Regulation of epithelial sodium channel by prostasin and its inhibitor in human middle ear epithelia

Abstract

The aim of this study was to determine the role of prostasin and HAI-1 in otitis media with effusion (OME). Normal human middle ear epithelium (NHMEE) is essentially an extension of the respiratory tract covered with airway surface liquid (ASL). The epithelial sodium channel (ENaC) plays a key role in the regulation of ASL, and its activity is regulated by serine protease (SP) and serine protease inhibitor (SPI). Among the SPs, prostasin is a well known ENaC activator and is inhibited by hepatocyte growth factor activator inhibitor-1 (HAI-1). I studied this mechanism via a multidirectional approach of molecular biology, physiology and proteomics.Prostasin and HAI-1 were identified in NHMEE by reverse transcription polymerase chain reaction (RT-PCR). The effect of SP and SPI on the ENaC-dependent current was measured via the Ussing chamber. Trypsin, a serine protease similar to prostasin, increased the ENaC-dependent current. Aprotinin, a kunitz type inhibitor similar to HAI-1, gradually decreased the ENaC-dependent current. In 3-dementional ASL volume measurement, ASL volume was found to be markedly increased under aprotinin treatment compared with control. In western blot for middle ear effusion, prostasin and HAI-1 were detected. 2-dimensional gel electrophoresis (2-DE) was performed with middle ear effusion, and several spots were selected according to the molecular weight and pI as candidates of prostasin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI- TOF MS) was performed for the candidates, and SP and SPI were detected. After IL-1ß treatment, prostasin was upregulated, and HAI-1 was not changed in real-time PCR.Therefore, we propose the idea that the regulation of ENaC by prostasin and HAI-1 is as a pathway to compensate for the decreased function of ENaC after the acute stage of infection.