation Spectroscopy (FCS) studies with a fluorescent agonist (ABEA‐X‐BY630) demonstrated that both wild‐type and mutant receptors bind agonist with high affinity but in subsequent downstream signaling assays the R108A mutation abolished agonist‐mediated inhibition of cAMP production and ERK phosphorylation. In further FCS studies, both A3AR and A3AR R108A underwent similar agonist‐induced increases in receptor density and molecular brightness which were accompanied by a decrease in membrane diffusion after agonist treatment. Using bimolecular fluorescence complementation, experiments showed that the R108A mutant retained the ability to recruit β‐arrestin and these receptor/arrestin complexes displayed similar membrane diffusion and organization to that observed with wild‐type receptors. These data demonstrate that effective G protein signaling is not a prerequisite for agonist‐stimulated β‐arrestin recruitment and membrane reorganization of the A3AR.

Delta opioid receptor activation modulates affective pain and modality-specific pain hypersensitivity associated with chronic neuropathic pain.

Delta opioid receptor (DOR) agonists alleviate nociceptive behaviors in various chronic pain models, including neuropathic pain, while having minimal effect on sensory thresholds in the absence of injury. The mechanisms underlying nerve injury-induced enhancement of DOR function are unclear. We used a peripheral nerve injury (PNI) model of neuropathic pain to assess changes in the function and localization of DORs in mice and rats. Intrathecal administration of DOR agonists reversed mechanical allodynia and thermal hyperalgesia. The dose-dependent thermal antinociceptive effects of DOR agonists were shifted to the left in PNI rats. Administration of DOR agonists produced a conditioned place preference in PNI, but not in sham, animals, whereas the DOR antagonist naltrindole produced a place aversion in PNI, but not in sham, mice, suggesting the engagement of endogenous DOR activity in suppressing pain associated with the injury. GTPγS autoradiography revealed an increase in DOR function in the dorsal spinal cord, ipsilateral to PNI. Immunogold electron microscopy and in vivo fluorescent agonist assays were used to assess changes in the ultrastructural localization of DORs in the spinal dorsal horn. In shams, DORs were primarily localized within intracellular compartments. PNI significantly increased the cell surface expression of DORs within lamina IV-V dendritic profiles. Using neonatal capsaicin treatment, we identified that DOR agonist-induced thermal antinociception was mediated via receptors expressed on primary afferent sensory neurons but did not alter mechanical thresholds. These data reveal that the regulation of DORs following PNI and suggest the importance of endogenous activation of DORs in regulating chronic pain states.

Discovery of Environment-Sensitive Fluorescent Agonists for α1-Adrenergic Receptors.

A series of novel fluorescent agonists were well developed herein with turn-on switch for α1-adrenergic receptors (α1-ARs) by conjugating the environment-sensitive fluorophore 4-chloro-7-nitrobenzoxadiazole with phenylephrine. Overall, these probes exhibited efficient binding and apparent fluorescence intensity changes (up to 10-fold) upon binding with α1-ARs. Moreover, these probes have been successfully applied for selectively imaging α1-ARs in the living cells. The dynamic process of α1-ARs internalization was traced successfully with these newly designed fluorescent agonists. Fluorescence polarization assay demonstrated specific interactions between these probes and α1-ARs. With these new probes, a bioluminescence resonance energy transfer binding assay has been well established and applied to the high-throughput screening of unlabeled α1-ARs agonist and antagonist. It is expected that these environment-sensitive fluorescent turn-on agonists may provide useful new tools in studying pharmacology and physiology of α1-ARs during drug discovery.

Probe dependence of allosteric enhancers on the binding affinity of adenosine A1 -receptor agonists at rat and human A1 -receptors measured using NanoBRET.

