Copper-induced release of immunoreactive alpha-melanotropin from isolated hypothalamic granules

Previously, we demonstrated that copper chelates stimulate the release of luteinizing hormone releasing hormone (LHRH) from isolated hypothalamic granules. To assess the generality of the copper-stimulated release process, we determined the effects of copper on the release of immunoreactive alpha-melanotropin (alpha-MSHi) from isolated granules. When granules were incubated with various copper complexes, CuATP stimulated alpha-MSHi release by 54 +/- 6% (mean +/- S.E.), Cu tartarate by 56 +/- 4%, CuBSA by 32 +/- 5% and Cu histidine by 29 +/- 2%. CuATP-stimulated alpha-MSHi release from granules incubated under N2 was 57% of that incubated under air. Furthermore, the reducing agent dithiothreitol (DTT) inhibited CuATP-stimulated alpha-MSHi release (p less than 0.01), whereas oxidized DTT did not do so. Pretreatment of granules with the thiol-blocking reagents iodoacetic acid or 5, 5'-dithiobis-(2-nitrobenzoic acid) inhibited CuATP-stimulated alpha-MSHi release by 52 +/- 3 and 38 +/- 4%, respectively. Thus, chelated copper, rather than ionic copper, is the active form of the metal and the action of copper involves the oxidation of thiols. These data are similar to those previously observed for the copper-stimulated release of LHRH. Hence, the effects of copper on the permeability of granule membranes may be a generalized phenomenon which underlies susceptibility of storage granules to the reduction-oxidation status of the cellular milieu.