Met-enkephalin secretion from mixed cultures of hypothalamic neurons and astrocytes.

Abstract

Release of the endogenous opioid pentapeptide, met-enkephalin, from primary cultures of dissociated fetal rat hypothalamic cells was studied using an assay system which could both measure and differentiate between free met-enkephalin and the larger enkephalin-containing peptides (ECPs), which are the processing intermediates of the enkephalin precursor. The cultures were maintained in fully defined, serum-free medium and contained both neurons and astrocytes. Free met-enkephalin was secreted from the cultures in significant quantities in response to nonspecific depolarisation with 56 mM potassium, by a mechanism dependent upon extracellular calcium. Under basal conditions, barely detectable amounts of free peptide were released, whereas ECPs were secreted in significant quantities which were not reduced by the removal of extracellular calcium. As the period of culture increased, so did the quantitative importance of this constitutive ECP secretion, relative to the stimulated release of free peptide. Treatment of the cultures with the cytotoxic agent, cytosine arabinoside, attenuated this temporal increase of ECP secretion, whilst leaving the stimulated release of free met-enkephalin relatively unaffected. This suggested that the met-enkephalin secretion seen within the cultures reflected the presence of at least two distinct enkephalinergic cell types and that the change in the nature of the secreted enkephalin was at least in part, due to the proliferation of one of these cell populations. These results are consistent with secretion of met-enkephalin from both neurons and astrocytes within these cultures. We propose that the neurons secreted essentially fully processed peptide in a regulated manner, whilst the mitotic glial cells constitutively secreted non- or partially processed precursor peptides.(ABSTRACT TRUNCATED AT 250 WORDS)