Effects of Glycogen Synthase Kinase 3 and Cyclin-Dependent Kinase 5 Inhibitors on Morphine-Induced Analgesia and Tolerance in Rats

Repeated administration of morphine is associated with the development of tolerance, yet the mechanism underlying this phenomenon is still poorly understood. Recent evidence implicating glycogen synthase kinase 3 (GSK3) in opioid receptor signaling pathways has prompted us to investigate its role in morphine tolerance. Administration of 10 mg/kg morphine i.p. to Wistar rats twice daily for 8 days resulted in complete tolerance to its analgesic effects as measured by the tail-flick test. When injections of morphine were preceded by intrathecal (i.t.) administration of either an inhibitor of GSK3 [(3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) or 6-bromoindirubin-3 oxime] or an inhibitor of cyclin-dependent kinase (Cdk), roscovitine, development of tolerance to morphine analgesia was completely abolished. In addition, a single i.t. injection of either kinase inhibitor was able to restore in a dose-dependent manner the analgesic effect of morphine in morphine-tolerant rats. None of the inhibitors in doses used in the present study had analgesic effects of their own nor an effect on the analgesic potency of morphine. Repeated i.t. administration of either inhibitor had caused an increase in abundance of GSK-3 phosphorylated at Ser in the dorsal lumbar part of the spinal cord of rats that were chronically treated with morphine. Furthermore, reversal of morphine tolerance by a single injection of either inhibitor was always associated with increased abundance of phospho-GSK3 . In conclusion, our data indicate that chronic morphine treatment activates a highly efficient pathway by means of which Cdk5 regulates GSK3 activity. Adaptations in cellular signaling evoked by repeated morphine administration that lead to development of tolerance are still unclear. Binding of morphine to its main target, the -opioid receptor, results in the activation of a Gi/o protein, inhibition of adenylate cyclases, and downregulation of the cAMP second messenger pathway. Prolonged exposure to morphine or specific -opioid receptor agonists has an opposite effect and leads to up-regulation of the cAMP pathway, possibly through the actions of the subunits of Gi/o proteins (Tan et al., 2003), although the involvement of regulators of G protein signaling (RGS proteins) was also indicated (Gold et al., 2003). It was reported that morphine could activate the mitogen-activated protein kinase cascade (Berhow et al., 1996; Bohn et al., 2000; Bilecki et al., 2004; Muller and Unterwald, 2004) with the involvement of the phosphoinositol-3-kinase and protein kinase C kinases (Tan et al., 2003; Belcheva et al., 2005). Activation of the phosphoinositol-3-kinase also led to phosphorylation of Akt and other down-stream effectors (Polakiewicz et al., 1998; Iglesias et al., 2003; Muller and Unterwald, 2004). Recent evidence points to the possibilThis research was supported by statutory funds (BP) by Grant PBZ-KBN033/P05/2000 (to J.R.P., I.O., A.W.B., and R.P.) from the Ministry of Scientific Research and Information Technology (Warsaw, Poland) and partially by European Union Grant GENADDICT LSHM-CT-2004-005166 (to W.M.). I.O. received a contribution from the Foundation for Polish Science. J.R.P. and I.O. contributed equally to this work. Parts of the work were presented at The 15th Annual Meeting of the European Neuropeptide Club; 2005 May 19–21; Riga, Latvia; The 11th World Congress on Pain; 2005 Aug 18–19, 2005; Sydney, Australia; and The 7th International Congress of the Polish Neuroscience Society, 2005 Sept 7–10, 2005; Kraków, Poland. 1 Current affiliation: Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.107581. ABBREVIATIONS: GSK, glycogen synthase kinase; Cdk, cyclin-dependent kinase; DMSO, dimethyl sulfoxide; SB216763, 3-(2,4-dichlorophenyl)4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; roscovitine, 2-(R)-[[9-(1-methylethyl)-6-[(phenylmethyl)amino]-9H-purin-2-yl]amino]-1-butanol; BIO, (2 Z,3 E)-6-bromoindirubin-3 -oxime; PP1, protein phosphatase 1; DARPP-32, dopamineand cAMP-regulated phosphoprotein of Mr 32,000; NMDA, N-methyl-D-aspartate. 0022-3565/06/3192-832–839$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 319, No. 2 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 107581/3147477 JPET 319:832–839, 2006 Printed in U.S.A. 832 at A PE T Jornals on O cber 7, 2017 jpet.asjournals.org D ow nladed from ity, that the glycogen synthase kinase (GSK) 3 may be involved in opioid signaling. GSK3 is one of the primary targets of the Akt kinases, and in vivo induction of Akt phosphorylation by morphine was found (Muller and Unterwald, 2004). Accordingly, it was found that cardioprotection afforded by morphine was indeed associated with GSK3 inhibition (Gross et al., 2004). In addition, lithium salts, which act primarily as GSK3 inhibitors (Gould and Manji, 2005), were found to modulate morphine effects, but depending on dose and administration procedure, both enhancement (You et al., 2001) and reduction (Dehpour et al., 1994; Johnston and Westbrook, 2004) of morphineinduced analgesia were reported. Finally, our expression profiling of gene expression in the amygdala after chronic morphine self-administration in rats has implicated possible alterations in the GSK3/Wnt signaling (Rodriguez Parkitna et al., 2004). Here we report that spinal administration of GSK3 or Cdk5 inhibitors abolished tolerance to morphine analgesia. Furthermore, reversal of tolerance was always associated with an increase in abundance of GSK3 phosphorylated at Ser. These findings suggest the involvement of Gsk3 and Cdk5 in opioid antinociception and tolerance and indicate a novel signaling pathway linking Cdk5 to GSK3 in the mechanism of long-term adaptations to morphine action. Materials and Methods