Novel Medical Treatments for Hypertension and Related Comorbidities.

PURPOSE OF REVIEW: The purpose of this review is to summarize the most recent data available on advances in development of novel medical treatments for hypertension and related comorbidities. RECENT FINDINGS: Approximately half of all hypertensive patients have not achieved goal blood pressure with current available antihypertensive medications. Recent landmark studies and new hypertension guidelines have called for stricter blood pressure control, creating a need for better strategies for lowering blood pressure. This has led to a shift in focus, in recent years, to the development of combination pills as a means of achieving improved blood pressure control by increasing adherence to prescribed medications along with further research and development of promising novel drugs based on discovery of new molecular targets such as the counter-regulatory renin-angiotensin system. Fixed-dose combination pills and novel treatments based on recently discovered pathogenic mechanisms of hypertension that have demonstrated promising results as treatments for hypertension and related comorbidities will be discussed in this review.

Na+, K+-ATPase inhibition induces neuronal cell death in rat hippocampal slice cultures: Association with GLAST and glial cell abnormalities.

Na+, K+-ATPase is a highly expressed membrane protein. Dysfunction of Na+, K+-ATPase has been implicated in the pathophysiology of several neurodegenerative and psychiatric disorders, however, the underlying mechanism of neuronal cell death resulting from Na+, K+-ATPase dysfunction is poorly understood. Here, we investigated the mechanism of neurotoxicity due to Na+, K+-ATPase inhibition using rat organotypic hippocampal slice cultures. Treatment with ouabain, a Na+, K+-ATPase inhibitor, increased the ratio of propidium iodide-positive cells among NeuN-positive cells in the hippocampal CA1 region, which was prevented by MK-801 and d-AP5, specific blockers of the N-methyl-d-aspartate (NMDA) receptor. EGTA, a Ca2+-chelating agent, also protected neurons from ouabain-induced injury. We observed that astrocytes expressed the glutamate aspartate transporter (GLAST), and ouabain changed the immunoreactive area of GFAP-positive astrocytes as well as GLAST. We also observed that ouabain increased the number of Iba1-positive microglial cells in a time-dependent manner. Furthermore, lithium carbonate, a mood-stabilizing drug, protected hippocampal neurons and reduced disturbances of astrocytes and microglia after ouabain treatment. Notably, lithium carbonate improved ouabain-induced decreases in GLAST intensity in astrocytes. These results suggest that glial cell abnormalities resulting in excessive extracellular concentrations of glutamate contribute to neurotoxicity due to Na+, K+-ATPase dysfunction in the hippocampal CA1 region.

Carnosine prevents necrotic and apoptotic death of rat thymocytes via ouabain-sensitive Na/K-ATPase.

It is known that ouabain, a selective inhibitor of Na/K-ATPase, not only can cause the activation of signal cascades, which regulate the cell viability, but also can cause the accumulation of free radicals, which can evoke the oxidative stress. We have shown that the nanomolar concentrations of ouabain result in the temporary increase in the level of intracellular free radicals, but the millimolar concentration of ouabain induces a stable intracellular accumulation of free radicals in rat thymocytes. The increasing level of free radicals resulting from both low and high concentrations of ouabain can be attenuated by the antioxidant, carnosine. Moreover, the long-term incubation with ouabain leads to the cell death by necrosis and apoptosis. Ouabain-mediated apoptosis and necrosis were also abolished by carnosine.

Interaction of atrial natriuretic peptide and ouabain in the myocardium.

Natriuretic peptides and digitalis-like compounds serve as regulators of homeostasis, including control of volume expansion and blood pressure. The aim of the present study was to explore possible interactions between atrial natriuretic peptide (ANP) and ouabain in the heart. ANP (1 nmol/L) had no effect in papillary muscle preparations from guinea pigs. Ouabain (1 µmol/L) induced positive inotropic effect. The addition of ANP prior to ouabain resulted in a significant decrease in the ouabain-induced positive inotropic effect, manifested as an attenuated increase in twitch maximal upward force slope and resting muscular tension. In addition, ANP caused an increase in Na⁺-K⁺-ATPase activity in heart microsomal preparations. The effect of ouabain on Na⁺-K⁺-ATPase activity was shown in a biphasic manner. Ouabain (0.01-1 nmol/L) had a small but significant increase on pump activity, but higher doses of ouabain inhibited activity. ANP attenuated ouabain-induced Na⁺-K⁺-ATPase activity. Furthermore, ouabain (50 nmol/L) or ANP (10 nmol/L) alone induced Akt activation in cardiomyocytes. However, ANP blocked ouabain-induced Akt activation. These results point to the existence of interactions between ANP and ouabain on Na⁺-K⁺-ATPase signaling and function in the heart, which may be mediated by regulation of Na⁺-K⁺-ATPase activity and (or) signal transduction mechanisms.

