On the binding of the bisquaternary ammonium compound paraquat to melanin and cartilage in vivo

The bisquaternary ammonium compound paraquat has been shown to accumulate in melanin-containing tissues and in cartilage in vivo. The present study was intended to elucidate if ionic binding is involved in the mechanism for the affinity of paraquat to these tissues in vivo. Ionic binding has previously been shown to be involved in the binding of paraquat to melanin in vitro and in the binding of other bisquaternary ammonium compounds to cartilage in vitro. [¹⁴C]paraquat was given to mice. Whole-body tissue sections were taken and incubated in solutions with different cationic composition. The ability of the cations to displace [¹⁴C]paraquat from melanin of the eye and intervertebral cartilages was then determined by autoradiography, using densitometric measurements for the quantitations. It was found that the cations in the incubation solutions were able to displace [¹⁴C]paraquat from both melanin and cartilage. The [¹⁴C]paraquat was more effectively displaced from cartilage than from melanin. In both tissues divalent cations were more effective than K⁺ and Na⁺; and H⁺ was also effective. The results indicate that ionic binding is involved in the binding in vivo of paraquat both to melanin and cartilage. The binding sites may be carboxyl groups present in the subunits in melanin and ester sulphate and carboxyl groups of chondroitin sulphate in cartilage. The stronger binding of paraquat to melanin may depend on extra interactions due to conjunctions of the aromatic rings of the paraquat and the melanin polymer.     

Interaction of ouabain with (Na+ + K+)ATPase from human heart and from guinea-pig heart.

(Na⁺ + K⁺)ATPase (ATP phosphohydrolase, EC 3.6.1.3.) has been prepared from human heart and guinea-pig heart, with respective specific activities of 10–15 μmol P_i · mg^?1 · h^?1 and of 25–30 μmol P_i · mg^?1 · h^?1. Residual Mg²⁺—ATPase activities were about 5 per cent. The parameters of (Na⁺ + K⁺)ATPase activity and of ouabain-interaction have been compared: (1) Half-maximal activity concentrations and Hill coefficients of Na⁺, K⁺, Mg²⁺ and ATP were similar for the two species. The apparent activation energies calculated from Arrhenius plots were also similar. A transition was observed at about 23°C. (2) Human heart (Na⁺ + K⁺)ATPase was 10 times more sensitive to ouabain-inhibition than that of guinea-pig. Hunter-Downs plots showed a competitive inhibition for K⁺ at low K⁺ concentration and noncompetitive inhibition at high concentration. (3) The Scatchard plot for [³H]ouabain binding was upward-concave with human heart and linear with guinea-pig heart. (4) The dissociation kinetics of [³H]ouabain from human preparations studied by an isotopic dilution technique indicated two classes of binding sites with k_d of 0.058 min^?1 and 0.0092 min^?1. The dissociation kinetics with guinea-pig heart indicated one single class of binding sites with a k_d of 0.43 min^?1. (5) The time-course of 0.2 μM [³H]ouabain binding showed pseudo-first order association kinetics in man and in guinea-pig. k_a for the two classes of binding sites in man were therefore similar, respectively equal to 3.4 × 10⁶ min^?1 · M^?1 and to 3.7 × 10⁶ min^?1 · M^?1 · k_a for guinea-pig heart was equal to 2.3.10⁶ min^?1 · M^?1. (6) In guinea-pig heart, K_D c from Scatchard plot and from k_d/k_a ratio were equal to the inhibition constant K_i calculated from Hunter-Downs plot indicating that the binding sites were closely related to (Na⁺ + K⁺)ATPase inhibition. (7) In human heart, K_D of the low affinity binding sites was close to K_i, whereas K_D of the high affinity binding sites was several times lower. This suggests that only low affinity binding sites might be involved in (Na⁺ + K⁺)ATPase inhibition by ouabain.     

Digitalis structure-activity relationship analyses: Conclusions from indirect binding studies with cardiac (Na+ + K+)-ATPase

We have performed direct and indirect binding studies with [³H]ouabain or [³H]digitoxin on beef or guinea pig cardiac (Na⁺ + K⁺)-ATPase to measure the potencies of a broad range of cardiotonic steroids for structure-activity relationship (SAR) studies for comparison with previously determined positive inotropic potencies. The positive inotropic potencies of twelve compounds on contracting guinea pig left atria correlated well with the equilibrium dissociation constants (K_D values) from the inhibition of [³H]ouabain binding to guinea pig cardiac (Na⁺ + K⁺)-ATPase (r = 0.98 for seven 5β-compounds, r = 0.95 for five 5α-compounds). Further we calculated K_D values from the inhibition of [³H]ouabain binding data for a total of 33 digitalis derivatives on the digitalis-sensitive beef cardiac (Na⁺ + K⁺)-ATPase. For the 27 compounds tested on both beef cardiac (Na⁺ + K⁺)-ATPase and guinea pig left atria, the potencies showed a significant correlation (r = 0.92 for 22 5β-compounds, r = 0.96 for five 5/ga-compounds. For seven compounds, K_D values were measured on beef cardiac (Na⁺ + K⁺)-ATPase using inhibition of binding of [³H]digitoxin. These values correlated well (r = 0.99) with the K_D values from the [³H]ouabain studies.These results show that: (1) The significant correlation observed between K_D values on guinea pig cardiac (Na⁺ + K⁺)-ATPase and positive inotropic potency in guinea pig left atria is further evidence that the pharmacological receptor for inotropy is part of the enzyme, (2) Inhibition of the binding of [³H]ouabain or [³H]digitoxin can be used to determine the relative potencies of unlabelled digitalis derivatives. The similar relative potencies on beef and guinea pig cardiac (Na⁺ + K⁺)-ATPase of a broad range of digitalis derivatives indicate that the binding site is similar for both species; and (3) SAR studies indicate that functional groups on these steroids have the same influence on potency on either the positive inotropy or cardiac (Na⁺ + K⁺)-ATPase studies.     

Electrophysiological studies of myotoxin a, isolated from prairie rattlesnake (Crotalus viridis viridis) venom, on murine skeletal muscles

S. J. Hong and C. C. Chang. Electrophysiological studies of myotoxin a, isolated from prairie rattlesnake (Crotalus viridis viridis) venom, on murine skeletal muscles. Toxicort23, 927–937, 1985. — Myotoxin a reduced the resting membrane potential of mouse and rat diaphragms from about ?80 mV to ?60 mV, induced spontaneous repetitive firing and enhanced the contractile force in response to single stimulations. The depolarizing effect was reversed noncompetitively by tetrodotoxin, local anesthetics or low Na⁺ solution, but was augmented by ouabain or low Cl^? solution while being unaffected by high K⁺ solution or electrical stimulation of the muscle. The duration of muscle action potential was prolonged by only 20–30%, whereas the rate of rise (dV/ dt) was unaffected. About a 40% increase of membrane conductance was observed, which could be abolished by the Na⁺-channel blocker tetrodotoxin. By contrast, K⁺ conductance was unaffected. Effects on caffeine-induced contracture, quantal release of neurotransmitter and the amplitude of miniature endplate potential were not appreciably affected. These effects of myotoxin a indicate that the toxin affects the muscle, but not the nerve, by acting specifically on the Na⁺-channel of the sarcolemma or T-tubule, like crotamine, rather than on the sarcoplasmic reticulum. The effects of sea anemone toxin II on the Na⁺-channel (marked depolarization and prolongation of action potential) could not be prevented by saturating the muscle with myotoxin a. On the other hand, the effect of veratridine, a member of another group of toxins acting on the Na⁺-channel, was enhanced. These results suggest that myotoxin a acts on the Na^?-channel at a site which is discrete from those of tetrodotoxin, veratridine and sea anemone toxin II.     

