Dihydroouabain Research Articles

Electrogenic Na+/K(+)-transport in human endothelial cells.

Na+/K+ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca2+]i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na+] did not induce significant changes of [Ca2+]i. Superfusing the cells with K(+)-free solutions also did not significantly affect [Ca2+]i. Reapplication of K+ after superfusion of the cells with K(+)-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mumol/l dihydroouabain (DHO) and was therefore identified as a Na+/K+ pump current. During block and reactivation of the Na+/K+ pump no changes in [Ca2+]i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mumol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10-1000 U/l) did not affect the pump currents. An increase of the intracellular Na+ concentration ([Na+]i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na+]i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K+ was 2.4 +/- 1.2 mmol/l, and 0.4 +/- 0.1 and 8.7 +/- 0.7 mmol/l for Tl+ and NH4+ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than -150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular sodium activity and its regulation in guinea-pig atrial myocardium.

1. Intracellular Na+ activity (aNai) and membrane resting potential were studied in quiescent guinea-pig atrial and papillary muscles by means of Na(+)-sensitive and conventional microelectrodes. The effects of the cardioactive steroid dihydroouabain (DHO) on aiNa, force of contraction and sarcolemmal Na+, K(+)-ATPase activity were also investigated. 2. In thirty atria and twenty-two papillary muscles, aNai amounted to 8.0 +/- 0.2 and 4.7 +/- 0.3 mM, respectively (mean +/- S.E.M.). When both tissues were from the same animal, with the same ion-sensitive microelectrode mean aNai values of 7.9 +/- 0.2 and 5.1 +/- 0.5 mM (P < 0.01) were obtained from eight atrial and eight papillary muscles, respectively. 3. Membrane resting potentials (Em) were significantly (P < 0.001) more negative in the papillary muscles (-83.5 +/- 0.7 mV; n = 8) than in the atrium (-78.1 +/- 0.5 mV; n = 8). Deviation of Em from EK (determined by K(+)-sensitive microelectrodes) was 3.0 +/- 0.2 mV in ventricular (P < 0.05) and 6.1 +/- 0.3 mV in atrial preparations (P < 0.05). 4. Inhibition of the Na+ pump by DHO increased aNai of the atrium within 10 min by 0.6 +/- 0.1 (n = 7), 1.3 +/- 0.1 (n = 5) and 3.2 +/- 0.2 mM (n = 5) at 5, 10 and 30 microM, respectively. In the papillary muscle, 10 microM DHO was without effect while aNai rose by 1.0 +/- 0.1 (n = 5) and 2.9 +/- 0.2 mM (n = 6) at 30 and 120 microM DHO. 5. Consistent with the aNai measurements, the potency of DHO to increase force of the isometric contraction was three times higher in atrium than in papillary muscle (stimulation frequency 0.2 Hz). 6. Hydrolytic activity of sarcolemmal Na+,K(+)-ATPase isolated from atria amounted to only one third of that detected in ventricles (0.07 +/- 0.01, n = 6, versus 0.2 +/- 0.01 mumol phosphate released min-1 (g tissue)-1, n = 5). The inhibitory potencies of DHO on sarcolemmal Na+,K(+)-ATPase preparations were found to be identical in the enzymes from either tissue. 7. It is concluded that a lower Na+ pump density is responsible for the higher aNai and for the lower resting membrane potential in atrial as compared to ventricular cells. The regulation of cellular Na+ homeostasis in atrial muscle appears to be closer to the limits of its capacity than in ventricle, explaining the higher sensitivity of the atrium to interventions which impede Na+ pump activity.

