Research Of The Mechanisms Of Muscarinic Receptor-Mediated Cholinergic Inhibition In Type ⅡVestibular Hair Cells Of Guinea Pigs

PART ONE The Presence of Functional m2Cholinergic Receptor on Type Ⅱ Vestibular Hair Cells of Guinea pigsObjective:To study the properties of Cholinergic Receptor in Type Ⅱ Vestibular Hair Cells of Guinea pigsMethods:A several of cholinergic antagonists and agonists were applied to the ACh-sensitive type Ⅱ hair cells, respectively by patch clamp whole cell recording.Results:①Extracellular200nM IBTX potently inhibited the ACh-mediated outward current.②Extracellular30μM MLA,1μM strychnine had no effect on the ACh-mediated BK current.③Extracellular m2-selective antagonist methoctramine100nM potently inhibited the ACh-mediated BK current, but not by300nM pirenzepine (ml-selective),10nM4-DAMP (m3-selective) and300nM tropicamide (m4-selective).④Extracellular30μM CCh (classical muscarinic agonist) and1μM APE (potent muscarinic agonist exhibiting slight selectivity for m2subtype) mimicked the effect of ACh, and were potently inhibited by300nM methoctramine each. Conclusion:The BK-type cholinergic inhibition on VHCs Ⅱ should be mediated by m2mAChR subtype. PART TWO The Intracellular signal Transduction Mechanisms of m2Muscarinic Receptor-Mediated Cholinergic Inhibition on Type II Vestibular Hair Cells of Guinea pigsObjective:We have demonstrated that m2mAChR mediated the cholinergic inhibition, we will further investigate its signal transduction pathway in type Ⅱ hair cells.Methods:A several of signal transduction reagents were applied to the ACh-sensitive type II hair cells, respectively by patch clamp whole cell recording.Results:①Intracellular application of GTPγS mimicked the ACh response. Inhibitor of a subunits of Gi-proteins GAPnt-2failed to suppress the ACh response, but inhibitor of a subunits and βγ subunits of Gi-proteins PTX completely abolished the ACh-sensitive BK current. db-cAMP mimicked the effect of ACh. These results suggested Giβγ subunits play a dominant role in the signal transduction pathway.②db-cAMP, a membrane analog of cAMP, mimicked the effect of ACh, and the adenylcyclase inhibitor2,5-dd-Ado totally abolished the ACh-sensitive BK current. These results suggested AC/cAMP is involved in the BK-type cholinergic inhibition.