Fig. 6.

Desensitization of PAR₁, PAR₃, and PAR₄ with thrombin has no effect on A. alternata-induced Ca²⁺ changes in human airway epithelial cells. A and B: average [Ca²⁺]_i of 80–110 cells in a field of view are plotted over time. Application of 100 nM thrombin to 16HBE14o- cells resulted in a minimal increase in [Ca²⁺]_i. Second and third applications of thrombin caused little [Ca²⁺]_i change. Application of 2.5 μM of the PAR₂ specific agonist 2-furoyl-LIGRLO-NH₂ (2f-pep; A) or A. alternata filtrate (Alt; B) resulted in rapid and large [Ca²⁺]_i changes throughout the cell culture. C and D: quantification of thrombin desensitization experiments. The first treatment with thrombin resulted in a small Ca²⁺ response (20.7 ± 6.2% in C and 16.0 ± 5.6% in D) consistent with low expression of PAR₁, PAR₃, and PAR₄. Following recovery to baseline [Ca²⁺]_i, a second treatment with thrombin resulted in a smaller Ca²⁺ response (8.7 ± 2.3 and 7.4 ± 4.5%), whereas a third application did not induce a [Ca²⁺]_i change, consistent with desensitization of PAR₁, PAR₃, and PAR₄. Subsequent application of 2.5 μM 2-furyol-LIGRLO-NH₂ or A. alternata filtrate resulted in a full Ca²⁺ response (99.8 ± 0.2 and 94.3 ± 1.9%, respectively). These data are consistent with PAR₂ as the protease target of A. alternata proteases. Traces in A and B represent >85 cells in a single experiment and are graphed ± SE; n = 5 for each experimental protocol.