Abstract:
In the inner ear, complex extracellular purinergic signalling pathways regulate cochlear homeostasis, sound transduction and auditory neurotransmission. In other tissues, these pathways are regulated through hydrolysis of extracellular nucleotides by membrane-bound ectonucleotidases (E-NTPDases). Purpose: This study investigated expression and distribution of two intracellular members of the E-NTPDase family, NTPDase5 and NTPDase6, in adult and developing cochlea, and their release into cochlear fluids under quiescent and stressed (noise) conditions. Methods: Inner ear tissues from Wistar rats were excised. Transcript levels of NTPDase5 and 6 were determined using quantitative RT-PCR and the enzyme distribution analysed with immunoperoxidase and confocal immunofluorescence. Cochlear perfusate was incubated in vitro with nucleotide substrates followed by RP-HPLC analyses to assess the presence of soluble enzymes. Results: mRNA expression of both enzymes was confirmed in rat cochlear tissues. Adult cochleae (n=5) showed strong NTPDase5 localisation in supporting Deiters cells and spiral ganglion neurons, while NTPDase6 was confined to the inner hair cells. Noise exposure (n=5) upregulated NTPDase5 mRNA and neuronal protein expression (p<0.05). NTPDase6 showed prominent expression in the developing hair cell bundles of embryonic and early postnatal cochleae (n=4 for each age point). RP-HPLC analysis provided evidence for the release of soluble NTPDase5 and NTPDase6 into perilymph. Conclusions: Spatial and temporal expression of NTPDase5 and NTPDase6 in adult and developing cochlear tissue provides support for their role in regulation of P2Y receptor signalling. Our study also supports a role of NTPDase5 in cochlear response to noise and NTPDase6 in hair cell development.