The effect of K+ concentration on the energy metabolism in perfused rat heart

From experiments at various perfusion pressures in hemoglobin-free perfused rat hearts, oxygen consumption and redox shift of pyridine nucleotide were found to vary linearly with cardiac work. This relation was used for analysis of the energy metabolism associated with ion pumps. Mechanical activities such as left ventricular pressure and heart rate varied with the extracellular K+ concentration. Ion-pump dependent changes in oxygen consumption and redox state of pyridine nucleotide, estimated as the difference of the values at normal (4.7 mM) and various other extracellular K+ concentrations with corrections for the change due to mechanical work, were found to vary linearly with the K+ concentration. The slope for oxygen consumption was about 0.1 mumol/min/g X wet wt per mM K+. Lactate release changed markedly but transiently, about 1 min after changing the extracellular K+ concentration, and its amount varied linearly with the K+ concentration. In the steady state, however, lactate release was almost independent of the extracellular K+ concentration, although oxidized pyridine nucleotide increased with increasing K+ concentration. Coronary flow increased with the extracellular K+ concentration. Heart rate changed little between 1 and 12 mM K+, but decreased sharply above 12 mM K+. At 20 mM K+, heart beat was arrested and approximately 40% of myoglobin was deoxygenated. The intracellular oxygen concentration was estimated to be about 10 microM even during aerobic perfusion. Similarly, Ca2+-free arrested heart was found to be in a hypoxic state. The results showed that oxygen entry into cardiac tissue is facilitated by the cardiac cycle.     

Contribution of an electrogenic pump to the resting membrane polarization in a crustacean heart.

1. In the neurogenic heart of the isopod crustacean Porcellio dilatatus, external K+ removal depolarized the membrane (K0 effect) whereas subsequent restoration of K+ resulted in a rapid hyperpolarization (K1 effect). 2. The amplitude of the K1 effect depended on the duration of the prior K+ deprivation and on the subsequent K+ concentration. 3. The membrane resistance slightly increased during the K0 effect; during the K1 effect, it only returned to its control value. 4. Ouabain, cooling and replacement of external Na+ by Li+ also produced depolarization. 5. The K1 effect was suppressed by ouabain and markedly depressed by lowering the temperature to 4-6 degrees C. It was abolished if Li+ replaced Na+ during the prior privation of K+; moreover Li+ was unable to act as a substitute for external K+ in generating the K1 effect if used at equivalent concentration, but enhanced the effect at high concentration. 6. The findings are consistent with the presence of an electrogenic sodium pump in the myocardium of Porcellio contributing to the resting membrane potential. 7. Changes in the spontaneous rhythm observed during K0 and K1 are further suggestive of the presence of an electrogenic Na+ pump in the pacemaker neurons of the cardiac ganglion. Another explanation is also proposed. 8. The magnitude of the spontaneous contractions of the heart was increased during the K0 effect and markedly decreased during the K1 effect. An indirect effect of the changes in internal Na+ concentration on the contractile processes is suggested.     

Training increases the concentration of [3H]ouabain-binding sites in rat skeletal muscle

Exercise is associated with a net loss of K+ from the working muscles and an increased plasma K+ concentration, indicating that the capacity for intracellular reaccumulation of K+ is exceeded. Training reduces the exercise-induced rise in plasma K+, and an increased plasma [K+] may interfere with physical performance. Since the clearing of K+ from the extracellular space depends on the capacity for active K+ uptake in skeletal muscle, the effects of training and inactivity on the total concentration of (Na+ + K+)-ATPase was determined. Following 6 weeks of swim training, the concentration of [3H]ouabain-binding sites in rat hindlimb muscles was up to 46% (P less than 0.001) higher than in those obtained from age-matched controls. Whereas muscle Na+, K+ contents remained unchanged, the concentration of citrate synthase increased by up to 76% (P less than 0.001). Training induced no change in the [3H]ouabain-binding-site concentration in the diaphragm, but in the heart ventricles, the K+-dependent 3-O-methylfluorescein phosphatase activity increased by 20% (P less than 0.001). Muscle inactivity induced by denervation, plaster immobilisation or tenotomy reduced the [3H]ouabain-binding-site concentration by 20-30% (P less than 0.02-0.001) within 1 week. In conclusion, training leads to a significant and reversible rise in the concentration of (Na+ + K+)-ATPase in muscle cells. This may be of importance for the beneficial effects on physical performance by improving the maximum capacity for K+ clearance.     

