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PubMed1995

Quantitative evaluation of myelinated nerve fibres and hair cells in cochleae of humans with age-related high-tone hearing loss.

In this study 9 human temporal bones from 8 individuals were fixed with Karnovsky solution by perilymphatic perfusion within 1-3 h after death and examined using the "block-surface method' (Spoendlin and Brun, 1974; Spoendlin and Schrott, 1987) and the "micro-dissection method' (Johnsson and Hawkins, 1967). The audiogram of 7 individuals showed high-tone hearing loss, typical for sensory-neural presbycusis. The inner (IHC) and outer hair cells (OHC) and the myelinated nerve fibers in the osseous spiral lamina were counted to correlate audiometric curves with hair-cell and nerve-fibre densities. The "block-surface' method allows accurate hair-cell and myelinated nerve-fibre enumeration with maximal preservation of cochlear structures. The most significant change in the cochlea was not the expected loss of hair cells but an evident loss of nerve fibres in the spiral lamina along the entire length of the cochlea. This loss of nerve fibres was found to be age-related. Reductions up to 30-40% in comparison to normal-hearing middle-aged persons were found in cochleae from persons older than 60 years. In 2 cases only 13% of the fibres remained in some regions of the cochlea. The hair-cell counts showed a reduction of approximately 80% of the OHCs, mainly in the apical parts of the cochlea, and only little differences in the number of IHCs as compared with a group of normal-hearing middle-aged persons. We conclude that neither loss of hair cells nor primary degeneration of nerve fibres alone can fully explain the high-tone loss. Probably injuries of hair cells or neuronal elements at the cellular level can cause threshold elevation.

Aged↗

PubMed1995

Nuclear transition of heat shock protein in guinea pig cochlea after hyperthermia.

The main reaction of heat shock protein (hsp) 70 family to heat shock is 2-fold, one is an increased synthesis in the cytoplasm and the other is a transition from the cytoplasm to the nucleus. Although the former has been already reported in the cochlea by several authors, there has been no description as to the latter. The present study was designed to determine whether this nuclear transition of hsp70 family is also present in the cochlea as in the other organs. Albino guinea pigs subjected to hyperthermia treatment (42 degrees C, 10 min) were killed at 0, 6, 18 or 24 h after the cessation of hyperthermia treatment. Immunohistochemical studies in the cochlea of the untreated animals revealed anti-hsp70 family immunoreactivity mainly in the cytoplasm of the various cells in the cochlea, including the interdental cells, the spiral ganglion cells or the outer hair cells. However, immunoreactivity remarkably increased in the nucleus immediately after the cessation of hyperthermia treatment and this increased immunoreactivity disappeared at 6 h or later. It is concluded that the nuclear transition of hsp70 family also takes place under hyperthermal stress in the cells of the cochlea as in other organs.

Animals↗

PubMed2006

Effect of geranylgeranylacetone on gentamycin ototoxicity in rat cochlea culture.

OBJECTIVE: Geranylgeranylacetone (GGA), an anti-ulcer drug, has been reported to induce heat shock proteins (HSPs) in several animal organs. Purpose of this study was to investigate whether GGA has protective effect on gentamycin (GM) ototoxicity and whether it induces HSP70 in rat cochlea. METHODS: We used cochlea explant culture from postnatal 5-day rat. Explants were pre-incubated with GGA, then incubated with GM and GGA. The number of surviving outer hair cells (OHCs) labeled by phalloidin was counted to evaluate the protective effect of GGA. The expression of HSP70 in whole cochlea tissue was investigated by immunoblot analysis. RESULTS: The number of surviving OHCs was significantly high in GGA 10(-5)M group compared to GM only group and GGA 10(-6)M group. In the immunoblot analysis, HSP70 levels in GGA added groups were slightly high compared to simple culture group, but much lower than those in heat shock group. CONCLUSION: It was suggested that GGA-induced HSP70, and had partial protective effects on GM ototoxicity in the cochlea. GGA has possibility to be safe and useful treatment drug for cochlea disorder.

Animals↗

PubMed2004

Noise overstimulation induces immediate early genes in the rat cochlea.

