Tetracycline adsorption on kaolinite: pH,metal cations and humic acid effects

Contamination of environmental matrixes by human and animal wastes containing antibiotics is a growing health concern. Because tetracycline is one of the most widely-used antibiotics in the world, it is important to understand the factors that influence its mobility in soils. This study investigated the effects of pH, background electrolyte cations (Li⁺, Na⁺, K⁺, Ca²⁺ and Mg²⁺), heavy metal Cu²⁺ and humic acid (HA) on tetracycline adsorption onto kaolinite. Results showed that tetracycline was greatly adsorbed by kaolinite over pH 3–6, then decreased with the increase of pH, indicating that tetracycline adsorption mainly through ion exchange of cations species and complexation of zwitterions species. In the presence of five types of cations (Li⁺, Na⁺, K⁺, Ca²⁺ and Mg²⁺), tetracycline adsorption decreased in accordance with the increasing of atomic radius and valence of metal cations, which suggested that outer-sphere complexes formed between tetracycline and kaolinite, and the existence of competitor ions lead to the decreasing adsorption. The presence of Cu²⁺ greatly enhanced the adsorption probably by acting as a bridge ion between tetracycline species and the edge sites of kaolinite. HA also showed a major effect on the adsorption: at pH < 6, the presence of HA increased the adsorption, while the addition of HA showed little effect on tetracycline adsorption at higher pH. The soil environmental conditions, like pH, metal cations and soil organic matter, strongly influence the adsorption behavior of tetracycline onto kaolinite and need to be considered when assessing the environmental toxicity of tetracycline.     

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.     

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.     

Enhancing the potency of lithospermate B for inhibiting Na+/K+-ATPase activity by forming transition metal ion complexes

Aim:

To determine whether replacing Mg²⁺ in magnesium lithospermate B (Mg-LSB) isolated from danshen (Salvia miltiorrhiza) with other metal ions could affect its potency in inhibition of Na⁺/K⁺-ATPase activity.

Methods:

Eight metal ions (Na⁺, K⁺, Mg²⁺, Cr³⁺, Mn²⁺, Co²⁺, Ni²⁺, and Zn²⁺) were used to form complexes with LSB. The activity of Na⁺/K⁺-ATPase was determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP. Human adrenergic neuroblastoma cell line SH-SY5Y was used to assess the intracellular Ca²⁺ level fluctuation and cell viability. The metal binding site on LSB and the binding mode of the metal-LSB complexes were detected by NMR and visible spectroscopy, respectively.

Results:

The potencies of LSB complexed with Cr³⁺, Mn²⁺, Co²⁺, or Ni²⁺ increased by approximately 5 times compared to the naturally occurring LSB and Mg-LSB. The IC₅₀ values of Cr-LSB, Mn-LSB, Co-LSB, Ni-LSB, LSB, and Mg-LSB in inhibition of Na⁺/K⁺-ATPase activity were 23, 17, 26, 25, 101, and 128 μmol/L, respectively. After treatment of SH-SY5Y cells with the transition metal-LSB complexes (25 μmol/L), the intracellular Ca²⁺ level was substantially elevated, and the cells were viable for one day. The transition metals, as exemplified by Co²⁺, appeared to be coordinated by two carboxylate groups and one carbonyl group of LSB. Titration of LSB against Co²⁺ demonstrated that the Co-LSB complex was formed with a Co²⁺:LSB molar ratio of 1:2 or 1:1, when [Co²⁺] was less than half of the [LSB] or higher than the [LSB], respectively.

Conclusion:

LSB complexed with Cr³⁺, Mn²⁺, Co²⁺, or Ni²⁺ are stable, non-toxic and more potent in inhibition of Na⁺/K⁺-ATPase. The transition metal-LSB complexes have the potential to be superior substitutes for cardiac glycosides in the treatment of congestive heart failure.     

