[¹²⁵I]lodomelatonin-Binding Sites in the Chicken Kidney: Characterization and Comparison to Other Avian Species

Abstract

The existence of [¹²⁵I]iodomelatonin-binding sites in the kidney of the chicken and other avian species has been demonstrated in the present study. [¹²⁵I]Iodomelatonin-binding sites in membrane preparations of the chicken kidney were reversible, saturable, specific and of high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (K_d) of 30.3 ± 5.1 pmol/l (n = 5) and a total number of binding sites (B_max) of 2.89 ± 0.24 fmol/mg protein (n = 5) at the middle of the light period (mid-light). The Hill coefficient approached 1.0, suggesting a single class of [^I25I]iodomelatonin-binding sites in the chicken kidney. The diurnal variation study showed that the B_max was 52% higher at mid-light than that in the middle of dark period(1.90 ± 0.33 fmol/mg protein, n = 4; p < 0.05), with no significant variation in K_d. The K_d value determined from kinetic analysis was 10.8 pmol/l in the mid-light birds, which was comparable to values determined from equilibrium studies. The competition inhibitor experiments demonstrated that [¹²⁵I]iodomelatonin-binding sites were highly specific for melatonin with the following order of potency: 2-iodomelatonin > melatonin, 6-chloromelatonin > 6-hydroxy-melatonin > N-acetylserotonin ≫ 5-methoxytryptamine, 5-methoxytryptophol, 5-hydroxyindole-3-acetic acid, 5-hydroxytiyptamine, 1-acetylindole-3-carboxaldehyde, 3-acetylindole, tryptamine, 5-methoxyindole-3-acetic acid, L-tryptophan, harmaline, acetylcholine, epinephrine and norepinephrine. Studies on the relative binding density of [¹²⁵I]iodomelatonin to the kidney in different species of birds showed the following descending order: duck > chicken > pigeon > quail. It has been reported that [^I25I]iodomelatonin-binding sites exist in the duck kidney and the possible direct action of melatonin on renal function suggested. The identification of [^l25I]iodomelatonin-binding sites in the chicken and other avian kidneys supports this hypothesis and suggests that [¹²⁵I]iodomelatonin-binding sites are universal in the kidneys of birds.

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).…”

Ontogeny of 2-[125I]iodomelatonin binding sites in the chicken (Gallus domesticus) kidney and spleen

“…Previous reports s f 2- [1251]iodomelatonikl binding sites in the chicken kidney (Song and Pang 1992;Song et al 1993a) and spleen P w n et al 1994) indicated that these binding sites were reversible, saturable, specific, and of high affinity. Reported vdues of Kd and B,,, of 2-[~251]iodomelatomin binding sites in the kidney of 808-to 1100-g chickens (about 2 -3 months old, & = 38.3 + 5.1 pmol/L, B,,, = 2.89 f 0.24 fmollmg protein; Song and Pang 1992) are comparable with animals with similar ages in the present study.…”

Comparison of the Pharmacological Characteristics of 2-[¹²⁵I]Iodomelatonin Binding Sites in the Lung, Spleen, Brain and Kidney of Chicken

“…Briefly, aliquots of membrane preparations were incubated with 2-[125I]iodomelatonin (concentrations ranging from 6 to 136 pM for saturation studies) with or without unla belled melatonin (1 \u\i) in a total volume of 350 pi per tube at 37°C for 50 min. These binding assay condi tions were used because previous studies indicated binding reached a kinetic steady state in less than 50 min of incubation time at 37°C [7,8,10,11,25). Membranes with bound 2-[l2SI]iodomelatonin were washed with assay buffer (4 x 3.5 ml) followed by vac uum filtration on glass fiber filters (Whatman GF/B, pore size 1 pm).…”