Comparison of the inhibitory effect of ouabain and dihydroouabain on the Na +-K + ATPase from frog skin

1. The effects of ouabain and dihydroouabain (DHO) on Na +-K + ATPase from frog skin were investigated. 2. Ouabain seemed to be a more effective inhibitor, reaching near maximum inhibition at 10 −5 M while DHO reached near maximum at 10 −3 M. 3. Both inhibitors acted within the first few minutes, but ouabain inhibited more strongly over virtually the whole 2 hr considered. 4. Pre-incubation of the enzyme with ATP or Na + reduced the level of inhibition due to subsequent ouabain or DHO, but preincubation with K + did not.

Charge movement by the Na/K pump in Xenopus oocytes.

Pre-steady-state transient currents (1986. Nakao, M., and D. C. Gadsby. Nature [Lond.]. 323:628-630) mediated by the Na/K pump were measured under conditions for Na/Na exchange (K-free solution) in voltage-clamped Xenopus oocytes. Signal-averaged (eight times) current records obtained in response to voltage clamp steps over the range -160 to +60 mV after the addition of 100 microM dihydroouabain (DHO) or removal of external Na (control) were subtracted from test records obtained before the solution change. A slow component of DHO- or Na-sensitive difference current was consistently observed and its properties were analyzed. The quantity of charge moved was well described as a Boltzmann function of membrane potential with an apparent valence of 1.0. The relaxation rate of the current was fit by the sum of an exponentially voltage-dependent reverse rate coefficient plus a voltage-independent forward rate constant. The quantity of charge moved at the on and off of each voltage pulse was approximately equal except at extreme negative values of membrane potential where the on charge tended to be less than the off. The midpoint voltage of the charge distribution function (Vq) was shifted by -24.8 +/- 1.7 mV by changing the external [Na] in the test condition from 90 to 45 mM and by +14.7 +/- 1.7 mV by changing the test [Na] from 90 to 120 mM. A pseudo three-state model of charge translocation is discussed in which Na+ is bound and occluded at the internal face of the enzyme and is released into an external-facing high field access channel (ion well). The model predicts a shift of the charge distribution function to more hyperpolarized potentials as extracellular [Na] is lowered; however, several features of the data are not predicted by the model.

The kinetics of the inhibition by dihydroouabain of the sodium pump current in single rabbit cardiac Purkinje cells.

The kinetics of the inhibition by dihydroouabain (DHO) of the Na pump current were studied in isolated rabbit cardiac Purkinje cells by means of whole-cell recording. A fast exchange of the extracellular solution at the membrane of the cell studied was performed via two multi-barrelled pipettes nearby. Judging from the steady-state inhibition of the Na pump current at various DHO concentrations half maximal inhibition occurred at 1 x 10(-5) mol/l DHO in a medium containing 2 mmol/l K+ and at 3.5 x 10(-5) mol/l DHO in a solution containing 10.8 mmol/l K+. Assuming a reversible one-to-one binding reaction, the time course of DHO binding and unbinding was analysed under a variety of conditions. It was shown that the antagonistic effect of K+ on the inhibition of the pump current was entirely due to a decrease of the association rate constant of DHO binding to the sodium pump at higher K+ concentrations. The association rate constant amounted to 1 x 10(4) (mol/l)-1.s-1 at 2 mmol/l K+ and to 2.8 x 10(3) (mol/l)-1.s-1 at 10.8 mmol/l K+. The dissociation rate constant of DHO unbinding remained unchanged (approximately 0.06 s-1). The equilibrium dissociation constant KD for the inhibition of the pump current by DHO decreased by a factor 3 to 5 if the Na+ concentration of the patch pipette solution was augmented from 5 to 50 mmol/l. Increasing DHO concentrations inhibited the pump current increasingly faster and to a larger extent. The KD values derived showed little variation with the concentration of DHO applied.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular Na+ activity and positive inotropic effect of sulmazole in guinea pig ventricular myocardium. Comparison with a cardioactive steroid.

