Srdjan M. Vlajkovic

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.

Mutation of the ATP-gated P2X2 receptor leads to progressive hearing loss and increased susceptibility to noise

Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X2 receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X2 receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X2 receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2-null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X2 function is required for life-long normal hearing and for protection from exposure to noise.

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.

ATP-gated ion channels mediate adaptation to elevated sound levels

The sense of hearing is remarkable for its auditory dynamic range, which spans more than 1012 in acoustic intensity. The mechanisms that enable the cochlea to transduce high sound levels without damage are of key interest, particularly with regard to the broad impact of industrial, military, and recreational auditory overstimulation on hearing disability. We show that ATP-gated ion channels assembled from P2X2 receptor subunits in the cochlea are necessary for the development of temporary threshold shift (TTS), evident in auditory brainstem response recordings as sound levels rise. In mice null for the P2RX2 gene (encoding the P2X2 receptor subunit), sustained 85-dB noise failed to elicit the TTS that wild-type (WT) mice developed. ATP released from the tissues of the cochlear partition with elevation of sound levels likely activates the broadly distributed P2X2 receptors on epithelial cells lining the endolymphatic compartment. This purinergic signaling is supported by significantly greater noise-induced suppression of distortion product otoacoustic emissions derived from outer hair cell transduction and decreased suprathreshold auditory brainstem response input/output gain in WT mice compared with P2RX2-null mice. At higher sound levels (≥95 dB), additional processes dominated TTS, and P2RX2-null mice were more vulnerable than WT mice to permanent hearing loss due to hair cell synapse disruption. P2RX2-null mice lacked ATP-gated conductance across the cochlear partition, including loss of ATP-gated inward current in hair cells. These data indicate that a significant component of TTS represents P2X2 receptor-dependent purinergic hearing adaptation that underpins the upper physiological range of hearing.

NOISE EXPOSURE INDUCES UP-REGULATION OF ECTO-NUCLEOSIDE TRIPHOSPHATE DIPHOSPHOHYDROLASES 1 AND 2 IN RAT COCHLEA

Extracellular ATP acting via P2 receptors in the inner ear initiates a variety of signaling pathways that may be involved in noise-induced cochlear injury. Nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39 and NTPDase2/CD39L1 are key elements for regulation of extracellular nucleotide concentrations and P2 receptor signaling in the cochlea. This study characterized the effect of noise exposure on regulation of NTPDase1 and NTPDase2 expression in the cochlea using a combination of real-time RT-PCR, immunohistochemistry and functional studies. Adult Wistar rats were exposed to broad band noise at 90 dB and 110 dB sound pressure level (SPL) for 72 h. Exposure to 90 dB SPL induced a small and temporary change of auditory thresholds (temporary threshold shift), while exposure to 110 dB SPL induced a robust and permanent change of auditory thresholds (permanent threshold shift). NTPDase1 and NTPDase2 mRNA transcripts were upregulated in the cochlea exposed to 110 dB SPL, while mild noise (90 dB SPL) altered only NTPDase1 mRNA expression levels. Changes in NTPDases expression did not correlate with levels of circulating corticosterone, implying that the up-regulation of NTPDases expression was not stress-related. Semi-quantitative immunohistochemistry in the cochlea exposed to 110 dB SPL localized the increased NTPDase1 and NTPDase2 immunostaining in the stria vascularis and up-regulation of NTPDase2 in the intraganglionic spiral bundle. In contrast, NTPDase1 was down-regulated in the cell bodies of the spiral ganglion neurones. Distribution of NTPDases was not altered in the cochlea exposed to 90 dB SPL. Functional studies revealed increased ectonucleotidase activities in the cochlea after exposure to 110 dB SPL, consistent with up-regulation of NTPDases. The changes in NTPDases expression may reflect adaptive response of cochlear tissues to limit ATP signaling during noise exposure.

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.

Distribution of ectonucleoside triphosphate diphosphohydrolases 1 and 2 in rat cochlea

Extracellular ATP and other extracellular nucleotides acting via P2 receptors in the inner ear initiate a wide variety of signalling pathways important for regulation of hearing and balance. Ectonucleotidases are extracellular nucleotide-metabolising enzymes that modulate purinergic signalling in most tissues. Major ectonucleotidases in the cochlea are likely members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family. In this study, we provide a detailed description of NTPDase1 and NTPDase2 distribution in cochlear tissues using immunocytochemistry. E-NTPDase immunoreactivity was not equally distributed in the tissues bordering scala media. It was observed in the organ of Corti, including sensory and supporting cells, but was notably absent from Reissners membrane and most of the marginal cells of the stria vascularis. NTPDase1 expression was most prominent in the cochlear vasculature and cell bodies of the spiral ganglion neurones, whereas considerable NTPDase2 immunoreactivity was detected in the stria vascularis. Both E-NTPDases were expressed in the cuticular plates of the sensory hair cells and nerve fibres projecting from the synaptic area underneath the inner and outer hair cells. E-NTPDase localisation corresponds to the reported distribution of some P2X receptor subunits (P2X(2) in particular) in sensory, supporting and neural cells and also P2Y receptor distribution in the vasculature and secretory tissues of the lateral wall. The role for E-NTPDases in purinergic signalling is most likely to regulate extracellular nucleoside triphosphate and diphosphate levels and thus provide termination for extracellular ATP signalling that has been linked to control of cochlear blood flow, electrochemical regulation of sound transduction and to neurotransmission in the cochlea.

