maitotoxin and Insulinoma

Maitotoxin (MTX) activates a Ca2+-dependent non-selective cation current (ICa-NS) in insulinoma cells whose time course is identical to non-selective cation currents activated by incretin hormones such as glucagon-like peptide-1 (GLP-1), which stimulate glucose-dependent insulin secretion by activating cAMP signaling pathways. We investigated the mechanism of activation of ICa-NS in insulinoma cells using specific pharmacological reagents, and these studies further support an identity between MTX- and GLP-1-activated currents. ICa-NS is inhibited by extracellular application of genistein, econazole, and SKF 96365. This inhibition by genistein suggests that tyrosine phophorylation may play a role in the activation of ICa-NS. ICa-NS is not inhibited by incubation of cells in glucose-free solution, by extracellular tetrodotoxin, nimodipine, or tetraethylammonium, or by intracellular dialysis with 4-aminopyridine, ATP, ryanodine, or heparin. ICa-NS is also not significantly inhibited by staurosporine, which does, however, partially inhibit the MTX-induced rise of intracellular Ca2+ concentration. These effects of staurosporine suggest that protein kinase C may not be involved in the activation of ICa-NS but that it may regulate intracellular Ca2+ release. Alternatively, ICa-NS may have a small component that is carried through separate divalent cation-selective channels that are inhibited by staurosporine. ICa-NS is neither activated nor inhibited by dialysis with KF, KF + AlF3 or GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), suggesting that GTP-binding proteins do not play a major role in the activation of this current.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cricetinae; Egtazic Acid; Glucagon-Like Peptide 1; Imidazoles; Insulinoma; Islets of Langerhans; Marine Toxins; Membrane Potentials; Oxocins; Pancreatic Neoplasms; Peptides; Staurosporine; Tumor Cells, Cultured

Partial structures of maitotoxin (MTX) were deduced by extensive two-dimensional NMR measurements, showing that the toxin had carbon-carbon double bonds in both termini of the molecule, and a primary alcohol group at the end. These functionalities were used for preparation of a radioligand of MTX; [3H]H-MTX and [3H]benzoyl-MTX. Both radioligands had high levels of non-specific binding to tissues. The binding of [3H]MTX to rat glioma C6 cells was inhibited by a didesulfo-MTX, suggesting the presence of the specific site for MTX-binding on the external surface of the cell membrane.

Topics: Animals; Cricetinae; Glioma; Insulinoma; Ligands; Magnetic Resonance Spectroscopy; Marine Toxins; Molecular Structure; Oxocins; Rats; Tritium; Tumor Cells, Cultured

In hamster insulinoma (HIT) cells, maitotoxin (MTX) induces a time-dependent and concentration-dependent release of insulin that requires the presence of extracellular calcium. The response is nearly completely blocked by cinnarizine and cadmium, but is not inhibited by the L-type calcium channel blocker nifedipine or by manganese. MTX induces 45Ca+ uptake in these cells in a dose-dependent mode, and the uptake is blocked with cinnarizine, nifedipine and cadmium, and is partially inhibited by manganese. MTX induces phosphoinositide breakdown in HIT cells, and the response is partially blocked by cadmium, but is not affected by nifedipine, cinnarizine or manganese. High concentrations of potassium ions also induce insulin release and calcium uptake in HIT cells. Both effects of potassium are blocked partially by nifedipine, cadmium and cinnarizine. High concentrations of potassium do not induce phosphoinositide breakdown in HIT cells. The results suggest that MTX-elicited release of insulin is attained by two mechanisms: 1) a nifedipine-sensitive action, which results from MTX-induced activation of L-type calcium channels, which can be mimicked with high potassium concentrations; and 2) a nifedipine-insensitive action, which may be initiated by the activation of phosphoinositide breakdown by MTX. Such an activation of phospholipase C would result in the formation of 1,4,5-inositol trisphosphate, a release of intracellular calcium and then release of insulin to the extracellular space. Cinnarizine is proposed to block both MTX-elicited mechanisms, the first by blockade of calcium channels and the second by blocking 1,4,5-inositol trisphosphate-induced release of internal calcium. Either mechanism alone appears capable of eliciting release of insulin.

Topics: Animals; Cadmium; Calcium; Calcium Channel Blockers; Calcium Radioisotopes; Cinnarizine; Cricetinae; Guinea Pigs; Insulin; Insulin Secretion; Insulinoma; Manganese; Marine Toxins; Nifedipine; Oxocins; Pancreatic Neoplasms; Phosphatidylinositols; Swine; Tumor Cells, Cultured