Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1993 Apr;463:227–244. doi: 10.1113/jphysiol.1993.sp019592

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

S Kakehata 1, T Nakagawa 1, T Takasaka 1, N Akaike 1
PMCID: PMC1175341  PMID: 7504105

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)

Full text

PDF
227

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Art J. J., Crawford A. C., Fettiplace R., Fuchs P. A. Efferent modulation of hair cell tuning in the cochlea of the turtle. J Physiol. 1985 Mar;360:397–421. doi: 10.1113/jphysiol.1985.sp015624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ashmore J. F. A fast motile response in guinea-pig outer hair cells: the cellular basis of the cochlear amplifier. J Physiol. 1987 Jul;388:323–347. doi: 10.1113/jphysiol.1987.sp016617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bascands J. L., Emond C., Pecher C., Regoli D., Girolami J. P. Bradykinin stimulates production of inositol (1,4,5) trisphosphate in cultured mesangial cells of the rat via a BK2-kinin receptor. Br J Pharmacol. 1991 Apr;102(4):962–966. doi: 10.1111/j.1476-5381.1991.tb12284.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bobbin R. P., Konishi T. Acetylcholine mimics crossed olivocochlear bundle stimulation. Nat New Biol. 1971 Jun 16;231(24):222–223. doi: 10.1038/newbio231222a0. [DOI] [PubMed] [Google Scholar]
  5. Bobbin R. P., Konishi T. Action of cholinergic and anticholinergic drugs at the crossed olivocochlear bundle-hair cell junction. Acta Otolaryngol. 1974 Jan-Feb;77(1):56–65. doi: 10.3109/00016487409124598. [DOI] [PubMed] [Google Scholar]
  6. Bonner T. I., Buckley N. J., Young A. C., Brann M. R. Identification of a family of muscarinic acetylcholine receptor genes. Science. 1987 Jul 31;237(4814):527–532. doi: 10.1126/science.3037705. [DOI] [PubMed] [Google Scholar]
  7. Bonner T. I., Young A. C., Brann M. R., Buckley N. J. Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron. 1988 Jul;1(5):403–410. doi: 10.1016/0896-6273(88)90190-0. [DOI] [PubMed] [Google Scholar]
  8. Brown D. A. G-proteins and potassium currents in neurons. Annu Rev Physiol. 1990;52:215–242. doi: 10.1146/annurev.ph.52.030190.001243. [DOI] [PubMed] [Google Scholar]
  9. Buckley N. J., Bonner T. I., Buckley C. M., Brann M. R. Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol Pharmacol. 1989 Apr;35(4):469–476. [PubMed] [Google Scholar]
  10. Canlon B., Cartaud J., Changeux J. P. Localization of alpha-bungarotoxin binding sites on outer hair cells from the guinea-pig cochlea. Acta Physiol Scand. 1989 Dec;137(4):549–550. doi: 10.1111/j.1748-1716.1989.tb08795.x. [DOI] [PubMed] [Google Scholar]
  11. Comis S. D., Leng G. Action of putative neurotransmitters in the guinea pig cochlea. Exp Brain Res. 1979 Jun 1;36(1):119–128. doi: 10.1007/BF00238472. [DOI] [PubMed] [Google Scholar]
  12. Dulon D., Zajic G., Schacht J. Increasing intracellular free calcium induces circumferential contractions in isolated cochlear outer hair cells. J Neurosci. 1990 Apr;10(4):1388–1397. doi: 10.1523/JNEUROSCI.10-04-01388.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eybalin M., Pujol R. Cochlear neuroactive substances. Arch Otorhinolaryngol. 1989;246(5):228–234. doi: 10.1007/BF00463561. [DOI] [PubMed] [Google Scholar]
  14. Flock A., Flock B., Ulfendahl M. Mechanisms of movement in outer hair cells and a possible structural basis. Arch Otorhinolaryngol. 1986;243(2):83–90. doi: 10.1007/BF00453755. [DOI] [PubMed] [Google Scholar]
  15. Fu T., Okano Y., Nozawa Y. Bradykinin-induced generation of inositol 1,4,5-trisphosphate in fibroblasts and neuroblastoma cells: effect of pertussis toxin, extracellular calcium, and down-regulation of protein kinase C. Biochem Biophys Res Commun. 1988 Dec 30;157(3):1429–1435. doi: 10.1016/s0006-291x(88)81035-0. [DOI] [PubMed] [Google Scholar]