In mammals, exposure to intense noise produces a permanent hearing loss called permanent threshold shift (PTS), whereas a moderate noise produces only a temporary threshold shift (TTS). Little is known about the molecular responses to such high intensity noise exposures. In this study we used gene arrays to examine the early response to acoustic overstimulation in the rat cochlea. We compared cochlear RNA from noise-exposed rats with RNA from unexposed controls. The intense PTS noise induced several immediate early genes encoding both transcription factors (c-FOS, EGR1, NUR77/TR3) and cytokines (PC3/BTG2, LIF and IP10). In contrast, the TTS noise down-regulated the gene for growth hormone. The response of these genes to different noise intensities was examined by quantitative RT-PCR 2.5 h after the 90-min noise exposure. For most genes, the extent of induction correlates with the intensity of the noise exposure. Three proteins (EGR1, NUR77/TR3, and IP10) were detected in many regions of the unexposed cochlea. After exposure to 120 dB noise, these proteins were present at higher levels or showed extended expression in additional regions of the cochlea. LIF was undetectable in the cochlea of unexposed rats, but could be seen in the organ of Corti and spiral ganglion neurons following noise. NUR77/TR3 was a nuclear protein before noise, but following noise translocated to the cytoplasm. These studies provide new insights into the molecular response to noise overstimulation in the mammalian cochlea.

Acoustic Stimulation↗

PubMed2006

Expression of the kidney injury molecule 1 in the rat cochlea and induction by cisplatin.

Cisplatin is a widely used chemotherapeutic agent whose dose-limiting side effects include ototoxicity and nephrotoxicity. Recent evidence indicates that cisplatin induces the expression of a novel protein, kidney injury molecule-1, in the renal proximal tubular epithelium to aid in regeneration. In this study, we determined whether kidney injury molecule-1 is expressed in the cochlea and is induced by cisplatin. Using reverse transcriptase polymerase chain reaction techniques, we have now identified kidney injury molecule-1 in the rat cochlea and in three different mouse transformed hair cell lines. Administration of cisplatin to rats produced hearing loss and induced kidney injury molecule-1 mRNA in the rat cochlea. Pretreatment of rats with lipoic acid, a scavenger of reactive oxygen species, significantly reduced cisplatin-induced hearing loss and kidney injury molecule-1 expression. Cisplatin also increased the expression of cochlear NOX3 mRNA, a member of the superoxide generating NADPH oxidase family of proteins recently identified in the cochlea, inhibition of which decreased kidney injury molecule-1 expression. Polymerase chain reaction performed on different regions of the cochlea indicated the presence of kidney injury molecule-1 mRNA in the lateral wall, organ of Corti and spiral ganglion. This distribution was confirmed by immunocytochemistry. Taken together, these data identify kidney injury molecule-1 as a novel cochlear injury molecule, whose expression is regulated by reactive oxygen species generated via the NADPH oxidase pathway.

Animals↗

PubMed1999

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the cochlea of guinea pigs after intratympanical endotoxin-treatment.

Since NO is believed to be involved in cochlear physiology, presence of the constitutive isoforms of nitric oxide synthase (NOS), and the target enzyme of NO, soluble guanylyl cyclase (sGC) in structures of the mammalian cochlea have been demonstrated. To date, no reports have been published regarding the detection of the inducible isoform (NOS II) in the cochlea. In order to show the capability of iNOS expression in cochlear tissue, a mixture of proinflammatory bacterial lipopolysaccharides (LPS) and tumor necrosis factor alpha (TNF-alpha) was injected into the tympanic cavity of guinea pigs, vs. saline-solution as control. Paraffin sections of LPS/TNF-alpha treated and saline-treated cochleae (6 h) were examined immunohistochemically with specific antibodies to neuronal, endothelial and inducible NOS and to sGC. Initiated expression of iNOS in the cochlea was observed in the wall of blood vessels of the spiral ligament (SL) and the modiolus, in supporting cells of the organ of Corti, in the limbus, in nerve fibers and in a part of the perikarya of the spiral ganglion after LPS/TNFalpha-treatment. iNOS was not detected in saline-treated control tissue. Expression of both constitutive NOS-isoforms (endothelial and neuronal NOS) and of sGC showed no significant differences in both experimental groups. Endothelial eNOS and neuronal bNOS were detected co-localized in ganglion cells, in nerve fibers, in cells of the SL and in supporting cells of the organ of Corti, but not in sensory cells. Strong labeling for bNOS became evident in the endosteum of the cochlea, while in the endothelium of blood vessels and in the epithelium of the limbus only eNOS could be labeled. sGC could be detected in SL, in supporting and sensory cells of the organ of Corti, in nerve fibers, ganglion cells, in the wall of blood vessels and in the limbus-epithelium. While small amounts of NO, generated by bNOS and eNOS, seem to support the cochlear blood flow and auditory function as well as neurotransmission, high amounts of iNOS-generated NO could have dysregulative and neurotoxic effects on the inner ear during bacterial and viral infections of the middle and inner ear.

