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.

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.

(+/-)-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.

(+/-)-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. Veratridine (5 mumol/I) enhanced basal [Na+]i 6.6-fold from 11.3 to 74.1 mmol/l Na+. Incubation of synaptosomes for 100 sec with (+/-)-kavain was sufficient to reduce dose dependently the stimulated increase of [Na+]i with an IC50 value of 86.0 mumol/l, and almost complete inhibition of Na(+)-channels was attained with 400 mumol/l) reduced veratridine-elevated [Na+]i to 30.4% and 7.9% of control whereas the centrally acting muscle relaxant mephenesin (400 mumol/l) was without any effect. Postapplication of 400 mumol/l (+/-)-kavain or 10 mumol/l TTX immediately diminished veratridine-elevated [Na+]i to nearly basal levels with a half life time of 69.7 and 41.8 sec, respectively. To study the influence of (+/-)-kavain on non stimulated synaptosomes, an increase in [Na+]i was induced by 200 mumol/l ouabain, which enhanced [Na+]i hyperbolically with an initial rate of 18.4 mmol Na+/l min. Preincubation of synaptosomes with 400 mumol/l (+/-)-kavain or 10 mumol/l TTX partly prevented Na(+)-influx for both compounds to the same extent of about 57% of control. The presented data indicate a fast and specific inhibition of voltage-dependent Na(+)-channels by (+/-)-kavain.

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. The viability of the frozen synaptosomes was estimated by the recovery of basal and stimulated respiration after short-term storage (1 h) in liquid nitrogen. With regard to basal, FCCP- and veratridine-induced respiration, best recovery revealed controlled-frozen synaptosomes resuspended in iso-osmotic sucrose buffer (con/iso group). Basal respiration of this group recovered completely, whereas veratridine- and FCCP-induced oxygen uptake was decreased to 87.7% and 82.4% of control, respectively. Further investigations performed with the con/iso group revealed complete recovery of anaerobic and aerobic lactate synthesis, and unaffected synaptosomal integrity, as judged by the amount of released L-lactate dehydrogenase before and after the cryopreservation procedure. Long-term storage of the con/iso group in liquid nitrogen up to 88 days did not have any influence on synaptosomal viability, as evaluated by the recovery of anaerobic lactate production and synaptosomal respiration. Therefore, based on the results of respiration, synaptosomal integrity, and lactate synthesis, metabolically active synaptosomes could be obtained after cryopreservation and storage in liquid nitrogen for at least 88 days.

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 17.7 nmol lactate/min/mg protein indicating an anaerobic ATP turnover of 17.7 nmol ATP/min/mg protein. This value accounts for 80% of ATP synthesized during oxygenated conditions and seems to cover the energetic demand of anoxic synaptosomes. This assumption was supported by linearity of lactate production throughout anoxia (90 min), by unaffected synaptosomal integrity and by complete recovery of postanoxic respiration after 90 min of anoxia. Stimulated synaptosomes: stimulation of oxygenated synaptosomes with 10(-5) mol/l veratridine enhanced ATP turnover 5-fold, due to activation of Na+/K+ ATPase, as a result of veratridine-induced Na+ influx. Consequently, if not limited in capacity, anaerobic ATP synthesis should be enhanced after addition of veratridine during anoxia. However, the opposite effect was observed. Veratridine reduced anaerobic glycolysis in a concentration-dependent manner. This inhibitory effect could be prevented by tetrodotoxin applied 5 min prior to veratridine. Inhibition of anaerobic glycolysis was independent of extrasynaptosomal glucose (1-30 mmol/l) and Ca2+ concentration (Ca(2+)-free and 1.2 mmol/l Ca2+). Veratridine stimulation of anoxic synaptosomes reduced also the recovery of postanoxic respiration. The data indicate that Na+ load inhibits anaerobic ATP synthesis, the only energy source during anaerobic conditions. To our knowledge, inhibition of anaerobic glycolysis due to increased Na+ influx has not been shown so far.

Ouabain-induced changes of calcium and potassium in slices of hippocampus of the rat: comparison to hypoxia and effect of R 56865.

Simple and reliable in vitro models of cerebral ischaemia are important for the identification of antiischaemic/antihypoxic compounds. Alterations of the concentrations of potassium and calcium were recorded in slices of hippocampus of the rat. The slices were subjected to hypoxia in the presence and absence of intoxication with glucose or ouabain (1 mmol/l). Normoxic slices of hippocampus showed an extracellular space of 57% and a tissue concentration of potassium of 45 mmol/kg wet wt. A cellular concentration of potassium of 92 mmol/kg was calculated. Hypoxia, in the presence of glucose, only slightly reduced tissue concentrations of potassium and did not influence concentrations of calcium. Omission of glucose during hypoxia led to tissue concentrations of potassium below 10 mmol/kg, within 10-30 min of hypoxia. Concentrations of calcium only increased from 3.3 to 3.5 mmol/kg after 30 min of hypoxia, without glucose. Intoxication with ouabain is proposed as alternative experimental model of ionic movements, associated with cerebral ischaemia/hypoxia. Tissue concentrations of potassium fell rapidly to values below 10 mmol/kg, within 5 min and concentrations of calcium rose to 5.2 mmol/kg, within 30 min of intoxication with ouabain. In quantitative terms, the model for cerebral ischaemia with intoxication with ouabain is suggested to be superior to the model based on hypoxia without glucose. To verify intoxication with ouabain as an experimental model for ischaemic/hypoxic insults, the effect of an investigational drug with antiischaemic/hypoxic properties (R 56865) was evaluated in the model. The drug R 56865 produced dose-dependent attenuation of the fall in tissue concentrations of potassium, between 3 x 10(-7) and 5 x 10(-6) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptosomal respiration: a potent indicator of veratridine-induced Na influx.

Inhibition of the voltage-dependent Na channel by tetrodotoxin (5 mumol/l) decreased by about 86% the stimulation of the respiration of rat brain synaptosomes induced by veratridine (10 mumol/l). A similar effect was achieved by blocking Na(+)-K(+)-ATPase with ouabain (1 mmol/l). Pharmacological manipulations of Ca homeostasis suggested that the veratridine-induced stimulation of respiration did not depend upon Ca entry. These data suggest that the veratridine-induced stimulation of synaptosomal respiration may result primarily from an increase in intracellular Na+. They provide the basis for an indirect method, which only requires recording of synaptosomal respiration, to test drugs for their possible interaction with excessive Na influxes.