Journal: Frontiers in Immunology

Article Title: Neuronal substance P-driven MRGPRX2-dependent mast cell degranulation products differentially promote vascular permeability

doi: 10.3389/fimmu.2024.1477072

Figure Lengend Snippet: Binding of compound 48/80 or ciprofloxacin to MRGPRX2 strongly promotes PMC degranulation and murine vascular permeability more than the binding to Mrgprb2. (A) Surface expression levels of FcεRIα and c-Kit (upper panel) and MRGPRX2 (middle panel) and expression levels of tdTomato (lower panel) in the PMCs from WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) mice. Control staining is shown in the shaded histograms. (B, D, F) Percentages of surface CD63-positive cells in WT, Mrgprb2-KO, and MRGPRX2-KI PMCs and BMMCs (B) and PMCs (D, F) after treatment with the indicated concentrations of compound 48/80 (B) , 10 μg/mL ciprofloxacin (D) , or 10 μg/mL icatibant (F) . Data are representative of three independent experiments and indicate the mean ± SD. * P < 0.05 and ** P < 0.01. (C, E, G) Quantification of the Evans blue dye that extravasated into the ear skin in WT, Mrgprb2-KO, and MRGPRX2-KI mice after treatment with indicated amounts of compound 48/80 (C) , ciprofloxacin (E) , and 1.75 μg icatibant (G) or phosphate-buffered saline (PBS). n = 5-9; ± SD. * P < 0.05 and ** P < 0.01.

Article Snippet: Compound 48/80 and capsaicin (Sigma-Aldrich, St Louis, MO), SP (Peptide Institute, Inc., Osaka, Japan), Cetirizine (TCI AMERICA, Portland, OR), TY-51469 (MedChemExpress, Monmouth Junction, NJ), RWJ-56110 (Tocris Bioscience, Ellisville, MO), AZ3451, I-191, AMG-517 (Selleck Chemicals LLC), AMG-9810 (Cayman Chemical, Ann Arbor, MI), DAPI (4’,6-diamidino-2-phenylindole) (FujifilmWako, Osaka, Japan), and Piperine (TCI, Tokyo, Japan) were used.

Techniques: Binding Assay, Permeability, Expressing, Control, Staining, Saline

Journal: Molecules

Article Title: Formononetin Inhibits Mast Cell Degranulation to Ameliorate Compound 48/80-Induced Pseudoallergic Reactions

doi: 10.3390/molecules28135271

Figure Lengend Snippet: FNT inhibits C48/80-induced cell degranulation in RBL-2H3. ( A ) Chemical structure of FNT. ( B ) Cell viability of RBL-2H3 cells treated with 0–100 µM FNT was determined using CCK-8 assays. ( C – F ) The inhibitory effect of FNT on the secretion of β-Hex ( C ), histamine ( D ), and the transcriptional level of proinflammatory factors TNFα ( E ) and IL-13 ( F ) in C48/80-mediated RBL-2H3 cells. The data are expressed as the mean ± SDs (n = 3). ### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the model group. FNT, formononetin; β-Hex, β-hexosaminidase; HIS, histamine; Dexa, dexamethasone.

Article Snippet: C48/80 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: CCK-8 Assay

Journal: Molecules

Article Title: Formononetin Inhibits Mast Cell Degranulation to Ameliorate Compound 48/80-Induced Pseudoallergic Reactions

doi: 10.3390/molecules28135271

Figure Lengend Snippet: FNT inhibits C48/80-mediated morphological changes in RBL-2H3 cells. RBL-2H3 cells were pre-treated with or without the indicated doses of FNT for 2 h and then stimulated with C48/80 for 10 min. ( A , B ) Representative morphological changes in RBL-2H3 cells were shown via toluidine blue staining. Red arrows indicate the cells that become irregular in shape and release purple particles. ( C , D ) FITC-phalloidin staining showed morphological changes in cells. F-actin cytoskeleton was stained with green fluorescence. Red arrow indicates oval cells in response to F-actin cytoskeleton changes. Means ± SDs (n = 3) are shown; ### p < 0.001 vs. the control group; ** p < 0.01, and *** p < 0.001 vs. the model group. Abbreviations as in .

