Expression of preproenkephalin mRNA by cultured astrocytes and neurons.

Abstract

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...

Cellular localization of proenkephalin mRNA and enkephalin peptide products in cultured astrocytes

“…The present results confirm and extend previous reports by localizing proenkephalin mRNA in astrocytes at the cellular level by in situ hybridization, and thereby provide direct evidence that the enkephalin mRNA previously identified in Northern blots is expressed by astrocytes and not by a small number of contaminating non-astrocytic cells which are present in the astrocyte-enriched cultures reported in previous studies 37, 43,52 . In particular, cells double-labeled for both proenkephalin mRNA and GFAP intermediate filaments provide first direct evidence that astrocytes express the proenkephalin gene during development.…”

“…These and other experiments suggest that endogenous opioids are normally available to cells in the developing CNS in sufficient quantities to tonically inhibit growth 17,18,41,[54][55][56] . In support of this hypothesis, opioid peptide levels and opioid binding are greatly increased in the rat cerebellum 48,49 28,32,43,52 . In the present study, proenkephalin (proenkephalin A) mRNA and enkephalin peptide expression were examined by in situ hybridization and immunocytochemistry, respectively, in primary cultures of the developing CNS (i) to identify the specific cellular sites of proenkephalin gene and peptide expression, and (ii) to determine whether opioids are expressed by replicating astrocytes prior to their reaching confluence.…”

“…Lastly, an additional control was performed by hybridizing with an [ 35 S]-labeled cRNA probe (5 × 10 4 cpm/slide) to prodynorphin mRNA10 which has some sequence homology to proenkephalin mRNA 10,22,39,50 . Vilijn et al 52 and Low et al 28 have previously demonstrated the absence of prodynorphin mRNA in astrocyte-enriched cultures by Northern analysis.…”

“…Recent reports have demonstrated the presence of proenkephalin mRNA by Northern analysis of confluent glial cultures that are enriched in astrocytes (i.e., 95-97% astrocytes) 28,32,43,52 . In the present study, proenkephalin (proenkephalin A) mRNA and enkephalin peptide expression were examined by in situ hybridization and immunocytochemistry, respectively, in primary cultures of the developing CNS (i) to identify the specific cellular sites of proenkephalin gene and peptide expression, and (ii) to determine whether opioids are expressed by replicating astrocytes prior to their reaching confluence.…”

A new species of enkephalin precursor mRNA with a distinct 5′‐untranslated region in haploid germ cells

“…Two pairs of direct repeats of 26 bases (92% homology) and 21 bases (81070 homology) in the 5'-terminal region are underlined and overlined, respectively. The nucleotides displayed above the mRNA sequence are mismatches seen in rat brain ppEnk cDNA (nucleotides -2, 33, 34 and 829) [8] Like mature enkephalinergic neurons, C6 glioma cells [11] and astroglial cells in primary culture [14] contain a large amount of ppEnk mRNA. This raises the possibility that the ppEnk gene is potentially expressed by immature ceils during early periods of neuron-glia development.…”