Functional Roles Of Cholinergic Neurotransmitter In The Vestibular Peripheral Efferent Nerve System And The Protelytic Mechanisms Underlying The Changes Of Currents Mediated By AChR

Part I The signal transduction pathways of M2muscarinic ACh receptors (m2mAChR) sensitive BK channels mediate cholinergic inhibition of type II vestibular hair cellsObjective:Most studies had demonstrated that ACh played an important role in the vestibular efferent system. Our recent studies suggested that efferent release of ACh activates m2muscarinic ACh receptor (m2mAChR) with subsequent opening of a big conductance KCa current (BK) in VHCs II of guinea pig. M2mAChR is a G-protein-coupled muscarinic AChRs (mAChRs) which act through second-messenger systems. Therefore, the signal transduction pathways of M2muscarinic ACh receptors (m2mAChR) sensitive BK channels mediated cholinergic inhibition of type II vestibular hair cells is still unclear. Methods:Type II vestibular hair cells (VHCs II) was isolated by collagenase type IA. By the whole-cell patch clamp recording, the signal transduction pathways were investigated.Results:1)100μM GTPγS, a non-hydrolyzable GTP analogous, was activated a sustained outward current developed and reached to the peak amplitude (225.8±42.6pA, n=4) within1-2min.2) we investigated the role of Gi-protein by using the inhibitor of Gia subunit GAPnt-2. Pretreatment with10mM GAPnt-2for1min failed to affect the current amplitude (ACh,117.6±21.5pA, GAPnt-2+ACh111.6±19.3pA, p=0.14, n=5). However, PTX would catalyze the ADP-ribosylation of the Gi-protein, which could inhibit the activities of a subunit and βγ subunits. The ACh response was potently inhibited by intracellular application of400ng/ml PTX, and the maximum inhibition effect of85.3±6.6%(n=5) was obtained within4min.3)1mM db-cAMP, a membrane analogous of cAMP, co-applied with100mM ACh and the current amplitude was increased to about1.3times (n=5). Then,2,5-dideoxyadenosine (2,5-dd-Ado), an cell-permeable inhibitor of AC was applied. ACh-sensitive BK current was potently inhibited by pretreatment with100mM2,5-dd-Ado for1min; the inhibition effect was65.9±8.8%(n=5) and the recovery time was about3min (n=6).Conclusion:In the present study, the activation of m2mAChRs may stimulate Giβγ-mediated excitation of AC/cAMP activities and lead to the phosphorylation of Ca2+channels, resulting in the influx of Ca2+and opening of the BK channel. Part II Two distinct channels mediated by m2mAChR and a9nAChR co-exist in type II vestibular hair cells of guinea pigObjective:Acetylcholine (ACh) is the principal vestibular efferent neurotransmitter among mammalians. It is now established that efferent release of ACh activates a small conductance Ca2+-activated K+channels (KCa) current (SK2), which is mediated by a9-containing nicotinic ACh receptor (α9nAChR) in mammalian type II vestibular hair cells (VHCs II). However, our recent studies suggested that efferent release of ACh activates m2muscarinic ACh receptor (m2mAChR) with subsequent opening of a big conductance KCa current (BK) in VHCs II of guinea pig. To better elucidate the correlation between these two distinct channels in VHCs II of guinea pig, this study was designed to verify whether these two channels and their corresponding AChR subtypes co-exist in the same VHCs II by whole-cell patch clamp recordings.Methods:Type II vestibular hair cells (VHCs II) was isolated by collagenase type IA and trypsin. By the whole-cell patch clamp recording, the correlation between these two distinct channels in VHCs II of guinea pig was investigated.Results:We found that m2mAChR sensitive BK currents were activated in VHCs II isolated by collagenase IA, while a9nAChR sensitive SK2currents were activated in VHCs II isolated by trypsin. Interestingly, after brief exposure the patched cells isolated by trypsin to collagenase IA for3min, the a9nAChR sensitive SK2currents were abolished, while m2mAChR-sensitive BK currents were activated.Conclusion:Our findings provide evidence that the two distinct channels and their corresponding AChR subtypes may co-exist in the same VHCs II, and the alternative presence of these two ACh receptors-sensitive currents was dependent on the isolating preparation with different enzymes. Part Ⅲ Different expression patterns of SK2and BK channel in the rat vestibular epithelia and Scarpa’s ganglionObjective:Acetylcholine (ACh) is the principal vestibular efferent neurotransmitter among mammalians. It has been reported that efferent release of ACh activates a small conductance Ca2+-activated K+channels (KCa) current (SK2), which is mediated by a9-containing nicotinic ACh receptor (a9nAChR) in mammalian type II vestibular hair cells (VHCs II). However, our recent studies suggested that efferent release of ACh activates m2muscarinic ACh receptor (m2mAChR) with subsequent opening of a big conductance KCa current (BK) in VHCs II of guinea pig. While the distribution of SK2and BK channel in the adult rat vestibular epithelia and Scarpa’s ganglion is still unknown.Methods:The cochlea of normal SD rat was embedded with OCT and frozen section, the cryosection were examined with immunofluorescence to investigate the expression patterns of SK2and BK channel in the adult rat vestibular epithelia and Scarpa’s ganglion.Results:1) SK2channel and BK channel are expressed in adult rat vestibular hair cells and the region of vestibular ganglion.2) BK channel and SK2channel is expressed in the three types vestibular ganglion neurons (SGN) in the adult rat.3) SK2channel is expressed both in the vestibular calyx, dimorphic afferents and type I vestibular hair cell, but BK channel is localized in type I vestibular hair cell but not in the vestibular calyx afferents.4) SK2channel is expressed both in the vestibular bouton afferents and type II vestibular hair cell, but BK channel is localized in type II vestibular hair cell but not in the vestibular bouton afferents. Conclusion:SK2channel and BK channel are expressed in adult rat vestibular hair cells and vestibular ganglion neuron. These results reveal that SK2channel and BK channel not only involve in the cholinergic inhibition of type II vestibular hair cells, but also play an important role in neuronal excitability of type I vestibular hair cells and vestibular ganglion neurons.