Potassium aspartate inhibits SH-SY5Y cell damage and apoptosis induced by ouabain and H2O2.

The present study aimed to investigate the effects of L-aspartic acid potassium salt (potassium aspartate, K-asp) on SH-SY5Y cells treated with ouabain and H2O2. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate the effects of K-asp on SH-SY5Y cell death induced by ouabain. Nissl staining was used to demonstrate the morphological changes of the SH-SY5Y cells. Light microscopy and 4',6-diamidino-2-phenylindole (DAPI) staining were performed to visualize apoptosis in SH-SY5Y cells incubated with ouabain for 6, 24 and 48 h. Transmission electron microscopy was used to observe the effect of K-asp on ultrastructural changes of the SH-SY5Y cells following incubation with ouabain for 24 and 48 h. An annexin V-fluorescein isothiocyanate/propidium binding assay and flow cytometry were performed successively to investigate how K-asp affected the H2O2-induced cell apoptosis. The MTT assay demonstrated that K-asp attenuated the cytotoxicity of the SH-SY5Y cells following treatment with ouabain, in a dose-dependent manner. The cell survival rates following 48 h incubation in the K-asp (15 mM) and K-asp (25 mM) groups were higher compared with the KCl and MK801 groups. Nissl staining demonstrated that the severity of cell injury in the KCl and K-asp (25 mM) groups were alleviated. In the DAPI staining and transmission electron microscopy analyses, KCl and K-asp (25 mM) reduced the rate of ouabain-induced apoptosis. Flow cytometry revealed that K-asp (25 mM) reduced H2O2 -induced apoptosis. These results demonstrated that K-asp (25 mM) inhibited the ouabain and H2O2-induced SH-SY5Y cell damage and apoptosis, possibly by supplementing levels of intracellular K(+).

Folic acid administration prevents ouabain-induced hyperlocomotion and alterations in oxidative stress markers in the rat brain.

OBJECTIVE: Bipolar disorder (BD) is a chronic, prevalent, and highly debilitating psychiatric illness. Folic acid has been shown to have antidepressant-like effects in preclinical and clinical studies and has also been suggested to play a role in BD. The present work investigates the therapeutic value of folic acid supplementation in a preclinical animal model of mania induced by ouabain. METHODS: Male Wistar rats were treated twice daily for seven days with folic acid (10, 50, and 100 mg/kg, p.o.) or the mood stabilizer lithium chloride (LiCl) (45 mg/kg, p.o.). One day after the last dose was given, the animals received an i.c.v. injection of ouabain (10 microM), a Na(+),K(+)-ATPase-inhibiting compound. Locomotor activity was assessed in the open-field test. Thiobarbituric acid-reactive substance (TBARS) levels, glutathione peroxidase (GPx), and glutathione reductase (GR) activities were measured in the cerebral cortex and hippocampus. RESULTS: Ouabain (10 microM, i.c.v.) significantly increased motor activity in the open-field test, and seven days of pretreatment with folic acid (50 mg/kg, p.o.) or LiCl (45 mg/kg, p.o.) completely prevented this effect. Ouabain treatment elicited lipid peroxidation (increased TBARS levels) and reduced GPx activity in the hippocampus. GR activity was decreased in the cerebral cortex and hippocampus. These effects were prevented by pretreatment with folic acid and LiCl. CONCLUSIONS: Our results show that folic acid, similarly to LiCl, produces a clear antimanic action and prevents the neurochemical alterations indicative of oxidative stress in an animal model of mania.

Altered levels of nodal excitability by rate-dependent inhibitory effects of essential oil of Citrus aurantium on the electrophysiological properties of isolated perfused rabbit AV-Node: protective role in the prevention of ouabain toxicity / Alteración de los niveles de excitabilidad nodal por efectos inhibitorios velocidad-dependiente del aceite esencial de Citrus aurantium sobre las propiedades electrofisiológicas del nodo AV Aislado y perfundido de conejo: papel protector en la prevención de toxicidad por ouabaína