Background and PurposeAdenosine is a local mediator that regulates a number of physiological and pathological processes via activation of adenosine A1‐receptors. The activity of adenosine can be regulated at the level of its target receptor via drugs that bind to an allosteric site on the A1‐receptor. Here, we have investigated the species and probe dependence of two allosteric modulators on the binding characteristics of fluorescent and nonfluorescent A1‐receptor agonists.Experimental ApproachA Nano‐luciferase (Nluc) BRET (NanoBRET) methodology was used. This used N‐terminal Nluc‐tagged A1‐receptors expressed in HEK293T cells in conjunction with both fluorescent A1‐receptor agonists (adenosine and NECA analogues) and a fluorescent antagonist CA200645.Key ResultsPD 81,723 and VCP171 elicited positive allosteric effects on the binding affinity of orthosteric agonists at both the rat and human A1‐receptors that showed clear probe dependence. Thus, the allosteric effect on the highly selective partial agonist capadenoson was much less marked than for the full agonists NECA, adenosine, and CCPA in both species. VCP171 and, to a lesser extent, PD 81,723, also increased the specific binding of three fluorescent A1‐receptor agonists in a species‐dependent manner that involved increases in B max and pK D.Conclusions and ImplicationsThese results demonstrate the power of the NanoBRET ligand‐binding approach to study the effect of allosteric ligands on the binding of fluorescent agonists to the adenosine A1‐receptor in intact living cells. Furthermore, our studies suggest that VCP171 and PD 81,723 may switch a proportion of A1‐receptors to an active agonist conformation (R*).

Editorial

Editorial

Fluorescent Agonists of the α7 Nicotinic Acetylcholine Receptor Derived from 3‐Amino‐Quinuclidine

Here, we investigated whether fluorescence labeled small molecule agonists of the α7 nicotinic acetylcholine receptor (nAChR) might be identified to enhance receptor studies. Enantiomerically pure 3‐amino‐quinuclidines appended with fluorophores at the 3‐amino group were synthesized and tested by electrophysiology on human α7 nAChR in Xenopus oocytes, uncovering (R)‐4 and (R)‐9 as the first examples of fluorescent α7 nAChR agonists. These molecules elegantly incorporate the fluorescent reporter group as part of the pharmacophore itself and provide a new class of tool compounds for the study of these ligand‐gated ion channels.

The Fluorescence Methods to Study Neurotransmitters (Biomediators) in Plant Cells.

Fluorescence as a parameter for analysis of intracellular binding and localization of neurotransmitters also named biomediators (acetylcholine and biogenic amines such as catecholamines, serotonin, histamine) as well as their receptors in plant cells has been estimated basing on several world publications and own experiments of the author. The subjects of the consideration were 1. application of reagents forming fluorescent products (for catecholamines - glyoxylic acid, for histamine - formaldehyde or ortho-phthalic aldehyde) to show the presence and binding of the compounds in cells, 2. binding of their fluorescent agonists and antagonists with cell, 3. effects of the compounds, their agonists and antagonists on autofluorescence, 4. action of external factors on the accumulation of the compounds in cells. How neurotransmitters can bind to certain cellular compartments has been shown on intact individual cells (vegetative microspores, pollens, secretory cells) and isolated organelles. The staining with reagents on biogenic amines leads to the appearance blue or blue-green emission on the surface and excretions of intact cells as well in some DNA-containing organelles within cells. The difference between autofluorescence and histochemically induced fluorescence may reflect the occurrence and amount of biogenic amines in the cells studied. Ozone and salinity as external factors can regulate the emission of intact cells related to biogenic amines. After the treatment of isolated cellular organelles with glyoxylic acid blue emission with maximum 460-475nm was seen in nuclei and chloroplasts (in control variants in this spectral region the noticeable emission was absent) and very expressive fluorescence (more than twenty times as compared to control) in the vacuoles. After exposure to ortho-phthalic aldehyde blue emission was more noticeable in nuclei and chloroplasts. Fluorescent agonists (muscarine, 6,7-diOHATN, BODIPY-dopamine or BODIPY-5HT) or antagonists (d-tubocurarine for acetylcholine, yohimbine for dopamine and norepinephrine, inmecarb for serotonin) of neurotransmitters that bound with animal receptors fluorescent in blue (460-480nm) or blue-green (490-530nm) and usually are bound with the plasmatic membrane of intact cells or with membrane of the isolated organelles studied. In some model cells autofluorescence (belonging to chlorophyll or not, for example secondary metabolites) may be stimulated by exogenous biogenic amines or their agonists and, on the contrary, be inhibited by certain antagonists. The fluorescence data may be applied for the testing in ecological monitoring, medicine and pharmacology.

