Biomedical subjects

P R Thorne

Publications and source records attributed to P R Thorne.

At least 19 recordsLinked to original sources

PubMed2004

Ensemble spontaneous activity in the guinea-pig cochlear nerve.

Spectral analysis of electrical noise recorded from the round window (RW) of the cochlea is referred to as the ensemble spontaneous activity (ESA) of the cochlear nerve. The ESA is considered to represent the summed spontaneous activity of single fibers of the auditory nerve and changes in the spectral characteristics of the ESA have been observed in humans with tinnitus. Experiments were undertaken to determine the relationship of the ESA to auditory neurotransmission. The ESA consisted of energy centered at approximately 900 Hz, similar to the spectral peak of single auditory neuron discharges. The amplitude of the ESA was correlated with good auditory sensitivity in the 12-30 kHz region of the cochlea. Constant pure tones of 12-22 kHz suppressed the ESA reducing its amplitude in a frequency and intensity dependent manner implying that the ESA recorded at the RW is generated or dominated by neurons in the basal region of the cochlea. The ESA was significantly suppressed by round window perfusion of the P2X receptor agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) (10 mM) the glutamate receptor antagonist 6-7-dinitroquinoxaline-2,3-dione (DNQX) (1 mM), and the sodium channel antagonist tetrodotoxin (TTX) (20 microM). Following intravenous furosemide injection (40 mg/kg) reduction and recovery of the ESA correlated with similar changes in the endocochlear potential (EP). Following DNQX and ATPgammaS an additional spectral peak at 200 Hz was often observed. This peak has been postulated to be a correlate of tinnitus in humans but had not previously been observed in a guinea-pig model of tinnitus. These data confirm the spectral characteristics of the ESA in guinea-pigs and show it is dependent on the sensitivity of the auditory nerve and intact auditory neurotransmission. In addition these experiments support the view that the ESA represents summed spontaneous neural activity in the cochlea and provide a platform for studies of the influence of ototoxic compounds on the spontaneous neural outflow of the cochlea as a model of tinnitus.

Acoustic Stimulation↗

PubMed2004

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.

Acoustic Stimulation↗

PubMed2001

Transient expression of P2X(1) receptor subunits of ATP-gated ion channels in the developing rat cochlea.

The expression pattern of the ATP-gated ion channel P2X(1) receptor subunit was studied in the developing rat cochlea by riboprobe in situ hybridisation and immunohistochemistry. Embryonic (E12, E14, E16 and E18) and postnatal (P0, P2, P4, P6, P10 and adult) rat cochleae were examined. Both mRNA and protein localisation techniques demonstrated comparable P2X(1) receptor expression from E16 until P6 but this expression was absent at later developmental stages. P2X(1) receptor mRNA expression was localised within the otic capsule and associated mesenchyme (from E16 to P6), spiral limbus (from P0 to P6) and within the spiral ligament adjacent to the insertion of Reissner's membrane (from P2 to P6). P2X(1) receptor protein had a similar distribution based upon immunoperoxidase localisation. P2X(1) receptor-like immunoreactivity was detected in the otic capsule and the surrounding mesenchyme (from E16 to P6), spiral limbus (from P0) and epithelial cells of Reissner's membrane (from P2 to P6). The spiral ganglion neurones showed the earliest P2X(1) receptor expression (from E16 to P6). This became associated with immunolabelling of their afferent neurite projections to the base of the developing inner and outer hair cells (observed from E18 and peaking at P2). Immunolabelling of the efferent nerve fibres of the intraganglionic spiral bundle (from E18 to P6) within the spiral ganglion was also observed. The results suggest that ATP-gated ion channels assembled from P2X(1) receptor subunits provide a signal transduction pathway for development of afferent and efferent innervation of the sensory hair cells and purinergic influence on cochlear morphogenesis.

Adenosine Triphosphate↗

PubMed2001

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 Student's 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 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, 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; Student's 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.

Adenosine Triphosphate↗

PubMed2000

Purinergic signalling: an experimental perspective.

