Biochemical regulation of circadian 2-125 I-iodomelatonin binding in chick optic tectum

Abstract

The avian optic tectum (Teo), a visual relay area, exhibits specific binding of 2-[1211]iodomelatonin (IMEL) in a circadian rhythmic fashion. Samples of Teo representing different circadian times (CTs) (CT2, CT6, CTIO, CT14, CT18, CT22) were incubated with 50 pM IMEL in radioreceptor (RRA) or autoradiographic assays. The RRA expressed a binding rhythm with a biphasic pattern of high values in late subjective day (SD) (CTIO: 29.86 fmol/mg) and mid subjective night (SN) (CT18: 29.7 fmol/mg) and low values in late SN (CT22: 3.76 fmol/mg). Conversely, autoradiography showed a binding rhythm with high values only during mid and late SD (average of 5.01 fmol/mg at CT6 and CT10) and low values in SN (an average of 0. 69 fmol/mg from CT 1 4-22). The hypothesis that the circadian rhythm of IMEL binding is due to an association and dissociation of the receptor with its G-protein was tested. Incubation of Teo tissue from different CTs in concentrations of IMEL ranging from 16 pM to 1 nM in the presence or absence of the non-hydrolyzable GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP-yS) was done. Analysis of the data found that saturation was reached in the lower concentrations (up to 250 pM) of IMEL for all CTs during SD and mid SN, corresponding to the high affinity binding site. The total number of receptors available (B.a,) was 80 fmol/mg during SD and decreased significantly during late SN (CT 22: 7.39 fmol/mg). The binding affinities (Kd) ranged from 89.29 pM at mid SD (CT6) to 236 pM (CT 14) and remained low during SN (CT22: 169.2 pM). Samples incubated with 100 /AM GTP-YS, had a decrease of 20-25% in the number of receptors during SD and 3570% at mid night and a non-significant change in binding affinities. Moreover, the rhythm in binding densities and the affinities of the receptor for the ligand was not abolished. In all, these data suggest that IMEL binding site is coupled to a G protein. However, the circadian rhythm in IMEL binding may not be driven by the association and dissociation of the receptor with its G protein.