The binding of inorganic and organic cations and H+ to cartilage in vitro

The binding of H⁺, Na⁺, Ca²⁺, Ni²⁺, Ce³⁺, chlorpromazine, chloroquine, hexamethonium, paraquat and gentamicin to decalcified cartilage from bovine nasal septum has been studied in vitro. The results indicated that the chondroitin sulphate is the binding material in the cartilage and that a stoichiometric binding occurs to the carboxyl and ester sulphate groups of the chondroitin sulphate. An analysis of the binding by the method of Scatchard was performed. The H⁺ ions were bound to two groups of sites, one representing the carboxyl, the other representing the ester sulphate groups. It was shown that the Scatchard association constants for H⁺ may be converted to pK_a values. Such transformation showed that the carboxyl groups of the chondroitin sulphate had a pK_a value of 4.57 and the ester sulphate groups a pK_a value of 2.60. The data for Na⁺ indicated a binding to three groups of sites. Two of these may represent the carboxyl and ester sulphate groups. The third, which was a small group with a very strong affinity, may represent a specific localised link with a few groups on the chondroitin sulphate. The experiments with Ca²⁺, Ni²⁺, Ce³⁺ and the organic substances indicated that, in contrast to H⁺ and Na⁺, only one binding class was present, which implies that the binding to the carboxyl and ester sulphate groups for these ligands occurs with a similar strength. The affinity of these cations to the cartilage was related to their positive charge, which is the general characteristic for an electrostatic interaction.     

Influence of alkali metal counterions on the glass transition temperature of amorphous indomethacin salts

Purpose. To test the hypothesis that the choice of counterion in salt formation would generally result in a change in the glass transition temperature, T_g, in relation to the nature of the interaction between the ionized parent structure and its counterion. Methods. Various alkali metal salts of indomethacin (IMC), lithium (Li⁺), sodium (Na⁺), potassium (K⁺), rubidium (Rb⁺), and cesium (Cs⁺) IMC were prepared as amorphous solid powders by lyophilization. The T_g was measured using differential scanning calorimetry or modulated-temperature differential scanning calorimetry. The spectroscopic properties of these salts were analyzed using Fourier transform-Raman and Fourier transform-infrared spectroscopy. Results. A comparison of the T_g values of the five salts showed T_g values in the order of Li⁺ > Na⁺ > K⁺ > Rb⁺ > Cs⁺, which is consistent with an order of decreasing ionic radius, and hence an increased charge density and an increased electrostatic interaction energy between the carboxylate ion and the alkali metal cation. Spectroscopic data support this interpretation by showing that the different salts exhibit spectral differences only in the region of the carboxylate group. Conclusions. Changes in T_g due to salt formation for alkali metal salts of IMC mainly result from differences in ionic interaction between the oppositely charged ions that appear to be related to the size/charge ratio of the counterion.     

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     

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.     

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。     

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.     

The relationship between Na+, K+-ATPase inhibition and cardiac glycoside-induced arrhythmia in dogs

Summary In order to determine if there is a relationship between Na⁺, K⁺-ATPase inhibition and cardiac glycoside-induced arrhythmia, the time course of the onset and offset of the arrhythmia induced by the semi-synthetic glycoside, actodigin, and the enzyme activity during arrhythmia and following reversion to normal sinus rhythm was studied in the intact, anesthetized dog. An infusion of actodigin (AY 22,241) at the rate of 0.1 mol/kg/min for 30 min induced a severe and persistent arrhythmia within 13.1±1.2 min in 9 dogs. Upon termination of the actodigin infusion, the arrhythmia spontaneously converted to sinus rhythm within 17.5±2.3 min. Left ventricular tissue was taken from dogs sacrificed at the peak of the actodigin-induced arrhythmic periods or from the dogs that were allowed to recover from the actodigin-induced arrhythmia. These samples were homogenized and the membrane-containing fraction was passed through a Millipore filter. The membrane fraction trapped in the filter was the assayed for Na⁺+K⁺ stimulate, Mg²⁺ dependent ATPase activity. The results showed that, in comparison to the time matched control dogs, the cardiac microsomes prepared from the arrhythmic dogs had a markedly reduced Na⁺, K⁺-ATPase activity. On the other hand, actodigin-treated dogs that were allowed to recover from the arrhythmic episode had Na⁺, K⁺-ATPase activity that was not significantly different from the control values.The amount of ³H-actodigin bound by the cardiac muscle microsomal fraction was also investigated. The microsomes from left ventricle were isolated with a slight modification of the method of Dutta et al. (1968). The microsomal binding of ³H-actodigin was maximum at 30 min (26.6 pmol/mg protein) when the sample was prepared from the dogs at the peak of the arrhythmic effect. However, the binding was significantly reduced (11.5 pmol/mg protein) in the microsomal fraction from hearts that had returned to sinus rhythm. These data provide direct evidence that inhibition of Na⁺, K⁺-ATPase and cardiac glycosideinduced arrhythmia may have some cause and effect relationship.This investigation was supported in part by the United States Public Health Services Research Grant HE 07051 and The Central Ohio Heart Association GrantA report of this study has been presented in the spring meetings of FASEB, April, 1974, Atlantic City, New Jersey and submitted by J. H. Zavecz in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Ohio State University     

