maitotoxin and Brain-Neoplasms

1. The aim of the present study was to determine the effects of maitotoxin on nerve growth factor production and the Ca2+ influx in clonal rat glioma cells (C6-BU-1). 2. Maitotoxin (1 - 10 ng ml-1) induced a profound increase in 45Ca2+ influx in an extracellular Ca2+-dependent manner. However, high KCl had no effect at all. These effects were supported by the results from the analysis of intracellular Ca2+ concentration using fura 2. 3. The maitotoxin-induced 45Ca2+ influx was inhibited by inorganic Ca2+ antagonists, such as Mg2+, Mn2+ and Co2+. The inhibitory effect of Co2+ was antagonized by increasing the extracellular Ca2+ concentrations. 4. Maitotoxin (3 ng ml-1) as well as A-23187 (1microM) and dibutyryl cyclic AMP (0.5 mM) caused an acceleration of nerve growth factor (NGF) production in C6-BU-1 cells, as determined by NGF enzyme immunoassay. 5. Reverse transcription polymerase chain reaction (RT - PCR) analysis showed that maitotoxin (10 ng ml-1) enhanced the expression of NGF mRNA, which was abolished by the removal of extracellular Ca2+. A-23187 also accelerated its expression. 6. These results suggest that maitotoxin activates a voltage-insensitive Ca2+ channel and accelerates NGF production mediated through a Ca2+ signalling pathway in C6-BU-1 glioma cells.

Topics: Animals; Brain Neoplasms; Bucladesine; Calcimycin; Calcium Channel Agonists; Calcium Channels; Cell Line; Fluorescent Dyes; Fura-2; Glioma; Immunoenzyme Techniques; Marine Toxins; Neoplasm Proteins; Nerve Growth Factors; Oxocins; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stimulation, Chemical; Tumor Cells, Cultured

Maitotoxin (MTX) at 0.3 nM elicited a 10-20-fold increase in the level of Ca(2+) influx in rat glioma C6 cells. At higher doses (3-30 nM), MTX induced marked Ca(2+) influx in human erythrocyte ghosts when monitored with the fluorescent dye Fura-2. Although the ghosts were not as susceptible to MTX as intact erythrocytes or other cell lines, Fura-2 experiments under various conditions suggested that the MTX-induced entry of ions into the ghosts was mediated by a mechanism similar to that reported for cells or tissues. These ghosts are the simplest system known to be sensitive to MTX and thus may be suitable for research on the direct action of MTX. Gangliosides GM1 and GM3, glycosphingolipids which have a sialic acid residue, strongly inhibited MTX-induced Ca(2+) influx in C6 cells, while the inhibitory action by asialo-GM1, which lacks a sialic acid residue, was somewhat weaker. Their inhibitory potencies were in the following order: GM1 (IC(50) approximately 2 microM) > GM3 (IC(50) approximately 5 microM) > asialo-GM1 (IC(50) approximately 20 microM). GM1 (3 microM) completely blocked MTX (30 nM)-induced Ca(2+) influx in human erythrocyte ghosts. When C6 cells were pretreated with tunicamycin, an antibiotic which inhibits N-linked glycosylation, or concanavalin A, a lectin which exhibits a high affinity for cell-surface oligosaccharides, MTX-induced Ca(2+) influx was significantly potentiated. This suggests that removal of oligosaccharides from the cell surface by tunicamycin or capping of sugar chains on plasma membranes by concanavalin A can potentiate the action of MTX.

Topics: Animals; Anti-Bacterial Agents; Brain Neoplasms; Calcium; Calcium Radioisotopes; Concanavalin A; Erythrocyte Membrane; Fluorescent Dyes; Fura-2; Gangliosides; Glioma; Marine Toxins; Membrane Lipids; Membrane Potentials; Models, Molecular; Molecular Conformation; Oxocins; Rats; Tumor Cells, Cultured; Tunicamycin

Since maitotoxin, a potent marine toxin, is known to cause not only Ca2+ influx but also phosphoinositide hydrolysis, we investigated the Ca2+ dependency of maitotoxin-induced phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Maitotoxin elicited inositol 1,4,5-trisphosphate accumulation in a time-dependent manner. In [3H]inositol-labeled cells, maitotoxin stimulated phosphoinositide hydrolysis in an extracellular Ca2+-dependent manner. Maitotoxin also caused an intracellular Ca2+ elevation, which was abolished by an intracellular Ca2+ chelater BAPTA-AM. Interestingly, maitotoxin still caused phosphoinositide hydrolysis in the BAPTA-AM-treated cells. These results indicate that maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in 1321N1 human astrocytoma cells.

Topics: Astrocytoma; Brain Neoplasms; Calcimycin; Calcium; Chelating Agents; Egtazic Acid; Extracellular Space; Humans; Hydrolysis; Inositol 1,4,5-Trisphosphate; Ionophores; Marine Toxins; Oxocins; Phosphatidylinositols; Tumor Cells, Cultured