Investigation of the multiple roles of extracellular nucleotides in the cochlea has developed from analysis of ATP-activated conductances in single sensory hair cells. Molecular probes such as radiolabelled ATP analogues and radiolabelled mRNA for ATP-gated ion channel subunits (P2X receptors) rapidly revealed the extensive nature of ATP signalling in this sensory organ. This has provided a foundation for physiological investigations which put extracellular nucleotides at the centre of homeostatic regulation of the driving force for sound transduction, modulation of mechanical tuning, control of cochlear blood flow and auditory neurotransmission. The purinergic signal transduction pathways associated with these processes have several novel features of significance to the broader field of purinergic neuroscience. In turn, these studies have benefited from the recent experimental advances in the field of purinergic signalling, a significant component of which is associated with the work of Professor Geoffrey Burnstock.

Adenosine Triphosphate↗

PubMed2000

Immunohistochemical localization of adenosine 5'-triphosphate-gated ion channel P2X(2) receptor subunits in adult and developing rat cochlea.

Substantial in vitro and in vivo data support a role for extracellular adenosine 5;-triphosphate (ATP) and associated P2 receptors in cochlear function. However, the precise spatiotemporal distribution of the involved receptor protein(s) has not been determined. By using a specific antiserum and immunoperoxidase labeling, the tissue distribution of the P2X(2) subunit of the ATP-gated ion channel was investigated. Here, we describe the first extensive immunohistochemical mapping of P2X(2) receptor subunits in the adult and developing rat cochlea. In the adult, immunoreactivity was observed in most cells bordering on the endolymphatic compartment (scala media), particularly in the supporting cells. Hair cells were not immunostained by the P2X(2) antiserum, except for outer hair cell stereocilia. In addition, weak immunolabeling was observed in some spiral ganglion neurons. P2X(2) receptor subunit protein expression during labyrinthine ontogeny was detected first on embryonic day 19 in the spiral ganglion and in associated nerve fibers extending to the inner hair cells. Immunostaining also was observed underneath outer hair cells, and, by postnatal day 6 (P6), intense immunolabeling was seen in the synaptic regions of both types of hair cell. Supporting cells of the sensory epithelium were labeled at P0. This labeling became most prominent from the onset of cochlear function (P8-P12). Conversely, expression in the vascular stria declined from this time. By P21, the pattern of immunolabeling was similar to that found in the adult. The localization and timing of P2X(2) immunoreactivity suggest involvement of extracellular ATP and associated ATP-gated ion channels in important physiological events, such as inner ear ontogeny, sound transduction, cochlear micromechanics, electrochemical homeostasis, and auditory neurotransmission.

Adenosine Triphosphate↗

PubMed2000

Apoptosis in the developing rat cochlea and its related structures.

Mammalian development involves proliferation and programmed cell death (apoptosis). This study was undertaken to analyse the spatial and temporal organisation of apoptosis in developing rat cochlear and associated tissues using in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling of DNA fragments (TUNEL), and light and electron microscopy. Embryonic (E12-E19 days) and postnatal rats (P0-P21 days) were studied. Fixed tissues were stained for apoptosis using TUNEL technique and the cytomorphology of apoptosis was confirmed by light and electron microscopy. Apoptotic cells were detected predominantly during the embryonic and early postnatal development of the cochlea. Apoptosis occurred in embryonic precursors of the cochlear duct epithelium, mainly in the region of its outgrowth between E12 and E16. In the periotic mesenchyme, apoptosis occurred in areas committed to develop into the middle ear cavity (peaking at E16) and perilymphatic compartments (peaking around E18-E19). Apoptosis in the VIIIth nerve (statoacoustic) ganglion was detected throughout the embryonic and early postnatal periods, peaking at E18-E19, around the time when the cochlear neural connections are being established. At later postnatal days, apoptosis was seen only occasionally in cochlear tissues, predominantly in tissues lining the middle ear cavity and sporadically in cells of the otic capsule. Therefore, apoptosis appears to occur in areas of remodeling, in areas of cavitation and in areas of differentiation. These findings provide a template for studying the molecular mechanisms involved in the development of the rat inner ear.

Animals↗

PubMed1999

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.

Adenosine Triphosphatases↗

PubMed1999

Expression of the P2X(2) receptor subunit of the ATP-gated ion channel in the cochlea: implications for sound transduction and auditory neurotransmission.

