Middle ear cell line that maintains vectorial electrolyte transport.

The middle ear epithelium plays a major role in keeping the temporal bone cavities fluid-free and air-filled, which is a mandatory condition to allow optimum transmission of the sound vibrations from the tympanic membrane to the inner ear. Previous works have recently established the absorptive function of the middle ear epithelium, using primary cultures derived from Mongolian gerbil (Meriones unguiculatus). Because of the paucity of cells as obtained by enzymatic digestion, we developed a middle ear cell line (MESV) using wild-type SV40 infection of primary culture of Mongolian gerbil's middle ear epithelial cells. Transformation was attested by nuclear expression of SV40 large T antigen, prolonged in vitro passages (presently beyond 50 passages), and tumor-inducing ability when subcutaneously injected in athymic mice. Transport properties were evaluated after the fifteenth passage. MESV cells retained most cardinal properties of the original middle ear epithelial cells: cell polarization was evidenced by the presence of mature junctional complexes that separate the cell membrane in two distinct domains, with apical microvilli at the luminal side, and by vectorial sodium transport responsible for the transepithelial lumen-negative potential difference (-9.3 +/- 0.14 mV in culture conditions (n = 9), -2.1 +/- 0.25 mV after overnight growth factors and serum deprivation). Short-circuit current was, like in primary cultures, mainly related to a sodium transport occurring through amiloride-sensitive apical sodium channels, since apical addition of amiloride (10(-5) M) reduced ISC from 7.0 +/- 1.4 to 0.6 +/- 0.1 microA/cm2 (P < 0.01, n = 6). Cellular cAMP content was increased by isoproterenol and prostaglandin E2 from 40.5 +/- 5.6 to 258.5 +/- 17.3 and 55.6 +/- 6.2 pmol/mg protein per 5 min, respectively (P < 0.05, n = 10). Isoproterenol and prostaglandin E2 increased ISC with very similar maximal effects: isoproterenol (10(-4) M) increased ISC from 5.73 +/- 0.31 to 12.77 +/- 0.39 microA/cm2, while prostaglandin E2 increased ISC from 5.47 +/- 0.21 to 12.87 +/- 0.42 (n = 3). Since amiloride (10(-5) M) abolished this stimulation, this may be related to an increase of the electrogenic sodium transepithelial transport. The MESV cell line could provide an interesting tool as a model of middle ear epithelial cells for the study of pathophysiological modulations of ion transport.