Dependence of myocardium injury on extracellular K+ concentration during calcium paradox

It is well-known that the first stage of the calcium paradox involves decreasing of Na+ gradient. The decreased sodium gradient is a cause of activation of the Na(+)-Ca+ exchange and formation of cardiac injury during the calcium repletion. Potassium ions are natural extracellular activators of Na(+)-pump. It has been shown that heart perfusion by Ca(2+)-free medium evoked extrusion from cells of hydrophilic amino acids whose transport-depends on sodium gradient. The heart reperdusion with Ca(2+)-containing agent leads to myofibrillar contracture and extensive myoglobin release. The simultaneous events are: elevation in tissue water contents, decreasing of intracellular concentration of adeninnucleotides, uncoupling of oxidation and phosphorylation in mitochondria. The decreasing of K+ level to 0.5 mM exacerbates myocardial damage during the calcium paradox, despite absence of myocardial contracture. The elevation of K+ (to 10 mM or 20 mM) attenuated the calcium paradox development in the heart. The elevated K+ concentration protected isolated heart from extensive myoglobin release, development of myocardial contracture. The high K+ concentrations alleviate mitochondrial damage and elevate contents of adeninnucleotide in the tissue. The positive effect of the elevated K+ concentration can be completely blocked by strophanthine, the selective Na+, K(+)-pumb blocker.     

Effect of perfusate potassium concentration on the contraction rate of the isolated rat heart during profound hypothermia

Effect of different concentration of K+ in perfusion fluid ([K+]) (5.9 mM, 3.6 mM, 2.38 mM) and the heart temperatures of 20 degrees C and below on the rat heart rate in the Langendorf preparations, were examined in conditions of retrograde perfusion with a modified Krebs-Henseleit buffer at constant perfusion volume. The lowering of [K+] diminished the temperature/heart rate ratio and depressed the heart standstill temperature from 12.3 +/- 0.6 degrees C at [K+] 5.9 mM (n = 12) to 6.7 +/- 0.6 degrees C at [K+] 3.6 mM (n = 5) and to 2.24 +/- 0.40 degrees C at [K+] 2.38 mM (n = 5). Temperature of the cold heart standstill had the liner relationship to Ig[K+]. Change the perfusion fluid with 5.9 mM K+ after heart cold standstill by the perfusion fluid with 3.6 mM K+ restored the heart beats to the rate of 40-50 min-1 in some experiments. The second heart standstill was at the mean temperature 3.6 degrees C lower than the first one.     

Long chain fatty acid inhibition of sodium plus potassium-activated adenosine triphosphatase from rat heart.

Long chain fatty acids were found to inhibit (Na+ + K+)-ATPase prepared from rat heart. Unsaturated and polyunsaturated fatty acids were more inhibitory than saturated fatty acids with myristic acid being the most inhibitory saturated fatty acid tested and linoleic the most inhibitory unsaturated fatty acid. As an example of fatty acid modification of the enzyme, inhibition of (Na+ + K+)-ATPase by oleate was examined. When compared to ouabain, inhibition of (Na+ + K+)-ATPase by oleate was found to be similar in that both were dependent on K+ concentration, but, in contrast to the almost instantaneous inhibition by ouabain, oleate inhibition was a slow process requiring over 20 min incubation at 37 degrees to produce maximum inhibition. Inhibition of rat heart (Na+ + K+)-ATPase by oleate was found to be readily reversible by washout. In the presence of albumin an oleate/albumin molar ratio greater than 7.5 was required for inhibition to occur. The activity of rat heart (Na+ + K+)-ATPase had a temperature optimum above 40 degrees and a discontinuous Arrhenius' plot with a transition temperature of 25 degrees. In the presence of oleate, however, the enzyme's optimum temperature decreased to below 40 degrees, the activation energy of the reaction at temperatures below 25 degrees was lowered from 24.7 kcal/mol to 12.6 kcal/mol and the enzyme had a linear Arrhenius' plot. The possibility of in vivo inhibition of cardiac (Na+ + K+)-ATPase under conditions of elevated fatty acids is discussed.     

