Interactions of maitotoxin with voltage-sensitive calcium channels in cultured neuronal cells.

Abstract

The dinoflagellate toxin maitotoxin (MTX) stimulated 45Ca2+ uptake in cultured NG108-15 neuroblastoma x glioma cells. Depolarizing stimuli (e.g., 50 mM K+) produced an immediate stimulation in Ca2+ uptake, whereas that produced by MTX One of the most important aspects of research on voltagesensitive sodium channels (VSNaC) has been the discovery and development of chemical probes that selectively alter different facets of channel function (1). Thus, toxins such as tetrodotoxin (TTX), batrachotoxin (BTX), and scorpion venom have been shown to interact with separate sites on the channel molecule (2). These toxins have been invaluable in studies directed at isolating the channel molecule and in understanding its properties at a molecular level. Clearly, similar agents would be helpful in advancing our understanding of other channels, such as those for potassium and calcium.Maitotoxin (MTX) is a water-soluble toxin of unknown structure isolated from the poisonous dinoflagellate Gambierdiscus toxicus (3). It is one of the entities responsible for the syndrome known as ciguatera poisoning, which is common in tropical and subtropical areas. The purified toxin has a positive inotropic effect (4), contracts smooth muscle (5, 6), stimulates transmitter release from sympathetic neurons (6) and cultured PC12h pheochromocytoma cells (7,8), and releases prolactin from primary pituitary cultures (9). It has been suggested that MTX is an "activator" of voltage-sensitive calcium channels (VSCC) (5-9). However, no experimental data have been obtained that directly examine the effect of MTX on VSCC.We have previously demonstrated that several cell lines including the neuroblastoma x glioma hybrid NG108-15 express a type of VSCC when grown under the appropriate conditions (10). The pharmacology of these channels has been characterized in detail (10). They appear to be extremely similar to those occurring in smooth muscle. Thus, they are blocked by nanomolar concentrations of dihydropyridines, such as nifedipine, and are activated by the novel dihydropyridines BAY K8644 and CGP 28392 (10, 11). In the present series of studies, we have investigated the interaction of MTX with VSCC in these cultured neuronal cells. Ca2+ Uptake Studies. 45Ca2+ uptake studies were carried out exactly as described by Freedman et al. (10). Briefly, NG108-15 cells were grown as monolayers in 5% C02/95% air in Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine. Cells were subcultured onto 60-mm tissue culture plates. To induce cellular differentiation, growth medium was supplemented with 10 ,M prostaglandin E1 and 50 AM 3-isobutyl-1-methylxanthene. Supplemented medium was replaced every 2 days or when required. 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. During the assay, tissue culture plates were supported in an open air water bath at 37°C. Cells were incubated for 5 min at 37°C. The uptake of 45Ca2+ was measured for increasing periods of time in...

Differential effects of maitotoxin on ATP secretion and on phosphoinositide breakdown in rat pheochromocytoma cells

“…The observed inhibitory effect of the calcium channel blocker nifedipine ( fig.2) is also consistent with results [1] showing inhibition by nifedipine of MTX-evoked release of norepinephrine in PCI2 cells. Furthermore, stimulatory effects of MTX on release of neurotransmitters and hormones are also inhibited in other cell types by calcium channel blockers [6,10]. Thus, it appears that at concentrations >2 ng/ml, used in the present study and in other reports [1,5,6], MTX interacts directly with the Lsubclass of calcium channels that are sensitive to dihydropyridines, and thereby triggers calciumdependent release processes.…”

“…In cultured neurons release is seen only at higher concentrations, ranging between 100 and 1000 ng/ml [5]. In NG108-15 cells MTX-induced calcium uptake is observed at concentrations in the range 30-100 ng/ml [6], while in pancreatic islet cells MTX stimulates calcium uptake and evokes insulin release at relatively low concentrations of 0.03-1 ng/ml [4]. The chemical structure of MTX is as yet unknown, and it is uncertain whether the purity and activity of preparations of MTX from different laboratories are identical.…”

“…figs 1,3), suggesting that two independent mechanisms are involved in these responses. Indeed, prior literature would also indicate that systems involved in phosphoinositide breakdown [11,12] are much more sensitive to MTX than are systems involved in stimulation of calcium uptake, muscle contraction and neurotransmitter release [1,5,6]. However, the range of concentrations of MTX that have been reported to be required to elicit biological responses is quite broad.…”

“…MTX increases resting tension in cardiac muscle [2], causes contraction of smooth muscle [3], induces insulin release [4] and stimulates quantal release of neurotransmitters [1,5,6]. These effects of MTX require the presence of calcium in the extracellular medium.…”

“…These effects of MTX require the presence of calcium in the extracellular medium. Furthermore, MTX-induced release of neurotransmitters is inhibited by dihydropyridine calcium channel blockers [1,6]. Thus, it has been proposed [1] that MTX is a direct calcium channel activator.…”