Cellular mechanism of acetylcholine-induced response in dissociated outer hair cells of guinea-pig cochlea.
1. The acetylcholine (ACh)-induced currents (IACh) in dissociated outer hair cells (OHCs) of guinea-pig cochlea were investigated using the whole-cell patch-clamp technique, in both conventional and nystatin perforated-patch configurations. 2. ACh and carbamylcholine (CCh) induced outward currents at a holding potential (VH) of -60 mV in the perforated-patch configuration. The IACh increased in a sigmoidal fashion over the concentration range between 3 x 10(-6) and 10(-3) M. The dissociation constant (KD) was 1.7 x 10(-5) M and the Hill coefficient (n) was 2.7. The KD and n for CCh were 8.7 x 10(-5) M and 2.2, respectively. Neither nicotine nor muscarine induced any detectable current up to a concentration of 10(-3) M. 3. Various muscarinic agonists such as oxotremorine-M, McN-A-343 and oxotremorine could also induce the outward currents, although these current amplitudes were about one-third that of ACh, indicating that they were partial agonists. 4. The muscarinic antagonists atropine, 4-DAMP, AF-DX 116 and pirenzepine inhibited the IACh in a concentration-dependent manner. The half-inhibitory concentrations (IC50) for atropine, 4-DAMP, AF-DX 116 and pirenzepine were 4.8 x 10(-6), 6.2 x 10(-6), 2.1 x 10(-5) and 2.9 x 10(-4) M, respectively. 5. When the extracellular Ca2+ concentration ([Ca2+])o) was reduced to lower than 1 mM, the amplitude of IACh, abruptly decreased. In a nominally Ca(2+)-free external solution ACh did not induce any current. The increase of [Ca2+]o beyond 1 mM did not change the IACh. 6. When OHCs were perfused intracellularly with a pipette solution containing 10 mM BAPTA in the conventional whole-cell mode, ACh could not induce outward K+ currents. The Ca2+ ionophore A23187 induced an outward current. These results indicate that intracellular Ca2+ is involved in the ACh response. 7. Calmodulin inhibitors such as chlorpromazine, W-7 and trifluoperazine inhibited the IACh in a concentration-dependent manner. 8. When OHCs were dialysed with either 100 microM GDP beta S or 1 micrograms/ml pertussis toxin (PTX) through the patch pipette at a VH of -60 mV, the IACh diminished within 10 min, whereas the IACh of the control remained steady for over 20 min, suggesting that a PTX-sensitive G-protein is involved in the ACh response.(ABSTRACT TRUNCATED AT 400 WORDS)
Kakehata
S
S
Department of Neurophysiology, Tohoku University School of Medicine, Sendai, Japan.
Nakagawa
T
T
Takasaka
T
T
Akaike
N
N
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002412:Cations / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D003051:Cochlea / Q000166:cytology / Q000187:drug effects*; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006168:Guinea Pigs; D018072:Hair Cells, Auditory, Outer / Q000187:drug effects*; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D009434:Neural Pathways / Q000187:drug effects; D010276:Parasympatholytics / Q000494:pharmacology; D010277:Parasympathomimetics / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
D002147:Calmodulin; D002412:Cations; D002614:Chelating Agents; D007473:Ion Channels; D010276:Parasympatholytics; D010277:Parasympathomimetics; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D002118:Calcium