Brevetoxins cause acute excitotoxicity in primary cultures of rat cerebellar granule neurons.
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Brevetoxins (designated PbTx-1 to -10) are potent lipid-soluble polyether compounds that are known to bind to and modulate voltage-gated sodium channel activity. To investigate whether brevetoxins produce direct central nervous system neurotoxic effects, cultured rat cerebellar granule neurons were exposed to brevetoxins in Locke's buffer for 2 h at 22 degrees C. Neuronal injury was quantified by assaying lactate dehydrogenase activity in the exposure buffer and in conditioned growth media collected at 22 h after brevetoxin exposure. Brevetoxins produced acute neuronal injury and death in neurons with a rank order potency of PbTx-1 (EC50 = 9.31 +/- 0.45 nM) > PbTx-3 (EC50 = 53.9 +/- 2.8 nM) > PbTx-2 (EC50 = 80.5 +/- 5.9 nM) > PbTx-6 (EC50 = 1417 +/- 32 nM), which is similar to their previously determined rank order potency for brevetoxin-induced icthyotoxicity and binding to [3H]PbTx-3-labeled sodium channels on synaptosomes. The neurotoxic response could be prevented by coapplication of the sodium channel antagonist tetrodotoxin or by the competitive or noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists D-AP5 and MK-801, ketamine, dextromethorphan, and dextrorphan, respectively. NMDA receptor antagonists afforded neuroprotection with rank order potencies comparable to those measured previously for protection against glutamate-induced excitotoxic responses. Further analysis revealed that brevetoxins induced a concentration-dependent release of L-glutamate and L-aspartate into the exposure buffer. These data indicate that brevetoxin-induced injury in cultured rat cerebellar granule neurons is mediated by NMDA receptors that are activated indirectly as a consequence of PbTx-induced sodium channel activation and attendant excitatory amino acid release. (+info)
Studies on maitotoxin-induced intracellular Ca(2+) elevation in chinese hamster ovary cells stably transfected with cDNAs encoding for L-type Ca(2+) channel subunits.
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The aim of the present study was to characterize the role played by different L-type Ca(2+) channel subunits in [Ca(2+)](i) increase induced by maitotoxin (MTX). In the presence of 5 mM extracellular K(+), MTX (0.01-0.5 ng/ml) induced a significant concentration-dependent increase in Fura-2-monitored [Ca(2+)](i) in single Chinese hamster ovary (CHO) cells expressing the alpha(1c) (CHOCalpha9 cells) or the alpha(1c)beta(3)alpha(2)delta (CHOCalpha9beta3alpha2/delta4 cells) subunits of voltage-gated Ca(2+) channels (VGCCs), whereas the effect was much reduced in wild-type CHO cells lacking VGCCs. In addition, MTX effect on CHOCalpha9, CHOCalpha9beta3alpha2/delta4, and GH(3) cells (0.01-0.1 ng/ml) was inhibited by the selective L-type Ca(2+) channel entry-blocker nimodipine (10 microM); a nimodipine-insensitive component was still present, particularly at high (>1 ng/ml) toxin concentrations. In CHOCalpha9beta3alpha2/delta4 cells, depolarizing concentrations of extracellular K(+) (55 mM) reinforced the [Ca(2+)](i) increase induced by MTX (0.1 ng/ml), and this effect was prevented by nimodipine (10 microM). Finally, patch-clamp experiments in CHOCalpha9beta3alpha2/delta4 cells showed that low MTX concentrations (0.03 ng/ml) induced the occurrence of an inward current at -60 mV, which was completely prevented by Cd(2+) (100 microM) and by nimodipine (10 microM), whereas the same dihydropyridine concentration (10 microM) failed to prevent the electrophysiological effects of a higher toxin concentration (3 ng/ml). In conclusion, the results of the present study showed that MTX-induced [Ca(2+)](i) elevation involves two components: 1) an action on L-type VGCCs at the pore-forming alpha(1c) subunit level, which is responsible for the greatest rise of [Ca(2+)](i); and 2) a VGCC-independent mechanism that is present both in excitable and in nonexcitable cells and is responsible for a lower elevation of [Ca(2+)](i). (+info)
Maitotoxin-induced nerve growth factor production accompanied by the activation of a voltage-insensitive Ca2+ channel in C6-BU-1 glioma cells.
