Haeme oxygenase-1 overexpression via nAChRs and the transcription factor Nrf2 has antinociceptive effects in the formalin test Javier Egea a,b, * , Angelo O. Rosa a,b,1 , Silvia Lorrio a,b , Laura del Barrio a,b , Antonio Cuadrado a,c , Manuela G. López a,b a Instituto Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4. 28029 Madrid, Spain b Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo, 4. 28029 Madrid, Spain c Dpto Bioquímica, Instituto de Investigaciones Biomédicas and Ciber de Enfermedades Neurodegenerativas (Ciberned), Facultad de Medicina, UAM, Spain article info Article history: Received 24 August 2008 Received in revised form 3 June 2009 Accepted 7 July 2009 Keywords: Epibatidine Haeme oxygenase-1 Pain Inflammation abstract Epibatidine has shown antinociceptive effects in various pain models, being 200-fold more potent than morphine. Previous results from our laboratory demonstrated that HO-1 overexpression has an antinoci- ceptive effect in the formalin test. Furthermore, epibatidine was able to induce haeme oxygenase-1 (HO-1). So, the aim of this study was to investigate the effect of HO-1 overexpression induced by epibati- dine in nociception elicited by formalin injection in the mice hindpaw. Administration of epibatidine (4 lg/kg) 24 h before the test reduced the nociceptive response during the first phase and second phase of the formalin test. This effect was prevented by treatment with tin protoporphyrin (SnPP, an inhibitor of HO-1 activity) administered via intraplantar 5 min before the test, suggesting a main role of HO-1. Western blot analysis revealed that epibatidine treatment increased by 2-fold HO-1 expression in the paw; this effect was lost in knockout mice for nuclear factor-erythroid 2-related factor 2 (Nrf2) and was accompanied by the loss of its antinociceptive effect. Furthermore, the antinociceptive effect of epi- batidine was related to the activation of alpha7 and/or alpha9 nAChRs since methyllycaconitine (MLA) and mecamylamine but not dihydro-b-erythroidine (DHbE) reverted this effect. Finally, we showed by flow cytometry and by immunofluorescence that white blood cells of the animals injected with epibati- dine expressed more HO-1 than control animals, and this expression was also reverted by MLA pre-treat- ment. These findings demonstrate that HO-1 induction by epibatidine has antinociceptive and anti-inflammatory effects by the activation of MLA-sensitive nAChRs. Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. 1. Introduction Epibatidine, an alkaloid isolated from the skin of the Equatorian poison-arrow frog Epipedobates tricolour, was found to be a potent non-opioid antinociceptive compound [41], 200-fold more potent than morphine. Epibatidine has shown antinociceptive effects in various models of pain such as the tail-flick test, hot plate test [4], and formalin test in the hind paw [6] and in the orofacial ver- sions [12]. Moreover, epibatidine has shown potent antinocicep- tive activity in some acute and chronic pain models when administered intrathecally or intraspinally in rats [15] or mice [7,29], and suppressing hyperalgesia and allodynia induced by nerve injury and capsaicin [17]. The antinociceptive mechanism of action of epibatidine remains unclear but is believed to be re- lated with the activation of neuronal nicotinic acetylcholine recep- tors (nAChRs) since mecamylamine, but not naloxone, an opioid antagonist, reverts its antinociceptive effects [2]. nAChRs subtypes are involved in different physiological func- tions, depending on their location in tissues [20,26]. Hence, there are evidences that point to the a4b2 nAChRs as the key receptor in antinociception in central nervous system (CNS) [4,14,21,23]. On the other hand, a7 nAChRs, which are distributed extensively in CNS and peripheral sites, are involved in many other important functions [5,36,52], including neuroprotection [9] and antinocicep- tion. In fact, choline, the precursor of acetylcholine (ACh), which is a selective endogenous agonist for a7 nAChRs [1], has antinocicep- tive effects against acute and inflammatory pain [53]. Furthermore, a7- and a9-containing nAChRs were localized in the plasma mem- brane of lymphocytes and macrophages and have been proposed as an essential regulator of inflammation [48,51]. Haeme oxygenase (HO) is the rate-limiting enzyme that de- grades the pro-oxidant heme group and produces equimolecular quantities of carbon monoxide, iron, and biliverdin. Biliverdin is 0304-3959/$36.00 Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2009.07.007 * Corresponding author. Address: Instituto Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4. 28029 Madrid, Spain. Tel.: +34 91 4975387; fax: +34 91 4975380. E-mail addresses: javier.egea@terra.es, javier.egea@uam.es (J. Egea). 1 Present address: Department of Pharmacology, Medical University of South Carolina, 173 Ashley Avenue BSC324, Charleston, SC 29425-5050, USA. PAIN Ò 146 (2009) 75–83 www.elsevier.com/locate/pain