Micromolar concentrations of veratridine activate sodium channels in embryonic cockroach neurones in culture

The mode of action of the alkaloid veratridine has been reinvestigated on cultured cockroach neurones, which are normally inexcitable and do not have a detectable fast sodium current. The whole-cell and cell-attached configurations of the patch-clamp technique were used to record the macroscopic and single channel currents, respectively. Concentrations of veratridine ranging from 10^–8 to 10^–5 M were found to induce a small tetrodotoxin (TTX)-sensitive inward current, which peaked around + 10 mV and reversed around + 55 mV. This current exhibited a pronounced plateau and was insensitive to changes in the holding potential. Bath application of veratridine induced typical TTX-sensitive inwardly-directed single-channel activity, falling into two (apparently coupled) categories of events: first, relatively large events (1 pA at a hyperpolarized potential of –125 mV relative to rest) of short duration and, second, small bursting events (0.4 pA under similar conditions) of slightly longer duration. Pipette application of similar concentrations of veratridine had similar effects in that two categories of events were observed: first, bursts of large events with multiple conductance states and, second, small events of very long duration. The current/ voltage relationship of these events was linear for the voltage range studied and the (extrapolated) reversal potential approximated + 110 mV. These results support the hypothesis that veratridine, in small concentrations, induces a slow voltage-dependent activation of TTX-sensitive sodium channels, independent of the fast activating and inactivating sodium channels involved in action potential generation.     

Veratridine-induced intoxication in the isolated left atrium of the rat: Effects of some anti-ischemic compounds

Summary Veratridine-induced Na⁺ and Ca²⁺ uptake was used as a simulation of ischemia-induced Na⁺ and Ca²⁺ uptake. Therefore, electrically driven (1 Hz) isolated left atria of the rat were intoxicated with veratridine and the ⁴⁵Ca²⁺ uptake was determined. Veratridine (10^–4 mol/l) increased the ⁴⁵Ca²⁺ uptake from 575±13 to 2320±86 dpm/mg ww (n=20). The total tissue content of ⁴⁵Ca²⁺ was elevated from 4328±132 to 5136 ±303 dpm/mg ww (n = 13). The veratridine-induced ⁴⁵Ca²⁺ uptake was completely suppressed by tetrodotoxin (10^–7 and 10^–6 mol/l), whereas amiloride (6·10^–6 mol/1) and phentolamine (10^–6 and 10^–5 mol/l) exhibited no effect on the veratridine-induced ⁴⁵Ca²⁺ uptake. Nifedipine (10^–7 and 10^–6 mol/l) was ineffective on veratridine-induced ⁴⁵Ca²⁺ uptake. Verapamil (10^–5 mol/l) suppressed the veratridine-induced ⁴⁵Ca²⁺ uptake, but the ⁴⁵Ca²⁺ uptake in the absence of veratridine was also suppressed by verapamil (10^–6 and 10^–5 mol/l). The novel anti-ischemic compounds R 56865 (10^–8–10^–5 mol/l) and R 59494 (10^–8 -10-5 mol/l) totally abolished veratridine-induced ⁴⁵Ca²⁺ uptake.It is speculated that Ca²⁺ enters the cell via a Na⁺ channel which changes its selectivity upon veratridine treatment. Consequently, R 56865 and R 59494 could display their protective effect by either inhibiting the modified Na⁺ channel or preventing the transition of the normal Na⁺ channel to its altered state. As ischemia- and veratridine-induced Na⁺ and Ca²⁺ uptake share some similarities, it is proposed that veratridine-induced ⁴⁵Ca²⁺ uptake of the isolated left atrium of the rat could be used to study the mechanism of action of novel antiischemic drugs. Send offprint requests to D. Wermelskirchen at the above address     

Systemic morphine reduces GABA release in the lateral but not the medial portion of the midbrain periaqueductal gray of the rat

Neuroanatomical, electrophysiological and pharmacological studies have provided indirect evidence indicating that GABAergic neurons play a key role in opiate analgesia mediated by the midbrain periaqueductal gray (PAG) and ventromedial medulla. Although these studies suggest that systemic administration of opiates inhibits GABA release in the PAG, there have been no investigations to date that have directly examined this issue. The present study was thus designed to determine whether systemic morphine injection inhibits GABA release in the PAG of awake, freely moving rats using in vivo microdialysis and subsequent HPLC analysis. Extracellular levels of GABA, glutamate, aspartate, glycine, homocysteic acid and taurine were monitored with the microdialysis technique in either the lateral or medial portion of the ventrocaudal PAG in unanesthetized, unrestrained rats. Amino acid release was induced by infusing veratridine (75 microM, a sodium channel activator) directly through the dialysis probe. The effect of veratridine alone and the effect of veratridine in the presence of systemic morphine on the concentrations of amino acids in the PAG dialysate were determined. There were no significant differences in the basal concentrations of GABA, taurine, aspartate, glutamate, homocysteic acid and glycine between dialysates collected from the medial versus the lateral ventrocaudal PAG. Glycine, taurine and glutamate were present in the highest concentrations in dialysis samples both before and after treatment with veratridine, whereas GABA, homocysteic acid and aspartate were present in the lowest concentrations. Perfusion of veratridine into the ventrocaudal PAG resulted in significant elevation of all amino acids investigated. Except for taurine, no significant difference in veratridine-induced release between the lateral and medial PAG was observed. Tetrodotoxin (TTX) significantly blocked veratridine-induced release of GABA, aspartate, glutamate, glycine and taurine but not homocysteic acid. When rats were injected with morphine (10 mg/kg i.p.), veratridine-induced release of GABA was selectively and significantly decreased in the lateral but not the medial PAG as compared to control rats injected with saline followed by veratridine perfusion. Systemic injection of morphine or saline caused no significant change in the basal concentration of amino acids in PAG dialysate samples. These findings are consistent with the proposed mechanism of action of morphine in the lateral ventrocaudal PAG and offer the first direct evidence that systemic opiates decrease GABA release in this midbrain region.     

Ability of some plant extracts, traditionally used to treat ciguatera fish poisoning, to prevent the in vitro neurotoxicity produced by sodium channel activators.

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators.     

A neurally mediated inotropic effect of veratridine and cevadine on isolated guinea-pig papillary muscle

Summary The positive inotropic effect of veratridine and cevadine was investigated in the isolated, isometrically contracting guinea-pig papillary muscle. The increase of the force of the rested-state contraction, elicited after incubation of the resting muscle with veratridine or cevadine, served as a measure of the neurally mediated, sympathomimetic effect of these alkaloids. Concentrations exceeding 5 mol/l veratridine or 15 mol/l cevadine produced concentration-dependent increases of the force of the rested-state contraction. These concentrations are larger than those causing the maximum positive inotropic effect on muscles contracting continually at a rate of 1 Hz. The positive inotropic effect of 30 mol/l veratridine as manifested by the rested-state contraction was absent in the presence of 100 nmol/l tetrodotoxin and in muscles from reserpine-pretreated animals. It was significantly inhibited by 50 nmol/l (–)-propranolol, but not by the same concentration of (+)-propranolol. The effect of 30 or 60 mol/l cevadine was likewise absent in catecholamine-depleted preparations. It is concluded that the indirect inotropic effect of veratridine or cevadine, which is attributed to their noradrenaline-releasing effect on intracardiac nerves, requires higher concentrations than the direct positive inotropic effect, which is a consequence of the increased transsarcolemmal influx of Na ions into the myocardial cell.These results were communicated to the Deutsche Pharmakologische Gesellschaft (Honerjäger 1977)     

Donepezil attenuates excitotoxic damage induced by membrane depolarization of cortical neurons exposed to veratridine

Long-lasting membrane depolarization in cerebral ischemia causes neurotoxicity via increases of intracellular sodium concentration ([Na+]i) and calcium concentration ([Ca2+]i). Donepezil has been shown to exert neuroprotective effects in an oxygen-glucose deprivation model. In the present study, we examined the effect of donepezil on depolarization-induced neuronal cell injury resulting from prolonged opening of Na+ channels with veratridine in rat primary-cultured cortical neurons. Veratridine (10 microM)-induced neuronal cell damage was completely prevented by 0.1 microM tetrodotoxin. Pretreatment with donepezil (0.1-10 microM) for 1 day significantly decreased cell death in a concentration-dependent manner, and a potent NMDA receptor antagonist, dizocilpine (MK801), showed a neuroprotective effect at the concentration of 10 microM. The neuroprotective effect of donepezil was not affected by nicotinic or muscarinic acetylcholine receptor antagonists. We further characterized the neuroprotective properties of donepezil by measuring the effect on [Na+]i and [Ca2+]i in cells stimulated with veratridine. At 0.1-10 microM, donepezil significantly and concentration-dependently reduced the veratridine-induced increase of [Ca2+]i, whereas MK801 had no effect. At 10 microM, donepezil significantly decreased the veratridine-induced increase of [Na+]i. We also measured the effect on veratridine-induced release of the excitatory amino acids, glutamate and glycine. While donepezil decreased the release of glutamate and glycine, MK801 did not. In conclusion, our results indicate that donepezil has neuroprotective activity against depolarization-induced toxicity in rat cortical neurons via inhibition of the rapid influx of sodium and calcium ions, and via decrease of glutamate and glycine release, and also that this depolarization-induced toxicity is mediated by glutamate receptor activation.     

Quantal stores of excitatory transmitter in nerve-muscle synapses of crayfish evaluated from high-frequency asynchronous quantal release induced by veratridine or high concentrations of potassium

At single voltage-clamped opener muscle fibres of crayfish claw, 10–100 mol/l veratridine increased within a few seconds the rate of asynchronous quantal release, ñ, of excitatory transmitter from ñ<1 quantum/s to ñ10,000 quanta/s. Thereafter ñ declined exponentially either with a single, (2)50 s, or with two time constants (1)19 s, (2)50 s. In total (t), about 0.3 million quanta were released by veratridine in a single short fibre of about 1 mm length. These values were estimated by means of the noise analysis technique and they agreed with equivalent parameters of release when 100 mmol/l K⁺ were used as release stimulus. Strong quantal release could be elicited only once in a single muscle by veratridine. Furthermore, the effect of veratridine on quantal release could be completely prevented by pretreatment with tetrodotoxin. In another nerve-muscle preparation of crayfish, the abdominal superficial extensor muscle, up to 3 million excitatory quanta could be released by veratridine in a single fibre. In the latter muscle veratridine-induced asynchronous quantal release was strongly dependent on the extracellular concentration of Ca²⁺ whereas in the claw opener dependence of quantal release on extracellular Ca²⁺ was negligible.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Pharmacological characterization of the voltage-dependent sodium channels of rainbow trout brain synaptosomes

Batrachotoxin, aconitine, and veratridine, alkaloid activators of voltage-dependent sodium channels, stimulated 22Na+ uptake by rainbow trout brain synaptosomes. The potency and efficacy of activation by these compounds decreased in the following order: batrachotoxin greater than aconitine much greater than veratridine. Aconitine-stimulated sodium uptake was completely inhibited by tetrodotoxin, a specific blocker of voltage-dependent sodium channels. Polypeptide toxins in the venom of the scorpion, Leiurus quinquestriatus, and the insecticide DDT enhanced veratridine-dependent sodium uptake but had no effect on non-specific uptake. These studies identify appropriate conditions for measuring sodium channel-dependent 22Na+ uptake in trout brain synaptosomes and characterize some of the pharmacological properties of trout brain sodium channels. Trout sodium channels differed from those in rat and mouse brain in their responses to batrachotoxin, aconitine, veratridine, and DDT but not to tetrodotoxin and Leiurus venom toxins. These results suggest that the specificity of some of the neurotoxin-binding domains of the trout brain sodium channel may differ from those of sodium channels in mammalian brain.     

Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels.

Brevetoxins and ciguatoxins are two classes of phycotoxins which exert their toxic effect by binding to site-5 of voltage-gated sodium channels. Sodium channels, a family of at least 10 structurally different proteins, are responsible for the rising phase of the action potential in membranes of neuronal, cardiac and muscular excitable cells. This work is a comparative study of the binding properties and the cytotoxic effects of ciguatoxins and brevetoxins on human embryonic cells (HEK) stably expressing either the skeletal muscle (Na(v)1.4), or the cardiac (Na(v)1.5) sodium channel alpha-subunit isoforms. We report that type A (PbTx-1) and type B (PbTx-3 and PbTx-2) brevetoxins as well as ciguatoxins target both cardiac and muscle channels; type B brevetoxins show isoform selectivity, presenting a lower affinity for the heart than the skeletal muscle channel. The lower selectivity of type B brevetoxins for heart sodium channels may result from a more rigid backbone structure than is found in type A brevetoxins and ciguatoxins.     

Calcium-dependent, sustained enhancement of excitability during washout of aconitine in rat hippocampal slices

 Extracellular recording of the stimulus-evoked population spike was performed in the CA1 area of rat hippocampal slices in order to investigate delayed effects of the plant alkaloids aconitine and veratridine. Veratridine (1 μM and 10 μM) suppressed the orthodromic and antidromic population spike. After washout of the drug, only a partial recovery was obtained. Aconitine (1 μM) exerted the same inhibitory action as veratridine. However, after washout, the spike amplitude was enhanced compared with the control. This enhancement of the spike amplitude was dependent on the concentration of aconitine and was maintained during the observation period of at least 2 h. Lowering the Ca²⁺ concentration of the bathing medium from 2.5 mM to 1.25 mM during application of aconitine attenuated recovery and prevented the enhancement observed during washout of the drug. Application of aconitine in the presence of CdCl₂ as well as in the presence of inhibitors of protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II prevented the increase in spike amplitude during washout with standard artificial cerebrospinal fluid. In contrast, the N-methyl-d-aspartate (NMDA) receptor antagonists D-AP5 and MK-801 as well as the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione were ineffective in abolishing the aconitine-induced enhancement. These data support the conclusion that different modes of action are involved in the effects of aconitine but not veratridine. It is concluded that the aconitine-induced increase in neuronal activity is mediated by intracellular Ca²⁺-dependent mechanisms leading to an activation of Ca²⁺-dependent protein kinases. This effect is independent of Ca²⁺ entrance through NMDA and non-NMDA receptors. Received: 3 July 1996 / Accepted: 14 November 1996