u46619  (Tocris)

Journal: bioRxiv

Article Title: Syncytial coupling of mid-capillary pericytes underlies seizure-associated electro-metabolic signaling

doi: 10.64898/2026.03.16.711912

Figure Lengend Snippet: (A) Changes in pericyte membrane potential after norepinephrine and UDP-glucose application. (B) The thromboxane analogue U46619 depolarizes and constricts pericytes. Upper left corner: The amplitude of the depolarization induced by U46619 depended on the intracellular chloride concentration. See the orange trace (34 mM Cl⁻) and the light purple trace (8 mM Cl⁻). Lower left and right panel: The plateau phase was reversed by the TMEM16 inhibitor Ani9 (blue trace), indicating the involvement of Ca 2+ -activated Cl - channels (Kruskal-Wallis test: p = 0.00016, post-hoc pairwise Dunn test: significant differences between group 1 and 3, p = 0.0003). (C) Upper panel: Recording of the resting membrane potential (Vm) of a pericyte and a neuron immediately after establishing the whole cell configuration with a CsCl-containing intracellular solution. In contrast to neurons, pericytic depolarization is moderate even after 10 min. Lower panel: Quantification of pericyte membrane potential immediately after rupturing the membrane and after 10 min using CsCl intracellular solution. (D) Representative recording of a voltage ramp (-100 mV to 60 mV) of a neuron (turquoise) and pericyte (red), indicating the presence of voltage gated inward currents in neurons but not in pericytes. (E) Relative fluorescence change of OGB-1 in response to depolarization steps starting from a holding potential of -100 mV. Neurons showed a marked increase in fluorescence for steps above -50 mV while pericytes solely presented with continuous slight baseline increase. (F) Comparison of the percentage change in vessel diameter, pericyte length, and fluorescence change upon i. control (n = 11), ii. different depolarizing current injection protocols (depolarization to 0 mV (n = 6), depolarization to -20mV (n = 13), recurrent depolarization steps (n = 11) or iii. 200 nM U46619 (n = 14)). Pericyte length, vessel diameter and Ca 2+ concentration changed significantly upon U46619 (OGB-1 fluorescence increase: 42.77 ± 7.33 %, p = 0.0001**, pericyte length change: -7.15 ± 1.61 %, p = 0.0002**, vessel diameter change: -33.92 ± 5.54 %, p = 0.0001**) but remained unchanged upon depolarization (current injection to 0 mV: p = 0.44, p = 0.56, p = 0.31; current injection to -20 mV: p = 0.86, p = 0.68, p = 0.19; recurrent depolarization steps: p = 0.12, p = 0.97, p = 0.03). Bonf.:*=significant. Scale bar: 10 µm. (G) Representative recording of a pericyte exhibiting spontaneous Ca 2+ fluctuations in the presence of the VGCC activator BAY-K-8644 (100 nM). (H) Change in OGB-1 fluorescence upon depolarization of the pericyte (on the excerpt) to 0 mV under BAY-K-8644. Slow increase in fluorescence was observed in 7 out of 9 cells.

Article Snippet: To constrict capillaries UDP-glucose (100 μM, Merck, CAS-Nr. 117756-22-6), norepinephrine (10 μM Sigma A7256) and the thromboxane agonist, U46619 (Stock solution: 2 mM, Tocris bioscience, diluted in DMSO) were applied via the perfusion.

Techniques: Membrane, Concentration Assay, Fluorescence, Comparison, Control, Injection

Journal: bioRxiv

Article Title: Syncytial coupling of mid-capillary pericytes underlies seizure-associated electro-metabolic signaling