Animals↗

PubMed2002

Simultaneous HPLC quantification of monoamines and metabolites in the blood-free rat cochlea.

Monoamine quantification in peripheral sensory receptors, such as the cochlea, is of major interest since monoamines could play a role in neurotransmission. A three-step biochemical protocol was developed to analyze monoamine content within the cochlea. Removal of the blood by aortic perfusion was carried out with an anticoagulant solution prior to the dissection of the cochlea from the temporal bone. The cochlear monoamines and some of their metabolites were then quantified, from homogenated cochlear tissue, by a new application of high performance liquid chromatography coupled to electrochemical detection. This method demonstrated enough sensitivity to detect norepinephrine (NE), dopamine (DA), serotonin (5-HT) and some of their metabolites (3,4-dihydroxyphenylacetic acid, DOPAC; homovanillic acid, HVA; and 5-hydroxyindole-3-acetic acid, 5-HIAA). Furthermore, it enabled the demonstration of noise-induced changes in the cochlear concentrations of NE, DA, DOPAC and HVA. In addition, the aortic perfusion allowed removal of the blood-borne 5-HT from the cochlea without inducing systemic alterations or monoamine degradation, as shown by the absence of effects on NE, DA, DOPAC, HVA or 5-HIAA concentrations. The present methodology may constitute a useful strategy to analyze monoamine turnover in the cochlea and other peripheral sensory receptors.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1997

Identification of preferentially expressed cochlear genes by systematic sequencing of a rat cochlea cDNA library.

107 expressed sequence tags (ESTs) from a rat cochlea cDNA library were identified by systematic sequencing coupled to database selection and RT-PCR analysis of novel sequences. This approach led us to select a clone, pCO8, showing no significant homology with any database sequence, that corresponds to a mRNA whose expression is restricted to the cochlea, except for traces detected in brain. Additional clones with novel sequences enriched in the cochlea were also found. ESTs bearing significant homologies with database sequences (63 out of 107) were classified according to the putatively encoded protein. They include tissue-specific genes not previously described in the cochlea as well as known genes from other species. We performed in situ hybridization in cochlear tissues to localize the pCO8 mRNA and that of clone pCO6 which is 100% homologous to the delayed rectifier potassium channel drk1. We found that both mRNAs were exclusively expressed in the cellular body of the primary auditory neurons from the spiral ganglion of the cochlea. These results indicate that this approach is an efficient way to identify novel genes that could be of importance in cochlear function.

Animals↗

PubMed1997

Mechanical responses of the mammalian cochlea.

Recent findings in auditory research have significantly changed our views of the processes involved in hearing. Novel techniques and new approaches to investigate the mammalian cochlea have expanded our knowledge about the mechanical events occurring at physiologically relevant stimulus intensities. Experiments performed in the apical, low-frequency regions demonstrate that although there is a change in the mechanical responses along the cochlea, the fundamental characteristics are similar across the frequency range. The mechanical responses to sound stimulation exhibit tuning properties comparable to those measured intracellularly or from nerve fibres. Non-linearities in the mechanical responses have now clearly been observed at all cochlear locations. The mechanics of the cochlea are vulnerable, and dramatic changes are seen especially when the sensory hair cells are affected, for example, following acoustic overstimulation or exposure to ototoxic compounds such as furosemide. The results suggest that there is a sharply tuned and vulnerable response related to the hair cells, superimposed on a more robust, broadly tuned response. Studies of the micromechanical behaviour down to the cellular level have demonstrated significant differences radially across the hearing organ and have provided new information on the important mechanical interactions with the tectorial membrane. There is now ample evidence of reverse transduction in the auditory periphery, i.e. the cochlea does not only receive and detect mechanical stimuli but can itself produce mechanical motion. Hence, it has been shown that electrical stimulation elicits motion within the cochlea very similar to that evoked by sound. In addition, the presence of acoustically-evoked displacements of the hearing organ have now been demonstrated by several laboratories.