  16. Fuchs P. A., Murrow B. W. A novel cholinergic receptor mediates inhibition of chick cochlear hair cells. Proc Biol Sci. 1992 Apr 22;248(1321):35–40. doi: 10.1098/rspb.1992.0039. [DOI] [PubMed] [Google Scholar]
  17. Fuchs P. A., Murrow B. W. Cholinergic inhibition of short (outer) hair cells of the chick's cochlea. J Neurosci. 1992 Mar;12(3):800–809. doi: 10.1523/JNEUROSCI.12-03-00800.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Galley N., Klinke R., Oertel W., Pause M., Storch W. H. The effect of intracochlearly administered acetylcholine-blocking agents on the efferent synapses of the cochlea. Brain Res. 1973 Dec 21;64:55–63. doi: 10.1016/0006-8993(73)90170-4. [DOI] [PubMed] [Google Scholar]
  19. Ghosh T. K., Eis P. S., Mullaney J. M., Ebert C. L., Gill D. L. Competitive, reversible, and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin. J Biol Chem. 1988 Aug 15;263(23):11075–11079. [PubMed] [Google Scholar]
  20. Giovannelli A., Grassi F., Mattei E., Mileo A. M., Eusebi F., Giovanelli A. Acetylcholine induces voltage-independent increase of cytosolic calcium in mouse myotubes. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10069–10073. doi: 10.1073/pnas.88.22.10069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guiramand J., Mayat E., Bartolami S., Lenoir M., Rumigny J. F., Pujol R., Récasens M. A M3 muscarinic receptor coupled to inositol phosphate formation in the rat cochlea? Biochem Pharmacol. 1990 Jun 15;39(12):1913–1919. doi: 10.1016/0006-2952(90)90609-o. [DOI] [PubMed] [Google Scholar]
  22. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  23. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  24. Hidaka H., Tanaka T. Transmembrane Ca2+ signaling and a new class of inhibitors. Methods Enzymol. 1987;139:570–582. doi: 10.1016/0076-6879(87)39113-x. [DOI] [PubMed] [Google Scholar]
  25. Horn R., Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol. 1988 Aug;92(2):145–159. doi: 10.1085/jgp.92.2.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Housley G. D., Ashmore J. F. Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea. Proc Biol Sci. 1991 May 22;244(1310):161–167. doi: 10.1098/rspb.1991.0065. [DOI] [PubMed] [Google Scholar]
  27. Hu J. R., el-Fakahany E. E. Selectivity of McN-A-343 in stimulating phosphoinositide hydrolysis mediated by M1 muscarinic receptors. Mol Pharmacol. 1990 Dec;38(6):895–903. [PubMed] [Google Scholar]
  28. Katada T., Ui M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci U S A. 1982 May;79(10):3129–3133. doi: 10.1073/pnas.79.10.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kijima H., Kijima S. Cooperative response of chemically excitable membrane. II. Two-state models and their limitations. J Theor Biol. 1980 Feb 7;82(3):425–463. doi: 10.1016/0022-5193(80)90248-9. [DOI] [PubMed] [Google Scholar]
  30. Klinke R., Galley N. Efferent innervation of vestibular and auditory receptors. Physiol Rev. 1974 Apr;54(2):316–357. doi: 10.1152/physrev.1974.54.2.316. [DOI] [PubMed] [Google Scholar]
  31. Klinke R. Neurotransmission in the inner ear. Hear Res. 1986;22:235–243. doi: 10.1016/0378-5955(86)90100-0. [DOI] [PubMed] [Google Scholar]
  32. Klinke R. Neurotransmitters in the cochlea and the cochlear nucleus. Acta Otolaryngol. 1981 May-Jun;91(5-6):541–554. doi: 10.3109/00016488109138540. [DOI] [PubMed] [Google Scholar]
  33. Konishi T. Action of tubocurarine and atropine on the crossed olivocochlear bundles. Acta Otolaryngol. 1972 Oct;74(4):252–264. doi: 10.3109/00016487209128447. [DOI] [PubMed] [Google Scholar]
  34. Kubo T., Fukuda K., Mikami A., Maeda A., Takahashi H., Mishina M., Haga T., Haga K., Ichiyama A., Kangawa K. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature. 1986 Oct 2;323(6087):411–416. doi: 10.1038/323411a0. [DOI] [PubMed] [Google Scholar]
  35. Marty A., Neher E. Potassium channels in cultured bovine adrenal chromaffin cells. J Physiol. 1985 Oct;367:117–141. doi: 10.1113/jphysiol.1985.sp015817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mitchelson F. Muscarinic receptor differentiation. Pharmacol Ther. 1988;37(3):357–423. doi: 10.1016/0163-7258(88)90005-8. [DOI] [PubMed] [Google Scholar]
