Characterization of melatonin high‐affinity binding sites in purified cell nuclei of the hamster (Mesocricetus auratus) harderian gland

Abstract

: In the present paper, binding of melatonin to purified cell nuclei from harderian glands of male and female hamsters was assessed. Binding of 125I‐melatonin to cell nuclei fulfills the criteria for binding to a receptor site. Binding kinetics exhibit properties such as dependence on time and temperature as well as reversibility, saturability, high affinity and specificity. The dissociation constants (KD) and the number of binding sites (Bmax) for the binding of 125I‐melatonin to harderian gland nuclei were 260 ± 56 pm and 12.2 ± 0.8 fmol/mg protein in male glands, and 280 ± 43 pm and 9.8 ± 0.6 fmol/mg protein in female glands, respectively. Competition experiments showed IC50 values for melatonin of 250 ± 45 pm and 290 ± 68 pm in male and female glands, respectively. Other indoleamines such as N‐acetylserotonine and 5‐metoxytryptamine showed IC50 values in the micromolar range, suggesting that the binding sites are specific for melatonin. Hill analyses of the data show nH values of 0.96–0.98, suggesting the existence of a single class of binding sites. These data indicate that specific 125I‐melatonin binding sites exist in the cell nuclei of Harderian glands in male as well as in female hamsters, without significant differences between them. The KD and Bmax values obtained from the binding in both sexes correlates well with the concentration of melatonin described in these respective Harderian glands. It is hypothesized that the nuclear binding sites of melatonin here described could be a physiological melatonin receptor, which may be involved in the genomic‐dependent antioxidant effects of melatonin on hamster Harderian glands elsewhere reported.

Coexpression of MT1 and RORα1 melatonin receptors in the Syrian hamster Harderian gland

“…On the contrary, the presence of ROR α 1 in male and female HGs has been shown by our results. This fact supports that this member of the RZR/ROR orphan receptor subfamily could be the recently postulated physiological nuclear melatonin receptor found in nuclei of HG cells [8]. Although ROR α 1 is a nuclear receptor, we have observed an unexpected cellular localization of ROR α 1 receptor, as immunopositivity was observed also in the cytoplasm.…”

“…The existence of a partially characterized melatonin membrane receptor, which presents a circadian rhythm, has been reported in the HG of Syrian hamsters [7]. On the contrary, as a nuclear melatonin binding has been recently described in nuclei of HG cells, the existence of a physiological nuclear melatonin receptor has been postulated [8]. Nevertheless, the identity of these receptors in HG has not been elucidated.…”

A proposed mechanism to explain the stimulatory effect of melatonin on antioxidative enzymes

“…This activation is a consequence of antioxidant enzyme mRNA synthesis and eventually enzyme stimulation. These processes probably involve melatonin receptors [24,[28][29][30], although this remains unknown.…”

Anatomy, histology, histochemistry and fine structure of the Harderian gland in the South American armadillo Chaetophractus villosus (Xenarthra, Mammalia)

“…In some animals the HG contains melatonin and the enzymes needed for its synthesis Djeridane and Touitou 2001;Coto-Montes et al 2003). We could detect melatonin in the lacrimal gland, nictitans gland and HG of the armadillo C. villosus (Aldana Marcos 1996).…”