Acoustic Stimulation↗

PubMed2001

Capsaicin stimulation of the cochlea and electric stimulation of the trigeminal ganglion mediate vascular permeability in cochlear and vertebro-basilar arteries: a potential cause of inner ear dysfunction in headache.

Trigeminal neurogenic inflammation is one explanation for the development of vascular migraine. The triggers for this inflammation and pain are not well understood, but are probably vasoactive components acting on the blood vessel wall. Migraine-related inner ear symptoms like phonophobia, tinnitus, fluctuation in hearing perception and increased noise sensitivity provide indirect evidence that cochlear blood vessels are also affected by basilar artery migraine. The purpose of this investigation was to determine if a functional connection exists between the cochlea and the basilar artery. Neuronally mediated permeability changes in the cochlea and basilar artery were measured by colloidal silver and Evans Blue extravasation, following orthodromic and antidromic stimulation of the trigeminal ganglion innervating the cochlea. Capsaicin and electrical stimulation induced both dose- and time-dependent plasma extravasation of colloidal silver and Evans Blue from the basilar artery and anterior inferior cerebellar artery. Both orthodromic and antidromic activation of trigeminal sensory fibers also induced cochlear vascular permeability changes and significant quantitative differences between the treated and control groups in spectrophotometric assays. These results characterize a vasoactive connection between the cochlea and vertebro-basilar system through the trigeminal sensory neurons. We propose that vertigo, tinnitus and hearing deficits associated with basilar migraine could arise by excitation of the trigeminal nerve fibers in the cochlea, resulting in local plasma extravasation. In addition, cochlear "dysfunction" may also trigger basilar and cluster headache by afferent input to the trigeminal system.

Animals↗

PubMed2000

Evidence for active, nonlinear, negative feedback in the vibration response of the apical region of the in-vivo guinea-pig cochlea.

The transverse vibration response of the organ of Corti near the apical end of the guinea-pig cochlea was measured in vivo. For cochleae in good physiological condition, as ascertained with threshold compound action potentials and the endocochlear potential, increasing amounts of attenuation and phase lag were found as the intensity was decreased below 80 dB SPL. These nonlinear phenomena disappeared post mortem. The data suggest that an active, nonlinear damping mechanism exists at low intensities at the apex of the cochlea. The phase nonlinearity, evident at all frequencies except at the best frequency (BF), was limited to a total phase change of 0.25 cycles, implying negative feedback of electromechanical force from the outer hair cells into a compliant organ of Corti. The amplitude nonlinearity was largest above BF, possibly due to interaction with a second vibration mode. The high-frequency flank of the amplitude response curve was shifted to lower frequencies by as much as 0.6 octave (oct) for a 50-dB reduction of sound intensity; the reduction of BF was 0.3 oct, but there was no change of relative bandwidth (Q(10 dB)). Detailed frequency responses measured at 60 dB SPL were consistent with non-dispersive, travelling-wave motion: travel time to the place of BF (400 Hz at 60 dB SPL) was 2.9 ms, Q(10 dB) was 1.0; standing-wave motion occurred above 600 Hz. Based on comparison with neural and mechanical data from the base of the cochlea, amplitudes at the apex appear to be sufficient to yield behavioural thresholds. It is concluded that active negative feedback may be a hallmark of the entire cochlea at low stimulus frequencies and that, in contrast to the base, the apex does not require active amplification.

Acoustic Stimulation↗

PubMed2000

Characteristics of the travelling wave in the low-frequency region of a temporal-bone preparation of the guinea-pig cochlea.

This study provides a detailed quantitative description of the acoustically evoked vibration responses in the low-frequency region of the in vitro guinea-pig cochlea. Responses of the basilar membrane, the reticular lamina and Hensen cells were measured with a laser Doppler vibrometer, without the need for introducing artificial light reflectors. The apex of the cochlea was opened, leaving the helicotrema intact. Two response components were detected: a 'fast' component, which was probably caused by the hole in the cochlea, and a 'slow' component, which shared the features of a classical travelling wave. The velocity response of the 'slow' component exhibited a relatively flat low-frequency slope (15 dB/oct) and a much steeper high-frequency roll-off (third turn: -47 dB/oct; fourth turn: -35 dB/oct). The group delay was dependent on the characteristic frequency. In the fourth turn, the sharpness of the velocity tuning curves (Q(10 dB): 1.0) was similar to those of in vivo mechanical and neural recordings, whereas in the third turn the tuning (Q(10 dB): 1.1) was much less than for in vivo recordings. The results indicate that cochlear amplification, which is responsible for the high sensitivity and sharp tuning in the basal part of the cochlea, is much less pronounced in the apical turn of the cochlea.

