Selective expression of aquaporin 1, 4 and 5 in the rat middle ear
Introduction
Regulation of fluid homeostasis in the middle ear is crucial for maintaining the appropriate humidity in the cavity of the middle ear. The amount of fluid in the middle ear cavity is regulated by elimination through the Eustachian tube as well as the secretion and absorption in the middle ear epithelium. The impairment of these processes results in secretory otitis media (SOM), which exhibits effusion behind the tympanic membrane without acute signs or symptoms. Approximately 80% of children are considered to be affected by SOM to some degree. To study the causal mechanism of the SOM is important, because epidemiological studies have indicated that all types of chronic otitis media originate from SOM (Tos et al., 1984).
Transepithelial Na+ absorption (Herman et al., 1993) and Cl− secretion (Furukawa et al., 1997) in the middle ear have recently been characterized. Fluid absorption occurs as the consequence of an osmotic gradient generated by vectorial Na+ transport, responsible for the transepithelial lumen-negative potential difference; Na+ is actively transported via amiloride-sensitive Na+ channels located at the apical membrane, and extruded by oubain-sensitive Na+, K+-ATPase at the basal membrane. This transport of Na+ in turn drives a passive Cl− absorption through the paracellular pathway, which generates a major transepithelial osmotic gradient (Herman et al., 1998). However, the pathways for epithelial water transport remain to be fully elucidated.
Aquaporins (AQPs) have been discovered initially as water channels in the plasma membranes, which consist of at least 10 subtypes in mammals (Deen and van Os, 1998). Among them, only AQP1–5 have been well characterized; they are different with respect to the permeability to glycerol and urea, sensitivity to mercury diuretics and distribution, suggesting that they have their own distinct functional roles. Little is known, however, about the expression of AQP in the middle ear epithelium. In the present study, we investigated the expression and cellular localization of AQP1–5 subtypes in the middle ear by using reverse transcription-polymerase chain reaction (RT-PCR), immunoblot and immunohistochemical analyses.
Section snippets
RT-PCR
Three male Sprague–Dawley rats (200–300 g body weight) were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/100 g), and exanguinated by transcardial perfusion with physiological saline (37°C). The kidney, submandibular gland and distal lung were removed, and middle ear epithelia were taken off from six bullae oticae under a stereomicroscope. Total cellular RNA of these tissues was isolated by the acid guanidine thiocyanate–phenol–chloroform extraction method using
Results
The expression of AQP1–5 mRNAs in the middle ear was studied by RT-PCR (Fig. 1); the identity of each AQP subtype was confirmed by the parallel study in kidney, submandibular gland and distal lung. The middle ear epithelium expressed AQP1, 4 and 5, but not detectable levels of AQP2 and 3 subtypes. Failure of PCR amplification in the absence of RT (Fig. 1) indicates that the PCR products were derived from the transcribed cDNAs, but not from the genomic DNA in the tissues. When the sequence of
Discussion
It has been assumed that water homeostasis in the cavity of the middle ear is passively regulated by a Na+-dependent osmotic gradient across apical membranes of the epithelial cells, and the osmotic gradient is generated by Na+ influx via the epithelial Na+ channels and Na+ extrusion via Na+, K+-ATPase at the basal membrane of the epithelial cells (Herman et al., 1998). In our present study, however, RT-PCR and immunoblot analysis provided the first direct evidence that AQP1, 4 and 5 subtypes
Acknowledgements
The authors thank Dr. Shinji Takechi for advice on the analysis of DNA sequencing, and Mr. Keizo Masumoto, Ms. Keiko Kawabata and Ms. Masako Yamamoto for technical assistance.
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