Fig. 7.

Desensitization of PAR₂ eliminates A. alternata-induced Ca²⁺ changes in human airway epithelial cells. Average [Ca²⁺]_i of 80–110 cells in a field of view are plotted over time. A: application of 100 μM trypsin to 16HBE14o- cells resulted in a rapid and large increase in [Ca²⁺]_i that recovered to baseline in 3–4 min. A second application of trypsin did not induce further changes in [Ca²⁺]_i. Application of A. alternata filtrate (Alt) also resulted in no change in [Ca²⁺]_i. However, application of ATP resulted in a full [Ca²⁺]_i response. B: application of the PAR₂ selective peptidomimetic agonist 2-furoyl-LIGRLO-NH₂ (100 μM; 2f-pep) resulted in a rapid increase in [Ca²⁺]_i. Subsequent addition of A. alternata filtrate did not result in an increase in [Ca²⁺]_i, whereas application of ATP resulted in increased [Ca²⁺]_i. C: quantification of PAR₂ desensitization by trypsin. The first treatment with trypsin resulted in a full Ca²⁺ response (100% in all cultures), whereas a second application of trypsin (no Ca²⁺ response) or subsequent application of A. alternata filtrate (0.4 ± 0.4%) resulted in negligible Ca²⁺ responses. However, application of ATP, an agonist that works independently of PAR₂ to raise [Ca²⁺]_i, demonstrated a full Ca²⁺ response (76.9 ± 15.3%). D: quantification of PAR₂ desensitization by 2-furoyl-LIGRLO-NH₂ (2f-pep). The first treatment with 2-furoyl-LIGRLO-NH₂ resulted in a full Ca²⁺ response (100% in all cultures). Subsequent application of A. alernata filtrate (0.52 ± 0.52%) resulted in negligible Ca²⁺ responses. As seen in the trypsin desensitization, application of ATP resulted in a full Ca²⁺ response (92.0 ± 1.8%). These data are consistent with PAR₂ as the protease target of A. alternata proteases. Traces in A and B represent >85 cells from a single experiment and are graphed ± SE at each time point; n = 3 for each experimental protocol.