No abstract

The existence of 125I-labelled iodomelatonin-binding sites in chicken ovaries and testes was investigated. The specific binding of 125I-labelled iodomelatonin to chicken ovarian and testicular tissue satisfies all the criteria for a binding site. It was rapid, stable, saturable, reversible, specific and of high affinity. Equilibrium studies showed that total and non-specific binding increased over a range of 5-150 pmol 125I-labelled iodomelatonin/l tested, with specific binding reaching saturation towards the middle range of radioligand concentrations. Scatchard analyses indicated a dissociation constant (Kd) of 36.5 +/- 5.3 pmol/l (means +/- S.E.M.) in the membrane preparations of chicken testes at the middle point in the period of light and a maximum number of binding sites (Bmax) of 0.93 +/- 0.40 fmol/mg protein (n = 6). In membrane preparations of chicken ovaries, the Kd was 102.2 +/- 27.3 pmol/l and the Bmax was 2.77 +/- 0.38 fmol/mg protein (n = 6). Equilibrium and kinetic dissociation constants in the picomolar range indicate high-affinity and physiologically relevant 125I-labelled iodomelatonin-binding sites. Competitive inhibition studies determined the following order of relative potency for inhibition of 125I-labelled iodomelatonin-binding to chicken gonadal membranes: 6-chloromelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, 1-acetylindole-3-acetic acid, 5-hydroxyindole-3-acetic acid and L-tryptophan. The presence of 125I-labelled iodomelatonin-binding sites suggests a direct pineal-gonadal connection in the chicken.

Through the synthesis and secretion of the hormone, melatonin, the pineal has been assigned the role of synchronizing a reproductive response to appropriate environmental conditions. Theoretical melatonin target sites may occur at several levels of the hypothalamic-pituitary-gonadal hierarchy, including a direct action on the gonads. The availability of a biologically active radioligand, 2-[¹²⁵I]iodomelatonin, has provided the opportunity to examine the possible direct melatonin action on the gonads. 2-[¹²⁵I]Iodomelatonin binding sites were identified in the testes and ovaries of chickens, ducks and quail but were not measurable in mammalian gonads, with the exception of tree shrew testes. The avian gonadal 2-[¹²⁵I]iodomelatonin binding sites were stable, saturable, reversible, specific and of high affinity. 2-[¹²⁵I]Iodomelatonin appeared to label a single class of binding sites as evidenced by the linearity of Rosenthal analysis of the specific binding data, the Hill coefficients close to unity and the monophasic competition curves. The high affinity on the gonadal 2-[¹²⁵I]iodo-melatonin binding sites, characterized by apparent equilibrium dissociation constants in the low picomolar range, was in accordance with circulating levels of melatonin suggesting that they may be physiologically relevant. Autoradiography indicated that these 2-[¹²⁵I]iodomelatonin binding sites were widely distributed throughout the testes but localized in ovarian follicles in the birds studied. Specific inhibition of testicular 2-[¹²⁵I]iodomelatonin binding by a guanine nucleotide analog has provided evidence that the 2-[^l25I]iodomelatonin binding sites in chicken testes may be coupled to a guanine nucleotide binding protein-effector system, thus promoting the idea that testicular 2-[¹²⁵I]iodomelatonin binding sites may mediate a cascade of intracellular events. Although no circadian rhythm in the density or affinity of 2-[¹²⁵I]iodomelatonin binding in chicken ovaries was found, there was a decrease in 2-[^I25I]iodomelatonin binding affinity at middark in chicken testes with no change in the number of testicular 2-[¹²⁵I]iodomelatonin binding sites. The present evidence is in line with the hypothesis of a direct melatonin action on the gonads and further investigations on the above problem will be rewarding.

Effects of 10 µmoI/l guanosine 5''-O-(3-thiotriphosphate) (GTPγS), a non-hydrolyzable analog of guanosine 5''-triphosphate, on 2-[¹²⁵I]iodomelatonin binding were investigated. In the chicken lung, 10 µmol/l GTPγS significantly increased (p < 0.05) the equilibrium dissociation constant (K_d) values, but did not affect the maximum number of binding sites (B_max). Conversely, in the chicken brain, GTPγS significantly depressed (p < 0.05) the B_max, but did not change the K_d of the 2-[¹²⁵I]iodomelatonin binding sites in the brain tissue. A third variation was observed in the chicken kidney with GTPγS altering (p < 0.05) both the K_d and the B_max of 2-[¹²⁵I]iodomelatonin binding sites. The reason underlying the different effects of GTPγS on 2-[¹²⁵I]iodomelatonin binding in the tissue preparations is not clear. However, we would like to hypothesize that they may represent distinct subtypes of the ML-1-type melatonin receptor with different receptor-G-proteins-effector complex. The group represented by the 2-[¹²⁵I]iodomelatonin binding sites found in the chicken lung, which is downregulated by GTPγS with a consequent increase in the K_d value, has been designated ML-1α. The second group, exemplified by the brain 2-[¹²⁵I]iodomelatonin binding sites, which respond to GTPγS with a change in B_max, has been labelled ML-1β. The third group, characterized by GTPγS -mediated alterations in both B_max and K_d and found in the chicken kidney, has been called ML-1γ. Different subtypes of melatonin receptors may address the issue of the different physiological actions of melatonin reported in individual tissues within the same species or similar tissues but different species. Specialized responses could be generated depending on the predominant subtype of ML-1 receptors associated with the target tissue.

[^I25I]Iodomelatonin binding sites have been identified and characterized in kidneys of birds and mammals. These binding sites in the kidneys of guinea pig, duck and chicken were found to be stable, reversible, saturable, specific and of high affinity. The binding densities (B_max) of [¹²⁵I]iodomelatonin binding sites in the kidneys of guinea pig, duck and chicken ranged from 1.07 to 6.43 fmol/mg protein and the equilibrium dissociation constants (K_d) from 19.2 to 44.6 pmol/l at the middle of the light period (mid-light). It appears that [¹²⁵I]iodomelatonin binding in the kidneys of mammalian species may have lower densities compared with birds. In the mammals studied, the guinea pig kidney showed the highest [¹²⁵I]iodomelatonin binding. Pharmacological data indicated that the [¹²⁵I]iodomelatonin binding to kidneys of guinea pig, duck and chicken was highly specific to melatonin, 2-iodomelponin and 6-chloromelatonin. Diurnal variations in the B_max of [¹²⁵I]iodomelatonin binding sites were detected in the kidneys of duck and chicken with no difference in affinity. However, there was no diurnal variation in the K_d or B_max in the guinea pig kidneys. The density of [¹²⁵I]iodomelatonin binding sites in the cortex of guinea pig kidney was more than 8-fold higher than the binding in the medulla. Guanosine 5''-O-(3-thiotriphosphate) (GTPγS; 10 µmol/1) reduced the B_max and increased the K_d of [¹²⁵I]iodomelatonin binding sites in the chicken kidney. However, in the membrane preparations of the guinea pig kidney, co-incubation with GTPγS (15 µmol/l) increased the K_d with no effect on the B_max of the [¹²⁵I]iodomelatonin binding. The effects of GTPγS on the kidney [¹²⁵I]iodomelatonin binding suggest that these binding sites may couple to a G protein. The identification of [¹²⁵I]iodomelatonin binding sites in the kidneys of mammals and birds supports the possibility of melatonin acting directly on the renal system.