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

Abstract

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.

A ligand-specific action of chelated copper on hypothalamic neurons: stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants.

“…Copper is present in the hypothalamus in concentrations much greater than in blood (14,15), and in hypothalamic homogenates, copper is highly concentrated in synaptosomes (pinched-off axonal terminals) (16). We noted (4,5) …”

“…Tsou et al (2) observed increased levels of luteinizing hormone-releasing hormone (LHRH) in the pituitary stalk of the rabbit after the systemic administration of cupric acetate. We showed (3)(4)(5) that copper, at a concentration within the range required for the activity of several copper-dependent enzymes (6)(7)(8)(9), markedly stimulates the release of LHRH from isolated hypothalamic granules. Based on these findings, we proposed (4) that copper plays a role in regulating LHRH release from LHRH neurons in the following manner.…”

Effects of Cupric Acetate on Hypothalamic Gonadotropin-Releasing Hormone Release in Intact and Ovariectomized Rabbits

“…4). Furthermore, coincubation of iso lated hypothalamic granules with copper salts stimulates the release of GnRH [2,5].…”

“…Copper salts initiate pituitary luteinizing hormone (LH) release, probably by discharging gonadotropin-releasing hormone (GnRH) from the hypo thalamus, since systemic injection of copper acetate (CuAc) increases GnRH levels in pituitary stalk plasma of anesthe tized rabbits [42], and addition of copper salts to isolated hypothalamic granules stimulates GnRH release in vitro [2,5], Ovarian steroids are involved in copper-induced ovula tion in rabbits, since estrogen facilitates [38,41] and proges terone may inhibit [12] the action of copper. However, since steroids can act at both central and pituitary sites [1,6,34,40], their effects on GnRH release after CuAc administra tion are unclear.…”

Castration of Male Rats Reduces the Capacity of Granules Isolated from the Median Eminence to Secrete Luteinizing Hormone-Releasing Hormone in Response to Copper

“…13] to study some of the molecular events involved in secretion. We [2,5,15,16] have previously demonstrated that copper is an extremely potent stimulator of the release of LHRH from isolated LHRH granules. The characteristics of this release process are such that release follows Michaelis-Menten equations for enzyme kinetics (the apparent Km for copper is 4-6 \iM and the V,mix is about 60% of the granule content of LHRH released in 6 min), and that chelated copper (particularly copper-amino acid complexes) rather than ionic copper is the active form of the metal.…”