Binding Sites for Melatonin in Bovine Pineal Gland

Abstract

High-affinity binding of melatonin to crude membrane preparations of bovine pineal gland was examined by a rapid filtration procedure through Whatman GFB paper. Maximal melatonin binding was attained in 60 min at 37 °C, in 2 h at 25 °C and in 5 h at 0 °C; at 25 and 37 °C it was 36 and 42% of that at 0 °C. Specific binding was thermolabile and decreased following incubation with trypsin; it was also pH dependent, the maximum being observed at physiological pH. Melatonin binding was inhibited by the addition of monovalent or divalent ions to the incubation buffer. Subcellular fractionation studies indicated that 39 and 50% of binding was located in the pellets at 900 and 27,000 g whereas 11% was detected in the microsomal pellet. Scatchard analysis revealed a single population of binding sites with K_d= (7.0 ± 1.5) 10^-7M (mean ± SEM, n = 4); binding site concentration ranged from 185 to 356 fmol/mg of protein. When various melatonin analogues were tested for their ability to inhibit (³H)-melatonin binding, the following Ki values (10^-7M), were obtained: N-acetyl-serotonin 120, serotonin 130, 2-methyl indole 154, 5-hydroxytryptophol 218, 5-methoxytryptamine 266, 5-methoxytryptophol 660, tryptamine 1,740, 5-hydroxyindole acetic acid 3,455, 5-methoxyindole acetic acid 12,690, 5-hydroxytryptophan 13,600, and 6-hydroxymelatonin 55,550. These results suggest that melatonin receptors may be present in the pineal gland.

Use of 2-[125I]iodomelatonin to characterize melatonin binding sites in chicken retina.

“…[3H]Melatonin also binds to membranes of bovine pineal gland (29) and trout and frog retinas (10,20 (10,20 Using a functional measurement of melatonin activity, Dubocovich (17) showed that the efficacy of melatonin and related indoles in inhibiting the calcium-dependent release of [3H]dopamine from rabbit retina is determined by the moiety (methoxy) on carbon 5 of the indole nucleus, whereas the affinity for the receptor is determined primarily by the moiety (acetamidoethyl) on carbon 3. In support of this conclusion, In addition to its role in the retina, melatonin has been implicated in the regulation of seasonal reproduction in mammals (1,2) and in the control of circadian rhythms in birds and reptiles (3)(4)(5) (30).…”

Melatonin: an endogenous negative modulator of 12-lipoxygenation in the rat pineal gland

“…Although to our knowledge there is no concrete evidence for the presence of melatonin receptors in the rat pineal gland, binding of melatonin to rat and bovine pineal glands has been observed previously [33][34][35], suggesting that melatonin receptors might exist in pineal glands. In order to clarify whether specific receptor subtypes are indeed involved in the regulation of 12-LOX expression, further investigation will be necessary.…”

Age-Related Changes in 24-Hour Rhythms of Norepinephrine Content and Serotonin Turnover in Rat Pineal Gland: Effect of Melatonin Treatment

“…Binding sites for melatonin have been reported in the pineal gland [38,39], and melatonin treatment affects pineal 5-HT content [40,41], densecored vesicles [42], annulate lamellae in pinealocytes [43] and tubulin content and enzymes in the melatonin biosynthetic pathway [43]. Part of these effects was observed after the addition of melatonin to pineal organ cultures [44].…”