Journal: Cell Biochemistry and Function

Article Title: Protease‐Activated Receptor 2 Activation Provokes an Increase in Intracellular Calcium and Serotonin Secretion in a Human Enteroendocrine Cell Line

doi: 10.1002/cbf.70132

Figure Lengend Snippet: P‐STS cells respond with an increase in [Ca 2+ ] i to the PAR2 agonist SLIGRL‐NH 2. (a) [Ca 2+ ] i responses to SLIGRL‐NH 2 (time courses of individual experiments are shown, n = 8). (b) The PAR2 antagonist I‐191 inhibits the [Ca 2+ ] i response to SLIGRL‐NH 2 (10 µM) while PAR2‐IN‐1 has no inhibitory effect ( n = 4 for each inhibitor concentration, medium as well as SLIGRL‐NH2). (c) I‐191 also inhibits the [Ca 2+ ] i response to trypsin (110 nM, n = 12). (d) Inhibition of the [Ca 2+ ] i response to SLIGRL‐NH 2 and trypsin by pre‐incubation (5 min) with I‐191 (same experiments as shown in b and c). Relative increase in fluorescence 40 s (trypsin) or 50 s (SLIGRL‐NH 2 ) after addition of agonist. * p < 0.05 (two‐tailed binominal test).

Article Snippet: ACh chloride, G‐418 sulfate solution, histamine dihydrochloride, hydrogen peroxide (30% w/w with stabilizers, freshly diluted before use) and nifedipine were from Sigma Aldrich (St. Louis, Missouri, USA); mibefradil dihydrochloride was from Tocris Bioscience (Bristol, United Kingdom); Gibco trypsin‐EDTA (0.05%) was from Thermo Fisher (Schwerte, Germany); the PAR2 agonist peptide SLIGRL‐NH2 was from HelloBio (Dunshaughlin, Ireland); I‐191 and PAR2‐IN‐1 were from MedChemExpress (Monmouth Junction, New Jersey, USA); Fluo‐4AM was from AAT Bioquest (Pleasanton, California, USA); rabbit anti‐phospho‐p38 antibody (Thr 180/Tyr 182) was from Santa Cruz Biotechnology (Dallas, Texas, USA); mouse anti‐human RelA/NF‐κB p65 (clone 532301) was from R&D Systems (Minneapolis, Canada), goat Alexa Fluor 488‐labeled secondary fluorescent antibodies were from Life Technologies (Carlsbad, California, USA) and the serotonin Elisa kit was from ImmuSmol (Bordeaux, France).

Techniques: Concentration Assay, Inhibition, Incubation, Fluorescence, Two Tailed Test

Journal: Cell Biochemistry and Function

Article Title: Protease‐Activated Receptor 2 Activation Provokes an Increase in Intracellular Calcium and Serotonin Secretion in a Human Enteroendocrine Cell Line

doi: 10.1002/cbf.70132

Figure Lengend Snippet: PAR2 activation does not desensitize P‐STS cells against activation with histamine or ACh. (a–c) Time courses of [Ca 2+ ] I in different experiments after addition of trypsin (final concentration 150 nM) to Fluo4‐AM‐labelled cells, followed by addition of a) medium ( n = 11, in three experiments the reaction to trypsin was delayed (open circles), (b) histamine (final concentration 10 µM, n = 8) or c) ACh (final concentration 0.1 µM, n = 6) 110 s later without trypsin removal. (d) Time courses of [Ca 2+ ] I experiments with immediate reaction to trypsin, including the experiments shown in a–c and an additional experiment with ACh added to a final concentration of 0.25 µM. (e and f) Cells activated by trypsin (150 nM) can respond to histamine (e, final concentration 10 µM) or Ach (f, final concentration 0.25 µM) added 110 s after trypsin.

