Palytoxin binds to and inhibits kidney and erythrocyte Na+, K+-ATPase

Summary Hog kidney Na⁺, K⁺-ATPase, purified to the microsomal stage and activated with detergent, binds palytoxin, as shown by the nearly complete competition of the toxin with ³H-ouabain. The K _i-values of palytoxin, but not of ouabain, depend on the protein concentration; this indicates additional binding sites for the toxin on kidney membranes. — Palytoxin inhibits the enzymatic activity of the detergent-activated preparation nearly completely (IC₅₀ 8·10^–7 mol/l). Inhibition of ATPase activity and of ouabain binding are promoted by borate, a known activator of palytoxin. — Palytoxin also inhibits the Na⁺, K⁺-ATPase of erythrocyte ghosts in the same dose range.The data are discussed in context with the hypothesis (Chhatwal et al. 1983) that palytoxin raises the cellular permeability by altering the state of Na⁺, K⁺-ATPase or its environment.Part of the thesis (Dr. rer. nat.) of H. Böttinger     

Antibacterial activity of six novel peptides from Tityus discrepans scorpion venom. A fluorescent probe study of microbial membrane Na permeability changes

Six novel peptides (named bactridines) were isolated from Tityus discrepans scorpion venom. From mass spectrometry molecular masses were 6916, 7362, 7226, 7011, 7101 and 7173 Da (bactridines 1–6). Bactridines 1 and 2 were sequenced by Edman degradation. The sequences and in silico analysis, indicated that they are positively charged polypeptides comprised of 61 and 64 amino acids (AA), respectively, bactridine 1 and bactridine 2 containing 4 disulfide bridges. Bactridine 1 was only toxic to cockroaches and crabs, and bactridine 2–6 were only toxic to mice. Bactridine 1 has a 78% sequence identity with ardiscretin. Ardisctretin is an insect specific sodium toxin which also produces a small depolarization and induces repetitive firing in squid axons resembling those of DDT [1,10(pchlorobenzyl) 2-trichloretane] in its ability to slow down action potential, to induce repetitive firing. Measured as the minimal inhibitory concentration, bactridines had high antibacterial activity against a wide range of Gram positive and Gram negative bacteria. Complete bacterial growth inhibition occurred at concentrations from 20 to 80 μM depending on the bacteria and peptide tested. Effects on membrane Na⁺ permeability induced by bactridines were observed on Yersinia enterocolitica loaded with 1 μM CoroNa™ Red. CoroNa™ Red fluorescence leakage from bacteria was observed after exposure to 0.3 μM of any bactridine tested, indicating that they modified Na⁺ membrane permeability. This effect was blocked by 10 μM amiloride and by 25 μM mibefradil drugs that affect Na⁺ and Ca²⁺ channels respectively. We found no evidence of changes of K⁺ or Ca²⁺ concentrations neither inside nor outside the bacteria in experiments using the fluorescent dyes Fluo 4AM (10 μM) and PBFI (20 μM).     

Effects of monovalent cations on cardiac Na+, K+-ATPase activity and on contractile force

Summary The relationship between Na⁺, K⁺-ATPase inhibition by monovalent cations and their inotropic effect was studied in guinea pig hearts. The activity of partially purified cardiac enzyme was assayed in the presence of 5.8 mM KCl and either 20 or 150 mM NaCl. Rb⁺ and Tl⁺ inhibited Na⁺, K⁺-ATPase activity, the magnitude of the inhibition by these cations being greater in the assay media containing lower Na⁺ concentrations. Tl⁺ produced a dose-dependent inhibition of Na⁺, K⁺-ATPase activity in the presence of 20 mM Na⁺ and 75 mM K⁺, a cationic condition similar to that of intracellular fluid. Other monovalent cations such as K⁺, Cs⁺, NH₄ ⁺, Na⁺ or Li⁺ produced essentially no effect on the Na⁺, K⁺-ATPase activity or slightly stimulated it. In left atrial strips stimulated with field electrodes and bathed in Krebs-Henseleit solution (5.8 mM K⁺ and 145 mM Na⁺), addition of Cs⁺ failed to alter the isometric contractile force significantly. NH₄ ⁺ and K⁺ caused a transient positive inotropic effect which was partially blocked by propranolol. The positive inotropic response to K⁺ was followed by a negative inotropic response. Rb⁺ produced a sustained, dose-dependent inotropic response reaching a plateau at 1–2 min, whereas Tl⁺ produced a dose-dependent positive inotropic effect which developed slowly over a 30-min period. The positive inotropic effects produced by Rb⁺ and Tl⁺ were insensitive to propranolol pretreatment. Concentrations of Tl⁺ and cardiac glycosides which produce similar inotropic effects appear to cause the same degree of Na⁺-pump inhibition. The onset of the positive inotropic response to Rb⁺ or Tl⁺ was not dependent on the number of contractions which is in contrast to the cardiac glycoside-induced inotropic response. Substitution of 20 mM LiCl for an equimolar amount of NaCl in Krebs-Henseleit solution produced a significantly greater inotropic response than that observed when sucrose was substituted for NaCl. It appears that, among monovalent cations, only sodium pump inhibitors produce a sustained positive inotropic response.     

The saluretic effect of the thiazide diuretic bemetizide in relation to the glomerular filtration rate

The effect of the thiazide diuretic, bemetizide, on the excretion of Na⁺, K⁺, Cl^–, Ca²⁺, and Mg²⁺ in relation to the glomerular filtration rate (GFR) was studied in 17 subjects whose creatinine clearances ranged from 133 to 5 ml·min^–1.After a 2-day fluid and salt balanced control period, 25 mg bemetizide given orally induced natriuresis and kaliuresis which lasted for 24 h and were proportional to the GFR of the patients. The ratio of bemetizide-induced K⁺/Na⁺ excretion was always 0.17 irrespective of individual GFR. In renal failure, bemetizide increased the fractional Na⁺ excretion from 3% to about 10%. Kaliuresis was associated with magnesiuria, whereas bemetizide-induced calciuresis was insignificant. The thiazide reversibly lowered GFR in all subjects.     

Development of an in vitro drug screening system for Mycobacterium leprae based on the determination of the intrabacterial sodium to potassium ratio of individual bacterial organisms

In vitro drug effects on Mycobacterium leprae (M. leprae) in a cell-free system have been monitored by mass spectrometric determination of the ratio of the intrabacterial concentrations of the sodium and potassium ions (Na⁺, K⁺ ratio) of a limited number of individual bacteria per sample. From the drug-induced increase of the median values of the distributions of the Na⁺, K⁺ ratio, information on the concentration and time dependence of drug effects as well as on antagonistic or synergistic interactions of drugs has been obtained. Moreover, absolute values for the percentage of killed bacteria (% kill) have been derived from the distribution of the Na⁺, K⁺ ratios within a bacterial population. For this, the limiting value of the Na⁺, K⁺ ratio (up to which bacteria are viable) —which had been determined as 0.45 for cultivable bacteria — has been presumed to be valid also for M. leprae. Highest killing rates have been observed for fusidic acid and clarithromycin, followed by rifabutine, rifampin, and clofazimine. Minocycline and dapsone have shown only moderate killing effects and isoniazid and — probably due to the restricted metabolism of M. leprae in a cell-free medium — ofloxacin have been completely inactive. Strong ofloxacin effects, however, have been observed for cultivable mycobacteria and intracellular M. leprae phagocytized by a murine macrophage cell line.     

Biochemical basis for the low sensitivity of the rat heart to digitalis

Summary The concentration of cardiac glycosides to produce positive inotropic effects in the rat heart is markedly higher than that in other species. Such a low digitalis sensitivity of the rat heat is attributed to the low affinity of cardiac Na⁺, K⁺-ATPase for digitalis in this species. In the present study the biochemical cause which is responsible for the formation of the unstable complex between the glycosides and Na⁺, K⁺-ATPase or positive inotropic, receptor in the rat heart was examined using Na⁺, K⁺-ATPase preparations obtained from rat hearts, guinea-pig hearts and rat brains as well as isolated, electrically stimulated atrial preparations obtained from these animals. Monensin, which alters transmembrane Na⁺ movements without interacting with the cardiotonic sites on Na⁺, K⁺-ATPase, had equivalent potencies in guinea-pig and rat hearts. Cassaine, which lacks a lactone ring but interacts with cardiotonic sites on Na⁺, K⁺-ATPase, increased the force of contraction in guinea-pig hearts at low, but in rat hearts only at high, concentrations. AY-22,241 (Actodigin) and prednisolone-3,20-bisguanylhydrazone (PBGH) bind to cardiotonic sites on Na⁺, K⁺-ATPase and had a similar spectrum as cassaine in these two species. Actodigin has an altered lactone ring resulting in a marked reduction of the inotropic potency, and PBGH is devoid of this structure. With the latter agent, the rabbit was as insensitive as the rat, although both rabbit and guinea-pig are equally sensitive to digitalis. K⁺ delayed the development of the positive inotropic action of ouabain with a minimal effect on the plateau response in guinea-pig hearts. In rat hearts, however, K⁺ markedly lowered the plateau response without affecting the time course of the response. These results indicate that the low sensitivity of the rat heart to digitalis is due to a difference in the glycoside binding sites on Na⁺, K⁺-ATPase; but the difference cannot be explained by the lack of a lactone ring complementary binding sites. The difference seems to result from the absence of lipid barrier which regulates the rate of release of cardiac glycosides from their binding sites on Na⁺, K⁺-ATPase.This work was supported by U.S. Public Health Service grant, HL-16052 and by the Michigan Heart Association     

