Denise Greenwood

Purinergic Regulation of Sound Transduction and Auditory Neurotransmission

In the cochlea, extracellular ATP influences the endocochlear potential, micromechanics, and neurotransmission via P2 receptors. Evidence for this arises from studies demonstrating widespread expression of ATP-gated ion channels (assembled from P2X receptor subunits) and G protein-coupled receptors (P2Y receptors). P2X2 receptor subunits are localized to the luminal membranes of epithelial cells and hair cells lining scala media. These ion channels provide a shunt pathway for K+ ion egress. Thus, when noise exposure elevates ATP levels in this cochlear compartment, the K+ conductance through P2X receptors reduces the endocochlear potential. ATP-mediated K+ efflux from scala media is complemented by a P2Y receptor G protein-coupled pathway that provides coincident reduction of K+ transport into scala media from the stria vascularis when autocrine or paracrine ATP signalling is invoked. This purinergic signalling likely provides a basis for a reactive homoeostatic regulatory mechanism limiting cochlear sensitivity under stressor conditions. Elevation of ATP in the perilymphatic compartment under such conditions is also likely to invoke purinergic receptor-mediated changes in supporting cell micromechanics, mediated by Ca2+ influx and gating of Ca2+ stores. Independent of these humoral actions, ATP can be classified as a putative auditory neurotransmitter based on the localization of P2X receptors at the spiral ganglion neuron-hair cell synapse, and functional verification of ATP-gated currents in spiral ganglion neurons in situ. Expression of P2X receptors by type II spiral ganglion neurons supports a role for ATP as a transmitter encoding the dynamic state of the cochlear amplifier.

Expression of the P2X2 receptor subunit of the ATP-gated ion channel in the retina.

THE site of extracellular ATP signalling in the retina was investigated by examining expression of the P2X2 receptor (P2X2R) subunit which assembles to form ATP-gated ion channels. Indirect in situ RT–PCR in situ hybridization localized the presence of mRNA for the P2X2R subunit within the soma of photoreceptors, inner nuclear layer neurones and the retina ganglion cells. Use of an antiserum specific for the P2X2R subunit confirmed the expression of the protein by these cells and demonstrated a particularly dense immunolabelling within the inner plexiform layer containing the dendritic processes of the retina ganglion cells. The outer segment of the photoreceptors also exhibited P2X2R-like immunoreactivity. The extensive expression of ATP-gated ion channel protein within the retina suggeststhat extra-cellular ATP plays diverse neurohumoral roles in regulation of visual processing and cellular homeostasis.

Evidence for alternative splicing of ecto-ATPase associated with termination of purinergic transmission.

Ectonucleotidases provide the signal termination mechanism for purinergic transmission, including fast excitatory neurotransmission by ATP in the CNS. This study provides evidence for ectonucleotidase expression in the rat cochlea, brain and other tissues. In addition to detection of rat ecto-ATPase and ecto-ATPDase in these tissues, we identify a novel ecto-ATPase splice variant arising from the loss of a putative exon (193 bp) in the C-terminal coding region. This is the first evidence of alternative splicing in the ecto-ATPase gene family. Splicing of the 193-bp putative exon containing a stop codon extends the open reading frame and provides translation of an additional 50 amino acids compared with the isoform isolated earlier from the rat brain (rEATPase(A); GenBank accession #Y11835). The splice variant (rEATPase(B); GenBank accession #AF129103) encodes 545 amino acids with a predicted protein molecular mass of 60 kDa. rEATPase(B) contains a long cytoplasmic tail (62 amino acids) with three potential protein kinase CK2 phosphorylation sites not present in rEATPase(A). Co-expression of two ecto-ATPase isoforms with different regulatory sites suggests that the extracellular ATP signal levels may be differently influenced by intracellular feedback pathways.

P2X2 receptor expression by interstitial cells of Cajal in vas deferens implicated in semen emission

