REGULATION OF W-OPIOID RECEPTORS, G-PROTEIN-COUPLED RECEPTOR KINASES AND L-ARRESTIN 2 IN THE RAT BRAIN AFTER CHRONIC OPIOID RECEPTOR ANTAGONISM A. DI Ł AZ, A. PAZOS, J. FLO Ł REZ, F. J. AYESTA, V. SANTANA and M. A. HURLE Ł Ã Department of Physiology and Pharmacology, School of Medicine, University of Cantabria, E-39011 Santander, Spain AbstractöThe aim of this study was to analyse the biochemical and behavioural consequences of chronic treatment with opioid receptor antagonists in rats. We have evaluated the respiratory depressant and antinociceptive e¡ects of the W- opioid agonist sufentanil, the density of brain W-opioid receptors, and the expression of G-protein-coupled receptor kinases and L-arrestin 2 in cerebral cortex and striatum, following sustained opioid receptor blockade. Our results demonstrate that 24 h after interruption of 7 days chronic infusion of naltrexone (120 Wg/h), the respiratory depressant potency of the W-opioid receptor agonist sufentanil was increased to a similar extent as the antinociceptive potency (about three-fold). This was accompanied by W-opioid receptor up-regulation in several areas of the rat brain associated with opioid control of pain perception and breathing. Moreover, chronic treatment with either naltrexone (120 Wg/h) or naloxone (120 Wg/h) caused signi¢cant increases in the expression levels of G-protein-coupled receptor kinases types 2, 3, and 6, and of L-arrestin 2 in brain cortex and striatum. Together our data suggest an increased constitutive receptor activity secondary to W-opioid receptor up-regulation following chronic antagonist treatment. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Keywords: autoradiography, analgesia, breathing, naloxone, naltrexone. Opioid receptors (W, N, and U) are G-protein-coupled receptors (GPCRs) that, like other GPCRs (see Fergusson, 2001), undergo adaptations such as desensi- tisation, internalisation, and down-regulation in response to agonist activation (D| ¤az et al., 2000; see Law et al., 2000). It is known that the molecular mechanisms responsible for agonist-dependent desensitisation of W-opioid receptors expressed in several cell lines involve G-protein-coupled receptors kinase (GRK)-mediated phosphorylation, and L-arrestin-dependent sequestration in endocytotic vesicles (Kovoor et al., 1997, 1998; Zhang et al., 1998; Wang, 2000). In living animals this regula- tory mechanism contributes to the receptor down-regu- lation and loss of analgesic potency observed after long- term opioid treatment, and to the addictive potential of this family of drugs (Terwilliger et al., 1994; Ozaita et al., 1998; Bohn et al., 1999, 2000; Hurle ¤, 2001). Con- versely to what occurs under the in£uence of agonists, opioid receptors undergo up-regulation and supersensi- tivity following the interruption of long-term treatment with opioid receptor antagonists such as naloxone and naltrexone, in both cultured cells and living animals (Morris et al., 1988; Yoburn et al., 1986, 1989; Unterwald et al., 1995). To date, there are not available data on the regulation of GRKs and L-arrestin expres- sion associated to these phenomena. The respiratory depressant e¡ect of opioids is a severe and life-threatening adverse reaction when these drugs are used as therapeutic agents in analgesia. Apnoea is also known to be the most frequent cause of death after opiate overdose in heroin addicts (see Flo ¤rez and Hurle ¤, 1993). Since naltrexone is being used as anti-crav- ing agent for opiate addictive processes (Kreek, 1996; Scha¡er and Naranjo, 1998), it is deemed of interest to assess whether the chronic administration of opioid receptor antagonists could modify the sensitivity to the respiratory depressant e¡ect of opioid receptor antago- nists as it occurs with the analgesia. In this study we have chronically administered opioid receptor antagonists in order to assess the changes in the expression levels of brain GRK2, GRK3, GRK6 and L-arrestin 2 that are associated with W-opioid receptor up-regulation. We have also evaluated the ability of chronic naltrexone to potentiate the respiratory depres- sant e¡ect of the W-opioid receptor agonist sufentanil in comparison with its antinociceptive e¡ect. 345 *Corresponding author. Tel.: +34-942-201961; fax: +34-942- 201903. E-mail address: hurlem@unican.es (M. A. Hurle ¤). Abbreviations: ANOVA, analysis of variance; DAMGO, [D-Ala(2)- MePhe(4)-Gly(5)-ol]enkephalin; GPCR, G-protein-coupled receptor; GRK, G-protein-coupled receptor kinase; PAGE, poly- acrylamide gel electrophoresis; PVDF, polyvinylidene di£uoride; SDS, sodium dodecyl sulphate; TBST, Tris-bu¡ered saline with Tween 20. NSC 5525 24-5-02 www.neuroscience-ibro.com Neuroscience Vol. 112, No. 2, pp. 345^353, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(02)00073-8 0306-4522 / 02 $22.00+0.00