Rostafuroxin: an ouabain-inhibitor counteracting specific forms of hypertension.

An innovative approach to the therapy of essential hypertension (EH) and the related complications has been pursued by our group with the aim of defining specific genetic-molecular mechanisms underlying the disease in sub-sets of patients. This approach is anticipated to have a major effect on the clinical practice, diagnostics and development of new drugs able to selectively target such mechanisms. The final achievement is the definition of biomarkers for identifying patients who more likely should benefit for a given therapy both in terms of efficacy and reduction of the adverse reactions. Among many, two mechanisms have been defined and addressed:Both alterations lead to hypertension, organ hypertrophy, negative vascular remodeling and increased cardiovascular risk by affecting the renal Na(+) handling, through the up-regulation of the Na(+)-K(+) pump and the activation of the Src-dependent signal transduction pathway. A novel antihypertensive agent, rostafuroxin (PST2238), has been selected and developed for its ability to correct the renal Na(+)-K(+) pump abnormalities sustained by the mutant adducin and EO-dependent mechanisms. It is endowed with high potency and efficacy in reducing blood pressure (BP) and preventing organ hypertrophy in animal models representative of both adducin and EO mechanisms. At molecular level, in the kidney, rostafuroxin normalizes the enhanced activity of the Na(+)-K(+) pump induced by mutant adducin and antagonizes the EO triggering of the Src-EGFr-dependent signaling pathway leading to renal Na(+)-K(+) pump and ERK phosphorylation and activation. In the vasculature, it normalizes the increased myogenic tone caused by ouabain. A very high safety ratio and the absence of interaction with other mechanisms involved in BP regulation, together with evidence of high tolerability and efficacy in hypertensive patients indicate rostafuroxin as the first example of a new class of antihypertensive agents designed to antagonize adducin and EO-hypertensive mechanisms. A recently concluded Phase II clinical trial (OASIS) has provided the proof of concept that such a compound is effective in the subset of patients where these two mechanisms are at work.

Ouabain inhibitor rostafuroxin attenuates dextromethorphan-induced manic potential.

Dextromethorphan (DM) abuse produces mania-like symptoms in humans. ERK/Akt signaling activation involved in manic potential can be attenuated by the inhibition of ouabain-like cardiac steroids. In this study, increased phosphorylations of ERK/Akt and hyperlocomotion induced by DM (30 mg/kg, i.p./day × 7) were significantly protected by the ouabain inhibitor rostafuroxin (ROSTA), suggesting that DM induces the manic potential. ROSTA significantly attenuated DM-induced protein kinase C δ (PKCδ) phosphorylation, GluN2B (i.e., MDA receptor subunit) expression, and phospho-PKCδ/GluN2B interaction. DM instantly upregulated the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent system. However, DM reduced Nrf2 nuclear translocation, Nrf2 DNA binding activity, γ-glutamylcysteine mRNA expression, and subsequent GSH/GSSG level and enhanced oxidative parameters following 1-h of administration. ROSTA, PKCδ inhibitor rottlerin, and GluN2B inhibitor traxoprodil significantly attenuated DM-induced alterations in Nrf2-related redox parameters and locomotor activity induced by DM in wild-type mice. Importantly, in PKCδ knockout mice, DM failed to alter the above parameters. Further, ROSTA and traxoprodil also failed to enhance PKCδ depletion effect, suggesting that PKCδ is a critical target for the anti-manic potential of ROSTA or GluN2B antagonism. Our results suggest that ROSTA inhibits DM-induced manic potential by attenuating ERK/Akt activation, GluN2B/PKCδ signalings, and Nrf2-dependent system.

Inhibition of endogenous ouabain by atrial natriuretic peptide is a guanylyl cyclase independent effect.

BACKGROUND: Endogenous ouabain (EO) and atrial natriuretic peptide (ANP) are important in regulation of sodium and fluid balance. There is indirect evidence that ANP may be involved in the regulation of endogenous cardenolides. METHODS: H295R are human adrenocortical cells known to release EO. Cells were treated with ANP at physiologic concentrations or vehicle (0.1% DMSO), with or without guanylyl cyclase inhibitor 1,2,4 oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cyclic guanosine monophosphate (cGMP), the intracellular second messenger of ANP, was measured by a chemiluminescent immunoassay and EO was measured by radioimmunoassay of C18 extracted samples. RESULTS: EO secretion is inhibited by ANP treatment, with the most prolonged inhibition (90 min vs ≤ 60 min) occurring at physiologic ANP concentrations (50 pg/mL). Inhibition of guanylyl cyclase with ODQ, also reduces EO secretion. The inhibitory effects on EO release in response to cotreatment with ANP and ODQ appeared to be additive. CONCLUSIONS: ANP inhibits basal EO secretion, and it is unlikely that this is mediated through ANP-A or ANP-B receptors (the most common natriuretic peptide receptors) or their cGMP second messenger; the underlying mechanisms involved are not revealed in the current studies. The role of ANP in the control of EO synthesis and secretion in vivo requires further investigation.

Widespread occurrence in frogs and toads of skin compounds interacting with the ouabain site of Na+, K+-ATPase.

Amphibians of the family Bufonidae contain high levels of skin compounds that both inhibit Na+- and K+-dependent adenosinetriphosphatase and antagonize the binding of ouabain to the enzyme. In species of Bufo and Atelopus, these compounds are relatively nonpolar bufodienolides, whereas Dendrophryniscus and Melanophryniscus contain more polar compounds of unknown structure. Skin extracts from 30 of 48 species of frogs representing an additional eight families contained relatively low levels of compounds that inhibit binding of ouabain to Na+,K+-adenosinetriphosphatase. The widespread occurrence of low levels of inhibitory compounds is consonant with the role for these compounds as physiological regulators of Na+,K+-adenosinetriphosphatase in amphibian skin; high levels in the Bufonidae probably also serve as a defense against some predators.

Reduction in endogenous cardiac steroids protects the brain from oxidative stress in a mouse model of mania induced by amphetamine.

OBJECTIVES: Bipolar disorder (BD) is a severe mental illness characterized by episodes of mania and depression. Numerous studies have implicated the involvement of endogenous cardiac steroids (CS), and their receptor, Na+, K+ -ATPase, in BD. The aim of the present study was to examine the role of brain oxidative stress in the CS-induced behavioral effects in mice. METHODS: Amphetamine (AMPH)-induced hyperactivity, assessed in the open-field test, served as a model for manic-like behavior in mice. A reduction in brain CS was obtained by specific and sensitive anti-ouabain antibodies. The level of oxidative stress was tested in the hippocampus and frontal cortex by measuring the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as the levels of antioxidant non-protein thiols (NPSH) and oxidative damage biomarkers thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC). RESULTS: AMPH administration resulted in a marked hyperactivity and increased oxidative stress, as manifested by increased SOD activity, decreased activities of CAT and GPx, reduced levels of NPSH and increased levels of TBARS and PC. The administration of anti-ouabain antibodies, which reduced the AMPH-induced hyperactivity, protected against the concomitant oxidative stress in the brain. CONCLUSIONS: Our results demonstrate that oxidative stress participates in the effects of endogenous CS on manic-like behavior induced by AMPH. These finding support the notion that CS and oxidative stress may be associated with the pathophysiology of mania and BD.

Diltiazem, a L-type calcium channel antagonist, suppresses ouabain-enhanced dopamine efflux by 1-methyl-4-phenylpyridinium ion (MPP+) in rat striatum.

The present study was examined whether diltiazem, a L-type Ca2+ channel antagonist, could suppresses 1 methyl-4-phenylpyridinium ion (MPP+)-induced dopamine (DA) in extracellular fluid of rat striatum. Ouabain (100 microM; 100 microM or 100 pmol/microl per min) significantly enhanced the level of DA by MPP+. However, in the presence of diltiazem (100 microM) significantly suppressed the level of DA release by ouabain and MPP+. These results suggest that diltiazem suppresses Ca2+ -dependent release of DA by ouabain-induced Ca2+ overload.

