Elsevier

Hearing Research

Volume 149, Issues 1–2, November 2000, Pages 167-177
Hearing Research

Morphine inhibits an α9-acetylcholine nicotinic receptor-mediated response by a mechanism which does not involve opioid receptors

https://doi.org/10.1016/S0378-5955(00)00180-5Get rights and content

Abstract

Nicotinic acetylcholine (nACh) receptors are known to be targets for modulation by a number of substances, including the opiates. It is known that acetylcholine (ACh) coexists with opioid peptides in cochlear efferent neurons, and such a colocalization has been proposed for the vestibular system. In the present study we test the hypothesis that morphine, an opioid receptor agonist with a broad spectrum of selectivity, modulates α9nACh receptor-mediated responses in frog vestibular hair cells. Morphine dose-dependently and reversibly inhibited ACh-induced currents as recorded by the perforated patch-clamp method. In the presence of morphine the ACh dose–response curve was shifted to the right in a parallel fashion, suggesting a competitive interaction. However, naloxone did not antagonize the inhibition produced by morphine. To test the hypothesis that morphine could interact with the α9nACh receptor without the involvement of opioid receptors, experiments were performed using Xenopus laevis oocytes injected with the α9nACh receptor cRNA. The currents activated by ACh in Xenopus oocytes, a system that lacks opioid receptors, were also dose-dependently inhibited by morphine. We conclude that morphine inhibits the α9nACh receptor-mediated response in hair cells and Xenopus oocytes through a mechanism which does not involve opioid receptors but may be a direct block of the α9nACh receptor.

Introduction

The functions of many ionotropic neurotransmitter receptors can be modulated by substances other than their natural ligands. Nicotinic acetylcholine (nACh) receptors are known to be targets for modulation by a number of chemically different substances (Arias, 1998), as well as by several neuropeptides including dynorphin (Oka et al., 1998) and met-enkephalin (Kumakura et al., 1980, Zhang et al., 1993). The modulatory effects of neuropeptides can be mediated either by their own G-protein coupled receptors, or by direct interaction with the nACh receptor. The latter may have particular importance when the receptors for neuropeptides are not present in the vicinity of their release sites. Such ‘mismatches’ have been described for opioid peptides and their receptors (MacMillan et al., 1998).

It is generally accepted that ACh is the major efferent transmitter of auditory and vestibular efferents (Guth et al., 1998). The response to ACh in outer hair cells of the mammalian cochlea, chick and turtle papilla, and frog saccular hair cells, is mediated by an α9-containing nACh (α9nACh) receptor (Fuchs and Murrow, 1992, Erostegui et al., 1994), which has an unusual pharmacological profile (Elgoyhen et al., 1994, Rothlin et al., 1999) and a restricted pattern of expression (Elgoyhen et al., 1994, Hiel et al., 1996, Athas et al., 1997, Park et al., 1997).

In addition to ACh, the opioid peptides were localized in the efferent neurons of the cochlea (Fex and Altschuler, 1981, Hoffman et al., 1984, Altschuler et al., 1988), and such a colocalization has been proposed for vestibular efferents (Guth et al., 1998). The coexistence of the enkephalins with ACh in peripheral sympathetic neurons have been reported previously (Kondo et al., 1985, Morales et al., 1995), suggesting that opioid peptides may modulate the effects of the classic neurotransmitters. We therefore decided to test the hypothesis that the broad-spectrum opioid receptor agonist, morphine, modulates α9nACh receptor-mediated responses in vestibular hair cells. In our study, the perforated-patch variant of the patch-clamp method was used to investigate the effects of morphine on ACh-evoked currents in frog saccular hair cells. Additionally, the experiments with Xenopus oocytes, a system which lacks opioid receptors, were performed to test the alternative hypothesis that morphine modulates the α9nACh receptor without involvement of the opioid receptors. Xenopus oocytes are a useful model for investigation of functional expression of nACh receptors after injection of cRNA encoding nACh receptor subunits (Boulter et al., 1987). When expressed in Xenopus laevis oocytes, α9 forms a homomeric receptor–channel complex that is activated by ACh and demonstrates similar pharmacological properties to those described for the cholinergic receptor that mediates synaptic transmission between efferent cholinergic fibers and cochlear outer hair cells and vestibular saccular hair cells (Elgoyhen et al., 1994).

