Autoradiographic labelling of P 2 purinoceptors in the guinea-pig cochlea

Abstract

Two different radioligands were used to identify extracellular ATP binding sites specific to P 2 purinoceptors in guinea-pig cochlear tissue. Deoxyadenosine 5′-(α-[ 35S]thio)triphosphate ([ 35S]dATPαS; 10 nM) provided a high activity probe for the P 2y purinoceptor subtype on the basis of selective block by 2-methylthio-ATP (2MeSATP; 100 μM). [ 3H]α,β-methylene-ATP (10 nM), a high affinity probe for a P 2x purinoceptor subtype was selectively blocked by inclusion of the related compound β,γ-methylene-ATP (100 μM). Both probes labelled the organ of Corti, stria vascularis and spiral prominence regions. The P 2x purinoceptor probe also bound to lateral wall tissue below the spiral prominence and insertion point of the basilar membrane within the scala tympani compartment, a region which failed to show significant binding using [ 35S]dATPαS. Frozen sections of whole cochlea permitted analysis of radioligand binding to the cell body region (spiral ganglion in Rosenthal's canal) of the primary auditory afferents and the auditory nerve itself, which lies within the central region of the modiolus of the cochlea. Both these regions exhibited 2MeSATP blockable [ 35S]dATPαS binding whereas specific [ 3H]α,β-methylene-ATP binding was absent from spiral ganglion and minimal in the auditory nerve region. These results demonstrate a mixed P 2 purinoceptor distribution in cochlear tissues and suggest that complex purine-mediated neurohumoral mechanisms may influence cochlear function at a number of sites.