Ouabain-specific antibodies: immunochemical properties and reversal of Na + , K + -activated adenosine triphosphatase inhibition.

Antibodies with high affinity and specificity for the cardiac glycoside ouabain were raised in rabbits. The antigen used was a conjugate of ouabain linked through its rhamnose moiety to terminal alpha-amino groups of poly D,L alanyl-human serum albumin. Ouabain-specific antibodies were present as early as 3 wk, and rose steadily in titer over the initial 20-33 wk of immunization. Levels as high as 6.5 mg specific immunoglobulin per ml antiserum were reached in one rabbit at the end of 45 wk. The average intrinsic association constants for ouabain were 1.3 x 10(9) M(-1) and 1.6 x 10(9) M(-1) in antisera studied in detail, and there was evidence of restricted heterogeneity of binding site affinities. A high degree of specificity was demonstrated. Significant cross-reactivity occurred only with other cardioactive steroid compounds such as acetyl strophanthidin, digoxin, and digitoxin, while endogenous steroids did not cross-react even when present in 1000-fold excess. A rapid and convenient radioimmunoassay procedure for plasma or urine ouabain concentrations was developed using these antibodies. Competition between ouabain-(3)H tracer and unlabeled ouabain for specific antibody binding sites allowed the measurement of ouabain concentrations as low as 0.1 ng/ml or less without need for extraction procedures. The high association constants observed in these studies permit antibody reversal of established myocardial effects of ouabain. Both blockade and reversal of ouabain inhibition of canine myocardial microsomal Na(+), K(+)-activated ATPase by antibody were documented, suggesting a possible mechanism for reversal of cellular effects.

Molecular mechanisms linking sodium to hypertension: report of a symposium.

There is abundant clinical and epidemiologic data linking excess body sodium with hypertension. The mechanism(s) at the molecular level to explain this relationship are unknown. Recent studies by multiple investigators, have identified several ion transport mechanisms in the vascular wall that interact to control vascular tone and contractility. These new data include 1) biochemical, pharmacologic, and molecule structural studies, 2) experiments in transgenic and knockout mice, and 3) results in clinical hypertension. The overall results provide compelling evidence for the concept that salt-dependent hypertension involves the secretion of endogenous ouabain (EO), an adrenal steroid synthesized with the same initial steps as aldosterone and secreted by the zona glomerulosa. Circulating EO inhibits arterial smooth muscle Na+ pumps with alpha 2 subunits. These are functionally coupled to the type 1 Na/Ca exchanger (NCX1). Thus when a2 Na pumps are inhibited in arterial smooth muscle, the resulting subplasma membrane increase in Na+ concentration triggers, via NCX1 Ca2+ entry, a rise in cytosolic Ca2+ concentration and increased myogenic tone and contractility. The ultimate result is a rise in peripheral vascular resistance-the hemodynamic hallmark of hypertension. The elucidation of this pathway has facilitated the development of pharmacologic agents that have therapeutic potential for hypertension and other cardiovascular diseases. These include agents that compete with EO for binding to the Na+ pump and inhibitors of NCX1.

Effect of salt acclimation on digitalis-like compounds in the toad.

Digitalis-like compounds (DLC) were shown to be a normal constituent of the skin and plasma of toads. In order to assess the possible physiological role of these compounds in the toad, their levels were determined in the brain, plasma and skin following acclimation in different NaCl solutions. We demonstrate that an increase in salt concentrations in the animal medium from 0 to 1.2% decreased the levels of DLC in the brain by 50% without altering significantly its levels in the plasma and skin. An increase in medium salt concentration to 1.5% resulted in a 50% increase of DLC levels in the skin without changing its levels in the plasma or brain. These results suggest that skin and brain DLC may participate in the long-term salt and water homeostasis in the toad, while the plasma compound either participates in the short-term regulations of salt and water homeostasis or have some other, unknown, function.

Reduced glutathione protect cells from ouabain toxicity.

