Veratridine-induced Cell Death Research Articles

Depolarization preconditioning produces cytoprotection against veratridine-induced chromaffin cell death

The hypothesis that K + channels and cell depolarization are involved in neuronal death and neuroprotection was tested in bovine chromaffin cells subjected to two treatment periods: the first period (preconditioning period) lasted 6 to 48 h and consisted of treatment with high K + solutions or with tetraethylammonium (TEA), a K + channel blocker; the second period consisted of incubation with veratridine for 24 h, to cause cell damage. Preconditioning with high K + (20–80 mM) or TEA (10–30 mM) for 24 h caused 20–60% cytoprotection against veratridine-induced cell death in bovine chromaffin cells. The absence of Ca 2+ ions during the first 9 h of an 18-h preconditioning period abolished the cytoprotection. Preconditioning with K + or TEA increased by 2.5-fold the expression of brain-derived neurotrophic factor and by nearly 2-fold the expression of the antiapoptotic protein Bcl-2. However, preconditioning did not modify the veratridine-evoked Ca 2+ signal. High K + shifted the Em by about 10 mV and TEA evoked a transient burst of action potentials superimposed on a sustained depolarization. We conclude that preconditioning may protect chromaffin cells from death by blocking K + channels that depolarize the cell and cause a cytosolic Ca 2+ signal, leading to enhanced expression of BDNF and Bcl-2.

Activation of p53 and the pro-apoptotic p53 target gene PUMA during depolarization-induced apoptosis of chromaffin cells

The pathogenesis of non-glutamatergic, depolarization-induced cell death is still enigmatic. Recently, we have shown that veratridine induces apoptosis in chromaffin cells, and we have demonstrated protective effects of antioxidants in this system, suggesting a role for Na + channels and oxidative stress in depolarization-induced cell death. We examined the possible contribution of p53, a transcription factor that has a major role in determining cell fate, and the mitochondrial apoptosis pathway in veratridine-induced cell death of cultured bovine chromaffin cells. Nuclear condensation and fragmentation were detected several hours after a 60-min exposure to 30 μM veratridine. Apoptosis was associated with a transitory increase in p53 protein levels. Veratridine induced transcription of the pro-apoptotic p53 target gene PUMA, but not of bax or pig3. Using transient transfection experiments, we found that wild-type p53, but not the mutant form p53-273H, was sufficient to induce cell death in the chromaffin cells, which was caspase-9 dependent. The down-regulation of either p53, by overexpressing p53-273H, or caspase-9 activity using a dominant-negative caspase-9 mutant protected chromaffin cells against veratridine-induced toxicity. Our data demonstrate the importance of p53 and the downstream activation of the mitochondrial apoptosis pathway in depolarization-induced apoptosis.

Minimal sodium channel pore consisting of S5-P-S6 segments preserves intracellular pharmacology

We studied the properties of a sodium channel comprised only of S5-P-S6 region of the rat sodium channel α-subunit Nav1.4 (μ1pore). Results obtained in HEK cell lines permanently transfected with the sodium channel α-subunit or with the μ1pore were compared with data of the native HEK cells. Sodium channel blockers, tetrodotoxin and tetracaine, protect cells transfected with the complete sodium channel against death produced by incubation with veratridine. Veratridine-induced cell death in cell lines expressing the μ1pore construct is antagonised by tetracaine, but not by tetrodotoxin. Whole-cell conductance also increases in the presence of veratridine in μ1pore transfected cells and tetracaine inhibits these currents. Our pharmacological and electrophysiological data suggest that μ1pore keeps binding sites for veratridine and tetracaine, but not for TTX, and reconstitutes the permeation pathway for Na + ions.