Magnesium: Effects on reperfusion arrhythmias and membrane potential in isolated rat hearts

Myocardial Na+,K+-ATPase was studied in patients with aortic valve disease, and myocardial Na+,K+- and Ca2+-ATPase were assessed in spontaneously hypertensive rats (SHR) and hereditary cardiomyopathic hamsters using methods ensuring high enzyme recovery. Na+,K+-ATPase was quantified by [3H]ouabain binding to intact myocardial biopsies from patients with aortic valve disease. Aortic stenosis, regurgitation and a combination hereof were compared with normal human heart and were associated with reductions of left ventricular [3H]ouabain binding site concentration (pmol/g wet weight) of 56, 46 and 60%, respectively (p < 0.01). Na+,K+ and Ca2+-ATPases were quantified by K+- and Ca2+-dependent p-nitrophenyl phosphatase (pNPPase) activity determinations in crude myocardial homogenates from SHR and hereditary cardiomyopathic hamsters. When SHR were compared to age-matched Wistar Kyoto (WKY) rats an increase in heart-body weight ratio of 75% (p < 0.001) was associated with reductions of K+- and Ca2+-dependent pNPPase activities (mol/min/g wet weight) of 42 (p < 0.01) and 27% (p < 0.05), respectively. When hereditary cardiomyopathic hamsters were compared to age-matched Syrian hamsters an increase in heart-body weight ratio of 69% (p < 0.001) was found to be associated with reductions in K+- and Ca2+-dependent pNPPase activities of 50 (p < 0.001) and 26% (p = 0.05), respectively. The reductions in Na+,K+- and Ca2+-ATPases were selective in relation to overall protein content and were not merely the outcome of increased myocardial mass relative to Na+,K+- and Ca2+-pumps. In conclusion, myocardial hypertrophy is in patients associated with reduced Na+,K+-ATPase concentration and in rodents with reduced Na+,K+- and Ca2+-ATPase concentrations. This may be of importance for development of heart f in hypertrophic heart disease.     

Changes in autorhythmic heart frequency elicited by redox agents

In isolated frog heart it was established that methylene-blue (MB, an oxidizing agent) decreased, while ascorbate (ASC, a reducing agent) increased the frequency of autorhythmic heart contractions. After MB treatment, in parallel with this phenomenon, the extracellular K+ concentration [K+]o showed a slow increase, but following ASC application a slow decrease occurred. Since these correlations are in good accordance with the idea that the pacemaking ability of heart, among other properties, depends on the voltage and time-dependent decrease in potassium conductance following the spike, changes in [K+]o might be one mechanism by which oxidizing and reducing agents modulate heart frequencies. On the basis of the effect of insulin (INS) and K-strophantoside (STR) on these modulatory influences, it is presumed that the changes in slow delta [K+]o transients might result, at least partly, from the effect of redox agents on the active transport system. In light of the increase in passive K+ fluxes after oxidant treatment and the decrease in this parameter following reductant treatment an effect of redox agents on the characteristics of the K+-channel is also postulated.     

Effect of prostaglandins on rat heart sarcolemmal ATPases

The ability of prostaglandins (PG) D2, E1, E2, F2 alpha and I2 (2.8 X 10(-11) to (2.8 X 10(-7) M) to modify Ca2+, Mg2+ and (Na+ + K+)-ATPase activities of rat heart sarcolemmal membrane fractions was examined. Administration of PGE2, PGF2 alpha, and PGI2 reduced basal (Na + + K+)-ATPase activity by up to 30, 80, and 80%, respectively. PGE1 and PGD2 were ineffective at any concentration. Neither Mg2+ -ATPase nor Ca2+ -ATPase was affected by PG treatment. Kinetic analysis revealed that the (Na+ + K+)-ATPase activity reducing ability of PGE2, PGF2 alpha and PGI2 was of a complex nature involving a reduction in Vmax and an elevation of the respective K values for either substrate or activator. These results demonstrate that some PG's are potent inhibitors of rat heart (Na+ + K+)-ATPase. These PG's produced varied inotropic influences on isolated heart preparations and it is uncertain whether their myocardial actions are dependent on enzyme inhibition.     

Ca2+-mediated activation of phosphofructokinase in perfused rat heart.

Perfusion of the isolated rat heart with Ca2+ concentrations exceeding 3 mM activated phosphofructokinase and phosphorylase, and decreased the concentration of cyclic AMP. Half-maximal activation of phosphofructokinase occurred at 5 mM-CaCl2; significant activation of phosphorylase did not occur until the concentration of CaCl2 exceeded 12 mM. The time course for the activation of phosphofructokinase at 12 mM-CaCl2 indicated that maximal activation occurred within 2 min; when the perfusion-medium Ca2+ concentration was re-adjusted to 3 mM, the phosphofructokinase activity returned to pre-activation values within 30 s. The addition of Ca2+ to extracts of heart did not activate phosphofructokinase. The activation of phosphofructokinase by sub-maximal doses of adrenaline and Ca2+ were not additive. The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol and by 1 microM-isoprenaline was inhibited by high concentrations of K+ (22-56 mM). The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol, 12 mM-CaCl2 and by 1 microM-isoprenaline was blocked by the slow Ca2+-channel blocker nifedipine. These findings suggest that both the beta- and alpha-adrenergic mechanisms for the activation of rat heart phosphofructokinase involve an increase in the myoplasmic Ca2+ concentration. This increase may result from an inhibition of Ca2+ efflux or a stimulation of Ca2+ influx.     

Role of K+, Na+, and Ca2+ ions in the cardiodepressive action of blood plasma in burn shock and the crush syndrome

Ion-selective electrodes were employed to measure the concentration of K+, Na+ and Ca2+ in blood plasma of rabbits with burn shock or crush syndrome (CS). No significant changes in the plasma concentration of Na+, and Ca2+ were found under both pathological conditions. The plasma concentration of K+ in burn shock significantly increased from 3.06 +/- 0.73 (control) to 5.28 +/- 2.65 mM (n = 10), whereas in CS from 3.42 +/- 1.03 to 4.92 +/- 1,29 mM (n = 8). The rise of K+ concentration in the control plasma to the maximal values seen in the "burn" and "syndrome" plasma led to an increase in the duration of intracellular action potentials (AP) but did not substantially change the amplitude of isometric contractions of the papillary muscles of rabbit heart. Meanwhile the similar rise of the duration of intracellular AP during perfusion of the papillary muscles with the "burn" and "syndrome" plasma was accompanied by an appreciable drop of the amplitude of isometric contractions. It is suggested that elevation of K+ concentration in blood plasma, inducing an increase in the duration of intracellular AP of cardiocytes may be responsible for changes in the ECG in burn and CS. At the same time inhibition of myocardial contractility in burn shock and CS is virtually not linked with hyperkalemia.     

Influence of pH and sodium on the inhibition of guinea-pig heart (Na+ + K+)-ATPase by calcium.

The inhibition of guinea-pig heart (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) by calcium has been studied at pH 7.4, 6.8 and 6.4. 1. A decrease in pH reduced the threshold inhibitory concentration of calcium and the calcium concentration producing an inhibition of 50% of the enzyme activity. 2. Calcium reduced the apparent affinity of the enzyme of Na+, this effect occurred only at pH 7.4. 3. Calcium increased the apparent affinity of the enzyme for K+, this effect was enhanced at acidic pH. 4. Activation of the enzyme by Na+ for a constant Na+ : K+ ratio has been studied at pH 7.4 and at pH 6.8 in the absence and in the presence of 3.10(-4) M Ca 2+; the results of this experiment indicate that Ca2+ effect at pH 7.4 was not influenced by Na+ -- K+ competition and was probably due to a Na+ -- Ca2+ interaction. 5. At pH 7.4, the calcium inhibitory threshold concentration and the concentration producing 50% inhibition were reduced when Na+ was low; at pH 6.8, the calcium inhibition was not markedly modified by the change of Na+ concentration. 6. The Ca2+ -activated ATPase of myosin B which is related to the contractile behaviour of muscle and the Ca2+ -ATPase of the sarcoplasmic reticulum which is related to the ability of this structure to accumulate calcium were activated in a range of calcium concentration producing an inhibition of (Na2+ + K+) -ATPase. The present results indicate that the increase by acidity of the (Na2+ + K+) -ATPase sensitivity to calcium might be due to a suppression of a Na+ -Ca2+ interaction. On the basis of these observations, it is proposed that calcium might inhibit the Na+ -pump during the repolarization phase of the action potential and that, by this effect, it might control cell excitability.     

