Biomedical subjects

G D Housley

Publications and source records attributed to G D Housley.

At least 37 records · Page 2Linked to original sources

PubMed1998

Expression of ATP-gated ion channels by Reissner's membrane epithelial cells.

Reissner's membrane forms a partition between the endolymphatic and perilymphatic cochlear compartments. Expression of the P2X2 receptor subunit which assembles to form ATP-gated ion channels was detected in guinea-pig Reissner's membrane using the reverse transcription polymerase chain reaction (RT-PCR). The P2X2 receptor subunit protein was localized to the epithelial cells which line the endolymphatic surface of Reissner's membrane using confocal immunofluorescence. The P2X receptor expression in Reissner's membrane was functionally confirmed using whole-cell voltage-clamp. An inwardly rectifying conductance was activated in Reissner's membrane epithelial cells in the presence of extracellular ATP (100 microM). This conductance had a pharmacological profile compatible with the P2X2 receptor designation, but exhibited substantial desensitisation which may be attributable to additional P2X receptor subunits. This study indicates that extracellular ATP, a humoral factor within scala media, acts via ATP-gated ion channels expressed by Reissner's membrane epithelial cells to decrease the driving force for sound transduction.

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

Localization of mRNA encoding the P2X2 receptor subunit of the adenosine 5'-triphosphate-gated ion channel in the adult and developing rat inner ear by in situ hybridization.

Localization of expression of the adenosine 5'-triphosphate (ATP)-gated ion channel P2X2 receptor subunit (P2X2R) in the rat inner ear at different stages of development was achieved by using in situ mRNA hybridization. In the adult, P2X2R mRNA was strongly expressed in many of the cells bordering the cochlear endolymphatic compartment. This included the interdental cells of the spiral limbus, all cells of the inner sulcus and organ of Corti, and cells of the spiral prominence. In the vestibular labyrinth, strong expression was noted in the transitional cells at the base of the crista ampullaris and in the sensory epithelium of the crista and maculae. During development, P2X2R mRNA expression was evident in the precursors of these structures at the earliest period studied, embryonic day 12 (E12). Expression increased during the ontogeny in both the cochlear and the vestibular end organs. In addition, both the spiral and vestibular ganglia showed developmental expression. In contrast to the supporting cells of the organ of Corti, both inner and outer hair cells exhibited P2X2R mRNA only after postnatal day 10 (P10) through P12, concomitant with the onset of hearing. P2X2R expression levels in all cells fell from a maximum at P12-P18 to lower levels in the adult. In the adult, P2X2R mRNA levels were modest in outer hair cells in the basal (high-frequency) encoding region of the cochlea, and inner hair cell labeling was low throughout the cochlea. Reissner's membrane, which maintains an electrochemical barrier between scala vestibuli and scala media, showed considerable expression of P2X2R mRNA in early postnatal development, and expression was maintained at moderate levels in the adult cochlea. These data are consistent with a role for the P2X2R subunit in the processes of labyrinthine development and the regulation of the electrochemical gradients supporting auditory and vestibular sensory transduction.

Adenosine Triphosphate↗

PubMed1998

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

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

Adenosine Triphosphate↗

PubMed1998

Extracellular nucleotide signaling in the inner ear.