Role and regulation of p53 in depolarization-induced neuronal death

The tumor suppressor gene p53 is a potent transcriptional regulator for genes involved in many cellular activities including cell cycle arrest and apoptosis. In this study, we examined the role of p53 in neuronal death induced by the sodium channel modulator veratridine. We also analyzed the involvement of Ca 2+, mitochondria and reactive oxygen species in p53 activation. Exposure of hippocampal neurons to veratridine (0.3–100 μM) resulted in a dose-dependent neuronal death, measured 24 h after treatment. p53-Like immunoreactivity, undetectable in neurons under control conditions, was observed in about 25% of neurons, 7 h after veratridine exposure. Treatments that modified the alkaloid-induced Ca 2+ influx including tetrodotoxin or Ca 2+ removal, prevented either veratridine-induced cell death or p53 immunoreactivity. Mitochondria were involved in veratridine-induced cell death, as the alkaloid collapsed inner transmembrane mitochondrial potential in a Ca 2+ influx dependent manner. Treatments of neuronal cultures with the permeability transitory pore blockers cyclosporin A and bongkrekic acid prevented veratridine-induced p53 immunoreactivity and neuronal death, placing mitochondria upstream of veratridine-induced p53 immunoreactivity. Reactive oxygen species also participated in veratridine-induced neurotoxicity and p53 activation. Antisense knockdown of p53 resulted in a significant increase in neuronal survival after veratridine treatment. This protective effect was maintained on N-methyl- d-aspartate or ischemia-induced death but not on staurosporine cytotoxicity. These results together suggest that p53-expression is involved in veratridine-induced neuronal death and that p53 might be a link between toxic stimuli of different types and neuronal death.

A “Minimal” Sodium Channel Construct Consisting of Ligated S5-P-S6 Segments Forms a Toxin-activatable Ionophore

The large size (six membrane-spanning repeats in each of four domains) and asymmetric architecture of the voltage-dependent Na+ channel has hindered determination of its structure. With the goal of determining the minimum structure of the Na+ channel permeation pathway, we created two stable cell lines expressing the voltage-dependent rat skeletal muscle Na+ channel (micro1) with a polyhistidine tag on the C terminus (muHis) and pore-only micro1 (muPore) channels with S1-S4 in all domains removed. Both constructs were recognized by a Na+ channel-specific antibody on a Western blot. muHis channels exhibited the same functional properties as wild-type micro1. In contrast, muPore channels did not conduct Na+ currents nor did they bind [3H]saxitoxin. Veratridine caused 40 and 54% cell death in muHis- and muPore-expressing cells, respectively. However, veratridine-induced cell death could only be blocked by tetrodotoxin in cells expressing muHis, but not muPore. Furthermore, using a fluorescent Na+ indicator, we measured changes in intracellular Na+ induced by veratridine and a brevotoxin analogue, pumiliotoxin. When calibrated to the maximum signal after addition of gramicidin, the maximal percent increases in fluorescence (deltaF) were 35 and 31% in cells expressing muHis and muPore, respectively. Moreover, in the presence of 1 microm tetrodotoxin, deltaF decreased significantly to 10% in muHis- but not in muPore-expressing cells (43%). In conclusion, S5-P-S6 segments of micro1 channels form a toxin-activable ionophore but do not reconstitute the Na+ channel permeation pathway with full fidelity.

Effects of dotarizine and flunarizine on chromaffin cell viability and cytosolic Ca 2+