Treatment of supraventricular arrhythmia includes a wide range of medical interventions. Herbal remedies are suitable alternatives to synthetic drugs due to their availability, minimal side effects and lower price. Pharmacological studies and traditional medical literature point to the cardiovascular effects of Citrus aurantium L. (Rutaceae) in many instances. In the present study we used isolated perfused AV-node of rabbit as an experimental model to determine the effect of various concentrations of essential oil of C. aurantium (0.1-0.3 v/v) on the nodal conduction time and refractoriness of an isolated rabbit AV-nodal preparations. Selective stimulation protocols were used to independently quantify AV nodal recovery, facilitation and fatigue in 18 rabbits. Our results showed concentration-dependent and rate-independent suppressive effects of essence of C. aurantium on the Wenchebach cycle length (WBCL), AV Conduction Time (AVCT) and effective and functional refractory periods (ERP & FRP). Functional properties such as facilitation and fatigue were significantly increased by this plant. Citrus aurantium plays a protective role against the toxic effects of ouabaine by increasing AV nodal conduction time and refractoriness. The above results indicated differential effects of C. aurantium on slow and fast pathways, with a dominant role on fast pathways. This research has explained the protective role of C. aurantium on ouabaine toxicity. All results indicated the potential anti-arrhythmic effects of C. aurantium in treating supraventricular tachyarrhythmia.

El tratamiento de la arritmia supraventricular incluye una amplia gama de intervenciones médicas. Los remedios herbarios son alternativas adecuadas a las drogas sintéticas debido a su disponibilidad, con escasos efectos secundarios y bajo precio. Estudios farmacológicos y la literatura médica tradicional señalan los efectos cardiovasculares de Citrus aurantium L. (Rutaceae) en muchos casos. En el presente estudio se usaron aislados perfundidos del nodo AV de conejo como modelo experimental para determinar el efecto de diferentes concentraciones de aceite esencial de C. aurantium (0,1-0,3 v/v) sobre en el tiempo de conducción nodal y refractariedad. Un protocolo de estimulación selectiva se utilizó para cuantificar de forma independiente la recuperación, la facilitación y la fatiga del nodo AV en 18 conejos. Nuestros resultados muestran efectos supresores dependientes de la concentración e independiente de la velocidad de la esencia de C. aurantium sobre la duración del ciclo Wenchebach (WBCL), tiempo de conducción AV (AVTC) y períodos refractarios eficaz y funcional (PRE y PRF). Propiedades funcionales tales como la facilitación y la fatiga se incrementaron de manera significativa por esta planta. La Citrus aurantium juega un papel protector contra los efectos tóxicos de ouabaína al incrementar el tiempo de conducción AV nodal y la refractariedad. Los resultados indican efectos diferenciales de C. aurantium sobre las vías lentas y rápidas, con un papel dominante en las vías rápidas. Esta investigación ha explicado el papel protector de C. aurantium sobre la toxicidad ouabaine. Todos los resultados indican los posibles efectos anti-arrítmicos de C. aurantium en el tratamiento de taquiarritmias supraventriculares.

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 novel toxicity-based assay for the identification of modulators of voltage-gated Na+ channels.

Voltage-gated Na+ channels are promising drug targets. Screening of large numbers of putative modulators, however, can be demanding and expensive. In this study, a simple, cheap, and robust assay to test the pharmacological modulation of Na+ channel function is presented. The assay makes use of the fact that the intracellular accumulation of Na+ ions can be cytotoxic. The toxicity of the Na+ channel activator veratridine in the presence of an inhibitor of the Na+/K+ ATPase (ouabain) in a Nav1.2a (rat brain IIA alpha) expressing cell line is assessed. Na+ channel blockers should reduce toxicity in this model. CHO cells which recombinantly expressed rat Nav1.2a subunits were seeded in 96-well plates, and cell survival was tested after 24 h incubation in medium containing veratridine and ouabain in the presence or absence of Na+ channel blockers. Propidium iodide fluorescence was used as toxicity readout. Veratridine (100 microM) or ouabain alone (500 microM) were not toxic to the cells. In the presence of 500 microM ouabain, however, veratridine induced halfmaximal cell death with an EC50 value of 15.1 +/- 2.3 microM. Ouabain's EC50 was 215.3 +/- 16.7 microM (with 30 microM veratridine). The effects of a number of Na+ channel blockers were tested and compared with their Na+ channel blocking activity measured in voltage-clamp experiments. Blockers from various chemical classes reduced toxicity half maximally with IC50 values ranging from 11.7 +/- 1.4 nM (tetrodotoxin) to 280.5 +/- 48.0 microM (lamotrigine). There was a linear relationship between the log IC50 values obtained by the two methods (slope: 1.1 +/- 0.08; correlation coefficient: 0.93). In summary, these data show that this novel toxicity assay is well suited to test Na+ channel blockers.

A galanin-mastoparan chimeric peptide activates the Na+,K(+)-ATPase and reverses its inhibition by ouabain.