Design, synthesis, pharmacological characterization of a fluorescent agonist of the P2Y₁₄ receptor.

The P2Y14R is a G(i/o)-coupled receptor of the P2Y family of purinergic receptors that is activated by extracellular UDP and UDP-glucose (UDPG). In an earlier report we described a P2Y14R fluorescent probe, MRS4174, based on the potent and selective antagonist PPTN, a naphthoic acid derivative. Here, we report the design, preparation, and activity of an agonist-based fluorescent probe MRS4183 (11) and a shorter P2Y14R agonist congener, which contain a UDP-glucuronic acid pharmacophore and BODIPY fluorophores conjugated through diaminoalkyl linkers. The design relied on both docking in a P2Y14R homology model and established structure activity relationship (SAR) of nucleotide analogs. 11 retained P2Y14R potency with EC50 value of 0.96 nM (inhibition of adenylyl cyclase), compared to parent UDPG (EC50 47 nM) and served as a tracer for microscopy and flow cytometry, displaying minimal nonspecific binding. Binding saturation analysis gave an apparent binding constant for 11 in whole cells of 21.4±1.1 nM, with a t1/2 of association at 50 nM 11 of 23.9 min. Known P2Y14R agonists and PPTN inhibited cell binding of 11 with the expected rank order of potency. The success in the identification of a new P2Y14R fluorescent agonist with low nonspecific binding illustrates the advantages of rational design based on recently determined GPCR X-ray structures. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands.

Direct visualisation of internalization of the adenosine A3 receptor and localization with arrestin3 using a fluorescent agonist

Fluorescence based probes provide a novel way to study the dynamic internalization process of G protein-coupled receptors (GPCRs). Recent advances in the rational design of fluorescent ligands for GPCRs have been used here to generate new fluorescent agonists containing tripeptide linkers for the adenosine A3 receptor. The fluorescent agonist BY630-X-(D)-A-(D)-A-G-ABEA was found to be a highly potent agonist at the adenosine A3 receptor in both reporter gene (pEC50 = 8.48 ± 0.09) and internalization assays (pEC50 = 7.47 ± 0.11). Confocal imaging studies showed that BY630-X-(D)-A-(D)-A-G-ABEA was internalized with A3 linked to yellow fluorescent protein, which was blocked by the competitive antagonist MRS1220. Internalization of untagged adenosine A3 could also be visualized with BY630-X-(D)-A-(D)-A-G-ABEA treatment. Further, BY630-X-(D)-A-(D)-A-G-ABEA stimulated the formation of receptor–arrestin3 complexes and was found to localize with these intracellular complexes. This highly potent agonist with excellent imaging properties should be a valuable tool to study receptor internalization.This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology’.

Design, synthesis, pharmacological characterization of a fluorescent agonist of the P2Y 14 receptor

Design, synthesis, pharmacological characterization of a fluorescent agonist of the P2Y 14 receptor

Validation of Alexa-647-ATP as a powerful tool to study P2X receptor ligand binding and desensitization

Ion channel opening and desensitization is a fundamental process in neurotransmission. The ATP-gated P2X1 receptor (P2X1R) shows rapid and long-lasting desensitization upon agonist binding. This makes the electrophysiological investigation of its desensitization process, agonist unbinding, and recovery from desensitization a challenging task. Here, we show that the fluorescent agonist Alexa-647-ATP is a potent agonist at the P2X1R and a versatile tool to directly visualize agonist binding and unbinding. We demonstrate that the long-lasting desensitization of the P2X1R is due to both slow unbinding of agonist from the desensitized receptor and agonist mediated receptor internalization. Furthermore, the unbinding of the agonist Alexa-647-ATP from the desensitized receptor is accelerated in the continuous presence of competitive ligand. Modeling of our data indicates that three agonist molecules are required to drive the receptor into desensitization. Direct visualization of ligand unbinding from the desensitized receptor demonstrates the cooperativity of this process.

