Properties Of Cholinergic Receptor-Mediated Ion Channels On Type Ⅰ Vestibular Hair Cells Of Guinea Pigs

PART ONE Modified dissociation methods for typeâ… vestibular hair cells from guinea pigsObjective: To approach a modified method of dissociating vestibular typeâ… hair cells (VHCâ… )from guinea pigs, which could provide more cells with longer survival time and better cell activity . Methods: VHCsâ… were dissociated from guinea pigs separately by 0.25% collagenaseâ…£(according to the general dissociation methods)and 0.05% trypsin with two different dissociation methods and were investigated, identified and evaluated by the ways of microscopy, patch clamping and calcium imaging techniques. Results: General dissociation methods with 0.25% collagenaseâ…£: Averagely 25 VHCsâ… were isolated every otocyst, about 14 and only 8 cells survived at the end of 3h and 6h, the RP was -55±10mV and the intracellular Calcium concentration was 105±15nmol/ L at the end of 3h, conglomerate cell aggregates could be found easily by light microscope; General dissociation methods with 0.05% trypsin: Averagely 40 VHCsâ… were isolated every otocyst, about 19 and 6 cells survived at the end of 3h and 6h, the RP was -60±10mV and the intracellular Calcium concentration was 95±10nmol/ L at the end of 3h; Modified dissociation methods with 0.25% collagenaseâ…£: Averagely 24 VHCsâ… were isolated every otocyst, about 17 and 11 cells survived at the end of 3h and 6h, whose RP was -55±10mV and whose intracellular Calcium concentration was 95±15nmol/ L at the end of 3h; Modified dissociation methods with 0.05% trypsin: Averagely 42 VHCsâ… were isolated every otocyst, about 32 and 23 cells survived at the end of 3h and 6h, whose RP was -60±10mV and whose intracellular Calcium concentration was more stable average to 90±10 nmol/ L at the end of 3h, conglomerate cell aggregates could be rarely found by light microscope. Conclusions: Modified dissociation methods with trypsin digestion followed by trituation could supply sufficient amount of VHCsâ… with satisfactory survival time compared with general methods, which is more adequate for the experiments that desire better cell activity just as patch clamping.PART TWO Cholinergic receptors locate on partial typeâ… vestibular hair cells of guinea pigsObjective: To discuss the existence of cholinergic receptors on typeâ… vestibular hair cells of guinea pigs. Methods: electrophysiological responses of isolated typeâ… vestibular hair cells to external ACh were examined by the means of whole-cell patch-clamp recordings. Results: 7.5%(21/279)VHCsâ… were found to be sensitive to ACh(10～1000μmol/L). ACh generated an outward current in a steady, slow, dose-dependent (EC50=63.78±2.31μmol/L) manner, ACh at the level of 100μmol/L triggered an 170±15pA current at -50 mV. Conclusions: Cholinergic receptors locate on partial typeâ… vestibular hair cells of guinea pigs and extracellular dosage of ACh (10～1000μmol/L)could trigger a outward current in a steady, slow and dose-dependent manner.PART THREEProperties of cholinergic receptors-mediated ion channel on typeâ… vestibular hair cells of guinea pigsObjective: To study the properties of the cholinergic receptor-mediated ion channels on VHCsâ… Methods: electrophysiological responses of isolated typeâ… vestibular hair cells to external ACh were examined by the means of whole-cell patch-clamp recordings. Results: ACh generated an outward current in a steady, slow, dose-dependent (EC50=63.78±2.31μmol/L) and non-voltage-dependent manner. In standard extracellular solution, ACh at the level of 100μmol/L triggered an 170±15pA current at -50 mV. The time interval for the next complete activation of ACh-sensitive current was no less than 1 minute. The ion channels did not shut off even when they were exposed to ACh for an extended period of time. Conclusions: Dose-dependent, calcium-dependent and non-voltage-dependent cholinergic receptors were located on a few of the VHCsâ… of vestibular epithelium of guinea pigs. The cholinergic receptors did not show desensitization to ACh, while a slow and steady current was activated by ACh(10～1000μmol/L)applied extracellularly. This work reveals the existence of efferent neurotransmitter receptors on VHCsâ… and helps in understanding of the function of vestibular efferent nervous system, and may provide some useful information on guiding