Regular articleTwo distinct forms of desensitization of G- protein coupled inwardly rectifying potassium currents evoked by alkaloid and peptide μ-opioid receptor agonists
Introduction
Repetitive applications of opioid alkaloids such as morphine (MS), heroin, and methadone (MD) invariably induce physical dependence, which is defined by a stereotypical withdrawal syndrome following the termination of application. The induction of dependence needs the activation of μ-opioid receptors (MORs), as mice with the targeted disruption of this receptor do not show any withdrawal syndrome when challenged with naloxone after chronic morphine administration (Matthes et al., 1996). The cellular and molecular mechanisms of dependence, although still elusive, are believed to be associated with a form of neuroadaptation of MOR -signaling Berke and Hyman 2000, Nestler and Aghajanian 1997, particularly in response to alkaloid agonists.
The prototypical agonist to induce dependence is the alkaloid morphine, whereas peptide agonists like [d-Ala2, NMe-Phe4, Gly5ol]enkephalin (DAMGO) are claimed to be much less efficient Noble et al 1994, Stevens and Yaksh 1989. These two groups of agonists also lead to very different responses when G-protein coupled inwardly rectifying potassium (GIRK) currents are monitored. Whereas Met-enkephalin (ME), endomorphine-1, and DAMGO at saturating concentrations elicit maximal GIRK currents that desensitize substantially within 15 min, the alkaloid morphine gives rise to smaller, but sustained currents (Blanchet and Lüscher, 2002). This observation has received considerable attention, since DAMGO, but not MS triggers endocytosis of MORs heterologously expressed in mammalian cells within 15–30 min (Keith et al., 1996). It may therefore be possible that internalization could protect neurons from overstimulation and thus prevent adaptive processes eventually leading to dependence (reviewed in Kieffer and Evans, 2002). This possibility is at the origin of the RAVE (relative activity versus endocytosis) model, which predicts abuse-liability of a given agonist by the ratio of the relative efficacy and its capability to trigger receptor internalization (Whistler et al., 1999). Here, we submit this hypothesis to further scrutiny by monitoring GIRK responses of neurons in acute slices of the locus coeruleus (LC) elicited by the alkaloid agonists MD and fentanyl (Ftn). We also searched for functional roles of rapid receptor redistribution on MOR-activated GIRK currents by interfering with endocytosis of clathrin-coated vesicles. Our results suggest that MOR-induced desensitization of GIRK currents is independent of receptor redistribution. Furthermore, we provide evidence that alkaloids lead to an apparent form of desensitization through a MOR-independent inhibition of GIRK channels.
Section snippets
Normal desensitization with impaired endocytosis
In LC neurons, DAMGO-elicited GIRK responses desensitized substantially within 15 min while morphine-induced responses did not (Blanchet and Lüscher, 2002). Interestingly, this dissociation for desensitization parallels the reported capability of these MOR agonists to drive internalization through clathrin-coated vesicles (Whistler et al., 1999), suggesting that desensitization may in fact be caused by a decreasing number of MORs at the cell surface, due to activity-driven endocytosis after
Discussion
Desensitization of MOR-evoked GIRK currents, broadly defined as a decrease in the response in the continuous presence of an agonist, is a form of neuroadaptation and may therefore be associated with dependence. As alkaloid agonists are considered to have a higher abuse liability compared to peptide agonists, any difference in signaling and desensitization may have implications for the understanding of dependence. Our results now indicate that desensitization occurs with both groups of agonists,
Electrophysiology in acute slices
Horizontal pontine slices (300 μm thick, VT1000 vibratome, Leica) were prepared from P10–P21 Sprague–Dawley rat brains (after decapitation according to the guidelines of the Cantonal Veterinary Office of Geneva) in cooled artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 119, KCl 2.5, MgCl2 1.3, CaCl2 2.5, NaH2PO4 1.0, NaHCO3 26.2, and glucose 11, and continuously bubbled with 95% O2 and 5% CO2. Slices were progressively warmed up to 32–34°C, left to recover for at least 1 h, and
Acknowledgements
We thank Michel Mühlethaler and Matt Frerking for critically reading prior versions of the manuscript. We acknowledge the help of members of the Lüscher and Muller labs, particularly Camilla Bellone and Tatiana Ivanova as well as Gisèle Gilliéron and Francçoise Loctin for technical support. C.L. is supported by a SCORE A award of the Swiss National Science Foundation and a young investigator’s grant of the Human Science Frontier Project.
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