Animals↗

PubMed2001

Na,K-ATPase expression in the mouse cochlea is not dependent on the mineralocorticoid receptor.

This study was performed in order to test the hypothesis that the mineralocorticoid hormone stimulates the expression of Na,K-ATPase in the cochlea of the mouse. Immunohistochemistry was used to investigate the distribution of the mineralocorticoid receptor (MR) in the cochlea of the C57Bl/J6 mouse at different ages between gestational day 19 and postnatal day 30, and the occurrence and distribution of Na,K-ATPase in the inner ear of a mouse with a null mutation of the MR. Adult patterns of staining for MR were found as early as on gestational day 19 in the cochlea, with small changes thereafter. MR was detected in the same structures in the cochlea as Na,K-ATPase in earlier studies, where the amount of Na,K-ATPase increased after postnatal day 4. Thus there is latency between the increase of MR and the increase of Na,K-ATPase. In the cochlea of the MR deficient mouse, antibody labelling of Na,K-ATPase showed no significant difference as compared to the control wild type mouse. The hypothesis that mineralocorticoid hormone alone via MR stimulates the formation of Na,K-ATPase in the inner ear could not be confirmed by this study, and other regulating mechanisms must be considered.

Animals↗

PubMed2003

Synaptophysin immunohistochemistry in the human cochlea.

Light microscopy and immunohistochemical analyses of a freshly prepared human cochlea, removed at meningioma skull base surgery, were performed with particular emphasis on synaptophysin (SY) reactivity. Synaptophysin, a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is a useful marker for sites of synaptic transmission of the efferent olivocochlear system in the cochlea. Following fixation and decalcification, cryosections of 30 microm were prepared. To introduce immunostaining, free-floating sections were exposed to monoclonal SY antibody. Positive SY immunostaining was solely restricted to the neural and sensory structures and did not include supporting cells of the organ of Corti. Dense reaction products were noted around the hair cells, especially at the basal portion of the inner and outer hair cells and their neural poles, as well as around the inner spiral bundle, tunnel spiral bundle, outer spiral bundle and upper tunnel crossing fibers. The majority of spiral ganglion cells stained positively. An intermingling network of thin unmyelinated nerve fibers stained densely, especially at the basal portions of the cochlea. The spiral limbus, inner and outer sulcus cells, basilar membrane, myelinated nerve fibers, spiral ligament and the stria vascularis were unstained. Human cochlea obtained during surgery offers excellent conditions for immunohistochemical analysis. In the basal cochlea in the organ of Corti, outer hair cell area, there may be alterations due to noise trauma from the drilling procedure.

Adult↗

PubMed1997

Histopathologic observations of the aging gerbil cochlea.

Age-related histopathologic changes were examined in cochleas from 17 gerbils born and kept in a quiet environment until near the end of their life expectancy. Hearing loss varied greatly as did the loss of outer hair cells (OHC). Inner hair cells (IHC) were seldom missing even in cochleas with severe hearing losses. Flask- and spherical-shaped OHCs were frequently seen in the apical turn. Stereocilia were usually present and orderly on OHCs, but the tallest row of stereocilia on IHCs was often disarrayed and sometimes missing. Alterations in supporting cells were sometimes present in regions of extensive OHC loss. Although pillar cells were seldom missing, the nuclei of outer pillar cells were commonly displaced from their normal basal position. The density of radial fibers appeared similar to that in young gerbils except in the apical turn of one old ear where a marked loss of radial fibers occurred without an attendant loss of IHCs. All of the quiet-aged cochleas showed a characteristic clustering of epithelial cells lining the scala media surface of Reissner's membrane. This structural rearrangement was not accompanied by a significant decrease in the total number of cells forming Reissner's membrane and did not appear to be associated with hearing loss. The findings confirm and extend earlier work showing that several different types of cells are susceptible to histopathologic changes in old ears. The extent of histopathologic changes varied widely as did the degree of hearing loss in animals with a restricted genetic background and maintained under carefully controlled environmental conditions. It was not possible, based on these initial findings, to relate specific structural to specific functional changes in the aging cochlea. Further light and electron microscopic analysis of other regions from these aged cochleas may provide more conclusive data.