Extracellular nucleotides, particularly adenosine 5'-triphosphate (ATP), act as signaling molecules in the inner ear. Roles as neurotransmitters, neuromodulators, and as autocrine or paracrine humoral factors are evident. The diversity of the signaling pathways for nucleotides, which include a variety of ATP-gated ion channels (assembled from different subtypes of P2X-receptor subunit) and also different subtypes of G protein-coupled nucleotide receptors (P2Y receptors) supports a major physiological role for ATP in the regulation of hearing and balance. Almost invariably both P2X and P2Y receptor expression is apparent in the complex tissue structures associated with the inner-ear labyrinth. However P2X-receptor expression, commonly associated with fast neurotransmission, is apparent not only with the cochlear and vestibular primary afferent neurons, but also appears to mediate humoral signaling via ATP-gated ion channel localization to the endolymphatic surface of the cochlear sensory epithelium (organ of Corti). This is the site of the sound-transduction process and recent data, including both electrophysiological, imaging, and immunocytochemistry, has shown that the ATP-gated ion channels are colocalized here with the mechano-electrical transduction channels of the cochlear hair cells. In contrast to this direct action of extracellular ATP on the sound-transduction process, an indirect effect is apparent via P2Y-receptor expression, prevalent on the marginal cells of the stria vascularis, a tissue that generates the standing ionic and electrical gradients across the cochlear partition. The site of generation of these gradients, including the dark-cell epithelium of the vestibular labyrinth, may be under autocrine or paracrine regulation mediated by P2Y receptors sensitive to both purines (ATP) and pyrimidines such as UTP. There is also emerging evidence that the nucleoside adenosine, formed as a breakdown product of ATP by the action of ectonucleotidases and acting via P1 receptors, is also physiologically significant in the inner ear. P1-receptor expression (including A1, A2, and A3 subtypes) appear to have roles associated with stress, acting alongside P2Y receptors to enhance cochlear blood flow and to protect against the action of free radicals and to modulate the activity of membrane conductances. Given the positioning of a diverse range of purinergic-signaling pathways within the inner ear, elevations of nucleotides and nucleosides are clearly positioned to affect hearing and balance. Recent data clearly supports endogenous ATP- and adenosine-mediated changes in sensory transduction via a regulation of the electrochemical gradients in the cochlea, alterations in the active and passive mechanical properties of the cells of the sensory epithelium, effects on primary afferent neurons, and control of the blood supply. The field now awaits conclusive evidence linking a physiologically-induced modulation of extracellular nucleotide and nucleoside levels to altered inner ear function.

Animals↗

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↗

PubMed1997

P2 receptor excitation of rodent hypoglossal motoneuron activity in vitro and in vivo: a molecular physiological analysis.

The role of P2 receptors in controlling hypoglossal motoneuron (XII MN) output was examined (1) electrophysiologically, via application of ATP to the hypoglossal nucleus of rhythmically active mouse medullary slices and anesthetized adult rats; (2) immunohistochemically, using an antiserum against the P2X2 receptor subunit; and (3) using PCR to identify expression of P2X2 receptor subunits in micropunches of tissue taken from the XII motor nucleus. Application of ATP to the hypoglossal nucleus of mouse medullary slices and anesthetized rats produced a suramin-sensitive excitation of hypoglossal nerve activity. Additional in vitro effects included potentiation of inspiratory hypoglossal nerve output via a suramin- and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS)-sensitive mechanism, XII MN depolarization via activation of a suramin-sensitive inward current, decreased neuronal input resistance, and a slow-onset theophylline-sensitive reduction of inspiratory output likely resulting from hydrolysis of extracellular ATP to adenosine and activation of P1 receptors. Immunohistochemically, P2X2 receptors were detected in inspiratory XII MNs that were labeled with Lucifer yellow. These data, combined with identification of mRNA for three P2X2 receptor subunit isoforms within the hypoglossal nucleus (two of which have not been localized previously in brain) and the previous demonstration that P2X receptors are ubiquitously expressed in cranial and spinal motoneuron pools, support not only a role of P2 receptors in modulating inspiratory hypoglossal activity but a general role of P2 receptors in modulating motor outflow from the CNS.

Adenosine↗

PubMed1997

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

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

Adenosine Triphosphate↗

PubMed1997

Variation in expression of the outer hair cell P2X receptor conductance along the guinea-pig cochlea.

