5-MCA-NAT does not act through NQO2 to reduce intraocular pressure in New-Zealand white rabbit Introduction Melatonin is involved in a wide range of physiological functions [1–4]. Two melatonin receptors, MT 1 and MT 2 , have been cloned from different tissues and species [5–9]. Also, another melatonin binding site, designated MT 3 and firstly described in the Syrian hamster brain [10], has been subsequently identified in the brain and peripheral tissues of different species [11]. In contrast to the MT 1 and MT 2 melatonin receptors subtypes, MT 3 has a specific pharmacological profile with affinity for prazosin and 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) [10]. An important organ where melatonin exerts its physio- logical actions is the eye. In this sense, ocular tissues possessing MT 3 melatonin receptors have not been specif- ically identified, but there is a functional evidence for MT 3 receptors in the eye. Thus, the melatonin MT 3 receptor has been showed to be involved in the regulation of intraocular pressure (IOP) [12, 13] in a dose-dependent manner [14, 15]. Specifically, the melatonin analogue, 5-MCA-NAT, mark- edly reduces IOP of normotensive rabbits [14, 15] and glaucomatous monkeys [16] acting through MT 3 melatonin receptors. An MT 3 melatonin binding site has been purified from hamster kidney and characterized as the human quinone reductase 2 (known as QR2, NQO2 or EC 1.10.99.2) [17]. Supporting this, the generation of NQO2 ) / ) mice, in which all tissues are depleted of 2-[ 125 I]-MCA-NAT binding, has confirmed the molecular identity between NQO2 and MT 3 in that species [18]. However, the apparent affinity of NQO2/MT 3 for melatonin and the analogue 5-MCA-NAT does not explain the role of these substances in the NQO2 function. Also, 5-MCA-NAT has a weak structural simi- larity with either NQO2 substrates or inhibitors known to date [17]. In addition, NQO2 may detoxify quinones because of its sequence homology with QR1 [19]. If melatonin binds to MT 3 and activates NQO2, this could justify the antioxidant role of melatonin but not explain the ocular hypotensive action of melatonin and 5-MCA-NAT. There are other cases in which it has not been possible to match the presence of NQO2 and the putative MT 3 melatonin receptor [11]. Moreover unexpectedly, the bind- ing of melatonin and derivates to NQO2 shows a poor inhibition capacity for the activity of the enzyme when the natural substrate menadione is present, although all occupy the enzyme active site [11, 18]. The idea that arises is that binding approaches for melatonin and derivatives on the NQO2/MT 3 receptor are not sufficient to preclude the existence of another receptor with a clear physiological action. Considering this, the purpose of this experimental work was to study the relationship between MT 3 /NQO2 and the role of the MT 3 -agonist, 5-MCA-NAT, in the regulation of IOP in the albino rabbit eye and to check if the MT 3 melatonin binding site indeed is the enzyme NQO2 in this animal model. Abstract: Solid data support the idea that the MT 3 melatonin binding site is an enzyme, quinone reductase 2 (NQO2), rather than a membrane melatonin receptor. However, the melatonin analogue, 5-methoxycarbonylamino-N- acetyltryptamine (5-MCA-NAT), reduces intraocular pressure (IOP) via MT 3 melatonin receptors. Therefore, the aim of this work was to test whether the melatonin binding site, MT 3 , is indeed the enzyme NQO2 in New Zealand rabbit eyes. To investigate this, the action of several substrates and inhibitors for NQO2 was compared to 5-MCA-NAT in their ability to modify IOP. Also, the effect of 5-MCA-NAT on IOP produced after NQO2 silencing by means of a siRNA was determinated. Altogether, the results led us to conclude that the in vivo effect of the MT 3 ligand 5-MCA-NAT on IOP is not mediated by the enzyme NQO2, suggesting the existence of another melatonin receptor. Pilar Alarma-Estrany*, Almudena Crooke* and Jesu ´ s Pintor Departamento Bioquı ´mica, E.U. O ´ ptica, Universidad Complutense de Madrid, C/Arcos de Jalo ´n s/n, Madrid, Spain Key words: 5-methoxycarbonylamino-N- acetyltryptamine, intraocular pressure, melatonin, MT 3 melatonin receptor, quinone reductase 2 Address reprint requests to Dr Jesu ´ s Pintor, Departamento Bioquı ´mica, E.U. O ´ ptica, Universidad Complutense de Madrid, C/Arcos de Jalo ´n s/n, 28037 Madrid, Spain. E-mail: jpintor@vet.ucm.es *Both authors have contributed equally to this work. Received April 29, 2009; accepted June 12, 2009. J. Pineal Res. 2009; 47:201–209 Doi:10.1111/j.1600-079X.2009.00702.x Ó 2009 The Authors Journal compilation Ó 2009 John Wiley & Sons A/S Journal of Pineal Research 201 Molecular, Biological, Physiological and Clinical Aspects of Melatonin