Effects of serine/threonine protein phosphatase inhibitors on morphine-induced antinociception in the tail flick test in mice Ana Moncada, Cruz Miguel Cenda ´n, Jose ´ M. Baeyens, Esperanza Del Pozo * Department of Pharmacology and Neuroscience Institute, Medical School, University of Granada, Avenida de Madrid 11, 18012 Granada, Spain Received 4 November 2002; received in revised form 5 February 2003; accepted 11 February 2003 Abstract The aim of this study was to evaluate the effects of serine/threonine protein phosphatase (PP) inhibitors on morphine-induced antinociception in the tail flick test in mice, and on [ 3 H]naloxone binding to the forebrain crude synaptosome fraction. Neither okadaic acid nor cantharidin (1 – 10 000 nM) displaced [ 3 H]naloxone from its specific binding sites, which indicates that they do not interact at the opioid receptor level. The i.c.v. administration of very low doses of okadaic acid (0.001– 1 pg/mouse) and cantharidin (0.001 – 1 ng/mouse), which inhibit PP2A, produced a dose-dependent antagonism of the antinociception induced by morphine (s.c.). However, L-norokadaone (0.001 pg/ mouse –1 ng/mouse, i.c.v.), an analogue of okadaic acid lacking activity against protein phosphatases, did not affect the antinociceptive effect of morphine. On the other hand, high doses of okadaic acid (10 ng/mouse, i.c.v.) and cantharidin (1 Ag/mouse, i.c.v.), which also block PP1, and calyculin-A (0.1 fg/mouse–1 ng/mouse, i.c.v.), which inhibits equally both PP1 and PP2A, did not modify the morphine-induced antinociception. These results suggest that the activation of type 2A serine/threonine protein phosphatases may play a role in the antinociceptive effect of morphine, and that PP1 might counterbalace this activity. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Antinociception; Morphine; Serine/threonine protein phosphatase; Okadaic acid; Cantharidin; Calyculin-A; L-Norokadaone; [ 3 H]Naloxone 1. Introduction The reversible phosphorylation and dephosphorylation of proteins, catalysed by protein kinases and protein phospha- tases, respectively, play an important role in the control of intracellular cell events. Serine/threonine protein phospha- tases (PPs) dephosphorylate serine and threonine residues in proteins (Van Hoof et al., 1996). Numerous types of PPs are present in the brain, but the best characterized are type 1 (PP1) and type 2 (PP2) (Price and Mumby, 1999). Type 2 protein phosphatases can be further subdivided into metal ion-independent PP2A, Ca 2+ -calmodulin-dependent PP2B, and Mg 2+ -dependent PP2C (see Van Hoof et al., 1996, for a review). Several drugs can inhibit the functions of these phosphatases. Okadaic acid and cantharidin are cell perme- able agents that inhibit PP2A in vitro at much lower concentrations than PP1 (Ishihara et al., 1989; Cohen et al., 1989, 1990; Hardie et al., 1991; Suganuma et al., 1992; Li et al., 1993; Honkanen, 1993). Calyculin-A is another cell permeable protein phosphatase inhibitor, which is equally potent against PP1 and PP2A (Ishihara et al., 1989; Suganuma et al., 1992; Honkanen et al., 1994; Girault, 1994). It has been reported that PPs modulate several biochem- ical and electrophysiological processes occurring after A- opioid receptor activation. Okadaic acid completely reverses the inhibitory effects of sufentanil upon cAMP formation in guinea pig longitudinal muscle (Wang et al., 1996). Okadaic acid also decreases the rate of recovery from desensitisation of currents induced by stimulating A- and y-opioid receptors (Osborne and Williams, 1995; Morikawa et al., 1998). However, there are few studies on the role of the PPs on opioid-induced effects in vivo. Bernstein and Welch (1998) have evaluated the effects of okadaic acid in tail flick test in naive and morphine-tolerant mice, and have reported that relatively high doses of this drug do not modify the effect of morphine in naive animals and increase the antinociception induced by morphine in animals which have received chronic treatment with this opioid. The effects of other inhibitors of PP1 or PP2A on morphine-induced antinoci- ception have not been tested. In the present study we evaluated the effects of three inhibitors of PPs on the antinociception induced by the acute 0014-2999/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0014-2999(03)01461-4 * Corresponding author. Tel.: +34-958-243539; fax: +34-958-243539. E-mail address: edpozo@ugr.es (E. Del Pozo). www.elsevier.com/locate/ejphar European Journal of Pharmacology 465 (2003) 53 – 60