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.     

Cardiac glycosides: Correlations among Na+, K+-ATPase,sodium pump and contractility in the guinea pig heart

Summary Relationships among positive inotropic response to cardiac glycosides, Na⁺,K⁺-ATPase inhibition and monovalent cation pump activities were studied using paced Langendorff preparations of guinea-pig heart. Na⁺,K⁺-ATPase activity was estimated from the initial velocity of (³H)-ouabain binding in ventricular homogenates, and cation pump activity from ouabain-sensitive ⁸⁶Rb uptake of ventricular slices. These parameters were assayed in control, ouabain- or digitoxintreated hearts either at the time of inotropic response to the cardiac glycosides or during the course of drug washout. Development and loss of the inotropic response during ouabain or digitoxin perfusion and washout was accompanied by reduction and subsequent recovery of the initial ouabain binding velocity, respectively. If homogenates from glycoside-treated hearts were incubated at 37°C for 10 min during ouabain-binding studies, the levels of binding were not different from those of control hearts, indicating a rapid dissociation of the glycosides from cardiac Na⁺,K⁺-ATPase in this species. Despite differences in the time course of the loss of inotropic responses produced by ouabain or digitoxin, the relationship between Na⁺,K⁺-ATPase inhibition and inotropic responses were similar. Inotropic responses to digitoxin during perfusion, and subsequent los during washout, also were accompanied by a reduction and subsequent recovery of ⁸⁶Rb uptake. A correlation between inhibition of cation pump activity and positive inotropy has hitherto not been demonstrated. Thus, it appears that with cardiac glycosides, a relationship exists among contractility, cardiac Na⁺,K⁺-ATPase and monovalent cation pump activities.     

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)     

Palytoxin: Membrane mechanisms of action

Palytoxin is a marine toxin originally isolated from the zoantharians of the genus Palythoa, but now is found in marine organisms ranging from dinoflagellates to fishes. With a MW of 2680, it is one of the largest nonpolymeric natural products ever found. Its complex structure has been elucidated and total synthesis has been achieved. With an LD₅₀ of 25 ng/kg for rabbits (the most sensitive species), it is one of the most lethal marine toxins. It binds to the Na,K-ATPase specifically with a K_D of 20 pM. It has a unique action on the Na,K-ATPase, converting the pump into an ion channel and resulting in K⁺ efflux, Na⁺ influx and membrane depolarization. As a result palytoxin causes a wide spectrum of secondary pharmacological actions. By acting like a key to unlock the internal structure of the Na,K-ATPase, palytoxin holds promise as a useful tool for investigation of the pump molecule.     

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     

Suitability of the Neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins)

Palytoxin and related compounds are neurotoxic phycotoxins produced by benthic microalgae belonging to the genus Ostreopsis. For several years this family of phycotoxins has been posing a threat to human health since they can bioaccumulate in shellfish. With the aim of replacing current biological assays, such as the mouse or hemolytic assays, we investigated using the Neuro-2a neuroblastoma cell line to detect palytoxin and related compounds. Cell death induced by the effects of PlTX and analogues on Na⁺, K⁺-ATPase were measured using the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay for mitochondrial reductase activity as a surrogate for cell number. The specificity of the Neuro-2a cell-based assay for palytoxin detection was confirmed by using ouabain, which also acts on Na⁺, K⁺-ATPase. Pre-treatment of the Neuro-2a cells with ouabain minimizes the effects of palytoxin. The specificity of the Neuro-2a assay was confirmed by the finding that cell death was not detected when Neuro-2a cells were exposed to other phycotoxins with unrelated cellular targets. When the Neuro-2a assay was used to detect palytoxin in mussel extracts spiked with levels of palytoxin around the proposed regulatory value of 250 μg palytoxin/kg shellfish, a good correlation was observed between the levels found and the expected values.We conclude by proposing an experimental design for functional assays using the Neuro-2a cell line for the specific detection of four neurotoxic phycotoxin families: saxitoxins, brevetoxins, ciguatoxins and palytoxins.     

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.     

Editorial Review: Palytoxin: Mechanism of Action of a Potent Marine Toxin

Abstract

Palytoxin, the most potent marine toxin known, causes death at doses as low as 0.025 μg/kg. Its chemical structure contains no identifiable subunits, making it one of the most complex natural products known. It exhibits a wide variety of pharmacological actions Including stimulating release of K⁺ from various cell types; depolarization of excitable membranes; contraction of smooth muscle; cardiovascular effects; hemolysis; histamine, prostaglandin and norepinephrine release; platelet aggregation and bone resorption. It also exhibits Inhibition of sperm motility, anti-tumor activity and tumor-promoting activity. Studies on the molecular mechanism of action have led to several proposed mechanisms. The most extensively studied is the proposal that palytoxin interacts with (Na⁺,K⁺)-ATPase at the ouabain binding site, but rather than inhibiting the ion pump as ouabain does, it converts it to an open ion channel, resulting in collapse of ion gradients across the cell membrane. Other possible mechanisms include a similar effect on the acetylcholine receptor, other types of ionophore activity, and release of an endogenous mediator. Many of the pharmacological effects of palytoxin are of types that are mediated by elevated intracellular Ca²⁺. Palytoxin causes elevated intracellular Ca²⁺, but it is not known if it is a direct result of opened ion channels or an indirect effect of altered Na⁺ concentrations.     