Recent studies suggest that inhibition of Na+,K(+)-ATPase may contribute to the positive inotropic action of the imidazopyridine sulmazole. Therefore, we investigated the effect of sulmazole and its stereoisomers and for comparison the effect of the cardioactive steroid dihydroouabain (DHO) on intracellular Na+ activity by means of Na(+)-sensitive microelectrodes. In the resting papillary muscle of the guinea pig, (+/-)-sulmazole increased intracellular Na+ activity (aiNa) within 15-20 minutes by 0.5 +/- 0.1 (n = 3), 1.3 +/- 0.1 (n = 7), 2.7 +/- 0.2 (n = 6), and 4.9 +/- 0.5 (n = 6) mM at 60, 100, 300, and 1,000 microM, respectively. (+)-Sulmazole was more effective than the racemate; aiNa was increased by 1.2 +/- 0.3, 2.1 +/- 0.3, and 4.0 +/- 0.2 mM at 60, 100, and 300 microM, respectively (n = 2 for each concentration). In the contracting papillary muscle (0.2 Hz), (+)- and (+/-)-sulmazole (600 and 1,000 microM) produced a maximum positive inotropic effect that exceeded that of DHO by 11% and 8%, respectively. As an inotropic agent, (+)-sulmazole was almost twice as potent as the racemate. The maximum direct inotropic effect of (-)-sulmazole (1,000 microM) amounted to only 14% of the DHO maximum and was, in contrast to the racemate and (+)-sulmazole, antagonized by 3 microM carbachol. (-)-Sulmazole did not affect aiNa.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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 Ipump inhibition by dihydroouabain (DHO) in guinea pig ventricular myocytes through use of wide-pore patch pipettes to "fix" internal Na+ activity at approximately 30 mM and to voltage clamp at -40 mV (T = 34 degrees C). DHO was found to have no effect on membrane conductance at a holding potential of -40 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 microM) 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 KD values of 0.05 microM and 64.5 microM. This is consistent with previously reported 3H-ouabain binding studies in guinea pig myocardium. These findings indicate that the electrogenic properties of the Na-K pump can be inhibited by glycoside binding to both high- and low-affinity sites.

Unaffected electrogenic Na-K pump activity in "diseased" human atrial fibers, as assessed by intracellular K+ activity.

To investigate the role of the electrogenic Na-K pump in the resting membrane of "diseased" or "depolarized" human atrial muscles, intracellular K+ activity (aiK) and resting membrane potential (Vm) were simultaneously measured using double-barreled K(+)-selective microelectrodes. Under perfusion with normal Tyrode's solution (37 degrees C) containing 5.4 mM [K]o, Vm averaged -43.9 +/- 1.4 mV, and aiK was 99.7 +/- 1.3 mM (mean +/- S.E., n = 33). The aiK was comparable to that of atrial muscles obtained from other intact mammalian species. In 5.4 mM [K]o, dihydro-ouabain (DHO) at concentrations of 10(-6) and 10(-5) M significantly decreased aiK and depolarized Vm. Similar decreases in aiK were observed when [K]o was decreased from 5.4 to 0.5 mM or when the temperature of the perfusing solution was decreased from 37 to 22 degrees C. Upon returning [K]o from 0.5 to 5.4 mM at 37 degrees C, aiK increased, Vm hyperpolarized markedly for about 3 min, and this was followed by less marked levels of hyperpolarization in the steady state. The high [K]o-induced increases in aiK were inhibited in the presence of DHO, and at low temperature (22 degrees C). Isoproterenol (10(-7) M) increased aiK and hyperpolarized Vm. Acetylcholine (10(-5) M) hyperpolarized Vm with no change in aiK. The rate of reduction of Na(+)-efflux during application of DHO (10(-5) M) was calculated based on the change in aiK and surface-to-volume ratio of the cell measured electronmicroscopically in the same tissue, and estimated to be 2.6 to 3.8 pmol/(cm2.s), close to the value reported for Purkinje fibers excised from intact animals. We conclude that the Na-K pump functions normally even in "diseased" human atrial muscles, thereby keeping aiK within a physiological range.

Voltage dependence of Na/K pump current in Xenopus oocytes.

Stage V and VI (Dumont, J.N., 1972, J. Morphol. 136:153-180) oocytes of Xenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3 mM K Barth's solution and measuring membrane current-voltage (I-V) relationships before and after the addition of 10 microM dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membrane I-V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (-40 mV). The resulting membrane I-V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pump I-V relationship must be free of hysteresis, the presence of hysteresis in DHO difference I-V curves can be used as a criterion for excluding such data from consideration as a valie measure of the Na/K pump I-V relationship. DHO difference I-V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90 mM) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0-1.5 microA cm-2, without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pump I-V relationships do not show a region of negative slope over the voltage range -110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump.

Voltage dependence of the Na-K ATPase: measurements of ouabain-dependent membrane current and ouabain binding in oocytes of Xenopus laevis.

Ouabain- or dihydroouabain(DHO)-sensitive membrane currents and binding of 3H-ouabain were investigated under voltage-clamp conditions in full-grown prophase-arrested oocytes of Xenopus laevis. (1) The ouabain-sensitive current is outwardly directed and usually exhibits a maximum at about +20 mV. The occurrence of the maximum is not affected by application of blockers for passive K+ currents. In the presence of Ba2+ as a K+ channel blocker, the KI value for inhibition of the Na-K ATPase by ouabain is reduced by an order of magnitude, the number of binding sites is not affected. In K+-free solution (which inhibits the normal reaction cycle of the Na-K ATPase), addition of DHO has no significant effect on the remaining currents. (2) The voltage-dependence of the ouabain-sensitive current can be modulated. Reduction of extracellular Na+ increases the pump current at the resting potential and reduces the positive slope of the I-V curve. Simultaneously, the number of binding sites for ouabain is reduced by about 25%. Seasonal variations of an unknown factor affect the negative slope. (3) Modulation of the voltage-clamp conditions has no effect on the number of binding sites for ouabain, on current inhibition by ouabain, or on ouabain binding at different concentrations of the inhibitor. It is concluded that the ouabain-sensitive current is not significantly affected by passive permeabilities and that its current-voltage dependence reflects the voltage dependence of current generated by the Na-K pump. Since ouabain binding, also at non-maximal binding concentrations, is not affected by membrane potential, steps that affect the probability of the E2P state should be voltage-insensitive.

Dihydroouabain, a reversible inhibitor of the sodium pump in frog skin

In many studies of the sodium pump in epithelia, a readily reversible analog of ouabain would be most useful. This would enable studies of pump activity to be made under control and experimental conditions on the same tissue. Of three compounds examined on the basolateral membrane of the isolated epithelia of frog skin, dihydroouabain (DHO) had characteristics very similar to ouabain except that it was apparently much more reversible. DHO (1 mmol/l) inhibited short circuit current (Isc) and transepithelial Na flux (JNa13) in a fashion similar to ouabain. Isc was inhibited from 17.0 +/- 2.5 to 10.2 +/- 1.0 microA/cm2 in 2-4 min while JNa13 was decreased from 16.8 +/- 1.9 to 4.7 +/- 0.8 microA/cm2 in the same time interval. After 60 min of washout, Isc and JNa13 recovered to about 70% of control values and were nearly equal. In another set of experiments, the washout of DHO and ouabain were compared directly on the same tissue. Sodium flux recovered four times faster after removal of DHO when compared to ouabain. Pretreatment of tissues with DHO prior to ouabain greatly increased the rate of Na flux recovery after washout of both drugs suggesting that DHO competes for ouabain sites. These data suggest that DHO can be used as a reversible analog for ouabain in studies of the Na pump in frog skin.

Properties of an electrogenic sodium‐potassium pump in isolated canine Purkinje myocytes.

1. Purkinje myocytes were isolated from canine Purkinje strands by collagenase exposure and gentle trituration. The myocytes were studied by a switched single-micro-electrode voltage-clamp technique at 37 degrees C in Tyrode solution containing 8 mM-K+ and 2 mM-Ca2+. 2. The dose-response relation for the cardiotonic steroid dihydroouabain (DHO) was obtained by measuring the change in membrane current caused by application of concentrations of 1-100 microM. The KD obtained in fourteen experiments was 3.7 +/- 1.1 microM (mean +/- S.E. of mean). 3. We employed 100 microM-DHO (a concentration more than 25-fold greater than the KD) to estimate the resting pump current (Ip) in the isolated myocytes. A value of 0.27 +/- 0.02 microA microF-1 (mean +/- S.E. of mean, n = 32) was obtained. 4. Myocytes were also exposed to K+-free solution for a period of 200 s. On return to K+-containing Tyrode solution there was a slowly decaying outward current. The time constant of decay of this pump current transient was 87 +/- 8 s (mean +/- S.E. of mean, n = 8). The integral beneath this transient was used to obtain a second estimate of the resting pump current. In four preparations where exposures in DHO and in K+-free solutions were employed the ratio Ip, DHO/Ip, K-free was 1.76 +/- 0.15 (mean +/- S.E. of mean). 5. From the magnitude of resting pump current, in the presence of total pump blockade the Na+ activity should rise at a rate of 1.3 mM min-1. 6. Reducing [K+]o from 8 to 1 mM reduced Ip by more than 40% initially. Ip then slowly increased over the next 30 min. These results suggest that the steady-state inward background current is not greatly altered by changes in [K+]o, and that [Na+]i rises to a new level. The changes in Ip obtained at early times following reduction of [K+]o to 1 or 0.5 mM (t less than 1.75 min) were used to estimate the Km for external K+; a value of 0.8 mM was obtained. 7. The results suggest that the properties of the Na+-K+ pump in isolated canine Purkinje myocytes are similar to those in canine Purkinje strands. This argues against major distortions of measured pump properties in the canine Purkinje strand and for the physiological state of the Na+-K+ pump in the isolated Purkinje myocyte.

Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes of Xenopus laevis.

Electrophysiological experiments were performed to analyze the Na+/K+-ATPase in full-grown prophase-arrested oocytes of Xenopus laevis. If the Na+/K+-ATPase is inhibited by dihydroouabain (DHO), the resting potential of the membrane of Na+-loaded oocytes may depolarize by nearly 50 mV. This hyperpolarizing contribution to the resting potential depends on the degree of activation of the Na+/K+-ATPase and varies with intracellular Na+ activity (aiNa) and extracellular K+ (K+o). It is concluded that variations of aiNa among different oocytes are primarily responsible for the variations of resting potentials measured in oocytes of X. laevis. Under voltage-clamp conditions, the DHO-sensitive current also exhibits dependence on aiNa that may be described by a Hill equation with a coefficient of 2. This current will be shown to be identical with the electrogenic current generated by the 3Na+/2K+ pump. The voltage dependence of the pump current was investigated at saturating values of aiNa (33 mmol/liter) and of K+o (3 mmol/liter) in the range from -200 to +100 mV. The current was found to exhibit a characteristic maximum at about +20 mV. This is taken as evidence that in the physiological range at least two steps within the cycle of the pump are voltage dependent and are oppositely affected by the membrane potential.

Inhibition of the sodium pump in guinea-pig ventricular muscle by dihydro-ouabain: effects of external potassium and sodium.

The inhibition of the electrogenic pump current in quiescent guinea-pig ventricular muscle by dihydro-ouabain (DHO) was studied with the three-micro-electrode voltage-clamp technique described previously (Daut, 1982c). From dose-response curves of the drug-induced current change (ID) the equilibrium dissociation constant of the binding of DHO to the Na-K pump (KD) and the electrogenic pump current flowing in the steady state (Ip) were inferred (Daut & Rüdel, 1982b). The external K concentration ([K]o) was varied between 2 and 4.5 mM (substituted by Na). KD was found to increase with increasing [K]o. A plot of log KD versus log [K]o gave a straight line with a slope of about 1.5. The time constants of the onset (tau on) and decay (tau off) of ID are supposed to represent the chemical kinetics of binding and unbinding of the drug (Daut & Rüdel, 1981, 1982b). Tau on was found to be inversely related to [K]o whereas tau off was found to be independent of [K]o. Ip was found to be independent of [K]o. This was interpreted to indicate that in the steady state Ip is mainly determined by the passive influx of Na into the cell, which may be relatively insensitive to small changes in [K]o. The effects of [K]o on the drug-induced current change are consistent with competitive inhibition of the binding of DHO to the Na-K pump. It is suggested that K ions and cardiac glycosides compete for extracellular binding sites on the same conformation of the Na-K pump. The external Na concentration ([Na]o) was varied between 147 and 49 mM (substituted by choline or Tris). Reduction of [Na]o produced a proportional decrease of Ip. This may be a consequence of the accompanying reduction of passive Na influx and the resulting decrease in intracellular Na activity (alpha iNa). Reduction of [Na]o markedly increased KD. This effect may be mediated by competition between Na and K at the K-loading sites of the pump and/or by separate modulatory Na-binding sites. It is concluded that the well known effects of external Na and K on the positive inotropic action of cardiac glycosides can be fully accounted for by the marked changes in the apparent binding affinity of the drug reported here.

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

1. 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 (I(D)) 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 I(D) were shown to be very small. From these findings and the results summarized below it was concluded that I(D) 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 of I(D) 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 of the drug to the Na-K pump under equilibrium conditions. From a Scatchard-type plot the equilibrium dissociation constant (K(D)) 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+/-0.26 muA/cm(2). It contributed 6.4+/-0.9 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 I(D) agreed with the chemical kinetics of binding and unbinding calculated for various DHO concentrations. The rate constant of unbinding (k(2)) was found to be 3.4 (+/-0.7) x 10(-2) S(-1) and the average rate constant of binding (k(1)) was 7.4 x 10(2) M(-1) S(-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: K(D) approximately 1.5 x 10(-6) M; k(1) approximately 4 x 10(3) M(-1) S(-1); k(2) approximately 6 x 10(-3) S(-1).8. An analysis of the transmembrane movements of Na and K in the steady state showed that the measured pump current density is consistent with a counter-transport of 3 Na and 2 K ions.

Relation between intracellular Na ion activity and tension of sheep cardiac Purkinje fibers exposed to dihydro-ouabain

The intracellular Na ion activity (aiNa) and the contractile tension (T) of sheep cardiac Purkinje fibers were simultaneously measured employing recessed-tip Na+-selective glass microelectrodes and a mechano-electric transducer. The aiNa of 6.4 +/- 1.6 mM (mean +/- SD, n = 56) was obtained in fibers perfused with normal Tyrode's solution. The changes in aiNa and T were measured during and after the exposure of fibers to a cardiac glycoside, dihydro-ouabain (DHO) in concentrations between 5 X 10(-8) M and 10(-5) M. The exposure time to DHO was 15 min. Both aiNa and T did not change in fibers exposed to 5 X 10(-8) M DHO, and the threshold concentration for the effect of DHO appeared to be around 10(-7) M. In DHO concentrations greater than the threshold, the increases in aiNa and T strongly correlated during the onset of DHO effects. The recoveries of aiNa and T were variable and slow, being dependent on the DHO concentration. In those fibers which recovered from the effects of DHO, the time-course of aiNa recovery was similar to that of T recovery. In fibers exposed to DHO of 5 X 10(-6) M or greater, the apparent toxic effects were observed in both action potential and contraction after an initial increase in T. The fibers manifesting the apparent toxic effects has a aiNa of approximately 30 mM or greater. The results of this study indicate that the increase in aiNa is associated with the positive inotropic action of the cardiac glycoside.

