The role of protease-activated receptor 2 in regulating ion transport in human middle ear epithelia

Abstract

Protease-activated receptor 2 (PAR2) has diverse functions in airway epithelia. We investigated the role of PAR2 in regulating epithelial surface liquid volume in human middle ear epithelia. The presence of PAR2 in human middle ear epithelial cells (HMEEC) was confirmed by reverse transcription- polymerase chain reaction (RT-PCR) and PAR2 expression was observed more prominently at the basal layer of inflamed middle ear mucosa by immunostaining. Hydrolysis of synthetic fluorescent PAR2 increased with the effusion of middle ear in dose-dependent manner and its activity decreased with trypin inhibitor, aprotinin. Furthermore known endogenous activator of PAR2, human airway tryptase (HAT) was identified in middle ear effusion. PAR2 was highly related with middle ear inflammation. Epithelial surface liquid volume was evaluated with confocal microscopy using Z-stack analysis. The height of surface liquid layer was 35.5 ± 1.6 μm with the treatment of 10 μM PAR2-AP (activating peptide) on apical side and 36.3 ± 4.6 μm with basolateral treatment of PAR2-AP. The increased surface liquid layer was smooth and evenly elevated compared with control (30.3 ± 2.4 μm). We also checked whether PAR2-AP could induce intracellular Ca2+ increasing and transepithelial current using fluorescent dye and vibrating probe. PAR2-AP induced intracellular Ca2+ increasing response similar to the response with ATP. And short circuit current was noted in the middle ear mucosa with the treatment of PAR2-AP. The increase of current density was prominent in the pretreatment of amirolide though, however it was blocked with 4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS). Therefore the activation of PAR2 can induce fluid secretion in the middle ear by increasing intracellular Ca2+ and PAR2 may be related with middle ear epithelial ion channel. These results imply that serine

protease such as HAT in the middle ear effusion has a role in regulating epithelial surface liquid volume by controlling chloride secretion via PAR2.