Studies on the expression of carboxypeptidase E and proenkephalin in cultured astrocytes

Abstract

The overall objective of this project was to study the expression of the neuropeptide precursor, proenkephalin (PE), and the neuropeptide processing enzyme, carboxypeptidase E (CPE), by cultured astrocytes.;Quantitative in situ hybridization studies demonstrated that cultured astrocytes, like neurons, demonstrate individual heterogeneity in their expression of CPE and PE mRNAs. Cultures contain at least two populations of cells which may be separated by statistical analysis. One population expresses high levels of these mRNAs and a second population expresses background levels of these mRNAs. The percentages of cells expressing these mRNAs and the average level of mRNA expression per cell varies depending on the brain region from which the astrocytes are derived. In addition, the level of expression of PE mRNA, but not CPE mRNA, was found to be inversely proportional to the culture density.;Using CPE as a marker for overall neuropeptide secretion, the neuropeptide secretory pathway in astrocytes was characterized via pulse-chase studies. TPA treatment of astrocytes causes an increase in CPE biosynthesis, as demonstrated by elevations in CPE mRNA levels. Quantitative in situ hybridization studies demonstrated that the increase in gene expression is largely due to an increase in percentages of astrocytes expressing high levels of CPE mRNA, rather than an increase in the average level of CPE mRNA expression per cell.;The amidating enzyme, peptidylglycine-{dollar}\alpha{dollar}-amidating monooxygenase (PAM), is present in most peptide producing tissues. Since certain PE-derived peptides are amidated, the expression of PAM mRNA by cultured astrocytes was also examined. The expression of PAM mRNA by astrocytes cultured from various brain regions demonstrates regional heterogeneity that is distinct from that of CPE and PE mRNA expression. Quantitative in situ hybridization analysis did not reveal any substantial individual heterogeneity in the levels of PAM mRNA expression per cell, however, the levels vary between brain regions.;These results indicate that cultured astrocytes demonstrate individual heterogeneity in their expression of PE and CPE, but not PAM, mRNAs; that astrocytes secrete CPE, and presumably neuropeptides, through a constitutive secretory pathway; and that astrocytes may alter their expression of PE and CPE in response to external stimuli. Thus, astrocytes may be involved in cell-signaling through the production of neuropeptides that are regulated by similar signal transduction pathways as neurons. (Abstract shortened by UMI.).