Article Snippet: C48/80 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Staining, Fluorescence

Journal: Molecules

Article Title: Formononetin Inhibits Mast Cell Degranulation to Ameliorate Compound 48/80-Induced Pseudoallergic Reactions

doi: 10.3390/molecules28135271

Figure Lengend Snippet: FNT inhibits C48/80-increased NF-κB activity in RBL-2H3 cells. RBL-2H3 cells were pre-treated (or not) with different doses of FNT for 2 h separately and then stimulated with C48/80 for 10 min, the whole cell lysate was taken. ( A ) Western blot analysis of NF-κB pathway signaling molecules (p-p65, p65, p-IκBα, and IκBα) in C48/80-stimulated RBL-2H3 cells treated with 10, 20, or 40 µM FNT. ( B , C ) Quantification of the proteins. ( D ) RBL-2H3 cells transfected with pNF-κB-Luc plasmid were treated with FNT for 4h, and the selective inhibition of NF-κB pathway via FNT was detected using dual luciferase reporter gene assay. Means ± SDs (n = 3) are shown; ### p < 0.001 vs. the control group; * p < 0.05, and *** p < 0.001 vs. the model group. Abbreviations as in .

Article Snippet: C48/80 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation, Inhibition, Luciferase, Reporter Gene Assay

Journal: Molecules

Article Title: Formononetin Inhibits Mast Cell Degranulation to Ameliorate Compound 48/80-Induced Pseudoallergic Reactions

doi: 10.3390/molecules28135271

Figure Lengend Snippet: FNT suppresses C48/80-induced cell degranulation in primary BMMCs. ( A ) Cell viability of BMMCs treated with 0–100 µM FNT was determined using CCK-8 assays. ( B – E ) The inhibitory effect of FNT on the secretion of β-Hex ( C ), histamine ( D ), and RBL-2H3 cells were pre-treated (or not) with different doses of FNT for 2 h separately and then stimulated with C48/80 for 2h. and the transcriptional level of inflammatory factors TNFα ( E ) and IL-13 ( F ) in C48/80-stimulated BMMCs. The data are expressed as the mean ± SDs (n = 3). ### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the model group. Abbreviations as in .

Article Snippet: C48/80 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: CCK-8 Assay

Journal: Molecules

Article Title: Formononetin Inhibits Mast Cell Degranulation to Ameliorate Compound 48/80-Induced Pseudoallergic Reactions

doi: 10.3390/molecules28135271

Figure Lengend Snippet: FNT attenuates C48/80-mediated pseudoallergic reactions in PCA Mice. ( A ) Representative images showed the diffusion of Evans blue from paws of PCA mice. ( B ) Representative pictures of pathological changes in PCA mice. H&E staining showed the degree of telangiectasia, and toluidine blue staining showed MC infiltration. Small blue particles indicated by arrows in toluidine blue staining are infiltrating mast cells. ( C ) Evans blue diffusion quantified at 620 nm. ( D ) Quantification of the change in paw thickness after stimulation. The data are shown as the mean ± SDs (n = 6); ## p < 0.01, ### p < 0.001 vs. the control group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the model group. Abbreviations as in .

Article Snippet: C48/80 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Diffusion-based Assay, Staining

Journal: Molecules

Article Title: Formononetin Inhibits Mast Cell Degranulation to Ameliorate Compound 48/80-Induced Pseudoallergic Reactions

doi: 10.3390/molecules28135271

Figure Lengend Snippet: FNT attenuated C48/80-mediated pseudoallergic reactions in ASA mice. ( A ) The protocol for ASA experiment. ( B ) Changes in rectal temperature after injecting with C48/80 were measured every 5 min over the next 30 min in each group. ( C ) Effect of FNT on the release of histamine in ASA mice (determined using ELISA). The data are shown as the mean ± SDs (n = 6); ## p < 0.01, ### p < 0.001 vs. the control group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the model group. Abbreviations as in .