1. Whole-cell patch-clamp recordings were used to determine the variation in the P2X receptor conductance, activated by extracellular ATP, in outer hair cells (OHCs) isolated from each of the four turns of the guinea-pig cochlea. 2. In standard solution (containing 1.5 mM Ca2+) slope conductances were determined in OHCs of known origin from current-voltage relationships obtained from voltage ramps applied between -100 and +50 mV. Membrane conductance throughout this voltage range was greatest in OHCs originating from the basal (high frequency encoding) region of the cochlea. This gradient in OHC conductance from apex to base of the cochlea can be attributed to variation in expression of both a negatively activated K+ conductance and a TEA-sensitive outwardly rectifying K+ conductance. OHC slope conductance measured about a membrane potential of -75 mV increased from a mean of 33.5 nS in the apical region (turn 4) to 96.8 nS in the basal region (turn 1) of the cochlea. 3. Removal of external Ca2+ reduced OHC conductance by an average of 25%, reflecting a Ca2+ dependence of the background conductances in these cells. In zero external Ca2+ the mean slope conductance measured at -75 mV in the apical turn was 25.0 nS compared with 73.8 nS in the basal turn. 4. In Ca(2+)-free solution both 2 mM and 4 microM ATP produced inward currents that were progressively larger in OHCs originating from more basal regions of the cochlea. The steady-state inward current elicited by 2 mM extracellular ATP increased from -1.44 to -3.26 nA for turns 4 and 1, respectively. 5. The P2X receptor conductance was determined between -100 and +50 mV by comparing voltage ramps in the presence and absence of extracellular ATP in Ca(2+)-free solution. The conductance was inwardly rectifying with a reversal potential close to 0 mV. Measured close to the resting membrane potential of the cells (-75 mV), 2 mM ATP elicited an average 300% increase in conductance in parallel with the systematic increase in background conductance which occurs in OHCs originating from the more basal regions of the cochlea. The conductance at -75 mV activated by 2 mM ATP increased from a mean of 59.6 nS in turn 4 OHCs to a mean of 166.2 nS in turn 1 OHCs. The conductance activated by 4 microM ATP was also greater in the basal turn OHCs (45.3 nS) than in the apical region OHCs (5.9 nS). 6. The number of ATP-gated ion channels on individual OHCs, presumed to be localized to the stereocilia, increases from approximately 6000 in turn 4 cells to 16,500 in turn 1 cells, based on estimates of unitary conductance and average maximum ATP-activated OHC conductance (2 mM ATP).

Adenosine Triphosphate↗

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↗

PubMed1995

Identification of a short form of the P2xR1-purinoceptor subunit produced by alternative splicing in the pituitary and cochlea.

A truncated form of the P2xR1 purinoceptor subunit (which we designate P2xR1-2) was detected in rat pituitary gland and the secretory epithelial tissue (stria vascularis) of the cochlea using RT-PCR of solid-phase cDNA libraries. PCR products corresponding to the P2xR1 purinoceptor subunit (1) were obtained from vas deferens, brain and microdissected cochlear sensory epithelial tissues including organ of Corti, sacculus and crista ampullaris. Cloning and sequencing revealed that the P2xR1-2 product included an 85-bp insertion in a region corresponding to a novel C-terminal end of the second membrane spanning domain and continuing as the cytoplasmic domain. A stop codon sequence after the first 51 bp of the insert effectively truncates this subunit, reducing the final cytoplasmic domain by 90% compared with the previously published P2xR1(-1) sequence, thereby reducing the overall peptide by approximately 25%. The region of the receptor lost in the truncated version coded for a number of serine/proline rich regions which may act as potential intracellular regulatory sites.

Alternative Splicing↗

PubMed1995

Autoradiographic labelling of P2 purinoceptors in the guinea-pig cochlea.

Two different radioligands were used to identify extracellular ATP binding sites specific to P2 purinoceptors in guinea-pig cochlear tissue. Deoxyadenosine 5'-(alpha-[35S]thio)triphosphate ([35S]dATP alpha S; 10 nM) provided a high activity probe for the P2y purinoceptor subtype on the basis of selective block by 2-methylthio-ATP (2MeSATP; 100 microM). [3H]alpha, beta-methylene-ATP (10 nM), a high affinity probe for a P2x purinoceptor subtype was selectively blocked by inclusion of the related compound beta, gamma-methylene-ATP (100 microM). Both probes labelled the organ of Corti, stria vascularis and spiral prominence regions. The P2x purinoceptor probe also bound to lateral wall tissue below the spiral prominence and insertion point of the basilar membrane within the scala tympani compartment, a region which failed to show significant binding using [35S]dATP alpha S. Frozen sections of whole cochlea permitted analysis of radioligand binding to the cell body region (spiral ganglion in Rosenthal's canal) of the primary auditory afferents and the auditory nerve itself, which lies within the central region of the modiolus of the cochlea. Both these regions exhibited 2MeSATP blockable [35S]dATP alpha S binding whereas specific [3H]alpha, beta-methylene-ATP binding was absent from spiral ganglion and minimal in the auditory nerve region. These results demonstrate a mixed P2 purinoceptor distribution in cochlear tissues and suggest that complex purine-mediated neurohumoral mechanisms may influence cochlear function at a number of sites.

Adenosine Triphosphate↗

PubMed1995

Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea: a possible source of extracellular ATP?

