Evidence that Copper-Amino Acid Complexes Are Potent Stimulators of the Release of Luteinizing Hormone-Releasing Hormone from Isolated Hypothalamic Granules*

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Abstract
Chelated Cu stimulates the release of LHRH from isolated hypothalamic granules. The chelator specificity, the kinetic constants, and the characteristics of Cu interaction with LHRH granules were evaluated. LHRH granules were isolated from the median eminence area of adult male rats and then incubated in a buffered medium at 37.degree. C. Release of LHRH into the incubation medium was assessed by RIA [radioimmunoassay] of LHRH remaining in the granules after incubation. CuHis as well as CuCys 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. 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 Cu was 4 .mu.M, and the maximal velocity was 65% of the granule content of LHRH released in 5 min. In addition, 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.degree. C. Cu, chelated to putative circulating chelators, markedly stimulates LHRH release. The apparent Km of 4 .mu.M for Cu in this process is within the concentration range for the physiological action of Cu. Blood-borne Cu apparently can interact rapidly with the LHRH granule in an energy-requiring fashion and consequent to this interaction, LHRH release occurs.