Abstract
We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1–3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca2+-dependent lysosomal exocytosis.
Highlights
The nature of ATP vesicles in the marginal cells was still unclear
ATP release in this setting is mediated by lysosomal-associated membrane protein 1 (LAMP1) and pannexin 1 (Panx1) -dependent lysosomal exocytosis
Given that the lysosome is ubiquitous across cell types, we suppose that lysosomal vesicles and ATP vesicles depicted by White and co-workers[11] in marginal cells of the stria vascular are the same, and that ATP release from the marginal cells is via Ca2+-dependent lysosomal exocytosis
Summary
The nature of ATP vesicles in the marginal cells was still unclear. Zhang and his colleagues[13] reported that lysosomes in the astrocyte contain abundant ATP that can be released in a stimulus-dependent manner. We report that quinacrine selectively labeled lysosomes in marginal cells and confocal imaging of quinacrine- or Mant-ATP[2′ -/3′ -O-(N′ -Methylanthraniloyl) adenosine-5′ -O – triphosphate] -labeled vesicles indicated that these were lysosomes. ATP release was measured in the extracellular fluid of marginal cells after glycyl-L-phenylalanineß-naphthylamide (GPN) treatment. These data offered solid evidence for lysosomal ATP storage in cochlear marginal cells of neonatal rats. Our results may provide new insight into mechanisms underlying intracellular ATP storage and release in marginal cells as well
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