Characterization, Guanosine 5′-O-(3-Thiotriphosphate) Modulation, Daily Variation, and Localization of Melatonin-Binding Sites in the Catfish (Silurus asotus) Brain

Abstract

Characteristics, guanosine 5′-O-(3-thiotriphosphate) (GTPγS) modulation, daily variation, and localization of melatonin-binding sites in the brain of a nocturnal teleost, the catfishSilurus asotus,were studied by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. The specific binding was rapid, stable, saturable, and reversible. The radioligand binds to a single class of receptor site with an affinity (Kd) of 30.7 ± 7.3 pMand total binding capacity (Bmax) of 9.76 ± 0.79 fmol/mg protein (mean±SE,n=5). The binding sites were highly specific for 2-iodomelatonin and melatonin. The specificity was almost identical to that of functional melatonin receptors in the dermal and epidermal melanophores in this species and that of ML-1 subtype melatonin receptors in vertebrates, including melatonin-binding sites in the goldfish brain. GTPγS treatment altered both theKdandBmaxvalues, indicating that melatonin-binding sites in the catfish brain are coupled to G protein. TheBmaxvalues exhibited no daily variation under light–dark cycles of 12 hr light:12 hr dark whereas plasma melatonin levels andKdfluctuated in a rhythmic fashion. The density of melatonin-binding sites in discrete brain areas was determined to be highest in optic tectum–thalamus and hypothalamus, intermediate in telencephalon, cerebellum, and medulla oblongata, and lowest in olfactory bulbs. These results suggest that melatonin secreted from the pineal organ and/or retina plays neuromodulatory roles in the catfish brain via G protein-coupled melatonin receptors. Characteristics of melatonin receptors seem to be highly conserved during evolution, although the density of melatonin receptors is not regulated by melatonin itself in this species.