Extracellular ATP has multimodal actions in the cochlea affecting hearing sensitivity. ATP-gated ion channels involved in this process were characterized in the guinea pig cochlea. Voltage-clamped hair cells exhibited a P2 receptor pharmacology compatible with the assembly of ATP-gated ion channels from P2X(2) receptor subunits. Reverse transcription-PCR experiments confirmed expression of the P2X(2-1) receptor subunit mRNA isoform in the sensory epithelium (organ of Corti); a splice variant that confers desensitization, P2X(2-2), was the predominant subunit isoform expressed by primary auditory neurons. Expression of the ATP-gated ion channel protein was localized using a P2X(2) receptor subunit-specific antiserum. The highest density of P2X(2) subunit-like immunoreactivity in the cochlea occurred on the hair cell stereocilia, which faces the endolymph. Tissues lining this compartment exhibited significant P2X(2) receptor subunit expression, with the exception of the stria vascularis. Expression of ATP-gated ion channels at these sites provides a pathway for the observed ATP-induced reduction in endocochlear potential and likely serves a protective role, decoupling the "cochlear amplifier" in response to stressors, such as noise and ischemia. Within the perilymphatic compartment, immunolabeling on Deiters' cells is compatible with purinergic modulation of cochlear micromechanics. P2X(2) receptor subunit expression was also detected in spiral ganglion primary afferent neurons, and immunoelectron microscopy localized these subunits to postsynaptic junctions at both inner and outer hair cells. The former supports a cotransmitter role for ATP in a subset of type I spiral ganglion neurons, and latter represents the first characterization of a receptor for a fast neurotransmitter associated with the type II spiral ganglion neurons.

Adenosine Triphosphate↗

PubMed1999

ATP-gated ion channel expression in primary auditory neurones.

Extracellular ATP acts via ionotropic P2X receptors to mediate fast neurotransmission in the central and autonomic nervous systems. Recent data, including identification of P2X2 receptor mRNA expression by spiral ganglion neurones, suggests that purinergic signalling may influence auditory neurotransmission via ATP-gated ion channels assembled from these subunits. Expression of the P2X2 receptor was localized to the region of the spiral ganglion neurone synapses with the inner hair cells using a P2X2 receptor specific antiserum. Whole-cell patch clamping of neurones cultured from post-natal day 3-5 spiral ganglia demonstrated a heterogeneity of ATP-activated conductances, consistent with the functional expression of P2X2 receptor subunit isoforms along with possible co-expression of additional P2X receptor subunits. These data provide substantive support for a purinergic transmission element at the peripheral auditory synapse.

Adenosine Triphosphate↗

PubMed1999

Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system.

The distribution of the P2X2 receptor subunit of the adenosine 5'-triphosphate (ATP)-gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X2 receptor-specific antisera and riboprobe-based in situ hybridisation. P2X2 receptor mRNA expression matched the P2X2 receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo-hippocampal and amygdalo-cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex. In the spinal cord, P2X2 receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X2 receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies. These data support the role for extracellular ATP acting as a fast neurotransmitter at pre- and postsynaptic sites in processes such as sensory transmission, sensory-motor integration, motor and autonomic control, and in neuronal phenomena such as long-term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Adenosine Triphosphate↗

PubMed1999

Modulation of cochlear blood flow by extracellular purines.

