Characterization and Mapping of Melatonin Receptors in the Brain of Three Mammalian Species: Rabbit, Horse and Sheep

Abstract

Melatonin receptors were characterized in the brains of three mammals (rabbit, horse and sheep) by an in vitro binding technique, using 2-[¹²⁵I]iodomelatonin as labelled ligand. Although binding sites for melatonin have been described recently in several vertebrate species (including the sheep), the rabbit and the horse have not been the subject of investigation so far. Apart from characterization, the present report describes receptor distribution in a number of brain regions, thus allowing for direct interspecies comparison under the same methodological conditions. 2-[¹²⁵I]iodomelatonin labelled high-affinity binding sites in crude membrane preparations from these species. A series of kinetic and saturation experiments revealed that the binding was rapid, stable, saturable, reversible, of high affinity (K_d in the low picomolar range) and low capacity (B_max between 1 and 20 fmol/mg protein). The competition studies showed that the relative order of potency of a variety of indoles for inhibition of 2-[¹²⁵I]iodomelatonin binding was as follows: 2-iodomelatonin > 6-chloromelatonin > melatonin > > > 5-methoxytryptophol > 5-methoxytryptamine, and that it was similar in the different brain regions. Prazosin, which has been reported as an extremely potent melatonin analog in the hamster brain, possessed no potency in all preparations from different regions in the three species under investigation. The regional distribution of the receptor showed insignificant species differences. Highest density was always recorded in the median eminence/pars tuberalis (ME/PT) area. Other regions (SCN, POA and certain cortical areas), showed lower, but significant, receptor content. Saturation and competition studies revealed that these binding sites were also of high affinity, low capacity and high specificity. These results demonstrate that melatonin receptor properties and distribution are similar in these species; the apparent receptor density, however, was highest in the sheep ME/PT. Most of the rabbit brain regions exhibited higher apparent B_max values, compared to the corresponding ones in the other two species.

2-[¹²⁵I]Iodomelatonin Binding Sites in the Testis and Ovary: Putative Melatonin Receptors in the Gonads

“…Progress to date in the efforts to demonstrate a cellular response to melatonin include receptor-mediated inhi bition of dopamine release in the rat hypo thalamus [52] and rabbit retina [53]; inhibi- 74 Ayre/Pang Melatonin Receptors in Gonads tion of prostaglandin synthesis in rat medial basal hypothalamus [54]; and modulation of cAMP formation in vitro in the hamster me dian eminence and pars tuberalis [55][56][57], mouse median eminence and pars tuberalis [58], rat pars tuberalis [59] and rabbit cortex [60]. Involvement of guanine nucleotide binding proteins in the melatonin signal transduction mechanism is also supported by evidence that guanine nucleotides dose-dependently and specifically inhibit 2-[125I]iodomelatonin binding in lizard brain homoge nates [61], rabbit cortex [60,62], sheep pars tuberalis [63], hamster median eminence and pars tuberalis regions [55], hamster hypothal amus [64], rat suprachiasmatic nuclei [65,66], rat area postrema [67], chicken brain [68,69] and chicken lung and kidney [69][70][71] fig. 1, 2).…”

“…Guanine nucleotide-binding pro teins (G proteins) are known to transduce 76 Ayrc/Pang Melatonin Receptors in Gonads [69], while a GTPyS-mediated decrease in Bmax with no change in Kd was reported in the ovine pars tuberalis [63], rat suprachiasmatic nuclei [66] and chicken brain [68,69]. A third variation was observed in the chicken lung [69,74], hamster brain [55], rat area postrema [67], rat suprachiasmatic nu clei [67] and rabbit cortex [62] with GTPyS modulating an increase in Kd but no change in Bmax. In tissues where both classes of high and low binding sites were investigated, GTPyS was reported to have no effect on the low affinity 2-[125I]iodomelatonin binding sites in lizard brain [61] or hamster hypothal amus [64].…”

Evidence for Gene Transcription of Adenohypophyseal Hormones in the Ovine Pars tuberalis

“…The autoradiographic method employed has been described in detail previously [25]. Briefly, slide mounted cryostat-cut sections were incubated with Tris-BSA (50 m.W Tris-HCl.…”

Expression and functional characterization of a melatonin‐sensitive receptor in Xenopus oocytes

“…Sections (20 p.m) were cut on a cryostat at -20°C and stored at -80°C until processed for autoradiography as described previously [14]. Briefly, sections were preincubated in Tris-BSA buffer (50 mmol/1 Tris-HCl, 4 mmol/l CaCI2, 0.05% BSA, pH 7.4 at 25°C) and then incubated in the same buffer containing 10(~120 pmol/l 2-[125j]iodomelatonin in the presence or absence of 0.1 gmol/1 unlabeled MEL.…”