Nitrendipine modulates the sodium binding site of cardiac (Na,K)-ATPase.

The present study deals with the effect of calcium entry blocking agent, nitrendipine, on the rat heart sarcolemmal (Na,K)-ATPase. Nitrendipine (1 mumol/l) exerted a stimulatory effect on (Na,K)-ATPase activity. Kinetic analysis of enzyme activation by increasing concentrations of sodium in the presence of nitrendipine revealed a 67% increase in Vmax value and a decrease of the apparent Ka value to 33% of that in control. The observed influence on (Na,K)-ATPase may contribute indirectly to the calcium antagonistic effect of nitrendipine.

Kinetic and thermodynamic properties of membrane bound Ca-ATPase with low affinity to calcium in cardiac sarcolemma; response to global ischemia of the heart.

Thermodynamic and kinetic properties of membrane bound Ca-ATPase with low affinity to calcium in cardiac sarcolemma were investigated with respect to the effect of global ischemia on the heart. Energy barrier for ATP hydrolysis catalyzed by the Ca-ATPase was slightly higher in hearts subjected to ischemia, as it was evident from increased values of activation energy. Ischemia also induced a time dependent decrease in the activity and maximum velocity (Vmax) value of Ca-ATPase. The depression of enzyme activity was evident as early as 15 minutes after the onset of ischemia. After 30 minutes of ischemia the decrease in Vmax slowed down, probably due to an "adaptational" decrease of the Km value for Ca-ATPase. This phenomenon may be interpreted as a mechanism by which the enzyme attempts to keep working in a situation when the supply of ATP is insufficient.

Free oxygen radicals contribute to high incidence of reperfusion-induced arrhythmias in isolated rat heart.

Early period of reperfusion of ischemic myocardium is associated with a high incidence of severe tachyarrhythmias including ventricular tachycardia and fibrillation (VT and VF). Free oxygen radicals (FOR) have been identified as one of the principal factors responsible for reperfusion-induced events. However, their role in arrhythmogenesis is not clear. In the present study, in isolated Langendorff-perfused rat hearts subjected to 30 min global ischemia, the onset of reperfusion induced 100% incidence of both VT and VF with their gradual cessation over 5 min of reperfusion. Generation of H2O2 in the myocardium in the first minutes of reperfusion was visualized by means of cerium cytochemistry and confirmed by X-ray microanalysis. The mechanism of the arrhythmogenic effect of FOR may involve inhibition of the sarcolemmal Na+/K+-ATPase, as demonstrated in the rat heart sarcolemmal fraction subjected to FOR-generating system (H2O2 + FeSO4).

Prevention of processes coupled with free radical formation prevents also the development of calcium-resistance in the diabetic heart.

Recently it was shown that besides their negative role in pathogenesis of diabetes, reactive oxygen species (ROS) and particularly the products of non-enzymatic glycation of proteins (NEGP) may also participate in some processes of adaptation of the myocardium to diabetes, such as in the mechanism of development of calcium resistance of the heart. Our study revealed that the hearts of rats with experimentally induced diabetes (single dose of streptozotocin, 45 mg/kg i.v., 6 U/kg insulin daily) develop considerable resistance against calcium overload (induced by means of Ca-paradox). On the day 63 after the beginning of experiment, when the diabetic cardiomyopathy became fully developed but the hearts were still not failing, their calcium resistance was increased to 83.33%. Our results provide evidence that, when applied in a special regimen, resorcylidene aminoguanidine (RAG, 4 mg/kg) prevented both, the formation of fructosamine (a source of ROS generation), and also that of the advanced Maillard products, in the heart sarcolemma of diabetic rats. The effect of RAG was accompanied by a decrease in calcium resistance in the group of rats with chronic diabetes (63 days) from 83.3 to 46.7%. It is concluded that NEGP and ROS formation are inevitably needed for development of calcium resistance in the diabetic hearts.

Sarcolemmal Ca2+-binding and enzyme activities in myocardium from hypothyroid rat.