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.     

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     

Cardiac glycoside receptor, (Na+ + K+)ATPase activity and force of contraction in rat heart

In order to elucidate the role of (Na⁺ + K⁺)ATPase and of the ouabain binding sites in the pharmacological effects of ouabain in the digitalis insensitive species rat, specific [³H]ouabain binding and (Na⁺ + K⁺)ATPase activity were measured simultaneously in a rat heart cell membrane preparation. Specific [³H]ouabain binding, ⁸⁶Rb⁺-uptake and force of contraction were also measured simultaneously in electrically stimulated contracting ventricular strips of rat heart. The following results were obtained: (1) Rat heart cell membranes exhibit two classes of [³H]ouabain binding sites with apparent dissociation constants (K_D) of the [³H]ouabain-receptor complex 1–2.3 × 10^?7 M and 2.8 × 10^?5 M. (2) (Na⁺ + K⁺)ATPase activity of rat heart cell membranes is half-maximally inhibited by ouabain at a concentration of 4 × 10^?5 M when assayed at the same conditions as [³H]ouabain binding. (3) Specific [³H]ouabain binding to electrically stimulated (1 Hz) contracting ventricular strips of rat heart exhibited only one class of receptors (K_D = 3 × 10^?7M). Force of contraction increased half-maximally at 3 × 10^?7 M ouabain when measured simultaneously and ⁸⁶Rb⁺uptake was inhibited half-maximally at 3 × 10^?5 M ouabain. Thus, there is a serious discrepancy between the effect of ouabain on (Na⁺ + K⁺)ATPase activity and ⁸⁶Rb⁺ uptake on one hand and on force of contraction on the other hand, whereas there is a good correlation between [³H]ouabain-receptor binding and increase in force of contraction. These results indicate that inhibition by ouabain of active cation transport is not a mandatory prerequisite of its positive inotropic effect, at least in the rat heart.     

Ionophorous properties of narasin, a new polyether monocarboxylic acid antibiotic, in rat liver mitochondria.

Narasin, a new polyether monocarboxylic acid antibiotic, inhibits ATPase and reverses swelling and H⁺ release from liver mitochondria after induction of cation transport. Among alkali metal cations, narasin shows ionophorous activities with a selectivity of Na⁺ K⁺ = Rb⁺ Cs⁺ Li⁺. However, narasin also induces permeability of mitochondria to the non-noble gas cation, Tl⁺ and the polyatomic cation, NH₄⁺. In fact, the polyether monocarboxylic acid ionophores, narasin and monensin, are more appropriate ionophores for NH₄⁺ than for alkali cations. Narasin inhibits 50 per cent of the ATP hydrolysis activated by dinitrophenol, valinomycin-K⁺ and monazomycin-K⁺-NH₄⁺ with ic₅₀ values of 2.5, 200 and 13 nM respectively. The corresponding ic₅₀ values for monensin are 7, 750 and 45 nM. The polyether monocarboxylic acid ionophores may play an important role in ionophore-producing organisms as NH₄⁺ carriers.     

Pals-associated tight junction protein functionally links dopamine and angiotensin II to the regulation of sodium transport in renal epithelial cells

Background and purpose:

Dopamine inhibits renal cell Na⁺,K⁺-ATPase activity and cell sodium transport by promoting the internalization of active molecules from the plasma membrane, whereas angiotensin II (ATII) stimulates its activity by recruiting new molecules to the plasma membrane. They achieve such effects by activating multiple and distinct signalling molecules in a hierarchical manner. The purpose of this study was to investigate whether dopamine and ATII utilize scaffold organizer proteins as components of their signalling networks, in order to avoid deleterious cross talk.

Experimental approach:

Attention was focused on a multiple PDZ domain protein, Pals-associated tight junction protein (PATJ). Ectopic expression of PATJ in renal epithelial cells in culture was used to study its interaction with components of the dopamine signalling cascade. Similarly, expression of PATJ deletion mutants was employed to analyse its functional relevance during dopamine-, ATII- and insulin-dependent regulation of Na⁺,K⁺-ATPase.

Key results:

Dopamine receptors and components of its signalling cascade mediating inhibition of Na⁺,K⁺-ATPase interact with PATJ. Inhibition of Na⁺,K⁺-ATPase by dopamine was prevented by expression of mutants of PATJ lacking PDZ domains 2, 4 or 5; whereas the stimulatory effect of ATII and insulin on Na⁺,K⁺-ATPase was blocked by expression of PATJ lacking PDZ domains 1, 4 or 5.

Conclusions and implications:

A multiple PDZ domain protein may add functionality to G protein-coupled and tyrosine kinase receptors signalling during regulation of Na⁺,K⁺-ATPase. Signalling molecules and effectors can be integrated into a functional network by the scaffold organizer protein PATJ via its multiple PDZ domains.     

Altered cardiac Na+,K+-ATPase activity in prehypertensive spontaneously hypertensive rat

Na⁺,K⁺-ATPase activity, Na⁺-dependent phosphorylation, and [³H]ouabain binding in sarcolemma prepared from 4 week old spontaneously hypertensive rat(SHR) ventricles were compared to the same parameters in sarcolemma from age matched normotensive Wistar-Kyoto (WKY) rat ventricles to examine whether the reduced myocardial Na⁺-pump activity in SHR is an inherited enzymatic defect or a second phenomenon due to sustained hypertension. The total body weights, ventricular weights, and blood pressures were the same for SHR and WKY. No significant differences in sarcolemmal protein content and protein recovery were noted between the two groups. Sarcolemma isolated from SHR ventricles showed significantly less Na⁺, K⁺-ATPase activity and number of phosphorylation sites when, compared to sarcolemma from the WKY ventricles. Equilibrium binding of [³H]ouabain and the tumover number of myocardial Na⁺,K⁺-ATPase, however, were the same for both groups. These results indicate that the low affinity (α, or α₁) isoform for ouabain is reduced in SHR compared to WKY but that the high affinity (α+, or α₂) α isoform is the same in ventricles of SHR and WKY. The reduced amount of isoform of the Na⁺,K⁺-ATPase in, prehypertensive SHR ventricles may play some role in the development of hypertension.     

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.     

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 heavy metals on transcription and enzyme activity of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase in the monogonont rotifer, <Emphasis Type="Italic">Brachionus koreanus</Emphasis>

Heavy metals can lead to osmotic stress by disrupting the regulation of sodium ion in aquatic organisms. In this study, gene expression patterns and enzymatic activities of Na⁺/K⁺-ATPase in the monogonont rotifer, Brachionus koreanus were measured after exposure to different Cd (7.5, 15, and 30 mg/L), and Pb (0.1, 0.5, and 1.0 mg/L), respectively. As results, a significant increase in Bk Na⁺/K⁺-ATPase activity was observed after exposure to Cd and Pb in a concentration-dependent manner. Bk Na ⁺/K ⁺-ATPase mRNA level was significantly upregulated in the Cd-exposed group, whereas its level was reduced in the Pb-exposed group. These findings indicate that heavy metals could induce osmotic stress in B. koreanus, and Na⁺/K⁺-ATPase may be involved in cellular ho-meostasis in response to heavy metal exposure. This study is helpful for the understanding of the molecular mode of action of B. koreanus in response to heavy metals.