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 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.     

Chronic digoxin treatment on canine myocardial Na+, K+-ATPase

Summary In order to determine whether a prolonged inhibition of cardiac Na⁺, K⁺-ATPase causes a compensatory or adaptive change in this enzyme, the relationships among serum digoxin concentration, binding of digoxin to the enzyme and cardiac Na⁺, K⁺-ATPase and sodium pump activity were studied in dogs chronically treated with digoxin. Digoxin was injected intravenously twice daily up to 4 weeks. Two hours after the injection of a single non-toxic dose of digoxin, Na⁺, K⁺-ATPase and sodium pump activities were inhibited quantitatively in a manner corresponding to the binding of digoxin to the enzyme. The magnitude of sodium pump inhibition was reduced 12 h after the digoxin injection, with simultaneous decreases in serum digoxin concentration and the binding of digoxin to the enzyme. After 1 or 4 weeks of digoxin treatment with non-toxic doses, the relationships among serum digoxin concentration, binding of digoxin to cardiac Na⁺, K⁺-ATPase and the degree of cardiac Na⁺, K⁺-ATPase or sodium pump inhibition remained unchanged. The magnitude of the inhibition was related to serum digoxin concentrations and digoxin binding to cardiac Na⁺, K⁺-ATPase, in a manner similar to that observed after a single digoxin injection. After 4 weeks of digoxin treatment with toxic doses, these relationships were also unaffected. It was concluded that prolonged digoxin treatment fails to alter the inhibition of myocardial Na⁺, K⁺-ATPase by this agent.This work was supported by U.S. Public Health Service Grant HL-16052.     

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na^＋/K^＋- ATPase

Aim:

To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain.

Methods:

The inhibitory potency of ouabain and the identified steroid-like compounds on Na⁺/K⁺-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na⁺/K⁺-ATPase.

Results:

All the examined steroid-like compounds displayed more or less inhibition on Na⁺/K⁺-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na⁺/K⁺-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K⁺ binding sites of Na⁺/K⁺-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na⁺/K⁺-ATPase.

Conclusion:

Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na⁺/K⁺-ATPase.     

Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na⁺/K⁺-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC₅₀=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 M)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H₁-receptors and -adrenoceptors, or neuronal Na⁺ channel blockade. The inhibition of ouabain contraction in tissues bathed in K⁺-free medium, and the inhibition by ouabain of the K⁺-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na⁺/K⁺-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 M)-induced contractions were abolished by a Ca²⁺-free solution but not by blockers of L-type Ca²⁺ channels. In human cultured bronchial smooth muscle cells, ouabain (10 M) produced a sustained increase in [Ca²⁺]_i (116±26 nM) abolished in Ca²⁺-free medium. Incubation with a Na⁺-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na⁺/Ca²⁺ exchange in ouabain contraction while selective inhibitors of Na⁺/H⁺-antiport, Na⁺/K⁺/Cl^–-antiport, or protein kinase C had no effect. Ouabain (10 M) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 M) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca²⁺ entry unrelated to L-type channels and involving the Na⁺/Ca²⁺-antiporter.     

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.     

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.     

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.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

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。     

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.     

Distribution of cardiac glycosides in heart and brain of dogs and their affinity to the (Na++K+)-ATPase

Summary The tissue distribution after repeated intravenous administration of tritium-labelled digoxin, -methyldigoxin and ouabain was examined in heart and brain of 6 beagle dogs. In addition, the (Na⁺+K⁺)-ATPase activity was measured in various heart and brain areas, and its affinity to the cardiac glycosides was determined. The glycoside concentrations in the atria are lower than in the ventricles, and the left heart areas show higher concentrations than the right areas. Significant differences in the (Na⁺+K⁺)-ATPase activity or its binding capacity in the various heart areas, which could be responsible for this characteristic distribution pattern, were not found. In agreement with its greater lipid-solubility, -methyldigoxin shows a higher accumulation in the brain than digoxin and ouabain. However, while -methyldigoxin is evenly distributed throughout all brain areas, concentration differences are found for digoxin and ouabain in the telencephalon, cerebellum and brain stem. This characteristic distribution of the more polar glycosides may be partly determined by the different structure of the capillaries in the central nervous system. In addition, the binding affinities for digoxin and ouabain also differ in the various crude brain preparations. In the diencephalon, pons, cerebellum and medulla the dissociation constants as a reciprocal measure of the binding affinity were lower for digoxin with 7.5 to 9.9×10^–9 than in the telencephalon, mesencephalon and spinal cord with dissociation constants of 1.1 to 1.45×10^–8 M. Since, in these brain areas higher glycoside concentrations per g wet weight were also measured, the glycoside accumulation in the various brain areas could be dependent on the higher receptor affinity of these brain areas. On the other hand, the binding affinities for -methyldigoxin were the same in all brain areas, with a mean dissociation constant of 1.45×10^–8 M.