the clinical rehabilitative treatment of vertigo.PART FOUR Contribution of cholinergic agonists to the intracellular calcium concentration of typeâ… vestibular hair cells of guinea pigsObjective: to discuss the existence and the subtypes of cholinergic receptors located on the membrance of type I vestibular hair cells(VHCs I) of guinea pigs by investigating the contribution of cholinergic agonists to the intracellular calcium concentration([Ca2+]i) of VHCs I. Methods: The effects of cholinergic agonists on the free intracellular calcium concentration of acutely isolated type I vestibular hair cells were investigated using fura-2 fluorescence digital imaging methods. Results:â‘ In normal extracellular solution, the non-selective cholinergic agonist acetylcholine (ACh) and the muscarinic agonist muscarine both caused the increace of [Ca2+]i in separately 21 out of 25 and 14 out of 18 VHCs I, while only 3 out of 20 and 2 out of 16 VHCs I had a weak increase of [Ca2+]i if investigated in calcium free extracellular solution; nicotine could increase the [Ca2+]i in 7 out of 32 VHCsâ… only at enough high concentration(up to 10 mmol/L) in normal extracellular solution, this [Ca²⁺]_i increase by nicotine at high concentration could not be investigated if at calcium free solution.â‘¡The [Ca²⁺]_i increase of VHCs I could be repeatedly caused by ACh, the desensitization of cholinergic receptor was not found.â‘¢The muscarinic antagonist atropine inhibited the [Ca²⁺]_i increase of VHCs I, the nicotinic antagonist d- tubocurarine (d-TC) could inhibited the [Ca²⁺]_i increase of VHCs I in a competitive way.â‘£Preincubation separately with m1-m4 muscarinic antagonists pirenzepine,methoctramine,4-DAMP and tropicamide could decrease the amplitude of [Ca²⁺]_i increase in VHCs I caused by ACh. Compared with the other three selective antagonists which had no significant contribution, the m2 muscarinic antagonist methoctramine could decrease 35±10% of the amplitude of [Ca²⁺]_i increase in VHCs I caused by 100μmol/L ACh. Conclusions: Muscarinic and nicotinic cholinergic receptors locate on VHCs I of guinea pigs, ACh increase the [Ca²⁺]_i of VHCs I mainly by agitating the m2 muscarinic receptors; the infux of extracellular calcium is the major route of [Ca²⁺]_i increase; the cholinergic receptors on VHCs I of guinea pigs show no desensitization to ACh.PART FIVE Intracellular mechanism of signal transduction of acetylcholine-mediated current on typeâ… vestibular hair cells of guinea pigsObjective: To approach the Intracellular mechanism of signal transduction of acetylcholine-mediated current on typeâ… vestibular hair cells of guinea pigs.Methods: electrophysiological responses of isolated typeâ… vestibular hair cells to external ACh were examined by the means of whole-cell patch-clamp recordings. Results: In standard extracellular solution, ACh at the level of 100μmol/L triggered a steady outward current at -50 mV. The Big-conductance calcium-dependent potassium channels(BK) blocker charybdotoxin(CTX) suppress the amplitude of IACh substantially, while the small-conductance calcium-dependent potassium channels(SK) blocker apamin had no significant contribution; the m2 selective muscarinic antagonist methoctramine inhibit the amplitude of IACh on isolated typeâ… vestibular hair cells, while the m1,m3 or m4 selective muscarinic antagonist, pirenzepine ,4-DAMP or tropicamide had no significant contribution; pertussis toxin(PTX), the G protein inhibitor, suppress the amplitude of IACh, GTP-γ-s could trigger a outward current in transient time and this current faded out gradually with the flush of normal extracellular solution with 100μmol/L ACh.PKA inhibitor H-89 suppress the amplitude of IACh obviously while the extracellular dosage of forskolin, the PKA activator, generated a IACh-like current .The IP3 receptor antagonist heparin, PKG inhibitor KT5823 and PKC selective inhibitor BIM had no significant contribution to IACh. Extracellular application of db-cAMP could trigger a IACh-like current, the PLC inhibitor U73122 had no significant contribution to IACh. Conclusions: ACh agitats the m2 muscarinic receptors on typeâ… vestibular hair cells of guinea pigs and trigger a BK current, hose Intracellular mechanism of signal transduction is mediated by the m2 cholinergic receptor coupled pathway, which is associated with G protein, cAMP and PKA.