doi: 10.64898/2026.03.16.711912

Figure Lengend Snippet: (A) Schematic of the mode of action for the drugs used to interfere with pre-seizure hyperpolarization. (B) Representative dual-patch recording of a coupled pericyte-endothelial cell pair showing membrane potential changes during seizure-like activity in rat OHSC. The initial depolarization is caused by the thromboxane analogue U46619, which additionally constricts pericytes. At the onset of 4AP-induced seizure-like activity, the pericyte repolarizes to values observed prior to U46619, followed by recurrent seizure-associated depolarizations (dual patch: n=4, recordings from single pericytes n=15). (C) Hyperpolarization of pericytes could not be inhibited by application of a nitric oxide synthase inhibitor L-NMMA and the NO scavenger carboxy-PTIO (n=9). (D, E, F) Blocking Kir2.x channels using 100 μM barium (n=16), application of the non-selective adenosine receptor agonist CGS 15943 (n=11) or the selective A2a receptor antagonist ZM 241385 (n=12) partially or completely inhibited pericyte hyperpolarization. (G) Hyperpolarization of pericytes was determined as the difference between resting Vm prior to U46619 and the maximum of the pre-seizure hyperpolarization. Delta values close to zero represent a complete repolarization upon epileptiform activity. (H) Quantification of the repolarization in the presence of different blockers. Kruskal-Wallis test followed by post-hoc Dunnett’s test (***p<0.001). (I) In human cortical brain slices, pericytes hyperpolarize after an increase in potassium from 2 mM to 5 mM. (J) Hyperpolarization of pericytes after 10 μM pinacidil indicates the presence of KATP channels in human tissue. (K) The higher the resting potential of the pericyte before application of pinacidil (purple) and 3 mM potassium on top of 2mM aCSF K + concentration (blue), the stronger the hyperpolarization (Linear regression: r = -0.775, p < 0.001).

Article Snippet: To constrict capillaries UDP-glucose (100 μM, Merck, CAS-Nr. 117756-22-6), norepinephrine (10 μM Sigma A7256) and the thromboxane agonist, U46619 (Stock solution: 2 mM, Tocris bioscience, diluted in DMSO) were applied via the perfusion.

Techniques: Membrane, Activity Assay, Blocking Assay, Concentration Assay

Journal: Poultry Science

Article Title: Novel insights into the regulation of NF2/SWH signaling in granulosa cells of prehierarchical follicle development in hen ovary

doi: 10.1016/j.psj.2026.106375

Figure Lengend Snippet: Effect of NF2/SWH signaling pathway on GC apoptosis via p38 signaling in chicken ovary. GCs were transfected with specific siRNA-NF2 and/or siRNA-YAP1, NC (scrambled siRNA, negative control), and no siRNA as a vehicle (blank control, BC), U46619, and SB203580. (A-B) The p-p38, p38 protein expression level in the GCs was analyzed using western blot. (C) Western blot analysis was employed to screen out the best processing time of U46619 (an activator of p38 MAPK phosphorylation) and SB203580 (an inhibitor of p38 MAPK phosphorylation). (D) Effects of different treatments on GC apoptosis. Cell apoptosis rate was measured through flow cytometry. (E) Expression of BCL2 and CASP3 mRNA and proteins in the GCs with different treatments were analyzed by RT-qPCR and western blot. Experimental procedures were performed in triplicate, and representative results report both means and standard deviations. For each group, the different superscript above the bar indicates that the difference was significant (* P < 0.05, ** P < 0.01).

Article Snippet: GCs that had been cultured for 24 h were washed twice with PBS and were then preincubated with or without 10 μM SB203580 (an inhibitor of p38 MAPK phosphorylation) or U46619 (an activator of p38 MAPK phosphorylation) for 1h and 2 h to screen out the best processing time of SB203580 (S1863, Beyotime, Jiangsu, China) and U46619 (sc-201242, Santa Cruz, Dallas, TX, USA) as described previously ( , , , ).

Techniques: Transfection, Negative Control, Control, Expressing, Western Blot, Phospho-proteomics, Flow Cytometry, Quantitative RT-PCR

Journal: Poultry Science

Article Title: Novel insights into the regulation of NF2/SWH signaling in granulosa cells of prehierarchical follicle development in hen ovary

doi: 10.1016/j.psj.2026.106375

Figure Lengend Snippet: Effect of NF2/SWH signaling pathway on GC mitochondria via p38 signaling. GCs were transfected with specific siRNA-NF2 and/or siRNA-YAP1, NC (scrambled siRNA, negative control), and no siRNA as a vehicle (blank control, BC), U46619, and SB203580. (A) Effects of different treatments on mitochondrial gene DNA copy number. (B) Effects of different treatments on ATP. (C) Effects of different treatments on cytochrome C. (D) Effects of different treatments on ROS. (E) Effects of different treatments on the membrane potential. Experimental procedures were performed in triplicate, and representative results report both means and standard deviations. For each group, the different superscript above the bar indicates that the difference was significant (* P < 0.05).

Article Snippet: GCs that had been cultured for 24 h were washed twice with PBS and were then preincubated with or without 10 μM SB203580 (an inhibitor of p38 MAPK phosphorylation) or U46619 (an activator of p38 MAPK phosphorylation) for 1h and 2 h to screen out the best processing time of SB203580 (S1863, Beyotime, Jiangsu, China) and U46619 (sc-201242, Santa Cruz, Dallas, TX, USA) as described previously ( , , , ).

