Inhibition of neuronal nitric oxide synthase activity by N 1 -acetyl-5- methoxykynuramine, a brain metabolite of melatonin Josefa Leo ´ n,* Germaine Escames,* Marı ´a I. Rodrı ´guez,* Luis C. Lo ´pez,* Vı ´ctor Tapias,* Antonio Entrena, Encarnacio ´n Camacho, Marı ´a D. Carrio ´n, Miguel A. Gallo,  Antonio Espinosa, Dun-Xian Tan,à Russel J. Reiterà and Darı ´o Acun ˜ a-Castroviejo* *Departamento de Fisiologı ´a, Instituto de Biotecnologı ´a, and  Departamento de Quı ´mica Farmace ´utica y Orga ´nica, Facultad de Farmacia, Universidad de Granada, Granada, Spain àDepartment of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA Abstract We assessed the effects of melatonin, N 1 -acetyl-N 2 -formyl- 5-methoxykynuramine (AFMK) and N 1 -acetyl-5-methoxyky- nuramine (AMK) on neuronal nitric oxide synthase (nNOS) activity in vitro and in rat striatum in vivo. Melatonin and AMK (10 )11 )10 )3 M), but not AFMK, inhibited nNOS activity in vitro in a dose–response manner. The IC 50 value for AMK (70 lM) was significantly lower than for melatonin (>1 mM). A 20% nNOS inhibition was reached with either 10 )9 M melatonin or 10 )11 M AMK. AMK inhibits nNOS by a non-competitive mechanism through its binding to Ca 2+ -calmodulin (CaCaM). The inhibition of nNOS elicited by melatonin, but not by AMK, was blocked with 0.05 mM norharmane, an indoleamine-2,3-dioxygenase inhibitor. In vivo, the potency of AMK to inhibit nNOS activity was higher than that of melatonin, as a 25% reduction in rat striatal nNOS activity was found after the administration of either 10 mg/kg of AMK or 20 mg/kg of melatonin. Also, in vivo, the administration of norharmane blocked the inhi- bition of nNOS produced by melatonin administration, but not the inhibition produced by AMK. These data reveal that AMK rather than melatonin is the active metabolite against nNOS, which may be inhibited by physiological levels of AMK in the rat striatum. Keywords: indoleamine-2, 3-dioxygenase, melatonin, N 1 - acetyl-5-methoxykynuramine, neuronal nitric oxide synthase, norharmane, rat striatum. J. Neurochem. (2006) 98, 2023–2033. Melatonin (aMT) exerts a variety of physiological functions in order to help cells adapt to their environment. aMT is produced by several organs and tissues in addition to the pineal gland (Stefulj et al. 2001; Hardeland et al. 2005), and production is found to decrease with age (Lardone et al. 2006). aMT is both a lipo- and hydrophilic molecule, and it readily crosses all physiological barriers including the blood– brain barrier and plasma membrane (Reiter 1991a; Costa et al. 1995), reaching any subcellular compartments inclu- ding the nucleus and mitochondria (Acun ˜ a-Castroviejo et al. 2001). As a typical hormone, aMT exerts some of its functions through protein G i -linked specific membrane receptors (Dubocovich and Markowska 2005), whereas other effects of the hormone are mediated by nuclear ROR/RZR, retinoic acid receptors (Acun ˜ a-Castroviejo et al. 1995; Wiesenberg et al. 1995). Although not classified as aMT Received February 14, 2006; revised manuscript received May 22, 2006; accepted May 22, 2006. Address correspondence and reprint requests to Professor Darı ´o Acun ˜ a-Castroviejo, Departamento de Fisiologı ´a, Facultad de Medicina, Avenida de Madrid 11, E-18012 Granada, Spain. E-mail: dacuna@ugr.es Abbreviations used: AFMK, N 1 -acetyl-N 2 -formyl-5-methoxykynur- amine; Alexa-CaM, AlexaFluor TM 594-conjugated CaM; AMK, N 1 - acetyl-5-methoxykynuramine; aMT, melatonin; BSA, bovine serum albumin; CaCaM, Ca 2+ -calmodulin; CaM, calmodulin; DTT, D,L-di- thiothreitol; H 4 -biopterin, 5,6,7,8-tetrahydro-L-biopterin dihydrochlo- ride; FAD, flavin adenine dinucleotide; HIAA, 5-hydroxyindole acetic acid; HT, 5-hydroxytryptophan; IDO, indoleamine-2,3-dioxygenase; MOPS, 3-(N-morpholino)propanesulfonic acid; MT, 5-methoxytrypta- mine; NAD, nicotinamide adenine dinucleotide; NAS, N-acetyl-sero- tonin; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; OHT, 5-hydroxytryptamine; PMSF, phenylmethylsulfonyl fluoride; RNS, reactive nitrogen species; ROS, reactive oxygen species; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Journal of Neurochemistry , 2006, 98, 2023–2033 doi:10.1111/j.1471-4159.2006.04029.x Ó 2006 The Authors Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 98, 2023–2033 2023