Abstract
BackgroundAdenosine triphosphate (ATP) plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats.MethodsSprague-Dawley rats aged 1–3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change.ResultsEvaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K+ and intra- and extracellular Ca2+. Furthermore, changes in the concentration of intracellular Ca2+ induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells.ConclusionWe confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K+ channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A2.
Highlights
Adenosine triphosphate (ATP) is a key signaling molecule in the cochlea, where it regulates sound transduction, hearing sensitivity, the active mechanical amplification by outer hair cells (OHCs), cochlear potential, cochlear homeostasis, and vascular tension [1,2,3]
Previous study confirmed the epithelial origin of cultured marginal cells in the stria vascularis by the expression of cytokeratin and vimentin [19]
We examined the purity of the cultured marginal cells using cytokeratin and vimentin antibodies
Summary
Adenosine triphosphate (ATP) is a key signaling molecule in the cochlea, where it regulates sound transduction, hearing sensitivity, the active mechanical amplification by outer hair cells (OHCs), cochlear potential, cochlear homeostasis, and vascular tension [1,2,3]. P2Y receptors, which are G-protein coupled receptors and elicit their effects through phospholipase C (PLC) to either release intracellular Ca2+ or activate adenylate cyclase, are present in hair cells and marginal cells of the stria vascularis [5,6,7]. This makes ATP an important candidate neurotransmitter for afferent nerves in the cochlea. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats
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