Article Snippet: C48/80 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: c48/80- and LPA-stimulated G protein activity in brain cryostat sections is mediated by LPA receptors, likely LPA1. (a) [35S]GTPγS autoradiography was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. c48/80 (100 μg ml−1; MP Biomedicals) or LPA (5 μM in 0.1% fatty acid-free BSA) was included in the [35S]GTPγS labeling step of developing (4-week-old) rat brain sections along with or without the LPA1/LPA3 receptor-selective antagonist Ki16425 (5 μM). Note that Ki16425 clearly abolishes [35S]GTPγS binding responses to c48/80 and LPA throughout the white matter tracts. Like 1-butanol, Ki16425 also suppresses basal G protein activity in the white matter regions, indicating tonic LPA receptor activity in the developing rat brain. Abbreviations: cca, corpus callosum. Scale bar=5 mm. (b) Quantitative autoradiography data on the corpus callosum, selected to represent the white matter regions. Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images for statistical analysis. Ki16425 dose-dependently decreases the basal [35S]GTPγS binding as well as that evoked by c48/80 (100 μg ml−1; MP Biomedicals) or LPA (5 μM) with IC50 values of 35±9, 59±59 and 87±19 nM, respectively. Values are mean±s.e.m. (or IC50±s.e.m.) representing six sections from six developing (4-week-old) animals.

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Activity Assay, Autoradiography, Labeling, Binding Assay, Radioactivity

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: [35S]GTPγS autoradiography of rat brain sections reveals LPA-mimicking and NEM-sensitive G protein activation in response to stimulation with c48/80. (a) [35S]GTPγS autoradiography was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. c48/80 (100 μg ml−1) from two suppliers (MP Biomedicals and Sigma-Aldrich) or LPA (50 μM in 0.1% fatty acid-free BSA) was present during the [35S]GTPγS labeling in step 3. Some sections were treated with the irreversible Gi/o protein inhibitor NEM (1 mM, included in step 1 of the protocol). In the control panel (left), the white matter anatomical loci where LPA typically activates G proteins are indicated. Note the highly restricted and LPA-mimicking distribution of c48/80-stimulated G protein activity throughout the white matter tracts, including the anterior commissure (aca), the corpus callosum (cca) and the fimbria of the hippocampus (fi). Other abbreviations: ctx, cerebral cortex; hip, hippocampal structures; str, striatum. Scale bar=5 mm. (b) Quantitative autoradiography data on selected brain regions and on whole brain for NEM-pretreated sections (inset). Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images for two white matter regions (corpus callosum (cca) and the fimbria of the hippocampus (fi)), two gray matter regions (cerebral cortex (ctx) and hippocampus (hip)), an area containing both white and gray matter (striatum (str)) and for whole section area, as detailed in the Methods section. Values are mean+s.e.m. representing sections from four individual animals. Risk level: *P<0.05 compared to basal in each specified brain region and #P<0.05 compared to NEM-pretreated basal on whole section radioactivity (one-way ANOVA with Tukey's multiple comparison).

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Autoradiography, Activation Assay, Labeling, Control, Activity Assay, Radioactivity, Comparison

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: c48/80 stimulates G protein activity in the developing white matter tracts through 1-butanol-sensitive mechanisms likely involving PLD. (a) [35S]GTPγS autoradiography of developing (4-week-old) and adult (9-week-old) rat brain sections was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. c48/80 (100 μg ml−1; MP Biomedicals) or LPA (50 μM in 0.1% fatty acid-free BSA) and the structural butanol isomers (2%, vol vol−1) were present during the [35S]GTPγS labeling step, as indicated. Note that the PLD inhibitor 1-butanol but not its inactive isomer tert-butanol selectively inhibits c48/80-stimulated G protein activity in the developing white matter tracts without affecting LPA-evoked responses. Note also age-dependent decline in basal and c48/80- or LPA-evoked [35S]GTPγS binding responses throughout the adult white matter tracts. Abbreviations: cca, corpus callosum; fi; the fimbria of the hippocampus; cbw, the cerebellar white matter (cbw). Scale bar=5 mm. (b) Quantitative autoradiography data on the corpus callosum of 4-week-old (left) and 9-week-old rats (right). Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images, as detailed in the Methods section. Values are mean+s.e.m. representing four sections from four (developing) or two (adult) individual animals. Risk level: *P<0.05 compared to the respective control treatment in the corpus callosum of 4-week-old rat (one-way ANOVA with Tukey's multiple comparison).

