MET-ENKEPHALIN SECRETION FROM MIXED CULTURES OF HYPOTHALAMIC NEURONS AND ASTROCYTES

Release of the endogenous opioid pentapeptide, met-enkephalin, from pr imary cultures of dissociated fetal rat hypothalamic cells was studied using an assay system which could both measure and differentiate betw een free metenkephalin and the larger enkephalin-containing peptides ( ECPs), which are the processing intermediates of the enkephalin precur sor. The cultures were maintained in fully defined, serum-free medium and contained both neurons and astrocytes. Free met-enkephalin was sec reted from the cultures in significant quantities in response to nonsp ecific depolarisation with 56 mM potassium, by a mechanism dependent u pon extracellular calcium. Under basal conditions, barely detectable a mounts of free peptide were released, whereas ECPs were secreted in si gnificant quantities which were not reduced by the removal of extracel lular calcium. As the period of culture increased, so did the quantita tive importance of this constitutive ECP secretion, relative to the st imulated release of free peptide. Treatment of the cultures with the c ytotoxic agent, cytosine arabinoside, attenuated this temporal increas e of ECP secretion, whilst leaving the stimulated release of free met- enkephalin relatively unaffected. This suggested that the met-enkephal in secretion seen within the cultures reflected the presence of at lea st two distinct enkephalinergic cell types and that the change in the nature of the secreted enkephalin was at least in part, due to the pro liferation of one of these cell populations. These results are consist ent with secretion of met-enkephalin from both neurons and astrocytes within these cultures. We propose that the neurons secreted essentiall y fully processed peptide in a regulated manner, whilst the mitotic gl ial cells constitutively secreted non- or partially processed precurso r peptides. There was also some evidence that the glial cells could at tenuate neuronal neuropeptide secretion.