Aging↗

PubMed1997

Up-regulation of adenosine receptors in the cochlea by cisplatin.

In a previous study, we have demonstrated the presence of two adenosine receptor (AR) subtypes, namely A1 and A3AR, in the chinchilla cochlea. One or both of these receptors couple to activation of antioxidant enzymes, with resulting decreases in lipid peroxidation. The chemotherapeutic agent, cisplatin, was shown to produce ototoxicity within a few days of administration presumably by generating reactive oxygen species (ROS) and thereby increasing lipid peroxidation. In this study, we focused on whether lipid peroxidation induces hearing loss by assessing the cochlear antioxidant defense system over a shorter time period (24 h) following cisplatin administration. Cisplatin was administered to anesthetized chinchillas by round window membrane application and hearing loss was determined by compound action potential (CAP) and endocochlear potential (EP) 24 and 72 h post-treatment. Elevations in CAP thresholds in response to click and to 2, 4, 8 and 16 kHz tones and decreases in EP were obtained within 24 h of cisplatin treatment. These changes persisted for at least up to 72 h. Measurements of antioxidant enzymes indicate no change in the activities of superoxide dismutase, catalase or glutathione peroxidase, either 24 or 72 h following cisplatin treatment. The levels of malondialdehyde obtained at these time points were equivalent to those obtained from the controls. Furthermore, no difference in cochlear morphology was detectable by scanning electron microscopy at the basal, middle or apical turns of the cochlea within 24 h. By 72 h, however, losses in both inner and outer hair cells were observed in the basal and middle turns of the cochlea. A major finding of this study is that exposure to cisplatin led to a 5-fold up-regulation of [125I]N6-2-[4-amino-3-phenyl]ethyladenosine binding in the cochlea within 24 h, reflecting increases in expression of AR(s) in this tissue. These data indicate a dissociation between cisplatin acute (within 24 h) ototoxicity and lipid peroxidation. Furthermore, up-regulation of AR(s) may represent a rapid compensatory mechanism by the cochlea to counter the toxic effects of increased ROS generated by cisplatin.

Acoustic Stimulation↗

PubMed1997

Cytotoxicity and mitogenicity of adenosine triphosphate in the cochlea.

Evidence is accumulating to indicate that extracellular adenosine 5'-triphosphate (ATP) may function as a neurotransmitter, neuromodulator, cytotoxin and mitogen. Many of the cells in the cochlea have ATP receptors, however, their function is unknown. The purpose of the present study was to test whether ATP may act as a cytotoxin in the cochlea. ATP was applied to acutely isolated outer hair cells (OHCs) and their shape changes monitored. In addition, ATP was applied into the cochlea by perfusion of the perilymph compartment for 2 h and the animals allowed to survive 3-4 weeks post drug application. At this time, sound-evoked cochlear potentials and distortion product otoacoustic emissions (DPOAEs) were monitored and the cochleas evaluated histologically. Results indicate that when applied to isolated OHCs, ATP (3-30 mM) induced a bleb formation in the infracuticular region of the cell that burst within a few minutes. Short OHCs were more sensitive to this effect of ATP than long OHCs. 3-4 weeks after the perilymph perfusion of ATP (60 mM; 2 h) cochlear potentials and DPOAEs were abolished, and histologically, cells in the organ of Corti and the stria vascularis were found to have been destroyed. In addition, there was loss of spiral ganglion cells and proliferating connective tissue filled varying proportions of the scala tympani and vestibuli. Application of sodium gluconate, a control, at the same concentrations had no effect either on the isolated OHCs or when applied in vivo. Results suggest that extracellular ATP or a metabolic product may act as a cytotoxin to some epithelial and neural elements in the cochlea and possibly as a mitogen to mesenchymal cells or fibrocytes.

Adenosine Triphosphate↗

PubMed1998

The pharmacology and kinetics of ecto-nucleotidases in the perilymphatic compartment of the guinea-pig cochlea.

This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.

5'-Nucleotidase↗