Analysis of NO synthase expression in neuronal, astroglial and fibroblast-like derivatives differentiating from PCC7-Mz1 embryonic carcinoma cells.
We studied the expression of the NO synthase isoforms in an in vitro model of neural development using RT-PCR, Western blot and immunohistochemistry. Murine PCC7-Mz1 cells (Jostock et al., Eur. J. Cell Biol. 76, 63-76, 1998) differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP along the neural pathway into neuron-like, fibroblast-like and astroglia-like cells. Undifferentiated cells showed immunofluorescent staining for neuronal-type NOS I and endothelial-type NOS III. This expression pattern was retained in those cells differentiating into neurofilament- and tau protein-positive neuronal cells. Thymocyte alloantigen (Thy1.2/CD 90.2)-positive fibroblasts, appearing around day 3, and glial fibrillary acidic protein (GFAP)-positive astroglial cells, appearing after day 6 of differentiation, stained negative for any NOS isoform. Starting at day 6 of differentiation, expression of inducible-type NOS II could be stimulated with cytokines in a subset of cells, which may represent activated astrocytes. NOS II was always undetectable in non-induced cultures. These data indicate that the ability of stem cells to express NOS I and NOS III is only retained when the cells differentiate along the neuronal lineage, while a small subpopulation of cells acquires the ability to express NOS II in response to cytokines.
Gath
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I
Department of Pharmacology, Johannes Gutenberg University, Mainz/Germany.
Steppuhn
A
A
Maelicke
A
A
Reinhardt
S
S
Förstermann
U
U
D000818:Animals; D001253:Astrocytes / Q000201:enzymology*; D015153:Blotting, Western; D003994:Bucladesine / Q000494:pharmacology; D018236:Carcinoma, Embryonal / Q000201:enzymology* / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005347:Fibroblasts / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D051379:Mice; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D016207:Cytokines; D014212:Tretinoin; D003994:Bucladesine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496321:Nos1 protein, mouse; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse