Ž . European Journal of Pharmacology 404 2000 89–93 www.elsevier.nlrlocaterejphar Blockade of dopamine transporter and tyrosine hydroxylase activity loss w 2 5 x by D-Ala , D-Leu enkephalin in methamphetamine-treated CD-1 mice Li-I Tsao, Teruo Hayashi, Tsung-Ping Su ) Cellular Pathobiology Unit, Molecular Neuropsychiatry Section, Intramural Research Program, National Institute on Drug Abuse, National Institute of Health, 5500 Nathan Shock DriÕe, Baltimore, MD 21224, USA Received 8 August 2000; accepted 11 August 2000 Abstract w 2 5 x Ž . D-Ala , D-Leu enkephalin DADLE has been previously reported to prolong the survival of tissues both in the periphery and in the central nervous system. Here, we show that DADLE was able to block the protein as well as the functional loss of dopamine transporter Ž . Ž . Ž DAT and tyrosine hydroxylase TH induced by methamphetamine. Male CD-1 mice received four injections of methamphetamine 10 . Ž . mgrkg, i.p. at 2-h intervals. DADLE 4 mgrkg, i.p. was given 30 min before each injection of methamphetamine. Western blotting and enzymatic assays showed that DADLE blocked the protein loss and functional impairment of DAT and TH induced by methamphetamine. q 2000 Elsevier Science B.V. All rights reserved. Žw 2 5 x . Keywords: DADLE D-Ala , D-Leu enkephalin ; Methamphetamine; Dopamine transporter 1. Introduction w 2 5 x Ž . D-Ala , D-Leu enkephalin DADLE can dramatically extend the organ survival time in a multiorgan block preparation, including the heart, lung, liver, spleen and kidney, from an average of 14–46 h — the longest in the Ž history of organ preservation Wu et al., 1996; Oeltgen et . al., 1996 . Further, DADLE can promote myocardial toler- ance to ischemia in isolated hearts in a fashion far superior Ž to the standard cardioplegic procedure Bolling et al., . 1997 . Recently, DADLE was demonstrated in an autora- diographic study to protect against methamphetamine-in- Ž . Ž duced dopamine transporter DAT loss in the brain Tsao . et al., 1998 . Thus, DADLE may be protective in both the periphery and the central nervous system. To further inves- tigate the protective properties of DADLE against metham- phetamine-induced neuronal damage, we examined in this study the protein levels and the functional properties of Ž . DAT and tyrosine hydroxylase TH after the DADLE and methamphetamine treatments in CD-1 mice. ) Corresponding author. Tel.: q 1-410-550-1519; fax: q 1-410-550- 1153. Ž . E-mail address: tsu@intra.nida.nih.gov T.-P. Su . 2. Materials and methods 2.1. Drug treatment Male CD-1 mice received four injections of metham- Ž . phetamine 10 mgrkg, i.p. or saline at 2-h intervals. Ž . DADLE 4 mgrkg, i.p. or saline was given 30 min before each methamphetamine administration. All drugs were pre- pared immediately before use and injected in a volume of 1 mlr100 g body weight. Mice were killed by cervical dislocation 2 weeks later. The striata were quickly dis- sected and used either fresh or frozen. Cares and treat- ments of animals were according to procedures approved by the Institutional Animal Care and Utilization Commit- tee. [ 125 ] 2.2. I RTI-121 binding assays q Ž The frozen striata were homogenized in Na buffer 10 q . mM Na phosphate and 120 mM NaCl with a TeflonrGlass Homogenizer and centrifuged at 750 = g for 10 min at 48C. The supernatants were further centrifuged Ž . at 14 000 = g for 20 min at 48C. The pellets P2 fraction were resuspended in Na q buffer. Binding assays were 0014-2999r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž . PII: S0014-2999 00 00616-6