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Expression of proenkephalin A mRNA and enkephalin-containing peptides in cultured fibroblasts
H Rosen1, R D Polakiewicz, R Simantov
1Department of Virology, Hebrew University of Jerusalem, Hadassah Medical School.
Abstract:
Proenkephalin A (PEA) gene was found to be expressed in primary, secondary and tertiary cultures of rat fibroblasts. The 1.4 kb PEA mRNA was detected by Northern blot analysis. The same cultures do not express detectable amounts of proenkephalin B (prodynorphin) or (POMC) mRNAs. Acidic cell extracts were purified on a C18 octadecyl Amprep column and analysed with a specific methionine enkephalin radioimmunoassay to detect whether PEA mRNA is translated. A significant amount of enkephalin immunoreactivity (178-185 fmol/mg protein) was observed upon trypsin and carboxypeptidase B digestion of fibroblast cell extracts, whereas only 3-5% of this amount was free enkephalin. It is therefore indicated that the PEA mRNA expressed in fibroblasts is indeed translated to the proenkephalin precursor protein, but the cells accumulate only a small quantity of the processed pentapeptides. The implication of these observations to the possible developmental role of PEA in various non-neuronal cells, including mesodermal lineages, is discussed.
Insights
Rat fibroblasts express the Proenkephalin A (PEA) gene, translating its mRNA into precursor proteins. However, these cells accumulate minimal processed pentapeptides, suggesting a potential developmental role in non-neuronal tissues.
Area of Science:
- Molecular Biology
- Neuroscience
- Cell Biology
Background:
- Proenkephalin A (PEA) gene expression is investigated in rat fibroblasts.
- Previous studies have not extensively explored PEA in non-neuronal cell types.
Purpose of the Study:
- To determine if Proenkephalin A (PEA) gene is expressed in rat fibroblasts.
- To investigate the translation of PEA mRNA and processing of its precursor in these cells.
Main Methods:
- Northern blot analysis to detect PEA mRNA.
- Radioimmunoassay (RIA) to quantify enkephalin immunoreactivity in cell extracts.
- Enzyme digestion (trypsin and carboxypeptidase B) to assess processed peptides.
Main Results:
- Proenkephalin A (PEA) mRNA (1.4 kb) was detected in primary, secondary, and tertiary rat fibroblast cultures.
- Fibroblast extracts showed significant enkephalin immunoreactivity after enzyme digestion, indicating precursor protein translation.
- Only a small fraction (3-5%) of free enkephalin was detected, suggesting limited accumulation of processed peptides.
Conclusions:
- Rat fibroblasts express and translate Proenkephalin A (PEA) mRNA into precursor proteins.
- These cells accumulate minimal processed enkephalin peptides, despite active translation.
- The findings suggest a potential developmental role for PEA in non-neuronal cells, including mesodermal lineages.