Techniques: Transfection, Negative Control, Control, Membrane

Journal: Nature Communications

Article Title: Structural and dynamic insights into agonist recognition and function of the thromboxane A 2 receptor

doi: 10.1038/s41467-026-69844-9

Figure Lengend Snippet: a Cartoon representation of the active TP structures bound to the agonist I-BOP and the mini-G q heterotrimer. The TP is coloured dark red, with mini-G αq in gold, G β and G γ in purple and green, respectively, and scFv16 in light grey. I-BOP is in olive green. Cutaway view of the U46619 ( b , blue) and I-BOP ( c , green) bound TP structures indicating that both ligands adopt an L-shaped conformation, while occupying a binding pocket that is buried deep within the receptor’s transmembrane core. The sealed cavity is proposed to protect the ligands from decomposition by hydration to the water-soluble TXB 2 . d Chemical structures of the endogenous TP agonist TXA 2 (left) and its derivative TXB 2 (right). e Chemical structures of the synthetic agonists used in this study: U46619 and I-BOP. In ( d ) and ( e ), the α- and ω-chains are labelled, the bicyclic ring that differs between the synthetic agonists and the endogenous TXA 2 is in yellow, and the iodobenzene group present in I-BOP is in green. f G q heterotrimer dissociation as measured by a loss of BRET after TP stimulation with U46619 (blue) or I-BOP (green) in HEK293 cells transiently co-expressing the wild-type receptor and TRUPATH biosensors for the full-length G q protein. Data are represented as mean ± SEM from 8 independent experiments for U46619 and 4 independent experiments for I-BOP, performed in triplicate, indicating EC 50 values of 11.22 ± 5.29 nM and 1.79 ± 1.08 nM (mean ± SEM) for U46619 and I-BOP, respectively.

Article Snippet: In the current study, we report the cryogenic electron microscopy (cryo-EM) structures of the TPα-G q complex bound to two potent and specific TXA 2 mimetics, U46619 and I-BOP.

Techniques: Binding Assay, Expressing

Journal: Nature Communications

Article Title: Structural and dynamic insights into agonist recognition and function of the thromboxane A 2 receptor

doi: 10.1038/s41467-026-69844-9

Figure Lengend Snippet: a Superposition of U46619 (blue) and I-BOP (green) in the cryo-EM structures. b BRET experiments were performed as above and EC 50 values plotted in response to mutations in the TP canonical binding pocket. EC 50 values are not available for mutants marked with N.A as their data did not fit dose-response curves. Data are presented as mean ± SEM from three independent experiments. * and *** indicate P < 0.05 and P < 0.001, respectively. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post-hoc tests, comparing the mutants treated with U46619 or I-BOP to the corresponding WT treated with U46619 or I-BOP. Shading indicates the following: WT-TP treated with U46619 and I-BOP (green); mutants of residues involved in agonist binding (blue); the T298 7.43 V mutant (peach); and mutants of residues involved in stabilizing the L-shaped agonist conformation (salmon). All mutants were treated with U46619. Residues surrounding the binding pocket of U46619 ( c ) and I-BOP ( d ). Residues are labelled and shown as sticks. All residues within 5 Å of the ligand are shown in grey or dark red. Residues that are discussed in the main text are highlighted in dark red. Dashed lines indicate hydrogen bonds with distances in Å. e Superposition of U46619 (blue) and I-BOP (green) in the binding pocket of the TP. Only the TP-U46619 receptor structure is shown for clarity. The ω-chain of I-BOP differs from that of U46619 and the natural agonist where the methylenes are substituted by iodobenzene. As a result, I-BOP sits deeper in the binding pocket when compared to U46619. f –h Water-mediated interactions, observed through MD simulations, between the bicyclic ring oxygen and T298 7.43 . A more detailed view of the simulation is provided in Supplementary Fig. . Simulations are shown for U46619 ( f , blue), I-BOP ( g , green), and the endogenous ligand TXA 2 ( h , orange).

Article Snippet: In the current study, we report the cryogenic electron microscopy (cryo-EM) structures of the TPα-G q complex bound to two potent and specific TXA 2 mimetics, U46619 and I-BOP.

