Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors

Abstract

Melatonin exerts a variety of physiologic activities that are mainly relayed through the melatonin receptors MT 1 and MT 2 Low expressions of these receptors in tissues have led to widespread experimental use of the agonist 2-[125 I]-iodomelatonin as a substitute for melatonin. We describe three iodinated ligands:, which are specific ligands at MT 2 receptors, and125 I]-iodomelatonin with slightly different characteristics. Here, we further characterized these new ligands with regards to their molecular pharmacology. We performed binding experiments, saturation assays, association/dissociation rate measurements, and autoradiography using sheep and rat tissues and recombinant cell lines. Our results showed that [125 I]-S70254 is receptor, and can be used with both cells and tissue. This radioligand can be used in autoradiography. Similarly, DIV880, a partial agonist [43% of melatonin on guanosine 59-3-O-(thio)triphosphate binding assay], selective for MT 2 , can be used as a tool to selectively describe the pharmacology of this receptor in tissue samples. The molecular pharmacology of both human melatonin receptors MT 1 and MT 2 , using a series of 24 ligands at these receptors and the new radioligands, did not lead to noticeable variations in the profiles. For the first time, we described radiolabeled tools that are specific for one of the melatonin receptors (MT 2 ). These tools are amenable to binding experiments and to autoradiography using sheep or rat tissues. These specific tools will permit better understanding of the role and implication in physiopathologic processes of the melatonin receptors.

Update on melatonin receptors: IUPHAR Review 20

“…Three new iodinated radioligands have been recently characterized for use in the pharmacological characterization and localization of MT receptors (Figure ). These radioligands are as follows: SD6 ( N ‐[2‐(5‐methoxy‐1Hindol‐3‐yl)ethyl]iodoacetamide), S70254 (2‐iodo‐ N ‐2‐[5‐methoxy‐2‐(naphthalen‐1‐yl)‐1H‐pyrrolo[3,2‐b]pyridine‐3‐yl])acetamide and DIV880 2‐(2‐[(2‐iodo‐4,5‐dimethoxyphenyl)methyl]‐4,5‐dimethoxy phenyl) (Legros et al, ; Legros et al, ). [ 125 I]‐SD6 has a similar pharmacological profile to that of 2‐[ 125 I]‐IMLT with the same affinity for MT 1 and MT 2 receptors.…”

“…The extensive pharmacological characterization of these three new radioligands in comparison with 2‐[ 125 I]‐IMLT and [ 3 H]‐MLT on membrane preparations from CHO‐K1 cell lines stably expressing hMT 1 or hMT 2 receptors showed that [ 125 I]‐S70254 and [ 125 I]‐DIV880 mainly differ from 2‐[ 125 I]‐IMLT in their dissociation kinetics, which are faster for [ 125 I]‐S70254 and [ 125 I]‐DIV880 than for 2‐[ 125 I]‐IMLT (Legros et al, ). Interestingly, [ 125 I]‐SD6 labelled only approximately half of the binding sites detected with 2‐[ 125 I]‐IMLT in cells expressing hMT 1 receptors while comparable amounts were detected in cells expressing hMT 2 receptors (Legros et al, ).…”

“…The MT 2 receptor‐specific [ 125 I]‐S70254 was successfully used for autoradiography studies in rat and sheep brain and retina slices (Legros et al, ). A similar labelling pattern to 2‐[ 125 I]‐IMLT (detecting MT 1 and MT 2 receptors) was observed in several areas but also distinct labelling in others.…”

Characterization of the various functional pathways elicited by synthetic agonists or antagonists at the melatonin MT1 and MT2 receptors

“…As described previously, CHO‐K1 cells stably expressing human MT 1 (hMT 1 ) or MT 2 (hMT 2 ) were grown to confluence, harvested in PBS buffer (Gibco, Invitrogen, Saint‐Aubin, France) containing 5 mmol/L EDTA, and centrifuged at 1000 g for 20 minutes at 4°C. The resulting pellet was suspended in 5 mmol/L Tris/HCl (pH 7.4) containing 2 mmol/L EDTA and homogenized using Kinematicapolytron.…”

The five dimensions of receptor pharmacology exemplified by melatonin receptors: An opinion

“…Indeed, combined with a radiotracer, such as the traditional 2‐[ 125 I]‐melatonin, or more recently, the triple‐tritiated melatonin, hundreds of melatonin‐like compounds have been described for their capacity to bind to melatonin receptors (see a chemical review of those molecules in Zlotos et al). More recent studies have given rise to a new generation of compounds that are more selective for one of the MT 1 or MT 2 subtypes . Interestingly, melatonin receptors are slightly different from other receptors, because we were essentially only seeking agonists, as opposed to the traditional search for antagonists as drugs for most of the other GPCRs.…”