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The mechanism of maitotoxin-induced elevation of the cytosolic free calcium level in rat cerebrocortical synaptosomes.

The present study was conducted to elucidate the mechanism of the maitotoxin (MTX)-induced increase in intrasynaptosomal free calcium level ([Ca2+]i). The MTX (1 ng/ml)-induced increase in [Ca2+]i was partially inhibited by the omission of extracellular Ca2+ (Ca2+e) or the addition of verapamil, but not by adding nifedipine, omega-agatoxin IVA, omega-conotoxin GVIA and omega-conotoxin MVIIC. An increase in [Ca2+]i in the absence of Ca2+e was sensitive to procaine, TMB-8, genistein and verapamil, but not to ryanodine and U-73122. These results may suggest that MTX increases [Ca2+]i by stimulating Ca2+ entry through voltage-independent nonselective cation channels and Ca2+ release from stores through a phospholipase C-gamma1-mediated pathway in rat cerebrocortical synaptosomes.

Maitotoxin (MTX), a water-soluble polyether (MW: 3424) isolated from the marine organism Gambierdiscus toxicus, is one of the most potent toxins known (1). This toxin increases calcium (Ca 2 + ) content and/ or Ca 2 + flux in a wide range of cell types (2, 3). Recent reports indicate that MTX primarily activates voltage-independent, nonselective cation channels (3 -6). MTX is also known to activate phosphoinositide hydrolysis (2,3,7,8). Recently, it has been proposed that MTX separately activates Ca 2 + influx and phosphoinositide hydrolysis in rabbit ciliatedtracheal epithelium and human astrocytoma cells (7,8). In rat brain synaptosomes, MTX increases intrasynaptosomal Ca 2 + and depolarizes synaptosomal plasma membranes through the inflow of extracellular Ca 2 + (Ca 2 + e) (9). The identity of the MTX-activated channels and the role of the intervening biochemical events (i.e., phosphoinositide hydrolysis) leading to an increase in the intrasynaptosomal free Ca 2 + level ([Ca 2 + ]i), however, remain unknown. The aim of this study is to elucidate the mechanism of MTX-induced increase in [Ca 2 + ]i using rat cerebrocortical synaptosomes.
Synaptosomes (P2B fraction) were prepared using the method described by Hajós (10) from the cerebral cortices of male and female Wistar rats (200 -300 g).
The levels of [Ca 2 + ]i were measured by monitoring the intensity of fura 2 fluorescence as described by Komulainen and Bondy (11).
Membrane potentials were measured by monitoring the intensity of rhodamine 6G fluorescence using the method described by Aiuchi et al. (12). An increase in the ratio of fluorescence intensity (measured as fs/ fi; fi and fs are the intensity of fluorescence in the absence and in the presence of synaptosomes, respectively) indicates depolarization of synaptosomal membranes.
Synaptosomal protein levels were quantified by using a kit from Bio-Rad (Richmond, CA, USA), as described by Bradford (13).
Statistical significance of differences was assessed by Student's t-test.
MTX (0.1 -10 ng /ml) caused the increase in [Ca 2 + ]i and membrane depolarization dose-dependently in rat cerebrocortical synaptosomes (data not shown). Removal of Ca 2 + from the medium inhibited these effects of MTX (1 ng /ml) by approximately 60% and 50%, respectively (data not shown). Decreasing to 6.2 mM the Na + in the medium or the application of TTX (1 m M) failed to alter these effects of MTX (data not shown). These results are similar to the reports of Taglialatela et al. (9) using rat whole brain. However, they suggested that MTX increased [Ca 2 + ]i through the inflow of Ca 2 + e because the MTX-induced increase in [Ca 2 + ]i was abolished in Ca 2 + -free medium containing 1 mM EGTA. In the case of synaptosomes, EGTA reduces [Ca 2 + ]i by a rapid and continuous efflux of Ca 2 + across the synaptic membrane (11). We could not measure the changes in [Ca 2 + ]i using agents that release Ca 2 + from intracellular stores in Ca 2 + -free medium containing 1 mM EGTA. Thus, it is not possible to measure [Ca 2 + ]i in Ca 2 +free medium containing EGTA. In our Ca 2 + -free experiments, synaptosomal preparations were suspended in Ca 2 +free medium containing 1 mM EGTA and subsequently resuspended in nominally Ca 2 + -free medium to wash out the EGTA. Then, [Ca 2 + ] i was measured in the nominally Ca 2 +free medium.
To identify the MTX-activated channels, the effects of MTX (1 ng / ml) on [Ca 2 + ]i and membrane potential were investigated in the presence of Ca 2 + e and Ca 2 + antagonists of different classes of voltage-dependent Ca 2 + channels. Verapamil (200 mM) inhibited both the [Ca 2 + ]i increase and depolarization induced by MTX by approximately 50% (Fig. 1: A and B). Nifedipine (10 mM), w-agatoxin IVA (200 nM), w-conotoxin GVIA (1 mM) and w -conotoxin MVIIC (500 nM) failed to modify the effects of MTX ( Fig. 1: A and B). SKF-96365 (30 mM), a receptor-mediated Ca 2 + -entry blocker, also had no effect (data not shown). The verapamil-induced inhibition may be due to a non-specific action rather than the inhibition of voltagegated Ca 2 + channels, since its concentration exceeds the specific range (14). Thus, the extracellular Ca 2 + -dependent MTX-induced increase in [Ca 2 + ]i may be due to Ca 2 + entry through voltage-independent nonselective cation channels.
To elucidate the Ca 2 + free-insensitive effect of MTX (1 ng/ml), we tested the effects of agents that can modify the release of Ca 2 + from intracellular stores on MTX (1 ng / ml)-induced increase in [Ca 2 + ]i in the absence of Ca 2 + e.
Procaine (8 mM), TMB-8 (100 m M) and genistein (110 mM, inhibitor of tyrosine kinase to induce tyrosine phosphorylation of phospholipase C-g1) markedly inhibited the increase in [Ca 2 + ]i, but not ryanodine (50 mM) and U-73122 (10 mM, inhibitor of activation of phospholipase C-b 1 through a trimeric G protein) (Fig. 2). MTX generates inositol 1,4,5-trisphosphate (IP3) through the hydrolysis of phosphatidylinositol by phospholipase C and IP3 releases Ca 2 + from intracellular stores (2). Concentrations of U-73122 and genistein were enough to induce an inhibition of IP3 production through specific sites, respectively (7). Thus, MTX-induced increase in [Ca 2 + ]i in the absence of Ca 2 + e may be mainly due to Ca 2 + release from stores through IP3 generated by the activation of phospholipase C-g1, which might imply phosphorylation on tyrosine residues (7, 15).
Verapamil (20 -200 mM) inhibited dose-dependently the effect of MTX in the presence or absence of Ca 2 + e (Fig. 3). In the absence of Ca 2 + e and the presence of verapamil, procaine or TMB-8 had no additive effect (data not shown). It is suggested that verapamil may inhibit Ca 2 + release from stores through membrane stabilizing action because the agent inhibited membrane depolarization partially and an increase in [Ca 2 + ]i by the same degree regardless of the presence or absence of Ca 2 + e (Figs. 1B and 3). This is supported by the report that blockade of 45 Ca entry into depolarized synaptosomes by relatively high concentrations of phenylalkylamines may result from a nonspecific membrane effect (14).