Effects of glibenclamide and tetracaine on 86Rb+ fluxes in mouse pancreatic β-cells

Summary Potassium transport was measured in -cell-rich islets from ob/ob-mice using the K⁺-analogue ⁸⁶Rb⁺. Both tetracaine (0.1 mM) and glibenclamide (0.1 M) reduced the oubain-resistant ⁸⁶Rb⁺ influx but did not significantly affect the oubain-sensitive portion (Na⁺/K⁺ pump). Tetracaine (0.5–1 mM) or glibenclamide (0.2 mM) decreased the ⁸⁶Rb⁺ equilibrium content and glibenclamide (1 M) transiently reduced the ⁸⁶Rb⁺ efflux rate but 0.1 mM tetracaine had only a slight effect on this flux rate. The results suggest that a change in ouabain-resistant (passive) K⁺ fluxes, but not the Na⁺/K⁺ pump, is involved in stimulation of insulin secretion by glibenclamide and tetracaine. Both drugs may exert similar effects on the -cell plasma membrane.     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

The interaction of canrenone with the Na+,K+ pump in human red blood cells

Summary Canrenone inhibits 30–40% of ouabain-sensitive Na⁺ efflux in human red cells. Half-maximal inhibition was obtained with a canrenone concentration=86±37 mol/l (mean±SD of 13 experiments). The partial inhibition of the Na⁺,K⁺ pump appears to be mediated at the digitalis receptor site with an apparent dissociation constant (K _C)=200±130 mol/l (mean±SD). Further evidence suggesting that canrenone is a partial agonist at the digitalis receptor site was obtained by the observation that it decreases the apparent affinity of the Na⁺,K⁺ pump for external K⁺. However, in contrast to ouabain, canrenone decreases the apparent pump affinity for internal Na⁺.Our results show that, at physiological cell Na⁺ levels canrenone is able to enhance the inhibition of the Na⁺,K⁺ pump by low doses of ouabain. Conversely, in cells treated with high concentrations of cardiac glycosides (in which cell Na⁺ content increases), canrenone is able to restimulate the blocked pumps.     

柴胡皂甙和甘草甜素抑制Na+,K+-ATP酶活性的构效关系

研究在离体条件下各种单体柴胡皂甙和甘草甜素抑制Na⁺,K⁺-ATP酶活性的构效关系。实验结果表明,各种柴胡皂甙抑制Na⁺,K⁺-ATP酶活性的作用强度依次为:b₁>d>b₂>b₄>a>b₃>e>c。柴胡皂甙化学结构中的C₂₃-OH,C₁₆-OH及C₁₁和C₁₃的共轭双烯可能对其抑制活性起重要作用。甘草甜素(GL),甘草次酸(GA)和生胃酮(18-β-甘草次酸半琥珀酸双钠盐,CX)抑制Na⁺,K⁺-ATP酶活性的作用强度依次为GA≥CX>GL。研究还证明,柴胡皂甙d对Na⁺,K⁺-ATP酶的抑制为非竟争性抑制。     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

Study of antidepressant-like activity of an enriched phloroglucinol fraction obtained from Hypericum caprifoliatum

Context: Hypericum caprifoliatum Cham &; Schlecht (Guttiferae) extracts have a potential antidepressant-like effect in rodents. However, the molecular mechanisms by which these extracts exert this effect remain unclear.