The effect of the neuropeptide galanin, the wasp venom toxin amphiphilic peptide toxin mastoparan and the chimeric peptide, galparan, consisting of N-terminal 13 amino acids of neuropeptide galanin linked at C-terminus to mastoparan amide (and its inactive analog Mas17) on the activity of Na+,K(+)-ATPase has been studied. Mastoparan inhibits the activity of the Na+,K(+)-ATPase with IC50 = 7.5 microM and also reduces the cooperativity for Na+ and K+, respectively, while galanin has no effect on the Na+,K(+)-ATPase activity. The chimeric peptide, galanin(1-13)-mastoparan amide (galparan), exhibits biphasic interaction with Na+,K(+)-ATPase, it activates the enzyme at maximal stimulating concentration of 4 microM followed by inhibition of the enzyme with IC50 of 100 microM. At maximum stimulating concentration (4 microM), galparan partly reduces the cooperativity only for Na+ and it also counteracts the inhibitory effect of oubain on Na+,K(+)-ATPase. Galparan's stimulatory effect was influenced by ATP. The chimeric peptide [19Lys,26Leu]-galparan, containing the inactive analog of mastoparan (Mas17), has no effects on rat brain Na+,K(+)-ATPase activity. Both chimeric peptides galparan and [19Lys,26Leu]-galparan are high-affinity galanin receptor ligands with IC50 of 6.4 nM and 0.71 nM, respectively, while galanin (1-13) and mastoparan alone have significantly lower affinity for the galanin receptor, IC50 of 125 nM and 1 microM, respectively. The ability of chimeric peptides to bind to galanin receptors does not correlate with their effects on the Na+,K(+)-ATPase.

Anti-arrhythmic profile of a garlic dialysate assayed in dogs and isolated atrial preparations.

The effects of garlic (Allium sativum L., Liliaceae) dialysate were studied on arrhythmias induced in anaesthetized dogs and on isolated left rat atria. Garlic dialysate suppressed premature ventricular contractions (PVC) and ventricular tachycardia (VT) in ouabain-intoxicated dogs as well as the ectopic rhythms induced by isoprenaline (10(-6) M) and aconitine (10(-8) M) on electrically driven left rat atria. The effective refractory period (ERP) and the sinus node recovery time (SNRT) of isolated rat atria were prolonged in a dose-dependent manner by the administration of this extract. Garlic dialysate decreased the positive inotropic and chronotropic effects of isoprenaline in a concentration-dependent manner. These last effects were increased by propranolol. The results suggest that garlic dialysate has a significant antiarrhythmic effect in both ventricular and supraventricular arrhythmias.

Testing for circadian differences in lethality for intravenous ouabain in male mice.

Using a randomized, balanced design and double-blind methodology, ouabain octahydrate was administered intravenously to male mice at six clock times. Eight runs were conducted using six constant dosage levels. All the dose-response curves at the clock times of 02:30, 06:30, 10:30, 14:30, 18:30 and 22:30 were parallel and no significant differences were noted between the respective LD50 determinations using nomograph methods. Independent chi-square analysis of all lethality data indicated no significant variation in response between clock times and between runs but a very highly significant difference between doses. Using regression methods, onset time for death was shown to vary inversely with log-dosage, but those periods of possible increased susceptibility could not be correlated with a shortened time to death. These findings are consistent with a random variation in lethality in regard to clock time rather than a true circadian pattern. The pooled (N = 576) intravenous LD50 for ouabain octahydrate was 3.75 mg/kg with 95% confidence limits of 3.60-3.90 mg/kg or, when calculated as anhydrous ouabain, 3.01 (2.89-3.13) mg/kg.

Mechanisms of age-related differences in the cardiotoxic action of digitalis.

Age-associated ouabain sensitivity was investigated in isolated perfused guinea pig heart and the findings were correlated with its receptor function. Ouabain (0.1 mumol L-1) produced average positive inotropic responses of 22% in 15-day-old, 25% in 4-6 month-old, and 34% in 18-24-month-old guinea pig hearts. The time required to produce the greatest positive inotropic response was 28 min in 15-day- to 6-month-old guinea pig hearts and 20 min in 18-24-month-old guinea pig hearts. To cause arrhythmias and cardiac arrest, 2.1 mumol L-1 ouabain was required in age groups of 15 days to 6 months, and 1.2 mumol L-1 in age groups of 12-24 months. The time of onset of arrhythmias and cardiac arrest was also significantly less in older than in younger animals. Cardiac membranes from 12-24-month-old guinea pig hearts had significantly lower Na+, K+ -ATPase activity per mg of sarcolemmal protein and numbers of [3H]ouabain binding sites than those from 28-day- to 6-month-old hearts. The enzyme inhibition by 0.1 mumol L-1 ouabain was, however, less in younger than in older groups. The difference in the inhibition of enzyme was reflected in lower ouabain binding affinity (Kd) in younger animals. The data confirm the existence of an age-related difference in ouabain sensitivity and suggest that these differences may be due to changes in receptor function.

