Chlorothiophenecarboxamides as P1 surrogates of inhibitors of blood coagulation factor Xa.

Neutral chlorothiophenecarboxamides bearing an amino acid and a substituted aniline were synthesized and investigated for their factor Xa inhibitory activity in vitro. From selected 2-methylphenyl morpholinones the solution properties were determined. The most soluble and active compounds were then investigated in different animal species to compare the pharmacokinetic parameters.

Halothiophene benzimidazoles as P1 surrogates of inhibitors of blood coagulation factor Xa.

Neutral weak halothiophene benzimidazole inhibitors of the serine protease factor Xa were identified via screening of a compound library. The X-ray crystal structure of representative 3a bound to human fXa confirmed the S1 binding mode. Starting from 3a a series of halothiophene benzimidazoles was synthesized and investigated for their factor Xa inhibitory activity.

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.

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.

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.

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.

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.

[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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

(+/-)-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.

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.

(+/-)-Kavain inhibits veratridine-activated voltage-dependent Na(+)-channels in synaptosomes prepared from rat cerebral cortex.

Kava pyrones are pharmacologically active compounds extracted from Piper methysticum Forst. Because kava pyrones were characterized by their anticonvulsive, analgesic and centrally muscle relaxing action, we investigated the influence of (+/-)-kavain, a synthetic kava pyrone, on veratridine-stimulated increase in intrasynaptosomal Na+ concentration ([Na+]i) of rat cerebrocortical synaptosomes. [Na+]i was measured spectrofluorometrically employing SBFI as Na+ sensitive fluorescence dye.

Anaerobic glycolysis and postanoxic recovery of respiration of rat cortical synaptosomes are reduced by synaptosomal sodium load.

Synaptosomes of rat cerebral cortex were used to study the effect of veratridine-induced Na+ load on postanoxic recovery of respiration and on aerobic and anaerobic ATP turnover, calculated from rates of oxygen consumption and lactate production. Non-stimulated synaptosomes: after onset of anoxia lactate synthesis of synaptosomes rose immediately from 0.8 to 17.

Cryopreservation of freshly isolated synaptosomes prepared from the cerebral cortex of rats.

In the present study, we established a cryopreservation method for freshly isolated synaptosomes prepared from the cerebral cortex of rats. Freshly prepared synaptosomes were either shock-frozen or frozen under temperature-controlled conditions using a programmable temperature controller. Each group was resuspended in iso-osmotic or hyperosmotic sucrose buffer prior to freezing, resulting in 4 different preservation protocols.