A comparison of inotropic actions of p-chloromercuribenzoate, ouabain, digitoxin and digitoxigenin

We have shown previously that sulfhydryl blocking agents, such as p-chloromercuribenzoate (PCMB), produce positive inotropic effects associated with inhibition of sarcolemmal Na⁺,K⁺ -ATPase or the sodium pump. Since these compounds and digitalis derivatives bind to the enzyme at the internal and external surface of the cell membrane, respectively, the difference in sites of interaction with Na⁺,K⁺ -ATPase may result in the development of the inotropic effect to be affected differently by conditions which alter properties of the cell membrane. Thus, inotropic actions of these agents were compared in the electrically stimulated left atrial preparation of guinea-pig heart. Propranolol delayed the development of the positive inotropic action PCMB to a greater extent than that of ouabain, digitoxin or digitoxigenin. The action of propranolol is due to membrane stabilization, rather than β-adrenoceptor blockade. The maximum inotropic effect achieved was not affected by propranolol. The onset of action of PCMB was influenced to a greater extent by the beat interval than was ouabain whose action was affected by the interval more than was digitoxin or digitoxigenin. The onset rate of the inotropic action of PCMB was slower and slower and that of ouabain or digitoxin was more rapid at higher temperature. The onset rate of the action of digitoxigenin was relatively temperature insensitive. The initial force of contraction is not the sole determinant of the onset rate of the action of PCMB, since a simultaneous decrease in extracellular sodium and calcium concentration delayed the onset rate without altering the developed force. Moreover, a decrease in the force of contraction caused by lower resting tension did not alter the onset rate. Thus, several conditions which affect properties of the cell membrane alter the onset rate of the inotropic action of PCMB to a greater extent than those of actions of digitalis derivatives; a finding which is consistent with the requirement of PCMB to penetrate the cell membrane for its action.     

Effects of lithium and thallous ions on sodium pump activity in the guinea-pig heart and their relationship to the positive inotropic action

Summary It has recently been demonstrated that both Tl⁺ and Li⁺ produce concentration-and time-dependent positive inotropic effects in guinea-pig atrial preparations although Tl⁺ inhibits and Li⁺ stimulates isolated Na⁺, K⁺-ATPase in vitro. In order to elucidate the mechanism of the positive inotropic actions of these cations, the effects of Tl⁺ and Li⁺ on sodium pump activity were studied. Active ⁸⁶Rb uptake in guinea-pig ventricular slices, an estimate of sodium pump activity, was highly sensitive to the inhibitory effect of the cardiac glycosides. Preincubation of slices with Tl⁺ caused a dose-and time-dependent inhibition of active ⁸⁶Rb uptake. Similar concentration-and time-dependent inhibition of active ⁸⁶Rb uptake was observed when Na⁺ in a Krebs-Henseleit solution was partially replaced with Li⁺. Lithium, however, stimulated a partially purified Na⁺, K⁺-ATPase in vitro. During heart slice incubation, Tl⁺ and Li⁺ accumulated in a time-dependent manner. This accumulation was not readily reversible when slices were transferred into Tl⁺-or Li⁺-free solutions. It appears that the inhibition of sodium pump activity is related to the positive inotropic action of these cations.This work was supported by United States Public Health Service Grants HL-16052 and HL-16055 and by the Michigan Heart Association. A part of this study was presented at the 60th Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, California, April 1976     

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.     

A possible effect of sulfhydryl reagents on the contractile activity of the rat detrusor muscle

We aimed to investigate the effect of sulfhydryl (SH) inactivating agents, ethacrynic acid and N-ethylmaleimide, on the contractile activity of rat detrusor muscle. Wistar Kyoto rats weighing 150-250 g were anaesthetized with ketamine and bled to death. The urinary bladders were surgically removed and detrusor strips were mounted under 0.5 g tension in organ baths. The responses were recorded with isotonic transducers on polygraph paper. After an equilibrium period, the tissues were contracted by electrical field stimulation, acetylcholine, ethacrynic acid or N-ethylmaleimide and the effects of l-cysteine, glutathione, verapamil, Ca²⁺-free solution, sodium nitroprusside or atropine were then examined on these contractions. Verapamil, Ca²⁺-free solution or atropine significantly reduced the contractions elicited by electrical field stimulation and acetylcholine whereas l-cysteine, glutathione or sodium nitroprusside had no effect on the contractions in response to these stimuli. l-Cysteine, glutathione, verapamil or Ca²⁺-free solution significantly inhibited the contractions induced by ethacrynic acid or N-ethylmaleimide. Sodium nitroprusside slightly inhibited only the contraction induced by ethacrynic acid but not that with N-ethylmaleimide. Atropine has no action on the contractions in response to these SH reagents. These findings suggest that SH reagents may play a role in the contractile activity of rat detrusor muscle and this action seems to be related to the gating of Ca²⁺ channels. Further experiments are needed to determine the cellular mechanism(s) of action by which these SH reagents act on the detrusor smooth muscle.     

Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats

Summary Stimulation of ₁-adrenoceptors evokes a different pattern of inotropic responses in atrial and ventricular heart muscle preparations from rats. The inotropic effects are accompanied by different changes in membrane potential. In an attempt to clarify the question whether or to which extent these events are causally related, the effects of phenylephrine on force of contraction, transmembrane potential, Ca²⁺ current (I_Ca) and K⁺ currents were comparatively studied in either tissue.In atrial preparations, phenylephrine 10 mol/l caused an increase in force of contraction, a marked prolongation of the action potential duration and a depolarization of the membrane at rest. In the ventricle, however, the addition of phenylephrine 10 mol/l produced first a decline in force of contraction associated with a hyperpolarization of the membrane and a reduction in the action potential duration. These changes were followed by an increase in force,of contraction and a slight prolongation of the action potential, whereas the resting membrane potential remained increased. The hyperpolarization was eliminated in the presence of ouabain 100 mol/l.In enzymatically isolated atrial and ventricular myocytes, the whole-cell voltage clamp technique was used to study membrane currents on exposure to phenylephrine. Phenylephrine 30 mol/l did not affect the magnitude of I_Ca in either cell type. Transient and steady state K⁺ outward currents, however, were significantly diminished to a similar extent in atrial and in ventricular myocytes.It is concluded that the positive inotropic effect of ₁-adrenoceptor stimulation in the rat atrium is related to an increase in action potential duration and a decrease in resting membrane potential due to a decrease in K⁺ currents. In the ventricle, phenylephrine additionally activates the Na⁺/K⁺ pump thereby hyperpolarizing the membrane. The rapid onset of pump stimulation seems to overwhelm, in the beginning, the phenylephrine-induced decrease in K⁺ conductance and therefore to evoke a transient negative inotropic effect.It is assumed that phenylephrine can alter the intracellular Ca²⁺ concentration due to changes in the action potential duration. The way how Ca 2+ enters the cell remains speculative, since direct changes of Ica were not detected. The more complicated changes in membrane potential in the ventricle suggest that also other mechanisms for the positive inotropic response to phenylephrine must be considered. Send offprint requests to H. Nawrath at the above address     

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.     

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.     

Na+/K+-ATPase α subunits as new targets in anticancer therapy

Background: The sodium pump (Na⁺/K⁺-ATPase) could be a target for the development of anticancer drugs as it serves as a signal transducer, it is a player in cell adhesion and its aberrant expression and activity are implicated in the development and progression of different cancers. Cardiotonic steroids (CS) are the natural ligands and inhibitors of the sodium pump and this supports the possibility of their development as anticancer agents targeting overexpressed Na⁺/K⁺-ATPase α subunits. Objectives: To highlight and further develop the concept of using Na⁺/K⁺-ATPase α1 and α3 subunits as targets in anticancer therapy and to address the question of the actual usefulness of further developing CS as anticancer agents. Conclusions: Targeting overexpressed Na⁺/K⁺-ATPase α subunits using novel CS might open a new era in anticancer therapy and bring the concept of personalized medicine from aspiration to reality. Clinical data are now needed to further support this proposal. Furthermore, future medicinal chemistry should optimize new anticancer CS to target Na⁺/K⁺-ATPase α subunits with the aim of rendering them more potent and less toxic.     