Objective: This study evaluated the effect of HC1, a fraction obtained from H. caprifoliatum enriched in phloroglucinol derivatives, on the Na⁺, K⁺ ATPase activity in mouse brain and verified the influence of veratrine on the effect of HC1 in the forced swimming test (FST).

Materials and methods: Veratrine (0.06?mg/kg) and HC1 (360?mg/kg) were given alone or combined i.p. 60 and p.o. 30?min, respectively, before FST. The effect of single and repeated administration (once a day for 3 consecutive days) of HC1 (360?mg/kg) on Na⁺, K⁺ ATPase activity was evaluated ex vivo in the cerebral cortex and hippocampus of mice subjected or not to FST.

Results: HC1 reduced the immobility time (103.15?±?18.67?s), when compared to the control group (183.6?±?9.51?s). This effect was prevented by veratrine (151.75?±?22.19?s). Mice repeatedly treated with HC1 presented a significant increase in Na⁺, K⁺ ATPase activity, both in cerebral cortex (46?±?2.41?nmol Pi/min?mg protein) and hippocampus (49.83?±?2.31?nmol Pi/min?mg protein), in relation to the respective controls (30?±?2.66 and 29.83?±?2.31?nmol Pi/min?mg protein respectively).

Discussion and conclusion: The HC1 antidepressant-like effect on FST might be related to its capacity to inhibit Na⁺?influx. HC1 increases hippocampal and cortical Na⁺, K⁺ ATPase activities possibly through long-term regulatory mechanisms.     

Inhibition of human colonic (Na++K+)-ATPase by arachidonic and linoleic acid

Summary The sodium pump, (Na⁺+K⁺)-ATPase, which is involved in the transport of cations and water movement by the colonic mucosa, may be decreased in various diarrhoeal states. In this study, we have measured ³H-ouabain binding and (Na⁺+K⁺)-ATPase activity in human colonic biopsy homogenates and the influence of various inflammatory and antiinflammatory compounds on these parameters. ³H-ouabain binds to one site of high affinity (K _D 1.9±0.2×10^–9 mol/l) with a maximal binding capacity of 7.5±0.8×10¹⁴ binding sites/g protein. Both arachidonic and linoleic acid inhibited (Na⁺+K⁺)-ATPase activity (IC₅₀ arachidonic acid: 7.5×10^–5 mol/l, linoleic acid: 6.5×10^–5 mol/l) and Mg²⁺-ATPase activity (IC₅₀ arachidonic acid: 9×10^–5 mol/l, linoleic acid: 4×10^–5 mol/l). Arachidonic acid inhibited ³H-ouabain binding, (IC₅₀ 3.2×10^–5 mol/l). The following antiinflammatory compounds, at concentrations up to 1×10^–3 mol/l, did not influence ATPase activity directly nor reverse the arachidonic acid-induced inhibition: indomethacin (cyclooxygenase inhibitor), nordihydroguaretic acid (lipoxygenase inhibitor), sulphasalazine and its metabolites: 5-aminosalicylic acid, N-acetylaminosalicylic acid and sulphapyridine.These results indicate that human colonic (Na⁺+K⁺)-ATPase is inhibited by the prostanoid precursors, arachidonic and linoleic acid. From a therapeutic point of view (effect on colonic (Na⁺+K⁺)-ATPase and perhaps diarrhoea), the suppression of the production of these prostanoid precursors by drugs may, therefore, be beneficial in the treatment of inflammatory bowel disease.Supported by DFG (Er65/4-4)     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Role of Na+-K+ ATPase enzyme in vascular response of goat ruminal artery

Objective:

To study the role of Na⁺, K⁺- ATPase enzyme in the vascular response of goat ruminal artery.

Materials and Methods:

Ruminal artery was obtained in chilled aerated modified Krebs-Henseleit solution (KHS) from a local slaughterhouse and transported in ice for further processing. The endothelium intact arterial ring was mounted in a thermostatically controlled (37 ± 0.5°C) organ bath containing 20 ml of modified KHS (pH 7.4) bubbled with oxygen (95%) and CO₂ (5%) under 2g tension. An equilibration of 90 min was allowed before addition of drugs into the bath. The responses were recorded isometrically in an automatic organ bath connected to PowerLab data acquisition system. In order to examine intact functional endothelium, ACh (10 μM) was added on the 5-HT (1.0 μM) - induced sustained contractile response. Similarly, functional characterization of Na⁺, K⁺-ATPase activity was done by K⁺-induced relaxation (10 μM-10 mM) in the absence and presence of ouabain (0.1 μM/ 0.1 mM), digoxin (0.1 μM) and barium (30 μM).