Visualizing trafficking of the adenosine‐A3 receptor using a fluorescent agonist: a tool for delineating intracellular signalling

Activation of many G protein‐coupled receptors (GPCRs) leads to β‐arrestin binding and sequestration of the receptor from the cell surface. Whilst internalisation of a GPCR leads to termination of some aspects of receptor‐mediated signalling, recent reports have shown that some GPCRs continue to signal even after internalisation. Here, we show using a mutated version of the adenosine A3 receptor (W243F‐A3AR), that persistent signalling to the cAMP pathway requires receptor internalisation. To investigate this further we developed a fluorescent A3AR agonist, consisting of the agonist NECA conjugated to BODIPY‐630/650‐X via a dipeptide linker. This fluorescent agonist was highly potent at the A3AR ( pEC50 = 9.06 ± 0.17, n=4) and acted as a full agonist in Chinese Hamster Ovary cells expressing this receptor. Confocal microscopy showed a high degree of co‐localisation of the fluorescent agonist with a YFP‐tagged A3AR on the cell surface after a short incubation period (5 min). After longer incubation (30 min), A3AR‐YFP was sequestered from the cell surface to intracellular granules where co‐localisation with the fluorescent agonist was still observed. This fluorescent probe thus allows us to visualise A3AR trafficking and investigate how the receptor continues to signal to second messenger pathways upon internalisation.We thank the Medical Research Council for financial support.

Identification of oligomer proanthocyanidins (F2) isolated from grape seeds as a formyl peptide receptor 1 partial agonist

Formyl peptide receptor 1 (FPR1) plays an important role in the rapid progression of glioblastoma and has been considered as a molecular target for the treatment. Previously, we have shown that oligomer proanthocyanidins (F2, degree of polymerization 2-15), isolated from grape seeds, inhibited FPR1-mediated chemotaxis of U-87 glioblastoma cells. In the present study, we investigated the capacity of F2 to interact with FPR1. The cross attenuation of chemotaxis revealed that F2 shared FPR1 with formyl-methionyl-leucyl-phenylalanine (fMLF), which is a prototype agonist of FPR1. F2 was chemotactic for U-87 cells, and the chemotactic response was abolished when FPR1 gene was silenced or FPR1 was competitively occupied. We further show that F2 specifically blocked the binding of fluorescent agonist to FPR1. Interestingly, F2 exhibited the characteristic of a partial agonist for FPR1, as shown by its capacity to activate FPR1-mediated PI3K-PKC-MAPK pathways. Meanwhile, F2 also attenuated fMLF-triggered MAPK activation, suggesting that F2 could antagonize the effect of an agonist. Furthermore, F2 abolished the invasion of U-87 cells induced by fMLF. Thus, we have identified F2 as a novel, partial agonist for FPR1, which may be useful for glioblastoma therapy.

Inhibitory effects of cytoskeleton disrupting drugs and GDP-locked Rab mutants on bradykinin B2 receptor cycling

The bradykinin (BK) B2 receptor (B2R) is G protein coupled and phosphorylated upon agonist stimulation; its endocytosis and recycling are documented. We assessed the effect of drugs that affect the cytoskeleton on B2R cycling. These drugs were targeted to tubulin (paclitaxel, or the novel combretastatin A-4 mimetic 3,4,5-trimethoxyphenyl-4-(2-oxoimidazolidin-1-yl)benzenesulfonate [IMZ-602]) and actin (cytochalasin D). Tubulin ligands did not alter agonist-induced receptor endocytosis, as shown using antibodies reactive with myc-tagged B2Rs (microscopy, cytofluorometry), but rather reduced the progression of the ligand–receptor–β-arrestin complex from the cell periphery to the interior. The 3 fluorescent probes of this complex (B2R-green fluorescent protein [B2R-GFP], the fluorescent agonist fluorescein-5-thiocarbamoyl-D-Arg-[Hyp3, Igl5, Oic7, Igl8]-BK and β-arrestin2–GFP) were condensed in punctuate structures that remained close to the cell surface in the presence of IMZ-602. Cytochalasin D selectively inhibited the recycling of endocytosed B2R-GFP (B2R-GFP imaging, [3H]BK binding). Dominant negative (GDP-locked)-Rab5 and -Rab11 reproduced the effects of inhibitors of tubulin and actin, respectively, on the cycling of B2R-GFP. GDP-locked-Rab4 also inhibited B2R-GFP recycling to the cell surface. Consistent with the displacement of cargo along specific cytoskeletal elements, Rab5-associated progression of the endocytosed BK B2R follows microtubules toward their (−) end, while its recycling progresses along actin fibers to the cell surface. However, tubulin ligands do not suppress the tested desensitization or resensitization mechanisms of the B2R.