Effects of hypothyroidism on heart sarcolemmal activities were examined by using membrane preparations obtained by two different methods from rats treated with propylthiouracil for 6 to 8 weeks. ATP-independent Ca2+ binding, sialic acid and phospholipid content, Ca2+ ATPase, Mg2+ ATPase and adenylate-cyclase were not altered in membranes isolated by the hypotonic shock-LiBr treatment method from hypothyroid hearts. On the other hand, depressed activities of ouabain sensitive Na+-K+ ATPase and 5'-nucleotidase were observed in this hypothyroid preparation. Sarcolemma isolated by the sucrose density gradient procedure from hypothyroid hearts exhibited lower ouabain-sensitive Na+-K+ ATPase and higher ATP-dependent Ca2+ binding as well as Ca2+ stimulated ATPase without any changes in the 5'-nucleotidase, adenylate cyclase and Mg2+-ATPase activities. The activation of ATP-dependent Ca2+ binding and Ca2+ stimulated ATPase by calmodulin in the hypothyroid preparation was greater than the control; these effects of calmodulin were blocked by trifluoperazine. The results suggest some specific changes in the heart sarcolemmal Ca2+-pump during the development of hypothyroidism.

The mechanism of cytolytic and cytostatic activity of benfluron.

Benfluron at concentrations of 0.26 and 0.52 mumol l-1 inhibited the formation of V79 colonies in a concentration-dependent way. Diminution of the size of colonies of the treated cells was accompanied by a decrease in protein content per cell. At the same time an increase in metabolic activity was observed. Benfluron blocked the V79 cells in S and G2 phases of the cell cycle and at higher concentrations induced cell lysis documented by alterations in cell membrane permeability and morphology. The in vitro membrane effect of benfluron involved a biphasic modulation of the cardiac sarcolemmal (Na+ + K+)-ATPase activity.

Enzyme kinetics and the activation energy of (Na,K)-ATPase in ischaemic hearts: influence of the duration of ischaemia.

Hearts from male rats were incubated at 37 degrees C for variable periods of global ischaemia. Estimation of kinetic parameters of (Na,K)-ATPase at 37 degrees C in the presence of increasing concentrations of ATP revealed a significant decrease of Vmax in the first 15 minutes of ischaemia with further stabilization at the lowest level in 45-60 minutes of ischaemia. The changes in ATP binding site occurred later after 45 minutes of ischaemia as showed by the decrease of the Km value. As to the activation energy, there were no significant differences between control and ischaemic hearts.

Enzyme kinetics and the activation energy of Mg-ATPase in cardiac sarcolemma: ADP as an alternative substrate.

Increasing concentrations of Mg within a range between 0.1-5.0 mmol/l step-by-step activated the Mg-dependent ATPase and ADPase in rat heart sarcolemma. Both Mg-dependent activities were influenced by NaN3 in a similar way. Also, activation of both enzymes by their substrates, ADP and ATP, were affected by NaN3 in a similar mode. It appears that both enzyme activities are secured by the same system which is capable of ADP hydrolysis during ATP insufficiency. In the absence of naN3 the enzyme revealed higher affinity to ATP than to ADP. The activation energy was lower for ATP hydrolysis. The above findings indicate that at non limiting concentrations of Mg2+ the enzyme is favoring ATP.

Heart sarcolemmal (Na+ + K+)-ATPase has an essential amino group in the potassium binding site on the enzyme molecule.

Selective modification of primary amino groups of (Na+ + K+)-ATPase by trinitrobenzene sulfonic acid (TNBS) resulted in a considerable inhibition of the specific activity of the enzyme. Investigation by means of enzyme and sorption kinetics of activation of heart sarcolemmal (Na+ + K+)-ATPase by its monovalent cationic ligands added simultaneously with TNBS revealed: a considerable competition between K+-ions and TNBS for the potassium binding site on the enzyme molecule; a non-competitive type of inhibition of Na+-induced activation of the enzyme. Both, potassium and sodium ions depressed, and magnesium ions enhanced the initial rate of TNBS-sorption; however, none of the above cations influenced the equilibrium value of TNBS sorption onto isolated sarcolemmal membranes. Ouabain, on the other hand, did not inhibit the initial rate and decreased the equilibrium value of TNBS sorption onto the membranes. The results obtained enabled the identification of an essential amino group in the potassium binding site of the (Na+ + K+)-ATPase molecule.

The membrane effect of benfluron: modulation of the heart sarcolemmal (Na+, K+)-ATPase and Mg(2+)-ATPase activities.

The effect of Benfluron on the heart sarcolemmal (Na+, K+)-ATPase and Mg(2+)-ATPase activities was studied in crude membrane fraction as well as in purified sarcolemmal membranes isolated from rat heart. Benfluron in concentration range 10(-7) -5 x 10(-5) mol.l-1 did not exert any effect on ATPase activities studied. 10(-4) mol.l-1 Benfluron was stimulatory towards (Na+, K+)-ATPase, while Mg(2+)-ATPase activity was depressed. Kinetic analysis of interaction of Benfluron with (Na+, K+)-ATPase revealed an increase in the Vmax and decrease in the K(m) values for ATP. The possible mechanism of interaction of the drug with (Na+, K+)-ATPase is discussed.