Article Snippet: ACh chloride, G‐418 sulfate solution, histamine dihydrochloride, hydrogen peroxide (30% w/w with stabilizers, freshly diluted before use) and nifedipine were from Sigma Aldrich (St. Louis, Missouri, USA); mibefradil dihydrochloride was from Tocris Bioscience (Bristol, United Kingdom); Gibco trypsin‐EDTA (0.05%) was from Thermo Fisher (Schwerte, Germany); the PAR2 agonist peptide SLIGRL‐NH2 was from HelloBio (Dunshaughlin, Ireland); I‐191 and PAR2‐IN‐1 were from MedChemExpress (Monmouth Junction, New Jersey, USA); Fluo‐4AM was from AAT Bioquest (Pleasanton, California, USA); rabbit anti‐phospho‐p38 antibody (Thr 180/Tyr 182) was from Santa Cruz Biotechnology (Dallas, Texas, USA); mouse anti‐human RelA/NF‐κB p65 (clone 532301) was from R&D Systems (Minneapolis, Canada), goat Alexa Fluor 488‐labeled secondary fluorescent antibodies were from Life Technologies (Carlsbad, California, USA) and the serotonin Elisa kit was from ImmuSmol (Bordeaux, France).

Techniques: Activation Assay, Concentration Assay

Journal: Cell Biochemistry and Function

Article Title: Protease‐Activated Receptor 2 Activation Provokes an Increase in Intracellular Calcium and Serotonin Secretion in a Human Enteroendocrine Cell Line

doi: 10.1002/cbf.70132

Figure Lengend Snippet: Activation with histamine does not desensitize P‐STS cells against PAR2 activation. (a) Time courses of [Ca 2+ ] I in different experiments after addition histamine (final concentration 10 µM, n = 11) to Fluo4‐AM‐labelled cells. In 3 experiments the reaction to histamine was delayed (open circles). (b) Cells activated by histamine (10 µM) can respond to trypsin (e, final concentration 150 nM) added 110 s after histamine, which was still present.

Article Snippet: ACh chloride, G‐418 sulfate solution, histamine dihydrochloride, hydrogen peroxide (30% w/w with stabilizers, freshly diluted before use) and nifedipine were from Sigma Aldrich (St. Louis, Missouri, USA); mibefradil dihydrochloride was from Tocris Bioscience (Bristol, United Kingdom); Gibco trypsin‐EDTA (0.05%) was from Thermo Fisher (Schwerte, Germany); the PAR2 agonist peptide SLIGRL‐NH2 was from HelloBio (Dunshaughlin, Ireland); I‐191 and PAR2‐IN‐1 were from MedChemExpress (Monmouth Junction, New Jersey, USA); Fluo‐4AM was from AAT Bioquest (Pleasanton, California, USA); rabbit anti‐phospho‐p38 antibody (Thr 180/Tyr 182) was from Santa Cruz Biotechnology (Dallas, Texas, USA); mouse anti‐human RelA/NF‐κB p65 (clone 532301) was from R&D Systems (Minneapolis, Canada), goat Alexa Fluor 488‐labeled secondary fluorescent antibodies were from Life Technologies (Carlsbad, California, USA) and the serotonin Elisa kit was from ImmuSmol (Bordeaux, France).

Techniques: Activation Assay, Concentration Assay

Journal: Cell Biochemistry and Function

Article Title: Protease‐Activated Receptor 2 Activation Provokes an Increase in Intracellular Calcium and Serotonin Secretion in a Human Enteroendocrine Cell Line

doi: 10.1002/cbf.70132

Figure Lengend Snippet: P‐STS cells respond with an increase in [Ca 2+ ] i to the PAR2 agonist SLIGRL‐NH 2. (a) [Ca 2+ ] i responses to SLIGRL‐NH 2 (time courses of individual experiments are shown, n = 8). (b) The PAR2 antagonist I‐191 inhibits the [Ca 2+ ] i response to SLIGRL‐NH 2 (10 µM) while PAR2‐IN‐1 has no inhibitory effect ( n = 4 for each inhibitor concentration, medium as well as SLIGRL‐NH2). (c) I‐191 also inhibits the [Ca 2+ ] i response to trypsin (110 nM, n = 12). (d) Inhibition of the [Ca 2+ ] i response to SLIGRL‐NH 2 and trypsin by pre‐incubation (5 min) with I‐191 (same experiments as shown in b and c). Relative increase in fluorescence 40 s (trypsin) or 50 s (SLIGRL‐NH 2 ) after addition of agonist. * p < 0.05 (two‐tailed binominal test).