Membrane Cholesterol Affects Stimulus‐Activity Coupling in Type 1, but not Type 2, CCK Receptors: Use of Cell Lines with Elevated Cholesterol

The lipid microenvironment of membrane proteins can affect their structure, function, and regulation. We recently described differential effects of acute modification of membrane cholesterol on the function of type 1 and 2 cholecystokinin (CCK) receptors. We now explore the regulatory impact of chronic cholesterol modification on these receptors using novel receptor-bearing cell lines with elevated membrane cholesterol. Stable CCK1R and CCK2R expression was established in clonal lines of 25RA cells having gain-of-function in SCAP [sterol regulatory element binding protein (SREBP) cleavage-activating protein] and SRD15 cells having deficiencies in Insig-1 and Insig-2 enzymes affecting HMG CoA reductase and SREBP. Increased cholesterol in the plasma membrane of these cells was directly demonstrated, and receptor binding and signaling characteristics were shown to reflect predicted effects on receptor function. In both environments, both types of CCK receptors were internalized and recycled normally in response to agonist occupation. No differences in receptor distribution within the membrane were appreciated at the light microscopic level in these CHO-derived cell lines. Fluorescence anisotropy was studied for these receptors occupied by fluorescent agonist and antagonist, as well as when tagged with YFP. These studies demonstrated increased anisotropy of the agonist ligand occupying the active state of the CCK1R in a cholesterol-enriched environment, mimicking fluorescence of the uncoupled, inactive state of this receptor, while there was no effect of increasing cholesterol on fluorescence at the CCK2R. These cell lines should be quite useful for examining the functional characteristics of potential drugs that might be used in an abnormal lipid environment.

CCR2 Acts as Scavenger for CCL2 during Monocyte Chemotaxis

BackgroundLeukocyte migration is essential for effective host defense against invading pathogens and during immune homeostasis. A hallmark of the regulation of this process is the presentation of chemokines in gradients stimulating leukocyte chemotaxis via cognate chemokine receptors. For efficient migration, receptor responsiveness must be maintained whilst the cells crawl on cell surfaces or on matrices along the attracting gradient towards increasing concentrations of agonist. On the other hand agonist-induced desensitization and internalization is a general paradigm for chemokine receptors which is inconsistent with the prolonged migratory capacity.Methodology/Principal FindingsChemotaxis of monocytes was monitored in response to fluorescent CCL2-mCherry by time-lapse video microscopy. Uptake of the fluorescent agonist was used as indirect measure to follow the endogenous receptor CCR2 expressed on primary human monocytes. During chemotaxis CCL2-mCherry becomes endocytosed as cargo of CCR2, however, the internalization of CCR2 is not accompanied by reduced responsiveness of the cells due to desensitization.Conclusions/SignificanceDuring chemotaxis CCR2 expressed on monocytes internalizes with the bound chemoattractant, but cycles rapidly back to the plasma membrane to maintain high responsiveness. Moreover, following relocation of the source of attractant, monocytes can rapidly reverse their polarization axis organizing a new leading edge along the newly formed gradient, suggesting a uniform distribution of highly receptive CCR2 on the plasma membrane. The present observations further indicate that during chemotaxis CCR2 acts as scavenger consuming the chemokine forming the attracting cue.