Selective and reversible inhibition of heart sarcolemmal (Na+ + K+)-ATPase by p-bromophenyl isothiocyanate. Evidence for a sulfhydryl group in the ATP-binding site of the enzyme.

Isothiocyanates are potent modifiers of thiol groups, and they have been successfully applied in studying the active site structure of renal (Na+ + K+)-ATPase. However, very little has been known on interactions of isothiocyanates with myocardial sarcolemmal ATPases. In the present study the mode of interaction and inhibitory effect of p-bromophenyl isothiocyanate (BPITC) on isolated rat heart sarcolemmal preparation ATPase activities not exhibiting (Mg-Ca)-ATPase activity was investigated. BPITC in concentrations of 10(-7)-10(-4) mol . l-1 inhibited selectively and non-competitively the (Na+ + K+)-ATPase activity in the sarcolemma with an ID50 around 2.10(-7) mol . l-1. The non-specific interaction of BPITC with bivalent cations, namely with Mg2+ and Ca2+, in the reaction system was eliminated by preincubation of membranes with BPITC keeping the ratio of inhibitor to membrane protein concentration constant. Under these conditions no considerable inhibitory effects were observed on Mg2+-ATPase or the low-affinity Ca2+-ATPase of sarcolemma. Preincubation of membranes with 2 mmol . l-1 ATP protected (Na+ + K+)-ATPase activity against inhibition by BPITC. The interaction of BIPTC with the sarcolemma proved to be reversible in the presence of beta-mercaptoethanol or dithiothreitol.

Effect of calcium on the structure-function relationship of (Na+ + K+)-ATPase in cardiac sarcolemma.

Calcium-induced changes in (Na+ + K+)-ATPase activity and structural changes of membrane bound proteins in rat heart sarcolemma were investigated. Increasing concentrations of Ca2+ (0.1-8.0 mmol.l-1) gradually inhibited the (Na+ + K+)-ATPase activity and decreased the alpha-helix content of sarcolemmal proteins. Mathematical and graphical analysis of observed data yielded a quantitative relationship between Ca2+-induced changes in (Na+ + K+)-ATPase activity and the secondary structure of membrane proteins in cardiac sarcolemma.

Quantitative relationship between the protein secondary structure in cardiac sarcolemma and the activity of the membrane-bound Ca2+-ATPase.

In the absence of ATP, increasing concentrations of calcium within a range between 0.1--8.0 mmol . 1(-1) gradually lowered the alpha-helix content of proteins in rat heart sarcolemma requiring no energy supply. In the presence of ATP, similar concentrations of calcium stepwise activated the sarcolemmal low-affinity Ca2+-ATPase. A mathematical analysis of the data obtained revealed a quantitative relationship between calcium-induced stimulation of the Ca2+-ATPase activity and a diminution of the alpha-helix contents of membrane proteins in cardiac sarcolemma. The cooperation between changes in protein conformation and energy consumption in relation to the supposed role of low-affinity Ca2+-ATPase in gating the calcium channel are discussed.

Exaprolol as a modulator of heart sarcolemmal (Na+ + K+)-ATPase. Evidence for interaction with an essential sulfhydryl group in the catalytic centre of the enzyme.

Beta-adrenoceptor blocking agents may have, in addition to their primary action, also ancillary effects on the cell membrane. In the present paper the non-specific interaction of exaprolol with the ATPase systems in isolated rat heart sarcolemmal membranes was investigated. When preincubated with sarcolemmal membranes in vitro, exaprolol in concentrations below 10(-4) mol.l-1 had no significant effect on sarcolemmal Mg2+-, Ca2+- and (Na+ + K+)-ATPase activities. At exaprolol concentration of 10(-4) mol.l-1 the Mg2+- and Ca2+-ATPase activities became inhibited whereas the (Na+ + K+)-ATPase activity was markedly stimulated. A kinetic analysis of these interactions revealed a non-competitive inhibition of Mg2+- and Ca2+-ATPase. In the case of (Na+ + K+)-ATPase a synergistic type of stimulation characterized by an exaprolol-induced conversion of an essential sulfhydryl group in the active site of the enzyme to the more reactive [S-] form has been observed thus increasing the affinity of the enzyme to ATP. Exaprolol concentrations exceeding 5 X 10(-4) mol.l-1 induced an overall depression of the investigated enzyme activities.

Postischemic reperfusion of the spinal cord: prolonged reperfusion alleviates the metabolic alterations induced by 25 min ischemia in the cervical and thoracolumbal segments.

