| objective: To study effect of hydrogen dioxide (oxygen free radidical donator) and vitamin C (oxygen free radical scavenger) on the electric current of large conductance calcium-activated potassium channels(BKCa channels) in isolated outer hair cells of old guinea pigs'cochlears, and to explore mechanism of action that oxygen free radical and oxygen free radical scavenger affect the electric current of BKCa channels in outer hair cells of old guinea pigs'cochlears, and how to regulate the function of outer hair cells, thus affecting the occurrence and development of presbycusis, which would provide theoretical basis and new ideas for clinical prevention and treatment to presbyacusia. Methods : Acute enzyme isolated to outer hair cells of old guinea pigs'cochlears, in which of BKCa channel's electric current were observed and recorded by whole-cell recording mode of patch-clamp. 1. Cell detachment Sixty choosen healthy diversicolor old guinea pigs (250-400g, Preyer's reflex sensitive) were quickly decapitated and taken out of acoustic capsule which would be inserted in cold and oxygenated extracellular fluid; dissecting bony shell of cochlea carefully by anatomical microscope, and disconnecting cochlear axis. the cochleas with bony shell removed were inserted in the extracellular fluid containing the typeⅣcollagenase (1mg/ml)for digestion with 12 minutes; and then shitf into the chamber which of bottom was preconditioned by purified fibrous joint protein for 1 hour and full of balneum , to terminate digestion. ligament of cochlea was dilacerated with plyers by anatomical microscope. Then outer hair cells after enzymatic digestion and some basal membrane which was digested insufficiently was falling off in the chamber. Dissecteting the basal membrane into pieces by anatomical microscope, standing 20 to 30 minutes so that isolated outer hair cells were 5 sticked to the bottom of the chamber after blown gently. Condition of All above operation was being at room temperature (20-25℃). 2. Recording of inon channels'electric current Drawing electrode, polishing electrode, replacing balneum, choosing a out hair cell which was adherent well, three-dimensional, typical morphous for high resistance seal. Negative pressure was exerted shortly to electrode, cell membrane adsorpted by electrode was disrupted so that forming the whole-cell recording mode of patch-clamp. Choosing -60mV as VH, depolarizating from -50mV to +50 mV, a step as 10mV, stimulus duration as 400ms, activating BKCa channels, observing and recording the electric current of BKCa channels. Signals of electric current were amplified by the patch-clamp amplifier, leaded in memory oscilloscope and 12 A / D, D / A converter, and then entered into the computer for the acquisition of electric current signals, the sampling frequency as 10kHz, low pass filtering as 2KHz. P-Clamp 9.0 private facilities was used for data analysis. Current amplitude (Am), current density, current - voltage curve (I-V Curve) as analysis items. 3. identifying and analyzing the characteristics of channels Changing VT, then observing and recording electrophysiological characteristics of electric current, including voltage-dependent of the open of channels,current amplitude, current density, current - voltage curve. And observing the activation amd inactivation time of electric current, such as whether the voltage-dependent. Observing effects of the specific blocker iberiotoxin (IbTX) of BKCa channel on the channels'activities. 4. Observation of drug action⑴after recording the stable and normal electric current of BKCa channels, dividing into groups to add H2O2 dilution (0.2mmol/L) 0, 10, 20, 40μl in the 2 ml chambers within freshly isolated outer hair cells so that the concentration of H2O2 in the balneum would be 0, 1, 2, 4μmol / L, observing and recording the effect of different concentration of H2O2 to electric current of BKCa channels. After Recording, eluting with fresh non-drugged balneum, and observing the recovery of electric current of BKCa channels.⑵dividing into groups to add Vitamin C solution (5mg/ml) 0, 10, 20, 40μl in the 2 ml chambers within freshly isolated outer hair cells so that the concentration of Vitamin C in the balneum would be 0, 25, 50, 100μg / ml, observing and recording the effects of different concentration of Vitamin C to electric current of BKCa channels.⑶Adding H2O2 dilution 40μl in the 2 ml chambers within freshly isolated outer hair cells so that the concentration of H2O2 would be 4μmol / L first, then dividing into groups to add in Vitamin C solution 10, 20, 40μl so that the concentration of Vitamin C in the balneum were 25, 50, 100μg / ml, observing and recording the effect of H2O2 and Vitamin C to electric current of BKCa channels. Results: 1. In the whole-cell mode of patch-clamp, electric current with a string of large amplitude, rapid activation, almost non—deactivation was recorded, the activation voltage is exceed -40 ~ -30mv, electric current enhanced with the increasing of membrane potential, electric current's amplitude increased continuously and performanced characteristics of outward rectification, the electric current was recorded without "rundown" phenomenon; when the concentration of IbTX was 100nmol / L, the activity of channels was completely blocked, confirming that it's BKCa channel's electric current. 2.⑴While the concentration of H2O2 as 1, 2, 4μmol / L, BKCa channel electric current performanced clearly excited about the H2O2 concentration-dependent, current amplitude and peak current density increases with the concentration, I-V curve rised also. The electric current of channels could be partial recovered after the elution of fresh balneum. Medication within three minutes, when VT was +50 mv, the BKCa channels'the maximum peak current densities of drug groups and control group were: 1μmol / L group compared to control group with rising from 22.09±0.27 PA / PF to 27.43±0.51PA / PF, amplification as 24.17%; 2μmol / L group compared to control group with rising from 22.09±0.27PA/PF to 35.81±0.74 PA / PF, amplification as 62.11%; 4μmol / L group compared to control group with rising from 22.09±0.27 PA / PF to 43.53±1.09PA/PF, amplification as 97.06%.⑵There was no significant effect to BKCa channels'electric current by using vitamin C alone.⑶In H2O2 4μmol / L + vitamin C with different concentrations as 25, 50, 100μg/ml groups, BKCa channels'electric current performanced about concentration-dependent inhibition, and electric current's amplitude and peak current density decreased with the increaseing concentration of vitamin C, I-V curves were descend . Medication within three minutes, when VT was +50 mv, the BKCa channels'the maximum peak current densities of drug groups and control group were: 25μg/ml vitamin C +4μmol/LH2O2 group compared to control group with dropping from 43.53±1.09PA/PF to 34.85±0.49PA/PF, amplification as -19.94%; 50μg/ml vitamin C +4μmol/L H2O2 group compared to control group with dropping from 43.53±1.09PA/PF to 30.63±0.56PA/PF, amplification as -29.64%; 100μg/ml of vitamin C +4μmol / L H2O2 group compared to control group with dropping from 43.53±1.09PA/PF to 25.79±0.58PA/PF, amplification as -40.75%. But still could not be recovered to normal levels before drugged. Conclusion: 1. The electric current with large-conductance, voltage-dependent, rapid activation, almost non-deactivation recorded in the experiment was BKCa channels'electric current, and sensitive to the specific blocker IbTX of BKCa channel. 2. H2O2 as donator of Oxygen free radical could activated BKCa channel electric current as dose-dependent, and the antioxidants vitamin C could inhibited the activated electric current as dose-dependent, but it could not recover to normal level before drugged. 3. The experimental results show that the way of oxygen free radical /BKCa was existed in the process that oxygen free radidical donator H2O2 affect to the BKCa channels'electric current in outer hair cells of old guinea pigs'cochlears, but vitamin C as oxygen free radical scavenger can reverse the process in a large extent. 4. the way of oxygen free radical /BKCa might take a negative feedback mechanism in inhibition of calcium overload in outer hair cells and damage to nerve cells, and anti-oxidant might lessen the effect of oxygen free radical to BKCa channels in outer hair cells.
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