It is widely accepted that a prolonged ouabain blockade of the Na(+),K(+)-ATPase makes cells detach from each other and from the substrate, leading to their death and that cellular resistance to ouabain is due to the presence of isoforms of Na(+),K(+)-ATPase with low affinity to this glycoside. In the present work the effect of reduced glutathione in the response of two types of renal cells to ouabain: MDCK, a ouabain-sensitive cell line and Ma104, a ouabain-resistant one, was studied. Glutathione protected MDCK cells from ouabain toxicity and inhibition of glutathione synthesis by L-buthionine-S,R-sulfoximine sensitized Ma104 cells to ouabain. As glutathione is involved with multidrug resistance (MDR) in cells expressing the multidrug resistance-related protein MRP1 and as Ma104 cells have a MDR phenotype, it was investigated whether Ma104 cells express this protein. The expression of the MRP1-mRNA in Ma104 cells was detected by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay, and the protein was detected by Western blotting and immunofluorescence. Treatment of Ma104 cells with ouabain increased MRP1-mRNA expression and altered the localization of MRP1 in these cells. Our results suggest that some cells may have mechanisms to protect themselves from ouabain toxicity and that MRP1 may have a role in controlling the toxic effects of ouabain.

Interaction of external alkali metal ions with the Na-K pump of human erythrocytes: a comparison of their effects on activation of the pump and on the rate of ouabain binding.

The effects of external alkali metal ions on the rate of ouabain binding and on the rate of the Na-K pump were examined in human red blood cells. In Na-containing solutions, K, Cs, and Li decreased the rate of ouabain binding. For K and Cs, the kinetics of this effect were similar to those for their activation of the pump. In Na-free (choline-substituted) solutions the rate of ouabain binding was decreased by K whereas it was promoted by Cs and Li. External Na increased the rate of ouabain binding whether or not external K was present, and the kinetics of this effect were not the same as those for inhibition of the pump by Na. These findings are interpreted to mean that not only do the cations affect ouabain binding at the external loading sites on the pump from which ions are translocated inward, but that there are additional sites on the external aspect of the pump at which cations can promote ouabain binding, and that these sites can be occupied by Li, Na, and Cs. It is postulated that these latter sites are those from which Na is discharged after outward translocation by the pump.

The kinetics of ouabain inhibition and the partition of rubidium influx in human red blood cells.

IN THE DEVELOPMENT OF OUABAIN INHIBITION OF RUBIDIUM INFLUX IN HUMAN RED BLOOD CELLS A TIME LAG CAN BE DETECTED WHICH IS A FUNCTION OF AT LEAST THREE VARIABLES: the concentrations of external sodium, rubidium, and ouabain. The inhibition is antagonized by rubidium and favored by sodium. Similar considerations could be applied to the binding of ouabain to membrane sites. The total influx of rubidium as a function of external rubidium concentration can be separated into two components: (a) a linear uptake not affected by external sodium or ouabain and not requiring an energy supply, and (b) a saturable component. The latter component, on the basis of the different effects of the aforementioned factors, can be divided into three fractions. The first is ouabain-sensitive, inhibited by external sodium at low rubidium, and requires an energy supply; this represents about 70-80% of the total uptake and is related to the active sodium extrusion mechanism. The second is ouabain-insensitive, activated by external sodium over the entire range of rubidium concentrations studied, and dependent on internal ATP; this represents about 15% of the total influx; it could be coupled to an active sodium extrusion or belong to a rubidium-potassium exchange. The third, which can be called residual influx, is ouabain-insensitive, unaffected by external sodium, and independent of internal ATP; this represents about 10-20% of the total influx.

Ouabain antagonists as antihypertensive agents.