Male reproduction is dependent upon seminal emission mediated by vas deferens contraction. This drives spermatic fluid to the prostatic urethra during ejaculation. We localize interstitial cells of Cajal (ICC), which express P2X2 receptor, subunits of ATP-gated ion channels, to rat, mouse and guinea-pig vas deferens submucosa. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of rat vas deferens resolved two functional splice variant transcripts of the P2X2 receptor subunit. The P2X2 receptor mRNA was localized principally within the lamina propria (submucosal) region of the rat vas deferens using in situ hybridization (ISH) and in situ RT-PCR-ISH. Immunohistochemistry using rat, mouse and guinea-pig vas deferens tissues confirmed expression of P2X2 receptor protein within the lamina propria, particularly within a dense column of small spindle-shaped cells adjacent to the columnar epithelial cells which line the lumen. This immunoreactivity was co-localized with neurone-specific enolase (NSE) and c-Kit protein expression, gene markers for ICC. Mucosal mast cells were distinguished from ICC by toluidine blue staining. Choline acetyltransferase immunoreactivity, a marker for post-ganglionic parasympathetic innervation, occurred on the lateral margin of the lamina propria and extended into the inner longitudinal muscle layer. P2X1 receptor immunolabelling was associated with sympathetic innervation of the smooth muscle in the outer longitudinal and circular muscle layers, but not the inner longitudinal layer. The physiological significance of the vas deferens ICC which express P2X2 receptors remains to be established. Possible roles include regulation of smooth muscle activity or mucosal secretion utilizing local ATP signaling, both of which would affect semen transport.

P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea

Type I and type II spiral ganglion neurons (SGN) innervate the inner and outer hair cells of the cochlea, respectively. This neural system is established by reorganization of promiscuous innervation of the hair cells, immediately before hearing is established. The mechanism for this synaptic reorganization is unresolved but probably includes regulation of trophic support between the hair cells and the neurons. We provide evidence that P2X receptors (ATP-gated ion channels) contribute such a mechanism in the neonatal rat cochlea. Single-cell quantitative RT-PCR identified the differential expression of two P2X receptor subunits, splice variant P2X2-3 and P2X3, in a 1:2 transcript ratio. Downregulation of this P2X2-3/3 receptor coincided with maturation of the SGN innervation of the hair cells. When the P2X2-3 and P2X3 subunits were co-expressed in Xenopus oocytes, the resultant P2X receptor properties corresponded to the SGN phenotype. This included enhanced sensitivity to ATP and extended agonist action. In P4 spiral ganglion explants, activation of the P2X receptor signaling pathway by ATPγS or α,βMeATP inhibited BDNF-induced neurite outgrowth and branching. These findings indicate that P2X receptor signaling provides a mechanism for inhibiting neurotrophin support of SGN neurites when synaptic reorganization is occurring in the cochlea.

Developmental regulation of neuron‐specific P2X3 receptor expression in the rat cochlea

ATP‐gated ion channels assembled from P2X3 receptor (P2X3R) subunits contribute to neurotransmission and neurotrophic signaling, associated with neurite development and synaptogenesis, particularly in peripheral sensory neurons. Here, P2X3R expression was characterized in the rat cochlea from embryonic day 16 (E16) to adult (P49–56), using RT‐PCR and immunohistochemistry. P2X3R mRNA was strongly expressed in the cochlea prior to birth, declined to a minimal level at P14, and was absent in adult tissue. P2X3R protein expression was confined to spiral ganglion neurons (SGN) within Rosenthals canal of the cochlea. At E16, immunolabeling was detected in the SGN neurites, but not the distal neurite projection within the developing sensory epithelium (greater epithelial ridge). From E18, the immunolabeling was observed in the peripheral neurites innervating the inner hair cells but was reduced by P6. However, from P2–8, immunolabeling of the SGN neurites extended to include the outer spiral bundle fiber tract beneath the outer hair cells. This labeling of type II SGN afferent fiber declined after P8. By P14, all synaptic terminal immunolabeling in the organ of Corti was absent, and SGN cell body labeling was minimal. In adult cochlear tissue, P2X3R immunolabeling was not detected. Noise exposure did not induce P2X3R expression in the adult cochlea. These data indicate that ATP‐gated ion channels incorporating P2X3R subunit expression are specifically targeted to the afferent terminals just prior to the onset of hearing, and likely contribute to the neurotrophic signaling which establishes functional auditory neurotransmission. J. Comp. Neurol. 484:133–143, 2005.

Potential Role of Purinergic Signalling in Cochlear Pathology

Adenosine triphosphate (ATP) is a major intercellular signalling molecule that is involved in neurotransmission in the central and autonomic nervous systems, regulation of blood flow, and neuroendocrine function. It is also a key signalling molecule involved in normal cochlear homoeostasis, regulating hearing sensitivity, controlling vascular tone and acting as a candidate neurotransmitter at the hair cell afferent synapses. It has also been established that extracellular ATP mediates some pathological processes such as inflammation, apoptosis and cell proliferation. Evidence for a profound influence of extracellular ATP on normal cochlear function offers the tantalising possibility that extracellular purine nucleotides may play a role in disease processes in the inner ear. This review draws on the current understanding of the pathophysiological role of extracellular ATP in tissues, and the evidence for the functional expression of purinergic signalling elements in the inner ear, to speculate on the potential role of purine nucleotides in cochlear pathology.

