ATP release mechanism from the supporting cells in the Kölliker organ in vitro in the cochlea of newborn rat

Abstract

The specific mechanism underlying in the Adenosine triphosphate (ATP) release from the Kölliker's organ is still unknown. The present study was designed to investigate whether the supporting cells in the Kölliker organ in vitro release ATP and to explore the mechanism of ATP releasing from these cells. Supporting cells in the Kölliker organ from P1 rats were isolated, purified and cultured with a combinatorial approach of enzymatic digestion and mechanical separation. Quinacrine staining was used to observe the cochlear membranous labyrinth and supporting cells. the bioluminescence assay was chosen to explore the release ATP from supporting cells in the Kölliker organ, when the ATP metabolism of the cells was influenced, the intracellular or extracellular Ca(2)+ concentration changed, the hemichannels blocked, and the phospholipase signaling pathways inhibited. There were intensely numerous star-like green spots of quinacrine staining in the cytoplasm of supporting cells. There was a strong log-linear relationship in the ATP standard curve generated by the bioluminescence assay. With increasing concentrations of bafilomycin A1, the ATP concentration in the culture medium of the supporting cells in the Kölliker organ decreased, while with adipic acid didecyl, it increased. In a certain concentration range, with increasing extracellular Ca(2)+ concentration, the supporting cells in the Kölliker organ releasing ATP decreased, while the intracellular Ca(2)+ concentration increased, the results showed the elevation of the amount of ATP release. Adding chelerythrine chloride or aristolochid acid into the culture medium of the supporting cells in the Kölliker organ could decrease the ATP release significantly via inhibiting the hemichannels. In addition, by reducing intracellular Ca(2)+ concentration, inhibition of intracellular signaling pathways phospholipase also decreased ATP release. This study demonstrated the presence and release of ATP from the supporting cells cultured in vitro. It showed that the changes of the intracellular and extracellular Ca(2)+ concentration could affect on the ATP release from the supporting cells in the Kölliker organ by regulating the hemichannels openings.