German cockroach extract induces [Ca 2+] oscillations from thapsigargin-sensitive Ca 2+ stores via protease activated receptor-2 in human airway epithelial cells

Abstract

Rationale The German cockroach has been reported to act as an allergen, which may be associated with a protease reaction in asthma. However, the molecular identity 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. We investigated the direct effect of GCE on Ca 2+ signaling in human airway epithelial cells and the type of PAR activated by GCE. Methods The Ca 2+–sensitive dye Fura2 was used to determine intracellular Ca 2+ concentration ([Ca 2+] i) by spectrofluorometry. Results GCE induced a baseline type of [Ca 2+] i oscillations in a dose-dependent manner. The oscillations persisted for long periods of time in the absence of Ca 2+ entry across the plasma membrane, suggesting that the observed [Ca 2+] i increases were due to Ca 2+ release from intracellular stores. Accordingly, after depleting endoplasmic recticulum (ER) Ca 2+ with thapsigargin, an ER-Ca 2+ ATPase inhibitor, the GCE-mediated [Ca 2+] I signals were abolished. Whereas desensitization of PAR-1, 3, 4 had no effect on GCE-mediated Ca 2+ mobilization, no GCE-mediated [Ca 2+] 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 [Ca 2+] i oscillations by Ca 2+ release from thapsigargin-sensitive Ca 2+ stores through activation of PAR-2.