Studies on porphyrin-heme biosynthesis in organotypic cultures of chick dorsal root ganglion. I. Observations on neuronal and non-neuronal elements.
Academic Article
Overview
abstract
Living, mature organotypic cultures of chick dorsal root ganglion maintained in culture for 3 weeks were incubated in medium containing various levels of a precursor of porphyrin and heme formation, delta-aminolevulinic acid (ALA), (0.5 mM to 10 mM) or a combination of ALA (10 MM) and a metal chelator, CaMg-EDTA (5 mM) for up to 48 hours. Although no morphologic changes occurred in the cultures incubated with these compounds as observed by bright-field or dark-field microscopy, fluorescence microscopic study at 12, 24, and 48 hours demonstrated an intense red fluorescence with in the non-neuronal cells of the cultures (Schwann cells, fibroblasts, and macrophages) but not in the nerve cells. Spectrofluorometric analysis of perchloric acid-methanol extracts of the cultures revealed an emission spectrum characteristic of porphyrins. Autoradiographic studies, using 14C-labelled ALA, indicated that ALA was taken up by all cells (nerve cells as well as non-neuronal cells) in the cultures. The cultures incubated with ALA plus the metal chelator CaMg-EDTA showed the same distribution of porphyrin fluorescence, but a 2-fold increase in the amount of porphyrins was generated, when compared to cultures incubated with ALA alone. This observation suggests that a considerable fraction of porphyrins may be utilized to form heme in these cells since CaMa-EDTA blocks ferrochelatase activity, the terminal enzyme in the heme biosynthetic pathway. This is the first demonstration of active porphyrin-heme biosynthesis from ALA in cultured nervous system cells. Our results indicate that this biosynthetic pathway remains active in 3-week old cultures of chick dorsal root ganglion, and further, that the pathway appears to be predominantly present in the non-neuronal cellular elements of the ganglion rather than in nerve cells.
