TY - JOUR
T1 - Cellular mechanism of acetylcholine‐induced response in dissociated outer hair cells of guinea‐pig cochlea.
AU - Kakehata, S.
AU - Nakagawa, T.
AU - Takasaka, T.
AU - Akaike, N.
N1 - Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.
PY - 1993/4/1
Y1 - 1993/4/1
N2 - 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)
AB - 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)
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U2 - 10.1113/jphysiol.1993.sp019592
DO - 10.1113/jphysiol.1993.sp019592
M3 - Article
C2 - 7504105
AN - SCOPUS:0027538406
SN - 0022-3751
VL - 463
SP - 227
EP - 244
JO - The Journal of Physiology
JF - The Journal of Physiology
IS - 1
ER -