Matrix magnesium and the permeability of heart mitochondria to potassium ion

Isolated beef heart mitochondria were treated with A23187 in the presence of different concentrations of Mg2+ or EDTA to establish varying levels of total mitochondrial Mg2+. The Mg2+ content was related to the rate of passive swelling of the mitochondria in potassium acetate and other potassium salts in which swelling is presumed to depend on K+ entry via an endogenous K+/H+ antiport. Swelling in these salts does not commence until Mg2+ has been depleted from an initial value of 36 nmol X mg-1 of protein to 8 nmol/mg-1, or less. Below this level, swelling increases linearly with decreasing Mg2+ to a maximum rate at 2 nmol of Mg2+ X mg-1. Rotenone-treated heart mitochondria suspended in 75 mM potassium acetate at pH 7.80 show no delta pH by 5,5-dimethyl-2,4-oxazolidinedione distribution. Distribution of methylamine also shows essentially no delta pH under these conditions when allowance is made for binding of [14C]methylamine by mitochondrial membranes under these conditions. Addition of A23187 results in a small and transient delta pH (delta pH less than 0.14, acid interior) as measured by methylamine distribution. Estimation of the maximum matrix free Mg2+ concentration from the maximum delta pH observed and the external free Mg2+ concentration at equilibrium with A23187 shows that swelling is not initiated until matrix free Mg2+ is decreased to below 150 microM. An independent estimate of free Mg2+ using a null-point procedure gives a lower, but quite similar value (50 microM) for maximum matrix free Mg2+ when swelling commences. The large depletion of total and free Mg2+ that is required to activate swelling in potassium acetate (and presumably K+/H+ antiport activity) does not appear to be compatible with previous indications that free Mg2+ acts as a "carrier brake" to regulate K+ extrusion from the mitochondrion on such an antiport (Garlid, K. D. (1980) J. Biol. Chem. 255, 11273-11279). The removal of a tightly bound component of mitochondrial Mg2+ is closely related to increased K+ permeability and increased passive swelling in potassium salts. This Mg2+ appears to play a role in the maintenance of mitochondrial membrane structure and integrity.     

Structural aspects of the sarcoplasmic reticulum K+ channel revealed by gallamine block.

We have studied single-channel conductance fluctuations of K+ channels present in the sarcoplasmic reticulum (SR) membrane systems of rabbit cardiac and skeletal muscle. K+ conductance through the channels is reversibly blocked by gallamine. Conductance block occurs only from the trans side of the channel and is resolved as a smooth reduction in the open state conductance. At a fixed K+ concentration, conduction decreases with increasing gallamine concentration and the data can be fitted to a single-site inhibition scheme. The degree of block seen at a constant gallamine concentration decreases as K+ concentration is increased, indicating competition between gallamine and K+. Gallamine block is voltage dependent, the degree of block increasing with increasing negative holding potential. Quantitative analysis of block yields a zero voltage dissociation constant of 55.3 +/- 16 microM and an effective valence of block of 0.93 +/- 0.12. We conclude that gallamine blocks by interacting with a site or sites located at an electrical distance 30-35% into the voltage drop from the trans side of the channel. This site must have a cross-sectional area of at least 1.2 nm2. The results of this study have been used to modify and extend our view of the structure of the channel's conduction pathway.     

Inhibition of cardiac Na+, K+-ATPase activity by dynorphin-A and ethylketocyclazocine

The effect of various opioids on Na+, K+ -ATPase partially purified from rat heart was examined. Dynorphin-A (1-13), dynorphin-A (1-17) and ethylketocyclazocine (EKC), which are k-type opiate agonists, markedly inhibited the enzyme activity in a dose-dependent manner; IC50 values were 12 microM, 21 microM and 0.38 mM, respectively. Morphine (mu-type agonist), methionine- and leucine-enkephalin (delta-type agonist) at the concentration of 1 mM did not affect the enzyme activity. The effect of dynorphin-A (1-13) and EKC was not antagonized by naloxone. Dynorphin-A (1-13) mainly decreased Vmax value without the change of Km value in the activation of Na+, K+-ATPase by ATP, Na+ and K+. Dynorphin-A(1-13) inhibited the partial reactions of Na+, K+-ATPase at the different degree of the potency; the inhibition of K+-stimulated phosphatase was greater than that of Na+-dependent phosphorylation. The present study suggests that dynorphin-A and EKC have an effect on cardiovascular system which is mediated by the inhibition of Na+, K+-ATPase in the heart.     

23Na and 39K nuclear magnetic resonance studies of perfused rat hearts. Discrimination of intra- and extracellular ions using a shift reagent.