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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. (+info)
Transient Ca2+-dependent activation of ERK1 and ERK2 in cytotoxic responses induced by maitotoxin in breast cancer cells.
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Treatment of MCF-7 breast cancer cells with the marine toxin maitotoxin (MTX) induces cell death. The cytotoxic effects are clearly detectable within 2-4 h after cell treatment with 10(-10)-10(-9) M concentrations of MTX. The response was found to depend on extracellular Ca2+, inasmuch as cell death was prevented when culture dishes received MTX, following addition of EGTA. MTX caused transient phosphorylation of extracellular signal-regulated kinase isoforms 1 and 2 (ERK1 and ERK2) mitogen-activated protein kinase isoforms in MCF-7 cells, which was maximal 15 min after toxin addition to culture vessels. The effect was dependent on influx of extracellular Ca2+, as it was abolished by EGTA, and was induced by ionophores, such as A23187 and ionomycin. Our findings show that signaling pathways involving Ca2+ ions may cause activation of ERK1 and ERK2 in cell death responses. (+info)
Maitotoxin activates a nonselective cation channel and a P2Z/P2X(7)-like cytolytic pore in human skin fibroblasts.
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Maitotoxin (MTX), a potent cytolytic agent, activates Ca(2+) entry via nonselective cation channels in virtually all types of cells. The identity of the channels involved and the biochemical events leading to cell lysis remain unknown. In the present study, the effect of MTX on plasmalemmal permeability of human skin fibroblasts was examined. MTX produced a time- and concentration-dependent increase in cytosolic free Ca(2+) concentration that depended on extracellular Ca(2+) and was relatively insensitive to blockade by extracellular lanthanides. MTX also produced a time- and concentration-dependent increase in plasmalemma permeability to larger molecules as indicated by 1) uptake of ethidium (314 Da), 2) uptake of YO-PRO-1 (375 Da), 3) release of intracellular fura 2 (636 Da), 4) uptake of POPO-3 (715 Da), and, ultimately, 5) release of lactate dehydrogenase (relative molecular weight of 140,000). At the single cell level, uptake of YO-PRO-1 correlated in time with the appearance of large MTX-induced membrane currents carried by the organic cation, N-methyl-D-glucamine (167 Da). Thus MTX initially activates Ca(2+)-permeable cation channels and later induces the formation of large pores. These effects of MTX on plasmalemmal permeability are similar to those seen on activation of P2Z/P2X(7) receptors in a variety of cell types, raising the intriguing possibility that MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore. (+info)
Maitotoxin and P2Z/P2X(7) purinergic receptor stimulation activate a common cytolytic pore.
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The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X(7) ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X(7) receptors or 2) MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-gamma, a maneuver known to upregulate P2Z/P2X(7) receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X(7) receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X(7) channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)); the initial increase reflects MTX-induced Ca(2+) influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca(2+)](i) and ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X(7) receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X(7) receptor, MTX-induced increases in [Ca(2+)](i) and ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca(2+)](i) had no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22 degrees C, a characteristic shared by the P2Z/P2X(7)-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X(7) receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable. (+info)
Maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in human astrocytoma cells.
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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. (+info)
Brevetoxin derivatives that inhibit toxin activity.
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BACKGROUND: The brevetoxins are marine neurotoxins that interfere with the normal functions of the voltage-gated Na(+) channel. We have identified two brevetoxin derivatives that do not exhibit pharmacological properties typical of the brevetoxins and that function as brevetoxin antagonists. RESULTS: PbTx-3 and benzoyl-PbTx-3 elicited Na(+) channel openings during steady-state depolarizations; however, two PbTx-3 derivatives retained their ability to bind to the receptor, but did not elicit Na(+) channel openings. alpha-Naphthoyl-PbTx-3 acted as a PbTx-3 antagonist but did not affect Na(+) channels that were not exposed to PbTx-3. beta-Naphthoyl-PbTx-3 reduced openings of Na(+) channels that were not exposed to PbTx-3. CONCLUSIONS: Some modifications to the brevetoxin molecule do not alter either the binding properties or the activity of these toxins. Larger modifications to the K-ring sidechain do not interfere with binding but have profound effects on their pharmacological properties. This implies a critical function for the K-ring sidechain of the native toxin. (+info)