Journal: Frontiers in Pharmacology

Article Title: Inhibition of rat locus coeruleus neurons by prostaglandin E 2 EP3 receptors: pharmacological characterization ex vivo

doi: 10.3389/fphar.2023.1290605

Figure Lengend Snippet: Effect of the EP3 receptor agonist sulprostone on the firing rate of LC neurons in the presence of the EP2 receptor antagonist PF-04418948 or the EP4 receptor antagonist L-161,982. (A,B) Representative examples of firing rate recordings of two LC neurons showing the effect of increasing concentrations of sulprostone in the presence of PF-04418948 (10 µM) (A) or L-161,982 (10 µM) (B) . The vertical lines represent the number of spikes recorded every 10 s and the horizontal bars the period of drug application. (C,D) Concentration-effect curves for sulprostone in control (filled squares) and in the presence of PF-04418948 (3 μM, open triangles or 10 μM, filled triangles) (C) or L-161,982 (3 μM, open diamonds or 10 μM, filled diamonds) (D) . The horizontal axes show the sulprostone concentration on a semi-logarithmic scale. The vertical axes express the reduction in firing rate of LC neurons as the percentage of the baseline. Data points are the mean ± SEM at each sulprostone concentration obtained from n number of experiments. The lines through the data are the theoretical curves in each group constructed from the mean of the individual concentration-effect curve parameters, as estimated by nonlinear regressions.

Article Snippet: For electrophysiological recordings, the following drugs were used (drug source): BaCl 2 (Sigma-Aldrich Química S.A., Madrid, Spain), γ-aminobutyric acid (GABA, Sigma-Aldrich), L-161,982 (Tocris Bioscience, Bristol, UK), L-798,106 (Tocris Bioscience), [Met]enkephaline acetate salt (Bachem, Weil am Rhein, Germany), misoprostol free acid (Cayman Chemical, Ann Arbor, MI, USA), pertussis toxin (Tocris Bioscience), PF-04418948 (Tocris Bioscience), prostaglandin E 2 (PGE 2 ) (Tocris Bioscience), SCH-23390 (Tocris Bioscience) and sulprostone (Cayman Chemical).

Techniques: Concentration Assay, Construct

Journal: Frontiers in Pharmacology

Article Title: Inhibition of rat locus coeruleus neurons by prostaglandin E 2 EP3 receptors: pharmacological characterization ex vivo

doi: 10.3389/fphar.2023.1290605

Figure Lengend Snippet: Effect of PGE 2 on the firing rate of LC neurons in the absence or presence of the EP3 receptor antagonist L-798,106 or a combination of the EP2 receptor antagonist PF-04418948 and the EP4 receptor antagonist L-161,982. (A,B) Representative examples of firing rate recordings of two LC neurons showing the effect of increasing concentrations of PGE 2 in the absence (A) and presence of L-798,106 (10 µM) (B) . The vertical lines represent the number of spikes recorded every 10 s and the horizontal bars the period of drug application. (C) Concentration-effect curves for PGE 2 in control (filled squares) and in the presence of L-798,106 (10 μM, filled circles) or PF-04418948 and L-161,982 (10 µM each, half-filled diamonds). The horizontal axis shows the PGE 2 concentration on a semi-logarithmic scale. The vertical axis expresses the reduction in firing rate of LC neurons as the percentage of the baseline. Data points are the mean ± SEM at each PGE 2 concentration obtained from n number of experiments. The lines through the data are the theoretical curves in each group constructed from the mean of the individual concentration-effect curve parameters, as estimated by nonlinear regressions. Note that the concentration-effect curve for PGE 2 is shifted to the right by the EP3 receptor antagonist and to the left by the EP2 and EP4 receptor antagonists.