Effect of citreoviridin, a mycotoxin from Penicillium citreoviride, on kinetic constants of acetylcholinesterase and ATPase in synaptosomes and microsomes from rat brain

The subcutaneous injection of acute (6 mg/kg) and subacute doses of citreoviridin, a toxin from Penicillium citreoviride NRRL 2579, inhibited brain synaptosomal (Na⁺ + K⁺)-ATPase whereas in microsomes, both (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities are significantly stimulated in a dose-dependent manner. In vitro, both Mg²⁺ and (Na⁺ + K⁺)-ATPase activities are inhibited in synaptosomal as well as in microsomal fractions. Kinetic parameters obtained from double reciprocal plots indicate that under in vivo conditions citreoviridin increases V_max and K_m values of microsomal (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase, whereas in synaptosomes Mg²⁺-ATPase, V_max and K_m are unaltered but (Na⁺ + K⁺)-ATPase V_max is decreased and K_m increased. In vitro, both Mg²⁺ and (Na⁺ + K⁺)-ATPase K_m values are increased and V_max values are decreased in the synaptosomal as well as in the microsomal fractions. On the other hand, AChE V_max values are decreased and K_m values are increased in the synaptosomes under both in vivo and in vitro conditions. In microsomes, in vivo, the V_max values are decreased and K_m values are unaltered while in vitro both are unaltered. These results indicate that citreoviridin neurotoxicity may be related to the altered enzymatic activities.     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

Effect of ethanol on the kinetics of rat brain (Na+ + K+) ATPase and K+-dependent phosphatase with different alkali ions.

(Na⁺ + K⁺)-dependent ATPase [(Na + K)-ATPase] and K⁺-dependent p-nitrophenyl phosphatase [pNPPase] activities in rat brain heavy microsomal fractions were studied in the presence of 120 mM Na⁺ and varied concentrations of K⁺, Rb⁺, Cs⁺, Li⁺ or NH₄⁺. Scatchard and Hill plots indicated non-hyperbolicity (cooperativity) with all except Li⁺, which supported a considerably lower activity than any of the other ions tested. Addition of 0.22 M ethanol to the incubation mixtures produced a formally competitive inhibition of ATPase activity with K⁺, Rb⁺ and Cs⁺, a non-competitive inhibition with Li⁺, and a mixed inhibition with NH₄⁺. The changes in pNPPase activity generally followed a similar but less clear-cut pattern. The values of the Hill constants were not changed for either enzyme activity. The findings are interpreted as evidence that ethanol inhibits ATPase activity by inducing conformational changes which alter the consequences of ion binding to the various receptor sites.     

Mechanism of inhibition of sodium- and potassium-dependent adenosine triphosphatase by the isoquinoline derivative BIIA: a specific interaction with sodium activation

An isoquinoline derivative, 3-benzylamino-5,6-dihydro-8,9-dimethoxy-imidazo-(5,1-a)-iso-quinoline hydrochloride, BIIA, with positive inotropic and antiarrhythmic actions, reversibly inhibited the Na⁺, K⁺-ATPase of deoxycholate and NaI treated musomes from guinea-pig heart, brain and kidney. The inhibition was pH dependent, increasing with increasing pH and the concentration of the highly lipid soluble, unprotonated molecule. BIIA inhibited Na⁺, K⁺-ATPase in a concentration range of 1–100 μmoles/l, in a manner uncompetitive with respect to ATP and K⁺, and competitive with respect to Na⁺, K⁺PNPPase of the same preparation was also inhibited by BIIA, albeit at higher concentrations. This inhibition was competitive with K⁺. Affinity for substrate, as measured with [¹⁴C]-ATP, was increased and labelling from AT³²P decreased competitive with Na⁺. BIIA decreases the affinity of the enzyme for (Mg²⁺ + P_i) supported ouabain binding, higher concentrations of BIIA also affect (Na⁺ ATP) supported binding. It is suggested that BIIA enters a hydrophobic environment of the Na⁺, K⁺-ATPase and interacts at or near the Na⁺-activation sites, inhibiting the formation of the phosphorylated intermediate.     

Na+ and K+ Dependence of Morphological Changes of Cultured Rat Cortical Astrocytes

Abstract: Cultured astrocytes display flattened, polygonal morphology in the absence of stimuli, and change into process‐bearing stellate cells in response to specific stimuli. In the present study, we investigated possible role of Na⁺, K⁺ and Ca²⁺ in this morphological change of cultured rat cortical astrocytes. Astrocyte stellation induced by dibutyryl cyclic AMP (1 mM), phorbol ester (100 nM) or amyloid β peptide (20 μM) was partly suppressed by replacing NaCl with choline chloride or LiCl in the extracellular medium or by adding KCl, and was completely abolished by replacing NaCl with KCl. Furthermore, the astrocyte stellation was blocked by the specific Na⁺‐K⁺ pump inhibitor ouabain. However, it was not significantly affected by removing CaCl₂ from the extracellular medium nor by adding the voltage‐dependent Ca²⁺ channel blocker nicardipine. These results suggest that Na⁺ and K⁺, but not Ca²⁺, electrochemical gradients across the plasma membrane are necessary for morphological changes of astrocytes. In addition, amyloid β25–35‐induced stellation was most susceptible to changing Na⁺ and K⁺ concentrations or ouabain, while phorbol ester‐induced stellation was least sensitive, demonstrating that the Na⁺ and K⁺ dependence differs among stimuli.     

Inhibition of Mg2+-Na+-K+-dependent ATPase system from human gastric mucosa by prostaglandins E1 and E2

The Mg²⁺-dependent and Na⁺K⁺-dependent (Na⁺K⁺-activated or transport) ATPase (EC 3.6.1.3.) were prepared from human fundic gastric mucosa and the effects of prostaglandins E₁ and E₂ on their activity studied.Significant inhibitions of Mg²⁺-dependent, total (Mg²⁺-dependent and Na⁺K⁺-dependent) and Na⁺K⁺-dependent ATPase activities were caused by prostaglandins E₁ and E₂, in concentrations of 10^?9 to 10 ^?6 M.A possible role of Mg²⁺Na⁺K⁺-dependent ATPase system is discussed in the inhibition of gastric secretion by type E prostaglandins.     

Studies on the mechanism of dog heart (Na+ + K+)-ATPase inhibition by D,L-aspartic acid and its K+ and Mg2+ salts.

d,l-aspartic acid as well as its K⁺ and Mg²⁺ salts have been found to be inhibitory to Na⁺ + K⁺)-ATPase activity of the dog heart microsomal fraction enriched with sarcolemma. According to intensity of the inhibitory effect (noncompetitive) they form the following sequel: mono-K⁺-aspartate $\text{?}\text{d,l}\text{-aspartic acid}\text{<}\text{mono-Mg}{}^{\mathrm{2+}}\text{-}\text{aspartate}\text{<}\text{mixture}$ of K⁺ and Mg²⁺-aspartate in a molar ratio of 1:1 (K⁺, Mg²⁺-ASP). The latter mixture is widely used as an agent in cardiac failure. For (Na⁺ + K⁺)-ATPase the salient effects of d,l-aspartic acid and/or its K⁺ and Mg²⁺ salts were: (i) decrease in V for ATP as substrate with unchanged K_m; (ii) for Na⁺ as an allosteric modifier of (Na⁺ + K⁺)-ATPase activity a decrease in V without any alteration in n as a measure of cooperativity between activating sites; (iii) for K⁺ a decrease in V and n as well as an increase in K_0.5. In the presence of Na⁺ and ATP the high affinity of the enzyme for K⁺ became reduced by d,l-aspartic acid, lowering at the same time the K_0.5 value.Effects like these have also been described for ouabain. The present data show that K⁺ and Mg²⁺ salts of d,l-aspartic acid act at a similar locus as does ouabain.     

