Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay.
An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.
Leung
F C
FC
Biology and Chemistry Department, Pacific Northwest Laboratory, Richland, Washington 99352.
D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000032:analysis; D002645:Chickens; D002940:Circadian Rhythm; D008550:Melatonin / Q000032:analysis* / Q000378:metabolism; D009924:Organ Culture Techniques; D010870:Pineal Gland / Q000378:metabolism*; D011863:Radioimmunoassay
D001191:Arylamine N-Acetyltransferase; D008550:Melatonin