Cellular mechanism of acetylcholine-induced response in dissociated outer hair cells of guinea-pig cochlea

J Physiol. 1993 Apr;463:227-44. doi: 10.1113/jphysiol.1993.sp019592.

Abstract

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)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / pharmacology*
  • Animals
  • Calcium / metabolism
  • Calcium / pharmacology
  • Calmodulin / antagonists & inhibitors
  • Cations / pharmacology
  • Chelating Agents / pharmacology
  • Cochlea / cytology
  • Cochlea / drug effects*
  • Electrophysiology
  • GTP-Binding Proteins / metabolism
  • Guinea Pigs
  • Hair Cells, Auditory, Outer / drug effects*
  • In Vitro Techniques
  • Ion Channels / drug effects
  • Ion Channels / metabolism
  • Neural Pathways / drug effects
  • Parasympatholytics / pharmacology
  • Parasympathomimetics / pharmacology
  • Signal Transduction / drug effects

Substances

  • Calmodulin
  • Cations
  • Chelating Agents
  • Ion Channels
  • Parasympatholytics
  • Parasympathomimetics
  • GTP-Binding Proteins
  • Acetylcholine
  • Calcium