[Stimulation and inhibition of the sodium pump by cardiac glycosides] (original) (raw)

Cardiac glycosides: Correlations among Na+, K+-ATPase, sodium pump and contractility in the guinea pig heart

Naunyn-schmiedebergs Archives of Pharmacology, 1974

l~elationships among positive inotropic response to cardiac glycosides, l~a+,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 (JH)-ouabain binding in ventrieular homogenates, and cation pump activity from ouabain-sensitive S6Rb uptake of ventrieular 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~ for i0 rain 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 perfnsion, and subsequent loss during washout, also were accompanied by a reduction and subsequent recovery of S6gb 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.

Independence of the positive inotropic effect of ouabain from the inhibition of the heart Na+/K+ pump

Proceedings of the National Academy of Sciences, 1980

Isolated left atria from guinea pigs were stimulated at 3.3 Hz and bathed at 30'C in Tyrode's solution containing 6 mM KCL. After equilibration, this solution was replaced by a low-K solution or by Tyrode's solution containing ouabain or dihydroouabain. These treatments evoked an increase in the contractility of the atria. The time to peak increase was about 30 min, and the inotropic effect was sustained for at least 40 min. After 30 min, 42K was added to the bathing solution in order to estimate the activity of the Na+/K+ pump. A linear

Sodium requirement for effects of ouabain on contraction of isolated guinea pig atria

Circulation Research, 1980

Reducing the sodium content of medium bathing isolated guinea pig atria produces a positive inotropic response associated with a marked decline in action potential amplitude and a decrease in relaxation velocity. The effects of sodium reduction appear to result from a decline in the rate of sodium-dependent calcium efflux across the sarcolemma. Consistent with this hypothesis, caffeine, which may inhibit calcium uptake by sarcoplasmic reticulum, produces a much more pronounced inhibition of relaxation velocity in the absence than in the presence of sodium. Cardiac glycosides also appear to inhibit sodium-dependent calcium efflux, possibly by increasing intracellular free sodium. In the presence of sodium, ouabain increased developed tension in association with a decline in the velocity of relaxation. High concentrations of ouabain (>10 JIM) increased resting tension in quiescent atria. In the complete absence of sodium, ouabain (100 JIM) had no effect on resting or developed tension. When sodium was restored to previously sodium-free medium, resting tension declined. If atria in sodium-free medium were pretreated with ouabain or deprived of potassium, sodium restoration caused an increase in resting tension. These effects can be related to inhibition of sodium-pump activity by ouabain or potassium depletion. We conclude that both sodium reduction and cardiac glycosides increase myocardial tension development by inhibiting sodium-dependent calcium efflux across the sarcolemma. The contractile effects of ouabain are dependent completely on the presence of sodium. Circ Res 46: 553-564, 1980 SODIUM and calcium metabolism appear to be linked in cardiac muscle. Developed tension apparently is controlled primarily by the amount of calcium delivered to the contractile proteins. Luttgau and Niedergerke (1958) found that contractile strength in frog hearts did not depend solely on the extracellular concentration of calcium [Ca 2+ ] o , but varied with the ratio [Ca 2+ ] o /[Na + ]i Thus, a reduction in [Na + ] o had a positive inotropic action. These authors pointed out that this relationship might be the result of a carrier in the sarcolemma which can bind and transport either two sodium ions or one calcium ion. Na +-Ca 2+ exchange activity resembling that in cardiac muscle has been characterized extensively in squid axon (Baker et al., 1969). According to the carrier model [Ca 2+ ]i is proportional to [Ca 2+ ] o ([Na + ]i/[Na + ] o) 2. Contractile strength also varies as [Ca 2+ ] o /[Na + ] 2 in mammalian cardiac muscle (Reuter and Seitz, 1968). In addition to accounting for the positive inotropic effect of extracellular sodium reduction, this Na +-Ca 2+ exchange model also predicts that a positive inotropic response should result from an elevation of intracellular sodium ([Na + ];). Cardiac glycosides (>10~7 M) appear to produce sodium pump inhibition.

Role of Na-H exchange in the inotropic action of Bay K 8644 and of ouabain in guinea-pig isolated atria

British Journal of Pharmacology, 1990

1 The inotropic effects of two concentrations of ouabain and of Bay K 8644 have been studied in isolated left atria of the guinea-pig in physiological solutions at pH lowered from 7.4 to 6.0 and in the presence of ethylisopropylamiloride (EIPA) an inhibitor of Na+/H' exchange. The low concentration of ouabain (300nM) was chosen to saturate the high affinity binding sites (it occupied about 7% of the low affinity sites). The high concentration of ouabain saturated both high and low binding sites. Bay K 8644 evoked a positive inotropic effect of a magnitude similar to ouabain (300 nM). 2 When comparing the positive inotropic effects of equi-effective concentrations of ouabain (300 nM) and of Bay K 8644 (100 nM), it was observed that extracellular acidification specifically depressed the inotropic effect of ouabain 300nm; the positive inotropic effect of the high concentration of ouabain (3#M) was barely affected by extracellular acidification. 3 EIPA 10puM depressed the positive inotropic effect of ouabain 300nm, but did not affect the peak response to Bay K 8644. The depressant action of EIPA on the positive inotropic effect of ouabain was concentration-dependent and was much more obvious on the effect of ouabain 300nm than on ouabain 3pM.

FACTORS AND AGENTS THAT INFLUENCE CARDIAC GLYCOSIDE‐Na+, K+‐ATPase INTERACTION*

Annals of the New York Academy of Sciences, 1974

The optimal conditions for the binding of cardiac glycosides, such as ouabain, to Na+, K+-ATPase in vitro are the simultaneous presence of ATP, Na+ and Mg++ or the presence of inorganic phosphate (Pi) and Mg++ (Reference 1). It has been postulated that in vitro binding of ouabain in the presence of ATP, Na+ and Mg++ reflects the binding of cardiac glycosides t o this enzyme system in intact animals,2 and it is this interaction that ultimately results in the production of the positive inotropic and/or arrhythmic effects3 Thus, studies on the factors and agents that influence the cardiac glycoside-Na', K+-ATPase interaction in vitro appear important for an understanding of the factors and agents that modify the magnitude of response t o cardiac glycosides in patients when the plasma concentrations of cardiac glycosides are maintained at a fixed level Moreover, such studies may shed light on the mechanism of the Na' , K+-ATPase reaction itself. Monovalent Cations Na+, K+-ATPase has been shown t o be an allosteric enzyme and its configurations are determined by monovalent cation and phosphate ligand~.~-' Therefore, it is reasonable t o assume that these ligands affect the ouabain-enzyme interactions. The binding of cardiac glycosides t o Na+, K+-ATPase in vitro in the presence of ATP, Na+ and Mg++ is markedly inhibited by K+ (Reference 1). Since K+ has been shown t o reverse cardiac glycoside-induced arrhythmias,l2> it was once assumed that K+ reduces the level of cardiac glycoside bound t o the Na+, K+-ATPase, presumably by reducing the rate of binding, increasing the rate of dissociation, or both. Supporting this hypothesis, a number of investigators has reported that K+ antagonized the ouabain-inhibition of the Na+, K+-ATPase activity in vitro, although the evidence does not quite fit simple competitive inhibition for ouabain with respect t o KCl (see Reference 14). The steady-state levels of the enzyme-bond ouabain at a given drug concentration in the medium are determined by two independent variables, i.e., the rate of binding and the rate of release. These two parameters can be studied separately. FIGURE 1A shows the results of an experiment in which the rate of [ 3 H ] ouabain binding was monitored. Na+, K+-ATPase preparations obtained from rat brain microsomal fractions following deoxycholic acid and NaI treatments were incubated at 37OC with 0.01 pM [ 3 H ] ouabain (specific activity; 13.2 Ci/mmol) in the presence of 20 mM NaCl, 5 mM Tris-ATP, and 50 mM Tris-HC1 buffer (pH 7.5). Aliquots were taken at the indicated times and the bound

Inhibition of Na-K pump current in guinea pig ventricular myocytes by dihydroouabain occurs at high- and low-affinity sites

Circulation Research, 1989

Binding of cardiac glycosides to the Na + ,K +-dependent ATPase has been shown to occur at both high-and low-affinity sites. However, recent reports suggest that glycoside-induced inhibition of electrogenic Na-K pump current occurs with simple first-order binding kinetics at relatively low-affinity sites. This implies that high-affinity binding sites have little to do with Na-K pump inhibition during exposure to cardiac glycosides. To better understand the role of the high-affinity site, we investigated the concentration dependence of I p^ inhibition by dihydroouabain (DHO) in guinea pig ventricular myocytes through use of wide-pore patch pipettes to "fix" internal Na + activity at-3 0 mM and to voltage clamp at-4 0 mV (T=34° C). DHO was found to have no effect on membrane conductance at a holding potential of-4 0 mV. Holding current was monitored and the difference between steady-state holding current before and during external exposure to nine concentrations (range, 0.01-1,000 /iM) of DHO was measured and normalized to cellular membrane capacitance. The concentration dependence of the inhibition of Na-K pump current was biphasic and well fitted to a two-binding site model with inhibitory K D values of 0.05 fiM and 64.5 fiM. This is consistent with previously reported 3 H-ouabain binding studies in guinea pig myocardium. These findings indicate that the electrogenic properties of the Na-K pump can be inhibited by glyeoside binding to both highand low-affinity sites.

Comparison of [3H]ouabain binding sites in intact cells and cell homogenates: apparent lack of glycoside receptors unrelated to sarcolemmal Na+,K+-ATPase in guinea-pig heart

European Journal of Pharmacology, 1988

In intact heart muscle cells incubated in a physiological solution, i.e. under the condition in which the cardiac glycosides produce pharmacological and toxicological effects, receptors for these actions of the glycosides should be available to ouabain. In cell homogenates, [3H]ouabain binding observed in the presence of Mg 2+ and inorganic phosphate represents binding of the glycoside to Na+,K+-ATPase. Therefore, numbers of these two types of [3H]ouabain binding sites were compared using viable myocyte preparations obtained from ventricular muscle of guinea-pig heart. The number of ouabain binding sites observed in viable myocytes in the absence of Ca 2÷ and K + was not different from the number of ouabain binding sites on Na÷.K+-ATPase observed with sodium dodecylsulfate-treated homogenates prepared from isolated myocytes. These results do not support the hypothesis that there are receptors for the pharmacological or toxic actions of ouabain other than those that are associated with sarcolemmal Na+,K +-ATPase.

Intracellular sodium affects ouabain interaction with the Na/K pump in cultured chick cardiac myocytes

Journal of General Physiology, 1990

Whether a given dose of ouabain will produce inotropic or toxic effects depends on factors that affect the apparent affinity (K0.5) of the Na/K pump for ouabain. To accurately resolve these factors, especially the effect of intracellular Na concentration (Nai), we have applied three complementary techniques for measuring the K0. 5 for ouabain in cultured embryonic chick cardiac myocytes. Under control conditions with 5.4 mM Ko, the value of the K0.s for ouabain was 20.6 _+ 1.2, 12.3 +-1.7, and 6.6 -+ 0.4 #M, measured by voltage-clamp, Na-selective microelectrode, and equilibrium [SH]ouabain-binding techniques, respectively. A significant difference in the three techniques was the time of exposure to ouabain (30 s-30 rain). Since increased duration of exposure to ouabain would increase Nal, monensin was used to raise Na~ to investigate what effect Na~ might have on the apparent affinity of block by ouabain. Monensin enhanced the rise in Na content induced by 1 #M ouabain. In the presence of 1 #M [SH]ouabain, total binding was found to be a saturating function of Na content. Using the voltage-clamp method, we found that the value of the K0.5 for ouabain was lowered by nearly an order of magnitude in the presence of 3/zM monensin to 2.4 _+ 0,2 #M and the magnitude of the Na/K pump current was increased about threefold. Modeling the Na/K pump as a cyclic sequence of states with a single state having high affinity for ouabain shows that changes in Na~ alone are sufficient to cause a 10-fold change in K0.5. These results suggest that N~ reduces the value of the apparent affinity of the Na/K pump for ouabain in 5.4 mM Ko by increasing its turnover rate, thus increasing the availability of the conformation of the Na/K pump that binds ouabain with high affinity.

Isoform-specific Stimulation of Cardiac Na/K Pumps by Nanomolar Concentrations of Glycosides

The Journal of General Physiology, 2002

It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (I P ) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of I P because of the following: (1) it was absent in 0 mM [K ϩ ] o , as was I P ;