In recent years, increasing amount of information has indicated that in some tissues the main damage due to oxidative stress does not occur during reperfusion but during the ischemic episode of the ischemia/reperfusion event. In this respect, serious doubts were also expressed about the origin of the increased amounts of free radicals which were believed to form and reported to appear in the perfusate during the first minutes of reperfusion. Moreover, speculative explanations were only available for a second increase in lipid peroxidation which was reported to occur after postischemic reperfusions exceeding 60 min. For this reasons, the present paper reports the results of investigation of ischemia/reperfusion injury to the cervical (CE) and thoracolumbal (ThL) segments of the spinal cord (SP) after an acute 25 min occlusion of the abdominal aorta, followed by 60-120 min reperfusion of the ischemic areas in rabbits. In CE and ThL segments of the SP, the ischemia induced: 1) a decrease in activities of superoxide dismutase (SOD), from 57.35+/-6.36 to 45.27+/-5.45 U x mg(-1) x min(-1) (S.E.M., 20.92%), p < 0.01, and from 58.36+/-5.45 to 33.00+/-4.55 U x mg(-1) x min(-1) (S.E.M., 43.46%), p < 0.001; 2) a significant decrease in gamma-glutamyl transpeptidase (gamma-GTP), from 114.66+/-1.45 to 99.88+/-4.4 micromol p-nitroaniline x mg(-1) x h(-1) (S.E.M. 12.89 %), p < 0.05 and from 112.24+/-1.20 to 95.09+/-2.40 micromol p-nitroaniline x mg(-1) x h(-1) (S.E.M., 16.26%), p < 0.05; 3) a considerable depression in Na,K-ATPase activity, from 7.14+/-0.58 to 5.08+/-0.32 micromol Pi x mg(-1) x h(-1) (S.E.M., 28.86%), p < 0.01, and from 7.23+/-0.11 to 5.09+/-0.31 micromol Pi x mg(-1) x h(-1) (S.E.M., 30.00%), p < 0.01. The Na,K-ATPase activity became decreased by ischemia and remained depressed significantly (all p < 0.01) throughout the experiment. After 60 min of reperfusion, SOD activity in the CE segment and that of gamma-GTP in the CE as well as ThL segments recovered, even slightly surpassing the control values, wheras SOD activity in the ThL segment became stabilized again close to its post-ischemic value. Prolonged, reperfusion for 120 min resulted in a further increase in gamma-GTP activity in the CE and ThL segments (to 132.79 and 132.30%, p < 0.01), and this was accompanied by a slight (p > 0.05) elevation in the content of conjugated dienes as well as by a new wave of depression of the SOD activity (p < 0.05) in both the CE and the ThL segment. From our results it could be concluded that all considerable damage to the spinal cord occurred during the ischemic period. In the period of reperfusion reparative changes started to predominate. This is in accordance with the recent discoveries indicating that, when coupled with an increase in tissue gamma-GTP activity, the post-ischemic reparative changes comprise a replenishment of the cell glutathione pool. This process is accompanied with a gradual increase in H2O2 production that results in repeatead inhibition of the SOD activity and a tendency to conjugated dienes formation.

Na,K-ATPase in the myocardium: molecular principles, functional and clinical aspects.

The role that Na,K-ATPase plays in Na+ and K+ antiport through the sarcolemma, in cation-homeostasis in cardiomyocytes as well as in excitation-contraction coupling and cell signalling in the myocardium is now widely recognized. It was its key importance for the cell membrane function that kept this enzyme intensively studied during the last three decades and finally brought to its discoverer the deserved Nobel Prize. Almost weekly are appearing new data concerning structure, function, regulation and role of the Na,K-ATPase in different physiological and pathological conditions. The special importance of the enzyme for heart function as well as the great amount of data that is concerned specifically with the heart Na,K-ATPase and accumulated since yet, started to call for setting them in order. The present paper updates basically important data on the cardiac Na,K-ATPase in relation to its specific properties, molecular mechanisms of function, mode of action, humoral and pharmacological modulation, adaptability, physiological role and clinical aspects.

High arrythmogenesis during early reperfusion of ischaemic myocardium: participation of oxygen free radicals.