<Emphasis Type="Italic">N</Emphasis>-Ethylmaleimide differentially inhibits substrate uptake by and ligand binding to the noradrenaline transporter

Using transfected HEK293 cells that express the human (h) noradrenaline transporter (hNAT), we show differential inhibitory effects of the thiol reagent N-ethylmaleimide (NEM) on [³H]NA uptake and [³H]nisoxetine binding. Irreversible inhibition of uptake by NEM was complete, faster, and occurred at lower concentrations. Furthermore, hNAT ligands (substrates and inhibitors) prevented NEM-induced inhibition of binding but not that of uptake, indicating different underlying mechanisms of inhibition. NEM-induced uptake inhibition was not primarily due to inhibition of the Na⁺/K⁺-ATPase since ouabain caused only partial inhibition. For the first time, we show that NEM at low concentrations causes a rapid and complete depletion of cellular adenosine triphosphate (ATP) not only in HEK293 cells but also in several other eukaryotic cell lines. Thus, while high NEM concentrations alkylate the NAT protein in a ligand-protectable manner, low concentrations inhibit substrate uptake through a loss of the Na⁺ and K⁺ gradient as a driving force by depleting cellular ATP.     

Die Haftung verschiedener Cardenolide am Papillarmuskel und einer mikrosomalen ATPase des Meerschweinchenherzens

Summary In experiments on isolated electrically stimulated guinea pig papillary muscles and on isolated cardiac Na⁺-K⁺-activated ATPase preparations the action and the reversibility of action of 3 different cardenolides-digitoxin, k-strophanthidin and strophanthidin-3-bromoacetate (SBA) (supposed to be an irreversible inhibitor of the transport ATPase)-were studied.The equieffective concentrations for maximum positive inotropic effects (around 90%) were 2×10^–6, 2×10^–5 and 4×10^–5 M, respectively. In washout experiments the positive inotropic action of all these substances was found to be completely reversible: the rates of decline of the positive inotropic effects were about 2.7%/min with digitoxin, 24%/min with strophanthidin and 22%/min respectivety 5.7%/0/min (two components) with SBA.The equieffective concentrations for maximum inhibition (90–95%) of the Na⁺-K⁺-activated ATPase by digitoxin, strophanthidin and SBA were 10^–4, 2×10^–4 and 10^–4 M respectively. In washout experiments (repeated centrifugations) different degrees of reversibility of these inhibitory effects were observed depending upon the experimental conditions. Preincubation of the enzyme with the cardenolides in theabsence of Na⁺, Mg²⁺ and ATP resulted in a persisting inhibition of the Na⁺-K⁺-ATPase of 14% with digitoxin, 10% with k-strophanthidin and- significantly higher (p < 0.05)-33% with SBA. Corresponding experiments with preincubation of the enzyme in thepresence of Na⁺, Mg²⁺ and ATP, however, demonstrated a full reversibility of the inhibitory action of all these substances.These results are in contrast, in certain respects, with those obtained in previous experiments on brain ATPase.It is concluded that SBA is able to inhibit irreversibly only the non-phosphorylated form of the cardiac Na⁺-K⁺-activated ATPase, whereas the phosphorylated intermediate of this enzyme seems to be protected against the irreversible inhibition by this substance. Assuming that the latter state of the enzyme is predominant in the intact heart muscle cell, a complete reversibility of the pharmacological action of SBA would be expected if the inotropic effect is mediated by an inhibition of the enzyme. Our results are compatible with this hypothesis.

Wir danken der Deutschen Forschungsgemeinschaft für die Unterstützung durch Sachbeihilfen und der Volkswagenstiftung für die Geräteausstattung.     

Quabain: Temporal relationship between the inotropic effect and the in vitro binding to,and dissociation from, (Na++K+)-activated ATPase

Summary The time course of the inotropic response to ouabain in Langendorff preparations was compared with that of the in vitro ATP-dependent (³H)-ouabain binding to cardiac (Na⁺+K⁺)-activated ATPase preparations, and subsequent dissociation, to determine the temporal relationship between the inotropic response and (Na⁺+K⁺)-activated ATPase inhibition.Species differences were minimal either in the onset of inotropic response or the (³H)-ouabain binding. The rates of both loss of the inotropic response to ouabain during washout and the dissociation of the ouabain-enzyme complex, however, were rapid in guinea pig and rabbit (relatively ouabain-insensitive species) and slow in cat and dog (ouabain-sensitive species). The half-time of the loss of the inotropic response was similar to the half-time of the dissociation of the ouabain-enzyme complex in each species.Since ATP-dependent binding of cardiac glycosides has been related to enzyme inhibition, it was concluded that the time course of the inotropic response to ouabain parallels the time course of (Na⁺+K⁺)-activated ATPase inhibition, and that the dissociation of ouabain from the enzyme may terminate the inotropic response.A part of this study was presented at the Fifth Annual Meeting of the International Study Group for Research in Cardiac Metabolism, Winnipeg, Manitoba, June, 1972.     

Discrimination by N-ethylmaleimide between the chronotropic and inotropic response to muscarinic receptor stimulation in rat atrium

Summary N-ethylmaleimide (NEM) rapidly blocked the negative chronotropic effect of carbachol on rat right atrium. In contrast, NEM did not reduce the negative inotropic response to muscarinic (M) receptor stimulation. Carbachol inhibited the specific binding of [³H]-N-methylscopolamine ([³H]-NMS) to membranes of rat atria as reflected by a shallow inhibition curve. Both guanosine triphosphate (GTP) and NEM shifted the [³H]-NMS inhibition curves of carbachol to the right. Pretreatment of the atrial membranes with NEM abolished the GTP-induced rightward shift. However, when instead of the membranes the intact atria were pre-incubated with NEM, no interaction between NEM and GTP in the membranal preparation was observed. The results indicate that NEM sharply discriminated between the inotropic and chronotropic effects to M-receptor stimulation in rat atria. The inhibitory effect of NEM on the M-receptor-mediated negative chronotropic effect in rat atrium cannot be explained by an interaction of the sulfhydryl reagent with GTP-binding proteins, like N_i or N_o.     

Amiloride analogues induce responses in isolated rat cardiovascular tissues by inhibition of Na+/Ca2+ exchange

Summary The role of inhibition of Na⁺/Ca²⁺ exchange in the positive inotropic, negative chronotropic and vasorelaxant responses to amiloride and some of its analogues was investigated in isolated cardiovascular tissues from female Wistar rats. The compounds tested were amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, a potent inhibitor of Na⁺/H⁺ exchange), phenamil and 2,4-dimethylbenzamil (DMB), both potent Na⁺ channel inhibitors with activity against Na⁺/Ca²⁺ exchange, and 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil (CBDMB), a potent inhibitor of Na⁺/Ca²⁺ exchange with reduced activity against Na⁺ channels compared with its parent compound DMB.Phenamil, DMB and CBDMB increased the force of contraction of right ventricular papillary muscles with similar potencies (-log EC₅₀ values: 4.77 ± 0.06, 5.09 ± 0.09, 4.97 ± 0.17 respectively), while amiloride and EIPA gave small negative inotropic responses. All compounds gave negative chronotropic responses at similar concentrations to those which exerted inotropic effects. Inhibition of KCl contraction of endothelium-free aortic rings was observed with all compounds tested. Phenamil, DMB and CBDMB but not amiloride or EIPA showed a shift to the left of the concentration-response curves in the presence of intact endothelium.These results provide further evidence for positive inotropic and endothelium-dependent vasorelaxant effects of amiloride analogues mediated by inhibition of Na⁺/Ca²⁺ exchange. Send offprint requests to J. R. Bourke at the above address     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Correlation between inhibition of (Na+, K+)-membrane-ATPase and positive inotropic activity of cardenolides in isolated papillary muscles of guinea pig

Summary Concentrations of 17 cardenolides, cardenolide glucuronides and sulfates producing halfmaximal inhibition of (Na⁺, K⁺)-membrane-ATPase from different organs and animal species were determined in vitro. In addition the concentrations that increased the contractility of guinea pig isolated papillary muscles to a particular level were investigated. Comparisons between ATPase-inhibiting and positive inotropic cardiac activities showed extensive parallelism: the correlation coefficients after log/log transformation were between 0.92 and 0.97. The same close correlations are found if dissociation constants of cardenolide receptor complexes and concentrations causing ⁸⁶Rb-uptake inhibition in human erythrocytes are examined.The concentrations necessary for inhibition of (Na⁺, K⁺)-membrane-ATPase of the guinea pig heart and the concentrations required to achieve a defined positive inotropic effect in guinea pig papillary muscle showed a log/log correlation coefficient of 0.97 (P<0.001). In both tests the potencies covered more than three orders of magnitude. The results support Repke's hypothesis on the digitalis receptor.     

Effects of sulphydryl inhibitors on frog sartorius muscle: P-chloromercuribenzoic acid and p-chloromercuribenzenesulphonic acid

1. Experiments were done on frog sartorius muscles to study the effects and mechanisms of action of the -SH inhibitors, p-chloromercuribenzoic acid (PCMB) and p-chloromercuribenzenesulphonic acid (PCMBS).

2. Both organomercurials produce a depolarization of the surface membrane which is associated with a period of asynchronous twitching and followed by inexcitability.

3. Only PCMB produces a unique fractionation of the electrically evoked twitch into an initial rapid and later slow phase.

4. PCMB and PCMBS increase the rate of ⁴⁵Ca efflux from whole muscle. Ethylenediamine tetraacetic acid (EDTA, 5 mM) causes only limited antagonism of the enhancement of ⁴⁵Ca efflux produced by PCMB whereas it completely antagonizes this same effect of PCMBS. EDTA selectively removes superficial calcium without penetrating into the intracellular space.

5. The results suggest that PCMB inhibits -SH groups in the terminal cisternae causing a fractionation of the twitch. PCMBS acts primarily at surface sites with limited access to the cisternae and sarcoplasmic reticulum.

     

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.     

Abnormal activation of Na<Superscript>+</Superscript>-K<Superscript>+</Superscript> pump in aortas from rats with endotoxaemia

A diminished reactivity to several vasoconstrictor agents is usually observed in blood vessels obtained from animals with endotoxic shock. The contractile state of vascular smooth muscle is influenced by the activity of the electrogenical sodium (Na⁺-K⁺) pump. Thus, we examined inhibitors and agonists of nitric oxide (NO)-guanosine 3':5'-cyclic monophosphate (cGMP) on contractions to phenylephrine (PE) and relaxations to potassium in isolated aortic segments from rats treated with bacterial endotoxin (lipopolysaccharide, LPS) for 6 h (i.e. to mimic a shock syndrome). Endotoxaemia for 6 h was associated with a severe hypotension and vascular hyporeactivity to noradrenaline and an increased plasma nitrate level in vivo. The PE-induced contraction was attenuated in aortic smooth muscle obtained from rats with endotoxic shock while the potassium-induced relaxation was greater in these preparations. Ouabain dose-dependently inhibited the potassium-induced relaxation in aortas from normal and endotoxaemic rats. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one enhanced the PE-induced contraction in endotoxaemic rats only, whereas it attenuated the difference of potassium-induced relaxation between normal and endotoxaemic rats. In contrast, in aortas obtained from normal rats, 8-bromo-cGMP reduced the PE-induced contraction and enhanced the potassium-induced relaxation to the level as seen in endotoxaemic animals. In aortas obtained from endotoxaemic rats, methylene blue further restored the PE-induced contraction to the normal and abolished the difference of potassium-induced relaxation between normal and endotoxaemic rats. These results suggest that the Na⁺-K⁺ pump in the vascular bed of animals with endotoxic shock is abnormally activated and this augmented activation is modulated by cGMP.     

The Na+/H+ exchanger modulates long-term potentiation in rat hippocampal slices

Although present in great variety in the brain, the role of Na⁺/H⁺ exchangers (NHEs) in hippocampal plasticity is still unknown and the effect of NHE inhibition on long-term potentiation (LTP) has not been studied yet. As it is conceivable that NHE inhibitors may severely affect mechanisms that are considered to underlie learning and memory we investigated whether the broad-spectrum NHE inhibitor 5′-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 μM) influences LTP induced by different stimuli based on a theta burst in interface hippocampus slices from 7–8-week-old Wistar and 30-month-old Fischer 344/Brown–Norway F1 hybrid (F344/BN) rats. EIPA did not affect basal synaptic transmission, paired pulse inhibition, or LTP induced by a weak stimulus, but improved the maintenance of the LTP of the population spike induced by a strong tetanus. Our data suggest that NHE activity serves as a negative feedback mechanism to control neuronal excitability and plasticity in both young and senescent animals.     

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