Article Snippet: ACh chloride, G‐418 sulfate solution, histamine dihydrochloride, hydrogen peroxide (30% w/w with stabilizers, freshly diluted before use) and nifedipine were from Sigma Aldrich (St. Louis, Missouri, USA); mibefradil dihydrochloride was from Tocris Bioscience (Bristol, United Kingdom); Gibco trypsin‐EDTA (0.05%) was from Thermo Fisher (Schwerte, Germany); the PAR2 agonist peptide SLIGRL‐NH2 was from HelloBio (Dunshaughlin, Ireland); I‐191 and PAR2‐IN‐1 were from MedChemExpress (Monmouth Junction, New Jersey, USA); Fluo‐4AM was from AAT Bioquest (Pleasanton, California, USA); rabbit anti‐phospho‐p38 antibody (Thr 180/Tyr 182) was from Santa Cruz Biotechnology (Dallas, Texas, USA); mouse anti‐human RelA/NF‐κB p65 (clone 532301) was from R&D Systems (Minneapolis, Canada), goat Alexa Fluor 488‐labeled secondary fluorescent antibodies were from Life Technologies (Carlsbad, California, USA) and the serotonin Elisa kit was from ImmuSmol (Bordeaux, France).

Techniques: Concentration Assay, Inhibition, Incubation, Fluorescence, Two Tailed Test

Journal: Cell Biochemistry and Function

Article Title: Protease‐Activated Receptor 2 Activation Provokes an Increase in Intracellular Calcium and Serotonin Secretion in a Human Enteroendocrine Cell Line

doi: 10.1002/cbf.70132

Figure Lengend Snippet: PAR2 activation does not desensitize P‐STS cells against activation with histamine or ACh. (a–c) Time courses of [Ca 2+ ] I in different experiments after addition of trypsin (final concentration 150 nM) to Fluo4‐AM‐labelled cells, followed by addition of a) medium ( n = 11, in three experiments the reaction to trypsin was delayed (open circles), (b) histamine (final concentration 10 µM, n = 8) or c) ACh (final concentration 0.1 µM, n = 6) 110 s later without trypsin removal. (d) Time courses of [Ca 2+ ] I experiments with immediate reaction to trypsin, including the experiments shown in a–c and an additional experiment with ACh added to a final concentration of 0.25 µM. (e and f) Cells activated by trypsin (150 nM) can respond to histamine (e, final concentration 10 µM) or Ach (f, final concentration 0.25 µM) added 110 s after trypsin.

Article Snippet: ACh chloride, G‐418 sulfate solution, histamine dihydrochloride, hydrogen peroxide (30% w/w with stabilizers, freshly diluted before use) and nifedipine were from Sigma Aldrich (St. Louis, Missouri, USA); mibefradil dihydrochloride was from Tocris Bioscience (Bristol, United Kingdom); Gibco trypsin‐EDTA (0.05%) was from Thermo Fisher (Schwerte, Germany); the PAR2 agonist peptide SLIGRL‐NH2 was from HelloBio (Dunshaughlin, Ireland); I‐191 and PAR2‐IN‐1 were from MedChemExpress (Monmouth Junction, New Jersey, USA); Fluo‐4AM was from AAT Bioquest (Pleasanton, California, USA); rabbit anti‐phospho‐p38 antibody (Thr 180/Tyr 182) was from Santa Cruz Biotechnology (Dallas, Texas, USA); mouse anti‐human RelA/NF‐κB p65 (clone 532301) was from R&D Systems (Minneapolis, Canada), goat Alexa Fluor 488‐labeled secondary fluorescent antibodies were from Life Technologies (Carlsbad, California, USA) and the serotonin Elisa kit was from ImmuSmol (Bordeaux, France).

