Purinergic Modulation of Cochlear Partition Resi

Authors: Peter R Thorne David J B Muñoz Gary D Housley

Publish Date: 2003/10/13

Volume: 5, Issue: 1, Pages: 58-65

PDF Link

Abstract

Introduction of adenosine 5′triphosphate ATP into the endolymphatic compartment of the guineapig cochlea decreases the endocochlear potential EP To determine if this is due to an ATPinduced change in compartment resistance the cochlear partition resistance CoPR was measured using constant current injections into scala media before during and after microinjection of ATP into the same compartment The CoPR mean = 313 ± 013 kΩ decreased with ATP in a dosedependent manner 251 ± 30 decrease in relation to baseline values and this was linearly correlated R 2 = 091 to the magnitude of the ATPinduced decline in EP 416 ± 70 decline in relation to the baseline Pyridoxalphosphate6azophenyl2′4′disulfonic acid PPADS a P2X receptor antagonist injected prior to ATP application blocked this ATPinduced reduction in EP and CoPR This indicates that ATPgated ion channels P2X receptors provide a latent shunt capable of regulating the majority of the electrical potential across the luminal surface of the sensory hair cells which is necessary for sound transduction The results suggest a novel sound transduction regulatory mechanism which via extracellular ATP has the capability of adjusting hearing sensitivityIdentification of P2 receptor subtypes in the mammalian cochlea supports the view that extracellular adenosine 5′triphosphate ATP plays a role in the regulation of cochlear function One important role may be in the regulation of the sound transduction process and thus cochlear sensitivity Thorne and Housley 1996 Housley 1998 Housley et al 1999 2002 Housley and Thorne 2000 Essential to sound transduction is the large 100–170 mV electrochemical driving force across the luminal surface of the organ of Corti provided by the positive endocochlear potential EP +60 to +100 mV and the negative resting membrane potential of the inner IHC and outer OHC hair cells −40 mV and −70 mV respectively Dallos 1985 Interestingly the introduction of ATP into the endolymphatic compartment causes a reversible decline in the EP in a dosedependent manner Muñoz et al 1995 1999 Kirk and Yates 1998 Pharmacological studies indicate that this is likely to be via activation of ATPgated ion channels assembled mainly from P2X2 receptor subunits Muñoz et al 1999 localized to cells lining the compartment Housley et al 1992 1998 1999 Mockett et al 1994 King et al 1998 Housley and Thorne 2000 particularly the hair cells and supporting cells of the organ of Corti and cells of Reissner’s membrane Evidence of free ATP in the endolymph under normal conditions and elevation of fluid ATP concentration with noise and hypoxia Muñoz et al 2001 implies that endogenous ATP may affect EP under normal and stress conditions Under this model the ATPgated conductance across the cells lining the endolymphatic compartment would regulate the driving force available for sound transduction by the hair cellsTo test this hypothesis we have studied the influence of exogenous ATP on the resistance of the endolymphatic compartment defined here as the cochlear partition resistance CoPR Here we demonstrate that ATP reduces the CoPR We propose that this arises from a shunt conductance that short circuits the endocochlear potential and coupled with the direct action of ATP to depolarize the hair cells leads to a significant compression in the electrochemical driving force for sound transductionMethods were approved by the University of Auckland Animal Ethics Committee Guinea pigs were anesthetized with 60 mg/kg IP NembutalTM and artificially ventilated with room air Endtidal O2 and CO2 and blood pressure were monitored continuously The cochlea was exposed and holes 40–100 µm in diameter were made in the bone overlying the scala media and scala tympani of the basal turn to insert micropipettes for EP and CoPR measurements and for current injection Fig 1AA Diagram showing the arrangement for microinjection of ATP into scala media and measurement of EP and CoPR SV = scala vestibuli SM = scala media ST = scala tympani B An example of ATPinduced reduction in CoPR ATP 500 nl 100 µM applied over 1 min reduced EP from 83 to 36 mV concomitant with a reduction in the magnitude of the 1 µA currentinduced voltage shift 3 s from 38 mV arrow a to 25 mV arrow b corresponding to a decrease of CoPR from 38 kΩ to 25 kΩ respectively After ATP injection CoPR returned to baseline levels arrow c The inset shows the voltage shifts increased in size for better illustration ATP injection produced a decrease in the EP C and CoPR D in every experiment Open symbols in C and D represent the mean EP and CoPR values respectively E The reduction in the magnitude of the EP was concomitant with a reduction in the CoPR in every experiment Each paired data point open symbol before the ATP pulse filled symbol during the ATP pulse represents one experiment Dotted line represents the bestfit interceptA doublebarreled glass micropipette was inserted into scala media for voltage measurement EP and to inject ATP A singlebarreled micropipette was inserted into scala media through an adjacent hole to deliver current The voltage and current pipettes each coupled to a Teflon®coated Ag–AgCl wire were filled with artificial endolymph and were separated by 04–05 mm Two Teflon®coated Ag–AgCl wires were placed in holes in scala tympani 07–08 mm separation adjacent 08–09 mm equidistant to the scala media electrodes to serve as ground references for the voltage measurement and current injection Fig 1A Different volumes see below of ATP 100 µM contained in artificial endolymph were microinjected into scala media using computercontrolled pressure pulses to produce declines in EP of different magnitude as reported previously Muñoz et al 1995 1999To ensure proper positioning of the current micropipette within scala media EP was monitored as the pipette was advanced and once a stable value was obtained the electrode was connected to the current source Current was generated by a twoelectrode voltage clamp amplifier GeneClamp 500 Axon Instruments Union City CA USA or a custommade constant current injector controlled by a D/A converter TL1 Scientific Solutions Inc Mentor OH USA using pClamp software pClamp 55 Axon Instruments Square current pulses 1 µA 3 s duration at 6 s intervals were continuously applied to the scala media electrode with the ground electrode in scala tympani ATP was then injected into scala media for 60 s to produce a steadystate reduction in EP Fig 1B As the extent of the ATPinduced reduction in EP is dosedependent Muñoz et al 1995 1999 the total amount of ATP injected was varied across experiments by changing the pressure to the injection pipette to produce a reduction in EP of different magnitudes The total volume of ATP solution applied varied across experiments from 250 to 1000 nl equivalent to 25 × 10−11 and 10 × 10−11 mol In another group of experiments the P2X receptor antagonist pyridoxalphosphate6azophenyl2′4′disulfonic acid PPADS 1 mM in artificial endolymph 50 nl was injected through the voltagesensing micropipette prior to the ATP pulse The effect of this postPPADS ATP pulse on EP and CoPR was compared with the effect produced by a prePPADS ATP pulse of the same amount baseline Control experiments were performed in the same manner using similar volumes of either artificial endolymph without ATP 250–1000 nl or with PPADS 1 mM 50 nlCurrentinduced voltage shifts in EP were measured before during and after the ATP and control pulses pClamp software and used to calculate the CoPR R = ΔV EP/I A minimum of three CoPR values were averaged to provide each CoPR data point Although EP can range from positive to negative under anoxia values in these studies the percentage change in EP assumed a 0 zero V reference EP changes in percentage were calculated relative to the initial EP measurement before the test or control pulses Results were expressed as the mean ± standard error of the mean Because insertion of the multiple electrodes into scala media caused some tissue damage which often resulted in lower EP values only experiments with EP values ≥55 mV were accepted

Keywords: