Copper Stimulation of LHRH Release from Median Eminence Explants

Abstract

Copper, complexed to histidine (CuHis), stimulates LHRH release from explants of the median eminence area (MEA). To gain further understanding of the mechanism of copper action, in this study, we assessed the Na⁺ and energy requirements for CuHis stimulation of LHRH release. MEA explants, obtained from adult male rats, were incubated at 37 °C for 15 min with 100 µM CuHis and then for 45 min in CuHis-free medium (Krebs-Ringer-phosphate buffer, pH 7.4). LHRH released into the medium was evaluated by RIA. When the incubation buffer contained 143 mM Na⁺, CuHis stimulated the release of LHRH from a basal level of 17.2 ± 1.26 (mean ± SEM, n = 7) to 74.5 ±6.2 pg/60 min per MEA. When [Na⁺] was reduced to 16 mM Na⁺ (by substituting with Li⁺), CuHis-stimulated LHRH release was inhibited by 80% (p< 0.001); indicating a requirement for Na⁺. In addition, we found that CuHis-stimulated LHRH release was a saturable function of Na⁺ concentration; saturation achieved with about 100 mM Na⁺. To assess the requirement for Na⁺ transport, we evaluated the effect of 1 mM ouabain, 10 µMtetrodotoxin (TTX), or 100 µM amiloride on CuHis stimulation of LHRH release. Ouabain inhibited CuHis stimulation of LHRH release by 80%, whereas TTX and amiloride were ineffective. In addition, we observed that CuHis did not stimulate LHRH release when incubation was carried out at 4 °C or at 37 °C in the presence of 5 mM KCN. In summary, these results indicate that the process of CuHis-stimulated release of LHRH from the median eminence has a requirement for extracellular Na⁺, for thermic and metabolic energy, for the activity of the enzyme Na⁺/K⁺ ATPase, and for Na⁺ transport by a limited number of sites. These requirements are supportive of the proposition that CuHis is taken up by the LHRH axonal terminal by co-transport with Na⁺, the driving force of which is the Na⁺ electrochemical gradient generated by NaVK⁺ ATPase.