Expression of preproenkephalin mRNA by developing glia and neurons was examined in cultures of embryonic and neonatal rat brain. Cultured glia from specific regions of embryonic day 17 and neonatal day 1 rat brain were identified as astrocytes on the basis of both morphology and expression of immunoreactivity for glial fibrillary acidic protein. The level of preproenkephalin mRNA in cultured neonatal hypothalamic astrocytes was comparable to levels present in cultured embryonic striatal and hypothalamic neurons. Levels of the mRNA were significantly higher in astrocytes derived from neonatal hypothalamus compared to astrocytes derived from other areas of the brain. Thus, there is heterogeneity among astrocytes with respect to preproenkephalin expression. Levels of preproenkephalin mRNA in cultured neonatal striatal astrocytes were only one-third as high as levels in embryonic striatal astrocytes; this observation suggests that glial expression of the gene may be down-regulated during development. Although cultured hypothalamic neurons contained substantial levels of prodynorphin mRNA, levels of this mRNA were not detectable in cultured astrocytes from any brain region or in cultured striatal neurons. Thus, glia do not express all opioid peptide genes during development. These observations suggest that expression of the preproenkephalin gene by astrocytes may play a role in development of the brain.Astrocytes are the most numerous cell type in the brain and appear to subserve multiple functions. Astrocytes may help to regulate the ionic environment of the extracellular space (1-3), to inactivate neurotransmitters by accumulating them (3-7), and to transfer nutrients and neurotransmitter precursors to neurons (8,9). Proliferation of astrocytes plays an integral part in the healing of brain injury, and interactions between neurons and astrocytes regulate neuronal differentiation and process elongation (9-13). Studies of cell surface molecules expressed by astrocytes have shown at least two identifiable subtypes (14), and there is some evidence that there are differences among astrocytes from different regions of the brain (10).Although astrocytes express receptors for neurotransmitters (6,(15)(16)(17)(18)(19) and contain systems for neurotransmitter uptake (3-7), they do not appear to synthesize neurotransmitters themselves. With the exception of the amino acid transmitters, it is generally accepted that neurotransmitters in brain are localized within neurons. However, neurotransmitter genes may be expressed by nonneuronal cells, particularly in the endocrine and immune systems (for a review, see ref. 20), and there is some evidence that glial precursors may express opioid peptides. Enkephalin-like immunoreactivity was detected in glia-like and neuron-like cells and in the germ layer of developing rat cerebellum but was not detected in fully differentiated cerebellar glia or neurons (21). Cells derived from the C6 glioma line contain abundant amounts of preproenkephalin mRNA and proenkephalin, although they...

Carboxypeptidase E (EC 3.4.17.10) is a carboxypeptidase B-like enzyme associated with the biosynthesis of many peptide hormones and neurotransmitters. Media collected from cultured astrocytes contain a carboxypeptidase E-like activity. Cultured astrocytes secrete approximately 73% of their cellular level of carboxypeptidase E per hour, and secretion is not substantially influenced by 35 mM KCl. In contrast, neurons secrete only 29% of their cellular carboxypeptidase E per hour, but secretion increases to 86% on stimulation with 35 mM KCl. Secretion of carboxypeptidase E activity from both neuronal and astrocyte cultures is relatively selective; neither acid phosphatase or acetylglucosaminidase is secreted in appreciable amounts. Cultured neurons and astrocytes express a carboxypeptidase E mRNA of a similar size. The levels of this mRNA differ in astrocytes cultured from different brain regions, with high levels in striatal, cortical, hippocampal, and hypothalamic astrocytes and low levels in cerebellar astrocytes. The level of carboxypeptidase E mRNA in hypothalamic astrocyte cultures is four- to fivefold higher than the level in hypothalamic neuronal cultures. These results indicate that cultured astrocytes express carboxypeptidase E mRNA and enzymatic activity and thus contain one of the enzymes required in the biosynthesis of many peptide hormones and neurotransmitters.