The electrogenic sodium pump in guinea‐pig ventricular muscle: inhibition of pump current by cardiac glycosides

Abstract

SUMMARY1. The inhibition of the electrogenic sodium pump in guinea-pig ventricular muscle by cardiac glycosides was studied with a voltage-clamp technique.2. Superfusion of the preparation with dihydro-ouabain (DHO) produced a reversible depolarization of up to 7 mV. When the membrane potential was clamped to a constant value near the resting potential application of DHO produced a corresponding current change in the inward direction which reached a steady state in less than 1 min.3. The drug-induced current change (ID) was found to be the result of a parallel shift of the current-voltage relation. The contributions of a change in extracellular K or intracellular Na to the measured ID were shown to be very small. From these findings and the results summarized below it was concluded that ID represents the blockage of the electrogenic pump current by DHO and that it is proportional to the number of drug molecules bound to the Na-K-ATPase in the intact cell.4. The dependence ofID on the concentration of DHO applied (5 x 10-6-8 x 10-4 M) was found to be consistent with the predictions of the law of mass action for reversible one-to-one binding ofthe drug to the Na-K pump under equilibrium conditions. From a Scatchard-type plot the equilibrium dissociation constant (KD) of DHO was determined to be 4-6 (± 2'3) x 10-5 M.5. The steady-state pump current in the resting preparation was calculated to be 0-81 ±026 #sA/cm2. It contributed 6-4±09 mV to the resting potential in Tyrode solution containing 3 mm-K.6. In the smallest preparations used the measured time course of the onset and decay of ID agreed with the chemical kinetics of binding and unbinding calculated for various DHO concentrations. The rate constant of unbinding (k2) was found to be 3-4 (± 0 7) x 10-2 s-1 and the average rate constant of binding (k1) was 7-4 x 102 M-1 -1 7. By comparing the effects of ouabain and DHO in the same preparation the following estimates of the chemical constants of ouabain binding to the Na-K pump were obtained: KD = 15 X 10-6 M; k, = 4 x 103 M-1 s-1; k2 = 6 x 10-3 s-1. * Present address: Abteilung fur Allgemeine Physiologie, Universitit Ulm.

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

“…No significant difference was found between the slopes as in the previous experiment indicating that the glycoside had no effect on steady-state membrane conductance at a holding potential of -40 mV. This finding agrees with earlier reports for intact guinea pig myocardium 5 and sheep Purkinje fibers.…”

“…120 - 21 In contrast, however, a previous study on the effect of DHO on electrogenic Na-K pump current in guinea pig ventricular tissue 5 reported that the concentration-inhibition relation can be fitted by a linear Scatchard plot suggesting that functionally significant binding occurs at sites with a single affinity (A^D=' 46 /iM) only. Similarly, despite findings of apparent binding of cardiac glycosides at both highand low-affinity sites in canine cardiac tissue, 2 -8 inhibition by DHO of electrogenic pump current reported in isolated canine Purkinje myocytes suggests that only binding at a single affinity site (Ap-3.7 fiM) is associated with Na-K pump inhibition.…”

“…For this analysis, the difference current was assumed to be proportional to the concentration of the DHO/Na-K pump complex. 5 Estimates of K D and V max were made using a nonlinear regression technique (BINKIN2, PROPHET Computer System; National Institutes of Health, Bethesda, Maryland). The curve depicted by the dashed line in Figure 4 plots the best-fitting theoretical doseresponse relation for a one-binding site model in which no cooperativity between binding sites is assumed (K D =234 /xM and V max =0.456 fiAJfiF, A=l).…”

Isoprenaline, Ca2+ and the Na(+)‐K+ pump in guinea‐pig ventricular myocytes.

“…Since there is net outward movement of one positive charge with each cycle, an outward (hyperpolarizing) current is generated. The steady-state Na+-K+ pump current was studied by selective blockade of the transport process using the cardiotonic steroid dihydroouabain (DHO) (Isenberg & Trautwein, 1974;Daut & Rudel, 1982;Cohen, Falk & Mulrine, 1987), chosen because of its rapid onset and reversibility. The rate of Na+-K+ transport is regulated by the intracellular sodium concentration, [Na+]i, and the extracellular potassium concentration, [K+]0.…”

Two functional Na+/K+‐ATPase isoforms in the left ventricle of guinea pig heart

“…One functional enzyme form may exist according to the following different approaches : the electrophysiological properties of isolated myocytes [4], the Na+/K+-ATPase activity [5,6], the [3H]ouabain-binding sites on microsomal preparations with low Na+/K +-ATPase activity [7], gel electrophoresis mobility and immunoblotting with specific antibodies [8, 91, and the force of isometric contraction of ventricular strips induced by ouabain [5]. The highly controversial aspect of these data is reflected by the scattering of the IC50 values: the apparent sensitivity of the enzyme (or site) to ouabain has been estimated as 0.130 pM 171, 2.2 pM [6] and 46 pM [4]. …”