Enhancement of triggered activity in ischemic Purkinje fibers by ouabain: a mechanism of increased susceptibility to digitalis toxicity in myocardial infarction.

Enhanced susceptibility to toxic arrhythmias by digitalis administration has been reported in clinical and experimental myocardial infarction. To investigate the mechanism responsible for this phenomenon, the effects of superfusion with normal Tyrode's solution and superfusion with Tyrode's solution containing 4 X 10(-8)M of ouabain in ischemic Purkinje fibers were compared. Ischemic Purkinje fibers of small endocardial preparations from 1 day old myocardial infarcts in 18 dogs were used for the study. During control conditions, these endocardial preparations demonstrated delayed afterdepolarizations and triggered activity. Superfusion with normal Tyrode's solution resulted in a gradual increase in maximal diastolic potential and action potential amplitude, a decrease in delayed afterdepolarizations amplitude and slowing and termination of triggered activity. Superfusion for 90 minutes with Tyrode's solution containing ouabain resulted in: 1) an increase in the magnitude of delayed afterdepolarizations in preparations demonstrating subthreshold delayed afterdepolarizations, 2) sustainment of triggered activity in preparations showing nonsustained triggered activity, and 3) shortening of cycle lengths of the triggered activity in preparations demonstrating sustained triggered activity before superfusion with ouabain. These effects occurred despite the gradual increase in maximal diastolic potential and action potential amplitude. Superfusion of normal Purkinje fibers with Tyrode's solution containing 4 X 10(-8)M of ouabain for 90 minutes did not result in delayed afterdepolarizations or triggered activity. Thus, ouabain at a concentration that has no toxic effect on normal Purkinje fibers may enhance arrhythmias in ischemic Purkinje fibers by increasing the magnitude of delayed afterdepolarizations and enhancing triggered activity.

Amiloride: relationship between cardiac effects and inhibition of Na+/Ca2+ exchange.

The potassium sparing diuretic amiloride at concentrations ranging between 0.1-0.8 mM inhibited the Na+/Ca2+ exchange in sarcolemmal vesicles isolated from beef heart. The rate of exchange activity was 50% reduced by 0.35 mM amiloride. In spontaneously beating atria isolated from normal and reserpinized guinea-pigs, amiloride produced a concentration-dependent positive inotropic effect and negative chronotropic effect (EC50 = 0.7 mM). Amiloride protected spontaneously beating atria and left atria driven at 1 Hz from digitalis cardiotoxicity assessed in terms of a raised end-diastolic tension. It is suggested that the positive inotropic effect, negative chronotropic effect of amiloride and heart protection against digitalis toxicity are related to the observed inhibition of sarcolemmal Na+/Ca2+ exchange activity.

Histaminergic mechanisms involved in the centrally mediated effects of ouabain.

The role of central histaminergic mechanisms in cardiac glycoside toxicity was evaluated in chloralose-urethane anesthetized cats. Cardiac rhythm, blood pressure and sympathetic nerve activity were monitored during continuous i.v. infusion of ouabain (1 microgram/kg/min). Histamine administered into the fourth cerebral ventricle decreased significantly the dose of ouabain necessary to produce ventricular arrhythmias and fibrillation. Toxicity potentiation was associated with enhancement of sympathetic efferent activity. Dimaprit, a specific H2 receptor agonist, produced a similar, but slightly greater, potentiation of ouabain activity than histamine when similarly administered. Intracerebroventricular (i.c.v.) pretreatment of cats with cimetidine, an H2 receptor antagonist, provided a dose-dependent protective effect against ouabain-induced toxicity. Central administration of diphenhydramine, an H1 receptor antagonist, had no effect on cardiotoxic actions. These results suggest a role for central histaminergic mechanisms in cardiac glycoside toxicity which is mediated through an H2 receptor process. Furthermore, it is suggested that ouabain stimulates central histaminergic neurons which impinge on hypothalamic areas and such excitement results in a centrogenic activation of sympathetic centers which is responsible for cardiac glycoside cardiotoxicity.

Ouabain induced seizures: site of production and response to anticonvulsants.