Techniques: Activation Assay, Concentration Assay

Journal: Cell Biochemistry and Function

Article Title: Protease‐Activated Receptor 2 Activation Provokes an Increase in Intracellular Calcium and Serotonin Secretion in a Human Enteroendocrine Cell Line

doi: 10.1002/cbf.70132

Figure Lengend Snippet: Activation with histamine does not desensitize P‐STS cells against PAR2 activation. (a) Time courses of [Ca 2+ ] I in different experiments after addition histamine (final concentration 10 µM, n = 11) to Fluo4‐AM‐labelled cells. In 3 experiments the reaction to histamine was delayed (open circles). (b) Cells activated by histamine (10 µM) can respond to trypsin (e, final concentration 150 nM) added 110 s after histamine, which was still present.

Article Snippet: ACh chloride, G‐418 sulfate solution, histamine dihydrochloride, hydrogen peroxide (30% w/w with stabilizers, freshly diluted before use) and nifedipine were from Sigma Aldrich (St. Louis, Missouri, USA); mibefradil dihydrochloride was from Tocris Bioscience (Bristol, United Kingdom); Gibco trypsin‐EDTA (0.05%) was from Thermo Fisher (Schwerte, Germany); the PAR2 agonist peptide SLIGRL‐NH2 was from HelloBio (Dunshaughlin, Ireland); I‐191 and PAR2‐IN‐1 were from MedChemExpress (Monmouth Junction, New Jersey, USA); Fluo‐4AM was from AAT Bioquest (Pleasanton, California, USA); rabbit anti‐phospho‐p38 antibody (Thr 180/Tyr 182) was from Santa Cruz Biotechnology (Dallas, Texas, USA); mouse anti‐human RelA/NF‐κB p65 (clone 532301) was from R&D Systems (Minneapolis, Canada), goat Alexa Fluor 488‐labeled secondary fluorescent antibodies were from Life Technologies (Carlsbad, California, USA) and the serotonin Elisa kit was from ImmuSmol (Bordeaux, France).

Techniques: Activation Assay, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Gaussian white noise stimulation as an alternative method to excite sensory neurons

doi: 10.3389/fphar.2025.1561905

Figure Lengend Snippet: Action potential firing elicited by current pulse injections in DRG neurons after one, three, or 7 days in vitro (DIV). DRG neurons were recorded in perforated current-clamp mode; inflammatory soup (i.s.: ADP 1 μ M, ATP 1 μ M, bradykinin 30 nM, histamine 1 μ M, PAR2 agonist 2-Furoyl-LIGRLO-NH2 1 μ M, prostaglandin E2 300 nM, serotonin 300 nM, substance P 10 nM) was present for 30 s before and during triggering of action potentials. (A) After determination of the rheobases, five depolarizing current pulses with amplitudes of 1 x, 1.5 x, 2 x, 2.5 x and 3x rheobases (here 15–45 pA) were injected into a DIV 1 neuron for time periods of 2 s each. The traces shown were obtained either in solvent (black traces) or in presence of inflammatory soup components (blue traces). (B) Currents were injected as in A (here 220–660 pA) into a DIV 3 neuron. (C) shows exemplary voltage traces of a DIV 7 neuron injected with 5 current pulses ranging from 140 to 420 pA. (D) statistical analysis of action potential numbers in presence of solvent (0.1 % DMSO, black filled circles) and in presence of i.s (blue filled circles) recorded in DIV 1 (left panel, n = 9), DIV 3 (center panel, n = 9), and DIV 7 (right panel, n = 9) neurons (Wilcoxon matched-pairs signed rank test). (E) shows the statistical comparison of rheobases (left panel), input resistance ( R i n , center panel), and membrane potential ( V M , right panel) of DIV 1, DIV 3, and DIV 7 neurons (Kruskal–Wallis test, followed by Dunn’s multiple comparison post hoc test).

Article Snippet: Prostaglandin E 2 ( PGE 2 ) was from MedChemExpress LLC (Monmouth Junction, NJ, United States; distributed by THP Medical Products, Vienna, Austria), the PAR2 agonist 2-furoyl-LIGRLO-amide was purchased from TargetMol (Wellesley Hills, MA, United States; distributed by Eubio, Vienna, Austria), substance P was from HelloBio (Bristol, United Kingdom).

