David J.B. Muñoz

Vesicular storage of adenosine triphosphate in the guinea-pig cochlear lateral wall and concentrations of ATP in the endolymph during sound exposure and hypoxia.

Previous studies have revealed putative vesicular stores of adenosine triphosphate (ATP) in the marginal cells of the cochlear stria vascularis which may serve as a source of ATP for purinergic signalling. This study aimed to provide further evidence of ATP storage in the cochlea and to see whether ATP levels in the endolymph are affected by noise and hypoxia. Tissues from the lateral wall and organ of Corti of the guinea-pig cochlea were fractionated to obtain vesicular (VF) and mitochondrial (MF) fractions. Free and total ATP were then measured by the luciferase-luciferin reaction from which membrane-bound vesicular ATP was calculated. In the lateral wall, the VF contained 2.02+/-0.04 nmol ATP/mg protein (n = 5), significantly greater (p < 0.001; paired Students t-test) than the concentration of ATP in the MF (0.36+/-0.05). In the organ of Corti, the VF contained 0.69+/-0.08 nmol ATP/mg protein (n = 4), significantly smaller than the amount in the VF of the lateral wall tissues (p < 0.001; non-paired Students t-test). Small amounts of fumarase. an enzyme of the mitochondrial matrix, in the VF, excluded the possibility of mitochondrial ATP contamination. To investigate the effect of hypoxia and noise on the ATP concentrations in the endolymph, fluid samples were collected from the first (basal) cochlear turn of anaesthetized guinea-pigs. As a result of hypoxia (15 min, 13% F1O2), ATP concentrations (nM, mean +/- SEM) increased from 6.2+/-2.3 to 9.3+/-4.5 (n = 4), but the difference was not statistically significant. As a result of noise (15 min, 10 kHz, 110 dB SPL. broad band), the ATP levels increased significantly from 7.4+/-1.2 to 16.0+/-1.8 (p = 0.01; Students t-test: n = 4). This study has demonstrated the presence of a vesicular store of ATP in the stria vascularis of the cochlea and described an increase in the ATP levels in the endolymph during noise exposure. The findings suggest that ATP is actively secreted from the vesicular store under conditions of metabolic stress. The presence of ATP under basal conditions supports a role for ATP in the sound transduction process during normal function.Previous studies have revealed putative vesicular stores of adenosine triphosphate (ATP) in the marginal cells of the cochlear stria vascularis which may serve as a source of ATP for purinergic signalling. This study aimed to provide further evidence of ATP storage in the cochlea and to see whether ATP levels in the endolymph are affected by noise and hypoxia. Tissues from the lateral wall and organ of Corti of the guinea-pig cochlea were fractionated to obtain vesicular (VF) and mitochondrial (MF) fractions. Free and total ATP were then measured by the luciferase ± luciferin reaction from which membrane-bound vesicular ATP was calculated. In the lateral wall, the VF contained 2.0290.04 nmol ATP:mg protein (n3⁄45), signi® cantly greater (pB0.001; paired Student’s t-test) than the concentration of ATP in the MF (0.3690.05). In the organ of Corti, the VF contained 0.6990.08 nmol ATP:mg protein (n3⁄44), signi® cantly smaller than the amount in the VF of the lateral wall tissues (pB0.001; non-paired Student’s t-test). Small amounts of fumarase, an enzyme of the mitochondrial matrix, in the VF, excluded the possibility of mitochondrial ATP contamination. To investigate the effect of hypoxia and noise on the ATP concentrations in the endolymph, ̄ uid samples were collected from the ® rst (basal) cochlear turn of anaesthetized guinea-pigs. As a result of hypoxia (15 min, 13% FIO2), ATP concentrations (nM, mean9SEM) increased from 6.292.3 to 9.394.5 (n3⁄44), but the difference was not statistically signi® cant. As a result of noise (15 min, 10 kHz, 110 dB SPL, broad band), the ATP levels increased signi® cantly from 7.491.2 to 16.091.8 (p3⁄40.01; Student’s t-test; n3⁄44). This study has demonstrated the presence of a vesicular store of ATP in the stria vascularis of the cochlea and described an increase in the ATP levels in the endolymph during noise exposure. The ® ndings suggest that ATP is actively secreted from the vesicular store under conditions of metabolic stress. The presence of ATP under basal conditions supports a role for ATP in the sound transduction process during normal function.