Activation of TRPV1 by capsaicin induces functional Kinin B1 receptor in rat spinal cord microglia

BackgroundThe kinin B1 receptor (B1R) is upregulated by pro-inflammatory cytokines and oxydative stress, which are enhanced by transient receptor potential vanilloid subtype 1 (TRPV1) activation. To examine the link between TRPV1 and B1R in inflammatory pain, this study aimed to determine the ability of TRPV1 to regulate microglial B1R expression in the spinal cord dorsal horn, and the underlying mechanism.MethodsB1R expression (mRNA, protein and binding sites) was measured in cervical, thoracic and lumbar spinal cord in response to TRPV1 activation by systemic capsaicin (1-50 mg/kg, s.c) in rats pre-treated with TRPV1 antagonists (capsazepine or SB-366791), the antioxidant N-acetyl-L-cysteine (NAC), or vehicle. B1R function was assessed using a tail-flick test after intrathecal (i.t.) injection of a selective B1R agonist (des-Arg9-BK), and its microglial localization was investigated by confocal microscopy with the selective fluorescent B1R agonist, [Nα-bodipy]-des-Arg9-BK. The effect of i.t. capsaicin (1 μg/site) was also investigated.ResultsCapsaicin (10 to 50 mg/kg, s.c.) enhanced time-dependently (0-24h) B1R mRNA levels in the lumbar spinal cord; this effect was prevented by capsazepine (10 mg/kg, i.p.; 10 μg/site, i.t.) and SB-366791 (1 mg/kg, i.p.; 30 μg/site, i.t.). Increases of B1R mRNA were correlated with IL-1β mRNA levels, and they were significantly less in cervical and thoracic spinal cord. Intrathecal capsaicin (1 μg/site) also enhanced B1R mRNA in lumbar spinal cord. NAC (1 g/kg/d × 7 days) prevented B1R up-regulation, superoxide anion production and NF-kB activation induced by capsaicin (15 mg/kg). Des-Arg9-BK (9.6 nmol/site, i.t.) decreased by 25-30% the nociceptive threshold at 1 min post-injection in capsaicin-treated rats (10-50 mg/kg) while it was without effect in control rats. Des-Arg9-BK-induced thermal hyperalgesia was blocked by capsazepine, SB-366791 and by antagonists/inhibitors of B1R (SSR240612, 10 mg/kg, p.o.), glutamate NMDA receptor (DL-AP5, 10 μg/site, i.t.), substance P NK-1 receptor (RP-67580, 10 μg/site, i.t.) and nitric oxide synthase (L-NNA, 10 μg/site, i.t.). The B1R fluorescent agonist was co-localized with an immunomarker of microglia (Iba-1) in spinal cord dorsal horn of capsaicin-treated rats.ConclusionThis study highlights a new mechanism for B1R induction via TRPV1 activation and establishes a link between these two pro-nociceptive receptors in inflammatory pain.

Allosteric interactions across native adenosine‐A 3 receptor homodimers: quantification using single‐cell ligand‐binding kinetics

A growing awareness indicates that many G-protein-coupled receptors (GPCRs) exist as homodimers, but the extent of the cooperativity across the dimer interface has been largely unexplored. Here, measurement of the dissociation kinetics of a fluorescent agonist (ABA-X-BY630) from the human A1 or A3 adenosine receptors expressed in CHO-K1 cells has provided evidence for highly cooperative interactions between protomers of the A3-receptor dimer in single living cells. In the absence of competitive ligands, the dissociation rate constants of ABA-X-BY630 from A1 and A3 receptors were 1.45 ± 0.05 and 0.57 ± 0.07 min−1, respectively. At the A3 receptor, this could be markedly increased by both orthosteric agonists and antagonists [15-, 9-, and 19-fold for xanthine amine congener (XAC), 5′-(N-ethyl carboxamido)adenosine (NECA), and adenosine, respectively] and reduced by coexpression of a nonbinding (N250A) A3-receptor mutant. The changes in ABA-X-BY630 dissociation were much lower at the A1 receptor (1.5-, 1.4-, and 1.5-fold). Analysis of the pEC50 values of XAC, NECA, and adenosine for the ABA-X-BY630-occupied A3-receptor dimer yielded values of 6.0 ± 0.1, 5.9 ± 0.1, and 5.2 ± 0.1, respectively. This study provides new insight into the spatial and temporal specificity of drug action that can be provided by allosteric modulation across a GPCR homodimeric interface.—May, L. T., Bridge, L. J., Stoddart, L. A., Briddon, S. J., Hill, S. J. Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics.