Na+-H+ exchange in cardiac sarcolemmal vesicles

The transport of Na+ by a purified sarcolemmal vesicular preparation from canine ventricular tissue was studied as a function of both internal and external pH. The uptake of Na+ into sarcolemmal vesicles increased upon raising the extravesicular pH of the reaction medium. Half-maximal uptake of Na+ was observed at a pHo of about 8.1 and maximal uptake occurred at pH 8.6. The uptake of Na+ by sarcolemma was also dependent upon the intravesicular pH. Na+ uptake into sarcolemmal vesicles was greatly attenuated in the absence of a H+ gradient across the membrane. Transport of Na+ was potently inhibited by amiloride, a known blocker of Na+-H+ exchange. LiCl was also an effective inhibitor of Na+ transport. In the presence of optimal H+ gradients, Na+ uptake was linear for the first 5 seconds of the reaction and exhibited a Vmax of 290 nmol Na+/mg per min and a KNa of 3.5 mM. These experiments strongly indicate the presence of a Na+-H+ exchange system in cardiac sarcolemma. This activity appeared to be relatively specific for this membrane fraction. The identification of Na+-H+ exchange activity in a sarcolemmal vesicular fraction from the heart will permit extensive characterization of the regulation and kinetics of this antiporter in future investigations.     

Recent advances in cardiac glycoside-Na+,K+-ATPase interaction.

Na+,K+-ATPase has been purified from lamb kidney and consists of two polypeptide peaks on polyacrylamide gel electrophoresis with an enzyme activity of 1,000 mumole Pi/mg pro per hr. A scheme depicting the interaction of cardiac glycoside with the enzyme and ligand effects on binding has been constructed. Under all ligand conditions, ouabain binding tends to reach the same maximum if sufficient ouabain is present. Initial rates vary with ligand conditions. Using a chase method, the rate of dissociation of the glycoside from the enzyme is not influenced by the ligands present, although with separation of the enzyme-glycoside complex from the binding medium, differences are noted. The effect of ouabain on Na binding demonstrated two classes of sites, KD = 0.2 mM and KD = 18 mM. Denaturation decreased the high affinity sites. There was also a good correlation between ouabain binding and inhibition of Na binding. Clearly, ligands are critical in regulating cardiac glycoside interaction with the enzyme.     

Background K+ current in isolated canine cardiac Purkinje myocytes.

The current-voltage (I-V) relation of the background current, IK1, was studied in isolated canine cardiac Purkinje myocytes using the whole-cell, patch-clamp technique. Since Ba2+ and Cs+ block IK1, these cations were used to separate the I-V relation of IK1 from that of the whole cell. The I-V relation of IK1 was measured as the difference between the I-V relations of the cell in normal Tyrode (control solution) and in the presence of either Ba2+ (1 mM) or Cs+ (10 mM). Our results indicate that IK1 is an inwardly rectifying K+ current whose conductance depends on extracellular potassium concentration. In different [K+]0's the I-V relations of IK1 exhibit crossover. In addition the I-V relation of IK1 contains a region of negative slope (even when that of the whole cell does not). We also examined the relationship between the resting potential of the myocyte, Vm, and [K+]0 and found that it exhibits the characteristic anomalous behavior first reported in Purkinje strands (Weidmann, S., 1956, Elektrophysiologie der Herzmuskelfaser, Med. Verlag H. Huber), where lowering [K+]0 below 4 mM results in a depolarization.     

The slowing of Ca2+ signals by Ca2+ indicators in cardiac muscle.

The use of high-affinity fluorescent probes for monitoring intracellular free Ca2+ in cardiac muscle is now widespread. We have investigated the consequences of introducing intracellular buffers with the properties of Fura-2 or Indo-1 on the action potential, Ca2+ transient and contractile activity of the myocardium. Our theoretical results suggest that, at the high intracellular concentrations of these fluorescent probes used on occasion to improve the signal-to-noise ratio of the emitted fluorescence, modulation of action potential profile and attenuation of the amplitudes of the Ca2+ transient and contraction can occur, together with subtle changes in the kinetics of these events.     