Extracellular adenosine 5′-triphosphate (ATP) in the endolymphatic compartment influences cochlear function

There is strong evidence for the presence of P2 purinoceptors on cochlear tissues, but the role of extracellular ATP in cochlear function is still unclear. Our previous studies have determined the presence of ATP in the cochlear fluids and indicated that the purinoceptors are substantially localized to the tissues lining the endolymphatic compartment. This implies that extracellular ATP may have an humoral role confined to the endolymphatic space. In order to study the influence of extracellular ATP in the endolymphatic space, a series of studies were undertaken in which ATP (10 microM to 10 mM) in artificial endolymph (EL) (test solution: 2-12.5 nl) was injected into the scala media and the effect on the cochlear microphonic (CM) and endocochlear potential (EP) evaluated. A double-barrelled pipette, with one barrel containing the test solution and the other artificial EL (control solution) was inserted into scala media of the third turn of the guinea-pig cochlea. A known volume (2-12.5 nl) of test or control solution was then pressure-injected into the space. ATP had a significant dose-dependent suppressive effect on both EP and CM with a threshold of approximately 2 x 10(-14) mol; the response was readily reversible, also in a dose-dependent fashion. Artificial EL of the same volume had no effect on EP and CM. The ATP effect on EP was blocked by the P2 purinoceptor antagonists suramin and reactive blue 2 (RB2). Neither adenosine (2 x 10(-13) to 2 x 10(-11) mol) nor suramin or RB2 on their own had any effect on EP and CM. This study provides the first evidence for an effect of extracellular ATP in the endolymphatic compartment on cochlear function which is mediated via P2 purinoceptors. This provides supporting evidence for an humoral role for extracellular ATP in the modulation of cochlear function.

Purinergic Modulation of Cochlear Partition Resistance and Its Effect on the Endocochlear Potential in the Guinea Pig

Introduction of adenosine 5′-triphosphate (ATP) into the endolymphatic compartment of the guinea-pig cochlea decreases the endocochlear potential (EP). To determine if this is due to an ATP-induced change in compartment resistance, the cochlear partition resistance (CoPR) was measured using constant current injections into scala media before, during, and after microinjection of ATP into the same compartment. The CoPR (mean = 3.13 ± 0.13 kΩ) decreased with ATP in a dose-dependent manner (25.1 ± 3.0% decrease in relation to baseline values) and this was linearly correlated (R2 = 0.91) to the magnitude of the ATP-induced decline in EP (41.6 ± 7.0% decline in relation to the baseline). Pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, a P2X receptor antagonist) injected prior to ATP application blocked this ATP-induced reduction in EP and CoPR. This indicates that ATP-gated ion channels (P2X receptors) provide a latent shunt capable of regulating the majority of the electrical potential across the luminal surface of the sensory hair cells, which is necessary for sound transduction. The results suggest a novel sound transduction regulatory mechanism, which, via extracellular ATP, has the capability of adjusting hearing sensitivity.

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.

Ectonucleotidases and Purinoceptors in the Cochlea and Their Putative Role in Hearing

It is well established that extracellular adenine nucleosides and nucleotides (adenosine, ATP and ADP) can influence cellular function in a range of tissues1. Interacting through specific receptors, termed purinoceptors, these compounds are known to act as neurotransmitters, neuromodulators, and humoral and trophic factors. Purinoceptors are classified into two main classes: P1 purinoceptors (subtypes A1, A2a, A2b and A3) which are preferentially responsive to adenosine and P2 purinoceptors (subtypes P2x and P2y) which are responsive to purine nucleotides (ATP, ADP, AMP)1. Over the past five years considerable evidence has emerged for a complex signalling role for extracellular purines in the inner ear2. Purinoceptors (P2x and P2y) have been identified in neural, sensory and secretory structures of the inner ear. Functional studies have shown a significant effect of extracellular ATP on inner ear function3, 4 and more recent studies have identified putative stores of ATP in secretory tissues. In addition, there is evidence for endogenous release of ATP into the fluids5 and for the presence of ectonucleotidases in cochlear tissues6. Our studies have been directed at identifying the functional role of extracellular ATP in the cochlea, that part of the inner ear which transduces sound into neural activity in the eighth cranial nerve (the auditory nerve). In this paper we summarise our current evidence on the nature and distribution of P2 purinoceptors, the storage and release of ATP and the extracellular hydrolysis of ATP in the cochlea. On the basis of this evidence we speculate on the significance of a neuromodulatory and humoral role for extracellular ATP in cochlear function and hence our sense of hearing.

Evidence for Ectonucleotidases in the Guinea-Pig Cochlea

A number of studies have indicated that adenosine 5′-triphosphate (ATP) acts as a signalling molecule in the cochlea of the inner ear modulating the process of hearing through its interaction with P2 purinoceptors (reviewed by Thorne and Housley1). Determining the terminating mechanism for extracellular ATP in the cochlea thus appears to be highly relevant. The first evidence for ATPase activity in cochlear tissues has been provided by fluorescence imaging of isolated cochlear hair cells2. In that study, binding of the fluorescent ATP analogue 2′-(or -3′)-0-(trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) to the basolateral region of the hair cells was quenched in the absence of divalent cations, Ca2+ and Mg2+, indicative of ecto-ATPase activity. The present work provides biochemical evidence for ectonucleotidase activity in the cochlea of the guinea-pig. Our in vivo study has demonstrated Ca2+ and Mg2+-dependent hydrolysis of exogenous ATP and production of metabolites in perfused cochlear tissues3. Further in vitro experiments have provided evidence for dephosphorylation of ADP and AMP and measures of ecto-APase, ecto-ADPase and ecto-5′-nucleotidase activity in the cochlear perilymphatic compartment.