Techniques: In Vitro, Injection, Solvent, Comparison, Membrane

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: Specificity and potency of anti-PAR2 monoclonal antibody MEDI0618 . ( A ) Live staining of PAR2-expressing (1321N1-hPAR2.cl8) or non-expressing (1321N1 parental) cell lines with MEDI0618 directly conjugated to Alexa Fluor 647. Scale bar = 20 µm. ( B ) Flow cytometry of hPAR2 overexpressing cells (1321N1-hPAR2.cl8) or non-expressing cells (1321N1 parental cell line) or A549 cells endogenously expressing hPAR2 live labelled with MEDI0618. ( C and D ) Calcium imaging from 1321N1-hPAR2 cells pretreated with MEDI0618 hIgG or an isotype control protein. ( C ) Exemplar raw calcium trace from a single well pretreated with MEDI0618 or isotype control antibody both at 1 nM, followed by PAR2 agonist stimulation with matriptase (10 nM). ( D ) Antibody titration of the anti-PAR2 antibodies MEDI0618 or PAR650097 or an isotype control antibody. Data show the matriptase (10 nM)-mediated calcium signal after preincubation with antibody and normalized to the matriptase response in the absence of antibody treatment. ( E ) Calcium imaging from A549 cells pretreated with MEDI0618, isotype control protein or PAR1 inhibitors (ATAP2 + WEDE15 mAbs) followed by 10 nM thrombin (PAR1 agonist) stimulation. Data are presented as mean ± standard error of the mean, n = 4. The concentration of the inhibitor is shown on the x -axis. Data are normalized to the peak thrombin calcium response in the absence of inhibitor pretreatment.

Article Snippet: PAR2 agonists Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLIGRL-NH2), 2-Furoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2 (LIGRLO) and the reverse peptide control 2-Furoyl-Orn-Leu-Arg-Gly-Ile-Leu-NH2 (OLRGIL) (Tocris) were diluted in PBS + 0.1% BSA to generate a 10 mM stock solution prior to final dilution in assay buffer.

Techniques: Staining, Expressing, Flow Cytometry, Imaging, Control, Titration, Concentration Assay

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: PAR2 functional expression in human and mouse cells relevant to migraine . Whole well calcium imaging from primary human dural fibroblasts (HDuF), human dural microvascular endothelial (HDuMEC) and mouse brain endothelial (bEnd.3) cells. ( A , D and G ) PAR2 agonists concentration response curve in ( A ) HDuF, ( D ) HDuMEC and ( G ) bEnd.3 cells. ( B , E and F ) Effect of MEDI0618 and isotype control protein (IgG) on inhibition of matriptase-induced calcium signalling at 30 nM in ( B ) HDuF, ( E ) HDuMEC and ( H ) bEnd.3 cells. ( C , F and I ) Representative calcium imaging traces of 30 nM matriptase-evoked activity following MEDI0618 or isotype control protein preincubation in ( C ) HDuF, ( F ) HDuMEC and ( I ) bEnd.3 cells. ( J and K ) Single-cell calcium imaging from mouse trigeminal neuron cultures. ( J ) Pseudocolour images of fura-2 ratio intensity show a subset of trigeminal neurons activated by treatment with 10 µM LIGRLO in comparison to 20 mM KCl treatment. Scale bar = 20 µm. ( K ) Representation of fura-2 traces recorded from two individual neurons during acute LIGRLO (10 µM) or KCl (20 mM) treatment.

Article Snippet: PAR2 agonists Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLIGRL-NH2), 2-Furoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2 (LIGRLO) and the reverse peptide control 2-Furoyl-Orn-Leu-Arg-Gly-Ile-Leu-NH2 (OLRGIL) (Tocris) were diluted in PBS + 0.1% BSA to generate a 10 mM stock solution prior to final dilution in assay buffer.

Techniques: Functional Assay, Expressing, Imaging, Concentration Assay, Control, Inhibition, Activity Assay, Comparison