The early period of reperfusion of ischaemic myocardium leads to a high incidence of severe tachyarrhythmias including ventricular fibrillation (VF), accompanied by a sudden transitional dysfunction. Oxygen free radicals (OFR) have been identified as one of the principal factors responsible for reperfusion-induced events. However, direct evidence for participation of OFR in the arrhythmogenic mechanisms upon reperfusion is still lacking. In the present study, in isolated Langendorff-perfused rat hearts subjected to 30 min global ischaemia, the onset of reperfusion induced 100% incidence of both ventricular tachycardia (VT) and VF with their gradual cessation during 5 min of reperfusion. Generation of H2O2 in the myocardium in the first minutes of reperfusion was demonstrated by means of cerium cytochemistry. There was an increased density of cerium perhydroxide precipitate distributed throughout the myocytes and endothelial cells, confirmed by X-ray microanalysis. The mechanism of the arrhythmogenic effect of OFR may involve the inhibition of the sarcolemmal Na+/K+ ATPase activity, as was revealed by subjecting the isolated sarcolemmal fraction of rat heart to the action of an oxy-radical generating system (H2O2 + FeSO4).

[Protective effect of 7-oxo-prostacyclin on myocardial calcium overload in the isolated rat heart]. / Protektívny úcinok 7-oxo-prostacyklínu na pret'azenie myokardu vápnikom v izolovanom srdci potkana.

The protective effect of a stable derivative of prostacyclin (7-oxo PGI2) was studied on the model of calcium overload (Ca2+ paradox) 48 h after i.m. administration of the drug in the dose of 50 micrograms/kg. In the isolated rat heart perfused at 37 degrees C and a constant perfusion pressure of 75 Torr (Langendorff preparation) Ca2+ paradox was induced by 3 min perfusion with calcium-free Krebs-Henseleit solution and a subsequent 10 min perfusion with a normal calcium-containing solution. The late protective effect of 7-oxo PGI2 was manifested by improved recovery of heart function (increase of contractility by 50%) and by better preservation of the content of macroergic phosphates (70% sigma ADN) during the Ca2+ repletion phase and of myocardial ultrastructure (sarcolemma) already during the Ca2+ depletion phase. The protective effect of 7-oxo PGI2 can be accounted for by stimulation of Na, K-ATPase activity, otherwise decreased during calcium depletion phase, and by the consequent prevention of alterations in sodium and calcium homeostasis. (Tab. 1, Fig. 4, Ref. 41.)

Mechanism of action of estradiol on sodium pump in sarcolemma from the myocardium.

Today it is accepted that estrogens mitigate the consequences of ischemic heart disease. Preliminary experiments revealed an increase in heart sarcolemmal (Na+ + K+)-ATPase activity after application of estradiol in vivo. It is also well known the key role of latter enzyme for heart function. The facts mentioned above indicate that estradiol may act on the heart just via modulation of the (Na+ + K+)-ATPase activity. In present paper it is confirmed that 17-beta-estradiol stimulates the activity of sarcolemmal (Na+ + K+)-ATPase by allosteric manner, particularly by increasing positive cooperativity between the K(+)-binding sites of the enzyme. This effect is manifested by enhancement in functional capacity of the sodium pump in sarcolemma. Stimulatory effect of estradiol is bound to integrated myocytes: neither is it manifested in isolated sarcolemma in vitro nor exhibits any influence on the affinity of binding sites for cardiac glycosides or on total capacity of the sarcolemma to bind ouabain. Basing on the data obtained it was assumed that estradiol acts on the (Na+ + K+)-ATPase not directly but by means of a mediator released within the myocyte.

Influence of calcium antagonists on heart sarcolemmal (Na+ + K(+)-ATPase.

The effect of calcium entry blocking agents nitrendipine and flunarizine on Mg(2+)-ATPase, (Mg2+ + Na(+)-ATPase, (Na+ + K(+)-ATPase and (Mg2+ + Ca(2+)-ATPase activities was studied. Nitrendipine (1 mumol/l-1) exerted a stimulatory effect on (Mg2+ + Na(+)-ATPase activity. Kinetic analysis of this effect revealed a two-fold rise in Vmax value and lowered Km value for activation of the enzyme by Na+ ions. In concentrations 10(-7) and 10(-5) mol.l-1 flunarizine behaved as a non-specific inhibitor of all sarcolemmal ATPases investigated. Nevertheless, in concentration of 10(-6) mol.l-1 flunarizine inhibited selectively the (Mg2+ + Na(+)-ATPase and (Na+ + K(+)-ATPase activities of myocardial sarcolemma. These observations provided evidence that both flunarizine and nitrendipine, in the concentration 10(-6) mol.l-1, modulate the (Mg2+ + Na(+)-ATPase and (Na+ + K(+)-ATPase activities in cardiac sarcolemma. These side effects of the above drugs particularly that of nitrendipine might have potential physiological relevance.