FLUORESCENCE IMAGING OF EXTRACELLULAR PURINERGIC RECEPTOR-SITES AND PUTATIVE ECTO-ATPASE SITES ON ISOLATED COCHLEAR HAIR-CELLS

Fluorescence imaging of extracellular adenosine-5'-triphosphate (ATP) binding sites on inner and outer hair cells isolated from the guinea p ig organ of Corti was achieved using the fluorescent analog of ATP, or -3')-O-(trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP; 30-75 mu M) . This analog, which fluoresces on binding to these sites, was pressur e applied by micropipette while hair cells were viewed by fluorescence microscopy. Fluorescence imaging revealed a widespread distribution o f extracellular binding sites, including the stereocilia, cuticular pl ate, and the basolateral margins of the cells, but particularly in inf racuticular and infranuclear regions. In support of extracellular bind ing, simultaneous electrophysiological recordings demonstrated that ra pid washout of TNP-ATP-induced fluorescence was dependent upon cell in tegrity. Suramin, a nonselective P-2 purinoceptor antagonist, coapplie d with TNP-ATP, reduced the fluorescence observed on the stereocilia a nd apical surface of the cuticular plate only. This implies that bindi ng sites on the apical surface of hair cells are P-2 receptors, consis tent with previous electrophysiological evidence for localization of P -2 receptors to the apical surface of cochlear hair cells (Housley et al., 1992). Binding of TNP-ATP to P-2 purinoceptors was confirmed by i ts antagonism of the inward current elicited by ATP (10 mu M) in volta ge-clamped hair cells. Fluorescence from the basolateral margin was si gnificantly quenched when TNP-ATP was applied in divalent cation-free solution. Because divalent cations are required for ATPase activity, t his finding provides evidence for the presence of ecto-ATPases on the basolateral membrane of hair cells. The divalent cation-free condition had no significant effect on the ATP-gated P-2 purinoceptor conductan ce. We propose that there are two classes of ATP binding sites on coch lear hair cells: apically located P-2 purinoceptors gating nonselectiv e cation channels and basolaterally located ecto-ATPases that may be i nvolved in purine turnover.