P2X2 receptor subunit expression in a subpopulation of cochlear type I spiral ganglion neurones

SPIRAL ganglion neurones in rat cochlea express three different isoforms of the P2X2 receptor subunit which assemble into ATP-gated ion channels. Two of these P2X2R subunit isoforms have previously been detected in other auditory tissues. The third isoform (designated P2X2–3R) has not been described. This isoform lacks 39 bp immediately prior to the stop codon, corresponding to a 13 amino acid deletion of the extreme C-terminus domain. Using direct in situ RT-PCR, expression of P2X2R mRNA was confined to a subpopulation of type I spiral ganglion neurones. This study supports a role for extracellular ATP as a neurotransmitter for a discrete population of auditory neurones where variation in P2X2R isoform assembly may confer functional heterogeneity, including enhanced desensitization.

TRPC-like conductance mediates restoration of intracellular Ca2+ in cochlear outer hair cells in the guinea pig and rat.

Ca2+ signalling is central to cochlear sensory hair cell physiology through its influence on sound transduction, membrane filter properties and neurotransmission. However, the mechanism for establishing Ca2+ homeostasis in these cells remains unresolved. Canonical transient receptor potential (TRPC) Ca2+ entry channels provide an important pathway for maintaining intracellular Ca2+ levels. TRPC3 subunit expression was detected in guinea pig and rat organ of Corti by RT‐PCR, and localized to the sensory and neural poles of the inner and outer hair cells (OHCs) by confocal immunofluorescence imaging. A cation entry current with a TRPC‐like phenotype was identified in guinea pig and rat OHCs by whole‐cell voltage clamp. This slowly activating current was induced by the lowering of cytosolic Ca2+ levels ([Ca2+]i) following a period in nominally Ca2+‐free solution. Activation was dependent upon the [Ca2+]o and was sustained until [Ca2+]i was restored. Ca2+ entry was confirmed by confocal fluorescence imaging, and rapidly recruited secondary charybdotoxin‐ and apamin‐sensitive KCa currents. Dual activation by the G protein‐coupled receptor (GPCR)–phospholipase C–diacylglycerol (DAG) second messenger pathway was confirmed using the analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG). Ion substitution experiments showed that the putative TRPC Ca2+ entry current was selective for Na+ > K+ with a ratio of 1: 0.6. The Ca2+ entry current was inhibited by the TRPC channel blocker 2‐aminoethyl diphenylborate (2APB) and the tyrosine kinase inhibitor, erbstatin analogue. We conclude that TRPC Ca2+ entry channels, most likely incorporating TRPC3 subunits, support cochlear hair cell Ca2+ homeostasis and GPCR signalling.

Allosteric modulation of native cochlear P2X receptors: insights from comparison with recombinant P2X2 receptors.

Extracellular adenosine 5′-triphosphate (ATP)-gated ion channels assembled from P2X receptor subunits exhibit subunit-selective allosteric modulation by protons and divalent cations. In voltage-clamped guinea-pig cochlear outer hair cells (OHC) and Deiters’ cells (DC), H+ and Cu2+, but not Zn2+, enhanced the P2X receptor-mediated inward currents. Acid pH (6.5) potentiated OHC ATP-gated currents by 45%. Co-application of Cu2+ (1–40 µM) with ATP increased the response by 20%. In DCs, ATP-gated currents were potentiated 85% by acid pH, and 70% by Cu2+. Alkaline pH inhibited ATP-gated inward currents by 73% in OHCs and 85% in DCs. Zn2+ was either ineffective (1–10 µM) or inhibitory (40–400 µM). Recombinant rat P2X2 receptor-mediated inward currents in Xenopus oocytes displayed allosteric modulation that was different from the native guinea-pig cochlear P2X receptors. The oocyte ATP-gated inward current was potentiated 450% by shifting from pH 7.5 to pH 6.5, and 130% with 40 µM Cu2+. The enhanced response to ATP with acid pH and Cu2+ is a signature of the P2X2 subunit. In contrast to native guinea-pig cochlear cells, extracellular Zn2+ (40 µM) increased the recombinant ATP-gated inward current by 200% in oocytes. These results suggest that the positive allosteric modulation of cochlear OHC and DC ATP-gated ion channels by protons and Cu2+ arises in part from the P2X2 receptor subunit, with additional regulatory elements.