Effects of temperature on 2-[125I]-iodomelatonin binding to melatonin receptors in the neural retina of the frog Rana perezi.

The present study analyzes the effect of temperature-dependent modifications on the binding of the analog 2-[125I]-melatonin to melatonin receptors in isolated neural retina membranes from the greenfrog Rana perezi. Association and dissociation rate constants (K+1, K-1) were exponentially increased by the assay temperature. At 15 degrees C, association and dissociation required several hours; meanwhile, at 35 degrees C, rate constants were 100- and 34-fold faster, respectively. However, the Kd constant calculated as K-1/K+1 was unmodified by the assay temperature. When frogs were acclimated at either 5 or 22 degrees C for 1 month, K+1, and K-1 constants determined at 15 and 25 degrees C were identical in both cold- and warm-acclimated groups. Thus, the binding kinetics of melatonin receptors in frog retinas did not shown any thermal compensation. Results from saturation curves and pharmacological profiles of melatonin binding sites support a lack of effect of assay temperature on the affinity of melatonin receptors in the frog retina. The inhibition of [125I]Mel binding by GTPgammaS showed clearly that the coupling of melatonin receptors to G proteins is temperature-dependent. Higher concentrations of the GTP analog were needed to inhibit specific binding when temperature decreased. The temperature effect on binding kinetics and on the G protein coupling to melatonin receptors suggests that the melatonin signal could be transduced distinctly depending on the temperature. Thus, temperature plays a major role, not only on melatonin synthesis, but also in the transduction of melatonin signal in ectotherms.

2-[125I]-melatonin binding sites in the central nervous system and neural retina of the frog Rana perezi: regulation by light and temperature.

The objective of the present study is to test daily and seasonal changes in 2-[125I]-Melatonin ([125I]-Mel) binding in different brain areas and the retina of the frog Rana perezi as well as the possible effect of light and temperature on melatonin receptors. During the day-night cycle, binding of [125I]-Mel showed a clear rhythm in the optic tectum, diencephalon, telencephalon, and neural retina, the binding being higher in the light phase than in the dark phase. By contrast, melatonin receptors did not show any significant summer-winter differences in any of the four tissues studied. In the neural retina, but not in the brain, exposure of frogs to 24 h darkness for one week leads to significantly less [125I]-Mel binding than 24 h light exposure. This darkness-induced reduction of [125I]-Mel binding is not due to a desensitisation of binding sites by high melatonin levels. Thermal acclimation to either 5 or 22 degrees C for one month did not change the affinity (Kd) and density (Bmax) of [125I]-Mel binding sites either in the brain or the retina. All these results indicate that there is a daily rhythm in melatonin receptors in the frog brain and retina, and that the light/dark cycle can drive this rhythm in [125I]-Mel binding in the retina. Temperature apparently did not modify [125I]-Mel binding in frogs.

Characterization of melatonin binding sites in the brain and retina of the frog Rana perezi.

The aim of this study was to characterize 2-[125I]iodomelatonin binding sites in the neural retina and central nervous system (telencephalon, diencephalon, and optic tectum) of the anuran amphibian Rana perezi. Saturation and kinetic studies and pharmacological characterization revealed the existence of a unique melatonin-binding site that belongs to the Mel 1 receptor subtype. The affinity of this site is similar in all tissues studied (Kd, 10.5-12.8 pM), but the density varied from diencephalon and optic tectum, which exhibit the highest density, to telencephalon with the lowest. Neural retina showed an intermediate receptor density. This melatonin-binding site fulfills the requirements of a real hormone receptor; the binding is saturable, reversible, and inhibited by different melatonin agonists and antagonists. The affinity order of ligands is: 2-phenyl-melatonin = 2-I-melatonin > 6-Cl-melatonin = melatoninz >> luzindole. Additionally, specific binding is decreased by non-hydrolysable GTP analogue, sodium, and by pretreatment of membranes with pertussis toxin. All these results suggest the existence of a widely distributed and pharmacologically homogeneous melatonin receptor of the subfamily Mel 1 in the nervous system of Rana perezi coupled to a Gi/o protein.