Results:

ACh (10⁻⁵ M) did not produce any relaxing effect on 5-HT-induced sustained contractile response suggesting that vascular endothelium has no significant influence on the activation of sodium pump by extracellular K⁺ in ruminal artery. Low concentration of Ba²⁺ (30 μM) (IC₅₀: 0.479 mM) inhibited K⁺-induced relaxation suggesting K_ir (inward rectifier) channel in part had role in K⁺-induced vasodilatation in ruminal artery. Vasorelaxant effect of KCl (10 μM-10 mM) in K⁺-free medium is also blocked by ouabain (0.1 μM and 0.1 mM) (IC₅₀:0.398 mM and IC₃₅: 1.36 mM), but not by digoxin (0.1 μM) (IC₅₀ 0.234 mM) suggesting that ouabain sensitive Na⁺, K⁺-ATPase isoform is present in the ruminal artery.

Conclusion:

In the goat ruminal artery functional regulation of sodium pump is partly mediated by K⁺ channel and ouabain sensitive Na⁺, K⁺ ATPase.     

Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na⁺,K⁺-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na⁺,K⁺-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na⁺,K⁺-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (k_obs). From the representation of k_obs versus PLTX concentration, the kinetic equilibrium dissociation constant (K_D) for the PLTX-Na⁺,K⁺-ATPase association can be calculated. The value of this constant is K_D = 6.38 × 10⁻⁷ ± 6.67 × 10⁻⁸ M PLTX. In this way the PLTX-Na⁺,K⁺-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.     

Interaction of sanguinarine and its dihydroderivative with the Na/K-ATPase. Complex view on the old problem

The effects of sanguinarine (SG) and its metabolite dihydrosanguinarine (DHSG) on Na⁺/K⁺-ATPase were investigated using fluorescence spectroscopy. The results showed that the enzyme in E1 conformation can bind both charged and neutral (pseudobase) forms of SG with a K_D = 7.2 ± 2.0 μM or 11.7 ± 0.9 μM, while the enzyme in E2 conformation binds only the charged form of SG with a K_D = 4.7 ± 1.1 μM. Fluorescence quenching experiments suggest that the binding site in E1 conformation is located on the surface of the enzyme for both forms but the binding site in E2 conformation is protected from the solvent. We found no evidence for interaction of Na⁺/K⁺-ATPase and DHSG. This implies that any in vivo effect of SG attributable to inhibition of Na⁺/K⁺-ATPase can be considered only prior to SG → DHSG transformation in the gastro-intestinal tract and/or blood. Hence, Na⁺/K⁺-ATPase inhibition will be effective in SG topical application but its duration will be very limited in SG oral or parenteral administration.     

Die Haftung verschiedener Cardenolide am Papillarmuskel und einer mikrosomalen ATPase des Meerschweinchenherzens

Summary In experiments on isolated electrically stimulated guinea pig papillary muscles and on isolated cardiac Na⁺-K⁺-activated ATPase preparations the action and the reversibility of action of 3 different cardenolides-digitoxin, k-strophanthidin and strophanthidin-3-bromoacetate (SBA) (supposed to be an irreversible inhibitor of the transport ATPase)-were studied.The equieffective concentrations for maximum positive inotropic effects (around 90%) were 2×10^–6, 2×10^–5 and 4×10^–5 M, respectively. In washout experiments the positive inotropic action of all these substances was found to be completely reversible: the rates of decline of the positive inotropic effects were about 2.7%/min with digitoxin, 24%/min with strophanthidin and 22%/min respectivety 5.7%/0/min (two components) with SBA.The equieffective concentrations for maximum inhibition (90–95%) of the Na⁺-K⁺-activated ATPase by digitoxin, strophanthidin and SBA were 10^–4, 2×10^–4 and 10^–4 M respectively. In washout experiments (repeated centrifugations) different degrees of reversibility of these inhibitory effects were observed depending upon the experimental conditions. Preincubation of the enzyme with the cardenolides in theabsence of Na⁺, Mg²⁺ and ATP resulted in a persisting inhibition of the Na⁺-K⁺-ATPase of 14% with digitoxin, 10% with k-strophanthidin and- significantly higher (p < 0.05)-33% with SBA. Corresponding experiments with preincubation of the enzyme in thepresence of Na⁺, Mg²⁺ and ATP, however, demonstrated a full reversibility of the inhibitory action of all these substances.These results are in contrast, in certain respects, with those obtained in previous experiments on brain ATPase.It is concluded that SBA is able to inhibit irreversibly only the non-phosphorylated form of the cardiac Na⁺-K⁺-activated ATPase, whereas the phosphorylated intermediate of this enzyme seems to be protected against the irreversible inhibition by this substance. Assuming that the latter state of the enzyme is predominant in the intact heart muscle cell, a complete reversibility of the pharmacological action of SBA would be expected if the inotropic effect is mediated by an inhibition of the enzyme. Our results are compatible with this hypothesis.