Humoral adenosine 5'-triphosphate (ATP), adenosine and uridine 5'-triphosphate (UTP) have been shown to have a role in controlling local blood flow in a variety of tissues. The presence of P1 and P2 receptors in the cochlea, and particularly the highly vascular region, the stria vascularis, implies a vasoactive role for these compounds in the inner ear. To test the effect of extracellular purines and pyrimidines on cochlear blood flow, cochleae from anaesthetised guinea-pigs were perfused with ATP (1 microM-10 mM), adenosine (1 microM-10 mM) and UTP (1 mM) in artificial perilymph while blood flow through the cochlea was measured. An acute perilymphatic perfusion technique was established via tubing placed through a hole in the bone overlying scala tympani of the first cochlear turn, with an outlet hole in scala vestibuli of the fourth turn. Blood flow was measured by placing the probe of a laser Doppler blood perfusion monitor on the bone overlying the stria vascularis in the third cochlear turn. ATP and adenosine produced a significant dose dependent increase in cochlear blood flow (28.8-229.0% and 35.8-258.1%, respectively). The effect of ATP (100 microM) on cochlear blood flow was reduced in the presence of reactive blue 2 (1 mM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (1 mM). The blood flow response to adenosine (10 microM) was reduced in the presence of 1,3-dimethylxanthine (theophylline, 100 microM), but not with either 3,7-dimethyl-1-propargylxanthine (10 microM) or 8-cyclopentyl-1,3-dipropylxanthine (10 microM). UTP did not produce any change in the cochlear blood flow. To determine if the ATP effect was also mediated by adenosine derived from ectonucleotidase activity, the perilymphatic compartment was perfused with either ATP plus theophylline (100 microM) or with the non-metabolisable form of ATP, adenosine 5'-O-(3-thiophosphate) (ATPgammaS, 100 microM). The effect of ATP on cochlear blood flow was unaffected with the inclusion of theophylline while ATPgammaS produced an increase in cochlear blood flow similar to the one observed with ATP. These findings indicate that extracellular ATP and its metabolite adenosine have a modulatory role in cochlear blood flow possibly mediated by both P1 and P2 receptors.

Adenosine↗

PubMed1999

P2X receptor-mediated changes in cochlear potentials arising from exogenous adenosine 5'-triphosphate in endolymph.

Our previous studies have determined the presence of adenosine 5'-triphosphate (ATP) in the cochlear fluids and shown that extracellular ATP introduced into the endolymphatic compartment of the guinea pig cochlea has a significant dose-dependent suppressive effect on both endocochlear potential (EP) and cochlear microphonic (CM), which is mediated via P2 receptors. In the present study, the influence of P2 receptor agonists and antagonists on this suppressive effect was investigated to characterise the subtypes of P2 receptor mediating the ATP-induced effect on cochlear function. Using a double-barreled pipette attached to a pressure injector, small volumes (2-10 nl) of ATP (0.01-1 mM) and P2 receptor agonists or P2 receptor antagonists in artificial endolymph were introduced into the scala media of the first (basal) and third turns of the guinea pig cochlea, while the EP and CM were monitored. ATP and P2 receptor agonists (5x10(-14)-1x10(-11)cibacron blue. Neither adenosine nor uridine 5'-triphosphate (2x10(-13)-2x10(-11) moles) nor the P2 receptor antagonists on their own had any effect on EP and CM. The ATP effect on the potentials was greater at the third cochlear turn when compared to the first turn. These results provide evidence that in the endolymphatic compartment of the guinea pig, the extracellular ATP effect on cochlear function is likely mediated through an interaction with P2 receptors which assemble as ATP-gated ion channels.

Adenosine Triphosphate↗

PubMed1998

Ecto-nucleotidases terminate purinergic signalling in the cochlear endolymphatic compartment.

There is strong evidence for a purinergic signalling system in the inner ear which regulates auditory sensitivity. This study describes the terminating mechanism for purinergic signalling in the cochlear endolymphatic compartment via ecto-nucleotidases. Exogenous ATP was introduced into the scala media (SM) of the isolated, perfused guinea-pig cochlea, and the effluent was assayed for the adenine nucleotide metabolites by reverse-phase HPLC. Tissue viability was confirmed by fluorescence imaging of cochlear tissues. Extracellular ATP degradation to adenosine was Ca2+/Mg2+ dependent, and was not affected by inhibitors of intracellular ATPases and non-specific alkaline phosphatase. High azide concentration (5 mM) and suramin produced an inhibitory effect on ATP hydrolysis, consistent with inhibition of E-type ATPase activity. The Vmax of ATP hydrolysis (2564 mumol min-1 SM-1) was indicative of high ecto-ATPase activity. Our results support the role of ecto-nucleotidases as a principal mechanism for termination of purinergic signalling within SM, a compartment of the cochlea showing considerable P2X receptor expression.

Adenosine↗

PubMed1998

Fluorescence imaging of Na+ influx via P2X receptors in cochlear hair cells.

