Signal transduction in smooth muscle - Selected contribution: Tryptase-induced PAR-2-mediated Ca2+ signaling in human airway smooth muscle cells

Tryptase, the major mast cell product, is considered to play an important r ole in airway inflammation and hyperresponsiveness. Tryptase produces diffe rent, sometimes opposite, effects on airway responsiveness (broncho-protect ion and/or airway contraction). This study was designed to examine the effe ct of human lung tryptase and activation of protease-activated receptor (PA R)-2 by synthetic activated peptide (AP) SLIGKV-NH2 on Ca2+ signaling in hu man airway smooth muscle (HASM) cells. Immunocytochemistry revealed that PA R-2 was expressed by HASM cells. Tryptase (7.5-30 mU/ml) induced a concentr ation-dependent transient relative rise in cytoplasmic Ca2+ concentration ( [Ca2+](i)) that reached 207 +/- 32 nM (n = 10) measured by indo 1 spectrofl uorometry. The protease inhibitors leupeptin or benzamidine (100 muM) aboli shed tryptase-induced [Ca2+](i) increase. Activation of PAR-2 by AP (1-100 mM) also induced a concentration-dependent transient rise in [Ca2+](i), whe reas the reverse peptide produced no effect. There was a homologous desensi tization of the [Ca2+](i) response on repeated stimulation with tryptase or AP. U-73122, a specific phospholipase C (PLC) antagonist, xestospongin, an inositol trisphosphate (IP3)-receptor antagonist, or thapsigargin, a sarco plamic Ca2+-ATPase inhibitor, abolished tryptase-induced [Ca2+](i) response , whereas Ca2+ removal, in the additional presence of EGTA, had no effect. Calphostin C, a protein kinase C inhibitor, increased PAR-2 [Ca2+](i) respo nse. Our results indicate that tryptase activates a [Ca2+](i) response, whi ch appears as PAR-2 mediated in HASM cells. Signal transduction implicates the intracellular Ca2+ store via PLC activation and thus via the IP3 pathwa y. This study provides evidence that tryptase, which is increasingly recogn ized as an important mediator in airway inflammation and hyperresponsivenes s, is also a potent direct agonist at the site of airway smooth muscle.