Wir danken der Deutschen Forschungsgemeinschaft für die Unterstützung durch Sachbeihilfen und der Volkswagenstiftung für die Geräteausstattung.     

Functional role of sodium pump in human placental arteries

Summary Ouabain (10^–7 to 10^–4 M) elicited concentration-dependent contractile responses in human placental arteries. The contractions were reduced by 10^–4 M amiloride and Ca²⁺-free medium, but not affected by 10^–6 M nifedipine or 10^–6 M Bay-K-8644, which markedly reduced or potentiated 75 mM K⁺-induced contractions, respectively. After contracting the vessels with 10^–6 M prostaglandin F₂ in a K⁺-free medium, the subsequent addition of 7.5 mM K⁺ induced a marked relaxation, which was blocked by 10^–6 M ouabain. This glycoside (10^–8 to 10^–4 M) also produced a concentration-dependent reduction of ⁸⁶Rb⁺ uptake. Scatchard analysis of the [³H]-ouabain binding to membrane fractions from human placental arteries suggests a single class of binding sites with a KD of 88.3 nM and a B_max of 345 fmol/mg. 5-Hydroxytryptamine (5-HT; 10^–9 to 10^–5 M) caused concentration-dependent contractions. Single concentrations produced transient responses composed of an initial contraction, followed by a slow fall in tension. Ouabain (10^–8 to 10^–6 M), K⁺-free medium or the reduction of bath temperature (28°C) did not modify contractions but inhibited the relaxant phase of the response. 5-HT (10^–8 to 10^–6 M) increased both total and ouabain-insensitive ⁸⁶Rb⁺ uptake, but the difference between them was not modified. These data indicate that: (1) human placental arteries possess an important sodium pump activity, inhibition or stimulation of which markedly alters vascular tone, (2) ouabain-evoked contractions are produced by Ca²⁺ entry mainly through Na⁺-Ca²⁺ exchange, secondary to intracellular Na⁺ accumulation, (3) the relaxant component of 5-HT response is dependent on the activity of the sodium pump, (4) the activation of Na⁺,K⁺-ATPase activity by this amine is not apparently due to direct effect, and (5) the inhibition of the sodium pump can cause long lasting increases of placental vascular resistance in the presence of physiological concentrations of 5-HT. Send offprint requests to J. Marin at the above address     

Epigallocatechin-3-gallate is an inhibitor of Na,K-ATPase by favoring the E1 conformation

Four catechins, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, and epicatechin, inhibited activity of the Na⁺,K⁺-ATPase. The two galloyl-type catechins were more potent inhibitors, with IC₅₀ values of about 1 μM, than were the other two catechins. Inhibition by epigallocatechin-3-gallate was noncompetitive with respect to ATP. Epigallocatechin-3-gallate reduced the affinity of vanadate, shifted the equilibrium of E₁P and E₂P toward E₁P, and reduced the rate of the E₁P to E₂P transition. Epigallocatechin-3-gallate potently inhibited membrane-embedded P-type ATPases (gastric H⁺,K⁺-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase) as well as the Na⁺,K⁺-ATPase, whereas soluble ATPases (bacterial F₁-ATPase and myosin ATPase) were weakly inhibited. Solubilization of the Na⁺,K⁺-ATPase with a nonionic detergent reduced sensitivity to epigallocatechin-3-gallate with an elevation of IC₅₀ to 10 μM. These results suggest that epigallocatechin-3-gallate exerts its inhibitory effect through interaction with plasma membrane phospholipid.