The Inhibition Effect And Mechanism Of Gentamicin On The Signal Transduction Pathway In The Vestibular Cholinergic Efferent Nerve System Of Guinea Pig

Part I The effect of gentamicin on the ACh-induced calcium-dependent potassium current in guinea pig type II vestibular hair cellsObjective:It is generally accepted that ACh is the important inhibitory neurotransmitter of the mammalian vestibular efferent system. Our previous studies showed that ACh could activate the large conductance calcium-dependent potassium channel (BK) and small conductance calcium-dependent potassium channels (SK) in guinea pig type II vestibular hair cells, mediated by m2muscarinic ACh receptor (m2mAChR) and a9nAChR respectively. Gentamicin could block ACh-sensitive potassium current in guinea pig outer hair cells by competing with calcium ion at the cholinergic receptor. However, it remains poorly understood whether gentamicin can affect the potassium current induced by ACh in guinea pig vestibular hair cells.Methods:The vestibular epithelium was removed from anesthetized young guinea pigs, then vestibular hair cells were isolated by enzymolysis and mechanical dissociation. The whole-cell patch clamp technique was used when the VHCs in good condition were on the bottom of the experimental chamber coated with rat collagen.Results:1) The m2mAChR-mediated BK currents were recorded in guinea pig type II vestibular hair cells isolated by collagenase IA, and the BK currents could be potently blocked by methoctramine (an m2mAChR antagonist). Gentamicin could reversibly inhibit the ACh-induced BK currents in a dose-dependent and voltage-independent manner.2) The α9nAChR-mediated SK currents were recorded in guinea pig VHCs II isolated by trypsin, and the SK current could be potently blocked by strychnine (an a9nAChR antagonist). Gentamicin could reversibly inhibit the ACh-induced SK current in a dose-denpendent manner.Conclusion:We found that gentamicin could inhibit the ACh-induced BK and SK currents in guinea pig VHCs II reversibly in a dose-dependent manner, and gentamicin inhibition of the BK currents was in a manner of voltage-independent. Part II The inhibition mechanism of gentamicin on the ACh-induced BK current in guinea pig type Ⅱ vestibular hair cellsObjective:Our recent study found that gentamicin could inhibit the ACh-induced BK currents in guinea pig VHCs II reversibly, but the mechanism is still unknown.Methods:The vestibular epithelium were removed from anesthetized young guinea pig, then vestibular hair cells were isolated by enzyme and mechanical dissociation. The whole-cell patch clamp technique was used when the VHCs in good condition were on the bottom of the experimental chamber coated with rat collagen.Results:1) Increasing the ACh concentration had little influence on the gentamicin inhibition effect on the ACh-induced BK currents in guinea pig VHCs Ⅱ.2) Increasing the extracellular calcium concentration could antagonize the GM inhibition effect.3) In the different extracellular calcium concentration solutions, strychnine (a potent nAChR antagonist) did not affect the ACh-induced BK currents.4) Gentamicin could potently inhibite inward calcium currents evoked by (-)-Bay-K8644(an L-type calcium channel agonist).5) Gentamicin slightly inhibited the BK current evoked by NS1619(a BK channel activator) in guinea pig VHCs II at high concentration (300μM).Conclusion:Acute gentamicin application could inhibit the ACh-induced BK current in guinea pig VHCs II mainly by competing with calcium ion at the L-type voltage-dependent calcium channel, which results in the decrease of calcium influx and the subsequent reduction of the BK current, but not by direct affecting the m2mAChR. Gentamicin may slightly inhibit the BK channel in high concentration, but it could not be the main mechanism of the inhibition on the ACh-induced BK current. Part Ⅲ The inhibition mechanism of gentamicin on the ACh-induced SK current in guinea pig type II vestibular hair cellsObjective:Our recent study found that gentamicin could block the ACh-induced SK currents in guinea pig VHCs II reversibly, but the mechanism of this inhibition is still unknown.Methods:The vestibular epithelium were removed from anesthetized young guinea pig, then vestibular hair cells were isolated by enzyme and mechanical dissociation. The whole-cell patch clamp technique was used when the VHCs in good condition were on the bottom of the experimental chamber coated with rat collagen.Results:1) Increasing the ACh concentration could antagonize the gentamicin inhibition effect on the ACh-induced SK current in guinea pig VHCs II.2) Increasing the extracellular calcium concentration could antagonize the GM inhibition effect on the ACh-induced SK current in guinea pig VHCs II.Conclusion:Acute gentamicin application could inhibit the ACh-induced SK current in guinea pig VHCs II by competing with ACh and calcium at the α9nAChRs to decrease the SK current. It was different from the competition with calcium at the L-type voltage-dependent calcium channel withoutout affecting the binding of ACh and m2mAChRs in gentamicin inhibition on BK currents. Compared with gentamicin inhibition on the ACh-induced K+current in guinea pig OHC, we found that ACh might activate the BK channels and SK channels mediated by nAChRs at the same time. The hypothesis should be further investigated.