German cockroach extract activates protease-activated receptor 2 in human airway epithelial cells☆

Abstract 

Background

The German cockroach has been reported to act as an allergen that might be associated with a protease reaction in asthma. However, the molecular identities of the antigens in German cockroach extract (GCE) with protease activity and the protease-activated receptors (PARs) that are activated by GCE in human airway epithelial cells have not been characterized.

Objective

We investigated the direct effect of GCE on Ca²⁺ signaling in human airway epithelial cells and the type of PARs activated by GCE.

Methods

The Ca²⁺-sensitive dye Fura2 was used to determine intracellular Ca²⁺ concentration ([Ca²⁺]_i) by means of spectrofluorometry.

Results

GCE induced a baseline type of [Ca²⁺]_i oscillations in a dose-dependent manner. The oscillations persisted for long periods of time in the absence of Ca²⁺ entry across the plasma membrane, suggesting that the observed [Ca²⁺]_i increases were due to Ca²⁺ release from intracellular stores. Accordingly, after depleting endoplasmic reticulum Ca²⁺ with thapsigargin, an endoplasmic reticulum Ca²⁺ ATPase inhibitor, the GCE-mediated [Ca²⁺]_i signals were abolished. Whereas desensitization of PAR-1, PAR-3, and PAR-4 had no effect on GCE-mediated Ca²⁺ mobilization, no GCE-mediated [Ca²⁺]_i increase was observed after desensitization of PAR-2.

Conclusions

These results indicate that GCE has a direct effect on human airway epithelial cells, in particular generating [Ca²⁺]_i oscillations through Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores through activation of PAR-2.

Keywords:  German cockroach, Ca²⁺ signaling, protease-activated receptor, allergen, human airway epithelial cell

Abbreviations:  [Ca²⁺]_i, Intracellular Ca²⁺ concentration, ER, Endoplasmic reticulum, GCE, German cockroach extract, GPCR, G protein–coupled receptors, IP₃, 1,4,5-Tris-inositolphosphate, PAR, Protease-activated receptor, PAR-2AP, PAR-2 agonist peptide, PMCA, Plasma membrane Ca²⁺ pump, PSS, Physiologic salt solution