Regulation of Purinergic signalling in the Cochlea

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.