Modulation of Ca2+ channel-gated Ca2+ release by W-7 in cardiac myocytes.

Cardiac muscle excitation-contraction coupling is controlled by the Ca(2+)-induced Ca2+ release mechanism. The present study examines the effects of a calmodulin antagonist W-7 on Ca2+ current (ICa)-induced Ca2+ release in whole cell-clamped rat ventricular myocytes. Exposure of cells to W-7 suppressed ICa, but the intracellular Ca(2+)-transients showed a lesser degree of reduction, suggesting possible enhancement of Ca(2+)-induced Ca2+ release. The effects of W-7 on the efficacy of Ca2+ release were most prominent at negative potentials. At test potentials of -30 mV, 20 microM W-7 almost completely blocked ICa, but significant Ca(2+)-transients remained, thus causing a four to six-fold increase in the efficacy of Ca(2+)-induced Ca2+ release. The depolarization-dependent Ca(2+)-transients were eliminated in absence of extracellular Ca2+, blocked by Cd2+, and were absent when the sarcoplasmic reticulum was depleted of Ca2+, implicating dependency on Ca(2+)-signaling between the L-type channel and the ryanodine receptor. W-7 mediated increase in the efficacy of Ca(2+)-induced Ca2+ release was eliminated when myocytes were dialyzed with the internal solution containing gluathione (5 mM), suggesting the possible role of cellular redox state in the regulation of Ca2+ release by the calmodulin antagonist.     

Inactivation of single cardiac Na+ channels in three different gating modes.

In small cell-attached patches containing one and only one Na+ channel, inactivation was studied in three different gating modes, namely, the fast-inactivating F mode and the more slowly inactivating S mode and P mode with similar inactivation kinetics. In each of these modes, ensemble-averaged currents could be fitted with a Hodgkin-Huxley-type model with a single exponential for inactivation (tauh). tauh declined from 1.0 ms at -60 mV to 0.1 ms at 0 mV in the F mode, from 4.6 ms at -40 mV to 1.1 ms at 0 mV in the S mode, and from 4.5 ms at -40 mV to 0.8 ms at +20 mV in the P mode, respectively. The probability of non-empty traces (net), the mean number of openings per non-empty trace (op/tr), and the mean open probability per trace (popen) were evaluated at 4-ms test pulses. net inclined from 30% at -60 mV to 63% at 0 mV in the F mode, from 4% at -90 mV to 90% at 0 mV in the S mode, and from 2% at -60 mV to 79% at +20 mV in the P mode. op/tr declined from 1.4 at -60 mV to 1.1 at 0 mV in the F mode, from 4.0 at -60 mV to 1.2 at 0 mV in the S mode, and from 2.9 at -40 mV to 1.6 at +20 mV in the P mode. popen was bell-shaped with a maximum of 5% at -30 mV in the F mode, 48% at -50 mV in the S mode, and 16% at 0 mV in the P mode. It is concluded that 1) a switch between F and S modes may reflect a functional change of inactivation, 2) a switch between S and P modes may reflect a functional change of activation, 3) tauh is mainly determined by the latency until the first channel opening in the F mode and by the number of reopenings in the S and P modes, 4) at least in the S and P modes, inactivation is independent of pore opening, and 5) in the S mode, mainly open channels inactivate, and in the P mode, mainly closed channels inactivate.     

Activation of L-type Ca2+ currents in cardiac myocytes by photoreleased GTP.

L-type calcium currents (ICa) were recorded from isolated ventricular myocytes by using standard patch-clamp methods. In the absence of agonist, photorelease of GTP by flash photolysis of intracellularly applied caged-GTP rapidly increased the amplitude of ICa over a wide range of membrane potentials. Control experiments clearly demonstrated that this effect was not due to either the release of photolytic by-products or to the light flash itself. The timecourse for activation of ICa by photolysis of caged-GTP was markedly altered by intracellular application of either GDP beta S or GTP gamma S. Upon maximal stimulation of ICa by intracellular dialysis with cAMP, photoreleased GTP induced a small, rapid increase in ICa followed by a gradual inhibition. The presence of Rp-cAMPS intracellularly reduced both the magnitude of the response to photoreleased GTP and its time to peak. Similar effects were observed when protein kinase inhibitor dialysed the cell interior, suggesting that both cAMP-dependent and independent processes were involved in this effect. We conclude that rapid release of GTP within ventricular myocytes, in the absence of agonist, causes rapid activation of L-type Ca2+ current. Mechanisms underlying this effect include stimulation of adenylate cyclase, together with other, as yet uncharacterized, GTP-dependent pathways for increasing ICa in the heart.     

Cyclic AMP phosphodiesterases and Ca2+ current regulation in cardiac cells.

At least four different isoforms of phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP in cardiac cells. However, their distribution, localization and functional coupling to physiological effectors (such as ion channels, contractile proteins, etc.) vary significantly among various animal species and cardiac tissues. Because the activity of cardiac Ca2+ channels is strongly regulated by cAMP-dependent phosphorylation, Ca(2+)-channel current (ICa) measured in isolated cardiac myocytes may be used as a probe for studying cAMP metabolism. When the activity of adenylyl cyclase is bypassed by intracellular perfusion with submaximal concentrations of cAMP, effects of specific PDE inhibitors on ICa amplitude are mainly determined by their effects on PDE activity. This approach can be used to evaluate in vivo the functional coupling of various PDE isozymes to Ca2+ channels and their differential participation in the hormonal regulation of ICa and cardiac function. Combined with in vitro biochemical studies, such an experimental approach has permitted the discovery of hormonal inhibition of PDE activity in cardiac myocytes.     

Inhibition of cardiac sarcolemmal Na+/H+ antiporter by opioids.

In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the opioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,488H, a selective kappa-opioid agonist, we characterized the nature of interaction between opioids and the Na+/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+ antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-50,488H with the Na+/H+ antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 +/- 0.2 to 5.0 +/- 0.3 mM, while the Vmax (52.0 +/- 5.0 nmol.mg-1.min-1) remained the same. These results suggest that U-50,488H and Na+ compete for the same site on the antiporter. When testing the effect of U-50,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na+/Ca2+ antiporter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride, we found that U-50,488H (IC50 = 100 +/- 15 microM) was threefold more potent than amiloride (IC50 = 300 +/- 20 microM) but it was three-fold less potent than the amiloride analog (IC50 = 30 +/- 10 microM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-50,488H is more potent than amiloride, the inhibitory characteristics of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter are similar to amiloride.     

Rapid purification of canine cardiac sarcoplasmic reticulum Ca2+-ATPase.

A pure, enzymatically active Ca2+-dependent adenosine triphosphatase (Ca2+-ATPase) has been isolated from canine ventricular sarcoplasmic reticulum. In contrast to that derived from skeletal muscle, the Ca2+-ATPase from cardiac sarcoplasmic reticulum was more active when solubilization and subsequent purification took place in the presence of its substrates, Ca2+ and ATP. Cholate- or deoxycholate-solubilized Ca2+-ATPase is recovered following rapid glycerol dilution and centrifugation. The Ca2+-ATPase is stable and possesses hydrolytic capacities up to 4 mumol/mg/min. Sodium dodecyl sulfate-polyacrylamide gels reveal the presence of one protein in the range of 95,000 to 100,000 daltons. This method also yields purified Ca2+-ATPase from fast skeletal muscle of similar activities to those reported by other laboratories.     

Force regulation by Ca2+ in skinned single cardiac myocytes of frog.

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.     

Voltage-dependent properties of three different gating modes in single cardiac Na+ channels.

Three different modes of Na+ channel action, the F mode (fast inactivating), the S mode (slowly inactivating), and the P mode (persistent), were studied at different potentials in exceptionally small cell-attached patches containing one and only one channel. Switching between the modes was independent of voltage. In the F mode, the mean open time (tau o) at -30 and -40 mV was 0.14 and 0.16 ms, respectively, which was significantly larger than at -60 and 0 mV, where the values were 0.07 and 0.08 ms, respectively. The time before which half of the first channel openings occurred (t 0.5), decreased from 0.58 ms at -60 mV to 0.14 ms at 0 mV. The fit of steady-state activation with a Boltzmann function yielded a half-maximum value (V 0.5) at -48.1 mV and a slope (k) of 5.6 mV. The mean open time in the S mode increased steadily from 0.12 ms at -80 mV to 1.09 ms at -30 mV, but was not prolonged further at -20 mV (1.07 ms). Concomitantly, t 0.5 decreased from 1.61 ms at -80 mV to 0.22 ms at -20mV. Here the midpoint of steady-state activation was found at -61.2 mV, and the slope was 8.7 mV. The mean open time in the P mode increased from 0.07 ms at -60 mV to 0.45 ms at 0 mV and t 0.5 declined from 2.14 ms at -60 mV to 0.19 ms at +20 mV. Steady-state activation had its midpoint at -14.7 mV, and the slope was 10.9 mV. It is concluded that a single Na+ channel may switch among the F, S, and P mode and that the three modes differ by a characteristic pattern of voltage dependence of tau 0, t 0.5, and steady-state activation.     

Formation of planar and spiral Ca2+ waves in isolated cardiac myocytes.

A novel Nipkow-type confocal microscope was applied to image spontaneously propagating Ca2+ waves in isolated rat ventricular myocytes by means of fluo-3. The sarcolemma was imaged with di-8-ANEPPS and the nucleus with SYTO 11. Full frame images in different vertical sections were obtained at video frame rate by means of an intensified CCD camera. Three types of Ca2+ waves were identified: spherical waves, planar waves, and spiral waves. Both spherical waves and spiral waves could initiate a planar wave, and planar waves were not influenced by the presence of a nucleus. Spiral waves, however, were consistently found adjacent to a nucleus and displayed a slower propagation rate and slower rate of increase in Ca2+ concentration in the wave front than did spherical and planar waves. The planar waves were apparent throughout the vertical axis of the cell, whereas spiral waves appeared to have a vertical height of approximately 3 microm, less than the maximum thickness of the nucleus (5.0 +/- 0.3 microm). These results provide experimental confirmation of previous modeling studies which predicted an influence of the nucleus on spiral-type Ca2+ waves. When a spontaneous Ca2+ wave is small relative to the size of the nucleus, it appears that the Ca2+ buffering by the nucleus is sufficient to slow the rate of spontaneous propagation of the Ca2+ wave in close proximity to the nucleus. These findings thus support the idea that the nucleus can influence complex behavior of Ca2+ waves in isolated cardiac myocytes.     

On the effect of unstirred layers on K+-activated electrogenic Na+ pumping in cardiac Purkinje strands.

Many studies of electrogenic Na+ pumping in Purkinje strands have involved intracellular Na+ loading by exposure to 0 mM K+, followed by reexposure to K+. For sheep Purkinje strands the K+ concentration for half-maximal stimulation (K0.5) in such studies is higher than K0.5 of canine Purkinje strands. A model was developed to determine if gradients in the K+ concentration of extracellular fluid layers during enhanced pump activity can account for the discrepancy. Pump activity was assumed linearly dependent on [Na+]i and dependent on [K+]o, according to Michaelis-Menten kinetics. The model simulated diffusion of K+ across unstirred layers and both depletion and accumulation of K+ in extracellular clefts of Purkinje strands during changes in the K+ concentration of the tissue bath. Errors in estimates of K0.5 occurred when delay in achieving a steady state extracellular K+ concentration was simulated. The simulations suggested that a linear relationship between pump current and intracellular Na+, a monoexponential decay of pump current, independence of the rate constants for the current decay on the initial Na+ load and holding potential, and apparent Michaelis-Menten K+ kinetics is not sufficient evidence against pump-induced interstitial K+ depletion having introduced errors in determination of K0.5. It is concluded that interstitial K+ depletion may account for the difference between determinations of K0.5 in sheep and canine Purkinje strands.     

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding.

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Blockade of cardiac sarcoplasmic reticulum K+ channel by Ca2+: two-binding-site model of blockade.

Potassium countercurrent through the SR K+ channel plays an important role in Ca2+ release from the SR. To see if Ca2+ regulates the channel, we incorporated canine cardiac SR K+ channel into lipid bilayers. Calcium ions present in either the SR lumenal (trans) or cytoplasmic (cis) side blocked the cardiac SR K+ channel in a voltage-dependent manner. When Ca2+ was present on both sides, however, the block appeared to be voltage independent. A two-binding site model of blockade by an impermeant divalent cation (Ca2+) can explain this apparent contradiction. Estimates of SR Ca2+ concentration suggest that under physiological conditions the cardiac SR K+ channel is partially blocked by Ca2+ ions present in the lumen of the SR. The reduction in lumenal [Ca2+] during Ca2+ release could increase K+ conductance.     

Na+/Ca2+ exchange in cardiac myocytes. Effect of ouabain on voltage dependence.

Sarcolemmal sodium/calcium exchange activity was examined in individual chick embryonic myocardial cell aggregates that were loaded with quin 2. The baseline [Ca2+]i was 68 +/- 4 nM (n = 29). Abrupt superfusion with sodium-free lithium solution produced a fourfold increase in steady-state [Ca2+]i to 290 +/- 19 nM, which was reversible upon sodium restitution. Other methods of increasing [Ca2+]i such as KCl-depolarization or caffeine produced a dose-dependent increase in quin 2 fluorescence, accompanied by sustained contracture. The [Ca2+]i increase in zero sodium was linear, and its half-time (t1/2) of 15.1 +/- 0.1 s was similar to that of the sodium-free contracture (t1/2 = 14.4 +/- 0.5 s) under the same conditions. The sodium-dependent [Ca2+]i increase was not significantly greater when potassium served as the sodium substitute instead of lithium. This suggests that sodium/calcium exchange has little voltage dependence in this situation. However, in aggregates pretreated with ouabain (2.5 microM), the [Ca2+]i increase was almost threefold greater with potassium than with lithium (P less than 0.007). Ouabain therefore potentiated the effect of membrane potential on calcium influx. We propose that elevation of [Na2+]i is a prerequisite for voltage dependence of the sodium/calcium exchange under the conditions studied. Sodium loading will then drastically increase calcium influx during the action potential while inducing an outward membrane current that could accelerate repolarization.     

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Although the enzyme (Na+ + K+)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na+ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na+ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na+ transport sensitively. This technique relies on the presence of Na+-Ca2+ exchange and ATP-driven Na+ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na+ uptake is monitored by a subsequent Nai+-dependent Ca2+ uptake reaction (Na+-Ca2+ exchange) using 45Ca2+. We present evidence that the Na+-Ca2+ exchange will be linearly related to the prior active Na+ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na+ isotopes. Applying this method, we measure cardiac ATP-dependent Na+ transport and (Na+ + K+)-ATPase activities in identical ionic media. We find that the (Na+ + K+)-ATPase and the Na+ pump have identical dependencies on both Na+ and ATP. The dependence on [Na+] is sigmoidal, with a Hill coefficient of 2.8. Na+ pumping is half-maximal at [Na+] = 9 mM. The Km for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na+ pumping. This approach should allow other new investigations on ATP-dependent Na+ transport across cardiac sarcolemma.