Purinergic signalling and intercellular Ca²⁺ wave propagation in the organ of Corti

Abstract

Extracellular ATP is a key neuromodulator of visual and auditory sensory epithelia. In the rat cochlea, pharmacological dissection indicates that ATP, acting through a highly sensitive purinergic/IP₃-mediated signaling pathway with (little or) no involvement of ryanodine receptors, is the principal paracrine mediator implicated in the propagation of calcium waves through supporting and epithelial cells. Measurement of sensitivity to UTP and other purinergic agonists implicate P2Y₂ and P2Y₄ as the main P2Y receptor isoforms involved in these responses. Ca²⁺ waves, elicited under highly reproducible conditions by carefully controlling dose (1 μM) and timing of focal agonist application (0.2 s), extended over radial distance greater than 160 μm from the source, identical to those activated by damaging single outer hair cells. Altogether, these results indicate that intercellular calcium waves are a robust phenomenon that confers a significant ability for cell–cell communication in the mammalian cochlea. Further ongoing research will reveal the roles that such Ca²⁺ waves play in the inner ear.

Introduction

Extracellular signalling by purine nucleotides has long been associated with sensory systems, where ATP acts as a co-transmitter and/or neuromodulator [1]. Purinergic receptors of the G protein-coupled (P2YR) type, as well as of the ionotropic type (P2XR) [2] are widely distributed on inner ear structures [3], [4].

Currently, seven subtypes of the P2X family (P2X_1–7) and eight subtypes of the P2Y family (P2Y_1–2, P2Y₄, P2Y₆ and P2Y_11–14) have been cloned and functionally characterised, albeit typically in expression systems rather than native tissue [5]. As non-selective cation channels, P2XRs provide direct routes for Ca²⁺ entry into the cell cytoplasm, whereas P2YRs either activate phosholipase C (PLC) and release intracellular Ca²⁺ or affect adenylyl cyclase and alter cAMP levels [6].

In the mammalian cochlea, the organ of Corti comprises a polarized epithelium that contains sensory receptors, the hair cells, in which ATP has been shown to affect Ca²⁺ homeostasis [7]. Sensory hair cells are surrounded by various types of supporting cells, including Deiters’ [8], [9], [10], Hensen's [11] and pillar cells [12], which all respond to focal application of ATP with a transient elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Cochlear supporting cells and adjacent epithelial (Böttcher's and Claudius’) cells are considered essential constituents of an epithelial network that participates in the regulation of the unique ionic composition of cochlear extracellular fluids [13]. Hensen's and Deiters’ cells, the two main types of supporting cells in the organ of Corti [14], [15], are highly permeable to K⁺ [16], [17] which is found at unusually high concentration in endolymph, the fluid bathing the apical surface of the organ of Corti [18]. K⁺ ions flow into the organ of Corti as they are the major component of the hair cell mechanoelectrical transduction current [19], [20], [21], [22]. Purinergic signalling in the cochlea can affect several essential functions, from ion homeostasis to active mechanical amplification by the outer hair cells [23], [24]. Notably, the level of ATP in cochlear fluids increases in response to modest sound over-stimulation [25]. ATP release from marginal cells in the stria vascularis has been proposed to exert a homeostatic regulatory mechanism [26], promoting K⁺ efflux from scala media and a G protein-mediated reduction of K⁺ import into the endolymph from stria vascularis [27]. An expected outcome of both of these effects is a reduction in cochlear sensitivity in noisy environments.

Mechanical stimulation of many cell types induces transient rises in [Ca²⁺]_i that can spread from cell to cell in a wave-like pattern by the release of an extracellular nucleotide intermediate [28]. Indeed, nucleotides are frequently released into the extracellular space as a consequence of cell damage and, once in the pericellular milieu, they can activate P2 nucleotide receptors or be rapidly hydrolyzed by ecto-ATPases and ectonucleotidases [29]. Using neonatal organotypic cultures from rat inner ear [30], [31], [32], we have recently shown, in response to sensory hair cell damage, activation of Ca²⁺ oscillations and ATP-dependent propagation of Ca²⁺ waves through cochlear supporting and epithelial cells that surround the damaged hair cells. ATP concentrations above about 15 nM, similar to that measured in noise damaged cochlea, reliably elicit Ca²⁺ oscillations in the same organ preparation [33].

Extracellular ATP possesses all the properties of a bona fide fast-acting intercellular messenger: (a) it is released in a controlled fashion, (b) ligates specific plasma membrane receptors coupled to signal transduction and (c) is quickly degraded to terminate its actions [29]. However, an alternative concept has been proposed for the mechanism of the Ca²⁺ wave propagation between glial [34] and epithelial cells [35] that, like cochlear supporting cells [36], [37], [38], are highly coupled by gap junctions. In this paradigm, Ca²⁺ waves are sustained by the intercellular diffusion of second messengers through gap junctions, with subsequent release of Ca²⁺ from intracellular stores [39]. As Ca²⁺ may permeate gap junctions [40] but is heavily buffered in the cytosol [41], IP₃ has been proposed as a better intercellular messenger in epithelial cells [35], [42], hepatocytes [43] and glial cells [44], [45], [46], [47], [48]. In the retina, both mechanism seem active: Ca²⁺ wave propagation among astrocytes depends on gap junctional communication while propagation between astrocytes and Müller cells involves purinergic signalling [49].

The contribution of gap junction communication to the propagation of cochlear Ca²⁺ waves is not explored in this paper and will be dealt with in a separate publication. In this work, we have analyzed the responses to different types of stimuli capable of eliciting Ca²⁺ waves in the organ of Corti. Experiments were performed in low divalent cation solutions, thus direct contribution from Ca²⁺ entry through ionotropic P2X receptors can be excluded. Our results implicate P2Y₂ and P2Y₄ receptors acting via the PLC-IP₃ signal transduction pathway with little or no contribution from ryanodine-sensitive Ca²⁺ stores.