Article Snippet: For electrophysiological recordings, the following drugs were used (drug source): BaCl 2 (Sigma-Aldrich Química S.A., Madrid, Spain), γ-aminobutyric acid (GABA, Sigma-Aldrich), L-161,982 (Tocris Bioscience, Bristol, UK), L-798,106 (Tocris Bioscience), [Met]enkephaline acetate salt (Bachem, Weil am Rhein, Germany), misoprostol free acid (Cayman Chemical, Ann Arbor, MI, USA), pertussis toxin (Tocris Bioscience), PF-04418948 (Tocris Bioscience), prostaglandin E 2 (PGE 2 ) (Tocris Bioscience), SCH-23390 (Tocris Bioscience) and sulprostone (Cayman Chemical).

Techniques: Concentration Assay, Construct

Journal: Frontiers in Pharmacology

Article Title: Inhibition of rat locus coeruleus neurons by prostaglandin E 2 EP3 receptors: pharmacological characterization ex vivo

doi: 10.3389/fphar.2023.1290605

Figure Lengend Snippet: Effect of misoprostol on the firing rate of LC neurons in the absence or presence of the EP3 receptor antagonist L-798,106 or a combination of the EP2 receptor antagonist PF-04418948 and the EP4 receptor antagonist L-161,982. (A,B) Representative examples of firing rate recordings of two LC neurons showing the effect of increasing concentrations of misoprostol in the absence (A) and presence of L-798,106 (10 µM) (B) . The vertical lines represent the number of spikes recorded every 10 s and the horizontal bars the period of drug application. (C) Concentration-effect curves for misoprostol in control (filled squares) and in the presence of L-798,106 (10 μM, filled circles) or PF-04418948 and L-161,982 (10 µM each, half-filled diamonds). The horizontal axis shows the misoprostol concentration on a semi-logarithmic scale. The vertical axis expresses the reduction in firing rate of LC neurons as the percentage of the baseline. Data points are the mean ± SEM at each misoprostol concentration obtained from n number of experiments. The lines through the data are the theoretical curves in each group constructed from the mean of the individual concentration-effect curve parameters, as estimated by nonlinear regressions. Note that the concentration-effect curve for misoprostol is shifted to the right by the EP3 receptor antagonist.

Article Snippet: For electrophysiological recordings, the following drugs were used (drug source): BaCl 2 (Sigma-Aldrich Química S.A., Madrid, Spain), γ-aminobutyric acid (GABA, Sigma-Aldrich), L-161,982 (Tocris Bioscience, Bristol, UK), L-798,106 (Tocris Bioscience), [Met]enkephaline acetate salt (Bachem, Weil am Rhein, Germany), misoprostol free acid (Cayman Chemical, Ann Arbor, MI, USA), pertussis toxin (Tocris Bioscience), PF-04418948 (Tocris Bioscience), prostaglandin E 2 (PGE 2 ) (Tocris Bioscience), SCH-23390 (Tocris Bioscience) and sulprostone (Cayman Chemical).

Techniques: Concentration Assay, Construct

Journal: Frontiers in Molecular Neuroscience

Article Title: Inhibition of synaptic transmission by anandamide precursor 20:4-NAPE is mediated by TRPV1 receptors under inflammatory conditions

doi: 10.3389/fnmol.2023.1188503

Figure Lengend Snippet: Effect of CB 1 and TRPV1 antagonists application on 20:4-NAPE-induced inhibition of mEPSC frequency in naive slices. (A,C) The application of CB 1 antagonist PF 514273 (0.2 μM) significantly increased the frequency of the recorded mEPSC, as is evident in the native recording example (A) and the averaged data graph ( C , n = 15, *** p < 0.001 vs. control, ### p < 0.001 vs. SB 366791, one-way ANOVA on ranks, Kruskal-Wallis test, Dunn’s post hoc test). Subsequent co-application of PF 514273 (0.2 μM) and 20:4-NAPE (20 μM) did not change the mEPSC frequency compared to the antagonist pretreatment (D) . (B,C) TRPV1 antagonist SB 366791 application (10 μM) did not change the mEPSC frequency compared to the control level ( n = 11). Subsequent application of 20:4-NAPE (20 μM) in the presence of SB 366791 (10 μM) induced significant inhibition of the mEPSC frequency (B,D) . (D) The summary graph shows a comparison of 20:4-NAPE-induced inhibition with the effects of 20:4-NAPE co-application with CB 1 and TRPV1 antagonists. Data are expressed as a percentage of the pretreatment. PF 514273 prevented the 20:4-NAPE-induced inhibitory effect on mEPSC frequency, while SB 3667 did not (* p < 0.05, *** p < 0.001 vs. pretreatment (100%), Wilcoxon signed rank test; # p < 0.05 vs. co-application of PF 514273 + 20:4-NAPE, one-way ANOVA with Tukey post hoc test).

