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