Techniques: Cryo-EM Sample Prep, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Structural and dynamic insights into agonist recognition and function of the thromboxane A 2 receptor

doi: 10.1038/s41467-026-69844-9

Figure Lengend Snippet: a Chemical structures of the TP agonist, U46619 (left), and antagonist, ramatroban (right). The red outline traces the bonds connecting the carboxyl carbon in the α-chain to the last atom in the ω-chain. There are 16 bonds in the agonist and 14 in the antagonist, indicating that the agonist is the longer ligand. b Superposition of the agonists, U46619 (blue) and I-BOP (green), with the antagonist, ramatroban (salmon, PDB 6IIU), indicating that both ligand types adopt an approximate L-shaped conformation in the binding pocket. Agonists sit lower inside the TP binding pocket than antagonists (highlighted by the orange circle). c Superposition of U46619 (blue) and ramatroban (salmon, PDB 6IIU) in their respective binding pockets indicates that the antagonist, ramatroban, contacts the hydroxyl group of T81 2.57 and the backbone of L78 2.54 through the sulphonamide moiety. Hydrogen bonds are indicated as black dotted lines, with distances in Å. d Superposition of the active (dark red) and inactive (grey, PDB 6IIU) TP structures highlighting major structural rearrangements in TM1, TM6, TM7, and H8. Close up views of microswitch residues and residues that undergo large conformational rearrangements are shown in dashed outlines.

Article Snippet: In the current study, we report the cryogenic electron microscopy (cryo-EM) structures of the TPα-G q complex bound to two potent and specific TXA 2 mimetics, U46619 and I-BOP.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Structural and dynamic insights into agonist recognition and function of the thromboxane A 2 receptor

doi: 10.1038/s41467-026-69844-9

Figure Lengend Snippet: a U46619 (blue) in the binding pocket of the activated TP, with canonical toggle switch residues highlighted. b, c Modelled G116V/A substitutions in the TP sterically clash (red sphere) with the ω-chain of the ligand. d EC 50 values from BRET experiments performed with TP constructs mutated at residues that are part of the proposed activation mechanism. EC 50 values are not available for mutants marked with N.A as their data did not fit dose-response curves. Data are presented as mean ± SEM from three independent experiments. * indicates P < 0.05. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post-hoc tests, comparing the mutants treated with U46619 or I-BOP to the corresponding WT treated with U46619 or I-BOP. Shading indicates the following: WT-TP and mutants of residues involved in the activation mechanism treated with U46619 (blue) and I-BOP (green). e Superposition of active (dark red) and inactive (grey, PDB 6IIU) TP structures showing conformational changes in the residues involved in receptor activation. f The sulphonamide oxygen of ramatroban hydrogen bonds to the side chain amide of Q301 7.46 , which, in turn, enables the carbonyl oxygen to interact with the indole of W258 6.48 . g The ω-chain hydroxyl group of U46619 interacts with the carbonyl oxygen of Q301 7.46 , enabling W258 6.48 to interact with N300 7.45 and to form an extensive hydrogen bond network with neighbouring residues that drives receptor activation.

Article Snippet: In the current study, we report the cryogenic electron microscopy (cryo-EM) structures of the TPα-G q complex bound to two potent and specific TXA 2 mimetics, U46619 and I-BOP.

Techniques: Binding Assay, Construct, Activation Assay

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Prothrombotic Activation of Platelet Pannexin‐1 Channels in Antiphospholipid Syndrome

doi: 10.1002/art.70001

Figure Lengend Snippet: PANX1 activation in response to various platelet agonists. Purified platelets from healthy volunteers were pretreated with CBX (100 μ M ) for 15 minutes and stimulated with different platelet agonists, such as thrombin, U46619, and convulxin, for an additional 15 minutes (A–C), and released ATP was measured in the supernatant. (D–F) Treated platelets were fixed with 2% paraformaldehyde and analyzed for surface P‐selectin (CD62P) expression using flow cytometry by staining with Alexa Fluor 647–conjugated anti‐CD62P. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001; P values were calculated using one‐way analysis of variance followed by Tukey's correction for multiple comparisons. CBX, carbenoxolone; ns, nonsignificant.

Article Snippet: ATP release was also measured after preincubating purified platelets from healthy controls with 100 μ M carbenoxolone for 15 minutes, followed by stimulation with various platelet agonists for an additional 15 minutes at 37°C: 0.025 U/mL of thrombin (Chrono‐Log), 5 μ M U46619 (Tocris), and 10 ng/mL of convulxin (Santa Cruz).

Techniques: Activation Assay, Purification, Expressing, Flow Cytometry, Staining