Article Snippet: Capsaicin, SB 366791 - selectivity pA 2 = 7.71 ( ) - and PF 514273 - selectivity Ki = 1 nM ( ) - were purchased from Tocris Bioscience and dissolved in DMSO.

Techniques: Inhibition

Journal: Frontiers in Molecular Neuroscience

Article Title: Inhibition of synaptic transmission by anandamide precursor 20:4-NAPE is mediated by TRPV1 receptors under inflammatory conditions

doi: 10.3389/fnmol.2023.1188503

Figure Lengend Snippet: Effect of CB 1 and TRPV1 antagonists application on 20:4-NAPE-induced inhibition of mEPSC frequency under inflammatory conditions. (A,C) CB 1 antagonist PF 514273 (0.2 μM) application did not change the mEPSC frequency ( n = 10). Subsequent co-application of 20:4-NAPE (20 μM) and PF 514273 (0.2 μM) induced strong inhibition (D) . (B,C) The frequency of mEPSC did not change during the application of TRPV1 antagonist SB 366791 (10 μM, n = 11). Subsequent co-application of 20:4-NAPE (20 μM) and SB 366791 (10 μM) also did not change the mEPSC frequency significantly ( n = 11). (D) Data expressed as a percentage of pretreatment show that SB 366791 application ( n = 11) prevented the 20:4-NAPE-induced ( n = 12) inhibitory effect on mEPSC frequency under the inflammatory conditions, while PF 514273 did not ( n = 10, * p < 0.05, ** p < 0.01 versus pretreatment (100%) Wilcoxon signed rank test; # p < 0.05 vs. co-application of SB 366791 + 20:4-NAPE, one-way ANOVA with Tukey post hoc test).

Article Snippet: Capsaicin, SB 366791 - selectivity pA 2 = 7.71 ( ) - and PF 514273 - selectivity Ki = 1 nM ( ) - were purchased from Tocris Bioscience and dissolved in DMSO.

Techniques: Inhibition

Journal: eLife

Article Title: α 1 -Adrenergic receptor–PKC–Pyk2–Src signaling boosts L-type Ca 2+ channel Ca V 1.2 activity and long-term potentiation in rodents

doi: 10.7554/eLife.79648

Figure Lengend Snippet: ( A ) Neurons were preincubated with vehicle, the phorbol ester phorbol-12-myristate-13-acetate (PMA), and the indicated kinase inhibitors before seal formation. ( B ) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs with vehicle (0.06% DMSO; black) and 2 µM PMA either alone (red) or with the Pyk2 inhibitors PF-719 (1 µM; green) and PF-431396 (3 µM; orange), or the Src inhibitors PP2 (10 µM; blue) and SU6656 (10 µM; purple). ( C ) The increase in NPo by PMA was blocked by all inhibitors. F 5,130 = 6.530. DMSO vs. PMA, p = 0.0003; PMA vs. PF-719 + PMA, p = 0.0372, PMA vs. PF-431396 + PMA, p = 0.0009; PMA vs. PP2 + PMA, p = 0.0003; PMA vs. SU6656 + PMA, p = 0.0005. ( D ) Ensemble averages during depolarization. ( E ) The increase in ensemble average peak currents by PMA was blocked by all inhibitors. F 5,130 = 5.665. DMSO vs. PMA, p = 0.0003; PMA vs. PF-719 + PMA, p = 0.0303, PMA vs. PF-431396 + PMA, p = 0.0051; PMA vs. PP2 + PMA, p = 0.0003; PMA vs. SU6656 + PMA, p = 0.0051. ( C, E ) Data are presented as means ± standard error of the mean (SEM). n represents the number of cells (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; analysis of variance [ANOVA] with post hoc Holm–Sidak’s multiple comparisons test). Panel A was created using Biorender.com .