Decoupling of heart muscle cells: Correlation with increased cytoplasmic calcium activity and with changes of nexus ultrastructure

Purkinje fibers of the sheep heart were exposed to (a) 0.1mm dihydro-ouabain (DHO), followed by (b) 0.1mm DHO in Na-free solution or to (c) 1mm dinitrophenol (DNP). The degree of electrical decoupling was characterized in terms of the inside longitudinal resistancer i as measured with a 3-microelectrode voltage-clamp technique. Procedurea increasedr i by a factor of 3.7±1.1 (mean±sd),b by a factor of 9.8±2.2, whereas inc incomplete voltage control indicated nearly complete uncoupling. Intracellular calcium activity (aCa i ) was monitored with a microelectrode system. At control conditionsaCa i was below 0.1 μm. The procedures listed above increasedaCa i to (a) 4±1.5 μm, (b) 8±2 μm, and (c) 36±12 μm. The increase ofaCa i was in good correlation with the changes in core resistance. Effects on nexus ultrastructure, investigated with freeze-fracture techniques, are shown in histograms. At control conditions, the particle diameter distributed around a single peak (8.3±0.5 nm). Proceduresb andc induced a second population at 10.8 nm; increased decoupling reduced the control population in favor of the 10.8 nm population. Decoupling enlarged the width of the nexus gap by a factor of 1.6; again, the control population decreased in favor of a new population. In the decoupled state the height of the particle was smaller. Pits on the E-face displayed a more regular array and a nearly unchanged center-to-center spacing. Separation into several peaks was not possible due to scatter of the data. We interpret the findings to mean that elevatedaCa i induces a conformational change of the nexus subunits which corresponds to a transition from an open to a closed state. The conformational change can be formally described by a particle contraction which disrupts the continuity with the particle of the adjacent membrane. Purkinje fibers exposed to DNP for 1 hr showed thinned (7.7±0.5 mm) and elongated particles. We suggest that this is a secondary event and not a precursor of functional uncoupling.

Sarcolemmal sodium permeability and contractile force of guinea pig papillary muscle: effects of germitrine.

The action potential of guinea pig papillary muscle exposed to the ceveratrum alkaloid germitrine (2 mugM) is followed by a long-lasting after-depolarization (maximal amplitude, 8 mV; half-time of decay, 32 seconds; total duration, approximately 75 seconds). This after-depolarization interrupts the terminal phase of repolarization. During repetitive stimulation (0.1-1.0 Hz; 80 nM germitrine) the after-depolarizations that follow consecutive action potentials are summed, causing persistent depolarization of up to 10 mV. The after-depolarization is reversibly abolished by tetrodotoxin (TTX). Test contractions evoked at various times during or after the germitrine-induced after-depolarization reveal a phase during which the ability of the muscle to develop force is transiently increased. This positive inotropic influence reaches its maximum 1 minute after the conditioning stimulus and thereafter decays with a half-time 4.8 times longer than the half-time of decay of the after depolarization. It is reversibly abolished by TTX and augmented by dihydro-ouabain (DHO). We conclude: Germitrine induces an after-depolarization by prolonging dramatically the Na permeability component which is mediated by the fast Na channels and normally restricted to the first few milliseconds of the action potential. The germitrine-induced selective and persistent increase of sarcolemmal sodium permeability (PNA) causes a positive inotropic effect, probably because intracellularly accumulating Na ions exchange for extracellular Ca ions.

The effect of dihydro-ouabain and lithium-ions on the outward current in cardiac Purkinje fibers. Evidence for electrogenicity of active transport.

The effect of dihydro-ouabain (DHO), ouabain and Li+ on the membrane currents of short Purkinje fibers in response to depolarizing clamp steps (2.5 sec duration) was measured.