Neuropharmacology and Analgesia Reversible inhibition of intracellular calcium influx through NMDA receptors by imidazoline I 2 receptor antagonists Susan X. Jiang a , Rong-Yuan Zheng b, ⁎, Jin-Qi Zeng b , Xiao-Li Li b , Zhao Han b , Sheng T. Hou a,c,d, ⁎ a Experimental NeuroTherapeutics Laboratory, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6 b Department of Neurology, the First Affiliated Hospital and Research Institute of Experimental Neurobiology, Wenzhou Medical College, No. 2 Fuxue Lane, Wenzhou City, Zhejiang Province, 325000, PR China c Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada d Key Laboratory of Biotechnology, Pharmaceutical Engineering of Zhejiang Province, School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou City, Zhejiang Province, 325035, PR China abstract article info Article history: Received 27 August 2009 Received in revised form 12 November 2009 Accepted 23 November 2009 Available online 1 December 2009 Keywords: Imadazoline I 2 receptor α 2 -Adrenoceptor 2-BFI Idazoxan NMDA Glutamate Calcium Neuroprotection Excitotoxicity Intracellular calcium ([Ca 2+ ]i) influx through N-methyl-D-aspartic acid (NMDA) receptors in cortical neurons is central to NMDA receptor-mediated excitotoxicity. Drugs that uncompetitively modulate NMDA receptor-mediated [Ca 2+ ]i influx are potential leads for development to treat NMDA receptor-mediated neuronal damage since these drugs spare NMDA receptor normal functions. Ligands to α 2 -adrenoceptors and imidazoline I 2 receptors confer neuroprotection possibility through modulating NMDA receptor-mediated [Ca 2+ ]i influx. Here, we investigated the characteristics of several ligands to α 2 -adrenoceptors and imidazoline I 2 receptor, in inhibiting NMDA receptor-mediated [Ca 2+ ]i influx in cultured cortical neurons using a ratiometric calcium imaging technique. In contrast to MK801, which non-reversibly blocks NMDA receptor-mediated [Ca 2+ ]i influx, imidazoline I 2 receptor antagonists, Idazoxan, and 2-(2-benzofuranyl)-2- imidazoline (2-BFI)-mediated inhibition of [Ca 2+ ]i influx can be rapidly reversed when removed, in a manner similar to that of memantine, an uncompetitive antagonist to NMDA receptors. Interestingly, ligands to α 2 -adrenoceptors, including agmatine sulfate and yohimbine, and a ligand to the nicotinic receptor, levamisol, neither inhibited NMDA receptor-mediated [Ca 2+ ]i influx, nor provided neuroprotection against glutamate toxicity, suggesting selective inhibition of NMDA receptor activities. The inhibition of NMDA receptor by Idazoxan and 2-BFI also led to the suppression of NMDA receptor-mediated calpain activity as a result of blocking NMDA receptor activity, rather than through direct inhibition of calpain activity. Collectively, these studies demonstrated that imidazoline I 2 receptor antagonists transiently and reversibly block NMDA receptor-mediated [Ca 2+ ]i influx. These compounds are leads for further development as uncompetitive antagonists to NMDA receptor-mediated excitotoxicity. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. 1. Introduction Excessive activation of NMDA receptor-type glutamate receptors causes excitotoxicity, a process contributing to a wide range of neurological disorders including stroke, Alzheimer's disease, epilepsy, Huntington's disease, Parkinson's disease, spinal cord and head injury, HIV-associated dementia, multiple sclerosis and glaucoma (Mody and MacDonald, 1995; Hou and MacManus, 2002; Albensi et al., 2004; Villmann and Becker, 2007; Besancon et al., 2008; Hou et al., 2008). Excitotoxicity takes place primarily as the result of glutamate binding to NMDA receptors and, to a lesser extent, by binding to other receptor subtypes. Over-stimulation of NMDA receptors with L- glutamate or NMDA results in an excessive [Ca 2+ ]i influx leading to the activation of a plethora of potentially neurotoxic mechanisms, such as the early indication of a calcium-dependent protease, calpain, which cleaves intracellular structural proteins such as spectrin (Hewitt et al., 1998; Neumar et al., 2001). Pharmacological inhibition of NMDA receptors ameliorates excitotoxicity-mediated neuronal death and dysfunction. Therefore, it is believed that chemical molecules with an uncompetitive mechanism of action, a relatively fast off-rate and which only transiently block NMDA receptors, promise to be potential candidates as effective therapeutics in reducing excitotoxicity-evoked brain damage (Lipton and Chen, 2005; Lipton, 2006, 2007). A prototype of an uncompetitive, fast off-rate NMDA receptor blocker is memantine. The drug memantine blocks NMDA receptors preferentially when they are excessively open (hence open channel European Journal of Pharmacology 629 (2010) 12–19 ⁎ Corresponding authors. Experimental NeuroTherapeutics Laboratory, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6. Tel.: +1 613 993 7764; fax: +1 613 941 4475. E-mail addresses: zhengry@yahoo.com.cn (R.-Y. Zheng), sheng.hou@nrc-cnrc.gc.ca (S.T. Hou). 0014-2999/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.11.063 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar