cyclic-gmp and Teratocarcinoma

The human Ntera2 (NT2) teratocarcinoma cell line can be induced to differentiate into post-mitotic neurons. Here, we report that the human NT2 neurons generated by a spherical aggregate cell culture method express increasing levels of typical pre-synaptic proteins (synapsin and synaptotagmin I) along the neurite depending on the length of in vitro culture. By employing an antibody directed against the luminal domain of synaptotagmin I and the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide, we show that depolarized NT2 neurons display calcium-dependent exo-endocytotic synaptic vesicle recycling. NT2 neurons express the neuronal isoform of neuronal nitric oxide synthase and soluble guanylyl cyclase (sGC), the major receptor for nitric oxide (NO). We tested whether NO signal transduction modulates synaptic vesicle turnover in human NT2 neurons. NO donors and cylic guanosine-monophosphate analogs enhanced synaptic vesicle recycling while a sGC inhibitor blocked the effect of NO donors. Two NO donors, sodium nitroprusside, and and N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino) ethanamine evoked vesicle exocytosis which was partially blocked by the sGC inhibitor. The activator of adenylyl cyclase, forskolin, and a cAMP analog induced synaptic vesicle recycling and exocytosis via a parallel acting protein kinase A pathway. Our data from NT2 neurons suggest that NO/cyclic nucleotide signaling pathways may facilitate neurotransmitter release in human brain cells.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Cell Differentiation; Cell Line, Tumor; Colforsin; Cyclic GMP; Enzyme Inhibitors; Exocytosis; Humans; Isoquinolines; Microtubule-Associated Proteins; Neurons; Nitric Oxide; Nitric Oxide Synthase Type I; Nitroso Compounds; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Pyridinium Compounds; Quaternary Ammonium Compounds; Signal Transduction; Sulfonamides; Synapsins; Synaptic Vesicles; Synaptotagmin I; Teratocarcinoma; Time Factors

We have investigated the possible role of second messengers on inositol homeostasis in NT2-N cells, human central nervous system neurons obtained by terminal differentiation of teratocarcinoma precursors. Uptake of inositol into NT2-N neurons was inhibited approximately 10% by protein kinase C (PKC) activation but was unaffected by either the presence of cyclic nucleotide analogs or changes in the intracellular concentration of Ca2+. Efflux of inositol from NT2-N neurons was enhanced in hypotonic buffer but virtually eliminated by inclusion of the Cl- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, a result which indicates the involvement of a volume-sensitive organic osmolyte-anion channel. Volume-sensitive inositol efflux was stimulated approximately 30% following activation of PKC or elevation of the cytosolic Ca2+ concentration but was unaffected by protein kinase A activation. These results suggest that whereas inositol uptake into NT2-N neurons is relatively refractory to regulation, volume-sensitive inositol efflux may be significantly affected by intracellular signaling events.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Biological Transport; Calcium; Cell Differentiation; Cyclic GMP; Enzyme Activation; Humans; Hypotonic Solutions; Inositol; Ionomycin; Lithium; Neurons; Oxotremorine; Protein Kinase C; Second Messenger Systems; Signal Transduction; Teratocarcinoma; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured

Chlorpromazine is a phenothiazine with a structure similar to that of methylene blue. Since methylene blue is a well known inhibitor of nitric oxide-induced cyclic GMP accumulation, we investigated whether chlorpromazine had the same effect. Cyclic GMP accumulation, induced in a mouse teratocarcinoma cell line (P19) by sodium nitroprusside (a nitric oxide releasing agent), was inhibited by both methylene blue (IC50 0.34 microM) and chlorpromazine (IC50 35 microM). Chlorpromazine's action was probably directed specifically at soluble guanylate cyclase, since the drug had no effect on ADP-ribosylation in rat hippocampus, another nitric oxide-affected, but cGMP-independent event.

Topics: Adenosine Diphosphate Ribose; Animals; Antipsychotic Agents; Autoradiography; Cell Survival; Chlorpromazine; Cyclic GMP; Hippocampus; Immunoenzyme Techniques; Male; Methylene Blue; Mice; Nitric Oxide; Nitroprusside; Teratocarcinoma; Testicular Neoplasms; Tumor Cells, Cultured