Article Snippet: Drugs were used at the following concentrations: 2 µM PMA (Merck Millipore Cat#524400), 1 µM BK (Sigma-Aldrich Cat#05-23-0500), 3 µM PF-431396 (Tocris Cat#4278), 10 µM PP2 (Sigma-Aldrich Cat#P0042), 10 µM PP3 (Tocris Cat#2794), and 10 µM SU6656 (Sigma-Aldrich Cat#S9692).

Techniques:

Journal: bioRxiv

Article Title: Focal adhesions are controlled by microtubules through local contractility regulation

doi: 10.1101/2023.04.17.535593

Figure Lengend Snippet: (A) Representative images of Vinculin-mIFP-transfected HT1080 cells carrying the OptoKANK constructs before and after blue light illumination of the focal adhesion (yellow dotted line) for control cells (Ctrl), cells treated with FRAX1036, PF-57328, Kinesore, as well as in APC-depleted, αTAT1-depleted cells, and in FAK -/- knockout MEF cells. Graph shows the normalized mean vinculin intensity after the illumination of cells treated as indicated (Data are presented as mean ± s.e.m; Ctrl, n =18; Nocodazole, n = 12; FRAX1036, n = 15; PF-573228, n = 10; FAK KO -/- , n = 8; Kinesore, n = 11; siRNA APC, n = 18; αTAT1, n = 10). Immunoblots of GEF-H1, p-PAK1/2, APC and GAPDH are shown in the black box. (B) Representative images of Vinculin-mIFP-transfected HT1080 cells carrying the OptoKANK constructs before and after blue light illumination on the focal adhesion (yellow dotted line) for cells treated with FRAX1036, and cells treated with FRAX1036 simultaneously with the contractility activator CNO3. Graph shows the normalized mean vinculin intensity after the illumination for cells under these conditions (Data are presented as mean ± s.e.m; Ctrl, n = 18; Noco, n = 12; PAK inhibitor, n = 15, PAK inhibitor + CN03, n = 10). (C) Representative images of Vinculin-mIFP-transfected HT1080 cells carrying the OptoKANK constructs before and after blue light illumination of the focal adhesion (yellow dotted line) for cells treated with FAK inhibitor PF-57328, Kinesine-1 modulating drug Kinesore, and APC-depleted cells and in such cells treated with Rho activator CNO3. Graph shows the normalized mean vinculin intensity after the illumination for cells under treatments mentioned above (Data are presented as mean ± s.e.m; Ctrl, n =18; Nocodazole, CNO3, n = 8; Kinesore, n = 10; Kinesore + CN03, =n = 16; APC KD, n = 18; APC KD + CNO3, n = 14, FAK inhibitor; n = 10; FAK inhibitor + CNO3, n = 10).

Article Snippet: Pharmacological treatments were performed using the following concentrations of inhibitors or activators: 1 μM for Nocodazole (Sigma-Aldrich), 0.4 μM for Y-27632 dihydrochloride (Sigma-Aldrich), 20 mM for Acetyl-Calpastatin (Tocris, Cat. No. 2950), Monastrol (Merk, cat. No. 254753-54-3), 80 µM for Dynasore (Abcam Cat. No. 120192), Batimastat (Tocris Cat. No. 2961), Tubacin (Cat. No. 3402), MAP4K4-IN-3 (MedChemExpress, Cat. No.: HY-125012), 1 µM for FRAX1036 (MedChemExpress Cat. No. HY-19538), 50 µM for Kinesore (Cat. No. 6664), 5 µM for PF-57328 (Tocris, Cat. No. 3239), and 1 μg ml - for Rho activator II (CNO3, Cytoskeleton).

Techniques: Transfection, Construct, Knock-Out, Western Blot