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Activity Assay, Autoradiography, Labeling, Binding Assay, Radioactivity, Control, Comparison

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: (a) Known phospholipase 2 (PLA2) inhibitors, bromoenol lactone (BEL, calcium-independent PLA2), arachidonoyltrifluoromethyl ketone (ATFMK, cytosolic PLA2) and p-bromophenacyl bromide (BPB, secretatory PLA2), have no effect on the c48/80-induced [35S]GTPγS binding to 4-week-old rat brain white matter areas. [35S]GTPγS autoradiography was conducted in three steps with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. (b) Quantitative autoradiography data on the corpus callosum, selected to represent the white matter. The autoradiography images were digitized and bound radioactivity values were obtained from the digitized images for statistical analysis. Values are mean+s.e.m. representing four sections from four developing (4-week-old) animals. Scale bar=5 mm. c48/80 (100 μg ml−1; MP Biomedicals) was included in the labeling step of autoradiography along with the inhibitors (each at 50 μM) as indicated in the figure. No significant alterations were detected in c48/80-induced [35S]GTPγS binding when inhibitors were included. Risk level: P<0.05 compared to the respective control treatment in the corpus callosum.

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Binding Assay, Autoradiography, Radioactivity, Labeling, Control

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: In alkaline conditions, c48/80 activates rat brain heterotrimeric G proteins through the 5-HT1A receptors. (a) [35S]GTPγS autoradiography was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section except that buffer pH was 8.40. At 100 μg ml−1, c48/80 stimulates [35S]GTPγS binding to the hippocampal structures (hip), identical to those labeled by the selective 5-HT1A receptor agonist 8-OH-DPAT (1 μM). Both c48/80- and 8-OH-DPAT-evoked responses are reversed by the selective 5-HT1A receptor antagonist NAN-190 (1 μM). Scale bar=5 mm. (b) Quantitative autoradiography data on the hippocampal region. Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images, as detailed in the Methods section. Values are mean+s.e.m. representing sections from four individual animals. Risk level: *P<0.05 as compared to the basal and #P<0.05 as compared to the 8-OH-DPAT- or c48/80-induced response in the absence of NAN-190 (one-way ANOVA with Tukey's multiple comparison).

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Autoradiography, Binding Assay, Labeling, Radioactivity, Comparison

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: c48/80, unlike MP-7 or stimulation of G protein-coupled muscarinic acetylcholine receptors, fails to enhance the rate of [35S]GTPγS binding to rat forebrain membranes. Certain commercial batches of c48/80 were also found unsuitable for [35S]GTPγS-based G protein activation assays. (a) Classical [35S]GTPγS membrane binding assay was conducted, as detailed in the Methods section. Rat forebrain membranes were incubated for 90 min together with 100 μg ml−1 c48/80 from MP Biomedicals or Sigma-Aldrich in the presence of 0.15 nM [35S]GTPγS with or without 10 μM excess of non-labeled GTPγS. The data represent the percentage of bound radioactivity+s.e.m. from three independent experiments performed in duplicate. A batch of c48/80 from Sigma-Aldrich, but not from MP Biomedicals, causes a significant amount of [35S]GTPγS labeling, even when excess of non-labeled GTPγS is present, probably indicating, for example, chemical interaction between the guanine nucleotides and some portion of the c48/80 mixture. *P<0.05 compared to basal bound radioactivity at 90 min and #P<0.05 compared to Nsb in basal conditions. (b) Rat forebrain membranes were incubated in the presence of c48/80 (100 μg ml−1; MP Biomedicals), MP-7 (50 μM) or carbachol (100 μM) for the indicated times. Data are represented as percentage of bound radioactivity±s.e.m. from three independent experiments performed in duplicate. At all time points tested, MP-7 and carbachol, but not c48/80, significantly stimulated [35S]GTPγS labeling. Risk level: P<0.05 compared to basal condition in each time point.

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Binding Assay, Activation Assay, Membrane, Incubation, Labeling, Radioactivity