The evidence that high levels of endogenous ouabain (EO), a closely related isomer of ouabain, are implicated in human hypertension and cardiac hypertrophy and failure stimulated the pharmacological research for developing novel anti-hypertensive agents active as ouabain antagonists. The pathogenetic mechanisms through which increased EO levels affect cardiovascular system involve the modulation of Na-K ATPase, the key enzyme responsible for renal tubular sodium reabsorption and the activation of signalling transduction pathways implicated in growth-related gene transcription. By studying both genetic and experimental rat models of hypertension and comparing them with humans, our group has demonstrated that elevated levels of circulating EO and the genetic polymorphism of the cytoskeletal protein adducin associate with hypertension and high renal Na-K pump activity. Ouabain itself induces hypertension and up-regulates renal Na-K pump when chronically infused at low doses into rats (OS). In renal cultured cells, either incubated for several days with nanomolar concentrations of ouabain or transfected with the hypertensive adducin genetic variant, the Na-K pump results enhanced. Moreover, both EO and adducin polymorphism affect cardiac complications associated to hypertension, the former through the activation of a signalling transduction pathway. As a consequence, a compound able to interact with the cellular and molecular alterations, sustained by EO or mutated adducin, may represent the suitable treatment for those patients in whom these mechanisms are at work. A new antihypertensive compound, PST 2238, that selectively antagonises the pressor effect and the alteration of renal Na-K pump, sustained both by ouabain and adducin polymorphism, is described. A selective ability of PST 2238 to antagonise the ouabain-induced organ hypertrophy is also documented. The specificity of PST 2238 mechanism of action is supported by the absence of interactions with receptors or hormones involved in blood pressure regulation and by the lack of diuretic activity and diuretic-associated side effects. It is concluded that this compound could be useful for the treatment of those forms of essential hypertension in which renal Na handling alterations and cardiac complications are associated with either increased EO levels and/or adducin polymorphism.

OASIS-HT: design of a pharmacogenomic dose-finding study.

Experimental evidence and observations in humans strongly support an interactive role of mutated alpha-adducin, sodium (Na(+))/potassium (K(+))-adenosine triphosphatase (ATPase) activity and endogenous ouabain in Na(+) homeostasis and the pathogenesis of hypertension. The Ouabain and Adducin for Specific Intervention on Sodium in HyperTension (OASIS-HT) trial is an early Phase II dose-finding study, which will be conducted across 39 European centers. Following a run-in period of 4 weeks without treatment, eligible patients will be randomized to one of five oral doses of rostafuroxin consisting of 0.05, 0.15, 0.5, 1.5, or 5.0 mg/day. Each dose will be compared to a placebo in a double-blind crossover experiment with balanced randomization. Treatment will be initiated with the active drug and continued with placebo or vice versa. Each double-blind period will last 5 weeks. The primary end point is the reduction in systolic blood pressure defined as the average of three clinic readings with the patient in the sitting position. Secondary end points include the reduction in diastolic blood pressure on clinic measurement, the decrease in the 24-h blood pressure, and the incidence of end points related to safety. Secondary objectives are to investigate the dependence of the blood pressure-lowering activity on the plasma concentration of endogenous ouabain and the genetic variation of the enzymes involved in the metabolism of this hormone, and the adducin cytoskeleton proteins. Eligible patients will have Grade I or II systolic hypertension without associated conditions and no more than two additional risk factors. In conclusion, OASIS-HT is a combination of five concurrent crossover studies, one for each dose of rostafuroxin to be studied. To our knowledge, OASIS-HT is the first Phase II dose-finding study in which a genetic hypothesis is driving primary and secondary end points.

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.

Effects of resveratrol on delayed afterdepolarization and triggered activity induced by ouabain in guinea pig papillary muscles.

The purpose of this study was to investigate the effects of resveratrol on delayed afterdepolarization (DAD) and triggered activity (TA) induced by ouabain in guinea pig papillary muscles and the underlying mechanism. Action potentials were recorded using intracellular microelectrode technique. The results obtained are as follows: (1) DAD and TA induced by ouabain (1 micromol/L) were inhibited by pretreatment with resveratrol (30, 60, and 120 micromol/L) in a concentration-dependent manner; (2) Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), a nitric oxide (NO) synthase inhibitor, failed to abolish the above effect of resveratrol (60 micromol/L ); (3) 5 micromol/L 17beta-estradiol (E(2)) or 30 micromol/L resveratrol had no effects on DAD and TA, however, resveratrol combined with E(2) at the same doses exerted significant inhibitory effects on DAD and TA; (4) Pretreatment with tamoxifen (TAM, 10 micromol/L), an inhibitor of estrogen receptor, also did not blocked the effects of resveratrol (60 micromol/L) on DAD and TA induced by ouabain. All these results indicated that resveratrol exerted an inhibitory effects on DAD and TA induced by ouabain, possibly by reducing calcium influx, which might not be mediated by NO and estrogen receptor. The antiarrhythmic effects of resveratrol may contribute to its cardioprotective action.