Ouabain, an inhibitor of Na+ -K" -ATP'ase, has been administered intraventricularly to rats to study the effect of impairment of membrane transport mechanisms on the genesis of seizures. Running and leaping seizures occur rapidly after injection of ouabain in a low volume (10 microliter) when the maximal uptake of ouabain (39.8%) is the hippocampus. Generalized clonic-tonic seizures are induced by higher volume injections (50 microliter) associated with wider distribution of ouabain, including the cerebellum and brainstem. Ouabain was injected into cerebral cortex, caudate nucleus, dorsal hippocampus, fastigeal nucleus, ventrolateral mesencephalic reticular formation and cerebellar cortex. The cerebellar injections produced both running and leaping and generalized clonic-tonic seizures. It is suggested that this results from decreased inhibitory effect of vermal and paravermal Purkinje cells on intra-cerebellar nuclei, which alters cerebellar influence on the reticular formation and the limbic system. Diphenylhydantoin, phenobarbitone, phenacemide, carbamezepine and clonazepam but not ethosuximide are effective against generalized clonic-tonic seizures, suggesting that this is a model for "grand mal" but not "petit mal" seizure mechanisms. It is furthermore suggested that running and leaping are subcortical, probably limbic, seizures that are most relevant as a model for temporal lobe seizures.

[Experimental models for the pharmacologic study of anti-arrhythmia drugs]. / Modèles expérimentaux pour l'étude pharmacologique des anti-arythmiques.

In order to assess the value and therapeutic safety of an antiarrhythmic drug, it must be submitted to multiple tests, which reproduce as faithfully as possible the conditions observed in human pathology. The main experimental approaches are presented here: screening and control tests. For each test, the authors emphasize the various experimental factors limiting their interpretation and allowing the extrapolation to men.

[Animal experiments on the effect of triamterene and amiloride on heart and circulation and the toxicity of digoxin (author's transl)]. / Tierexperimentelle Untersuchungen über den Einfluss von Triamteren und Amilorid auf Herz, Kreislauf nud Toxizität des Digoxins

1. In anesthetized cats 2,4,7-triamino-6-phenyl-pteridine (triamterene) (5 mg/kg i.v.) causes a short-lasting increase in blood pressure, a decrease in heart rate, whereas the contractility (see article) remains unchanged. N-Amidino-3,5-diamino-6-chloropyrazinecarboxamide (amiloride) (5 mg/kg i.v.) causes a fall of blood pressure, an increase in heart rate, and an increase in contractility. 2. In cats the i.v. toxicity of digoxin is diminished by pretreatment with triamterene or amiloride; the arrhythmias and the cardiac arrest are significantly delayed in the course of an infusion of digoxin. In guinea-pigs a pretreatment with triamterene only delays the arrhythmias but not the lethal effect of digoxin or g-strophanthin. 3. In the isolated atria of the guinea-pig triamterene and amiloride (5 mug or 50 mug/ml) lowers the rate of contraction and prolongs the functional refractory period. The force of contraction is increased by triamterene and diminished by amiloride.

The possible relationship between cardiac glycogen levels, ouabain toxicity and the anti-arrhythmic effect of anacardium occidentale

Using the open chest dog with the heart in situ, the influences upon the cardiac glycogen by fasting, anacardium occidentale, and ouabain were studied. Fasting elevated the cardiac glycogen while intravenous infusion of glucose did not increase the cardiac glycogen. Anacardium occidentale elevated the cardiac glycogen after a slow intravenous infusion. The depletion of cardiac glycogen occurred concurrently with the onset of ectopic beats due to ouabain toxicity, and the toxic dose of ouabain was significantly increased in relation to the high cardiac glycogen. The stress of cardiac surgery significantly depleted the glycogen content of the human heart, and this depletion was not related to trauma or the time the patient was on cardio-pulmonary by-pass. It is concluded that: (a) anacardium occidentale protects against ouabain arrhythmias by elevating the cardiac glycogen, (b) there is a critical cardiac glycogen level below which ouabain readily becomes toxic, and (c) surgery on the heart in the human depletes its glycogen stores (AU)

Cardiac effects of sodium selenite.

The classical idea that selenium is toxic to the heart at levels higher than available in a balanced diet is not supported by experimental work. In mice, treatment with sodium selenite increased the LD50 of ouabain and 2,4-dinitrophenol, and increased the tolerance to nitrogen inhalation. Although sodium selenite had no effect on the dog heart with circulation intact, there was a reduction in coronary vascular resistance in the heart-lung preparation. In the isolated ventricular segment perfused with blood, the administration of sodium selenite caused a positive inotropic effect which appeared even after blockade of beta-adrenergic receptors and in segments perfused with a Krebs-bicarbonate solution that was deficient in oxygen. These results cannot be explained merely as the correction of a selenium deficiency but rather as a positive influence of sodium selenite on the heart that has been acutely stressed by oxygen lack, ouabain, or 2,4-dinitrophenol.