Evidence that copper-amino acid complexes are potent stimulators of the release of luteinizing hormone-releasing hormone from isolated hypothalamic granules.

Chelated copper has been previously shown to stimulate the release of LHRH from isolated hypothalamic granules. In this study, we evaluated the chelator specificity, the kinetic constants, and the characteristics of copper interaction with LHRH granules. LHRH granules were isolated from the median eminence area of adult male rats and then incubated in a buffered medium at 37 C. Release of LHRH into the incubation medium was assessed by RIA of LHRH remaining in the granules after incubation. It was found that CuHistidine (CuHis) as well as CuCysteine markedly stimulated LHRH release from the isolated granules, release being 56% and 63%, respectively, of the total LHRH content of granules incubated in buffer alone. In contrast, neither CuGly-His-Lys nor CuBSA stimulated LHRH release. The CuHis-stimulated release of LHRH was a saturable function of the concentration of CuHis. The Michaelis-Menten constants of this release process were estimated; the apparent Km for copper was found to be 4 microM, and the maximal velocity was 65% of the granule content of LHRH released in 5 min. In addition, we noted that CuHis-stimulated release of LHRH, assessed 6 min after CuHis, was completely abolished when dithiothreitol (DTT) was added immediately after CuHis, partially abolished when added 1 or 2 min after CuHis, and not affected at all when added 3 min after CuHis. This time course of DTT inhibition of LHRH release suggests that a period of 2-3 min of copper interaction with the granules is required for the 6-min manifestation of copper action. Furthermore, this DTT-inhibitable interaction of copper did not occur when granules were incubated at 4 C. In summary the findings that copper, chelated to putative circulating chelators, markedly stimulates LHRH release and that the apparent Km of 4 microM for copper in this process is within the concentration range for the physiological action of copper support the proposal that blood-borne copper can interact rapidly with the LHRH granule in an energy-requiring fashion and that, consequent to this interaction, LHRH release occurs.