Stimulation of Na+,K+-ATPase of isolated smooth muscle membranes by the Ca2+ channel inhibitors,nimodipine and nitrendipine

Nimodipine (0.015 to 1.5 μM) increased Na⁺, K⁺-ATPase activity by 70–120% in isolated smooth muscle membranes. At 0.015 μM, nitrendipine, but not nifedipine, verapamil or diltiazem, also activated this enzyme. Nimodipine stimulated this Na⁺, K⁺ATPase three times more than nitrendipine at 15 nM. Marked stimulation of Na⁺,K⁺-ATPase by nimopidine was seen in membranes from rat and guinea pig aorta and rat vas deferens, but not in membranes from guinea pig heart or brain. Although it is not known whether these results are applicable to intact cells, the results are consistent with the hypothesis that vasodilation produced by nimodipine and nitrendipine may be due not only to inhibition of Ca²⁺ entry but also to the stimulation of the Na^? pump.     

Effect of isoproterenol on the activity of Na+, K+-adenosine triphosphatase from dog hearts

The effect of isoproterenol on the activity of Na⁺,K⁺-stimulated ATPase, a membrane-bound enzyme, from dog hearts was studied. In isoproterenol-treated hearts the yield of the membrane fraction enriched in sarcolemma was increased, and Mg²⁺- and N⁺,K⁺-ATPase. 5^'-nucleotidase and succinic dehydrogenase activities were decreased. A decrease in the activity of marker enzymes in the fractions of heavy microsomes and mitocondria was also observed. Thus using marker enzymes as a measure of purity, no increase in mutual contamination of the isolated subcellular fractions could be detected. Investigation of special properties of Na⁺,K⁺-ATPase showed alteration in pH optimum, changed K_m value and increased sensitivity to HgCl₂, without any significant increase in the content of protein-bound sulfhydryl groups. These effects of isoproterenol can be attributed to destruction of membrane and/or other cellular proteins allowing more non-enzyme protein to sediment with the isolated fraction or to a more specific influence on the properties of Na⁺ ,K⁺-ATPase.     

Effect of methyl isocyanate on rabbit cardiac Na+, K+-ATPase

 The present study describes the effect of methyl isocyanate (MIC) on rabbit cardiac microsomal Na⁺, K⁺-ATPase. Addition of MIC in vitro resulted in dose-dependent inhibition of Na⁺, K⁺-ATPase, Mg²⁺-ATPase and K⁺-activated p-nitrophenyl phosphatase (K⁺-PNPPase). Activation of Na⁺, K⁺- ATPase by ATP in the presence of MIC showed a decrease in V_max with no change in K_m. Similarly, activation of K⁺ PNPPase by PNPP in the presence of MIC showed a decrease in V_max with no change in K_m. The circular dichroism spectral studies revealed that MIC interaction with Na⁺, K⁺-ATPase led to a conformation of the protein wherein the substrates Na⁺ and K⁺ were no longer able to bind at the Na⁺- and K⁺-activation sites. The data suggest that the inhibition of Na⁺, K⁺-ATPase was non-competitive and occurred by interference with the dephosphorylation of the enzyme-phosphoryl complex. Received: 3 November 1994/Accepted: 23 February 1995     

Effects of ethanol and lead ingestion on urinary sodium excretion and related enzyme activity in rat kidney

The present study was undertaken to examine lead-ethanol interaction with respect to previously reported lead enhancement of urinary Na⁺ excretion [Y. Suketa, S. Hasegawa and T. Yamamoto, Toxic. appl. Pharmac.47, 203 (1980)]. Simultaneous administration of ethanol reduced the enhancement due to lead alone. A decrease in serum Na⁺ concentration followed lead administration and the decrease appears to be less when ethanol was administered simultaneously. Renal (Na⁺ + K⁺ATPase activity was suppressed after lead administration, but it was stimulated after ethanol administration. the suppression of renal (Na⁺ + K⁺ATPase activity after lead administration was diminished after simultaneous administration of ethanol and lead. the diminution of the suppression appeared to be associated with a corresponding reduction in urinary Na⁺ excretion.     

Activation by reduced glutathione of methotrexate transport into isolated rat liver cells

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na⁺-free, Na⁺ and K⁺-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers (p-CMBS, 4-CMB and DIP²⁺) and ATP.It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K⁺. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na⁺/K⁺ ratio, both effects were partially overcome by GSH. After treatment by DIP²⁺, Na⁺/K⁺ ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K⁺ and accumulation of Na⁺ Here neither effect could be reversed by GSH; consequently, high cellular amounts of K⁺ and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake.It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.     

Comparative effects of substituted phenothiazines and their free radicals on (Na =, K + )-activated adenosine triphosphatase

The inhibitory effects of free radicals of various substituted phenothiazines on (Na⁺,K⁺)-activated ATPase [Mg²⁺-dependent, (Na⁺,K⁺)-activated ATP phosphohydrolase; EC 3.6.1.3] were studied in vitro. Enzyme preparations were obtained from rat brain microsomal fractions after deoxycholic acid and NaI treatments. Free radicals were produced by either ultraviolet (253.7 nm) irradiation or enzymatic oxidation with peroxidase. Without enzymatic or photo-oxidation, phenothiazine derivatives failed to inhibit (Na⁺,K⁺)-activated ATPase activity significantly. Photo-oxidative intermediates of thioridazine, triflupromazine and trifluoperazine were potent inhibitors of (Na⁺,K⁺)-activated ATPase activity. Those from chlorpromazine, perphenazine and promazine were less potent. Peroxidase-hydrogen peroxide treatment of promazine, thioridazine, perphenazine and chlorpromazine produced free radical intermediates which significantly inhibited (Na⁺,K⁺)-activated ATPase activity. The same treatment of triflupromazine and trifluoperazine, however, failed to produce detectable amounts of free radical intermediates. Concomitantly, no inhibition of (Na⁺,K⁺)-activated ATPase activity was observed under these conditions. It was concluded that free radical intermediates of various substituted phenothiazines are differentially potent inhibitors of (Na⁺,K⁺)-activated ATPase, and that the formation of free radicals from substituted phenothiazines is dependent upon the oxidizing conditions and nature of substituent groups.     

Stimulation of Erythrocyte Cell Membrane Scrambling by Nystatin

The antifungal ionophore nystatin dissipates the Na⁺ and K⁺ gradients across the cell membrane, leading to cellular gain of Na⁺ and cellular loss of K⁺. The increase of cellular Na⁺ concentration may result in Ca²⁺ accumulation in exchange for Na⁺. Increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) and loss of cellular K⁺ foster apoptosis‐like suicidal erythrocyte death or eryptosis, which is characterised by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. The present study explored whether nystatin stimulates eryptosis. Cell volume was estimated from forward scatter (FSC), phosphatidylserine exposure from annexin V binding and [Ca²⁺]_i from Fluo3‐fluorescence in flow cytometry. A 48‐hr exposure to nystatin (15 μg/ml) was followed by a significant increase of [Ca²⁺]_i, a significant increase of annexin V binding and a significant decrease of FSC. The annexin V binding after nystatin treatment was significantly blunted in the nominal absence of extracellular Ca²⁺. Partial replacement of extracellular Na⁺ with extracellular K⁺ blunted the nystatin‐induced erythrocyte shrinkage but increased [Ca²⁺]_i and annexin V binding. Nystatin triggers cell membrane scrambling, an effect at least partially due to entry of extracellular Ca²⁺.     

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.     

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.