We demonstrate that morphine inhibits the α9nACh receptor-mediated response in vestibular hair cells and Xenopus oocytes injected with α9 cRNA through a mechanism which does not involve opioid receptors and therefore could be a direct block of the α9nACh receptor.

Section snippets

Isolation of hair cells

Frog saccular hair cells were isolated enzymatically. The dissociation protocol, optimized for the purpose of observing reliable ACh responses, was previously described (Holt and Guth, 1999). Briefly, leopard frogs (Rana pipiens) were chilled, pithed and decapitated. Each side of the head was placed into a perilymph-like standard external solution containing (in mM): 105 NaCl, 2.5 KCl, 0.81 MgCl2·6H2O, 1.8 CaCl2·2H2O, 3.4 NaHCO3, 0.5 NaH2PO4·H2O, 2.5 Na2HPO4, 1 ascorbate, 4 glucose and 5

The effect of morphine on ACh-evoked currents in isolated saccular hair cells

In saccular hair cells, the application of ACh typically produced outward currents (Fig. 1A), presumably of the small-conductance, Ca2+-activated potassium currents known as SK (Xia et al., 1998). According to our previous data obtained using current-clamp (Holt and Guth, 1999), these outward currents correspond to a hyperpolarization of the saccular hair cells. The response to ACh is sensitive to apamin and is completely and reversibly blocked by strychnine, which enabled us to consider this

Discussion

We have demonstrated that morphine dose-dependently inhibited ACh-evoked currents in isolated frog saccular hair cells. ACh is known to be the predominant efferent transmitter in the majority of the synapses between efferent nerve terminals and vestibular hair cells (Guth et al., 1998). In the cochlea, the release of ACh upon efferent stimulation as well as the synthesis of ACh by efferents have been demonstrated previously (Guth et al., 1976), suggesting that most of the efferents in the

Acknowledgements

This work was supported by NIH Grant DC00303 (P.S.G.) and an International Research Scholar grant from the Howard Hughes Medical Institute (A.B.E.). The authors wish to thank Ms A. Puri for the excellent technical assistance.

References (50)

  • D.W. Hoffman et al.

    Proenkephalin and prodynorphin related neuropeptides in the cochlea

    Hear. Res.

    (1985)
  • H. Kondo et al.

    Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats

    Brain Res.

    (1985)
  • M.C. Liberman

    Efferent synapses in the inner hair cell area of the cat cochlea: an electron microscopic study of serial sections

    Hear. Res.

    (1980)
  • S.J. MacMillan et al.

    The release of β-endorphin and the neuropeptide-receptor mismatch in the brain

    Brain Res.

    (1998)
  • A. Merchan Perez et al.

    Choline-acetyltransferase-like immunoreactivity in the organ of Corti of the rat during postnatal development

    Brain Res. Dev. Brain Res.

    (1994)
  • C. Mignat et al.

    Affinity profiles of morphine, codeine, dihydrocodeine and their glucuronides at opioid receptor subtypes

    Life Sci.

    (1995)
  • B.J. Morley et al.

    Identification of the subunits of the nicotinic cholinergic receptors in the rat cochlea using RT-PCR and in situ hybridization

    Brain Res. Mol. Brain Res.

    (1998)
  • H.J. Park et al.

    Expression of the nicotinic acetylcholine receptor subunit, α9, in the guinea pig cochlea

    Hear. Res.

    (1997)
  • A.W. Scholtz et al.

    Immunohistochemical investigation of enkephalins in the human inner ear

    Hear. Res.

    (1998)
  • H. Suarez-Roca et al.

    Delta-opioid-receptor activation by [D-Pen2,D-Pen5]enkephalin and morphine inhibits substance P release from trigeminal nucleus slices

    Eur. J. Pharmacol.

    (1992)
  • D.E. Vetter et al.

    Role of α9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation

    Neuron

    (1999)
  • C. Zhang et al.

    The receptors activated by exogenous and endogenous opioids in the superior cervical ganglion of the cat

    Brain Res.

    (1993)
  • R.A. Altschuler et al.

    Colocalization of enkephalin-like and choline acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig

    J. Histochem. Cytochem.

    (1984)
  • R.A. Altschuler et al.

    Lateral olivocochlear neurons contain both enkephalin and dynorphin immunoreactivities: immunocytochemical co-localization studies

    J. Histochem. Cytochem.

    (1988)
  • G.B. Athas et al.

    Expression profiling of vestibular hair cells from the frog

    ARO Abstr.

    (1997)
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