There is accumulating evidence for a purinergic humoral system involved in the control of cochlear function. Evidence of specific P2 purinoceptors on cochlear tissues implies a role for extracellular adenosine triphosphate (ATP) in the cochlea. To further this hypothesis a study was undertaken to determine if there was any specific source of purine compounds in cochlear tissues. Cochlear tissues (the sensory epithelium and lateral wall) from the guinea pig were incubated with the acridine derivative quinacrine dihydrochloride (5 x 10(-6) M in phosphate-buffered saline for 30 min at room temperature) which fluoresces on binding to high concentrations of ATP. Most cochlear tissues showed a diffuse green fluorescence slightly above the background level. However, a region of the marginal cells of the stria vascularis showed a specific punctate fluorescence. Optical sectioning of these cells by confocal microscopy revealed that the fluorescent structures in these marginal cells was confined to a region up to 10 microns from their endolymphatic surface. Similar cells studied by transmission electron microscopy showed membrane-bound vesicles located in the same region of the cell. These data imply that purine compounds are localized in discrete structures, perhaps vesicles, within the marginal cells which could serve as a source of extracellular ATP in the cochlea.

Adenosine Triphosphate↗

PubMed1995

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.

Adenosine↗

PubMed1995

Adenosine 5'-triphosphate (ATP) concentrations in the endolymph and perilymph of the guinea-pig cochlea.

The concentration of adenosine 5'-triphosphate (ATP) in endolymph (EL), perilymph (PL) and cerebrospinal fluid (CSF), collected from anesthetized guinea pigs was determined using the luciferase-luciferin reaction. The cochlea was exposed by a ventrolateral approach and the bone overlying scala media of the third turn (EL) or scala vestibuli of the first turn (PL) was shaved to a thin layer and a small fenestrum made. For EL sampling, a double-barrelled pipette was inserted through the spiral ligament-stria vascularis complex. One barrel was filled with 150 mM KCl to record the endocochlear potential (EP) and upon the appearance of the positive EP, 0.12-1.22 microliter of fluid was aspirated into the other barrel by gentle negative pressure. For PL sampling, a single-barrelled pipette was advanced into scala vestibuli and 0.3-1.6 microliter of fluid was collected by capillarity. CSF (0.36-1.75 microliter) was obtained from the cisterna magna. The cochleae were removed and processed for light microscopy to determine the extent of tissue damage from the sampling procedure. ATP concentrations (mean +/- SEM, nM) for EL, PL and CSF were 12.95 +/- 2.4 (n = 10), 10.5 +/- 3.9 (n = 11) and 16.1 +/- 5.4 (n = 11) respectively. Differences in ATP concentrations among fluids were not statistically significant. To test the effect of hypoxia on ATP levels, a group of guinea pigs was subjected to a 90 s period of respiratory anoxia prior to sampling of EL, PL or CSF. ATP concentrations were 14.4 +/- 3.5 (n = 11), 20.7 +/- 4.1 (n = 10) and 13.5 +/- 4.6 (n = 4) for EL, PL and CSF, respectively; only PL ATP concentrations were statistically different (P = 0.018, Wilcoxon rank sum test) to basal conditions. This is the first study which demonstrates the presence of free ATP in cochlear fluids. The results indicate that ATP is present in cochlear fluids at concentrations close to those known to cause hair cell depolarization in vitro.

Adenosine Triphosphate↗

PubMed1994

Nicotinic acetylcholine receptor subunits expressed in rat cochlea detected by the polymerase chain reaction.

Poly(A)+ RNA was extracted from rat cochleae using guanidinium thiocyanate and oligo(dT)-cellulose, and converted into cDNA by reverse transcriptase using an oligo(dT) primer. Oligonucleotides complementary to conserved 5' and 3' regions of alpha and beta subunits of the neuronal nicotinic acetylcholine receptor subunit (nAChR) family were then used as primers to screen the cochlear cDNA via the polymerase chain reaction (PCR) procedure. PCR products of approximately 900 bp length, purified by agarose gel electrophoresis, were nick translated to produce [32P]-dCTP labelled probes for Southern Blot screening of nAChR cDNAs. Of the four alpha and three beta subunits screened, only alpha 5 and beta 4 nAChR cDNAs hybridized. The alpha 5 PCR product was cloned and sequenced and proved to be identical to published sequence for alpha 5. The detection of alpha 5 and beta 4 nAChR subunit expression in cochlear tissue supports previous electrophysiological and immunocytochemical evidence for nAChR-mediated centrifugal control of hearing function.

Animals↗