Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-ha7 cells by activating neuronal nicotinic acetylcholine receptors Jukka S. Pakkanen,* Heli Nousiainen,* Jari Yli-Kauhaluoma, Irene Kyla ¨nlahti,  Tommi Mo ¨ ykkynen,à Esa R. Korpi,à Jian-Hong Peng,§ Ronald J. Lukas,§ Liisa Ahtee* and Raimo K. Tuominen* ,   *Division of Pharmacology and Toxicology,  Viikki Drug Discovery Technology Center, Faculty of Pharmacy and àInstitute of Biomedicine, Pharmacology, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland §Division of Neurobiology, Barrow, Neurological Institute, Phoenix, Arizona, USA Abstract (–)-Methadone acts as an agonist at opioid receptors. Both (+)- and (–)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of a3b4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing a7 and a3* nAChR subtypes and SH-EP1-ha7 cells heterologously expressing human a7 nAChRs. Methadone potently inhibited binding of [ 3 H]methyl- lycaconitine to a7 nAChRs and that of [ 3 H]epibatidine to a3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH- SY5Y cells. Nicotine and both (+)- and (–)-methadone evoked increases in [Ca 2+ ] i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the a7 selective antagonist methyllycaconitine, suggesting effects of methadone as a7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl 2 , ry- anodine and xestospongin-c implicates voltage-operated Ca 2+ channels and intracellular Ca 2+ stores as downstream mod- ulators of elevated [Ca 2+ ] i . Collectively, our results suggest that methadone engages in complex and potentially pharma- cologically significant interactions with nAChRs. Keywords: addiction, calcium fluorometry, nicotine, opioid. J. Neurochem. (2005) 94, 1329–1341. Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels, composed of a or a and b subunit combinations from a family of nine a (a2– a10) and three b (b2–b4) subunits. nAChRs are widely distributed in the central nervous system as well as autonomic and sensory ganglia. In the brain, many of these receptors are presynaptic, and they modulate the release of a number of neurotransmitters (Wonnacott 1997). Activation of nAChRs causes behavioural changes, which can be related to these modulatory effects. Nicotine affects locomotor activity via altered dopaminergic activity, cognitive functions via cholinergic stimulation, and pain perception via altered opioidergic and serotonergic activity (Gotti et al. 1997). Nicotine shares the ability to activate the dopaminergic system with other addictive drugs, including opioids (Stolerman and Jarvis 1995). Although it is evident that these drugs have their independent mechanisms of action, which are Received March 11, 2005; revised manuscript received April 27, 2005; accepted April 29, 2005. Address correspondence and reprint requests to Jukka Pakkanen, Division of Pharmacology and Toxicology, Faculty of Pharmacy, Uni- versity of Helsinki, Helsinki, POB 56, FI-00014, Finland. E-mail: jukka.s.pakkanen@helsinki.fi Abbreviations used: a-bgt, a-bungarotoxin; AChR(s), nicotinic acetyl- choline receptor(s); [Ca 2+ ] i , amount of intracellular calcium; DHbE, dihydro-b-erythroidine; DMSO, dimethylsulfoxide; FCS, foetal calf serum; 5-HT, 5-hydroxytryptamine; lit, literature; MLA, methylly- caconitine; mp, melting point; Nal, naloxone; Nic, nicotinic; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; TSS, Tyrode’s salt solution. Journal of Neurochemistry , 2005, 94, 1329–1341 doi:10.1111/j.1471-4159.2005.03279.x Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 1329–1341 1329