Melatonin receptors in brain areas and ocular tissues of the teleost Tinca tinca: Characterization and effect of temperature M.A. Lo ´ pez Patin ˜ o, A.L. Alonso-Go ´ mez, A. Guijarro, E. Isorna, M.J. Delgado * Departamento de Fisiologı ´a (Fisiologı ´a Animal II), Facultad de Biologı ´a, Universidad Complutense, 28040 Madrid, Spain Received 25 July 2007; revised 5 November 2007; accepted 7 November 2007 Available online 22 November 2007 Abstract The aim of the present study was to characterize the central melatonin receptors in brain areas and ocular tissues of the teleost Tinca tinca. We investigated the temperature-dependence of 2-iodo-melatonin ([ 125 I]Mel) binding in the optic tectum-tegmentum area and the neural retina. The binding of [ 125 I]Mel showed a widespread distribution in brain and ocular tissues, with the highest density in the optic tectum-thalamus and the lowest in hindbrain. The [ 125 I]Mel affinity was similar in all the studied tissues, and it was on the order of the low pM range. Saturation, kinetic and pharmacological studies showed the presence of a unique MT 1 -like melatonin binding site. In addition, the non-hydrolysable GTP analog, the GTPcS, and sodium cations induced a specific binding decrease in the optic tectum and neural retina, suggesting that such melatonin binding sites in the tench are coupled to G protein. Thus, these melatonin binding sites in optic tectum and neural retina fulfil the requirements of a real hormone receptor, the specific binding is rapid, saturable, and reversible, and is inhibited by GTP analogs. Additionally, a clear effect of temperature on such central melatonin receptors was found. Temperature did not modify the B max and K d , but the kinetics of [ 125 I]Mel binding resulted in a highly thermosensitive process in both tissues. Both association and dissociation rates (K +1 and K À1 ) significantly increased with assay temperature (15–30 °C), but the K d constant (esti- mated as K À1 /K +1 ) remained unaltered. In conclusion, this high thermal dependence of the melatonin binding to its receptors in the tench central nervous system supports the conclusion that temperature plays a key role in melatonin signal transduction in fish. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Melatonin; Receptors; Brain; Retina; Temperature; Fish 1. Introduction Melatonin (N-acetyl 5-methoxytryptamine) is synthe- sized in the vertebrate pineal complex and the retina on a daily rhythmic pattern, with high levels during the dark phase and low levels during the light phase (Falco ´n, 1999). This daily profile exhibits seasonal changes that can synchronize a large number of rhythmic physiological and behavioural processes (Pe ´vet, 2003). In fish, melatonin has been involved in the control of seasonal reproduction, ocular functions, pigmentation pattern, motor activity, feeding regulation and osmoregulation (Zachmann et al., 1992; Ekstro ¨m and Meissl, 1997; Pinillos et al., 2001; Kulc- zykowska, 2002). In the last decade, concerted efforts have been made to characterize the receptors involved in the transduction of such melatonin effects. Radioligand studies using the high specific activity radi- oiodinated melatonin agonist, 2-[ 125 I]-iodo-melatonin ([ 125 I]Mel), have allowed the characterization of melatonin binding sites (putative melatonin receptors) in central and peripheral tissues of several fish species. In addition, some cloning studies have supported the existence of three sub- types of high affinity melatonin receptors in fish, MT1, MT2, and Mel 1c, belonging to the G-protein coupled receptors (Mazurais et al., 1999; Gaildrat et al., 2002; Park et al., 2007). Another binding site, MT3, with low affinity binding for [ 125 I]Mel, has been identified in mammals as the enzyme NRH: quinone oxidoreductase (Nosjean et al., 2000). Melatonin binding sites in fish brain have been charac- terized in several species, such as goldfish, Carassius aura- 0016-6480/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2007.11.011 * Corresponding author. Fax: +34 913944935. E-mail address: mjdelgad@bio.ucm.es (M.J. Delgado). www.elsevier.com/locate/ygcen Available online at www.sciencedirect.com General and Comparative Endocrinology 155 (2008) 847–856