Differential distribution of adenosine receptors in rat cochlea.

Adenosine is a constitutive cell metabolite that can be released from cells via specific bi-directional transporters and is an end-point for nucleotide hydrolysis. In the extracellular space, adenosine becomes a signalling molecule for P1 (adenosine) receptors that modulate physiological responses in a wide range of mammalian tissues. Whereas adenosine signalling has been implicated in the regulation of cochlear blood flow and in cochlear protection from oxidative damage, the potential roles for adenosine signalling in the modulation of sound transduction and auditory neurotransmission have not been established. We have characterised the expression and distribution of adenosine receptors in the rat cochlea. mRNA transcripts for all four subtypes of adenosine receptors (A1, A2A, A2B and A3) were detected in dissected cochlear tissue by using reverse transcription/polymerase chain reaction analysis. The protein distribution for the A1, A2A and A3 receptor subtypes was identified by immunoperoxidase histochemistry and confocal immunofluorescence labelling. These receptors were differentially expressed in the organ of Corti, spiral ganglion neurones, lateral wall tissues and cochlear blood vessels. The distribution of adenosine receptors in sensory and neural tissues and in the vasculature coincided with other elements of purinergic signalling (P2X and P2Y receptors, ectonucleotidases), consistent with the integrative regulation of many physiological processes in the cochlea by extracellular nucleotides and nucleosides. Our study provides a framework for further investigation of adenosine signalling in the inner ear, including putative roles in oxidative stress responses.

NTPDase1 and NTPDase2 immunolocalization in mouse cochlea: implications for regulation of p2 receptor signaling.

Cellular, molecular, and physiological studies have demonstrated an important signaling role for ATP and related nucleotides acting via P2 receptors in the cochlea of the inner ear. Signal modulation is facilitated by ectonucleotidases, a heterologous family of surface-located enzymes involved in extracellular nucleotide hydrolysis. Our previous studies have implicated CD39/NTPDase1 and CD39L1/NTPDase2, members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, as major ATP-hydrolyzing enzymes in the tissues lining the cochlear endolymphatic and perilymphatic compartments. NTPDase1 hydrolyzes both nucleoside triphosphates and diphosphates. In contrast, NTPDase2 is a preferential nucleoside triphosphatase. This study characterizes expression of these E-NTPDases in the mouse cochlea by immunohistochemistry. NTPDase1 can be immunolocalized to the cochlear vasculature and neural tissues (primary auditory neurons in the spiral ganglion). In contrast, NTPDase2 immunolabeling was principally localized to synaptic regions of the sensory inner and outer hair cells, stereocilia and cuticular plates of the outer hair cells, supporting cells of the organ of Corti (Deiters’ cells and inner border cells), efferent nerve fibers located in the intraganglionic spiral bundle, and in the outer sulcus and root region of the spiral ligament. This differential expression of NTPDase1 and 2 in the cochlea suggests spatial regulation of P2 receptor signaling, potentially involving different nucleotide species and hydrolysis kinetics.

Post exposure administration of A1 adenosine receptor agonists attenuates noise-induced hearing loss

Adenosine is a constitutive cell metabolite with a putative role in protection and regeneration in many tissues. This study was undertaken to determine if adenosine signalling pathways are involved in protection against noise injury. A(1) adenosine receptor expression levels were altered in the cochlea exposed to loud sound, suggesting their involvement in the development of noise injury. Adenosine and selective adenosine receptor agonists (CCPA, CGS-21680 and Cl-IB-MECA) were applied to the round window membrane of the cochlea 6h after noise exposure. Auditory brainstem responses measured 48h after drug administration demonstrated partial recovery of hearing thresholds (up to 20dB) in the cochleae treated with adenosine (non-selective adenosine receptor agonist) or CCPA (selective A(1) adenosine receptor agonist). In contrast, the selective A(2A) adenosine receptor agonist CGS-21680 and A(3) adenosine receptor agonist Cl-IB-MECA did not protect the cochlea from hearing loss. Sound-evoked cochlear potentials in control rats exposed to ambient noise were minimally altered by local administration of the adenosine receptor agonists used in the noise study. Free radical generation in the cochlea exposed to noise was reduced by administration of adenosine and CCPA. This study pinpoints A(1) adenosine receptors as attractive targets for pharmacological interventions to reduce noise-induced cochlear injury after exposure.