Effect Of Hydrogen Peroxide And α-tocopherol On High-conductance Calcium-activated Potassium Channels Of Outer Hair Cells Of Cochlea In Guinea Pigs

Objective:To study the effect and mechanics of hydrogen peroxide andα-tocopherol on High-conductance Calcium-activated Potassium Channels of outer hair cells of cochlea in aged guinea pig,and explore how the functions of outer hair cells were adjusted by hydrogen peroxide andα-tocopherol,which affect some aged inner diseases,so as to find some new way to prevent and cure aged inner diseases,for example presbyacusia. Methods: To use the patch-clamp technique of single channel research.1.OHC preparation.Healthy thirty six months old guinea pigs,and they accord with inferior condition including normal auricle form,no trauma of ear,sensitive auricle reflex,normal eardrum,otoacoustic emission in existence and so on.Guinea pigs were decapitated.The temporal bones were removed from the skull and placed in extracellular fluid.The lateral wall of bony cochlea was removed under a binocular microscope,the whole and integrated cochlea without bony lateral wall were moved into extracellular fluid containing collagenase(typeâ…£).After 10～15 min of incubation,the tissue was transferred into collagenase-free extracellular fluid for 3～5 min and then into a chamber treated by human plasma fibronectin purified protein,and removed spiral ligament under anatomical microscope.The cells were mechanically dispersed by gentle flux through a micropipette and allowed isolated OHCs to settle for 10～15 min.2.Channel current record.Isolated OHC were placed in a chamber with symmetrical high K+ solution on the stage of an inverted microscope.They were readily distinguished from other cells by their cylindrical shape and height.Single channel currents were recorded from cell-attached and inside-out patches using patch-clamp technique.Current were measured with a CEZ-2200 patch-clamp amplifier and stored on a digital audiotape recorder(P-clamp 10.1) for further analysis.The current was low pass filtered with a filter(3KHz).Choose some index to analysis applying P-clamp 10.1 software package,including Current Amplitude,Probability of Open,Mean Open Time,Mean Close Time.3. Channel of Identification and Analysis.BKCa channel were recorded using cell-attached and inside-out patch clamp through changing holding potential,the Ca²⁺ concentration from 1.0×10^-7mol/L to 1.0×10^-5mol/L in the bath solution and increasing the TEA concentration in the bath solution.4.Observation of drug action.(1) In 2ml bath solution of chamber,BKCa current were recorded using cell-attached patch clamp after Gigaohm Seal through changing the concentration of H₂O₂ from 100μmol/L to 400μmol/L in bath solution and then washout.(2) BKCa channel were recorded using cell-attached patch clamp after given 300μmol/L H₂O₂(no washout ) through changing the concentration of VitE from 50μmol/L to 200μmol/L. Results: 1.In symmetrical high K+ solution and inside-out patches,single channel BKCa currents were recorded and increased with the increasing of holding potential.The Po and To of single channel were increased with the increasing of depolarizing potential.Conductance of single channel current was 218.45±0.53pS.Membrane reversal potential (VreV) was 0mV.BKCa currents were recorded and increased with increasing of Ca²⁺ concentration.So was Po of single channel.BKCa currents were recorded and decreased with increasing of TEA,even blocked at 50mmol/L TEA.So was Am of single channel.2.BKCa currents were recorded and increased using cell-attached patch clamp at 300μmol/L H₂O₂,and decreased with follwing 150μmol/L VitE. Conclusions: 1.The single channel currents recorded from OHC in our experiments is BKCa,with voltage-dependence,Ca²⁺ concentration-dependence,and the conductance is 218.45±0.53pS(n=6).2.BKCa currents were recorded and increased with the change of H₂O₂ concentration,and then decreased with the change of VitE concentration.3.We could conclud that H₂O₂ and VitE affect the fuction of OHC possibly throuth intracellular mechnism.4.According to the experiments,we supposed that H₂O₂ and VitE participated in the onset of presbyacusia,which provide some theory support to antioxygen therapy of presbyacusia.