High-resolution 23Na and 39K nuclear magnetic resonance (NMR) spectra of perfused, beating rat hearts have been obtained in the absence and presence of the downfield shift reagent Dy(TTHA)3- in the perfusing medium. Evidence indicates that Dy(TTHA)3- enters essentially all extracellular spaces but does not enter intracellular spaces. It can thus be used to discriminate the resonances of the ions in these spaces. Experiments supporting this conclusion include interventions that inhibit the Na+/K+ pump such as the inclusion of ouabain in and the exclusion of K+ from the perfusing medium. In each of these experiments, a peak corresponding to intracellular sodium increased in intensity. In the latter experiment, the increase was reversed when the concentration of K+ in the perfusing medium was returned to normal. When the concentration of Ca2+ in the perfusing medium was also returned to normal, the previously quiescent heart resumed beating. In the beating heart where the Na+/K+ pump was not inhibited, the intensity of the intracellular Na+ resonance was less than 20% of that expected. Although the data are more sparse, the NMR visibility of the intracellular K+ signal appears to be no more than 20%.     

Contamination of a cardiac sarcolemmal preparation with endothelial plasma membrane

Preparation of sarcolemma from whole rabbit heart using the method of Jones et al. (Jones,L.R., Besch, H.R., Fleming, J.W., McConnaughey, M.M. and Watanabe, A.M. (1979) J. Biol. Chem. 254, 530-539) results in a 46-fold purification of the endothelial plasmalemma-specific marker angiotensin converting enzyme. This implies contamination of the sarcolemma with vascular endothelial plasmalemma. During preparation of sarcolemma from sheep heart, using the same method, angiotensin converting enzyme copurified with the general plasma membrane marker (Na+ + K+)-ATPase. The ratio of myocyte to endothelial plasma membrane in the final preparation is therefore similar to that in the whole heart homogenate. Ultrastructural analysis has shown that the myocyte/endothelial surface area is 70:30 in whole cardiac muscle. Comparison of angiotensin converting enzyme activity of an endothelial plasma membrane fraction with that of whole heart sarcolemma suggests an upper limit of 42% for endothelial contamination. Contamination by endothelial plasmalemma was dramatically reduced by preparing sarcolemma from myocytes produced by proteolytic disruption of whole hearts. Following disruption, myocytes were separated from non-muscle cells by sedimentation through 0.5 M sucrose. Sarcolemma prepared from sheep cardiac myocytes had approximately 15-fold less angiotensin converting enzyme activity than whole sheep heart sarcolemma but comparable ouabain-inhibitable (Na+ + K+)-ATPase activity.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells.

The K+ content and the K+ flux were measured in the cell lines ME2 and MF2 isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the seem K+ content and the same K+ steady state flux per unit of surface area. In ME2 cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K+ net movement. The (Na+ plus K+)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K+ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells. The K+ net flux is considerable increased when the intracellular K+ level is reduced after preincubation of the cells in a K+ -free medium. Thus, internal K+ seems to regulate the K+ influx.     

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.     

Effects of ATP on the intermediary steps of the reaction of the (Na+ + K+)-ATPase. IV. Effect of ATP on K0.5 for Na+ and on hydrolysis at different pH and temperature.

The pH optimum for (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) depends on the combination of monovalent cations, on the ATP concentration and on temperature. ATP decreases the Na+ concentration necessary for half maximum activation, K0.5 for Na+ (Na+ + K+ = 150 mM), and the effect is pH and temperature dependent. At a low ATP concentration a decrease in pH leads to an increase in K0.5 for Na+, while at the high ATP concentration it leads to a decrease. K0.5 for ATP for hydrolysis decreases with an increase in pH. The fractional stimulation by K+ in the presence of Na+ decreases with the ATP concentration, and at a low ATP concentration K+ becomes inhibitory, this being most pronounced at 0 degrees C. The results suggest that (a) ATP at a given pH has two different effects: it increases the Na+ relative to K+ affinity on the internal site (K0.5 for ATP at pH 7.4, 37 degrees C, is less than 10 microM); it increases the molar activity in the presence of Na+ + K+ (K0.5 for ATP at pH 7.4, 37 degrees , is 127 microM), (b) binding of the cations to the external as well as the internal sites leads to pK changes (Bohr effect) which are different for Na+ and for K+, i.e. the selectivity for Na+ relative to K+ depends both on ATP and on the degree of protonation of certain groups on the system, (c) ATP involves an extra dissociable group in the determination of the selectivity of the internal site, and thereby changes the effect of an increase in protonation of the system from a decrease to an increase in selectivity for Na+ relative to K+.