Morphine inhibits an α9-acetylcholine nicotinic receptor-mediated response by a mechanism which does not involve opioid receptors
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)
- et al.
Localization of enkephalin-like immunoreactivity in acetylcholinesterase-positive cells in the guinea-pig lateral superior olivary complex that project to the cochlea
Neuroscience
(1983) - et al.
Enkephalin-like immunoreactivity in the guinea pig organ of Corti: ultrastructural and lesion studies
Hear. Res.
(1984) - et al.
Opioid peptides as possible neuromodulators of the afferent synaptic transmission in the frog semicircular canal
Neuroscience
(1999) Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor
Biochim. Biophys. Acta
(1998)- et al.
Vestibular and cochlear efferent neurons in the monkey identified by immunocytochemical methods
Brain Res.
(1987) - et al.
Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells
Cell
(1994) - et al.
In vitro pharmacologic characterization of a cholinergic receptor on outer hair cells
Hear. Res.
(1994) - et al.
The vestibular hair cells: post-transductional signal processing
Prog. Neurobiol.
(1998) - et al.
Expression of nicotinic acetylcholine receptor mRNA in the adult rat peripheral vestibular system
Brain Res.
(1996) - et al.
Several distinct receptor binding enkephalins in olivocochlear fibers and terminals in the organ of Corti
Brain Res.
(1984)
Proenkephalin and prodynorphin related neuropeptides in the cochlea
Hear. Res.
Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats
Brain Res.
Efferent synapses in the inner hair cell area of the cat cochlea: an electron microscopic study of serial sections
Hear. Res.
The release of β-endorphin and the neuropeptide-receptor mismatch in the brain
Brain Res.
Choline-acetyltransferase-like immunoreactivity in the organ of Corti of the rat during postnatal development
Brain Res. Dev. Brain Res.
Affinity profiles of morphine, codeine, dihydrocodeine and their glucuronides at opioid receptor subtypes
Life Sci.
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.
Expression of the nicotinic acetylcholine receptor subunit, α9, in the guinea pig cochlea
Hear. Res.
Immunohistochemical investigation of enkephalins in the human inner ear
Hear. Res.
Delta-opioid-receptor activation by [D-Pen2,D-Pen5]enkephalin and morphine inhibits substance P release from trigeminal nucleus slices
Eur. J. Pharmacol.
Role of α9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation
Neuron
The receptors activated by exogenous and endogenous opioids in the superior cervical ganglion of the cat
Brain Res.
Colocalization of enkephalin-like and choline acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig
J. Histochem. Cytochem.
Lateral olivocochlear neurons contain both enkephalin and dynorphin immunoreactivities: immunocytochemical co-localization studies
J. Histochem. Cytochem.
Expression profiling of vestibular hair cells from the frog
ARO Abstr.
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Augmented breaths ('sighs') are suppressed by morphine in a dose-dependent fashion via naloxone-sensitive pathways in adult rats
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The efferent medial olivocochlear-hair cell synapse
2012, Journal of Physiology ParisCitation Excerpt :Thus, when expressed in Xenopus laevis oocytes, α9 forms homomeric (Elgoyhen et al., 1994), calcium-permeable (Katz et al., 2000), ACh-gated channels with the following pharmacological properties, which are largely indistinguishable from those reported for the native hair cell cholinergic receptor: it is not activated but blocked by nicotine and muscarine, by the nicotinic antagonist curare and by the neuronal nAChR antagonist α-bungarotoxin (in a reversible manner, different to the blockade of neuronal receptors), the muscarinic antagonist atropine, the glycinergic antagonist strychnine, the GABAergic antagonist bicuculline and the serotonin type 3 receptor antagonists ICS 205-930 and ondansetron (Elgoyhen et al., 1994; Rothlin et al., 1999, 2003; Verbitsky et al., 2000). Modulation of α9-containing receptors by opioid compounds (Lioudyno et al., 2000, 2002), ryanodine (Zorrilla de San Martin et al., 2007), ototoxic drugs such as quinine (Ballestero et al., 2005) and aminoglycoside antibiotics (Rothlin et al., 2000) and neramexane, a drug under investigation for the treatment of tinnitus (Plazas et al., 2007; Suckfull et al., 2011), has been also reported. Moreover, some α-conotoxins which are valuable tools to differentiate nicotinic receptors, have high affinity for α9-containing receptors (McIntosh et al., 2005, 2009; Ellison et al., 2006; Nevin et al., 2007).
Efferent System
2008, The Senses: A Comprehensive ReferenceFast cholinergic efferent inhibition in guinea pig outer hair cells
2006, Brain ResearchCitation Excerpt :α9-Containing nAChRs-mediated SK channels play a role in mediating the cholinergic efferent synapse inhibition (Vetter et al., 1999; Jagger et al., 2000; Katz et al., 2000; Oliver et al., 2000; Holt et al., 2001). OHCs, obtained from guinea pig cochlea in this study, displayed a rapidly activating IK(ACh), which was in high agreement with the profiles of the α9-containing nAChRs-mediated SK current in previous studies of mammalian OHCs and frog saccular hair cells (Yoshioda et al., 1994; Blanchet et al., 2000; Lioudyno et al., 2000; Holt et al., 2001). Resembled the classical properties of SK current in other kinds of cells, IK(ACh) in this study was potently blocked by apamin, but not inhibited by IBTX.