Melatonin Does Not Affect Luteinizing Hormone-Releasing Hormone Binding to Neonatal Rat Anterior Pituitary Membranes

Abstract

Melatonin suppresses luteinizing hormone-releasing hormone (LHRH)-stimulated release of luteinizing hormone (LH) and follicle-stimulating hormone from neonatal rat gonadotrophs. Since this effect occurs rapidly and selectively, we examined in this study the possibility that melatonin may inhibit LHRH-receptor binding. The physiologically active LHRH analog (D-Ala⁶, des-Gly¹⁰)-LHRH-N-ethylamide (D-Ala) was iodinated by a modification of a lactoperoxidase method and its concentration determined by self-displacement in the receptor binding assay. Crude anterior pituitary membranes from 10- to 12-day-old female rats were incubated with [¹²⁵I]iodo-D-Ala (¹²⁵I-D-Ala) either alone or in the presence of melatonin or a peptide LHRH antagonist at 4 °C in a volume of 200 µl. Specific binding was defined as the difference between the binding in the absence and in the presence of 50 or 100 µM D-Ala, as specified. Binding experiments were terminated by dilution and filtration through either Millipore EHWP or Whatman GF/C filters. Specific binding approximated equilibrium by 120 min. Scatchard analysis revealed an apparent K_D of 1.5 nM with the EHWP filters and 1.13 nM with the GF/C filters. In all experiments, use of either filter produced similar results; the only obvious difference was an apparent increase in the number of binding sites with the EHWP filters. Melatonin (1 µM) had no detectable effect on binding of ¹²⁵I-D-Ala. By contrast, the peptide LHRH antagonist at 10 and 100 nMinhibited binding of ¹²⁵I-D-Ala by 43 and 80%, respectively. However, in parallel studies with cultured anterior pituitary cells, both melatonin and the peptide antagonist inhibited the D-Ala-stimulated LH release. Melatonin (1 µM) suppressed the LH response to 10 pM D-Ala by 51%, and the peptide antagonist at 10 and 100 nM similarly inhibited release by 33 and 84%, respectively. These data clearly show that while melatonin and the LHRH peptide antagonist both suppress stimulated LH release by gonadotrophs in culture, melatonin does not antagonize the binding of D-Ala to the LHRH receptor.