Dotarizine (a novel piperazine derivative with antimigraine properties) and flunarizine (a Ca 2+ channel antagonist) were compared concerning: first, their ability to cause chromaffin cell damage in vitro; second, the possible correlation of their octanol/water partition coefficients and those of another 28 compounds (i.e., Ca 2+ channel antagonists, blockers of histamine H 1 receptors, antimycotics, β-adrenoceptor antagonists, neuroleptics), with their ability to cause cell damage; third, their capacity to protect the cells against the damaging effects of veratridine; and fourth, their capabilities to enhance the basal cytosolic Ca 2+ concentration in fura-2-loaded single chromaffin cells, or to modify the pattern of [Ca 2+] i oscillations elicited by veratridine. After 24-h exposure to 1–30 μM dotarizine, the viability of bovine adrenal chromaffin cells (measured under phase contrast or as lactate dehydrogenase, released into the medium) was similar to that of control, untreated cells; at 100 μM, 80% lactate dehydrogenase release was produced. At 1–3 μM flunarizine caused no cell damage; however 10 μM caused 20% lactate dehydrogenase release and 30 and 100 μM over 90% lactate dehydrogenase release. The time course of cell damage was considerably faster for flunarizine, in comparison to dotarizine. Out of 30 molecules tested (at 10 μM), having different octanol/water partition coefficients (log P), dotarizine was among the molecules causing no cell damage; flunarizine caused 20% cell loss, lidoflazine and verapamil over 50% cell loss, and penfluridol, draflazine, astemizole or nifedipine over 80% cell loss. No correlation was found between log P and cytotoxicity. Both dotarizine (10–30 μM) and flunarizine (3–10 μM) provided protection against veratridine-induced cell death; however, at 30 μM dotarizine afforded a pronounced protection while flunarizine enhanced the cytotoxic effects of veratridine. Dotarizine (30 μM) (but not flunarizine) caused a prompt transient elevation of the basal [Ca 2+] i. Both compounds abolished the K +-induced increases of [Ca 2+] i as well as the oscillations of [Ca 2+] i induced by veratridine. The blocking effects of dotarizine were readily reversed after washout, while those of flunarizine were long-lasting. These differences might be relevant to the clinical use of dotarizine as an antimigraine drug.

Degradation of NCAM by tPA-plasmin system in brain

The hypothesis that K+ channels and cell depolarization are involved in neuronal death and neuroprotection was tested in bovine chromaffin cells subjected to two treatment periods: the first period (preconditioning period) lasted 6 to 48 h and consisted of treatment with high K+ solutions or with tetraethylammonium (TEA), a K+ channel blocker; the second period consisted of incubation with veratridine for 24 h, to cause cell damage. Preconditioning with high K+ (20–80 mM) or TEA (10–30 mM) for 24 h caused 20–60% cytoprotection against veratridine-induced cell death in bovine chromaffin cells. The absence of Ca2+ ions during the first 9 h of an 18-h preconditioning period abolished the cytoprotection. Preconditioning with K+ or TEA increased by 2.5-fold the expression of brain-derived neurotrophic factor and by nearly 2-fold the expression of the antiapoptotic protein Bcl-2. However, preconditioning did not modify the veratridine-evoked Ca2+ signal. High K+ shifted the Em by about 10 mV and TEA evoked a transient burst of action potentials superimposed on a sustained depolarization. We conclude that preconditioning may protect chromaffin cells from death by blocking K+ channels that depolarize the cell and cause a cytosolic Ca2+ signal, leading to enhanced expression of BDNF and Bcl-2.

Vincamine and vincanol are potent blockers of voltage-gated Na + channels

The effects of three vinca derivatives on [ 3H]batrachotoxin binding in rat cortical synaptosomes, on the inhibition of whole-cell Na + currents evoked in voltage-clamped cortical neurones of the rat, on the protection against veratridine-induced cell death in cortical cultures and on the maximal electroshock-induced seizures in mice were compared. Vinpocetine, vincamine and vincanol reduced [ 3H]batrachotoxin binding with IC 50 values of 0.34, 1.9 and 10.7 μM, blocked Na + currents with IC 50 values of 44, 72 and 40 μM, and protected cortical cultures against veratridine-induced cell death with IC 50 values of 0.49, 26 and 33 μM, respectively. Upon i.p. administration, vinpocetine, vincamine and vincanol attenuated maximal electric shock-induced convulsions in a dose-dependent manner with ED 50 values of 27, 15.4 and 14.6 mg/kg, respectively. The present findings indicate that the three vinca derivatives are potent blockers of voltage-gated Na + channels, a mechanism that may contribute at least in part to the pharmacological/therapeutic benefit of these drugs.