  37. Murase K., Randic M., Shirasaki T., Nakagawa T., Akaike N. Serotonin suppresses N-methyl-D-aspartate responses in acutely isolated spinal dorsal horn neurons of the rat. Brain Res. 1990 Aug 13;525(1):84–91. doi: 10.1016/0006-8993(90)91323-9. [DOI] [PubMed] [Google Scholar]
  38. Murase K., Ryu P. D., Randic M. Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons. Neurosci Lett. 1989 Aug 14;103(1):56–63. doi: 10.1016/0304-3940(89)90485-0. [DOI] [PubMed] [Google Scholar]
  39. Nakagawa T., Akaike N., Kimitsuki T., Komune S., Arima T. ATP-induced current in isolated outer hair cells of guinea pig cochlea. J Neurophysiol. 1990 May;63(5):1068–1074. doi: 10.1152/jn.1990.63.5.1068. [DOI] [PubMed] [Google Scholar]
  40. Norris C. H., Guth P. S. The release of acetylcholine (ACH) by the crossed olivo-cochlear bundle (COCB). Acta Otolaryngol. 1974 May;77(5):318–326. doi: 10.3109/00016487409124631. [DOI] [PubMed] [Google Scholar]
  41. Peralta E. G., Ashkenazi A., Winslow J. W., Ramachandran J., Capon D. J. Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature. 1988 Aug 4;334(6181):434–437. doi: 10.1038/334434a0. [DOI] [PubMed] [Google Scholar]
  42. Plinkert P. K., Gitter A. H., Zimmermann U., Kirchner T., Tzartos S., Zenner H. P. Visualization and functional testing of acetylcholine receptor-like molecules in cochlear outer hair cells. Hear Res. 1990 Feb;44(1):25–34. doi: 10.1016/0378-5955(90)90019-l. [DOI] [PubMed] [Google Scholar]
  43. Rathinavelu A., Isom G. E. Regulation of ANF receptor internalization: involvement of extracellular calcium. Biochem Biophys Res Commun. 1991 Mar 29;175(3):1017–1022. doi: 10.1016/0006-291x(91)91666-z. [DOI] [PubMed] [Google Scholar]
  44. Robertson D., Johnstone B. M. Efferent transmitter substance in the mammalian cochlea: single neuron support for acetylcholine. Hear Res. 1978 Oct;1(1):31–34. doi: 10.1016/0378-5955(78)90006-0. [DOI] [PubMed] [Google Scholar]
  45. Schimerlik M. I. Structure and regulation of muscarinic receptors. Annu Rev Physiol. 1989;51:217–227. doi: 10.1146/annurev.ph.51.030189.001245. [DOI] [PubMed] [Google Scholar]
  46. Shigemoto T., Ohmori H. Muscarinic receptor hyperpolarizes cochlear hair cells of chick by activating Ca(2+)-activated K+ channels. J Physiol. 1991 Oct;442:669–690. doi: 10.1113/jphysiol.1991.sp018814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shirvan M. H., Pollard H. B., Heldman E. Mixed nicotinic and muscarinic features of cholinergic receptor coupled to secretion in bovine chromaffin cells. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4860–4864. doi: 10.1073/pnas.88.11.4860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Spoendlin H. Innervation patterns in the organ of corti of the cat. Acta Otolaryngol. 1969 Feb-Mar;67(2):239–254. doi: 10.3109/00016486909125448. [DOI] [PubMed] [Google Scholar]
  49. Steinacker A., Rojas L. Acetylcholine modulated potassium channel in the hair cell of the toadfish saccule. Hear Res. 1988 Sep 15;35(2-3):265–269. doi: 10.1016/0378-5955(88)90123-2. [DOI] [PubMed] [Google Scholar]
  50. Sugiyama H., Ito I., Hirono C. A new type of glutamate receptor linked to inositol phospholipid metabolism. Nature. 1987 Feb 5;325(6104):531–533. doi: 10.1038/325531a0. [DOI] [PubMed] [Google Scholar]
  51. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  52. Wiederhold M. L. Variations in the effects of electric stimulation of the crossed olivocochlear bundle on cat single auditory-nerve-fiber responses to tone bursts. J Acoust Soc Am. 1970 Oct;48(4):966–977. doi: 10.1121/1.1912235. [DOI] [PubMed] [Google Scholar]
  53. van Megen Y. J., Klaassen A. B., Rodrigues de Miranda J. F., Kuijpers W. Cholinergic muscarinic receptors in rat cochlea. Brain Res. 1988 Nov 22;474(1):185–188. doi: 10.1016/0006-8993(88)90682-8. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES