Effects of AL 107, a novel semisynthetic cardiac glycoside, on the cardiovascular system in anaesthetized beagle dogs with pentobarbital-induced cardiac insufficiency.

The inotropic efficacy, arrhythmogenicity and cardiohaemodynamic properties of AL 107 (3-alpha-methyl-digitoxigenin glucoside, CAS 62190-59-4), a novel cardiac glycoside, were studied in anaesthetized dogs with pentobarbital-induced acute cardiac insufficiency. Three groups of dogs received AL 107, ouabain or verhicle. The cardiac glycosides were infused intravenously in eight increasing dose levels which where given cumulatively. Slope of left ventricular pressure rise (LVdp/dtmax) increased in the AL 107- and ouabain-treated groups. At the end of the 6th dose level ouabain caused a significantly higher LVdp/dtmax (137 +/- 15% of the baseline value taken before induction of insufficiency) than AL 107 (94 +/- 9%). Further increase of the dose resulted in a reduction of LVdp/dtmax in ouabain-treated dogs, whereas AL 107 continuously increased LVdp/dtmax up to the highest dose infused, where 130 +/- 16% of the baseline value was reached. In ouabain-treated dogs, ECG abnormalities accompanied the decrease of LVdp/dtmax whereas ECG-changes did not interfere with the development of left ventricular contractility in AL 107-treated dogs. The 6th dose of ouabain provoked a maximal increase of cardiac output (CO) (up to 107 +/- 8% of baseline values) and stroke volume (SV) (up to 122 +/- 6% of baseline values) which decreased upon further dose elevation. In the dose-range studied AL-107 induced a continuous increase of both parameters which, however, hardly reached the baseline values. ECG abnormalities occurred in both substance-treated groups and showed quantitative but not qualitative differences. The ECG showed rapid recovery after cessation of AL 107 infusion but did not normalize during a postinfusional recovery period of 1 h after treatment with ouabain. In conclusion, AL 107 increased cardiac performance in an acute canine model of cardiac insufficiency. It was slightly less active than ouabain. However, the ECG disorders were more moderate in AL 107--than in ouabain-treated dogs, a difference which was most pronounced with respect to reversibility in the postinfusional period.

The alkaloid 6-benzoylheteratisine inhibits voltage-gated Na+ channels in rat brain synaptosomes.

The effects of the Aconitum alkaloid 6-benzoylheteratisine on the aconitine-, veratridine-, oubain- and KCl-induced alterations in free synaptosomal Na + ([Na+]i) and Ca2+ ([Ca2+]i) and the release of endogenous glutamate from rat cerebrocortical synaptosomes were investigated. [Na+]i and [Ca2+]i were fluorometrically determined employing SBFI and Fura-2 as the Na+ and Ca2+ sensitive dyes, respectively. Glutamate was detected by a continuous enzyme-linked fluorometric assay. The study revealed a concentration-dependent inhibitory effect of 6-benzoylheteratisine on aconitine-induced increases in [Na+]i, [Ca2+]i and the release of glutamate. The IC50 values were 4.1 microM (Na+), 4.8 microM (Ca2+) and 4.8 microM (glutamate release). Application of 100 microM 6-benzoylheteratisine after stimulation with 5 microM veratridine also reduced the induced [Na+]i and [Ca2+]i with half-lives of 72.1 and 44.7 s, respectively. Furthermore, 100 microM 6-benzoylheteratisine reduced the ouabain-induced Na+ influx to the same extent as the Na+ channel inhibitor tetrodotoxin, which points to an inhibition of non-activated Na+ channels by 6-benzoylheteratisine. Additionally, 100 microM 6-benzoylheteratisine failed to affect the release of glutamate and the increase in [Ca2+]i induced by 30 mM KCl, indicating that voltage-gated Ca2+ channels were not affected by 6-benzoylheteratisine. The data suggest an inhibitory effect of 6-benzoylheteratisine on voltage-gated Na+ channels as the only target, whereas mechanisms of Na+ and Ca2+ homoeostasis and pathways of glutamate release seem not to be affected by the drug.

Kavain, dihydrokavain, and dihydromethysticin non-competitively inhibit the specific binding of [3H]-batrachotoxinin-A 20-alpha-benzoate to receptor site 2 of voltage-gated Na+ channels.

The mode of action of the kava pyrones, kavain, dihydrokavain and dihydromethysticin on the specific binding of [3H]-batrachotoxinin-A 20-alpha-benzoate to epitope 2 of voltage-dependent Na+ channels was investigated by performing saturation experiments in the presence and absence of these kava pyrones. The tested compounds significantly decreased the apparent total number of binding sites (Bmax) for [3H]-batrachotoxinin-A 20-alpha-benzoate (control: 0.5 pmol/mg protein, kava pyrones: 0.2-0.27 pmol/mg protein) with little change in the equilibrium constants (KD) for [3H]-batrachotoxin-A 20-alpha-benzoate (control: 28.2 nM, kava pyrones: 24-31 nM). The results indicate for the kava pyrones a non-competitive inhibition of the specific [3H]-batrachotoxinin-A 20-alpha-benzoate binding to receptor site 2 of voltage-gated Na+ channels.

Regulation of enzymes involved in anthocyanin biosynthesis in carrot cell cultures in response to treatment with ultraviolet light and fungal elicitors.

The accumulation of anthocyanins in cell cultures of Daucus carota L. and the enzymes involved in their biosynthesis were investigated under growth in the dark, continuous irradiation with UV light, incubation with elicitors from Pythium aphanidermatum, and elicitor treatment of UV-irradiated cells. Upon UV irradiation, anthocyanin accumulation was strongly enhanced, and the enzymes of the phenylpropanoid and flavonoid pathways, including the "late" enzymes cyanidin galactosyltransferase, cyanidin galactoside xylosyltransferase, cyanidin triglycoside sinapoyltransferase and sinapic acid glucosyltransferase, all showed transient increases in their activities. The time courses of the enzyme activities exhibited successive maxima with an ordered sequence corresponding to their position in the biosynthetic pathway, suggesting a coordinated induction of the entire set of enzymes. The key enzymes phenylalanine ammonia-lyase and chalcone synthase are regulated on a transcriptional level. Incubation of dark-grown carrot cells with fungal elicitors led to a rapid and transient induction of phenylalanine ammonia-lyase corresponding to the formation of 4-hydroxybenzoic acid, but the amount of anthocyanin did not increase and there was no enhancement of any of the enzyme activities which are part of the anthocyanin pathway, including the enzymes catalyzing glycosylation and acylation reactions. Treatment with UV light and elicitors resulted in a rapid induction of the phenylpropanoid pathway, whereas the inducing effect of UV light on the anthocyanin content, on chalcone synthase and on the enzymes catalyzing the final steps of anthocyanin biosynthesis was suppressed. These results indicate a coordinated regulation of the enzymes involved in anthocyanin biosynthesis, an independent inducibility of the phenylpropanoid pathway, and a hierarchy of the different effectors, as shown by the dominating role of the elicitor-signal over the UV stimulus.

Mode of antinociceptive and toxic action of alkaloids of Aconitum spec..

Extracts of the plant Aconitum spec. are used in traditional Chinese medicine predominantly as anti-inflammatory and analgesic agents, the latter allegedly equally potent as morphine but without any habit-forming potential. As the only pharmacologically active compounds, the C19 diterpenoid alkaloid aconitine, and some of its derivatives, have been proven to be antinociceptive in different analgesic assays, but the mode of action is unknown. To elucidate the mode of action, ten aconitine-like derivatives were investigated with respect to their affinity for voltage-dependent Na+ channels, the action on synaptosomal Na+ and Ca2+ homoeostasis and their antinociceptive, arrhythmogenic and acute toxic properties. Since aconitine is known to bind to site II of Na+ channels, we determined the affinity of the aconitine-like derivatives in vitro to synaptosomal membranes by the [3H]-batrachotoxinin-binding assay and their properties on intrasynaptosomal concentrations of free Na+ and Ca2+ ([Na+]i and [Ca2+]i), both the latter determined fluorometrically with SBFI and Fura-2 respectively. Furthermore, the alkaloids' arrhythmogenic potential was investigated in guinea-pig isolated atria and the antinociceptive action on formalin-induced hyperalgesia and the acute toxic action estimated in mice. The results show that the alkaloids could be divided into at least three groups. The first is characterized by a high affinity to the site II of Na+ channels (Ki about 1.2 microM), the ability to enhance [Na+]i and [Ca2+]i (EC50 about 3 microM), a strong arrhythmogenic action that starts at about 30 nM, an antinociceptive effect (ED50 about 0.06 mg/kg) and high acute toxicity (LD50 values about 0.15 mg/kg). To this group belong aconitine, 3-acetylaconitine and hypaconitine. The second group, with lappaconitine as the only member, has an affinity to Na+ channels an order of magnitude lower (Ki = 11.5 microM), less acute toxicity (LD50 about 5 mg/kg), and a two orders of magnitude lower antinociceptive action (ED50 about 2.8 mg/kg) and lower cardiotoxicity (bradycardia observed at 3 microM). Additionally, lappaconitine suppresses the increase in [Ca2+]i of aconitine-stimulated synaptosomes and increases the excitation threshold of left atria, indicating an inhibition of Na+ channels. The other derivatives, i.e. delcorine, desoxydelcorine, karakoline, lappaconidine, lappaconine and lycoctonine, belong to the third group, which has hardly any effects. They have a low affinity to Na+ channels with Ki values in the millimolar range, show no effect on synaptosomal [Na+]i and [Ca2+]i, no arrhythmogenic potential up to 100 microM, no antinociceptive activity and low toxicity with LD50 values greater than 50 mg/kg. For the investigated alkaloids we suggest two different antinociceptive-like modes of action. Aconitine, hypaconitine and 3-acetylaconitine may induce a block of neuronal conduction by a permanent cell depolarisation, whereas lappaconitine might act like local anaesthetics. However, because of the low LD50/ED30 quotients of 2-6, the antinociceptive-like action of the Aconitum alkaloids seems to reflect severe intoxication rather than a specific antinociceptive action. The structure/activity relationship shows that alkaloids that activate or block Na+ channels have a benzoyl ester side chain in the C-14 or C-4 positions respectively, whereas the other compounds lack this group.

Influence of extracellular K+ concentration on the time-course of Na+/K+-ATPase inhibition by cardiac glycosides with fast and low binding kinetics.

The magnitude of the K+ antagonism of cardiac glycoside binding to Na+/K+-ATPase prepared from porcine heart, was estimated from the enzyme activities determined in the presence of different concentrations of K+ ([K+]), ouabain, and alpha-methyl-digitoxigenin-glucoside, the latter showing a 30 fold greater dissociation rate than ouabain. An increase of [K+] (3-20 mmol/l) prolonged the half-lives of Na+/K+-ATPase inhibition and caused a rightward shift of the cardiac glycoside's dose-response curves by the same factor, almost maximal (4 fold) at 14 mmol/l K+. These data could be verified from the cardiac glycoside-elevated intravesicular Na+ concentrations of rat brain vesicles. These concentrations declined rapidly in brain vesicles treated with alpha-methyl-digitoxigenin-glucoside but not with ouabain after K+ was increased from 3.5 to 14 mM. The results suggest that the magnitude of the K+ antagonism under physiological conditions is only limited by the lifespan of the cardiac glycoside-binding E2P enzyme conformation reduced by K+.

Effects of long-term administration of hypericum extracts on the affinity and density of the central serotonergic 5-HT1 A and 5-HT2 A receptors.

Extracts of St. John's wort, Hypericum perforatum L. (Hypericaceae), are used as a phytotherapeutic antidepressant. A number of clinical studies demonstrate that their antidepressive potency is comparable to tricyclic antidepressants (TCA). Although the therapeutic effect of hypericum extracts is well documented, very little is known about the molecular mode of action. As the improvement of the depressive symptoms with both TCA and hypericum extracts only occurs significantly after a lag phase of 10 to 14 days, it is assumed that the medication causes long-term adaptations within the central nervous system. In this context, serotonergic (5-HT) receptors are of special interest. Therefore, we investigated possible alterations in affinity and density of 5-HT1 A and 5-HT2 A receptors caused by long-term treatment of rats with St. John's wort. The brain without cerebellum and brain stem of rats, treated daily for 26 weeks with a commercially available hypericum extract (2700 mg/kg LI 160) were used for membrane preparations. Affinity (KD) and amount (Bmax) of serotonergic receptors were determined by employing receptor binding assays using 3 H-8-OH-DPAT and 3H-Ketanserin as selective radioligands for the 5-HT1 A and the 5-HT2 A receptors, respectively. We found that in hypericum-treated rats the number of both 5-HT1 A and 5-HT2 A receptors were significantly increased by 50% compared to controls, whereas the affinity of both serotonergic receptors remained unaltered. The data suggest an upregulation of 5-HT1 A and 5-HT2 A receptors due to prolonged administration of hypericum extracts. These results are consistent with a modification of the expression levels of serotonergic receptors caused by synthetic antidepressants.

Kava extract ingredients, (+)-methysticin and (+/-)-kavain inhibit voltage-operated Na(+)-channels in rat CA1 hippocampal neurons.

The action of synthetic kava pyrones, (+)-methysticin and (+/-)-kavain, on voltage-operated Na(+)-channels was studied in whole-cell patch-clamped CA1 hippocampal neurons. In doses of 1-400 microM, both compounds exerted a rapid and reversible inhibition of the peak amplitude of Na(+)-currents. Shifting holding membrane potential (Vhold) to more positive values enhanced their blocking effect. The drugs studied did not demonstrate use-dependent properties at 10 Hz stimulation but shifted H infinity curve toward more negative potentials, accelerated time-course of inactivation and slowed down the recovery from inactivation. Voltage-dependence of Na(+)-channel inhibition can be explained by interaction of (+)-methysticin and (+/-)-kavain with resting closed and inactivated states of Na(+)-channel.

Relaxation of evoked contractile activity of isolated guinea-pig ileum by (+/-)-kavain.

Kava pyrones are the pharmacologically active compounds of Piper methysticum Forst. In the present study, the effect of the synthetic kava pyrone (+/-)-kavain was investigated on evoked contractile activity of isolated guinea-pig ileum. (+/-)-Kavain (1 microM-1 mM) dose-dependently reduced contractions of ileum evoked by carbachol (10 microM), by BAY K 8644 (0.3 microM), or by substance P (0.05 microM). (+/-)-Kavain also inhibited the contractile responses induced by raising the extracellular K+ concentration from 4 to 20 mM and by blocking the K+ channel by barium chloride (1 mM) or 4-aminopyridine (0.3 mM). After pre-incubation with 1 microM nifedipine, carbachol (1 microM) evoked 18.2 +/- 14.3% of contraction at control (i.e. prior pre-incubation with nifedipine). This remaining response was completely abolished by high concentrations of (+/-)-kavain (400 microM). After treatment of the longitudinal ileum strips with pertussis toxin (PTX), carbachol (1 microM) evoked 27.0 +/- 6.2% of the control response in untreated ileum. These contractions were also blocked by (+/-)-kavain (400 microM). However, (+/-)-kavain had no effect on the caffeine-induced (20 mM) contractions of ileum strips, which were permeabilized with digitonin or beta-escin. Moreover, it failed to affect Ca(2+)-evoked contractions of skinned muscles. These results suggest that the kava pyrone (+/-)-kavain may act in a non-specific musculotropic way on the smooth muscle membrane.

Bicuculline-induced epileptiform activity in rat hippocampal slices: suppression by Aconitum alkaloids.

Alkaloids of Aconitum spec. (Ranunculaceae) are employed in traditional Chinese folk medicine as analgesics. The present study was designed in order to investigate the effects of the structurally related alkaloids aconitine, lappaconitine, and 6-benzoylheteratisine on experimentally induced epileptiform activity. Experiments were performed as extracellular recordings of stimulus evoked population spikes in rat hippocampal slices. Epileptiform activity was induced by bicuculline. All three alkaloids exerted an inhibitory action on excitability of hippocampal pyramidal cells in a frequency-dependent manner. The onset of inhibition was accelerated by increasing the frequency of electrical stimulation. Aconitine (1 microM) evoked a complete suppression of both normal and epileptiform activity, whereas lappaconitine (10 microM) and 6-benzoylheteratisine (10 microM) selectively diminished the epileptiform afterdischarges and the duration of the bursts, but spared the normal activity. The present findings suggest that the structurally related Aconitum alkaloids aconitine, lappaconitine, and 6-benzoylheteratisine possess an anticonvulsive potential. The predominant effect of these alkaloids is to suppress the spread of seizure activity, and they may therefore tend to distort epileptic events. However, despite their similar structure, they exert qualitatively and quantitatively different inhibitory effects.

Antithrombotic action of the kava pyrone (+)-kavain prepared from Piper methysticum on human platelets.

(+)-Kavain, a 4-methoxy-alpha-pyrone prepared from Piper methysticum Forst. (Piperaceae), was investigated regarding its assumed antithrombotic action on human platelets which was deduced from its ability to suppress arachidonic acid (AA)-induced aggregation, exocytosis of ATP, and inhibition of cyclooxygenase (COX) and thromboxane synthase (TXS) activity, the latter two effects being estimated from the generation of prostaglandin E2 (PGE2) and thromboxane A2 (TXA2), respectively. Exogenously applied AA (100 mumol/l) provoked a 90% aggregation of platelets, the release of 14 pmol ATP, and the formation of either 220 pg TXA2 or 43 pg PGE2, each parameter being related to 10(6) platelets. An application of (+)-kavain 5 min before AA, dose-dependently diminished aggregation, ATP-release, and the synthesis of TXA2 and PGE2 with IC50 values of 78, 115, 71, and 86 mumol/l, respectively. The similarity of the IC50 values suggest an inhibition of COX by (+)-kavain as primary target, thus suppressing the generation of TXA2 which induces aggregation of platelets and exocytosis of ATP by its binding on TXA2-receptors.

Aconitum sp. alkaloids: the modulation of voltage-dependent Na+ channels, toxicity and antinociceptive properties.

Alkaloids from Aconitum sp., used as analgesics in traditional Chinese medicine, were investigated to elucidate their antinociceptive and toxic properties considering: (1) binding to Na+ channel epitope site 2, (2) alterations in synaptosomal Na+ and Ca2+ concentration ([Na+]i, [Ca2+]i), (3) arrhythmogenic action of isolated atria, (4) antinociceptive and (5) acute toxic action in mice. The study revealed a high affinity group (Ki 1 microM) and a low affinity group (Ki 10 microM) of alkaloids binding to site 2. The compounds of the high affinity group induce an increase in synaptosomal [Na+]i and [Ca2+]i (EC50 3 microM), are antinociceptive (ED50, 25 microg/kg), provoke tachyarrhythmia and are highly toxic (LD50 70 microg/kg), whereas low affinity alkaloids reduce [Ca2+]i, induce bradycardia and are less antinociceptive (ED50 20 mg/kg) and less toxic (LD50 30 mg/kg). These results suggest that the alkaloids can be grouped in Na+ channel activating and blocking compounds, but none of the alkaloids seem to be suitable as analgesics because of the low LD50/ED50 values.

[3H]-monoamine uptake inhibition properties of kava pyrones.

Three kava pyrones, the natural compounds (+)-methysticine and (+)-kavain, and the synthetic racemate (+/-)-kavain, were tested concerning their action on in vitro uptake of monoamines in synaptosomes prepared from the cerebral cortex and hippocampus of rats. (+/-)-Kavain and (+)-kavain were found to potently inhibit the uptake of [3H]-noradrenaline. Uptake of [3H]-noradrenaline was inhibited in the following order of potency: (+/-)-kavain = (+)-kavain > (+)-methysticine, whereas none of the kava pyrones efficiently blocked the uptake of [3H]-serotonin. The results indicate a pyrone-specific non-stereo-selective inhibition of the [3H]-noradrenaline uptake which might be responsible for or, at least, contribute to the psychotropic properties of kava pyrones.

Kavain inhibits non-stereospecifically veratridine-activated Na+ channels.

The action of the natural kava pyrone, (+)-kavain, and its synthetic racemate, (+/-)-kavain, on voltage-dependent Na+ channels was investigated, while considering their stereospecific properties, on veratridine-induced increases in cytosolic free Na+ and Ca2+ ([Na+]i, [Ca2+]i) and the release of endogenous glutamate from cerebrocortical synaptosomes. Both compounds dose-dependently suppressed the veratridine-induced increase in [Na+]i, [Ca2+]i and glutamate release with IC50 values (+/- S.D.) of 71 +/- 22, 72 +/- 7, 120 +/- 37 micromol/l (+)-kavain and 77 +/- 21, 90 +/- 14, 92 +/- 23 micromol/l (+/-)-kavain, respectively. As judged from the dose-dependency, IC50 values, velocity and time course of action, both kava pyrones were equally effective suggesting a non-stereospecific inhibition of veratridine-activated Na+ channels.

Anticonvulsive action of (+/-)-kavain estimated from its properties on stimulated synaptosomes and Na+ channel receptor sites.

Kava pyrones are constituents of the intoxicating pepper (Piper methysticum Forst), which has been shown to be anticonvulsive. The question of how the excitability of neurons is affected was investigated by determining the interaction of (+/-)-kavain with epitopes (site 1, site 2) of voltage-dependent Na+ channels and the action of (+/-)-kavain on 4-aminopyridine-stimulated synaptosomes as model of repetitive firing neurons. [3H]Saxitoxin and [3H]batrachotoxin were used for radioligand-binding assays performed with synaptosomal membranes. Gultamate released from 4-aminopyridine-stimulated cerebrocortical synaptosomes and the cytosolic concentrations of Na+ and Ca2+ ([Na+]i, [Ca+]i) were detected fluorometrically by using an enzyme-linked assay, sodium-binding benzofuranisophthalate (SBFI) and Fura-2, respectively. (+/-)-Kavain failed to compete with [3H]saxitoxin up to 400 mumol/l but dose-dependently suppressed binding of [3H]batrachotoxin with an IC50 value of 88 mumol/l (Ki = 72 mumol/l) although displacement of [3H]batrachotoxin was restricted to 33% of control at 400 mumol/l (+/-)-kavain. In stimulated synaptosomes, 5 mmol/l 4-aminopyridine provoked an increase in [Na+]i and [Ca2+]i by 9 mmol/l Na+ and 235 nmol/l Ca2+. Comparable to the reduction in [3H]batrachotoxin binding, 400 mumol/l (+/-)-kavain suppressed the increase in [Na+]i and [Ca2+]i to 38 and 29% of control, respectively. Consistent with the increase in [Na+]i and [Ca2+]i, 5 mmol/l 4-aminopyridine provoked glutamate release (rate: 38 pmol/s*mg protein) which was dose-dependently diminished to 60% of control by 400 mumol/l (+/-)-kavain. KCl depolarization (40 mmol/l) provoked an increase in [Ca2+]i and glutamate release almost identical to the responses elicited by 4-aminopyridine but 400 mumol/l (+/-)-kavain suppressed only the rate of glutamate release by 9% of control. The data suggest an interaction of (+/-)-kavain with voltage-dependent Na+ and Ca2+ channels, thereby suppressing the 4-aminopyridine-induced increase in [Na+]i, [Ca2+]i and the release of endogenous glutamate.

Inhibition of neuronal activity in rat hippocampal slices by Aconitum alkaloids.

The structurally related Aconitum alkaloids aconitine, lappaconitine, and 6-benzoylheteratisine inhibited the orthodromic and antidromic population spike in hippocampal CA1 area in a frequency-dependent manner. Aconitine (1 microM) completely suppressed epileptiform activity induced by omission of Mg2+ as well as normal neuronal activity, whereas lappaconitine (10 microM) and 6-benzoylheteratisine (10 microM) diminished epileptiform activity by sparing normal neuronal activity.

Continuous enzyme-linked fluorometric detection of L-(+)-lactate released from rat brain vesicles under anoxic conditions.

A method is described for the on-line detection of L-(+)-lactate released from brain vesicles under physiological conditions. The principle of L-lactate detection is based on the reversible oxidation of L-lactate catalysed by L-lactate dehydrogenase (LDH, EC 1.1.1.27) employing 3-acetylpyridine-adenine-dinucleotide (APAD) as analogue of NAD according to the reaction: L-lactate + APAD reversible pyruvate + APADH. In practical terms, L-lactate synthesis of vesicles incubated in the presence of LDH and APAD was continuously followed by the fluorescence (490 nm) of APADH excited at 410 nm. Addition of a L-lactate standard (10 mumol/l) enhanced APADH fluorescence with a half-life of 6.0 +/- 0.6 s allowing us to uncover a short-term alteration of L-lactate synthesis. This method was applied to evaluate a prospective change of L-lactate generation caused by the anoxia-induced increase in intravesicular Na+ and Ca2+ concentration ([Na+]i, [Ca2+]i), both fluorometrically determined by SBFI and Fura, respectively. Upon anoxia, [Na+]i and [Ca2+]i increased continuously up to 40 mmol/l Na+ and 900 nmol/l Ca2+ within 400 s. Concurrently, intravesicular NADH ([NADH]i) and basal L-lactate synthesis were enhanced within a few seconds, the latter from 4.2 +/- 1.5 to 15.8 +/- 1.5 nmol L-lactate/min per mg protein. Incubation of vesicles in the presence of 10 mumol/l tetrodotoxin (TTX) suppressed the increase in [Na+]i and [Ca2+]i but failed to influence L-lactate synthesis. The data indicate a continuous Na+ influx via voltage-dependent Na+ channels accompanied by an increase in [Ca2+]i during anoxia which did not affect anaerobic L-lactate synthesis. The method of fluorometric L-lactate determination was confirmed to be suitable for the detection of L-lactate released under physiological conditions from brain vesicles and seems to be applicable to various cell models.

Aconitine inhibits epileptiform activity in rat hippocampal slices.

The effect of aconitine, an alkaloid neurotoxin known to bind at site 2 of the sodium channel, was investigated on epileptiform activity in hippocampal slices by use of extracellular recordings in CA1 pyramidal cell layer. Epileptiform activity was induced by bicuculline, picrotoxin, penicillin, pentylenetetrazol or by omission of magnesium from the bathing medium, respectively. In every case aconitine (0.1 and 1 microM) blocked the multiple population spikes representing the epileptiform activity. The onset of inhibition was shorter by use of an increased concentration of the epileptogenic drug. Epileptiform activity evoked by pentylenetetrazol and low magnesium was first increased by aconitine followed by a rapid inhibition, while the bicuculline-, picrotoxin-, and penicillin-induced epileptiform discharges were immediately abolished.

(+/-)-kavain inhibits the veratridine- and KCl-induced increase in intracellular Ca2+ and glutamate-release of rat cerebrocortical synaptosomes.

The action of (+/-)-kavain on the veratridine, monensin and KCl-depolarization evoked increase in free cytosolic Ca2+ concentration ([Ca2+]i), and its influence on the release of endogenous glutamate from rat cerebrocortical synaptosomes were investigated. [Ca2+]i was fluorimetrically determined employing FURA as the Ca2+ sensitive fluorophore, and glutamate was detected by a continuous enzyme-linked fluorimetric assay. The incubation of synaptosomes in the presence of (+/-)-kavain up to a concentration of 500 mumol/l affected neither basal [Ca2+]i nor spontaneous release of glutamate, but dose-dependently reduced both veratridine-elevated [Ca2+]i (IC50 = 63.2 mumol/l) and glutamate-release (IC500 = 116.4 mumol/l). The inhibition of these parameters, attained with 500 mumol/l(+/-)-kavain, could be overcome by inducing an artificial Na+ influx, using monensin as a Na+ ionophore, An application of (+/-)-kavain after veratridine caused a decrease in veratridine-elevated [Ca2+]i, which was similar to the action of tetrodotoxin (TTX) with regard to time course, half-life of [Ca2+]i decline and the final steady state level of [Ca2+]i. Concomitantly, veratridine-induced glutamate-release was blocked. The results indicate that specific inhibition of voltage-dependent Na+ channels is a primary target of (+/-)-kavain, thus preventing a [Na+]i provoked increase in [Ca2+]i and glutamate-release. However, pathways related to the elevation of [Ca2+]i by [Na+]i itself, and the processes involved in normalization of elevated [Ca2+]i and glutamate-release downstream to enhanced [Ca2+]i, seems to be unaffected by (+/-)-kavain. Using KCl-depolarized synaptosomes, 400 mumol/l (+/-)-kavain reduced, in analogy to Aga-GI toxin, KCl-evoked [Ca2+]i and diminished the part of glutamate-exocytosis which is related to external Ca2+ to about 75% of control. At a concentration of 150 mumol/l, which is above the IC50 value necessary to block voltage-dependent Na+ channels, (+/-)-kavain affected neither basal nor the KCl-induced increase in [Ca2+]i. These results might suggest that (+/-)-kavain at concentrations sufficient to block Na+ channels completely. moderately inhibits the non-inactivating Ca2+ channels located on mammalian presynaptic nerve endings.

The protective action of tetrodotoxin and (+/-)-kavain on anaerobic glycolysis, ATP content and intracellular Na+ and Ca2+ of anoxic brain vesicles.

Because recent reports point to Na+ channel blockers as protective agents directed against anoxia-induced neuronal damage including protection of anaerobic glycolysis, the influences of tetrodotoxin (TTX) and (+/-)-kavain on anoxic rat brain vesicles were investigated with respect to lactate synthesis, vesicular ATP content and cytosolic free Na+ and Ca2+ ([Na+]i, [Ca2+]i), both of the latter determined fluorometrically employing SBFI and FURA-2, respectively. After anoxia, basal lactate production was increased from 2.9 to 9.8 nmol lactate/min/mg protein. Although lactate synthesis seemed to be stable for at least 45 min of anoxia, as deduced from the linearity of lactate production, the ATP content declined continuously with a half life (tau 1/2) of 14.5 min, indicating that anaerobic glycolysis was insufficient to cover the energy demand of anoxic vesicles. Correspondingly, [Na+]i and [Ca2+]i increased persistently after anoxia by 22.1 mmol/l Na+ and 274.9 nmol/l Ca2+, determined 6.3 min after onset. An additional stimulation of vesicles with veratridine accelerated the drop of ATP (tau 1/2 = 5.1 min) and provoked a massive Na+ overload, which levelled off to 119 mmol/l Na+ within a few minutes. Concomitantly, [Ca2+]i increased linearly with a rate of 355 nmol Ca2+/l/min. Despite the massive perturbation of ion homeostasis, lactate production was unaffected during the first 8 min of veratridine stimulation. However, complete inhibition of lactate synthesis took place 30 min after veratridine was added. The Na+ channel blockers TTX and (+/-)-kavain, if applied before anoxia, preserved vesicular ATP content, diminished anoxia-induced increases in [Na+]i and [Ca2+]i and prevented both the veratridine-induced increases of [Na+]i and [Ca2+]i and the inhibition of lactate production. The data indicate a considerable Na+ influx via voltage-dependent Na+ channels during anoxia, which speeds up the decline in ATP and provokes an increase in [Ca2+]i. A massive Na+ and Ca2+ overload induced by veratridine failed to influence lactate synthesis directly, but initiated its inhibition.