Protection against veratridine toxicity in rat cortical cultures

Neuroprotection against 100 microM veratridine-induced cell death and inhibition of voltage-dependent sodium currents by phenytoin, carbamazepine, lidocaine and vinpocetine were studied in rat primary cerebrocortical cultures. Neuroprotective efficacies and sodium channel blocking potencies of these drugs failed to show a correlation, suggesting that (i) mechanisms other than sodium channel blockade may be involved in the neuroprotection, and/or (ii) inhibitory efficacy against veratridine- and voltage-activated channels may differ remarkably.

Neuroprotective profile of lifarizine (RS‐87476) in rat cerebrocortical neurones in culture

1. The ability of the neuroprotective agent, lifarizine (RS-87476), to mitigate veratridine-, cyanide- and glutamate-induced toxicity in rat embryonic cerebrocortical neurones in primary culture has been compared with that of tetrodotoxin (TTX), nitrendipine, (+)-MK-801 and (-)-MK-801. Lactate dehydrogenase (LDH) released into the culture medium was used as the indicator of cell viability. 2. Incubation of cultures for 16 h in a medium containing veratridine (10(-4) M), sodium glutamate (10(-3) M) or sodium cyanide (10(-3) M) resulted in consistent elevations of LDH activity in the culture medium. The ability of compounds to attenuate these elevations was expressed as the concentration required to inhibit the increases in LDH release by 50% (IC50). 3. Neurotoxicity induced by veratridine was inhibited by lifarizine (IC50 = 4 x 10(-7) M), TTX (IC50 = 3 x 10(-8) M) and nitrendipine (IC50 = 3 x 10(-5) M). In contrast, (+)-MK-801 (up to 3 x 10(-5) M) was ineffective against this insult. 4. Glutamate-induced neurotoxicity was inhibited by (+)-MK-801 (IC50 = 1.4 x 10(-8) M) and to a lesser extent by (-)-MK-801 (IC50 = 1 x 10(-7) M), but was unaffected by lifarizine, TTX or nitrendipine (up to 10(-6) M). 5. (+)-MK-801 was effective against sodium cyanide-induced neurotoxicity (IC50 = 1.9 x 10(-8) M), whereas lifarizine and TTX (up to 10(-6) M) and nitrendipine (up to 3 x 10(-6) M) were without protective activity against this insult. 6. The results demonstrate that lifarizine potently protects rat cortical neurones in vitro against a neurotoxic insult that requires activation of sodium channels for its expression, and that the compound is ineffective against insults mediated by N-methyl-D-aspartate receptor activation. The weak efficacy of nitrendipine against veratridine-induced cell death argues against the involvement of L-type calcium channels in this insult. These data are consistent with the notion that the neuroprotective activity oflifarizine observed in vivo may be mediated by inhibition of neuronal sodium currents.

Vinpocetine is a highly potent neuroprotectant against veratridine-induced cell death in primary cultures of rat cerebral cortex

The effects of vinpocetine and phenytoin against veratridine-induced cell death were investigated in primary cultures of rat cerebral cortex. Toxicity was evaluated by phase contrast microscopy and quantified by measuring lactic dehydrogenase leakage from damaged cells. Vinpocetine was highly potent in inhibiting the cell death evoked by veratridine. The concentrations of the drug evoking 50% protection (IC 50 values) against 100μM (maximal response) and 50μM (half-maximal response) veratridine were 490 nM and 63 nM, respectively. The protective efficacy of vinpocetine exceeded about 100-fold that of phenytoin (IC 50 = 44.2 μM against 100μM veratridine), a prototype sodium-channel blocker. These data suggest that the blockade of voltage-gated sodium channels is a possible mechanism of action for the well-known neuroprotective and anticonvulsant properties of vinpocetine.