Hair cells are the primary sensory receptors of the cochlea and vestibular systems. If hair cells are damaged or lost the surrounding support cells are triggered to divide to produce new hair cells. In some cases support cells are also thought to undergo phenotypic conversion to hair cells. Thus support cells are the progenitors of any newly replaced hair cells. Little is known about the physiology of vestibular support cells. We hypothesised that ATP might provide a trophic or mitogenic signal for support cells in damaged hair cell epithelia. Embryonic day 20 chicks were killed humanely, the utricles dissected out and treated with thermolysin to enable removal and culture of the epithelia sheet (Warchol, 2002). Cultures were loaded with fura-2 AM, and imaged during puff application of extracellular nucleotides at various concentrations. The UTP and ATP activated significant increases in the measured fura-2 ratio, proportional to increases in intracellular calcium ([Ca²⁺]_i). Dose-response curves showed that the rank order for agonist potency was UTP > ATP >> UDP ~ ADP. The EC₅₀ values for UTP and ATP were 0.5 ± 0.3 and 13.1 ± 2.5 µM, respectively (means ± S.D., n ▓ge│ 3 for each concentration). P2 receptors are classified into P2X and P2Y, the former being composed of ligand-gated ion channels and the latter being G-protein-coupled receptors (Ralevic & Burnstock, 1998). The increase in [Ca²⁺]_i we observe could therefore result from entry of calcium from the external medium via P2X receptors or release from intracellular stores activated via P2Y receptors. Pre-incubation with 1 µM thapsigargin prevented the changes in [Ca²⁺]_i activated by either UTP or ATP, suggesting P2Y receptor involvement. When both ATP and UTP were applied simultaneously at concentrations close to their EC₅₀ values, the increase in [Ca²⁺]_i was not significantly different from the increase observed with applications of either nucleotide alone. The latter result suggests that the action of both UTP and ATP is via the same receptor(s). Application of 10 µM pyridoxal-phosphate-6-azophenyl-2Ô,4Ô-disolfonate (PPADS) significantly reduced the [Ca²⁺]_i rise to 25 % of control values evoked by UTP (P < 0.05, n = 3, Student’s paired t test).

These results confirm the presence of P2Y receptors in support cells in chick utricular cultures. Given the profile we observe, we suggest that support cells express P2Y₄ and/or P2Y₆ receptors. The data also suggest a physiological role for signalling by extracellular nucleotides in the vestibular system.

This work was supported by The Wellcome Trust, The Royal Society and a Physiological Society vacation studentship to E.O.A.