The adenosine 5'-triphosphate (ATP)-activated membrane conductance, mediated by P2X receptors, was examined in isolated guinea-pig cochlear inner and outer hair cells. Photo-activated release of caged-ATP elicted a 30-ms latency inwardly rectifying non-selective cation conductance, blocked by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10-100 microM), consistent with the direct activation of ATP-gated ion channels. A K(Ca) conductance in the inner hair cells (IHC), activated by the entry of Ca2+ through the ATP-gated ion channels, was blocked by including 10 mM 1,2-his(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the internal solution. Real-time confocal slit-scanning fluorescence imaging of Na+ influx through the ATP-gated ion channels was performed using the dye Sodium Green with simultaneous whole-cell recording of membrane currents. The Na+ entry was localized to the endolymphatic surface, with the increase in [Na+]i detected within approximately 200 ms of the onset of the inward current response. Within 600 ms Na+ had diffused throughout the cell cytoplasm with the exception of the subnuclear region of the outer hair cells. Correlation of voltage-clamp measurements of Na+ entry with regional increases in Na+-induced fluorescence demonstrated ATP-induced increases in intracellular Na+ in excess of 45 mM within 4 s. These data provide direct evidence for the Na+ permeability of the ATP-gated ion channels as well as independent evidence for the localization of P2X receptors at the endolymphatic surface of the sensory hair cells. The localization of the ATP-gated ion channels to the apical surface of the hair cells supports an ATP-mediated modulation of 'silent' K+ current across the cochlear partition which could regulate hearing sensitivity by controlling the transcellular driving force for both mechanoelectrical and electromechanical transduction in hair cells.

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↗

PubMed1996

Localization of ATP-gated ion channels in cerebellum using P2x2R subunit-specific antisera.

The distribution of the P2x2 purinoceptor subunit protein, which forms ATP-gated ion channels by homo- and hetero-multimeric assembly, was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit. These antisera, the first available for labelling the P2x2R subunit protein, were validated by selective labelling of a fusion protein containing the target amino acid sequences, and in cerebellum, by peptide specific block of immunoreactivity and by comparison with the distribution of P2x2R mRNA. P2x2R-like immunoreactivity was seen in Purkinje cells, specifically the soma and dendrites, neurons in the granular and molecular layers and deep cerebellar nuclei. The identification of P2x2R-like immunoreactivity within the cerebellar neural circuitry is consistent with a role for extracellular ATP acting as a fast neurotransmitter in motor learning and coordination of movement. Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Adenosine Triphosphate↗

PubMed1996

Ectonucleotidase activity in the perilymphatic compartment of the guinea pig cochlea.

It has been clearly demonstrated that extracellular adenosine 5'-triphosphate (ATP) exerts a potent modulatory activity in the cochlea through its interaction with P2 purinoceptors. However, little is known regarding the metabolism of extracellular ATP in cochlear tissues via ectonucleotidases. This study provides evidence for the presence of ectonucleotidases in the perilymphatic compartment of the guinea pig cochlea. Using microperfusion, ATP (500 microM) was introduced into the cochlear perilymph through the basal turn scala tympani, and effluent was collected from the basal turn scala vestibuli. Samples were subsequently analysed for the presence of adenine metabolites using high performance liquid chromatography (HPLC). Cell viability was evaluated by the activity of the intracellular enzyme lactate dehydrogenase (LDH) in the perfusate. ATP was degraded to 122.8 +/- 9.9 microM (25.0 +/- 5.8%) during the passage through the cochlear perilymphatic compartment. Breakdown of ATP resulted in the formation of adenosine 5'-diphosphate (41.5 +/- 9.0 microM), adenosine 5'-monophosphate (201.3 +/- 15.5 microM), adenosine (108.6 +/- 8.3) and inosine (15.0 +/- 1.5 microM). The degradation of ATP was significantly (P < 0.001, Student's t-test) inhibited in the absence of divalent cations, Ca2+ and Mg2+ in the perfusate. In control experiments, no spontaneous degradation of ATP was observed in vitro. LDH activity was similar during ATP perfusions (2.9 +/- 0.9%) to control perfusions with artificial perilymph (4.2 +/- 1.0%) indicating well preserved cell integrity in the cochlear perilymphatic compartment. The degradation of extracellular ATP in the presence of intact tissues and its inhibition in the absence of divalent cations, a cofactor for ectonucleotidases, provides evidence for ectonucleotidase activity in the perilymphatic fluid space of the cochlea.

Adenine↗