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. 2021 Mar 8;11:629726. doi: 10.3389/fimmu.2020.629726

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Copyright © 2021 Abji, Rasti, Gómez-Aristizábal, Muytjens, Saifeddine, Mihara, Motahhari, Gandhi, Viswanathan, Hollenberg, Oikonomopoulou and Chandran

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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PAR2 and tryptase-6 trigger calcium signals in HEK-293 cells. Calcium signaling assay using the fluorescent indicator, Fluo-4 AM, was used to confirm the signaling potential of PAR2 and determine whether tryptase-6 can elicit a calcium response via PAR2 in HEK-293 cells. The concentration-effect curve for the PAR2 agonist 2fLI with an EC50 of 0.14 µM (A) and the concentration–inhibition curve for the PAR2 inhibitor I-191 with an IC50 of 2.1 nM (B) are shown. Stimulation of cells with 0.15 µM of 2fLI (C) was inhibited 99% by 10 nM of I-191 (D). Stimulation of cells with 10.0 FU/min/ng tryptase-6 isolated by antibody affinity chromatography from PsA SF caused an elevation of intracellular calcium (E) and 10 nM of I-191 caused a 34% inhibition of this signal (F). Calcium traces are shown with agonists (2fLI and tryptase-6) added at time zero with or without pre-treatment with I-191 (x-axis=time post addition of agonist, y-axis=units of fluorescence due to calcium release).