ELSEVIER Hearing Research 84 (1995) 177-193 HBIRIrlG RESFARCH Autoradiographic labelling of P2 purinoceptors in the guinea-pig cochlea Bruce G. Mockett a, Xuenong Bo b, Gary D. Housley a,., Peter R. Thorne a, Geoffrey Burnstock b a Department of Physiology, School of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand b Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK Received 20 June 1994; revised 27 December 1994; accepted 5 January 1995 Abstract Two different radioligands were used to identify extracellular ATP binding sites specific to P2 purinoceptors in guinea-pig cochlear tissue. Deoxyadenosine 5'-(a-[35S]thio)triphosphate ([35S]dATPaS; 10 nM) provided a high activity probe for the P2y purinoceptor subtype on the basis of selective block by 2-methylthio-ATP (2MeSATP; 100 /zM). [3H]a,/3-methylene-ATP (10 nM), a high affinity probe for a P2x purinoceptor subtype was selectively blocked by inclusion of the related compound /3,y-methylene-ATP (100 tzM). Both probes labelled the organ of Corti, stria vascularis and spiral prominence regions. The P2x 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]dATPaS. 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]dATPaS binding whereas specific [3H]a,/3-methylene-ATP binding was absent from spiral ganglion and minimal in the auditory nerve region. These results demonstrate a mixed P2 purinoceptor distribution in cochlear tissues and suggest that complex purine-mediated neurohumoral mechanisms may influence cochlear function at a number of sites. Keywords: Extracellular ATP; [3H]ct,/3-methylene-ATP; /3,y-methylene-ATP; [35S]dATPaS; 2-methylthio-ATP; Organ of Corti; Stria vascularis; Spiral ganglion; Hair cells I. Introduction Extracellular adenosine 5'-triphosphate (ATP) at low (/zM) concentrations has been shown to exert physio- logical responses in many different cells and tissues (see Gordon, 1986 for a review). There is now substan- tial evidence that these responses occur through the interaction of extracellular ATP with specific cell re- ceptors. These receptors have been termed P2 purinoceptors (Abbracchio et al., 1993 for a review) to distinguish them from receptors (PI) which are more responsive to the extracellular action of adenosine. The substantial number of reports make it apparent that extracellular ATP has a neurohumoral function in a variety of tissues including neurotransmission in the * Corresponding author. Fax: +64 (9) 3737-499; email: g.hous- ley@auckland.ac.nz. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0378-5955(95)00024-0 autonomic nervous system, particularly in the gastro- intestinal and urinogenital systems and blood vessels, and also in sensory and central neurons where it may act either as the principal neurotransmitter or a co- transmitter (Burnstock, 1978; Burnstock and Kennedy, 1985; Gordon, 1986; Friel, 1988; Dubyak, 1991; Ed- wards et al., 1992; Evans et al., 1992; Barnard, 1993; Burnstock, 1993; Abbracchio and Burnstock, 1994; Barnard et al., 1994). Based on physiological and pharmacological studies of the relative potencies of different analogues of ATP, P2 purinoceptors have been further subdivided into Pzx, P2y, P2z, P2t and P2u groups (O'Connor et al., 1991; Abbracchio et al., 1993). A revised classification of P2 purinoceptors, based on the original description by Burnstock and Kennedy (1985), has been adopted by the IUPHAR Committee on Receptor Nomenclature and Drug Classification (Abbracchio and Burnstock, 1994; Fredholm et al., 1994). This proposal, which we